Inflammation and Epilepsy: New Vistas (Progress in Inflammation Research, 88) 3030674029, 9783030674021

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Progress in Inflammation Research 88 Series Editors: Michael J. Parnham · Achim Schmidtko Thorsten J. Maier

Damir Janigro · Astrid Nehlig Nicola Marchi Editors

Inflammation and Epilepsy: New Vistas

Progress in Inflammation Research Volume 88

Series Editors Michael J. Parnham Institute of Clinical Pharmacology Goethe University Frankfurt Frankfurt am Main, Germany Achim Schmidtko Institute of Pharmacology and Clinical Pharmacy Goethe University Frankfurt Frankfurt am Main, Germany Thorsten J. Maier Federal Institute for Vaccines and Biomedicines Paul Ehrlich Institute Langen (Hessen), Hessen, Germany

This book series addresses all key topical aspects of basic research, therapy and its clinical implications in the field of inflammatory diseases. It provides a unique reference source for academic and industrial biomedical researchers, drug development personnel, immunologists, rheumatologists, cardiologists, allergologists and many other relevant clinical disciplines. Each publication supplies regular scientific updates on newest developments and allow providing access to state-of-the-art techniques and technologies. The series gathers knowledge from leading authorities on the multiple facets of inflammation research, making it a valuable asset for advanced students in biomedical sciences, early career investigators and for professionals in both basic and translational research and in the clinic. Each volume comprises a carefully selected collection of high-quality review articles on the respective field of expertise. They also introduce new investigators to the most pertinent aspects of inflammatory disease and allow established investigators to understand fundamental ideas, concepts and data on sub-fields that they may not normally follow. Thus chapters should not comprise extensive data reviews nor provide a means for authors to present new data that would normally be published in peer-reviewed journals. Instead, the chapters should provide a concise overview and guide to the most pertinent and important literature, thus reflecting a conceptual approach rather than a complete review of the particular field of research. Moreover, each chapter should be intelligible for less experienced researchers or even newcomers to the fields of pathology, mechanisms and therapy of inflammatory disease. To this end, authors should consider introducing PhD students or postdocs who are new to the laboratory to the major concepts and the most critical literature in their chosen field of research. More information about this series at http://www.springer.com/series/4983

Damir Janigro  •  Astrid Nehlig  •  Nicola Marchi Editors

Inflammation and Epilepsy: New Vistas

Editors Damir Janigro Department of Physiology and Biophysics Case Western Reserve University Cleveland, OH, USA

Astrid Nehlig INSERM Strasbourg, France

FloTBI Inc. Cleveland, OH, USA Nicola Marchi Cerebrovascular and Glia Research, Department of Neuroscience Institute of Functional Genomics (UMR 5203 CNRS – U 1191 INSERM, University of Montpellier) Montpellier, France

ISSN 1422-7746     ISSN 2296-4525 (electronic) Progress in Inflammation Research ISBN 978-3-030-67402-1    ISBN 978-3-030-67403-8 (eBook) https://doi.org/10.1007/978-3-030-67403-8 © Springer Nature Switzerland AG 2021, corrected publication 2022 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

For many years, an inflammatory etiology for seizures was only considered for specific types of epilepsies. Today, there is sufficient evidence to suspect an inflammatory involvement in the majority of seizure disorders. This book presents the most current concepts on inflammation and epilepsy. This information is conveyed by top researchers and clinicians who have joined forces to produce a state-of-the-art and balanced view on this topic. The book is a must-read for clinicians and scientists involved in the understanding and treatment of epilepsy, but also for those who are interested in alternative therapies such as the ketogenic diet, vagal nerve stimulation, and immunomodulation not only for seizures with a known inflammatory or infectious cause but for all types of seizures. The chapter by Drs. Janigro and Marchi serves as an introduction to the book and a historical summary of today’s research hot topics. The authors also summarize what type of therapeutic approaches can be envisioned to treat seizure disorders, emphasizing non-pharmacological means of treatment. A distinction is also made to underscore the differences and commonalities between infection and inflammation as causes of seizures. Finally, this chapter introduces the central role of the blood-­ brain barrier (BBB) in epileptogenesis, ictogenesis, and anti-epileptic drug pharmacokinetics. The chapter by Dr. Vezzani et  al. focus on emerging molecular mechanisms of neuroinflammation in seizure disorders. Molecular players such as cytokines, HMGB1, COX-2, and their inhibitors, TGF-β, and circulating or resident immune cells are discussed in the context of pathways that may influence neuronal excitability. The chapter by Drs. Audinat and Rassendren describe how microglia and astrocytes become reactive in brain regions experiencing seizures in human or experimental epilepsies. The authors review how dysregulation of astrocyte and microglia physiological functions and the emergence of specific reactive states impact epilepsy progression. The chapter by Dr. Van Vliet et al. acknowledges the central role of the BBB in epilepsy. Dysfunction of the BBB occurs in several CNS pathologies, including epilepsy in which it can contribute to disease progression and resistance to therapeutic drugs. The authors discuss molecular mechanisms involved in BBB dysfunction in epilepsy, based on preclinical and clinical studies. Dr. Bauer v

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discusses the role of systemic inflammation in seizure susceptibility and interictal-­ ictal transitions. Again, the central role of the BBB is underscored; circulating cytokines and T-lymphocytes may modulate both neuronal excitability and BBB function. It is suggested that clinical trials testing anticonvulsant effects of immunomodulatory drugs will eventually be required to clarify to what extent systemic immune function influences interictal-ictal transition. The chapter by Drs. Granata and Matriciardi describes the clinical manifestations and known mechanisms of encephalopathies with seizures as a prominent symptom that respond to immunomodulatory therapies. New autoantigens involved in aberrant immune response have been discovered; it is thus possible that the number of epilepsies associated with an autoimmune response will increase over time. Recent clinical guidelines for the diagnosis of autoimmune encephalitis have been published. These guidelines encourage clinicians to start early immunotherapy while awaiting results for auto-antibodies testing in “possible” and “probable” autoimmune encephalitis and provide criteria for the diagnosis of “autoantibody-­negative but probable autoimmune encephalitis,” which may benefit from immunomodulation. Drs. Thom and Koepp focus on a common comorbidity of epilepsy, that is, cognitive decline. Neuropathology of surgically resected tissues from patients with drug-resistant epilepsy report age-accelerated pTau accumulation and variable levels of β-amyloid that have been compared with known tauopathies, including chronic traumatic encephalopathy. This links epilepsy to other tauopathies and dementias. Dr. Boison’s focus is the ketogenic diet in the treatment of epilepsies. The ketogenic diet is a high-fat, low carbohydrate metabolic intervention that has been in use for the treatment of epilepsy for almost a hundred years. This metabolic intervention affects epilepsy through multiple mechanisms, which combine to exert potent anti-inflammatory activity. His chapter focuses on novel disease-modifying properties of ketogenic diet therapy. The chapter by Drs. Xu and Miller describes how the immune response is a process tightly controlled by a system of checks and balances to prevent aberrant inflammatory damage. In their review, they discuss the emerging knowledge of CD4+FoxP3+ regulatory T cells (Tregs) in neurological disorders, recruitment of thymus-derived natural Tregs (tTreg) to the central nervous system (CNS), generation of peripherally derived antigen-specific Tregs (pTregs), Treg markers, and the immunosuppressive mechanisms of Tregs. Dr. Pitkänen et  al. describe the current knowledge on post-traumatic epilepsy (PTE). The authors focus on post-traumatic inflammation, which, in addition to being a major component of the pathophysiology of TBI, may also be a key player in the development of PTE. The players involved are members of the neurovascular unit, including the blood-brain barrier, microglial cells, NG2-glial cells, pericytes, and astrocytes, all contributing to the post-traumatic inflammation and the development of PTE. Cleveland, OH, USA

Damir Janigro

Strasbourg, France

Astrid Nehlig

Montpellier, France

Nicola Marchi

Contents

 Proand Anti-inflammatory Neurovascular Processes in Epilepsy: A Fragile and Dynamic Equilibrium����������������������������������������    1 Damir Janigro and Nicola Marchi  Emerging Molecular Mechanisms of Neuroinflammation in Seizure Disorders ����������������������������������������������������������������������������������������   21 Silvia Balosso, Annamaria Vezzani, and Teresa Ravizza  Glial Mechanisms of Inflammation During Seizures�����������������������������������   45 Etienne Audinat and François Rassendren  Perivascular Inflammation and Extracellular Matrix Alterations in Blood-Brain Barrier Dysfunction and Epilepsy ��������������������������������������   71 D. W. M. Broekaart, A. Korotkov, J. A. Gorter, and E. A. van Vliet  Blood T cells and Cytokine Levels During Interictal-Ictal Transitions������������������������������������������������������������������������������  107 Sebastian Bauer  Autoantibodies, Encephalopathies, and Epilepsy ����������������������������������������  125 Sara Matricardi and Tiziana Granata  Tau Protein in Drug-Resistant Epilepsy and Cognitive Decline������������������  149 Maria Thom and Matthias Koepp  Ketogenic Diet, Inflammation, and Epilepsy������������������������������������������������  185 Detlev Boison  Role of Regulatory T cells in Epilepsy ����������������������������������������������������������  203 Dan Xu, Sookyong Koh, and Stephen D. Miller

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 Inflammation at the Neurovascular Unit in Post-traumatic Epilepsy������������������������������������������������������������������������������  221 Xavier Ekolle Ndode-Ekane, Jenni Kyyriäinen, and Asla Pitkänen Correction to: Inflammation and Epilepsy: New Vistas. . . . . . . . . . . . . . .   C1

The original version of this book was revised: This book was initially published with incorrect ISSN numbers which has been corrected now. The correction to this book is available at https://doi. org/10.1007/978-3-030-67403-8_11

Pro- and Anti-inflammatory Neurovascular Processes in Epilepsy: A Fragile and Dynamic Equilibrium Damir Janigro and Nicola Marchi

Abstract  The relationship between epilepsy and inflammation dates back to 1958 when inflammation in the epileptic brain was first recognized in Rasmussen’s encephalitis. In the last two decades, preclinical and clinical studies provided growing evidence to support a link between brain or peripheral inflammation, seizures, and epileptogenesis. Inflammation is activated to restore tissue homeostasis and to initiate repair programs by the activation of pro-­resolving mediators. Inflammation is based on early activation of innate immunity mechanisms (e.g., macrophages), which may successively recruit adaptive immunity effectors (e.g., lymphocytes). An incomplete or defective resolution of this complex homeostatic mechanism can lead to chronic inflammation, histological damage, and autoimmunity. In the brain, the inflammatory response to injuries involves brain-­ resident cells and leukocyte recruited from the bloodstream in complex crosstalk tightly regulated by the blood-­­ brain barrier (BBB). Two distinct inflammatory processes have been linked to seizures: neuroinflammation and systemic inflammation. Seizures can be triggered by a pathogen or dysregulated sterile immunity. Neuroinflammation is an intrinsic brain response that involves the activation of innate immunity mechanisms in glia, neurons, and the microvasculature. Systemic inflammation, whose effects on the brain are primarily mediated by impairment of BBB, can induce neuron hyperexcitability through loss of ionic and neurotransmitter homeostasis. It is traditionally considered that activation of innate immunity and brain-­resident cells leading to neuroinflammation has a pivotal role in structural epilepsies. In contrast, a prominent role of adaptive immunity in triggering or perpetuating the inflammatory

D. Janigro Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA FloTBI Inc., Cleveland, OH, USA N. Marchi (*) Cerebrovascular and Glia Research, Department of Neuroscience, Institute of Functional Genomics (UMR 5203 CNRS – U 1191 INSERM, University of Montpellier), Montpellier, France e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_1

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response relates to epileptic encephalitis and systemic or neurological autoimmune conditions associated with the development of epilepsy. In this chapter we will ­summarize current knowledge on systemic and neuroinflammation and on the BBB which, when intact, separates the periphery from the brain. Keywords  Infection · Sterile inflammation · Peripheral immune cells · Glia · Blood-brain barrier · Autoimmunity · Traumatic brain injury

1  Sterile Inflammation and Infection Epilepsy is a CNS disease associated with epileptic seizures and other neurobehavioral outcomes of this condition. In the past decades, inflammation has emerged as an etiological factor for epileptogenesis and ictogenesis [1, 2]. Several brain insults or systemic events cause activation of a variety of inflammatory cells and processes. A quick review of the numbers of PubMed hits for “inflammation AND epilepsy” shows an almost exponential growth of publications starting around 2000 (Fig. 1). A similar profile is obtained when querying “inflammation AND seizures,” suggesting that seizures and epilepsy are used in this context as synonyms. We further

Fig. 1  PubMed hits (performed 9/2020) querying terms associated with epilepsy and inflammation. Please see text for description and significance

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queried for “pilocarpine AND seizures” or “pilocarpine AND epilepsy” and found overlapping citation profiles despite of the fact that many experimental approaches use only acute status epilepticus (SE) as an endpoint, while others track the animals for longer times until spontaneous epileptiform seizures ensue. A different result is achieved by querying “infection AND epilepsy” or “infection AND seizures.” First, a steady number of publications is evident since the late 1960s, with a nearly exponential increase starting in the 1980s. In addition, “infection AND seizure” gives a different profile compared to “infection AND epilepsy,” suggesting that these two pools of manuscripts focus on distinct clinical or experimental problems. For example, acute seizures in COVID-­19 patients have been reported [3], while chronic epilepsy may develop in patients with malaria [4] or cysticercosis [5] without an acute prodromal seizure. Regardless of the reasons for these differences, we used this example to introduce the well-­known clinical dissimilarity between inflammation and infection ([6] and Figs. 2 and 3). By reading the definition of inflammation in Wikipedia, we learn that “Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molecular mediators. Infection describes the interaction between the action of microbial invasion and the reaction of the body’s inflammatory response—these two components are considered together when discussing an infection.” Herein, the term “sterile inflammation” is used to define non-­pathogen-­driven immune responses. In this book, we primarily discuss inflammation as a process independent from an infectious cause; however, it is important to note that the players involved in sterile inflammation (microglia, monocytes, T and B cells, innate immunity) are the very same involved in fighting infection and that their brain counterparts (astrocytes, neurons) are liable of microbial invasion while also capable of releasing inflammatory mediators (e.g., cytokines) in response to pathogens or sterile insults

INFLAMMATION Sterile

Pathogen-driven Central Nervous System Peripheral

Peripheral

Neuroinflammation

Systemic inflammation Fig. 2  Graphic representation of pathways connecting inflammation to epilepsies

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Infection

TBI

PAMP

Acute seizure

CNS

Auto antigen unmasking

peripheral

CNS

peripheral

BBBD

Inflammation

BBBD Tissue damage

DAMP + Microglia activation + Neuronal cell death + GABA and glutamate plasticity + Changes in neuronal microenvironment

Immune cell activation Chronic BBBD

Autoimmunity

EPILEPSY Fig. 3  Relationship between infection, inflammation, and epilepsy. The pathways leading to blood-­brain barrier-­induced seizures are highlighted in red. Traumatic brain injury is used as an example of traumatic, noninfectious trigger. PAMP pathogen-­associated molecular pattern, DAMP damage-­associated molecular pattern

(Fig. 2). A remarkable feature of inflammation in epilepsy is that seizures can be caused by overactive immune activity in the absence of a pathogen and that seizures themselves become the trigger for additional dysregulated immune activation (Chapters “Emerging Molecular Mechanisms of Neuroinflammation in Seizure Disorders”, “Glial Mechanisms of Inflammation During Seizures”, “Blood T-­­cells and Cytokine Levels During Interictal-­­ ictal Transitions” and “Autoantibodies, Encephalopathies and Epilepsy”; Figs. 2 and 3 and [2, 6]). Pathogen-­induced seizures and epilepsy are very common. In concert with the contribution of both peripheral (or systemic) and neuroinflammation to sterile brain inflammation, the development of epilepsy has been linked to infections of the central nervous system (CNS) but recently also to infections and inflammation outside of the CNS [7]. Thus, the risk of being diagnosed with epilepsy is increased not only following CNS infections but also following exposure to a broad range of peripheral pathogens. Peripheral infection may indirectly affect the brain through the initiation of immunologic cascades and brain-­reactive antibodies (Figs.  2 and 3; Chapter

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“Autoantibodies, Encephalopathies and Epilepsy”), increasing the risk of epilepsy. Infections and systemic inflammation increase the permeability of the blood-­brain barrier (BBB; e.g., [8]), which can be seizure-­promoting [9] or translate into epilepsy [10] (see also below). Thus, while during infection the immune system may err and induce an autoimmune response trying to fight a pathogen, in sterile inflammation the trigger for misguided immunity resides in debris or nuclear material from damaged or dead cells (e.g., [11]. In addition, in the case of paraneoplastic syndromes, the autoantigen belongs to tissue that assumes a non-­self role during neoplastic differentiation [12–14].

2  Inflammation During Brain Development As discussed in several chapters in this book, peripheral and CNS events of inflammatory nature can be both ictogenic and epileptogenic. In addition, seizures can directly follow an insult or appear during a variable-­length latent period. These processes, albeit different, occur prenatally, after birth, and during brain development or during adulthood and aging. Maternal infections or experimental use of lipopolysaccharide (LPS) increases postnatal seizure propensity [15, 16]. Activation of astrocytes appears to be a key aspect of the observed pathology. In prenatal studies, however, it is not always possible to determine the relative maternal vs. fetal involvement or whether the infectious agent affects both. Postnatally, a crucial developmental event for epileptogenesis is the “GABA switch,” consisting of a transition from depolarizing GABA responses to the mature, hyperpolarizing form [17–21]. GABA is an inhibitory transmitter released by interneurons. Both feedback and feed-­forward inhibitory synapses have been described [22–24]. The inhibitory actions of GABA in the mature brain consist of a hyperpolarization carried by an inward chloride (GABA-­A) or outward potassium current (GABA-­B). Developmental changes in chloride brain homeostasis convert the GABA-­A response to an outward, depolarizing chloride current. Perinatal environmental perturbations affect the expression of chloride transporters, delaying the developmental switch of GABA signaling; inflammatory cytokines, in particular interleukin-­1β, represent a critical causal factor [25]. This is one of the mechanisms of immature brain hyperexcitability and propensity to seizure-­like phenomena such as spreading depression [20, 26]. Interestingly spreading depression of cortical activity is a trigger for inflammation [27] mediated by Toll-­like receptors [28].

3  Fever and Seizures A common event involved in seizure generation is fever, a factor known to lower seizure threshold in children. Apart from febrile convulsions that in most cases are a benign and self-­ limited condition, fever may precipitate seizures in genetic

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epilepsies (e.g., Dravet syndrome, PCDH19). The role of inflammatory mechanisms in inducing hyperexcitability has not been definitively proven in these conditions. There are two more conditions in which fever is consistently associated with the dramatic onset of seizures in a previously healthy and that evolve into chronic drug-­­ refractory epilepsy: idiopathic hemiconvulsion-­ hemiplegia-­ epilepsy syndrome (IHHES) and febrile infection-­related epilepsy syndrome (FIRES). In IHHES after the unilateral SE, the child develops atrophy of one hemisphere, contralateral hemiparesis, and epilepsy. FIRES is a condition due to multifaceted etiology, characterized by the sudden onset of recurrent seizures, which evolve into super-­refractory status epilepticus in the turn of hours or few days [29, 30]. Following the acute phase, patients often develop a drug-­resistant type of epilepsy; a common prognosis is a cognitive disability. The period of ictal behavior typically follows a febrile respiratory or gastrointestinal infection. Routine diagnostics serology, cerebrospinal fluid analysis, and MRI are unremarkable in most patients [31, 32]. There is a distinct lack of typical inflammatory markers in the early phases of disease, nor obvious inflammation has been reported in the few postmortem studies on brain specimens. Nevertheless, the prodromal febrile stage, together with the evidence in patients of functional deficiency in endogenous interleukin-­1 receptor antagonist (that results in overactivity of IL-­1β, an important player in systemic inflammation and innate immunity), led to hypothesize that FIRES is a postinfective disease due to imbalance of pro-­and anti-­­ inflammatory mediators. The release of pro-­inflammatory agents (cytokines, chemokines, and danger signals) not properly hampered by anti-­inflammatory mediators activates the innate immune system, with the ensuing chain of events, including the release of brain-­derived pro-­inflammatory molecules. Based on these rationales, the use of anakinra, a recombinant interleukin-­1 receptor antagonist, seems to be a promising targeted treatment [29, 33].

4  BBB, Inflammation, and Epilepsy The BBB is a key component of the neurovascular unit (NVU; [34]; Chapters “Emerging Molecular Mechanisms of Neuroinflammation in Seizure Disorders”, “Perivascular Inflammation and Extracellular Matrix Alterations in Blood-­­Brain Barrier Dysfunction and Epilepsy” and “Inflammation at the Neurovascular Unit in Post-­­Traumatic Epilepsy”). As already mentioned and as shown in Fig.  3, BBB disruption (BBBD) may be a cause or consequence of seizures (for review, see [35–37]). Early interest in the BBB in epilepsy was related to its role in drug resistance and as the interface of GLU-­1 expression and glucose transport to the brain [36, 38–40]. The general idea (promoted in a well-­cited yet non-­referenced statement in [41]) was that ~98% of small molecules do not cross the BBB. This number, however, relates to estimates which may be further complicated by conditions brain disease such as brain edema [42], which can significantly affect brain penetration of small-­molecule AED [43–45]. In spite of the commonly believed dogma, brain

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levels of free lipophilic AED do not necessarily increase after BBB disruption as independently shown in experimental models and patients [42, 46, 47]. Thus, the role of a leaky BBB in drug delivery to CNS needs to be further understood. The mechanism of seizure-­induced BBB alterations is similarly not fully understood (Chapters “Emerging Molecular Mechanisms of Neuroinflammation in Seizure Disorders”, “Perivascular Inflammation and Extracellular Matrix Alterations in Blood-­­Brain Barrier Dysfunction and Epilepsy”, “Blood T-­­cells and Cytokine Levels During Interictal-­­ictal Transitions” and “Role of Regulatory T cells in Epilepsy”). Reactive oxygen species (ROS), matrix metalloproteinases (MMPs), angiogenic factors, inflammatory cytokines, autoantibodies, leukocyte adhesion, and immune cell extravasation have all been proposed as culprits [1, 6, 35, 48–52]. Regardless of the trigger for BBB disruption, studies have shown that serum albumin readily accumulates in the brain (Fig. 4). In addition, it has been shown that albumin acts as a pro-­epileptogenic factor by activation of a TGF-­β pathway in astrocytes [10, 53–55]. In addition to causing BBB dysfunction, seizures may induce, by an NMDA-­dependent mechanism, abnormal expression of AED transporters [56]. From the inflammation viewpoint, it is important to note the COX-­2 inhibitors were capable of reverting an abnormal expression of P-­glycoprotein (P-­ gp). The complex interactions between seizures and multiple drug resistance were recently reviewed [35]. In addition to the incorrect orthodoxy that predicts that a “leaky” BBB facilitates the passage of small-­molecule AED [42, 46, 47], it is widely believed that BBB disruption alone will lead to an increase in water entry into the brain (Fig.  4). However, the total osmolarity of blood and cerebrospinal fluid are equal (289 mOsm/L; [57]) which could not produce the dramatic movement of water into the brain that is characteristic of vasogenic edema. There are at least two possible mechanisms by which water moves into the brain parenchyma after BBBD: (1) K+ moves down its concentration gradient from blood into the brain resulting in a K+ concentration that is sufficient to depolarize neurons, trigger action potentials, and drive repolarization further elevating cerebral K+ levels [58]. This high K+ may lead to disruption of the osmotic homeostasis between the brain and blood, causing water to move into the brain. (2) Cellular damage from a traumatic insult or axonal degeneration (Chapter “Tau-­­ Protein in Drug Resistant Epilepsy and Cognitive Decline”) results in intracellular protein release into the brain parenchyma. As extracellular protein levels are normally kept low in the brain, the addition of such a large amount of protein would perturb the osmotic balance between the brain and blood, resulting in water influx into the brain. Yet another dogma predicts that leukocyte entry into CNS occurs when the BBB is breached. We have shown that BBBD protocols per se do not allow cell entry, which requires expression of adhesion molecules and endothelial activation [59, 60]. In other words, from the standpoint of drug delivery, water homeostasis, and leukocyte extravasation, the BBB is not a mechanical “door” that can be open or closed, but rather a complex cellular lining separating two entirely different environments. Leukocyte extravasation does, however, occur, and the consequences in epilepsy can be devastating. While a short-­lived “leak” does not translate into influx, changes

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Fig. 4  Cartoon depicting the proposed fallacy of common assumption on the role of the BBB in epilepsy. Under normal condition, top panel, the BBB promotes accumulation of a lipophilic, small-­molecule AED. This is achieved by the dissociation of protein-­bound AED (e.g., phenytoin [42, 47]) into free drug, which owing to its lipophilicity freely crosses the BBB. In contrast, both albumin (Alb) and albumin + AED cannot leave the vascular compartment. Similarly, inflammatory cells are restricted from extravasation by BBB endothelial cells. Water equilibrates across the BBB because the brain and vascular compartment have similar osmolarity. BBB disruption (middle panel) allows albumin and albumin + AED entry into the CNS where albumin is taken up by astrocytes. Neuronal activity is only slightly increased (synaptic response) by influx of vascular (5 mM) potassium in the brain (where potassium is ~2–3 mM). When the BBB and circulating blood cells become activated and adhesion molecules are expressed (bottom panel), cell extravasation occurs. In addition, epileptiform activity increases brain osmolarity allowing vasogenic edema to ensue

in endothelial cells and circulating leukocytes allow for the phenomenon of cell entry studied in-­depth in multiple sclerosis [61–66]. While BBBD can become ictogenic without apparent influx of circulating cells in clinical and experimental models [9, 67], it is widely accepted that lymphocytes play an essential role in epileptogenesis. Chapters “Blood T-­­cells and Cytokine Levels During Interictal-­­ ictal Transitions” and “Autoantibodies, Encephalopathies and Epilepsy” focus on these aspects of inflammation in epilepsy.

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5  Traumatic Brain Injury Traumatic brain injury (TBI; Chapter “Inflammation at the Neurovascular Unit in Post-­­Traumatic Epilepsy”) is an excellent example of a noninfectious event that can cause both acute ictogenesis and delayed epileptogenesis [68–74]. Traumatic brain injury (TBI) occurs in as many as 64–74 million people worldwide each year [75]. TBI severity ranges from mild to severe and may be followed by posttraumatic sequelae, including depression, cognitive, emotional, and behavioral deficits. TBI may also cause posttraumatic seizures (PTS), increase seizure susceptibility, and increase the incidence of epilepsy, a phenomenon known as posttraumatic epilepsy (PTE). Epidemiological data support a link between traumatic brain injury (TBI) and seizures. TBI accounts for approximately 16% of acute symptomatic seizures, which usually occur in the first week after trauma. Children are at higher risk for posttraumatic seizures than adults and experience greater morbidity and mortality from cerebral edema, which results from inflammation [76, 77]. To study how TBI leads to changes in neuronal excitability in human subjects, one needs to focus on the fact that posttraumatic seizures refer only to seizures that occur after TBI and are caused by TBI. Exacerbation of preexisting seizures is not a good clinical example of post-­TBI seizures. Another important preamble is the acknowledgment that the temporal relationship between the traumatic event and seizures is a key factor in the underlying mechanisms of ictogenesis and epileptogenesis. Early posttraumatic seizures (2–5% of all cases in mild TBI (mTBI); 10–15% in severe TBI [76, 78, 79]) are likely different in mechanism from late seizures and are defined as occurring within 1 week of trauma. Late seizures are most common after penetrating, war-­related events (53% in Vietnam vets with penetrating TBI [79]). Additionally, 82% of individuals who experience a late posttraumatic seizure will have another seizure within a year. This suggests that patients should be treated aggressively with anticonvulsant medication after a first unprovoked late seizure [80]. However, factors unrelated to TBI are at play in this population; these include infection, presence of foreign material in the brain parenchyma, uncontrolled bleeding, etc. Recurrent seizures are chronic events that occur many months or years after TBI; whether these events are due to or consequence of late or early seizures remains unclear. In contrast, most veterans in Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) who suffered from TBI did so from blast injury. The diagnosis of posttraumatic epilepsy was clinically confirmed only in a few veterans. On the other hand, the diagnosis of posttraumatic stress disorder (PTSD) was confirmed in 81% of the sample, and a diagnosis of psychogenic non-­epileptic seizures (PNES) was suspected in 44% of the sample [81]. While for PTE a neuroinflammatory response is supported by mounting evidence from human and animal studies support, a similar role in PTSD and PNES is only suspected. Although early seizures that occur within days from TBI are often managed by typical antiepileptic drugs (e.g., levetiracetam and phenytoin), these do not seem to decrease the risk to develop PTE [80].

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Recurrent spontaneous seizures in PTE are resistant to antiepileptic treatments in about one-­third of patients [82]. Experimental models and recent clinical research have focused on inflammation as a mechanism of epileptogenesis after TBI [69, 74, 83–86]. In particular, the role of BBB dysfunction has been shown to play a pivotal role [51, 87–93]. Virtually all brain cell types are involved in posttraumatic epileptogenesis. For example, it has been shown that astrocytes respond to trauma by altered expression of potassium buffering [94], glutamate uptake ([95], also involved in PTSD [96]), and an array of pathological changes (see [52, 97, 98]). Microglia activation most likely occurs in response to various pro-­inflammatory cytokines and chemokines and the release of danger-­associated molecular patterns (DAMPs, Fig. 2) by damaged cells [99]. Microglia also display abnormal polarization after TBI [100, 101], while neurons display a range of neurophysiological changes including loss of long-­term potentiation [102], hyperexcitability and synchronization, and lowering of seizure threshold [69, 84, 85, 103–107].

6  Therapeutic Concepts Developing new anti-­seizure solutions is clinically significant, considering drug-­­ resistant subjects and the occurrence of unwanted side effects in those who respond to antiepileptic drug therapy. Overwhelming experimental evidences point to inflammation as a prime therapeutic target in epilepsies. A decade or so ago, we suggested that several antiepileptic maneuvers commonly employed in the clinic were actually useful even in pathologies without a frank inflammatory component at least in part because of their anti-­inflammatory actions [2, 108–112]. Since then, targeting or counteracting specific pro-­inflammatory cytokines, chemokines, and their receptors has been examined as an approach to manage seizure burden in a broad range of seizure disorders. Furthermore, animal models of multiple drug-­­ resistant (MDR) seizures, together with clinical evidence for the presence of neuroinflammation in MDR epilepsy, support the hypothesis that drugs that modulate inflammatory pathways may overcome the problem of pharmacoresistant seizures [50]. We proposed that multiple drug resistance to antiepileptic drugs may be a pathology on its own, which coexists with a seizure disorder [43]. If this were the case, it would be important to develop treatments that revert an MDR patient into a drug responder. Given that the BBB plays a role in both seizure generation [9] and drug resistance [44], targeting BBB dysfunction with anti-­inflammatory drugs has been proposed as means to treat multiple drug-­resistant seizures even in pathologies where an inflammatory etiology is not suspected [2, 109, 110, 112]. Historically, AEDs have always been considered as compounds acting on the CNS.  In fact, the first AEDs (bromide and barbiturates) both act as agonists of GABA-­A chloride currents, albeit with different mechanism. More modern AEDs have targeted mechanisms of neuronal excitability, such as Na+ currents. The increasing number of non-­neuronal pharmacological strategies applicable to seizure disorders includes new and repurposed drugs, with clear anti-­inflammatory effects

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[113]. A proposed use for anti-­inflammatory or BBB-­repairing molecules is in association with neuronal antiepileptic drugs. We here provide a few examples based on recent literature. However, in addition to these “neurocentric” approaches, old remedies for seizures that are popular today have also a long history. The ketogenic diet (Chapter “Ketogenic Diet, Inflammation and Epilepsy”) is a high-­ fat, low-­­ carbohydrate metabolic regime that has been in use for the treatment of epilepsy for almost a hundred years. This metabolic intervention affects epilepsy through multiple mechanisms [30, 114–116], also including a potent anti-­inflammatory activity [117]. Another example of a nontraditional antiepileptic treatment is the vagal nerve stimulation (VNS; [118]). The vagus does not innervate cranial regions [119], making it an unlikely modulator of CNS function. Nevertheless, the first use of VNS was in the late 1900 to treat epilepsy which was believed to be due to excessive cerebral blood flow [119]. The vagus (X cranial nerve) is a perfect example of bidirectional communication between the CNS and periphery. In fact, it connects its nuclei (located in the CNS) with virtually every thoracic and abdominal organ. This bidirectional communication is made possible by efferent and afferent fibers, involved diverse physiological functions such as the control of sinoatrial (SA) node pacing, digestion, and vocalization. Among the several neurophysiological mechanisms affected by VNS, cholinergic, adrenergic/noradrenergic, as well as GABA-­and glutamatergic receptors are involved. However, in spite of a broad range of actions on neurons and other excitable cells (e.g., SA node myocytes), vagal stimulation also affects a substantial level of control on systemic immunity. By cooperating with sympathetic fibers at the level of the celiac ganglia, the vagus indirectly controls the splenic nerve. The splenic nerve terminals are closely juxtaposed to macrophages, B cells, and T cells. The spleen is ideally positioned within the circulatory system to detect, respond to, and protect against blood-­ borne antigens, damaging cytokine mediators from immune activation. The anti-­inflammatory response by vagal stimulation is mediated via α7ChR (α7 nicotinic cholinergic receptor), and mice lacking the α7 nicotinic receptor (α7ChR) subunit fail to generate an anti-­inflammatory response to intraperitoneal challenge with LPS [120]. However, vagally mediated suppression of LPS-­induced inflammation requires T lymphocytes, as vagal stimulation fails to suppress inflammation in nude mice, but can be partially restored by the adoptive transfer of T lymphocytes that produce acetylcholine (Ach) into nude mice [120]. Current knowledge suggests that VNS act as an antiepileptic treatment acutely because of its indirect actions on CNS neurons and at steady state as a neuromodulator [118, 119, 121]. Glucocorticoids, which activate a broad range of inflammation-­limiting mechanisms, are used in clinical cases of epileptic syndromes, encephalitis, or selected instances of drug-­resistant epilepsy as add-­on therapy [110, 122–124]. Nonetheless, glucocorticoid therapy bears significant side effects limiting their long-­term or continuous use [125]. Reports have indicated the beneficial effect of enhancing endogenous anti-­inflammatory mechanisms, specifically circulating T-­regulatory cells in temporal lobe epilepsy (Chapters ““Blood T-­­cells and Cytokine Levels During Interictal-­­ictal Transitions” and “Role of Regulatory T cells in Epilepsy”; [126])

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and the role of pro-­resolving cell membranes lipid to target experimental epileptogenesis [127]. Furthermore, the anti-­ inflammatory annexin-­ A1/glucocorticoid receptor system appears to be defective in experimental temporal lobe epilepsy. The latter results are interesting as approaches are needed to bypass glucocorticoid side effects as occurring in specific epileptic encephalopathies or drug-­resistant seizures, with the ultimate goals of a robust steroid-­receptor sparing targeting of inflammation [128]; this is also important to avoid glucocorticoid receptor desensitization [128–130]. As mentioned above, another strategy to reduce inflammation and restore brain homeostasis is to repair a “leaky” BBB [91, 109, 131]. Convincing evidence shows that increased BBB permeability is icto-­and epileptogenic [9, 51, 92, 132, 133]; conversely, seizures cause BBB damage ([1, 91]; Figs. 2 and 3). For instance, losartan, an antihypertensive molecule, was shown to reduce BBB permeability following experimental status epilepticus [134]. Anakinra, the recombinant form of the human IL-­1 receptor antagonist, afforded febrile seizure protection [29], with a possible experimental mechanism targeting the BBB [112]. Furthermore, the molecule IPW-­5371, a blocker of the transforming growth factor-­β receptor activated by downstream molecular events after BBB disruption, decreased hyperexcitability in a model of epilepsy, protecting BBB function [53]. Platelet-­derived growth factor subunits BB protect the endothelium-­pericyte structures in mouse models of status epilepticus [135]. Collectively, these data outline the contemporary research effort for developing pharmacological strategies targeting all the neuro-­glio-­vascular cells in seizure conditions. An obvious issue, mentioned earlier in this chapter, is the penetration across the BBB for AED. The introduction of the concept of inflammation as a trigger of icto-­ and epileptogenesis may, paradoxically, decrease the relevance of drug penetration in the brain. On the one hand, penetration of novel anti-­inflammatory antibodies (e.g., the immunoglobulin-­sized natalizumab) is facilitated by paracellular leaks in the BBB as seen in epilepsy. This is not in contrast to Fig. 4, which refers to small-­­ molecule, protein-­bound AED. Immunoglobulin entry into the brain has been used extensively to histologically map BBB disruption in epilepsy, and therapeutic monoclonal antibodies or other IgGs likely have an identical pharmacokinetic profile. On the other hand, and most importantly, if maintenance of epilepsy or initiation of seizures depends on systemic inflammation, there is no necessity for drugs to enter the brain. Examples are the IL-­1 receptor antagonist anakinra and the corticosteroid dexamethasone. Both are useful in the treatment of certain epilepsy or in the case of dexamethasone and ACTH of non-­inflammatory epilepsies, but neither has decent brain penetration. Anakinra is virtually impermeant to abnormal BBB (0.02% of plasma levels; [136–138]), and its action may be directed to peripheral Il-­1β [112], while dexamethasone is a P-­glycoprotein substrate with similarly poor brain penetration [139]. This is perhaps in specular analogy to the finding that the commonly used seizure-­promoting agent pilocarpine does not need to enter the brain to evoke status epilepticus, but rather acts on cholinergic immune synapses in the periphery [110, 112, 140, 141].

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In conclusion, understanding the role of peripheral and CNS inflammation on seizures and epilepsy has transformed the field of research and epilepsy pharmacology. Further development and new treatments will rapidly emerge when new and old knowledge are assimilated in an unbiased and patient-­and outcomes-­driven research.

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Emerging Molecular Mechanisms of Neuroinflammation in Seizure Disorders Silvia Balosso, Annamaria Vezzani, and Teresa Ravizza

Abstract  Unabated neuroinflammation plays a pathogenic role in various CNS diseases. In epilepsy, neuroinflammation is not a bystander phenomenon of the diseased brain but contributes to neuronal hyperexcitability underlying seizure generation, cell loss, and neurological comorbidities. Several molecules that constitute the inflammatory milieu in the epileptogenic area activate intracellular signaling pathways in neurons, glia, and cellular components of the blood-brain barrier, resulting in pathologic modifications of cell function. These molecular entities ultimately lead to alterations in synaptic transmission and plasticity. The mechanisms activated by inflammatory molecules include rapid posttranslational changes in voltage-gated and receptor-coupled ion channels and transcriptional regulation of immune and nonimmune gene expression. These modes of action highlight an emerging neuromodulatory role of inflammatory molecules that differs from their classical functions as immune activation effectors in infection or autoimmunity. We review the main actions exerted by inflammatory molecules on target cells by selecting those with a demonstrated role in experimental seizures and discuss their relevance for human epilepsy. Keywords  Cytokines · HMGB1 · COX-2 · Oxidative stress · TGF-β · Leukocytes

1  Introduction Clinical and experimental findings provide strong evidence favoring the involvement of neuroinflammation – the production and release of inflammatory mediators by resident brain cells  – in the pathogenesis of seizures, neuronal cell loss, and neurological comorbidities [1–4]. Neuroinflammation is a common hallmark of epileptic foci in various drug-resistant forms of epilepsy with differing etiologies and S. Balosso · A. Vezzani · T. Ravizza (*) Laboratory of Experimental Neurology, Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milano, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_2

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is not only associated with autoimmune disorders or active CNS infections [4, 5]. Importantly, several inflammatory mediators are endowed with specific effects on neuronal and synaptic function [6–8]; these may also alter glial cells and blood-­ brain barrier (BBB) properties [9–11]. After acute injuries or during seizures, inflammatory mediators are rapidly synthesized and released in the brain mostly by activated microglia and astrocytes and by neurons and BBB cellular constituents. Notably, neuroinflammation can be evoked by a pure increase in neuronal activity [neurogenic neuroinflammation [12]] and in the absence of overt neuropathology [13–15]. This intrinsic innate (immune-­ like) response includes a rapid release of proinflammatory cytokines (e.g., IL-1β, IL-6, and TNF) and danger signals (e.g., high-mobility group box 1 (HMGB1), ATP, extracellular matrix degradation products), upregulation of Toll-like receptors, the activation of the transcription factor NF-kB, chemokine and prostaglandin production, complement system activation, and increased expression of adhesion molecules [1, 5]. Neuroinflammation, together with the activation of peripheral leukocytes, which may be subsequently recruited [16–18], should reestablish brain tissue homeostasis and promote cell repair. In epilepsy, however, this response is not adequately controlled by endogenous resolving mechanisms [19–22], thus ultimately contributing to cell dysfunction. In the brain, activation of immune mechanisms typically triggers transcriptional factors (e.g., NF-kB, AP-1) which upregulate inflammatory genes; some molecules, however, are initially released by their constitutive pools, thus providing a rapid source of extracellular effector molecules. For example, caspase-1 cleaves the inactive IL-1β precursor in the cytosol of glial and neuronal cells, thus producing the mature and biologically active form of the cytokine that is subsequently released in the extracellular space [23]. Caspase-1 is rapidly activated by changes in intracellular K+, Ca2+, and Cl− ions [24] that occur during neuronal network hyperexcitability [25], while the danger signal HMGB1 is released upon nuclear-to-cytoplasmatic translocation [26], which requires increased Lys acetylation at two specific molecular sites. Upon their extracellular release, various inflammatory mediators can in turn modulate the expression of non-immune genes and related proteins involved in neurogenesis, cell death, and synaptic molecular reorganization and plasticity [27]. In addition to transcriptional activity, inflammatory mediators also activate non-­ conventional intracellular signaling pathways in brain cells acting as key communication elements between neuronal, glial, and vascular compartments (Fig. 1). In this chapter, we review the evidence describing the mechanisms by which neuroinflammation can promote brain cell dysfunction by focusing on molecules and pathways which are similarly altered in brain specimens from experimental models and human drug-resistant epilepsies and with a role in experimental seizures.

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Fig. 1  Various epileptogenic insults trigger brain inflammation, involving the production of an array of inflammatory mediators by resident cells (e.g., neuroinflammation). When this event is not adequately resolved by endogenous anti-inflammatory mechanisms, it induces pathologic modifications in neuronal and glial cell function and contributes to BBB permeability alterations. These phenomena alter synaptic transmission, enhance neuronal excitability, and reduce seizure threshold (see text for details) contributing to seizure generation and progression, neuronal cell damage, and neurological comorbidities

2  Cytokines, Chemokines, and Danger Signals 2.1  Interleukin-1 Receptor/Toll-Like Receptor Signaling IL-1R type 1 (IL-1R1) and Toll-like receptor (TLR) 4, and their prototypical endogenous ligands, namely, IL-1β and HMGB1, are induced in neuronal, glial, and BBB cells following various epileptogenic injuries in rodents and also present in surgically resected human epileptogenic foci. Pharmacological studies and genetic interference with this signaling demonstrated that its activation promotes seizure recurrence and is involved in epileptogenesis [13, 14, 28–32]. In models of status epilepticus, IL-1R1/TLR4 signaling also plays a role in neuronal cell loss and neurogenesis [29]. These receptors may mediate cognitive deficits and depression-like symptoms in models of status epilepticus as well as in naive animals or in offspring of dams exposed to IL-1R1/TLR4 ligands during gestation [3, 33–36]. A key feature of the pathologic effects of IL-1β in epilepsy is the inefficient induction of the

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endogenous IL-1 receptor antagonist (IL-1Ra) which acts to rapidly terminate IL-1β-mediated actions. In this frame, the administration of IL-1Ra drastically reduced seizures in animal models [32, 37, 38] and patients with drug-resistant seizures in the absence of known bacterial, parasitic, or viral triggers [39–42]. Interactions with the Glutamate System  The search of mechanisms underlying the ictogenic effects of IL-1R1/TLR4 signaling activation led to the identification of rapid posttranslational changes induced in NMDA receptors [14, 43, 44]. In particular, the activation of IL-1R1/TLR4 axis in hippocampal neurons by their endogenous ligands IL-1β and HMGB1 induces Src kinase-dependent phosphorylation of the NR2B subunit of the NMDA receptors, leading to enhanced neuronal Ca2+ influx [14, 43–45]. Pharmacological blockade of this molecular event in vivo, either by using a Src kinase inhibitor or a NR2B receptor antagonist, precludes the ictogenic activity of both IL-1β and HMGB1 [14, 44]. Thus, the involvement of increased NMDA function in mediating the ictogenic effects of IL-1R1 and TLR4 activation represents one of the molecular mechanisms underlying the pivotal role played by NMDA receptors in seizure generation and recurrence [46]. Additional mechanisms induced by the activation of IL-1R1/TLR4 axis have been described as follows. IL-1β increases CA1 pyramidal cell excitability by reducing NMDA-induced outward current. This action involves p38 mitogen-­ activated protein kinase (MAPK), which increases the phosphorylation of Ca2+dependent K+ channels [47]. Non-NMDA ionotropic glutamate currents are also activated by TLR4 in the dentate gyrus leading to increased excitation of granule cells which contributes to post-traumatic dentate hyperexcitability [48]. HMGB1 effects on neuronal excitability include a molecular (receptor-independent) interaction with presynaptic NMDA receptors resulting in enhanced glutamate release from presynaptic terminals evoked upon NMDA receptor stimulation [49]. Both IL-1β and HMGB1 can indirectly affect neuronal excitability by increasing the extracellular glutamate concentration by inhibiting the astroglial glutamate reuptake [50, 51]. HMGB1 can also induce glutamate release from hippocampal gliosome preparations implying that this molecule may increase gliotransmission [52]. The consequent increase in extracellular glutamate may contribute to neuronal excitability and excitotoxicity in injured brain regions. Interactions with the GABA System  Changes in neuronal transmission are also evoked by IL-1β interactions with GABAA receptors. This cytokine reduces synaptically mediated GABA inhibition in CA3 hippocampal region via still unidentified kinases [53, 54] and inhibits GABA-mediated Cl− influx into neurons [54]. Pathophysiological concentrations of IL-1β decreased the amplitude of GABA-­ evoked currents by up to 30% in human epileptic specimens [55]. This effect was shown by performing electrophysiological recording in oocytes transplanted with membranes from surgically resected hippocampal and cortical tissue of patients with temporal lobe epilepsy with or without hippocampal sclerosis. This effect was reproduced by patch-clamp recordings on neurons in entorhinal cortex slices from rats with chronic epilepsy, and was not observed in control slices, and was mediated by IL-1R1-dependent activation of protein kinase C (PKC) [55].

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Induced Channelopathies  Activation of IL-1R1/TLR4 signaling may induce “acquired channelopathies,” as shown, for example, by the downregulation of the HCN1 channel and the related Ih current in dendrites of hippocampal pyramidal neurons [8]. HCN1 channels are key regulators of the filtering properties of hippocampal pyramidal cell dendrites and their responses to excitatory inputs, and they are involved in theta rhythms, which have been linked to cognitive functions. These channels are downregulated in experimental and human epilepsy tissue and contribute to seizures [56, 57]. TLR4 activation by systemic lipopolysaccharide (LPS) during the first two postnatal weeks in rats induced TNF and IL-1β in activated microglia cells, then resulting in cognitive impairment and increased seizure susceptibility in adulthood [58]. These pathological outcomes were associated with persistent changes in the expression pattern of NMDA and AMPA receptor subunits in the cerebral cortex and hippocampus, as well as changes in K+-Cl cotransporter, predicting modifications in CNS excitability [58]. Epigenetic Modifications  In addition to its extracellular actions as a secreted protein, nuclear HMGB1 contributes to chromatin remodeling and facilitates the binding of various transcription factors to gene promoters [59, 60]. Macrophages secreting HMGB1 upon LPS exposure, or with a deletion of the HMGB1 gene, undergo epigenetic changes characterized by a decrease in histone content and in the number of nucleosomes. These changes result in transcriptional activation of genes associated with cellular stress and the inflammatory responses [61, 62]. It is conceivable, therefore, that HMGB1 cytoplasmatic translocation  – which occurs prominently in neurons and glia during epileptogenesis and in chronic epilepsy [14, 63, 64] – may provoke epigenetic changes in brain cells that profoundly alter cell phenotype, thereby affecting disease outcomes. The activation of IL-1R/TLR4 signaling induces microRNA (miR)146a acting as a negative regulator of this pathway. We found overexpression of miR146a in astrocytes and neurons both in experimental and human epileptogenic tissues [30, 65] which may represent a homeostatic mechanism for counteracting the inflammatory pathway activation and its pathologic consequences. Accordingly, intracerebral injection of a synthetic miR146a mimic significantly reduced neuronal excitability in the hippocampus and acute and chronic seizure recurrence and blocked the progression in spontaneous seizure frequency when injected in mice early after the onset of epilepsy [30]. Blood-Brain Barrier  The presence of IL-1β, HMGB1, IL-1R1, and TLR4 on endothelial cells of the brain vasculature and in perivascular astrocytes support the evidence that inflammation impairs the permeability function of the BBB. In this respect, IL-1β promotes the disassembling of the tight junctions and induces NO and the activation of matrix metalloproteinases in endothelial cells [66]. BBB dysfunction, in turn, has functional consequences for brain excitability and seizures (see Sect. 4).

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2.2  Inflammasome and P2X7 Receptors The inflammasome is a key driver of neuroinflammation. It consists of intracellular multi-protein complexes instrumental for the release of IL-1β [67, 68], HMGB1 [69, 70], and IL-18 [68], the first two inflammatory molecules being chiefly involved in seizure mechanisms [1, 71] (see Sect. 2.1). A rise in cytosolic Ca2+ and Na+ and a reduction in cytoplasmatic K+ triggers inflammasome activation [72], and these ionic modifications are provoked by sustained neuronal depolarization occurring during seizures [25]. Extracellular ATP is an inflammasome activator released by injured cells and during seizures. ATP activates P2X7 receptors that are ATP-gated ionotropic purinergic receptor predominantly expressed in microglia, but also in neuronal presynaptic terminals and to a lesser extent by astrocytes. The receptor expression is increased in these cell types in experimental and human epilepsy [73, 74]. Upon their engagement, P2X7 receptors promote changes in intracellular ionic concentration that trigger inflammasome activation [75]. The activation of P2X7 receptors in microglia induces multiple intracellular signaling pathways (i.e., p44/42 ERK kinase, p38 MAPK, c-Jun N-terminal kinase, AMP-activated protein kinase-α, mTOR/S6 kinase, NF-κB signaling), therefore contributing to both the generation and persistence of neuroinflammation [76]. The presynaptic neuronal P2X7 receptors modulate glutamate and GABA release as shown in hippocampal slices [77]. In mouse cortical astrocyte cultures, ATP induces glutamate release from astrocytes by activating P2X7 receptors [78]. Taken together, these data suggest that the overexpression of P2X7 receptors in epilepsy results in a dysregulation of neuronal activity contributing to the establishment of a hyperexcitable neuronal network. In accord, the administration of P2X7 receptor antagonists in mice reduced the severity of status epilepticus and the frequency of spontaneous seizures; these effects were associated with a reduction of glia activation [79–81].

2.3  Chemokines Besides their chemotactic role in leukocyte trafficking across the BBB [82], chemokines also represent a new class of neuromodulators. Indeed, chemokines increase voltage-gated Na+ currents and decrease Ca2+ currents in dorsal root ganglia neurons, while enhancing K+ currents in the rat hippocampal neurons [83–85]. Moreover, chemokines regulate neurotransmitter release [86, 87]. In this context, fractalkine (CX3CL1) acts as a positive modulator of GABAA receptors in human TLE brain specimens by reducing their use-dependent desensitization (rundown) [88]. This effect may be mediated by phosphorylation of GABAA subunits, thus leading to a “stabilization” of the receptor. The upregulation of fractalkine receptor in epileptogenic tissue may therefore represent a homeostatic attempt to reduce

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hyperexcitability by promoting GABA receptor function. In line with this set of data, CXCL12 reduced evoked excitatory synaptic transmission in mouse Purkinje cells, through a mechanism involving a decrease of NMDA-dependent glutamate release [89]. Conversely, several chemokines (e.g., CCL3, CCL5, CCL11, CCL17) by activating their G-protein-coupled receptors induce intracellular signaling including MAPK, Akt phosphorylation, and cAMP, ultimately resulting in increased intracellular Ca2+ concentrations [87] which in turn can contribute to hyperexcitability and seizure-induced neuronal cell loss.

2.4  Tumor Necrosis Factor In animal models of acute seizures and in kindling model, TNF is induced in microglia, astrocytes, and endothelial cells of the BBB together with a concomitant reduction in neuronal TNF receptor type (TNFR2) and an increase in neuronal and astrocytic TNFR1. These modifications were also found in human TLE specimens [90, 91]. TNF has either pro- or anti-ictogenic effects which are dependent on its brain concentration, and the receptor subtype predominantly activated in the diseased brain. Thus, mouse recombinant TNF injected into the mouse hippocampus significantly reduced seizures by activating TNFR2, while it promoted seizures by activating TNFR1 [92]. TNFR1 signaling components including TNFR-associated protein with death domain (TRADD), Fas-associated protein with death domain (FADD), and cleaved caspase-8 are upregulated in brain samples from TLE patients with intractable epilepsy and in mice exposed to status epilepticus [93]. This protein complex promotes cell damage, suggesting that TNFR1 activation may be a crucial event in neuronal cell death associated with seizures [94, 95]. Interactions with the Glutamate System  In animal models, TNF alters seizure susceptibility and affects neuronal cell survival by acting on its neuronal receptors or indirectly by activating receptors expressed by astrocytes and the microvasculature. In this context, a functional interaction between TNF and glutamatergic neurotransmission has been reported. Beattie et al. (2002) have shown that the astrocytic TNF/ TNFR1 axis induces a rapid increase in synaptic surface expression of AMPA receptors [96]. This leads to an increase in the mean frequency of AMPA-induced miniature excitatory postsynaptic currents indicating an enhancement in synaptic efficacy [96]. The newly expressed AMPA receptors lack the GluR2 subunit [97], a molecular conformation which favors neuronal Ca2+ influx. The activation of AMPA receptors alter synaptic strengths and contributes to excitotoxicity. TNF can exacerbate or reduce the excitotoxic neuronal damage induced by AMPA in organotypic slice cultures depending on concentration, tissue exposure time, and receptor activated [98]. Modifications in NMDA, AMPA, and KA receptor subunit expression were measured in TNF receptors knockout mice [99], indicating that TNF receptor signaling is involved in determining glutamate receptor subunit composition. These

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modifications were associated with changes in glutamatergic neurotransmission and seizure susceptibility in the same mice [92, 99]. TNF exerts also indirect effects on neuronal excitability by increasing the extracellular glutamate concentration via inhibition of the astroglial glutamate reuptake [50, 100] and by inducing glutamate release from activated astrocytes [101] and microglia [102]. Interactions with GABA Receptors  TNF induces endocytosis of GABAA receptors, thus reducing the inhibitory synaptic strength [103]. This decrease is mediated by TNFR1 and involves posttranslational mechanisms mediated by PI3K-Akt pathway in neurons [103]. Interestingly, using a computational neuron-glia interaction model, Savin and colleagues demonstrated that TNF overexpression by glial cells influences synaptic scaling that leads to increased seizure susceptibility [104]. Modulation of Voltage-Gated Ion Channels  TNF affects voltage-gated ion channels. This cytokine enhanced Na+ and Ca2+ channel currents in mouse cultured neurons, in the former instance by increasing the membrane expression of these channels [105, 106], and decreased inwardly rectifying K+ currents in cortical astrocytes [107]. Blood-Brain Barrier  TNF released by perivascular astrocytes or microglia can compromise the permeability properties of the BBB [108–110]. Weinber and colleagues showed that rats overexpressing TNF displayed a massive IgG extravasation in the brain parenchyma [91]. It has been demonstrated that BBB failure contributes to long-lasting hyperexcitability phenomena in surrounding brain tissue mediated by extravasated albumin that induces astrocyte dysfunctions (see Sect. 4).

2.5  Immunoproteasome The proteasome is the core of the ubiquitin proteasome system and degrades the large majority of the cytoplasm proteins [111]. The incorporation of the three inducible subunits β1i, β2i, and β5i into newly formed proteasomes results in the generation of the immunoproteasome. This isoform is induced during inflammation and plays a key role in regulation of both the adaptive and the innate immune responses [111]. The β1i and β5i subunits are induced in brain specimens of patients with pharmacoresistant mesial temporal lobe epilepsy and malformations of cortical development [112–114]. β5i subunit expression is also induced in experimental epilepsy, and its selective pharmacological inhibition reduced the incidence and delayed the occurrence of 4-aminopyridine-induced seizure-like events evoked in acute rat hippocampal/entorhinal cortex slices [115]. In line with these results, the reduction of (immuno)proteasome subunit expression induced in the hippocampus by rapamycin administered in SE-exposed rats was associated with a decreased number of spontaneous seizures in drug-treated vs vehicle-injected rats [114]. These

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findings suggest that the upregulation of the β5i subunit in epileptogenic tissue plays a role in increasing neuronal network excitability, therefore promoting seizure generation. The molecular mechanisms underlying these effects are unclear. The immunoproteasome activation in neurons may affect cell excitability by modulating synaptic transmission and plasticity [116–118] by regulating pre- and postsynaptic axonal density and the degradation of synaptic proteins [117] and the growth of new spines on dendrites of hippocampal pyramidal neurons [119]. Specific inhibition of the immunoproteasome β5i activity reduced inflammatory signaling in microglia isolated from mouse models of Alzheimer’s disease [120] and in human peripheral blood mononuclear cells (PBMC) [111, 121]. These data suggest that immunoproteasome activation in glia and immune cells may contribute to the inflammatory milieu in brain and promotes neuronal excitability. In turn, IL-1β can induce proteasome subunit expression in human astrocytes in vitro [113]. Immunoproteasome may regulate neuroinflammation also acting in the extracellular space and modulating proinflammatory functions of cytokines. A similar mechanism has been recently demonstrated in multiple sclerosis models where extracellular proteasome, by processing osteopontin, induced the release of osteopontin active fragments with enhanced chemotactic activity [122].

2.6  Oxidative Stress Oxidative stress and neuroinflammation are two phenomena intimately associated, since they are functionally interconnected and reinforce each other [123, 124]. Moreover, as in the case of neuroinflammation, in animal models oxidative stress is rapidly and persistently induced after epileptogenic injuries [125, 126]. Notably, markers of oxidative stress are increased in both the brain and blood in human epilepsy [127, 128]. Since the crosstalk between oxidative stress and neuroinflammation is complex, we will discuss only few selected examples. The best-characterized interaction is based on the evidence that inflammasome activation generates reactive oxygen species (ROS), and conversely, inhibition of ROS blocks inflammasome activation [129]. Zhou and colleagues identified ROS-­ induced thioredoxin-interacting protein (TXNIP) binding to NLP3 inflammasome, as the key molecular mechanism for inflammasome assembly and activation [130]. NADPH-oxidase (NOX) dependent generation of ROS is required to increase the surface expression of TLR4 in cultured macrophages exposed to LPS. This mechanism involves alterations in lipid raft’s annexin VI content, the activation of Ca2+dependent kinases, and the generation of ceramide [124]. Conversely, TLR4 signaling activates prooxidant enzymes such as NOX and iNOS, thus producing high levels of ROS [124], which in turn favor the membrane mobilization of TLR4 within the lipid rafts. This process – defined as “TLR-radical cycle” – may represent an important mechanism involved in the maintenance of inflammation, as it occurs in epileptogenic tissues.

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Finally, oxidative stress may induce various modifications within macromolecules (e.g., lipids and proteins), generating the so-called oxidation-specific epitopes which are increasingly recognized as inducers of inflammation [124]. An example of these pathological changes is represented by HMGB1. HMGB1 is stabilized in its disulfide isoform during oxidative stress which has proinflammatory activity by activating TLR4 [131, 132]. We have shown that disulfide HMGB1 is involved in seizures and neuronal cell loss by enhancing the NMDA-dependent neuronal Ca2+ influx [45], similarly to IL-1β (see paragraph 1.2.1). The transient treatment of rats exposed to SE with antioxidant drugs prevented the progression in spontaneous seizure frequency during epilepsy development and reduced neuronal cell loss and cognitive deficits [63, 133, 134, 188].

3  Arachidonic Acid-Related Pathways Cyclooxygenase Enzyme  Dichotomous effects of COX-2 blockade on seizures have been reported. Thus, the administration of COX-2 inhibitors before status epilepticus may lead to adverse effects (e.g., increased mortality during the first 2 weeks after brain injury) in various animal models, whereas their administration after SE may reduce spontaneous seizures depending on the animal model [1, 135]. These dual effects of COX-2 inhibition likely depend on the profile of induction of prostaglandins (PG) during seizures in the various experimental models as well as their dynamic changes in various phases of the disease [136]. In this respect, PGE2 has been shown to be proconvulsive and neurotoxic [137, 138], while PGF2 has inhibitory action on seizures [139]. Prostaglandin E2  The available mechanistic data mostly relate to the effects of PGE2 on synaptic transmission and neuronal excitability [140]. In particular, there is evidence that COX-2, which is expressed in postsynaptic dendritic spines, regulates neuronal activity via PGE2 synaptic signaling. Somatic and dendritic membrane excitability was significantly reduced in CA1 pyramidal neurons in hippocampal slices when endogenous PGE2 was reduced by a selective COX-2 inhibitor, and this effect was mediated by cannabinoid receptor type 1 (CB1) [141]. Moreover, the exogenous application of PGE2 to pyramidal CA1 neurons increases frequency of firing and excitatory postsynaptic potentials amplitude, most likely by reducing K+ currents in neurons [142]. COX-2-mediated PGE2 synthesis leads also to the production of free radicals as intermediate products that in turn can potentiate glutamate-mediated effects [143]. The production of PGE2 from TNF-activated astrocytes mediates astrocytic Ca2+-dependent glutamate release [101], thus contributing to ictal activity and excitotoxicity. Finally, PGE2 modulates voltage-gated ion channels function by increasing the expression of Na+ channels in isolated dorsal root ganglion cells, through a G-protein-dependent mechanism [144].

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Prostanoid Receptors  Since several COX-2 inhibitors have been withdrawn from the market due to side effects [145], an alternative strategy to increase target selectivity with milder adverse events was pursued by targeting downstream effector molecules in the COX-2 signaling cascade [146]. In this respect, the PGE2 EP1 and EP2 prostanoid receptor subtypes were considered. The activation of EP1 increased the probability to develop status epilepticus and exacerbated the associated neuronal cell loss and inflammatory response. These effects were mediated by phospholipase C, Ca2+, and PKC which potentiated heteromeric kainate receptors [147]. Differently, animals treated with EP2 antagonist immediately after status epilepticus termination showed moderate neuroprotection [148], while delayed and repeated administration of EP2 antagonist was associated with broader benefits including marked neuroprotection, less neuroinflammation, reduced mortality, prevention of BBB permeability dysfunction, and rescue of memory deficits [149–151]. It has been suggested that neuronal EP2 receptors promote neuroprotection via PKA-dependent signaling activation. By contrast, glia (especially microglia) EP2 receptor activation leads to neurotoxicity and neurodegeneration, in part via cAMP-exchange protein activated by cAMP signaling [148]. This pathway upregulates inflammatory mediators, including iNOS, NOX, COX-2, and various cytokines [152, 153]. Excessive EP2 activation can eventually kill activated microglia and thus might also participate in the resolution of inflammation. Monoacylglycerol Lipase  Serine hydrolase monoacylglycerol lipase (MAGL) is the enzyme that hydrolyzes the endocannabinoid 2-arachidonoylglycerol (2-AG) to provide a major brain source of arachidonic acid for proinflammatory eicosanoid synthesis [154]. We have recently shown that the severity and duration of benzodiazepine-­refractory status epilepticus and the consequent cell loss and cognitive deficits were significantly reduced in mice treated with a potent and selective irreversible MAGL inhibitor [155]. These therapeutic effects were mediated by a reduction of arachidonic acid availability to COX-2, thus providing anti-­ inflammatory effects [155]. In line with these results, 2-AG, which accumulates during MAGL inhibition, was shown to inhibit seizures and delay kindling epileptogenesis by decreasing afterdischarge duration in rodents [156]. These effects were attributed to a reduction of excitatory neurotransmission by 2-AG activation of CB1 receptors [157]. Pro-resolving Lipid Mediators  Arachidonic acid is also involved in the production of endogenous anti-inflammatory factors. Indeed, the biosynthesis of specialized pro-resolving lipid mediators – key molecules that mediate the active resolution of inflammation in peripheral tissues and in CNS [158]  – is generated from arachidonic acid and ω3-polyunsaturated fatty acids by the action of lipoxygenases [159]. However, these pathways appear to be defective in epilepsy [22]. Using LC-MS/MS analysis, we have recently shown that the pro-resolving lipidomic profile in the hippocampus is dysregulated during epileptogenesis. Moreover, the injection of protectin D1 lipid mediators in mice during epileptogenesis exerts anti-inflammatory

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effects, reduced the number and duration of spontaneous seizures, and rescued cognitive deficits [22]. Pharmacoresistance  COX-2 can also contribute to pharmacoresistance. Thus, seizure-associated overexpression of P-glycoprotein (P-gp) – the multidrug transporter limiting the brain penetration of some anti-seizure drugs – in brain vessels is dependent upon the release of glutamate and the subsequent activation of the NMDA receptor-COX-2 signaling pathway. Accordingly, COX-2 selective inhibition in vivo blocked Pgp upregulation and increased brain uptake of phenytoin in epileptic rats [160–162].

4  B  BB and Transforming Growth Factor-Beta (TGF-β) Signaling BBB permeability regulates the reciprocal blood-to-brain exchange of molecules and immune cells, and its integrity is instrumental for protecting the brain from the entry of xenobiotics or potentially dangerous molecules and cells. BBB dysfunction occurs in epilepsy as in other neurological disorders such as stroke and trauma [163, 164]. Inflammatory molecules can alter the permeability properties of the BBB, for example, by inducing the breakdown of tight-junction proteins in brain vessels via the activation of Src kinases [66, 165–168]. Increased transcytosis in endothelial cells may also occur in inflamed tissue [169–171]. These effects favor the brain extravasation of circulating cells as well as serum small molecules and macromolecules, which are normally excluded, such as albumin. Upon entering the brain, albumin is sensed by astrocytes via activation of transforming growth factor-beta receptors type 2 (TGF-β-R2), inducing inflammatory signaling in these cells [172]. This process induces a cascade of pathological changes within the neurovascular unit, including the activation of inflammatory genes in reactive astrocytes [173], and their reduced K+ and glutamate buffering [174] capacity. Among the pathologic consequences mediated by albumin, there is recent evidence of increased excitatory synaptogenesis (through ALK55-Smad2/3 intracellular signaling activation) [175] and degradation of perineuronal net [176] (PNN)  – a protective structure of the extracellular matrix that provides synaptic stability and restricts reorganization of inhibitory interneurons [177]. Some of these changes were documented across animal models of epilepsy and in human brain samples. They contribute to establish a hyperexcitable milieu in surrounding tissue [172, 178] and cause a lasting decrease in seizure threshold [179]. In line with these findings, the blockade of TGF-β signaling by losartan or its analogs prevented epileptogenesis by precluding the activation of the TGF-β signaling and its consequences [176, 180–183].

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5  Peripheral Immune Cells There is evidence for the involvement of the peripheral immune cells in human epilepsy [5]. The contribution of leukocytes to brain tissue inflammation differs depending on the nature of the epileptogenic trigger in animal models and the etiology of human epilepsy. If this phenomenon has some relevance for tissue hyperexcitability or neuropathology is still an open question since divergent findings were obtained [16, 17, 184]. Limited evidence is available on the role of brain-infiltrating monocytes. Varvel and colleagues showed that CCR2+ monocytes invade the hippocampus within days after status epilepticus in mice while occasional CD3+ T-lymphocytes were encountered [18]. Ccr2 knockout mice exposed to status epilepticus displayed reduced monocyte recruitment together with accelerated weight regain, reduced BBB dysfunction, and attenuated neuronal damage [18]. These effects in knockout mice were attributed to a reduced extent of neuroinflammation since monocytes import proinflammatory and pro-ictogenic cytokines (such as IL-1β and TNF) upon their brain entry [18]. These findings identified brain-infiltrating CCR2+ monocytes as a myeloid-cell subclass that contributes to neuroinflammation and morbidity during epileptogenesis. Lowering the expression of CCR5 in PBMC before status epilepticus induction in rats reduced seizures, BBB leakage, and the extent of neuroinflammation and afforded neuroprotection [185–187]. Inhibition of CCR5 in PBMC may decrease their interaction with endothelial cells, thus reducing leukocyte migration across the BBB, and consequently neuroinflammation and related pathologic events.

6  Conclusions Pharmacological interventions on neuroinflammatory pathways activated in experimental and human epilepsy have increased our understanding of this complex response to brain injury and helped to identify potential targets for attaining anti-­ ictogenic and anti-epileptogenic effects. The design of anti-inflammatory drug interventions in epilepsy requires a deep understanding of the type and dynamic changes of each specific neuroinflammatory pathway during disease development. This information allows to envisage the optimal therapeutic window for target-specific drug intervention. We also need to distinguish unequivocally between homeostatic and pathologic inflammatory signaling triggered by epileptogenic insults in order not to interfere with the mechanisms of tissue repair. The progress in elucidating the molecular mechanisms underlying the consequences of neuroinflammation for brain pathophysiology using experimental epilepsy, and their validation in human epilepsy brain, is critical for developing safe

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and effective drugs with disease-modifying, rather than purely symptomatic, therapeutic effects. Acknowledgments  The authors gratefully acknowledge their sources of support, namely, the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n°602102 (EPITARGET), Associazione Italiana Contro l’Epilessia (AICE-FIRE), and Fondazione Italo Monzino.

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Glial Mechanisms of Inflammation During Seizures Etienne Audinat and François Rassendren

Abstract  It is now clearly established that microglia and astrocytes become reactive in brain regions experiencing seizures in human or experimental epilepsies. The expression of these reactive phenotypes leads to the dysregulation of physiological functions normally fulfilled by these glial cells and to the acquisition of inflammatory properties that influence the activity and the fate of brain cells, including neurons, glia, and cells of the blood vessels. In this chapter, we review how dysregulation of astrocyte and microglia physiological functions and the emergence of specific reactive states impact epilepsy progression. Keywords  Astrocyte · Microglia · Temporal lobe epilepsy · Cytokine · Purinergic signaling · Gliotransmission · Inflammation

1  Introduction Glia of the central nervous system (CNS) comprises astrocytes, cells of the oligodendrocyte lineage, and microglia. Besides of their well-known neurochemical and metabolic support of neurons, it is now clear that glial cells also dynamically interact with neurons to integrate neuronal activity and, in turn, directly influence neuronal output. In the case of astrocytes and microglia, this modulation of neuronal activity can operate through the release of mediators, either classical transmitters such as glutamate, GABA, D-serine, and ATP or cytokines or growth factors. Under physiological conditions, “metabolic” and “information processing” functions of glia operate on a time scale ranging from milliseconds to hours or even days, when considering brain development and hormonal cycles, and are fundamental for brain function, including cognition [2, 11, 12, 42, 91, 118, 140, 158, 160, 185, 186, 193]. Under pathological conditions, microglia and astrocytes develop reactive E. Audinat (*) · F. Rassendren IGF, University of Montpellier, CNRS, INSERM, Montpellier, France e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2021 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_3

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phenotypes that result in (i) impairment of their homeostatic functions and (ii) upregulation of “immune” processes through which microglia and astrocytes orchestrate CNS inflammatory reactions. This remodeling of glial phenotype modifies their interactions with neurons and also with the vasculature (pericytes and endothelial cells), thereby leading to impaired neuronal activity and BBB dysfunction [38, 67, 69, 83, 139, 154, 157, 176, 199, 202, 211]. The relationships between inflammation, gliosis, seizures, and epilepsy emerged at the beginning of this century from clinical and experimental observations. The best-documented evidence for a direct causality between inflammation and human epilepsy comes from Rasmussen encephalitis, in which brain inflammation of unknown origin triggers focal motor seizure [197]. However, Rasmussen encephalitis remains an extreme condition that does not directly relate to other types of epilepsy. Twenty years later, there is a general agreement that brain inflammation represents a hallmark of seizures and epilepsy [16, 184], but whether reducing inflammation has any clinical benefit for patients remains an unanswered question. Another key question is whether inflammation and gliosis contribute to epileptogenesis and/or to the genesis of spontaneous recurrent seizures (ictogenesis). In other words, are these processes involving primarily non-neuronal cells a cause or a consequence of aberrant neuronal network activities? Answering these fundamental questions is difficult for different reasons. First, experimental models of epileptogenesis leading to chronic seizures, even when considering only models of temporal lobe epilepsy (TLE), are heterogeneous and often not directly comparable. Second, the evolution of the immune response during the progression of the disease (e.g., acute to chronic) remains poorly understood. Third, investigating central immune response in human subjects is limited to brain resections obtained from patients undergoing surgical removal of epileptic foci since noninvasive approaches such as PET imaging of inflammatory markers remain rather crude. In this chapter, we will first review the roles of astrocytes and microglia under homeostatic conditions. We then discuss gliosis or glial remodeling under non-­ homeostatic conditions, its link with inflammation, and how astrocytes and microglia influence the pathogenesis of epilepsies, focusing mostly on temporal lobe epilepsy (TLE).

2  Astrocytes and Microglia in Homeostatic Conditions 2.1  Astrocytes 2.1.1  Homeostasis of K+ and Water Under physiological conditions, astrocytes contribute to the maintenance of ion homeostasis and, in particular, regulate extracellular K+ ([K+]0). The accumulation of K+ in the extracellular space leads to sustained neuronal depolarization and hyperexcitability [74, 207]. Astrocytes control [K+]0 by two mechanisms: K+ uptake

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and spatial buffering. K+ uptake is mediated through Na+/K+ pumps, Na+/K+/Cl− cotransporters, and expression of inward-rectifier potassium (Kir) 4.1 channels; it also relies on the very negative resting potential of astrocytes [41, 99, 148, 167]. Remarkably, mice lacking Kir4.1 channels on astrocytes exhibit impaired K+ and glutamate uptake and are more seizure prone [32, 49]. Control of ion homeostasis by astrocytes also relies on spatial buffering [127], which is mediated by gap junctions (GJ), channels composed of connexin (Cx) 43 and Cx30 [119]. These channels allow the formation of a functional network between astrocytes (syncytium) and the diffusion of K+ following the electrochemical gradient: K+ is taken up by astrocytes where neuronal activity is high, diffuses through the syncytium, and is released at remote sites with lower [K+]0. In line with the spatial buffering concept, the physiological functioning of astroglial networks should have an anti-epileptic role, and indeed, a reduced threshold for generating epileptic activity was found in mice with coupling-deficient astrocytes [131, 205]. Another important function of astrocytes is the regulation of extracellular space (ECS) volume and fluid osmolarity through aquaporin 4 (AQP4). This channel mediates transmembrane water movements according to osmotic gradients and is expressed by astrocytes mostly at perisynaptic processes and perivascular endfeet [165]. Regulating water flux near synapses and blood vessels may be linked to K+ homeostasis [133]. 2.1.2  Neurotransmitter Buffering Another crucial role for astrocytes that directly impacts neuronal excitability is neurotransmitter buffering or uptake. The removal of neurotransmitters, such as glutamate and GABA, from the synaptic cleft is crucial for a correct functioning of synapses and an appropriate balance between excitation and inhibition (E/I balance). Alterations in these buffering mechanisms could lead to neuronal hyperexcitability and excitotoxicity [33, 112]. The astrocytic buffering of neurotransmitters is mediated by uptake through transporters and their metabolism through the glutamine – glutamate – GABA cycle [54]. Glutamate and GABA reuptake rely on specific transporters expressed by astrocytes: EAAT1 and EAAT2 (also known as GLAST and GLT1) for glutamate and GAT1 and GAT3 for GABA. Once internalized in astrocytes, glutamate is converted into glutamine by glutamine synthetase (GS), while GABA enters the Krebs cycle which produces alpha ketoglutarate, glutamate, and glutamine. Glutamine produced in astrocytes is then released, taken up by neurons and used as a precursor for both glutamate and GABA synthesis. Adenosine kinase (ADK) is another important astrocytic enzyme involved in E/I balance. By phosphorylating adenosine to adenosine monophosphate (AMP), it exerts a key role in regulating extracellular levels of adenosine [58, 179]. Adenosine dampens neuronal activity and its release exerts anticonvulsant effects [40, 101].

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2.1.3  Gliotransmission The concept of the “tripartite synapse” emerged from experiments demonstrating that astrocytes can sense neurotransmitters released into the synaptic cleft by presynaptic neurons but can also release gliotransmitters that regulate neuronal and synaptic activities [13, 142]. Several classical neurotransmitters have been also identified as gliotransmitters, among which glutamate, D-serine, ATP, adenosine, and GABA have been the most studied for their modulation of neuronal excitability and of the activity of excitatory and inhibitory synapses [10, 11, 72, 134, 143]. An increase in intracellular Ca2+ is often needed for the release of gliotransmitters [11, 17, 91, 156, 203] that can be mediated through exocytotic or non-exocytotic pathways [70, 198]. Yet, gliotransmitters can be released also through Ca2+-independent mechanisms involving connexin hemichannels or pannexin (PANX) channels [1], exchangers such as the cysteine-glutamate antiporter, or reversal of neurotransmitter transporters [109]. It should be noted, however, that in vivo studies on gliotransmission are still sparse and that the mechanisms and the functional consequences of gliotransmission are still matters of debates and controversies. These issues have been discussed recently in several reviews [11, 17, 43, 64, 91, 130, 134, 156, 162]. 2.1.4  Interactions with Blood Vessels Another important aspect of astrocyte function that is affected by gliosis is their close interaction with brain vasculature. Astrocytes contact blood vessels with their endfeet and under physiological conditions contribute to the maintenance of the blood-brain barrier (BBB) by increasing the expression of tight-junction proteins occludin and claudin-5 and by inhibiting the expression of chemokines and leukocyte adhesion molecules, which would favor BBB disruption [8, 217]. Astrocytes have been also proposed to control cerebral blood flow in response to neuronal activity and to contribute to power neurons by supplying energy substrate needed to sustain synaptic activity [65, 123, 125, 141].

2.2  Microglia Microglial cells are the resident macrophages of the CNS, and unlike the other mononuclear macrophages associated with the CNS (i.e., perivascular, meningeal, and choroid plexus macrophages), they are in direct contact with other cells and synapses in the CNS parenchyma [88, 120, 146, 163, 191, 212]. In line with their immune cell identity, microglial cells have been primarily studied under pathological conditions, and, until recently, our knowledge on these cells was mostly derived from analyses of CNS pathologies. It is now clear, however, that these immune cells

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also interact with their neighboring partners of the CNS parenchyma and fulfill specific functions in homeostatic conditions. 2.2.1  Never Resting Microglia During the last 15 years, the development of new tools for functional analysis and of new animal models has completely changed our view of normal microglia functions. In 2005, two in  vivo studies using two-photon microscopy in anesthetized knock-in CX3CR1-eGFP mice reported that microglial cells in physiological conditions are not resting, as previously thought, but are permanently extending and retracting their thin processes [44, 122]. This motility of microglial processes may help to detect more rapidly danger signals appearing in the parenchyma. These two articles actually showed that in response to laser lesions or to local applications of ATP, which is considered as an alarm signal [151], microglial cells rapidly extend their processes toward the lesion or the source of ATP, which acts here as a chemoattractant. Although other signaling pathways may control the oriented motility of microglial processes, purinergic P2Y12 receptors and adenosine A2A or P2Y13 receptors are instrumental for rapid process extension and retraction, respectively, occurring right after an insult to the CNS parenchyma [73, 115, 128]. 2.2.2  Monitoring and Modulating Neuronal Activity Following these initial observations, other groups showed that during their surveillance of the parenchyma, which involves a two-pore domain K+ channel [108], microglial cell processes contact synapses. The occurrence and duration of these contacts are regulated by neuronal activity and can influence the activity and the fate of the synapses [48, 60, 103, 144, 172, 190, 204]. Interestingly, microglial cells promote the formation of new spines in the cortex of animals engaged in a motor learning task [135]. Parallel studies also showed that mobilization of microglia-­ specific pathways could lead to the modulation of synaptic activity. For instance, the fractalkine signaling pathway regulates excitatory synaptic transmission and plasticity in the hippocampus [152, 166], microglial Toll-like receptor (TLR) 4 signaling controls glutamate release probability of presynaptic fibers indirectly through the mobilization of astrocytes [137], and microglial P2Y12 receptors are necessary for synaptic plasticity during the critical period of ocular dominance [172]. A recent study also revealed the existence of contact between microglial processes and neuronal soma. These junctions, which can be rapidly induced by neuronal activation, are organized in specialized nanodomains enriched in Kv2.1 potassium channels, mitochondria, and vesicular nucleotide transporter (Vnut) containing vesicles on the neuronal side and P2Y12 on the microglial side [39]. These junctions could, therefore, inform microglia on the excitability and metabolic state of the neuron. Curiously, these junctions, which are observed in 90% of neurons in different regions of the CNS, had not been noticed before. Altogether, these influential

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studies have led to the notion that microglial cells monitor and regulate synaptic and neuronal activity in the healthy brain. 2.2.3  Microglia Diversity Heterogeneity of microglia in physiological conditions was first evidenced during CNS development when their phenotype is continually evolving from amoeboid cells, when entering the CNS parenchyma at embryonic stages, to fully ramified cells at juvenile stages. Remarkably, at a given stage of development, microglial cells with different morphological and functional phenotypes are differentially distributed in different areas of the CNS. This reflects the dynamics of microglia colonization of the brain and their exceptional ability to adapt to their environment, which explains the tight coupling between their own maturation and the maturation of other components of the CNS [118]. This variety of phenotypes underlies a variety of microglia functions during development that have been reviewed recently [118, 132, 186]. More recent studies have also revealed the existence of different microglia populations in different regions of the CNS [15, 45, 171, 177]. Interestingly, microglia repopulating the CNS after a transient depletion in adult mice regain the specific phenotype that they normally express in a given brain area [45], indicating that at any given time microglial cells retain the ability to adapt their phenotype to local environmental cues.

3  Astrogliosis and Microgliosis The role of astrocytes and microglia in pathologies of the CNS is widely associated with reactive phenotypes developed by these cells in response to a breach in CNS homeostasis. The acquisition of these reactive phenotypes, called gliosis, is specific to each glial cell type but is also dependent on the nature, duration, and location of the pathological triggers. Because glial cells have homeostatic functions in non-­ pathological conditions (see above), profound changes of their phenotype upon appearance of pathological conditions have two consequences: (i) the loss of their homeostatic influence and (ii) the emergence of new functions associated with the acquisition of reactive, inflammatory phenotype.

3.1  Astrogliosis in Epilepsy Astrogliosis is particularly evident in epileptic syndromes associated with hippocampal sclerosis (HS) that is characterized by a glial scar [187]; this reactivity of astrocytes is commonly manifested by upregulation of GFAP expression. Astrogliosis, however, involves many more changes at the molecular, cellular, and

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functional levels [51], and these changes can precede GFAP upregulation (see below). The degree of alterations observed in reactive astrocytes varies according to the type of insult inducing the reaction and to the possibility of damage reversion [6, 173]. Transcriptomic analysis of reactive astrocytes has disclosed the occurrence of two broad phenotypes: a pro-inflammatory phenotype A1 having detrimental effects [104, 105, 215] and an immune-suppressive phenotype A2 with protective functions [7, 106]. Interestingly, astrocyte A1 phenotype can be induced by the co-release of IL-1α, TNFα, and C1q from reactive microglia [105]. Although the specific phenotype of astrocytes in different forms of epilepsy has not yet been firmly established, different studies point toward a deleterious A1 phenotype. For example, in epilepsy models, the release of interleukin-1beta (IL-1β) and high-mobility group box 1 by astrocytes induces the activation of IL-1R/TLR signaling pathways that favors the induction and maintenance of seizure activity [111, 201]. On the other hand, mechanisms that oppose to this deleterious astrocyte phenotype have been described [84, 100, 195], and transforming growth factor-beta (TGF-β) signaling, which is activated in astrocytes during epilepsy, reverts an A1 phenotype in vitro [105]. However, in most cases, the population of astrocytes has been considered as a whole without taking into consideration a possible diversity of the astrocyte reactivity (e.g., lesion vs. peri-lesional area). A still debated question is whether astrocyte reactivity is a cause or a consequence of seizures. In favor of a causative role, astrocyte-specific deletion of genes (tuberous sclerosis complex 1 or beta 1 integrin) or mutation of an astrocyte-specific gene (GFAP) induces astrogliosis and the occurrence of spontaneous seizures [114, 129, 150]. On the other hand, there were no signs of astrogliosis in non-lesional epileptogenic cortical areas of patients with focal cortical dysplasia [155]. Moreover, in a guinea pig model of temporal lobe epilepsy with hippocampal sclerosis (HS-MTLE; unilateral, intra-hippocampal kainate), astrogliosis and cell damage were observed only in the injected, and not in the contralateral, hippocampus despite the occurrence of epileptiform activities in both hippocampi [124]. The latter two examples suggest a dissociation between the occurrence of seizure and astrogliosis, as defined with the classical marker GFAP. However, they do not exclude the contribution of astrocytes to the functional remodeling that occurs during epileptogenesis and to the generation of seizures, especially in view of the many functions of astrocytes by which they influence the excitability of neuronal networks. 3.1.1  Homeostasis of K+ and Water Different studies, in humans and animal models, have highlighted a clear association between uncontrolled extracellular K+ increase and epilepsy [176]. In particular, measurements of extracellular K+, patch-clamp recordings of Kir currents in astrocytes, single-cell RT-PCR, and immunostaining of Kir proteins revealed a reduction in Kir-mediated current and a reduction of Kir4.1 expression in surgical specimens from patients suffering from MTLE as well as in rodent models of MTLE [26, 75, 77, 79, 85, 96, 164]. GJ communication within the astrocyte syncytium

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contributes to K+ homeostasis (see above) and is regulated by pro-inflammatory cytokines (IL-1β and TNFα) [113]. Evidence suggests that alterations in astrocyte coupling could be involved in the genesis and progression of epilepsy. In a mouse model of HS-MTLE (unilateral intracortical kainate injection), it was demonstrated that astrocyte coupling is impaired as early as 4 h after the induction of status epilepticus (SE), a mechanism probably related to the release of IL-1β and TNFα, and thus suggesting that impaired coupling represents a key event in epileptogenesis [18]. Moreover, the authors showed a complete lack of GJ coupling in sclerotic hippocampi of HS-MTLE patients. Mice lacking AQP4 display a mild ECS volume expansion, an elevated threshold, but prolonged duration for electrographic seizures induced in vivo by hippocampal electrical stimulation [23, 24]. Subsequent analysis of these mice, however, indicated that they also displayed an enhanced gap junctional coupling and an improved spatial [K+]0 buffering [178]. In human and in animal models of MTLE, there is a loss of astrocytic perivascular AQP4, which precedes the appearance of chronic seizures [4, 55]. This suggests that perturbed polarized expression of AQP4 during epileptogenesis, leading to altered [K+]0 disposition at the vascular interface, may be of pathophysiological relevance. 3.1.2  Neurotransmitter Buffering Alterations in neurotransmitter transporters have been reported for different neurological diseases, including epilepsy [37]. The involvement of alterations in neurotransmitter homeostasis in epilepsy is demonstrated by an increase in extracellular glutamate levels in the sclerotic hippocampus of MTLE patients [30, 31]. In experimental epilepsy, the deletion of GLT1 in astrocytes enhanced responses to subconvulsive doses of pentylenetetrazol [182]. Alterations in GABA extracellular levels can also contribute to hyperexcitability and epilepsy. Depending on the epileptic syndrome, both decreased extracellular GABA levels and increased GABA receptor-mediated activity can be detrimental for the maintenance of E/I balance. On the one hand, an increase in GAT3 astrocytic GABA transporter expression levels in TLE at time of seizure onset, leading to a reduction of extracellular GABA levels, has been reported [52, 102]. On the other hand, in absence epilepsy a dysfunction of the astrocytic GABA transporter GAT1 was found to be responsible for the generation of nonconvulsive seizures by determining an increase in GABA-A receptor-mediated inhibition in thalamocortical neurons [36, 145]. Reduction in GS function is involved in the pathogenesis of epilepsy by inducing extracellular glutamate accumulation, as reported in TLE patients [54, 56, 194]. This finding was also reported for animal models in which blocking GS function with methionine sulfoximine induced recurrent seizures, neuronal loss, and hippocampal atrophy [53, 208]. Considering that glutamine is fundamental also for GABA synthesis in interneurons, GS dysfunction could also be related to impaired

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inhibitory transmission, as shown in an experimental model of TLE in which the downregulation of GS in astrocytes was responsible for a compromised inhibitory synaptic transmission [129]. Astrogliosis occurring in epilepsy is known to be associated with increased levels of ADK both in humans and in experimental models [25]. The increase in ADK and the consequent reduction of extracellular adenosine levels are responsible for reducing seizure threshold [25]. Altogether these data clearly highlight how crucial neurotransmitter buffering is to maintain a functional synaptic activity and avoid the generation of a seizure-­ prone context. 3.1.3  Gliotransmission In vitro models of seizure showed that an excitatory loop involving astrocyte Ca2+ signaling, glutamate, and ATP signaling lowers seizure threshold in neurons [66]. Moreover, Ca2+ signals in astrocytes facilitate spread of epileptiform activity in vivo [78]; in the neocortex, increased astrocyte Ca2+ signaling and glutamate release contribute to neuronal cell death in vivo after pilocarpine-induced SE [47]. Astrocyte glutamate release occurs in different ways, one of which generates, in neurons, slow inward currents (SICs) resulting from the activation of extra-synaptic NMDA receptors and allowing the synchronization of small ensembles of neurons [9, 63, 136]. It has been proposed that SICs generated by astrocytes could be at the origin of hypersynchronous excitation of neurons in models of epilepsy [188], but further pharmacological experiments clearly showed a dissociation between ictal events and SICs [62]. ATP is an essential mediator of communication between astrocytes and other types of brain cells [28]. Expression of a dominant-negative SNARE that impairs the vesicular release of ATP from astrocytes [138] delays the onset of recurrent seizures, decreases epileptiform activity, and reduces hippocampal damage after a pilocarpine-induced SE [35]. Release of ATP through the other route constituted by pannexin 1 (PANX1) hemichannels appears to contribute to ictal discharges in human epileptic brain tissue and mouse models of MTLE (intra-hippocampal kainate) or SE (systemic kainate) [50, 161]. In a rat model of TLE (rapid kindling), activation of P2Y1 receptors induces astrocyte Ca2+ hyperactivity responsible for an increase in glutamate release from astrocytes. This glutamate release activates neuronal presynaptic mGluR5, which increases the release probability at the CA3-CA1 synapses. Blocking astrocyte Ca2+ signaling or inhibiting P2Y1 or mGluR5 receptors relieved this abnormal enhancement of synaptic strength [3]. Similarly, in a mouse model of TLE (unilateral intracortical kainate injection), enhanced astrocyte P2Y1 signaling increased astrocyte glutamate release. However, in the dentate gyrus, this glutamate release activated neuronal presynaptic NMDA receptors and enhanced excitatory synaptic transmission in granule cells of the dentate gyrus [121]. Remarkably, this enhanced P2Y1 signaling resulted from a constitutive

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activation of an autocrine loop involving astrocyte ATP release onto P2Y1 receptors [170]. This autocrine loop, which can be triggered by TNFα, was upregulated before the initiation of the chronic stage of this TLE model [121]. It should be remembered, however, that ATP released from astrocytes also activates P2Y1 expressed by inhibitory interneurons [90, 181] and that its degradation into adenosine inhibits the activity of excitatory pyramidal cells through A1 receptor activation [170, 181]. Altogether, the majority of the above cited observations point toward the conclusion that the actions of astrocyte purinergic signaling determined by the combined effects of ATP, adenosine, and glutamate can displace the equilibrium between excitation and inhibition and promote appearance of epileptiform activity. It is important to note that P2Y1 regulation of astrocyte Ca2+ signaling described in the context of epilepsy has been also reported in a mouse model of Alzheimer’s disease [46, 149] and in a model of traumatic brain injury [34]. In both cases, blocking this pathway had beneficial consequences on outcome of these models. Impaired astrocyte purinergic signaling could thus be a common signature of different CNS pathologies. Yet, additional in vivo experiments are needed to evaluate its weight on TLE progression. 3.1.4  Interactions with Blood Vessels Modifications of astrocyte phenotype during astrogliosis can contribute to BBB impairment that occurs in epilepsy through disruption of tight-junction coupling, increased expression of adhesion molecules on endothelial cells determining an increase of pro-inflammatory cytokines, and the extravasation of leukocytes in the brain parenchyma [110, 196]. In line with the idea that astrocytes contribute to perturbed BBB integrity is the observation of enhanced Ca2+ signaling in astrocyte endfeet during the latent phase of a mouse model of TLE (unilateral intracortical kainate (Szokol et al. [180])). This reactivity of astrocyte endfeet may be driven by the TNFα-induced endothelin-1 release that occurs after pilocarpine-triggered SE in rats which promotes the production of reactive oxygen species in astrocytes via endothelin receptor-B activation [94]. Furthermore, BBB disruption in epilepsy leads to extravasation of serum albumin, which activates TGF-β in astrocytes [76]. TGF-β signaling activation in astrocytes then leads to a series of modifications, including impaired K+, water, and glutamate buffering capacity [168], increased excitatory synaptogenesis [210], and degradation of perineuronal net around inhibitory interneurons [95], which may promote hyperexcitability [183]. Interestingly, reactive astrocytes are associated with microglia-pericytes clusters along leaky capillaries in human and experimental epilepsies and may contribute to the formation of a perivascular scar at loci of impaired blood-brain barrier [97, 98].

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3.2  Microgliosis in Epilepsy Microglial cells are involved in a large number of CNS diseases, and very often they are mobilized at early stages of these diseases. This mobilization leads to changes of their phenotype, which allows microglia to react and adapt in a specific manner to the new environment [71, 147, 169]. Microglial reactivity was first described as a morphological change: from a small soma with long and thin ramified processes in physiological conditions, they adopt a less ramified morphology with fewer and thicker processes in pathological conditions, which can eventually lead to an amoeboid morphology, without process, in the most severe cases. We know now that these morphological changes are accompanied by important functional modifications including changes in gene expression and motility properties, increased proliferation, phagocytic activity, and release of numerous pro- and anti-inflammatory mediators [71, 92, 147]. Yet, microglia activation is not an all-or-none process; it is progressive and depends on the actual pathological conditions and on the context in which the disease appears. There is therefore not a single reactive state but a diversity of phenotypes that are determined by a fine detection of environmental cues and that allows microglial cells to perform specific functions in different pathological conditions. Although microglial morphological alteration is a convenient mean to assess reactivity, it does not reflect the complexity of the functional remodeling of microglia associated with specific pathological context. This is best exemplified for P2Y12 receptors whose expression is either up- or downregulated in morphological similar reactive microglia but in different pathological context, status epilepticus or neuropathic pain, respectively [14, 73, 189]. The diversity of functional remodeling of reactive microglia was further demonstrated by transcriptional analyses performed in various pathological states. These studies firmly established that in a given pathology, microglial reactivity constantly evolves, that there is a diversity of microglial reactive states at a given time of the disease, and that the transcriptional profile adopted by microglia is disease specific [81]. Nevertheless, there is a core of genes that are commonly deregulated across different pathological states and may serve as unambiguous makers of microglial reactivity [80]. 3.2.1  Microglial Inflammatory Signaling Pathways in Epilepsy Recently, human microglial cells of patients suffering from MTLE and who underwent surgery were analyzed in depth, uncovering specific phenotypes in regions of sclerosis (CA1, CA3) versus areas with less neuronal damage [117]. These observations suggested that the repairing interleukin IL-10 regulates the microglial phenotype in these sclerotic regions. Moreover, this study also provides evidence for an early and transient expression of several cytokines, including CXCL8 and IL-1β, after each spontaneous seizure [117]. Increased expression and production of inflammatory cytokines in the hippocampus is well documented in experimental models of TLE and occurs rapidly

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following SE [200]. These cytokines can be produced by different cell types, including neurons, astrocytes, and microglia. However, in a mouse model of TLE induced by pilocarpine, microglial cells were shown to upregulate transiently pro-­ inflammatory cytokines IL-1β, IL-6, and TNFα mRNAs 3  days after SE [20]. Interestingly, at this stage anti-inflammatory markers arginase-1, chitinase-3-like protein (Ym1), and IL-4, but not IL-10, were also upregulated by microglia [20]. Because many of these cytokines are known to modulate neuronal excitability and astrocyte signaling, these observations suggest that microglial inflammatory mediators have a complex role during epileptogenesis. Surprisingly, the expression of these genes was not dysregulated in microglia 3 days post-SE in a model of intra-­ cerebroventricular injection of kainate [27]. These conflicting observations are likely due to differences between the two models and methodological differences (RNA-Seq of purified microglia versus qPCR of hippocampal tissue). Toll-like receptor (TLR) signaling could contribute to the production of cytokines and other mediators by microglial cells after SE.  Activation of TLR3 and TLR4 receptors in  vitro induces the production of inflammatory cytokines by microglia [126]. TLR3 deficiency reduces microglial reactivity, the level of pro-­ inflammatory cytokines TNFα and interferon-β, as well as spontaneous recurrent seizures [68]. Antagonizing TLR4 reduces acute and chronic seizure recurrence in a kainate model of TLE [111]. This effect of TLR4 could involve a signaling pathway including microglia, astrocytes, and neurons. Pascual and co-workers showed that activation of microglial TLR4 induces ATP release by microglia, which activates astrocyte P2Y1 receptors, promoting the release of glutamate by astrocytes which in turn increases excitatory synaptic transmission in CA1 [137]. Stromal cell-derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 also seem to play a role in TLE. Rats treated with a CXCR4 antagonist decreased spontaneous seizures and aberrant adult neurogenesis in a kainate model [174]. Interestingly, CXCR4 is upregulated in hippocampal microglia 3 days after kainate-­ induced SE [27]. Again, some of these effects could involve both microglia and astrocytes as SDF-1 has been shown to trigger the release from microglia of TNFα which stimulates astrocytic glutamate release [21]. Fractalkine (CX3CL1) is a neuronal chemokine that exclusively signals to microglia, which are the only CNS cells expressing the fractalkine receptor CX3CR1. This signaling pathway is thought to act as an off signal that opposes microglial reactivity [22, 29, 107]. Increased expression of fractalkine has been reported in the hippocampus and temporal neocortex of TLE patients and in a pilocarpine rat model [214]. Fractalkine application restored inhibitory GABAergic currents in excitatory neurons in brain slices from TLE patients [153]. Moreover, fractalkine signaling favors microglial process convergence toward neuronal axons and dendrites which increases after pilocarpine- or kainate-induced SE; CX3CR1 deficiency worsens seizure phenotype [59]. Interestingly, deficiency of P2Y12 receptors, which drive microglial process motility, also worsens kainate-induced seizure behaviors [60].

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Accumulating evidence favors the role of microglial purinergic receptors in experimental models of seizures. The transcripts of the four main microglial purinergic receptors, i.e., P2X4, P2X7, P2Y6, and P2Y12, are upregulated in the hippocampus in a mouse model of kainate-induced SE. Membrane currents induced by the activation of P2X7, P2Y6, and P2Y12 as well as microglial process motility toward an agonist of P2Y12 increased after SE [14]. In the same model, P2X4 proteins are upregulated in reactive microglia, and P2X4 deficiency reduces several aspects of microglial reactivity, the upregulation of several pro-inflammatory mediators, and neuronal death induced in the hippocampus by SE [192]. However, while microglial P2X4 receptor has been involved in BDNF-mediated disinhibition and generation of local hyperexcitability in the spinal cord [19], this receptor is also upregulated in neurons, and it is still not known whether P2X4 expressed by neurons, microglia, or both contributes to an inflammatory state after SE. P2X7 receptors are prominently expressed in different immune cells, are one of the main triggers of IL-1ß processing, and contribute to TNFα production. In the brain, P2X7 is expressed in homeostatic microglia, and its expression increases after SE. For these reasons, the potential involvement of P2X7 in epilepsy has been early investigated in different animal models using either pharmacological or genetic approaches to modulate receptor activity. However, opposite results were reported depending on the way SE was triggered. In the model of pilocarpine-­ evoked SE, inhibition of P2X7 activity using either specific antagonists or P2X7-­ deficient mice increases seizure susceptibility after pilocarpine injection, an effect that may be linked to PANX1 since siRNA-mediated downregulation of PANX1 also reduced the threshold for seizures [93]. On the contrary, in a model of intra-­ amygdala injection of KA, P2X7 activity seems to be pro-convulsive since P2X7-­ deficient mice show reduced electrographic seizure during SE and pretreatment with P2X7 antagonists reduced behavioral seizures and neuronal cell death and IL-1ß increase in the hippocampus. In this model, microRNA-22 represses the expression of P2X7 and thus has anticonvulsive effects [86]. Whether this regulation is also present in the human brain is unknown. The reasons for these opposite results are not clear. In both studies, the same pharmacological agents and the same strain of P2X7-deficient mice were used. However, the two experimental models of SE present notable differences, and it is possible that the peripheral administration of pilocarpine might trigger mechanisms which are not present in the intracranial KA injection model. Importantly, adhesion of circulating leukocytes, which express P2X7, on endothelial cerebral blood vessels cells is thought to be an initial and mandatory step of SE induction in the pilocarpine model [61]. This adhesion might depend on P2X7 activity and therefore be impaired by P2X7 antagonists. Recent studies using P2X7 antagonist that crosses the BBB showed in rats that administration of this molecule 3 months after systemic kainate injections decreased the severity of spontaneous recurrent seizures [5]. Although seizure frequency remained unaltered, this study further strengthens the idea that blocking P2X7 may have beneficial effects in regulating seizure during the acute and chronic phases of the disease.

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While rodent studies support that P2X7 may represent a potential therapeutic target to reduce seizure burden, experimental data in human subjects are still lacking. Particularly, all clinical trials testing the effectiveness of P2X7 antagonists in rheumatoid arthritis have failed to demonstrate any beneficial effect [89], despite the clear demonstration of the involvement of this receptor in animal models of the disease. Nevertheless, in specimens from patients suffering from focal cortical dysplasia (FCD) epilepsy, P2X7 mRNA and protein expression increased compared to control samples. This was accompanied by an increased immunoreactivity against IL-1ß in microglia and dysmorphic neurons and balloon cells, which are characteristic cells of FCD [209]. Other studies reported an increase of P2X7 expression in the brain of epileptic patients; however because the specificity of the antibodies remains questionable, these results have to be interpreted with caution. 3.2.2  Complex Contribution of Microglia in Epilepsy The above-described studies indicate that targeting different signaling pathways dysregulated (mostly upregulated) in microglia can have either pro- or anticonvulsive effects. There are certainly concerns regarding the cell-type specificity of some of these manipulations, but if we consider the most microglia-specific receptors that have been studied, i.e., CX3CR1 and P2Y12, it is interesting to note that microglial cells seem to have anticonvulsive actions. This is in agreement with other studies supporting the idea that, under pathological conditions, homeostatic interactions between microglia and neurons have an inhibiting effect on neuronal activities. This was initially described in the zebrafish embryo where microglial processes are attracted by active neurons, in an ATP-dependent manner. The resulting contacts reduced both spontaneous and evoked neuronal activities [103]. Another study reports dynamic interactions of microglial processes with the initial axonal segment in conditions of neuronal hyperexcitability leading to a reversal of cell depolarization [87]. Interestingly, recent experiments with microglia depletion in mouse models of epilepsy also support such a “calming” effect of microglia on neuronal activity. Using three different genetic strategies to deplete microglia, Wu and collaborators showed that the absence of microglia aggravates both acute and chronic seizures in kainate mouse models of SE or TLE [213]. In another study on seizure progression in a mouse model of viral encephalitis, pharmacological depletion of microglia using the antagonist of the Csf1 receptor PLX5622, maximum seizure occurrence was reached more rapidly in microglia-depleted mice. Still, the overall seizure incidence was not affected [206]. Depletion of microglia with PLX5622 induced seizures in a mouse model of low-level picornavirus infection of the CNS [159]. In contrast, the use of another Csf1 receptor antagonist, PLX3397, had anti-­ epileptic effects in mouse models of pilocarpine or intra-hippocampal kainate [175]. However, that study used a much lower concentration of PLX3397 than the one required to deplete microglia [57], and the authors indeed showed that microglia were not depleted [175]. These observations suggest that inhibition of microglial Csf1 receptors has anti-seizure effects, whereas microglial depletion worsens seizures.

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The signaling pathway through which microglia cells inhibit active neurons in physiological or epileptic conditions remains to be identified. Converging evidence, however, indicates that P2Y12 receptors are central in this calming effect. Indeed, pharmacological or genetic inhibition of P2Y12 receptors results in aggravated neuronal death following transient ischemia [39] or worsened seizure [60, 116]. While P2Y12 expression can be upregulated in specific reactive states associated with neuropathic pain [82] or shortly after SE [14], sustained microglial reactivity is often associated with total repression of P2Y12 expression [81]. Such a switch in the expression pattern of P2Y12 may contribute to the opposite effects of microglia during seizure progression, but many other pathways may also be involved. Interestingly, elevating mTOR signaling in mouse microglia induces a microglial reactive state without induction of pro-inflammatory cytokines and leads to the appearance of spontaneous recurrent seizures [216]. This study suggests that the loss of homeostatic functions in microglia may be more important than the acquisition of an inflammatory phenotype in promoting seizures’ appearance. However, the genetic strategy used in that study does not allow to exclude the possibility that border-associated macrophages residing in the meninges, the choroid plexus, and the perivascular spaces or even some CX3CR1-expressing monocytes contribute to the observed effect. Finally, the spatiotemporal dynamics of microglial reactivity within different parts of the epileptic hippocampus (e.g., sclerotic vs. non-sclerotic regions) generate a diversity of microglial phenotypes that need to be characterized to understand the complex roles of these immune cells in regulating the activity and fate of neurons and other brain cells during epileptogenesis and at a chronic stage of the disease.

4  Concluding Remarks In the CNS glial cells have homeostatic functions that support and also finely tune neuronal and synaptic activities. Yet, in pathological conditions where homeostasis is durably lost, glial cells become reactive. These reactive states evolve along with disease conditions and can be deleterious or protective. In the context of epilepsy, glial reactivity occurs only in brain areas experiencing seizures, and increasing evidence indicates that this reactivity contributes to disease progression. Major challenges in the field aim are identifying the triggers, the time course, and evolution of glial reactivity. However, better characterizing the consequences of the loss of glial homeostatic functions is certainly of equal importance. The urgent need for new therapeutic strategies will benefit from a better understanding of the signaling pathways through which glial cells regulate or dysregulate other brain cells and thereby contribute to the progression of different forms of epilepsy.

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Perivascular Inflammation and Extracellular Matrix Alterations in Blood-Brain Barrier Dysfunction and Epilepsy D. W. M. Broekaart, A. Korotkov, J. A. Gorter, and E. A. van Vliet

Abstract  The blood-brain barrier (BBB) is important to maintain brain homeostasis, which is crucial for the functionality of neuronal networks. BBB dysfunction is observed in several neurological diseases, including epilepsy. Prolonged BBB dysfunction and subsequent perivascular inflammation as well as alterations in the extracellular matrix (ECM) play an important role in epileptogenesis. In this chapter we aim to give an overview of the processes involved in BBB dysfunction and epileptogenesis and provide evidence from animal as well as from human studies. We will focus on modulators of BBB function including specific cell types, inflammatory mediators as well as ECM molecules. Furthermore, we will discuss new therapeutic targets and the results of intervention studies that are important for the development of novel therapeutic approaches in epilepsy. Keywords  Blood-brain barrier · Extracellular matrix · Brain inflammation · Epilepsy · Epileptogenesis · Therapy

D. W. M. Broekaart · A. Korotkov Department of (Neuro)pathology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands J. A. Gorter Swammerdam Institute for Life Sciences, Center for Neuroscience, University of Amsterdam, Amsterdam, The Netherlands E. A. van Vliet (*) Department of (Neuro)pathology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Swammerdam Institute for Life Sciences, Center for Neuroscience, University of Amsterdam, Amsterdam, The Netherlands e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_4

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1  The Blood-Brain Barrier The blood-­brain barrier (BBB) is a complex and highly selective barrier that separates the peripheral circulation from the central nervous system (CNS). The BBB is primarily formed by brain endothelial cells connected via tight junctions. They are ensheathed by astrocytic end feet, while pericytes and perivascular macrophages are embedded in the basement membrane of blood vessels between endothelial cells and astrocytes (Fig. 1). Together with neurons, they form the neurovascular unit, a dynamic system that tightly controls the movement of ions and molecules and prevents the entrance of cells from the bloodstream into the CNS which is important for brain homeostasis and regulating neuronal physiology [1]. After a stroke or an insult, a variety of cells (including endothelial cells, pericytes, and astrocytes) can produce inflammatory mediators and extracellular matrix (ECM) proteins, which modulate diverse molecular pathways that cause adaptation of the BBB to environmental changes that may lead to BBB dysfunction [2–6]. Dysfunction of the BBB is observed in several CNS pathologies, including epilepsy in which it can contribute to disease progression and resistance to therapeutic drugs [7–10]. We will discuss molecular mechanisms that are involved in BBB dysfunction in epilepsy, based on evidence from preclinical (Table 1) and clinical studies (Table 2), with a focus on specific inflammatory pathways and extracellular matrix (ECM) proteins. Furthermore, we will discuss the potential of novel therapeutic interventions (Tables 3 and 4).

2  M  odulators of Blood-Brain Barrier Function and Their Involvement in Epileptogenesis 2.1  Endothelial Cells A monolayer of tightly connected endothelial cells forms a physical barrier across the capillary walls that restricts the movement of ions such as Na+, K+, and Cl−, while at the same time it allows the diffusion of oxygen and CO2 [1]. In contrast to the periphery, endothelial cells within the CNS have very low rates of transcytosis, limiting the transport of solutes mediated by vesicles [109]. The control of vascular integrity of capillaries is mediated by cell-­cell and cell-­matrix interactions regulated by tight junctions and adherens junctions. The tight junction family includes claudins, junctional adhesion molecules, occludins, endothelial-­cell-­selective adhesion molecules, and nectins [110]. In vitro experiments have shown that tight junction proteins have a size-­selective permeability to uncharged molecules of up to 4 nm and low permeability to larger molecules, suggesting that larger molecules can only traverse when the integrity of the BBB is compromised or via a transport mechanism [111, 112]. Adherens junctions include vascular endothelial (VE)-­cadherin and platelet endothelial cell adhesion molecule 1 (PECAM1). These are linked to

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Fig. 1  Organization of the blood-­brain barrier and mediators involved in its dysfunction. The blood-­brain barrier (BBB) consists of several cell types working together to form a tight layer, regulating the influx and efflux of substances between the blood and the brain. The major cell types are tightly connected endothelial cells, surrounded by the basement membrane, pericytes, and perivascular macrophages that are embedded in the basement membrane of blood vessels, as well as astrocytes that ensheathe these cells with their endfeet. After an epileptogenic insult, these cells can, together with microglia/macrophages, leukocytes, and neurons, secrete pro-­inflammatory mediators and extracellular matrix proteins, which can disrupt the BBB and as a consequence lead to the entry of blood components (e.g., albumin) into the brain. Perivascular inflammation, BBB dysfunction, and remodeling of the extracellular matrix can enhance neuronal excitability and contribute to epileptogenesis

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Table 1  Experimental studies Marker Model NG2DsRed / Mouse intrahippocampal PDGFR-­β kainic acid SE model or intraperitoneal kainic acid SE model

MMPs/ TIMPs

Rat lateral fluid-­percussion injury model Rat angular bundle stimulation SE model

Rat kainic acid SE model

Rat kainic acid SE model

Rat kainic acid SE model Rat pilocarpine SE model Mouse kainic acid SE model Rat kainic acid SE model

Mouse pilocarpine SE model Mouse controlled cortical impact TBI model

IL-­1β

Rat angular bundle stimulation SE model Mouse intrahippocampal kainic acid SE model Rat pilocarpine SE model

Rat hippocampal stimulation SE model

Findings Hippocampus 1 d↑, 2 d↑, 3 d↑, 1 w↑, >4 w↑ Clustering with microglia Positive correlation with BBB dysfunction Hippocampus, cortex, thalamus, 2 d↑, 7 d↑, 3 m↑ Hippocampus and/or temporal lobe, Mmp2 1 d↑, 1 w↑, 3 m↑, Mmp3 1 d↑, 1 w↑, 3 m↑, Mmp9 1 d↑, 1 w↑ 3 m↑, MMP9 1 d↑, 1 w↑ 3 m↑, Mmp14 1 d↑, 1 w↑ Hippocampus, diencephalon, striatum, frontal cortex, midbrain, MMP2 3 d↑, 1 w↑ Hippocampus, diencephalon, striatum, frontal cortex, cerebellum, mid brain, MMP9 6 h↑, 12 h↑ Hippocampus, frontal cortex and temporal lobe MMP2 activity 3 d↑ Temporal lobe MMP2 activity 16 d↑ Frontal cortex, temporal lobe, MMP9 activity 12 h↑ Hippocampus Mmp14 1 d↑ Hippocampus Mmp3 48 h↑, 1 w↑, Mmp13 48 h↑, MMP13 1 w↑ Hippocampus MMP3 3 d↑, MMP9 3 d↑ MMP2 and MMP9 activity neurons 8 h↑; glia 8 h↑, 3 d↑; blood vessel 8 h↑, 3 d↑, 7 d↑, 15 d↑ Hippocampus MMP9 3 d↑ Cortex MMP9 30 min↑, 2 h↑, 6 h↑, 1 d↑ Hippocampus MMP9 30 min↑, 6 h↑, 1 d↑ Hippocampus and/or temporal lobe Timp1 1 d↑,1 w↑, Timp2 1 d↑, 1 w↑ Hippocampus Timp1, Timp2 15 d↑

References [10–12]

[12] [13–15]

[16]

[17]

[18] [19] [20, 21] [22]

[23] [24]

[14] [25]

Neocortex 2 h↑, 6 h↑, 24 h↑, piriform [26] cortex 2 h↑, 24 h↑, hippocampus 2 h↑, 6 h↑ Hippocampus, Il-­1β 2 h↑, 6 h↑, 60 d↑ [27] IL-­1β 18 h↑, 48 h↑, 1 w↑ (continued)

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Table 1 (continued) Marker

Model Rat angular bundle stimulation SE model Mouse soman SE model Rat febrile seizure model Rat pilocarpine SE model

Rat pilocarpine SE model TNF-­α

Rat amygdala kindling model Rat pilocarpine SE model Immature rat pilocarpine SE model Rat intrahippocampal kainic acid SE model Rat hippocampal stimulation SE model Rat subcutaneous kainic acid SE model Mouse soman SE model

TGF-­β

CCL2

Rat amygdala kindling model Rat angular bundle stimulation SE model Mouse pilocarpine SE model Mouse i.c.v. kainic acid SE model Rat angular bundle stimulation SE model

Rat pilocarpine SE model

CD163

Rat intrahippocampal kainic acid SE model Rat angular bundle stimulation SE model

Findings Entorhinal cortex 1 d↑, 1 w↑

References [28]

Cortex 1 h↑, 2 h↑, 6 h↑, 7 d↑, hippocampus 6 h↑, 24 h↑, 48 h↑, 7 d↑ Hippocampus 24 h↑ Frontoparietal cortex, entorhinal cortex, and hippocampus 4 h–36 h↑, 3–7 d ↑, 4 m↑ Dentate gyrus, entorhinal cortex 24 h↑ Hippocampus, entorhinal cortex 6 d↑ Blood↑, cortex↑, amygdala↑, hippocampus↑ Facilitates seizures Hippocampus 12 h↑, 24 h↑,1 w↑ Hippocampus 2–4 h↑, 8 w↑

[29] [30] [31]

[32] [33–35]

[26, 36] [37, 38]

Hippocampus 2 h↑, 6 h↑, 18 h↑, 1 d↑, 3 d↑ Hippocampus 2 h↑, 6 h↑, 18 h↑, 1 d↑

[27]

Hippocampus 3 h↑, 6 h↑, 12 h↑, 1 d↑

[40]

Cortex 2 h↑, 6 h↑, 1 d↑, 2 d↑ Hippocampus 6 h↑, 1 d↑, 2 d↑, 7 d↑ Cortex, amygdala, hippocampus 2 h↑

[29] [33]

Hippocampus 48 h↑, 1 w↑, 3 w↑

[41]

Hippocampus 2 h↑, 1 d↑,1 w↑, 2 m↑

[42]

Hippocampus 1 d↑, 3 d↑

[43]

Hippocampus 1 d ↑, 1 w↑, 6 w↑, 7–9 m↑ Positive correlation with seizure frequency Hippocampus 2 h↑, 6 h↑, 24 h↑, 5 d↑

[28, 44]

Hippocampus 2–4 h↑, 12–72 h↑ 21–45 d ↑ Hippocampus 1 d ↑, 1 w↑, 6 w↑, 7–9 m↑ Positive correlation with BBB dysfunction and seizure frequency

[39]

[26, 45, 46] [47, 48] [44]

(continued)

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Table 1 (continued) Marker CD68

Model Rat angular bundle stimulation SE model

miRNAs

Rat angular bundle stimulation SE model

Rat pilocarpine SE model Mouse pilocarpine SE model

Findings Hippocampus 1 d ↑, 1 w↑, 6 w↑, 7–9 m↑ Positive correlation with duration SE and seizure frequency Hippocampus miR-­146a 1 d ↑, 1 w↑ Negative regulation of inflammation Hippocampus miR-­155 1 d↑, 1 w↑, 3–4 m↑ Hippocampus miR-­155 1 d↑, 14 d↑, 30 d↑, 60 d↑ Hippocampus miR-­155 4 h↑, 12 h↑

References [44]

[15, 49]

[50] [51]

d day(s), h hour(s), m month(s), w week(s), ↑ increase, i.c.v. intracerebroventricular, SE status epilepticus, TBI traumatic brain injury

the cytoskeleton by binding catenins and thereby regulate junctional assembly and maintenance. In addition, endothelial cells secrete a variety of factors, including inflammatory mediators and ECM proteins, which modulate diverse molecular pathways that lead to adaptation of the BBB [2, 3].

2.2  Astrocytes Perivascular astrocyte endfeet ensheathe the abluminal side of the cerebral vessels and contains a distinct array of proteins including dystroglycan, dystrophin, aquaporin 4, and the ATP-­sensitive inward rectifier potassium channel Kir4.1 [113]. As astrocytic processes can also ensheathe neuronal processes, astrocytes provide coupling between blood vessels and the neuronal circuitry. As a consequence, astrocytes are involved in the regulation of blood flow and permeability of the barrier in response to neuronal activity [114–116]. It is proposed that astrocytes release soluble factors determining the fate of cerebral vascular endothelial cells. In vitro co-­ culture experiments have supported the hypothesis that astrocytes are mainly involved in maintaining the barrier, rather than barrier formation [117]. In addition, astrocytes secrete a variety of factors, with either BBB-­promoting or BBB-­disrupting effects, depending on the signals received from neurons, microglia, and/or endothelial cells [2, 3, 116]. These factors include pro-­inflammatory mediators and ECM proteins that modulate diverse molecular pathways, which will be discussed in the following paragraphs.

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Table 2  Human studies Marker Pathology PDGFR-­β TLE, FCD

References [10, 11, 52, 53]

MMP

[13, 54–56]

TIMP

IL-­1β

TNF-­α TGF-­β

CCL2

CD163 CD68

miRNAs

Findings Hippocampus, cortex↑ Clustering with microglia TLE MMP2↑, MMP3↑, MMP9↑, MMP14↑ hippocampus FCD, TSC MMP2↑, MMP3↑, MMP9↑, MMP14↑ cortex TSC MMP2↑, MMP11↑, MMP14↑, MMP15↑, MMP17↑, MMP19↑ SEGA SE MMP2↑, MMP3↑, MMP9↑, MMP14↑ hippocampus TLE TIMP1↑, TIMP2↑, TIMP3↑, TIMP4↑ hippocampus FCD, TSC TIMP1↑, TIMP2↑, TIMP3↑, TIMP4↑ cortex TSC TIMP1↑, TIMP2↑, TIMP3↑ SEGA SE TIMP1↑, TIMP2↑, TIMP3↑, TIMP4↑ hippocampus TLE Hippocampus↑ FCD, glioneuronal tumor Cortex↑ Correlated to seizure frequency TSC Cortex↑ TLE Hippocampus↑ High dose can induce seizures TSC Cortex↑ TLE Hippocampus↑ FCD Cortex↑ TLE pSmad2↑ SE, TLE Hippocampus↑ Temporal cortex↑ Positive correlation with duration of epilepsy FCD Cortex↑ Stroke, traumatic brain injury, Cortex↑ Rasmussen’s encephalitis Positive correlation with family history of epilepsy SE, TLE Hippocampus↑ SE, TLE Hippocampus↑ Positive correlation with duration of epilepsy TLE, TSC miR146a, miR-­21, miR-­155, miR-­147b, hippocampus↑, cortex↑

[54, 57, 58] [59]

[13] [13, 56] [57, 58] [59] [13] [60, 61] [62, 63] [64] [37, 61, 65, 66] [67] [61, 68, 69] [70] [71] [44, 72, 73]

[62, 73] [73]

[44] [44]

[15, 49, 50, 58, 74–76]

↑ increase,  FCD focal cortical dysplasia, SE status epilepticus, SEGA subependymal giant cell astrocytoma, TLE temporal lobe epilepsy, TSC tuberous sclerosis complex

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Table 3  Intervention studies in animals Target Integrins

Model Mouse pilocarpine SE model Glucocorticoid Rat pilocarpine SE receptor model

Intervention α4 or α4β1 antibody

IL-­1β

IL-­1β synthesis inhibitor (VX-­765) IL-­1β synthesis inhibitor (VX-­765) or pralnacasan IL-­1β synthesis inhibitor (VX-­765)

Rat hippocampal kindling model Mouse and rat intrahippocampal kainic acid SE model Genetic absence epilepsy rats from Strasbourg Rat hippocampus stimulation and pilocarpine SE model Mouse intra-­amygdala kainic acid SE model Mouse bicuculline/ kainic acid SE model

Rat intrahippocampal kainic acid SE model Mouse bicuculline SE model Guinea pig in vitro whole brain model

TNF-­α

TGF-­β

Dexamethasone

VX-­765 + IL1ra

VX-­765 + CyP

Findings BBB leakage↓ Ictogenesis↓ BBB leakage↓ Ictogenesis↓ Epileptogenesis↓ No kindling development Ictogenesis↓ Epileptogenesis↓ Number and duration of spike-­and-­waves↓ No effects on epileptogenesis Neuroprotection Epileptogenesis↓

References [77] [78]

[31] [79, 80]

[81]

[82]

[83]

Ictogenesis↓ TLR4 antagonists Epileptogenesis↓ (BoxA or Rhodobacter sphaeroides lipopolysaccharide) IL-­1ra Ictogenesis↓

[85]

IL-­1ra

[86]

IL-­1ra (anakinra)

Ictogenesis↓

BBB leakage↓ Bicuculline-­­ induced seizures↓ Rat kainic acid-­­ TNF-­α-­neutralizing DNA damage induced seizure model antibodies hippocampus↓ Rat pilocarpine SE Soluble TNF p55 Neuronal model receptor damage↓Vasogenic edema↓ TGF-­β RII antibody, Albumin and Deoxycholic acid or TGFβ-­induced albumin application on TGF-­β RI kinase epileptiform activity inhibitor the cortex in rats (SB431542), TGF-­β activity↓ inhibitor (losartan) Epileptogenesis↓ Intracerebroventricular ALK5/TGF-­β infusion of albumin or inhibitor (SJN2511) TGF-­β in mice

[84]

[87]

[88] [36]

[89–91]

[92]

(continued)

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Table 3 (continued) Target

CCL2

Model Intervention Intracerebroventricular TGF-­β inhibitor infusion of albumin in (losartan) mice

Albumin application on the cortex in rats

TGF-­β receptor blockers

Mouse lipopolysaccharide model

CCL2 transcription inhibitor (bindarit), CCR2 antagonist (RS102895), or CCL2 antibody CCL2 antibody or CCR2 −/− mice

Mouse i.c.v. kainic acid SE model MMPs

miRNAs

Mouse pentylenetetrazole seizure model Mouse controlled cortical impact TBI model Mouse middle cerebral artery occlusion model Mouse pentylenetetrazole seizure model Mouse kainic acid SE model Rat rapid kindling model Mouse intra-­amygdalar kainic acid SE model Mouse intracerebral lipopolysaccharide model Mouse pilocarpine SE model Mouse pilocarpine SE model Rat pilocarpine SE model

Findings Blockade of degradation of perineuronal nets around parvalbumin neurons ECM genes (Serpine1, Adamts1, MMP9, Serpina3n, Stat3, TIMP1, Ncan, TnC) ↓ Ictogenesis↓

References [71]

[71]

[93]

Mmp9 −/−

Brain infiltration of [43] monocytes↓, seizure severity↓ Epileptogenesis↓ [94, 95]

Mmp9 −/−

Epileptogenesis↓

[24]

Mmp9 −/−

BBB leakage↓

[96]

MMP9 inhibitor (DP-­b99)

Epileptogenesis↓

[97]

MMP2/9 inhibitor (IPR-­179) MMP2/9 inhibitor (IPR-­179) i.c.v. mimic miR-­146a Mmp3−/−

Ictogenesis↓ Epileptogenesis↓ Ictogenesis↓ Epileptogenesis↓ Ictogenesis↓ Epileptogenesis↓ BBB leakage↓

[13]

[98]

i.n. mimic miR-­146a i.c.v. antagomir of miR-­155 i.c.v. antagomir of miR-­155

Ictogenesis↓

[99]

Ictogenesis↓

[51]

Ictogenesis↓ Epileptogenesis↓

[50]

[13] [83]

↑ increase, ↓ decrease, BBB blood-­brain barrier, i.c.v. intracerebroventricular, IL-­1ra interleukin 1 receptor antagonist, i.n. intranasal, SE status epilepticus, TBI traumatic brain injury

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Table 4  Intervention studies in humans Target Glucocorticoid receptor

IL-­1β

TNF-­α

Pathology Refractory encephalopathy with continuous spike-­and-­­ wave during sleep Intractable childhood epilepsy TLE

Intervention Dexamethasone

Findings 7/15 patients responded

Dexamethasone

Seizure frequency ↓ [101] in 5/13 patients Seizure frequency↓ [102]

IL-­1β synthesis inhibitor (VX-­765) Super-­refractory SE IL-­1 receptor secondary to FIRES antagonist (anakinra) Drug-­resistant epilepsy IL-­1 receptor antagonist (anakinra) Rasmussen’s TNF-­α antibody encephalitis (adalimumab)

References [100]

Seizure frequency↓ [103, 104]

Seizure frequency↓ [105–107]

Seizure frequency ↓, sustained improvement in 5/11 patients

[108]

↑ increase,↓ decrease, FIRES febrile infection-­related epilepsy syndrome, SE status epilepticus, TLE temporal lobe epilepsy

2.3  Pericytes Pericytes are located within the basement membrane of brain arterioles and capillaries. They interact with astrocytic endfeet and participate in structural and homeostatic BBB functions [10]. Pericytes are responsive to stimuli from cerebral glial cells and from circulating leukocytes and participate to both the innate and the adaptive immune response [10]. Interestingly, several studies provide evidence that pericytes have the ability to interact with immune cells [10] and can acquire a microglial phenotype [118]. Furthermore, they may have macrophage-­like properties [119] and rapidly relay inflammatory signals from the periphery to the brain via Chemokine C-­C motif ligand 2 (CCL2) [6]. Pericytes can be detected using an antibody against platelet-­derived growth factor receptor beta (PDGFR-­β), and they can also be studied using NG2DsRed transgenic mice. Using these approaches, it has been shown that pericytes redistribute and participate in immune changes (a process that is called pericytosis) 24  h to 7 days after kainic acid-­induced status epilepticus (SE) in mice [11, 12], as well as in rats 2–7 days and 3 months after traumatic brain injury [12]. Recent evidence shows that pericytosis occurs during epileptogenesis in the intra-­hippocampal kainic acid mouse model of SE, during the latent phase, but also during the chronic epileptic phase [10]. Furthermore, hypertrophic pericytes that detach from the capillary basal lamina are also found in resected epileptogenic brain tissue from patients with temporal lobe epilepsy (TLE) or focal cortical dysplasia (FCD) [10, 11, 52,

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53]. The deattachment of pericytes may lead to vascular leakage [120]. Pericytes can cluster with perivascular microglia after SE, a process that is mediated by IL-­1β and associated with BBB damage [10]. The aggregation of pericytes with microglia may create an inflammatory perivascular niche. It has also been proposed that migration of pericytes into an injured brain region stabilizes leaky blood vessels and initiates glial scar formation after traumatic brain injury [12]. This points to a dual role of pericytes. Since the precise role of pericytes in the epileptogenic brain is not completely understood, this needs to be further investigated.

2.4  Basement Membrane Besides the several cell types that make up the BBB, the basement membrane plays an important role in vascular integrity as well. It provides both an extra physical barrier and an ideal location for many signaling processes [121]. The basement membrane consists of ECM that is produced by endothelial cells and pericytes, making up the inner vascular basement membrane, whereas astrocytes are mostly responsible for the parenchymal basement membrane [122, 123]. These two membranes have a slightly different composition, according to their function and cell associations, but are predominantly composed of laminin, collagen IV, nidogen, and heparan sulfate proteoglycans that support mutual interactions between endothelial cells, pericytes, and astrocytes [2, 123, 124]. Binding of these ligands by the matrix transmembrane receptors dystroglycan and integrins leads to the activation of various growth factors and signaling cascades. Deficiencies of ligand binding, on the other hand, can lead to altered brain vascularization [125, 126].

2.5  Extracellular Matrix and Matrix Metalloproteinases The ECM is the noncellular component within the brain, which does not only provide a physical scaffold but is also important for cellular growth, activity, and survival [127]. The ECM is produced intracellularly and secreted to form a dense network of proteins and glycans, occupying the parenchyma of virtually all cells. It accounts for 10–20% of the total brain volume and consists of highly organized molecular structures [127]. Besides ECM molecules, the extracellular space is also home to soluble growth factors and fragments of membrane-­bound molecules that are proteolytically generated [128]. The major component of the ECM is hyaluronan, which has a high molecular weight and provides the CNS with a structural framework on which several other ECM components can bind [129, 130]. The ECM is a highly dynamic structure that continuously undergoes controlled remodeling, mediated by specific proteolytic enzymes that are responsible for ECM degradation as well as by molecules that are produced by endothelial cells, pericytes, and astrocytes [131].

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One of the most important players in ECM degradation is the family of matrix metalloproteinases (MMPs) [131]. Containing around 25 family members, from which only a few are present in brain, these proteases are divided in different groups based on their substrate specificity, which is defined by their structure. They are either membrane-­bound or secreted into the ECM and require proteolytic removal of a pro-­peptide domain for activation [132]. Under normal conditions, MMPs execute functions in remodeling of the ECM, angiogenesis, neurogenesis, and synaptic plasticity [134]. However, under pathophysiological conditions, MMPs can be activated which promotes degradation of ECM components and contributes to neuroinflammation via activation of cell adhesion molecules and receptors, as well as cytokines such as interleukin 1β (IL-­1β), tumor necrosis factor-­α (TNF-­α), and transforming growth factor-­β (TGF-­β). The stimuli leading to transcriptional activation of MMPs include pro-­ inflammatory cytokines, reactive oxygen species, hypoxia, and alterations in pH [134–136]. Furthermore, MMPs can be activated through stimulation by proteases of the plasmin system, such as tissue plasminogen activator (t-­PA) and urokinase plasminogen activator (u-­PA) that are produced as a result of BBB disruption [137]. MMPs can degrade several tight junction proteins, such occludin and claudin, as well as several components of the basement membrane, including laminin, fibronectin, heparan sulfate, and type IV collagen. The activity of MMPs is further regulated by the endogenous tissue inhibitor of metalloproteinases (TIMPs). An imbalance between activation and inhibition of these proteases can disturb ECM homeostasis and result in a compromised BBB. In several SE models, higher gene and protein expression of MMP2, MMP3, MM9, MMP13 and/or MMP14, as well as MMP2 and MMP9 activity, is observed in the hippocampus within 24 h after SE as compared to controls [16, 17, 22], but also at later time points: from several days after SE (during the latent phase) to several weeks (during the chronic phase, when recurrent spontaneous seizures are evident) [13, 18–21, 23]. Using microarray and PCR analysis, it has been shown that MMP gene expression changes according to a specific pattern in the angular bundle stimulation SE rat model: Mmp2 peaks during the latent phase but is also higher during the acute and latent phase as compared to controls. Mmp3 expression peaks at the acute phase but is also higher at the latent and chronic phase as compared to controls. Mmp9 peaks during the chronic phase but is also higher during the acute and latent phase, and Mmp14 peaks during the acute phase and latent phase [13, 14]. Furthermore, gene expression of the endogenous MMP inhibitors Timp1 and Timp2 is higher 1  day and 1  week in the hippocampus and/or the temporal lobe of the angular bundle stimulation SE rat model as compared to controls [14] and 15 days after SE in the intrahippocampal kainic acid SE mouse model [25]. In the hippocampus of patients with TLE, higher gene expression of MMP2, MMP3, MMP14, TIMP2, and TIMP3 is observed in the hippocampus [13] as well as higher protein expression of MMP2, MMP3, MMP9, MMP14, TIMP1, TIMP2, TIMP3, and TIMP4 [13, 54–56] as compared to autoptic controls. In the cortex of patients with FCD or TSC, higher protein expression of MMP2, MMP3, MMP9, and TIMP2 is reported [54, 57]. In a recent study, higher protein expression of MMP2, MMP3, MMP9, MMP14, TIMP1, TIMP2, TIMP3, and TIMP4 is observed

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in neurons and/or glial cells within the hippocampus of patients who died after SE, as well as in epileptogenic brain tissue from patients with TLE [13] or tuberous sclerosis complex (TSC) [58] as compared to autoptic controls, which is associated with BBB dysfunction [58]. Furthermore, higher MMP2, MMP11, MMP14, MMP15, MMP17, MMP19, TIMP1, TIMP2, and TIMP3 gene expression is shown in subependymal giant cell astrocytomas of patients with TSC as compared to autoptic controls [59]. Higher MMP expression is associated with opening of the BBB in rats [135], via degradation of the basal lamina and tight junction proteins, promoting the infiltration of neutrophils in the brain parenchyma [98]. In Mmp3 knockout mice, disruption of the BBB after intracerebral injection of lipopolysaccharide is attenuated [98]. Although MMPs are expressed by endothelial cells and various cells of brain parenchyma, blood-­borne cells become an important source of MMPs when the BBB is compromised. In ischemic Mmp9 knockout mice, MMP9-­immunoreactive neutrophils and leukocytes are shown in the areas of blood vessel disruption, and leukocyte-­derived MMP9 is largely responsible for BBB disruption [138], which may be due to basal lamina type IV collagen degradation [139]. Moreover, in Mmp9 knockout mice, ZO-­1 degradation is reduced, and BBB disruption is attenuated after ischemia mice [96]. Traumatic brain injury leads to the disruption of the BBB mediated by MMP9 and aquaporin-­4 [140]; the latter is highly expressed in the end feet of astrocytes. Furthermore, MMP2, which is bound to the membrane and activated by MMP14, degrades tight junction proteins [141]. In Mmp9-­deficient mice, kindling-­induced epileptogenesis is delayed [94, 95], while in mice that overexpress MMP9, the opposite occurs [94]. Mmp9-­deficient mice also have less seizures after traumatic brain injury, while the opposite is found for mice that overexpress MMP9 [24], indicating an important role for MMP9 in epileptogenesis. Therefore, MMP inhibitors may have therapeutic potential in epilepsy. The general MMP inhibitor GM6001 attenuates induced capillary leakage ex vivo and in vitro by prevention of tight junction degradation [142, 143]. However, more than 50 MMP inhibitors have been tested during clinical trials, and they have low bioavailability and/or severe side effects [144]. The MMP9 inhibitor DP-­b99 delays the onset and severity of pentylenetetrazole-­induced seizures in mice [97]. However, despite encouraging preclinical and phase II clinical trial data, DP-­b99 shows no evidence of efficacy in treating human ischemic stroke [145]. Interestingly, a novel MMP inhibitor, IPR-­179, has a high affinity for MMP9 but also for MMP2 (although to a lesser extent). IPR-­179 has antiseizure and antiepileptogenic effects in the intrahippocampal kainic acid SE model and in the rat rapid kindling model and attenuated seizure-­induced cognitive decline  [13]. Furthermore, IPR-­179 is much more potent than the broad-­spectrum inhibitor minocycline, and side effects were not observed [13]. Therefore, this novel MMP inhibitor deserves further investigation in clinical trials. Another recent development is the use of nanoparticles for the delivery of TIMP1 to inhibit MMPs [146]. In response to injury, proteases have a dual role: they may cause further damage but are also involved in repair. The same enzymes that could be destructive in the early stages after injury may be beneficial for the healing process at later stages [147–149]. When using MMP inhibitors, one

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should therefore carefully consider the time of administration relative to an epileptogenic insult, the duration of the treatment, and the specificity of the inhibitor.

2.6  Immune Cells Endothelial cells express cellular adhesion molecules, including intracellular adhesion molecule-­1 (ICAM-­1) and vascular cell adhesion molecule 1 (VCAM-­1), which play a major role in the recruitment of leukocytes into the brain [150–152]. In control conditions, very low amounts of ICAM-­1 and VCAM-­1 are expressed. However, in various CNS pathologies (including epilepsy), leukocytes can gain entry to the brain due to increased expression of these adhesion molecules. Cells of the innate immune system (e.g., microglia, macrophages, monocytes, natural killer cells) closely interact with cells from the adaptive immune system (B-­and T-­lymphocytes) to maintain a proper immune balance in the brain. ICAM-­1 and VCAM-­1 are strongly upregulated following seizure induction, with the highest expression observed 24 h and 7 days after pilocarpine-­induced SE in mice. A prominent role for leukocytes in epilepsy has been suggested after pilocarpine-­­induced SE in rats [77, 153]. Furthermore, increased numbers of CD68-­ positive monocytes/macrophages, CD163-­positive perivascular macrophages, and other cells of the innate immune response (e.g., microglia) are evident during the acute (24  h post-­SE), latent (1  week post-­SE), and chronic phase (6  weeks and 7–9  months post-­SE) after angular bundle stimulation-­induced SE in rats [44]. Interestingly, the number of hippocampal CD163-­ positive perivascular macrophages is positively correlated to BBB dysfunction, as assessed with the BBB permeability marker fluorescein in rats [44]. Furthermore, the expression of CD68-­postive monocytes/macrophages, CCL2, and CD163-­positive perivascular macrophages is positively correlated to the duration of SE, as well as the number of spontaneous seizures [44]. In contrast, T-­lymphocytes are not detected in the hippocampus of controls or at 24  h, 1  week, or 2  months after angular bundle stimulation-­­induced SE in rats [44]. Comparable results are obtained after pilocarpine or kainic acid-­induced SE in rats, since B-­and T-­lymphocytes are rarely observed during epileptogenesis, while monocytes/macrophages are evident in the brain shortly after SE, which persisted during the chronic phase [60, 154, 155]. In resected hippocampal tissue from patients with TLE, higher expression of ICAM-­1 is observed as compared to controls, which is associated with BBB disruption [156]. Furthermore, CD68-­positive monocytes/macrophages, CD163-­positive perivascular macrophages, and other cells of the innate immune response (e.g., microglia) are persistently activated in resected hippocampal tissue from patients with TLE [44]. In humans, the duration of epilepsy positively correlates to the expression of CD68-­ postive monocytes/macrophages [44]. In contrast, CD3-­­ positive T-­lymphocytes, CD83-­positive immature and mature dendritic cells, and CD209-­positive mature dendritic cells are scarce in human control brain and are

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occasionally detected in brains of patients who died after SE and in patients with TLE [44, 60, 77, 157]. This suggests that these cells play a less prominent role than the dramatically increased activated cells of the innate immune response (e.g., monocytes, macrophages, and microglia). However, in patients with FCD or Rasmussen’s encephalitis, activation of both the adaptive and innate immunity is shown [62, 158]. In a recent study, flow cytometric analysis of inflammatory leukocytes in resected brain tissues from pediatric patients with genetic (FCD) or acquired (encephalomania) epilepsy demonstrates significant brain infiltration of blood-­­ borne inflammatory myeloid cells and activated memory CD4+ helper and CD8+ cytotoxic T-­lymphocytes [159]. Moreover, pro-­inflammatory IL-­17 producing ϒδ T-­lymphocytes are concentrated in the epileptogenic zone, and their numbers positively correlate with seizure severity, whereas the number of brain-­infiltrating regulatory T-­ lymphocytes inversely correlate with disease severity [159]. This is supported by experiments after kainic acid-­induced SE in mice, since IL17RA-­and ϒδ T cell-­deficient mice have less severe seizures, whereas autologous natural T-­regulatory lymphocyte depletion worsens and T-­regulatory lymphocyte supplementation dampens seizure susceptibility [159]. Inhibition of leukocyte-­vascular interactions by blocking antibodies to α4 or α4β1 reduces BBB leakage after pilocarpine-­induced SE in mice [77]. However, the use of FDA-­approved drugs (e.g., Tysabri or Gilenya), to prevent leukocyte entry into the brain as potential therapy for patients with epilepsy, is problematic because of potential severe side effects [159]. Anti-­inflammatory/immune-­suppressive therapy using the corticosteroid dexamethasone was given to 15 patients with refractory epileptic encephalopathy, of which 7 patients responded [100]. In another study, dexamethasone reduced the seizure frequency in 5/13 children with drug-­resistant epilepsy [101]. In animals, dexamethasone reduces BBB leakage, ictogenesis, and epileptogenesis after pilocarpine-­induced SE in rats [78]. However, the severe side effects of steroids have prevented long-­term or widespread use of these drugs [159]. In contrast to these findings, an anti-­epileptogenic and neuroprotective role for both innate and adaptive immune cells has been suggested after kainic acid-­induced SE in mice [160]. Taken together, the innate immune response is mainly activated in the epileptogenic brain, suggesting a prominent role during epileptogenesis, while activation of the adaptive immune response is not always evident. However, this may depend on the type of epilepsy, animal model, or species.

2.7  Inflammatory Mediators 2.7.1  Cytokines and Chemokines Cytokines and chemokines (chemotactic cytokines that direct the migration of cells that express the appropriate chemokine receptor) play an important role in the neuroinflammatory response. These inflammatory mediators can be released after an initial

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precipitating injury such as SE or traumatic brain injury by a variety of cells, including endothelial cells, astrocytes, microglia, neurons, as well as by infiltrating leukocytes [3]. They are not only involved in promoting local brain inflammation but can also affect BBB function and act as neuromodulators by directly affecting neuronal function and excitability [3]. We do not intend to give a complete overview of all cytokines and chemokines, but rather focus on specific cytokines and chemokines that have been suggested or shown to play a major role in BBB dysfunction and epilepsy. 2.7.2  Interleukin-1β The interleukin-­1 (IL-­1) family is the most important family of secreted cytokine mediators of inflammatory responses [161]. Out of the 11 family members, the most studied interleukin is IL-­1β. Although it was initially described as immune cell mediator in the periphery, produced by blood monocytes and tissue macrophages [162], IL-­1β has also been associated with several functions and dysfunctions in the brain [163]. It is mainly released by microglia and astrocytes [164]. Both IL-­1β and IL-­1α bind to the type 1 IL-­1β receptor (IL-­1R1), thereby eliciting a cascade of signaling events involving activation of the transcription factor NFκB and stress-­­ related mitogen-­activated protein kinases (MAPKs), leading to additional pro-­ inflammatory cytokine expression [162, 165]. One of the major factors involved in activation of IL-­1β is the Toll-­like receptor 4 (TLR4) [166]. While IL-­1 is present in low levels under physiological conditions, its increased expression and secretion are observed during various neurological disorders, including epilepsy [167]. After electrically or chemically induced SE, Il-­1β gene expression is rapidly upregulated (within 1 h), and the expression remains high 1 week after SE induction [27–29, 87]. Several hours after the increase of Il-­1β, an increase of IL-­1ra is observed, although never in excess to IL-­1β [27], indicating an ineffective control of IL-­1β synthesis after SE. After exposure to prolonged febrile seizures, a long-­­lasting induction of IL-­1β protein is observed in the rat hippocampus [30]. With the use of immunohistochemistry, several studies have shown the expression of IL-­1β and IL-­R1 during epileptogenesis. Both during the acute phase after SE and during the chronic phase  – when spontaneous seizures occur  – strong IL-­1β induction is observed in microglia and astrocytes in brain areas involved in seizure generation and propagation [32, 60]. Cytokine analysis after pilocarpine-­induced SE shows that the concentration of IL-­1β is higher 2, 4, and 24 h after SE in several brain regions, including the hippocampus and piriform cortex, as compared to control [26]. In patients with TLE, IL-­1β is higher in the hippocampus as compared to autoptic controls [60, 61]. IL-­1β and IL-­1R1 immunoreactivity is observed in neurons, microglia, and astrocytes, including perivascular astrocytic endfeet [60]. In patients with TSC, IL-­1β expression is high in activated microglia and astrocytes [64]. In patients with FCD, high IL-­1β expression is observed in glial cells, in neurons, and in giant cells [62]. In another study, in patients with FCD and in patients with glioneuronal tumors, IL-­1β and IL-­1R1 are highly expressed by more than 30% of

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neurons and glia [63]. On the other hand, the so-­called decoy receptor IL-­1R2, named due to its lack of downstream signaling, and the IL-­1R antagonist (IL-­1Ra) are only expressed in 10–20% of cells [63]. In pediatric patients with febrile seizures and adult patients with TLE, an increase in frequency and carriage of the interleukin-­1β (-­511) allele 2 was found, which is associated with increased production of IL-­1β [168, 169]. Furthermore, increased concentrations of IL-­1β are found in cerebrospinal fluid of children with febrile seizures [170]. During epileptogenesis, higher IL-­1β expression is observed in astrocytic endfeet and in endothelial cells of the microvasculature, suggesting their association with the BBB. In fact, changes in IL-­1β expression in these cells are associated with tissue extravasation of serum albumin [31]. Furthermore, transgenic hippocampal overexpression of IL-­1β leads to BBB leakage [171], while BBB disruption is attenuated after administration of IL-­ 1ra, together with a delayed SE onset after pilocarpine-­­induced SE [172]. IL-­1β is known to have several effects on the permeability properties of the BBB [165], as it affects the induction of CCL2 and ICAM-­1 and can lead to dramatic infiltration of leukocytes [171, 173, 174]. Besides that, IL-­1 can change the expression of MMPs and adhesion molecules and thereby potentially influence BBB integrity [175]. In patients with FCD, the number IL-­1β-­and IL-­1R1-­positive neurons is positively correlated to seizure frequency [63]. Furthermore, in preclinical models using chemoconvulsant drugs such as kainic acid or bicuculline, the pre-­application of IL-­1β can prolong the duration of seizures [85, 86]. It is known that IL-­1β is involved in increased neuronal excitability through the increase of extracellular glutamate levels in several ways: (i) by inhibition of glutamate reuptake, (ii) by enhanced release of glutamate from glia, or (iii) by enhancement of NMDA-­mediated glutamate release from synaptic terminals [176]. Furthermore, IL-­1β can induce the phosphorylation of the NR2B subunit of the NMDA receptor which causes enhanced calcium influx [177]. By inhibiting GABA-­mediated chloride fluxes, IL-­1β is able to reduce inhibitory transmission [178, 179]. Accordingly, overexpression of IL-­1Ra or administration of IL-­1Ra has anticonvulsant effects [39, 86, 172]. Taken together, the induction of IL-­1β leads enhances neuronal excitability. In several animal models of epilepsy using either electrical stimulation or chemoconvulsants to induce seizures, VX-­765 or pralnacasan (an inhibitor of IL-­1β-­­ converting enzyme) has shown to reduce the number of acute and chronic seizures [31, 79, 80]. Moreover, administration of VX-­765 in Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a model in which rats experience absence seizures, reduces the number and the duration of spike-­ and-­ waves discharges [81]. Administration of an IL-­1 receptor antagonist alone or in combination with BoxA (broad spectrum receptor antagonist of HMGB1) and/or ifenprodil (NMDA receptor inhibitor) also reduces the number and duration of seizures and seizure onset [84–86]. Furthermore, the TLR4 antagonist Rhodobacter sphaeroides lipopolysaccharide reduces the number and duration of seizures number in models of both acute symptomatic seizures and spontaneous recurrent seizures [84]. Several proof-­of-­concept clinical trials have been performed targeting the interleukin signaling pathway. The blockade of IL-­ 1β biosynthesis with IL-­ 1β

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converting enzyme (ICE)/Caspase-­ 1 inhibitors (VX-­ 765) has been tested in a 6-­week phase IIA randomized, double-­blind, placebo-­controlled study in drug-­ resistant focal-­­onset epilepsy patients. Compared to placebo, VX-­765 shows a delayed beneficial effect that even persisted for a period after drug discontinuation [102]. A case report of a child with super-­refractory SE secondary to febrile infection-­ related epilepsy syndrome (FIRES) treated with anakinra (a human recombinant IL-­1 receptor antagonist) shows considerable improvement in seizure control [103]. Beneficial effects are also seen using an add-­on therapy of anakinra in four adolescents with drug-­resistant epilepsy [105]. Subsequently, several studies followed, also showing positive effects of anakinra [104, 106, 107]. 2.7.3  Tumor Necrosis Factor-α (TNF-α) TNF-­α is a cytokine, associated with the innate immune response and involved in the activation, differentiation, proliferation, and infiltration of immune cells into the CNS [180]. Generally expressed in low levels during normal physiological conditions, TNF-­α can be rapidly upregulated in glia, neurons, endothelial cells, as well as peripheral circulating cells [181, 182] in various pathophysiological conditions, including epilepsy [183, 184]. Though it is commonly considered a pro-­inflammatory cytokine, TNF-­α also has a neuroprotective role, depending on its concentration and the predominantly activated receptor type. While injected nanomolar amounts of mouse recombinant TNF-­α and transgenic mice with low to moderate TNF-­α overexpression are protective to pathophysiological changes [185], high expression of TNF-­α is found to be pathological [186, 187]. TNF receptor type 1 (TNFR1 or p55) is thought to mediate pro-­convulsive effects due to an intracellular “death domain,” while TNF receptor type 2 (TNFR2 or p75) mediates anticonvulsive and neuroprotective effects [65, 188–190]. Higher expression of Tnf-­α is found 2 h after amygdala kindling in rats in several brain regions, including the parietal, prefrontal and piriform cortex, amygdala, and hippocampus [33] as compared to controls. Higher expression of TNF-­α is also evident 12 h, 24 h and 1 week within the hippocampus after pilocarpine-­induced SE in rats [26, 36]. Furthermore, TNF-­α expression is higher in the hippocampus 2–4 h and 8 weeks in the immature rat pilocarpine SE model [37, 38]. This is also found in several other SE models, in which Tnf-­α is rapidly induced, followed by a decline to basal levels within 72 h after SE onset [27, 29, 39, 40]. Immunohistochemical analysis shows expression in astrocytes, microglia, and in the BBB endothelium [26, 27, 36, 155]. Higher expression of TNF-­α is observed in the astrocytes and neurons within the hippocampus of patients with TLE or TSC as compared to autoptic control tissue [37, 61, 65, 67]. Several in vitro studies have shown that TNF-­α has a permeabilizing ability [191–195]. In addition, TNF-­α treatment of endothelial cells causes membrane delocalization, decreases the expression of tight junction proteins such as occludin, claudin-­5, and ZO-­1 [195–197], and increases the activity of MMP2 and MMP9 [198]. The effect of TNF-­α on BBB integrity is confirmed in various

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experimental disease models [199, 200]. Kim et al. (2013) have shown that after SE, TNF-­α-­mediated phosphorylation of NFκB results in extensive neutrophil infiltration and neuronal loss via TNFR2 [201]. Animal models of epilepsy support the mutual facilitative interaction between TNF-­α as pro-­inflammatory cytokine and seizure development, as electrical stimulation enhances TNF-­α levels in both blood and brain, while, in turn, administration of TNF-­ α after amygdala kindling facilitates behavioral seizures [34, 35]. Furthermore, transgenic mice with low/moderate overexpression of TNF-­α in astrocytes show decreased susceptibility to seizures [185], while mice with TNF-­α overexpression develop signs of neurological dysfunction, including seizures [186]. When used during immunotherapy, a high dose of TNF-­α is able to induce seizures in humans [66]. The effect of TNF-­α on brain excitability is presumed to be mediated by alterations on the assembly and expression of several receptors [202, 203]. TNF-­α can activate intracellular kinases that induce the expression of an extrasynaptic calcium-­permeable AMPA receptor [204, 205]. TNF-­α also promotes the induction of excitatory neuronal NMDA-­NR1 receptors [206] and the endocytosis of inhibitory GABA-­A receptors [205]. Moreover, glial TNF-­α can induce glutamate release and increase intracellular Ca2+ mobilization [207, 208]. Taken together, these studies provide evidence of decreased inhibitory strength and increased excitability due to TNF-­α. Pharmacological interventions targeting TNF-­α, such as neutralizing antibodies, can reduce the number of DNA-­damaged cells in the hippocampus after kainic acid-­­ induced SE in animals [88]. Neutralization of TNF-­α using soluble TNF p55 receptor attenuates SE-­ induced vasogenic edema and neuronal damages in kainic acid-­induced SE rat model [36]. Moreover, TNF-­α antibodies diminish ischemia-­­ induced apoptosis and attenuate enhanced activity of MMP2 and MMP9 and BBB damage through the upregulation of tight junction proteins in an in vitro model of human BBB [209]. In an open-­label study in 11 patients with Rasmussen’s encephalitis, it is shown that anti-­TNF-­α therapy using adalimumab was well tolerated and led to a reduction of the seizure frequency [108]. Five patients had sustained improvement over consecutive quarters in seizure frequency. Three of these five patients did not show further deterioration of neurocognitive function [108]. 2.7.4  Transforming Growth Factor-β (TGF-β) TGF-­β is a multifunctional cytokine that exists in four isoforms (TGF-­β1–β4) and plays an important role in a variety of processes, cell proliferation and differentiation, brain inflammation, and modulation of the ECM [71, 210]. TGF-­β dimers first activate the TGF-­β II receptor, which in turn phosphorylates the TGF-­β I receptor or activin-­like kinase 5 (ALK5). The phosphorylated ALK5 subsequently activates intracellular SMAD protein complexes and multiple mitogen-­ activated protein kinase (MAPK) pathways to regulate a wide range of downstream signaling pathways [180].

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In rats, Tgf-­β expression is higher in the cortex, amygdala, and hippocampus 2 h after amygdala kindling as compared to controls [33]. The expression of TGF-­β1 and TGF-­β2 isoforms is higher in neurons and glial cells within the hippocampus 2 days, 1 week, and 3 weeks after SE in rats as compared to controls [41]. In patients with TLE, TGF-­β1 is higher in the hippocampus [61, 68–70], as well as in the cortex of patients with cortical dysplasia [70] as compared to controls. Furthermore, the expression of pSmad2, a marker for TGF-­β receptor signaling, is higher in the hippocampus of patients with TLE as compared to autoptic controls [71]. Several preclinical studies indicate that TGF-­β signaling is involved in epileptogenesis after brain injury associated with BBB disruption. When albumin, the most common serum protein, leaks into the brain, it binds to TGF-­β receptors in astrocytes, and the TGF-­β pathway is activated through phosphorylation of predominantly SMAD2/3. This impairs spatial buffering of potassium and glutamate, increases expression of pro-­inflammatory cytokines, downregulates inhibitory neurotransmission, and induces excitatory synaptogenesis, resulting in increased neuronal excitability [89, 92, 211–213]. In a recent study, it is shown that TGF-­β-­regulated transcriptional activation is an early common response in various preclinical models (including three BBB disruption models, a photothrombotic cortical stroke model, and the partially isolated undercut cortex) after epileptogenic insults. Transcriptomic profiles that these models had in common include chemokine signaling, focal adhesion, and cytokine-­cytokine receptor signaling, indicating cell-­matrix-­cytokine and brain inflammation as the common thread in early response to different injuries accompanying albumin extravasation [71]. Blocking TGF-­β receptors suppresses brain inflammation and epileptiform activity in vitro and in vivo in rodents, as well as albumin-­induced expression of ECM genes including Serpine1, Adamts1, MMP9, Serpina3n, Stat3, Timp1, Ncan, and TnC [71, 89–91]. Furthermore, the TGF-­β inhibitor losartan reduces epileptogenesis in rats after cortical exposure to albumin or after cortical application of the bile acid deoxycholate [91] and blocks albumin-­induced degradation of perineuronal nets around parvalbumin-­positive neurons in mice [71]. More recently, it has been shown that SJN2511, a specific ALK5/TGF-­β inhibitor, prevents excitatory synaptogenesis and epilepsy in mice after intracerebroventricular injection of albumin or TGF-­β [92]. Although TGF-­β plays a detrimental role in epileptogenesis, in the context of stroke it may actually play a beneficial role by increasing BBB integrity via inhibition of tissue plasminogen activator [214], indicating that TGF-­β signaling is pleiotropic, complex, and context-­dependent. 2.7.5  Chemokine C-C Motif Ligand 2 (CCL2) CCL2 is a potent attractant for monocytes, therefore being previously denominated chemoattractant protein-­1 (MCP-­1) [45]. CCL2 binds to its receptor CCR2 which is present on endothelial cells, astrocytes, microglia, and neurons and can regulate

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migration and infiltration of monocytes, T-­lymphocytes, and natural killer cells to regions of inflammation as well as BBB permeability [45]. Higher expression of both CCL2 and CCR2 is observed in several rodent models of epilepsy. After pilocarpine-­induced SE, CCL2 expression is higher 2 h, 1 week, and 2 months post-­SE in the hippocampus of mice as compared to controls [42] and in the hippocampus, piriform cortex, and neocortex 2  h, 6  h, 1  day, 3  days, and 5 days post-­SE in rats [26, 45, 46]. Induction of SE with kainic acid, either intracerebroventricular (mice) or intrahippocampal (rat), results in higher hippocampal CCL2 expression 1 day and 3 days post-­SE in mice [43] and 2–4 h, 12–72 h, and 21–45 days post-­SE in the hippocampus, piriform cortex, and neocortex of rats [47, 48]. Finally, in the rat angular bundle stimulation SE model, higher CCL2 and CCR2 expression is evident 1  day, 1  week, 6  weeks, and 7–9  months  after SE [28, 44]. Higher expression of CCL2 is evident in resected brain tissue of patients with TLE (in glial cells and surviving neurons within the hippocampus and temporal lobe) [44, 72] and in patients with FCD, particularly in glial cells, neurons, and balloon cells as compared to autoptic control tissue [62, 73]. Increased expression of CCL2 can enhance BBB permeability [47, 215] by reducing the tight junction-­associated proteins ZO-­1, ZO-­2, occludin, and claudin-­5 [215, 216], sustain perivascular inflammation, and contribute to the progression of epilepsy [8]. Vice versa, recurrent seizures can enhance CCL2 release and brain inflammation. This is supported by our recent findings in resected brain tissue of patients with TLE. In this study, the duration of epilepsy positively correlates to the expression CCL2 [44]. Furthermore, a significant correlation exists between CCL2 expression and a family history of epilepsy [73]. In animals, CCL2 expression is positively correlated to the duration of SE, as well as the number of spontaneous seizures in the rat angular bundle stimulation SE model [44]. This suggests that increased CCL2 expression occurs as a consequence of seizure activity (such as during SE) but also that CCL2 can contribute to progression of seizures. The administration of a CCL2-­neutralizing antibody reduces the infiltration of macrophages into the brain after intracerebroventricular injection of kainic acid in mice [43]. Furthermore, the severity of seizures is reduced in Ccr2 −/− mice when a second dose of kainic acid was injected into the ventricle 14 days after the first injection [43]. In mice with chronic seizures, the systemic administration of either a CCL2 transcription inhibitor (bindarit) or a selective antagonist of the CCR2 receptor (RS102895), or the intracerebral injection of a CCL2 antibody, abrogated the worsening of spontaneous seizures provoked by a systemic inflammatory challenge induced by lipopolysaccharide [93].

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2.8  MicroRNAs It is now increasingly appreciated that the repertoire of inflammatory mediators in the brain includes non-­coding RNA molecules. MicroRNAs (miRNAs) is a class of small non-­coding RNAs, which has been most extensively studied in relation to neurological diseases, including epilepsy. miRNAs are 18–22 nucleotide long molecules with the ability to regulate gene expression at the posttranscriptional level. miRNAs navigate the RNA-­induced silencing complex (RISC) through complementary binding to the 3′-­untranslated regions (UTR) of the mature messenger RNA (mRNA) transcripts, which leads to a repression of the target gene translation [217]. miRNAs may have hundreds of direct targets within a cell and often simultaneously target several genes within a certain intracellular signaling pathway. A number of transcriptomic studies show that a great multitude of miRNAs are dysregulated in human TLE and experimental TLE models and with a very complex spatiotemporal expression patterns throughout epileptogenesis [218]. However, a meta-­analysis across these studies demonstrates that only a few miRNAs show strong and reproducible changes in expression level. These changes are dynamic throughout epileptogenesis and associated with such processes as inflammation, cell death, and the ECM remodeling [219]. Several miRNAs have gained a particular interest due to their ability to regulate innate immune response and inflammation in the brain [220, 221]. It has been shown that miR-­146a and miR-­155 expression is increased during epileptogenesis in the rat angular bundle stimulation SE model and in the hippocampus of patients with TLE [15, 49] and in the rat and mouse after pilocarpine-­induced SE [50, 51]. Furthermore, the expression of miR-­21, miR-­146a, miR-­147b, and miR-­155 is higher in tubers of patients with TSC as compared to autoptic control tissue and associated with intractable epilepsy and BBB dysfunction [74, 75]. These miRNAs are expressed in different cells of the brain, but their role as inflammatory modulators is especially intriguing in astrocytes and microglial cells [74, 75, 222]. These miRNAs are upregulated after IL-­1β stimulation in human astrocyte cultures and in cell cultures derived from surgically resected brain tissue of patients with TSC [74, 75]. Furthermore, miR146a and miR147b are identified as potent negative regulators of inflammatory signaling and as inhibitors of aberrant astrocytic proliferation and differentiation from the neuronal precursor cell pool. In a recent study it is shown that IL-­1β-­induced dysregulation of MMP3, TIMP2, TIMP3, and TIMP4 could be rescued by transfection of miR146a and miR147b in tuber-­derived TSC cultures [58]. In another in vitro study, transfection of miR-­320d reduces MMP2 expression in human fetal astrocyte cultures [59]. Several attempts have been made to modulate epileptogenesis in TLE models using miRNA-­based interventions. The inflammation-­associated miR-­146a is considered to be anti-­inflammatory, and increasing its level in the brain may be a therapeutic approach to treat epilepsy and alleviate associated neuropathological consequences, including inflammation at the BBB. Although the direct effects of this miRNA on the BBB are not investigated, it could also lead to restoration of the

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BBB, by reducing perivascular inflammation. Intraventricular administration of miRNA-­146a mimic reduces ictogenesis and epileptogenesis after intracerebral injection of kainic acid in mice [83]. In another study, intranasal administration of miR-­146a mimic prior to a pilocarpine-­induced SE extended the latency to generalized convulsions and reduced seizure severity in mice, which is associated with a reduction in expression of the inflammatory mediators TNF-­α, IL-­1β, and IL-­6 [99]. In contrast to miR-­146a, experimental evidence suggests that miRNA-­155 exerts a pro-­inflammatory type of action; thus its silencing may have a beneficial effect. Indeed, the inhibition of miR-­155 reduces seizure frequency after pilocarpine-­­ induced SE in rats [50] and mice [51]. Interestingly, inhibition of miR-­155 in vivo supports the integrity of endothelial tight junctions and reduces brain tissue damage after experimental ischemic stroke [223]. Moreover, miR-­155−/− mice show less BBB leakage in an acute model of systemic inflammation [224]. These findings indicate a possibility to use an antagomir of miR-­155 for the modulation of BBB leakage in epilepsy. Other studies suggest that miRNAs related to neuroinflammation can be used as therapeutic approach in epilepsy, e.g., inhibition of miR34a, miR-­132, and miR-­134 and/or overexpression of miR-­22, miR-­124, and miR-­128 [225, 226]. Functional studies should validate the therapeutic potential of these miRNAs. So far, multiple clinical trials (although not in patients with epilepsy) have been initiated using miRNAs or small interfering-­based compounds, of which some entered phase II clinical trials [227]. However, key challenges to overcome include adverse inflammatory side effects, toxicity, brain-­specific targeting, prevention of endosomal degradation, passage of the BBB, and non-­invasive administration. In this respect, studies in experimental epilepsy models using intranasal delivery of miRNAs show promising results [226]. In summary, BBB dysfunction is one of the key commonalities in epileptogenic processes that is observed in acquired but also in genetic epilepsies. About 30% of those patients cannot be adequately treated with antiepileptic drugs. Since BBB dysfunction can contribute to epileptogenesis, restoration of BBB function may be a novel therapeutic approach. Therefore, the identification of the underlying mechanisms that lead to BBB dysfunction and epilepsy is crucial. Sustained perivascular inflammation and degradation of the ECM by MMPs seem to play an important role in this respect, since interfering with these processes has antiseizure and/or antiepileptogenic effects in preclinical studies. However, clinical studies using such approaches are still rare, and therefore bridging preclinical and clinical research remains a major challenge. Collaborative preclinical and clinical drug discovery studies with academics, research institutes, and industry are needed to bridge the gap and to streamline the drug development process. Although this expensive and lengthy process presently may take several years, a joint approach could speed up the process and lead to the identification of therapeutic targets and development of new drugs and a novel therapeutic approach.

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Blood T cells and Cytokine Levels During Interictal-Ictal Transitions Sebastian Bauer

Abstract  It is largely unknown how and why acute epileptic seizures develop in a brain that does not seize most of the time. Not only local neuroinflammation but also systemic inflammation contributes to modulation of seizure susceptibility and can facilitate interictal-ictal transition. Vice versa, acute seizures influence systemic immunity by acting on circulating cytokines (in particular IL-6) and the distribution of blood leukocytes. For this bidirectional relation, the condition of the blood-brain barrier (BBB) is critical. Circulating cytokines may modulate both neuronal excitability and BBB function, while pronounced infiltration of circulating blood T cells into the brain appears only in certain etiologies. However, blood T cells and other circulating leukocytes appear to have strong effects on the BBB and might thereby indirectly affect seizure susceptibility. Clinical trials testing anticonvulsant effects of immunomodulatory drugs will eventually be required to clarify to what extent systemic immune function influences interictal-ictal transition. Keywords  Seizures · Ictogenesis · Ictal transition · Cytokines · T cells

1  I nterictal-Ictal Transition: Neuronal Mechanisms of Ictogenesis Epileptic seizures are defined as transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain [30]. Seizures can appear as acute symptomatic events within a certain timeframe during or after an acute systemic or brain injury. Alternatively, seizures can be “unprovoked” and appear “spontaneously” (i.e., without an obvious external trigger). These are the most prominent clinical hallmarks of epileptic seizures. Although epilepsies are S. Bauer (*) Epilepsy Center Frankfurt Rhine-Main, Department of Neurology, LOEWE Center for Personalized Translational Epilepsy Research (CePTER), Goethe-University Frankfurt, Frankfurt, Germany e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_5

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chronic conditions associated with enduring neurobiological, cognitive, psychological, and social consequences, a single seizure usually lasts only seconds to minutes [54]. Considering typical seizure frequencies, most epilepsy patients spend much less than 1% of their lifetime seizing. The process of transition from interictal “regular” neuronal activity to ictal (=seizure) activity is called ictogenesis, and its mechanisms are largely unknown. Proposed general factors involved in ictogenesis comprise increase of synchronization, reduced inhibition, and/or increased excitation. Newer modeling results point to time-varying ensemble interactions of different neurons rather than simple overexcitation or net lack of inhibition [126]. Indeed, recent experiments using optogenetics and designer receptors exclusively activated by designer drugs (DREADD) techniques suggest that ictogenesis begins with firing of relatively small subsets of highly connected interneurons, resulting in a depolarizing shift in GABA-mediated chloride reversal potential [65]. However, the effect of interneurons on ictogenesis depends not only on the respective interneuron subset and connectivity but also on the concurrent activity of other neurons: Ictal activation of interneurons can terminate seizures, while interictal activation of the same neurons can initiate seizures [6]. Still, the reasons for the occasional and sudden increases in interneuron firing leading to a seizure are not well understood. Inflammatory processes seem to play an important role in modulation of neuronal excitability.

2  Inflammation and Ictogenesis: General Concepts The brain has long been considered as a site of “immune privilege” [79], because it is separated from circulating immune effectors by the blood-brain barrier (BBB) and has a relatively low abundance of antigen presenting cells [68]. These findings explain, for example, the high tolerance of the CNS for allograft transplants. Nevertheless, pronounced interactions occur between the brain and the peripheral immune system. Therefore, two distinct concepts are used to classify immune responses in the CNS: • Neuroinflammation, usually regarded as chronic, CNS-specific, inflammation-­ like glial response which does not reproduce the classic characteristics of peripheral inflammation [111]. Neuroinflammation can exacerbate seizure severity and frequency even in isolated brains without contribution of systemic immunity [64, 75, 121]. • Systemic inflammation, which can affect neuronal excitability via communication with the BBB or by overcoming of a disrupted BBB which, in turn, is influenced by the brain. This chapter addresses systemic inflammatory mechanisms mediated by blood cytokines and T cells, while neuroinflammation is discussed in Chapter “Emerging Molecular Mechanisms of Neuroinflammation in Seizure Disorders” . It is obvious that the BBB has a central role in permitting or preventing influences of peripheral immunity on seizures (discussed in Chapter “Perivascular Inflammation and Extracellular Matrix Alterations in Blood-brain Barrier Dysfunction and Epilepsy”).

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Fig. 1  Relationships between seizures, pathologies of epilepsies, anti-epileptic drugs (AED), the peripheral immune system, and the BBB (see text). The red arrow indicates the topic of this chapter

The picture is complicated by the fact that anticonvulsants (“anti-epileptic” drugs, AED) can have not only an (intended) impact on ictogenesis but also on the immune system [41] and in few cases (e.g., everolimus in tuberous sclerosis) on the underlying pathology of the epilepsy (Fig. 1). Considering the associations depicted in Fig. 1, two questions arise naturally: Does Systemic Inflammation Cause Seizures?  There is good clinical and experimental evidence for a modulatory effect of systemic inflammation on ictogenesis. Severe systemic infections increase the likelihood of seizures in non-epileptic patients [88]. Systemic treatment with interferon α leads occasionally (1%) to seizures in humans [108]. Systemic injection of LPS into mice decreases the seizure threshold within 30 min ([102]; [106]), an effect probably mediated via IL-β [70] and CCL-2 [18]. Incidentally, the increased seizure susceptibility after a single systemic LPS injection in rats becomes permanent if applied during an early developmental state [34]. Seizure susceptibility also increases after induction of peripheral inflammation such as colitis, arthritis, or granulomas in rats [96]. In mice, maternal systemic immune activation during pregnancy enhances seizure susceptibility in the offspring [94]. A simple injection of LPS-activated leukocytes into the systemic circulation of epileptic mice leads to increased seizure frequency [66]. In summary, while systemic inflammation is not always sufficient to induce ictogenesis (and might not even be necessary for in vitro generation of epileptiform activity [90]), it can certainly facilitate interictal-ictal transition. Indeed, experimental peripheral or systemic inflammation can initiate subsequent neuroinflammation with microglia activation and enhanced intrahippocampal cytokine production ([45]; [98]), ­secondarily resulting in all the sequelae of neuroinflammation as described in Chapter “Emerging Molecular Mechanisms of Neuroinflammation in Seizure Disorders”.

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Do Seizures Cause Systemic Inflammation?  Plenty of clinical evidence demonstrates that acute seizures are associated with alterations of peripheral immune effectors such as circulating leukocytes and cytokines. Considering the time course of those alterations, it is obvious that seizures are indeed their cause. The following sections provide details, with a focus on circulating cytokines and T lymphocytes.

3  Acute Seizures and Circulating T Lymphocytes Seizure-related alterations of circulating blood leukocytes have been observed as early as 1937 [17]. Patients with psychiatric diseases such as schizophrenia or depression were treated with chemically or electrically induced seizures, and differential blood counts were performed. Results varied considerably between studies: Both increase and decrease of circulating lymphocytes were described [4, 17, 40]. Since the 1930s, cases of mild to moderate postictal CSF pleocytosis (>5 cells per μl) have been reported [59]. Because the findings could not be explained by infections, a transient BBB dysfunction (BBBD) was assumed as underlying pathomechanism [104]. Postictal pleocytosis appeared in 2–5% of patients presenting with acute seizures [25, 59]. Other authors found higher frequencies and differences in CSF cell types between seizure etiologies: Alcohol withdrawal seizures were associated with appearance of polymorphonuclear cells (granulocytes), while acute or remote cerebrovascular etiology involved mononuclear cells (lymphocytes and monocytes) [95]. In 1967, T lymphocytes were discovered as a specific entity [81]. Shortly thereafter, it became clear that T lymphocytes comprise different subclasses (including CD8+ and CD4+ T cells) with distinct functions [16]. The interictal blood distribution of CD4+ and CD8+ T cells in epilepsy patients was investigated for the first time in 1985 [26]. In the interictal state, patients with focal or generalized epilepsies have significantly fewer circulating CD4+ T lymphocytes and in many (but not all) studies also a significantly greater number of CD8+ T lymphocytes than healthy controls [13, 26, 76, 87, 124]. Similar findings were obtained in children with febrile seizures [82] and West syndrome [83]. However, another study found no differences in blood CD4+ T cells, CD8+ T cells, or Treg cells between TLE patients and controls [122]. Not only the number but also the function of leukocyte subsets can be altered by seizures. A reduced natural killer (NK) cell activity was found in epilepsy patients and their siblings [76, 124]. Peripheral blood mononuclear cells (PBMC) from children with febrile seizures (FS) produced more IL-1β than those isolated from children without seizures, even if the control children suffer from viral or bacterial infections; this alteration was shown to be transient [43]. CD4+ T cells from TLE patients contain more intracellular IFNγ, IL-6, and TNF-α than T cells from controls [122].

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Pilocarpine injection in rats leads to acute status epilepticus and reduces the CD4+/CD8+ ratio as compared to baseline values [71], demonstrating a systemic immunomodulatory impact of acute seizure activity. When number and distribution of blood leukocytes change during seizures (which usually last less than 2 minutes), they may rapidly return to baseline after the seizure ends. Therefore, video EEG monitoring (vEEG) is required for exact timing of peri-ictal blood investigations in humans. VEEG comprises continuous EEG and video sampling over several days or weeks in epilepsy patients and is performed for differential diagnostics as well as presurgical evaluation. Usually, the patients’ AED dosage is reduced, which increases the probability of seizure occurrence. Continuous EEG recording often allows to determine seizure onset time with a precision of seconds. An early video EEG study showed postictal leukocyte counts above the upper normal limit in 36% of patients with generalized tonic-clonic seizures (GTCS), but only in 7% of patients with complex partial seizures and none in patients with simple partial seizures [107]. In order to investigate the effect of interictal-ictal transition on blood leukocyte subsets, their distribution was investigated in TLE patients 10 min and 24 h after a seizure during vEEG and compared to baseline values of the same patients [7]. Absolute numbers of total leukocytes, neutrophils, and total lymphocytes as well as relative proportions of NK cells and NK-like T cells increased significantly after seizures as compared to baseline, while CD4+ T cells and the CD4+/CD8+ ratio decreased. Numbers of basophils, eosinophils, and monocytes as well as proportions of CD8+ T cells, B cells, and plasma cells remained unaffected. When interpreting such findings, one should keep in mind that a relative increase of a subpopulation can be actually caused by a decrease of another subpopulation and that changes in absolute cell counts can come along with unchanged relative proportions of subsets. Furthermore, decrease in the peripheral circulation may result from increased adhesion of leukocytes to the endothelium. The mechanisms of action leading to altered leukocyte distributions after seizures are speculative. An increase in total leukocytes, total lymphocytes, and NK cells is significant even after complex partial (automotor and dialeptic) seizures which are not associated with intense muscle contractions. Therefore, it is unlikely that the alterations appear simply due to seizure-related physical activity. The subset changes were more pronounced in patients with mesial TLE with hippocampal sclerosis as compared to other etiologies, indicating possible disease-specific mechanisms. Furthermore, the changes correlated with simultaneous systemic release of epinephrine. Intravenous epinephrine injection [10] and sepsis [38] can lead to similar changes in the distribution of lymphocyte subpopulations in humans. Hence, the effects of seizures on the distribution of leukocyte subpopulations might be mediated via sympathetic innervation after activation of the neuroendocrine axis. Considering the rapid kinetics of the observed changes, release from peripheral cell pools and/or pronounced adhesion to vessel endothelium are likely mechanisms of altered leukocyte numbers. Is the seizure-induced disturbance of blood leukocyte distribution a purely peripheral epiphenomenon or could it in turn impact the underlying epilepsy? Experimental findings support the latter hypothesis. Not only resident microglia but also peripheral leukocytes are required for seizure development in a virus-induced

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mouse model of epilepsy, and both cell populations mediate their proconvulsive effects via IL-6 [62]. Interestingly, experimental seizures trigger expression of endothelial adhesion molecules (ICAM-1, VCAM-1, P-selectin, E-selectin) resulting in enhanced leukocyte adhesion to CNS vessels [29, 63]. This mechanism indicates a possible “feedback” action of circulating leukocytes on the BBB or, after invasion into the parenchyma, on the brain. Thus, blood macrophages accumulate in the cerebral vasculature after electrically induced acute seizures in rats [52]. Strong presence of peripherally derived T cells in epileptogenic lesions caused by infectious or autoimmune encephalitis including Rasmussen encephalitis, in tuberous sclerosis complex, and in focal cortical dysplasia is well established [9, 12, 105, 128]. However, data on T-lymphocyte infiltration from the systemic circulation into the epileptogenic regions in other common epilepsies (which are not demonstrably inflammation-related) are contradictory. Some authors found no or only sparse infiltration in various animal models and/or resected human hippocampi from TLE patients [14, 69, 72, 97, 114]; others report more abundant presence of T cells in mice several weeks after pilocarpine-induced status epilepticus [86], in human hippocampal sclerosis [9, 33, 84, 130], and in the cortex and hippocampus 24 h after even single brief experimental seizures in non-epileptic mice [109]. However, when T cells are found, they are frequently located in perivascular spaces [44], suggesting that they act at the level of the BBB [72]. Interestingly, infiltrating CD8+ T cells seem to have a neuroprotective effect in a mouse model of kainate-induced TLE [24, 130]. On the other hand, there is solid evidence that perforin (expressed by CD8+ T lymphocytes and NK cells) is an important player in ictogenesis and the development of seizure-induced BBBD [73, 112]. Levetiracetam, a first-line AED which interacts with synaptic vesicle protein 2A (SV2A), inhibits not only ictogenesis but also perforin release from CD8+ T lymphocytes in vitro [61]. However, it is unknown if this mechanism contributes to its anticonvulsant efficacy.

4  Acute Seizures and Blood Cytokines Cytokines can directly influence neuronal excitability [35, 48, 116, 120]. In vitro studies revealed manifold effects of IL-1β and TNF-α on voltage-gated Na+, K+, and Ca2+ channels as well as on ligand-gated NMDA, AMP, GABA, and TRPV1 channels [103]. Interestingly, the effect of TNF-α (and possibly other cytokines) on Na+ currents depends on the neuron type, probably due to differential receptor distribution [55, 91]. IL-1β reduces GABA-mediated currents in hippocampal membranes from TLE patients but not in controls [100]. Similarly, IL-6 reduces the amplitude of inhibitory postsynaptic currents, possibly via a decrease of GABAA receptors [37], and enhances dendritic EPSPs and somatic population spikes in the CA1 region [85]. IL-1β, TNF-α, and IFNγ can induce neuronal synchronization in vitro [20]. Accordingly, IL-1β lowers the threshold for seizure-like activity in entorhinal cortex slices [19], and both IL-1β and TNF-α decrease the seizure threshold in rats

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after intracerebroventricular application [98]. While intrahippocampal injection of IL-1β alone does not induce seizures, it prolongs duration of seizures induced by subsequent kainate injection in a dose-dependent manner up to twofold [117]. Accordingly, IL-1β alone fails to alter neuronal Ca2+ concentrations in vitro but facilitates glutamate-induced increase of intracellular Ca2+ in neurons [125]. Sequelae of cytokine action on the brain can be extreme: Transgenic mice overexpressing IL-6 in the CNS develop severe neurologic symptoms including seizures which they eventually die from [15]. Ample evidence exists for an association of BBBD with acute seizures [50, 74]. Thus, it is obvious to assume that systemic inflammation, associated with elevated blood cytokine levels, could lead to altered neuronal excitability and possibly to ictogenesis. This assumption has been studied from various perspectives. In numerous studies, interictal blood levels of dozens of cytokines were investigated in patients with different epilepsy syndromes (e.g., [47]; [58]; [87])); most of them covered the well-known pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. A recent comprehensive meta-analysis of these studies showed significant upregulation of IL-6 and IL-17 serum levels in epilepsy patients versus healthy controls [23]. Incidentally, similar immune alterations have been described in epileptic dogs [80]. These findings document a “basic” interictal dysregulation of innate immune effectors in epilepsy patients as compared to healthy persons. Of note, the interictal IL-6 increase is markedly reduced after successful epilepsy surgery [67]. In order to asses if the transition from the interictal to the ictal state is associated with further alterations of blood cytokine concentrations in humans, patients have been studied within defined timeframes (usually ≤72 h) after admission to the hospital due to acute GTCS. Serum and CSF levels of IL-6 (but not IL-1β or TNF-α) were increased in several patients with recent GTCS as compared to epilepsy patients who had their last seizure at least 2 weeks ago and to control patients without seizures [92, 93]. Similar patterns were observed after febrile seizures [123], in patients with various epilepsy syndromes who were admitted within 24 h or 48 h after a seizure ([36, 110],) and in patients with daily seizures as compared to infrequent seizures [49]. Blood concentration of the soluble IL-6 receptor, which is probably required for mediating the effects of IL-6 on neurons, is lower in patients with recent seizures than in healthy controls [57]. The extent of IL-6 regulation appears to depend on seizure type (GTCS>focal) and seizure duration: The higher the cumulative seizure burden, the more IL-6 appears in the circulation [57]. The blood half-life of most cytokines is short [127]. Thus, the gold standard for assessing peri-ictal blood cytokine kinetics in humans is repeated blood sampling during vEEG.  In several studies, IL-6 (and sometimes IL-1 receptor antagonist) blood levels have been found to increase within minutes or hours after seizures [2, 3, 8, 58, 113] (meta-analysis in [129])), while IL-1β and TNF-α remained unchanged. Presence and extent of ictal IL-6 increase depend on various factors: • Seizure duration >100  s was associated with greater IL-6 release than shorter seizures [3].

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• Seizure semiology: GTCS were associated with higher IL-6 concentrations than focal seizures [3, 8]. • Localization of the epilepsy: IL-6 increase in TLE was higher than in extra-­ temporal epilepsies in one study [3], while an earlier trial did not reveal differences in IL-6 release between TLE, ETLE, and idiopathic generalized epilepsies [113]. • Site of seizure onset: Right temporal epilepsy was associated with higher IL-6 levels than left-sided TLE [8], confirming a suggested cerebral lateralization in the control of immune processes [78]. • Etiology of the underlying epilepsy: Postictal IL-6 increase was detectable in TLE of various etiologies (e.g., cavernomas), but absent in patients with hippocampal sclerosis [8]. Likewise, it depends on the model whether IL-6 appears in the blood after experimentally induced seizures: Acute status epilepticus after systemic pilocarpine injection but not after electrical brain stimulation is associated with elevated blood levels of IL-6  in rats [46]. In contrast to blood level alterations, no differences in hippocampal cytokine concentrations including IL-6 were detected interictally between TLE patients with and without hippocampal sclerosis [1]. • Previous seizure frequency: < 10 seizures/month in patients with TLE was associated with a postictal increase in IL-6 concentration [3]. • Baseline cytokine blood levels: Low baseline IL-6, low baseline IL-1Ra, and high baseline IL-1β levels were associated with higher postictal IL-6 increase [3, 113]. The concept of “epilepsy” is sometimes used in a very broad sense but comprises many different diseases with diverse etiologies and pathomechanisms. The clinical findings described above underline the importance of careful phenotyping in order to investigate reasonably homogenous patient groups. Studying patients who are treated with electroconvulsive therapy due to major depression allows to assess the effects of acute symptomatic seizures on blood cytokines without interference of “chronic epileptic” immunological influences. Interestingly, the pattern of blood cytokines (increase of IL-6, but not IL-1β) after electroconvulsive therapy is similar to the postictal pattern in epilepsy patients [53]. Experimental evidence suggests a causal role of interictal-ictal transition in the induction of IL-6. Depolarization of cultured cortical neurons as well as acute seizures induced in naïve mice via maximum electroshock leads to an increase of IL-6 mRNA by about 60–140% [101]. The origin of seizure-associated circulating cytokines, on the other hand, is unknown. They may stem directly from the brain or its vasculature. IL-1β, for example, is produced within hours not only in the seizure onset zone but also in cortex involved in seizure propagation and even in the spinal cord [42, 97]. Neurons, astrocytes, microglia, and endothelial cells are both sources and targets of ictal cytokine release [5, 115]. Seizure-induced increase of IL-1β, TNF-α, and IL-6 is mainly seen in microglia (but also in neurons), begins at 2 h after seizure induction, peaks at 6–12 h, lasts 65 years) have more significant cognitive deficits than non-epilepsy controls in both short- and long-term visual and verbal memory and executive functions [111]. Longitudinal neuropsychological studies have shown that chronic epilepsy is associated with progressive memory impairments and, furthermore, patients M. Thom (*) · M. Koepp Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology, London, UK e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_7

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with epilepsy are more likely to develop dementia compared to the general population [120]. These clinical associations could be a direct effect of duration of epilepsy, frequency of seizures but also influenced by anti-epilepsy treatments, the nature of the underlying brain pathology or other as yet unidentified risk factors. It is recognised that both severity and the trajectory of cognitive impairment may vary greatly between individuals with epilepsy suggesting heterogenic pathomechanisms including genetic risk factors. Indeed for some epilepsies, as progressive myoclonic epilepsies, mitochondrial disorders and autoimmune encephalitis, cognitive decline is inextricably linked with the underlying disease process. This chapter will focus on the role of tau in cognitive decline associated with focal and generalised epilepsy syndromes, particularly temporal lobe epilepsy (TLE). In patients with TLE, progressive memory impairment occurs in a significant proportion of patients [111]. In TLE, neuropsychological testing identifies focal deficits in language, memory and visuospatial functions in a subset of patients, with language-based cognitive task deficits in verbal memory, naming tests and verbal fluency usually associating with left temporal lobe epilepsy and visual memory dysfunction typically greater with right TLE.  More widespread deficits in cognitive function beyond the temporal lobe including general intelligence and executive function are recognised but with wide variability, some patients having minimal impairment. Patients with more severe deficits (about 30% of patients) were shown in one study to be older, have a longer duration of epilepsy and notably have a poorer prospective cognitive course as well as correlations with reduced grey and white matter brain volumes on imaging [51]. A recent study of 185 adult TLE patients with HS identified four clusters: (i) cognitive deficits across all domains, (ii) low executive functions and speed, (iii) deficits in language and memory and (iv) a minimally impaired group with no significant deficits [31]. In a further study with diffusion tensor imaging (DTI), different cognitive phenotypes in TLE, with and without HS, showed that both regional and widespread alterations of superficial and deep white matter tracts and networks were associated with language and memory impairment [104], but the underlying pathological basis was not clearly defined. The association of dementia and epilepsy may relate to common risk factors, including head injuries, cerebrovascular disease (CVD) risks and shared common pathophysiological and molecular pathways that with ageing continue to promote both seizures and neurodegeneration. Tau protein has recently emerged as a potential candidate player in neurodegeneration associated with epilepsy following several lines of evidence: Seizures increase tau phosphorylation [63], and several recent studies have shown accumulation of tau in resected specimens from patients with refractory epilepsy, correlating with poorer neuropsychometric performance [46, 121]. Accumulation and aggregation of abnormal forms of cellular hyperphosphorylated tau are one of the hallmark features of Alzheimer’s disease (AD), with resultant neuronal dysfunction [27]. New onset of epilepsy is not uncommon in the later disease stages of AD, particularly in hereditary forms [73] with evidence that seizures further accelerate cognitive decline [111]. Neuronal activity increases tau translation and potentially contributes to enhanced trans-synaptic propagation and accelerated disease progression [97]. Experimental studies have shown that tau reduction reduces seizures in both epilepsy and AD models [52]. This chapter aims

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to discuss the evidence for a primary role for tau in cognitive decline in epilepsy, particularly focal epilepsy.

2  Tau Protein, Normal Function and Brain Development Tau protein, encoded by the MAPT gene on chromosome 17 (17q21.31), is a microtubule-­associated protein (MAP), first identified in 1975. The physiological multifunctional roles of tau include the promotion of assembly and stabilisation of microtubules, tau being concentrated primarily in axons, particularly at axonal growth cones with tau mRNA present in axons. Tau also regulates dendrite polarity and axonal sprouting [17], and perikaryal tau has roles in maintaining the integrity of DNA and RNA, synaptic function and plasticity, with tau present at both pre- and postsynaptic sites (as reviewed in Sotiropoulos et al. [119]). The MAPT gene consists of 16 exons (150kb); alternative splicing of exons 2 and 3 creates variable N-terminal regions which contain both, one or no exons (2N, 1N 0N); splicing of exon 10 produces forms with either 3 or 4 microtubule-binding domains near the C terminus (three-repeat (3R) tau, four-repeat (4R) tau); this results in 6 isoforms of varying length [42]. The functional significance and relative distribution of the isoforms are not fully understood [119], but there is developmental regulation in their expression. In the adult human brain, there is equal representation of 3R and 4R isoforms but in the developing brain, only the shortest isoform (0N3R) is expressed. This foetal pattern persists until the first months of post-natal life when exons 2 and 10 show an abrupt shift in alternative splicing [48] with the 0N3R form being downregulated in the adult brain. These developmental switches likely reflect roles of tau in the maturation of neuronal cytoskeleton and cortical connectivity [48], with tau shown to regulate axon genesis and circuit formation [49]. Phosphorylation of tau (pTau) is recognised in the developing human brain particularly at Ser214, Ser202, Ser396 and Ser404 sites, differing from the common pathological phosphorylation sites in AD [49]. Tau phosphorylation is also developmentally regulated, remaining elevated until the end of synaptogenesis [17]. Interestingly, a common pattern of pTau labelling from 14 to 27 post-conception weeks (PCW) is restricted to the marginal zone/molecular layer with evidence of focal aggregation [49], a pattern which draws parallels with observations in epilepsy (see sections below).

3  Tau in Neurodegeneration Abnormal cellular aggregation of tau is the hallmark of several neurodegenerative diseases, collectively termed ‘tauopathies’. Tau undergoes several post-translational modifications including glycosylation, ubiquitination, glycation and truncation. The most important and disease-relevant post-translational modification of tau is hyperphosphorylation which influences tau self-assembly, aggregation and accumulation in the cell as inclusions, for example, in the form of paired helical filaments forming

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neurofibrillary tangles in AD.  It is still debated whether the neurodegenerative effects of tau are through a loss of normal function, impacting on microtubule stability, axonal transport and synaptic transmission [33], or a ‘toxic gain of function’ where hyperphosphorylation of tau drives aberrant cellular interactions including at the synaptic and dendritic compartments [47, 113].

3.1  Tau Phosphorylation and Hyperphosphorylation Aberrant phosphorylation is considered one of the critical events in neurodegeneration. Normal phosphorylation of tau is a dynamic and tightly regulated process, essential to normal cellular functions, including axonal transport and interaction with neurotransmitter receptors and synaptic function [17]. In AD, ageing and other tauopathies, tau becomes highly phosphorylated at up to 45 specific amino acid sites (‘hyperphosphorylated tau’) (Table  1). Phosphorylation is also accompanied by conformational changes of the tau molecule, including several truncation steps that promote its self-aggregation (Table  1). The chronology of these steps has been Table 1  Comparison of tau species and forms identified in Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE) and epilepsy MARKER

Target Epitope (Ser = Serine, Thr=Threonine)

Alzheimer’s Disease ( pale shade = earlier events)

Chronic Traumatic Encephalopathy (dark shade = stronger evidence)

PHOSPHORYLATION SITES (p) All stages of inclusions Experimental models

Tau 5 anbody (total Tau)

Recognizes all 6 Tau isoforms (Aa 215 and 235) irrespecve of phosphorylaon status

TOC1

Oligomeric Tau (209-224)

Early event

P175/231

pThr175, pThr231,

In tangles, NT, plaques (Moszczynski, Yang et al. 2017)

PHF1

pSer 396/Ser 404

Early phosphorylaon site (Mondragon-Rodriguez, Perry et al. 2014)

pS422

pSer 422

Early event – in Pretangles

AT8

AT100

pSer 199/Ser 202/Thr 205 (Triple phosphorylaon site pThr212/Ser 214/Thr 217

Early to late AD : Tangle, pretangles, neuropil threads (sensive marker) Late: NFT aggregates

PG5

pSer409

CP13

pSer202

RZ3

pThr231

Phosphorylaon site in AD (Duka, Lee et al. 2013) Phosphorylaon site in AD (Duka, Lee et al. 2013) Phosphorylaon site in AD (Duka, Lee et al. 2013)

S214

pSer214

AT180

pThr231

Yes ; also highly phosphorylated site in developing brain (Hei, Kim et al. 2019) Sensive marker, equivalent to AT8 (Nakano, Kobayashi et al. 2004)

Present in neurones/glia (Kanaan, Cox et al. 2016) In CTE and CTE-ALS (neurones > glia) Early phosphorylaon triggered by TBI (Moszczynski, Strong et al. 2018) Present in neurones/glia (many reports) e.g. (Arena, Smith et al. 2020)

Present in neurones/glia (Kanaan, Cox et al. 2016) Present in neurones/glia (many reports) Present in tangles (Arena, Smith et al. 2020)

Epilepsy (Dark shade = reported finding)* Increased in hippocampus in TLE (Gourmaud, Shou et al. 2020) and temporal cortex (Puvenna, Engeler et al. 2016) in some cases. No reports Increased in TLE (Liu, Ou et al. 2017) pSer396 in epilepsy neocortex (Puvenna, Engeler et al. 2016) and increased in TLE (Liu, Ou et al. 2017) No reports Several reports in TLE (surgical) and PM cases (see text) No reports No reports

Present (many reports) eg (Arena, Smith et al. 2020) Detected (Katsumoto, Takeuchi et al. 2019)

No reports Higher pTau/total Tau rao in epilepsy than CTE (Puvenna, Engeler et al. 2016) No reports

Increased in TLE (Gourmaud, Shou et al. 2020)

TLE temporal lobe epilepsy. *pTau181 also identified in CSF in TLE patients (elevated pTau181/ total tau to controls) [84]

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investigated in human tissue from AD by comparing dominant tau forms at different disease stages. Phosphorylation is likely to be an early event with specific epitopes (phosphorylation at Ser396-404) occurring very early [85] (Table 1). For aggregation of tau into filaments, conformational changes include a shift from a random coil to a compact state with the N terminus binding the microtubule-binding repeat region followed by C- and/or N-terminal truncation [9]. Misfolding of tau and hyperphosphorylation then leads to its oligomerisation as highly structured insoluble aggregates. Tau oligomers are therefore considered the initial step in aggregation, preceding the formation of fibrils. Tau oligomers are formed of two or more molecules of tau (non- phosphorylated or phosphorylated) existing as dimers, multimers and granules [103]. These spherical structures of molecular weight of 67–70kDa can be isolated from brain extracts as well as CSF in AD, including early disease stages as disease biomarkers. They are of considerable biological and physiological interest as (i) they can initiate the sequestration of tau into fibrillary aggregates [103]; (ii) they represent biologically neurotoxic tau forms, with evidence of mitochondrial dysfunction [113] and synaptic dysfunction [47]; and (iii) they may initiate the seeding of tau, implicated in the cell-to-cell (trans-synaptic) spread through exosomes (small membranous vesicles) [97].

3.2  Identification of Abnormal Cellular Tau in Tissues Cellular tau accumulation can be demonstrated in AD tissues with immunohistochemistry (IHC), in phosphorylated or non-phosphorylated forms and in early pre-­ tangle stages and tangles [27]. A pre-tangle is defined as neuronal tau accumulation before formation of an inclusion, showing granular cytoplasmic staining, but the nucleus intact and the overall morphology of the cell retained. Neuropil threads typically appear before the detection of tangles representing tau in both axons and dendrites. Neurofibrillary tangles (NFTs) contain mature aggregated cytoplasmic filamentous structures, and extracellular NFT in the neuropil are referred to as ‘ghost’ tangles. Many commercial antibodies recognise early and late tau phosphorylation sites and conformational changes (Table 1). The widely used AT8 antibody recognises a triple phosphorylation site (at Ser199, Ser202 and Thr205); is present in early pre-tangles as well as neuropil threads, neuritic plaques and mature tangles; and is a sensitive marker for disease detection and used for routine Braak staging of AD [13]. Other antibodies identify epitopes associated with tau conformational changes; for example, TNT1 and TNT2 identify conformational changes in tau which lead to exposure of a phosphatase-activating domain (PAD); these antibodies have been shown to detect tau polymers but not monomers and early pre-­ tangles but not late-stage tangles in AD [22]. Thus a panel of IHC markers can be used to evaluate the relative stages of tau accumulation in a sample; although extensively studied in AD and CTE (Table 1), tau forms have as yet not been studied in detail in epilepsy (see sections below).

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3.3  Tau Dynamics and Neuronal Activity Tau is a regulator of neuronal synaptic plasticity and function, influencing neuronal excitability [119]; increased neuronal activity rapidly increases extracellular tau in the brain [97]. It has long been recognised that glutamate induces rapid tau phosphorylation, detectable by AT8 labelling [116]. Phosphorylation of tau is also modified through NMDA receptor activation shown at AT180, PHF1 and AT8 epitope sites but not AT100; this effect was demonstrated for up to 120 min, suggesting that this is a reversible process [86]. Stimulation of neuronal activity at AMPA receptors also induces tau release from synapses [99], and experimentally, hyperexcitation of neurones increases synaptic tau protein [64]. In a further study, it was demonstrated, in neuronal cultures, that glutamate stimulation of AMPA and NMDA receptors induced dendritic microtubule protein translation in a dose-dependent manner with phosphorylation at an AD-relevant epitope (detected by AT8) and with the potential to trigger neurodegeneration [63]. Recently, intrinsic hyperexcitability of human neuronal iPSC with a tau mutation has been shown [66]. There is also substantial data from epilepsy models of functional alteration of tau including its phosphorylation [129]. Increased total tau and tau mRNA have been shown in mossy fibre axons 6 days following kainate injection [98]; after experimental axonal injury, tau accumulates at the sprouting growth cone [21]. In the intra-amygdala kainic acid model of status epilepticus, an increase in tau protein was shown at 4 h and significant increases in pTau (AT8, PHF1) at 4–8 h, including expression in mossy fibres co-localising with zinc transporter 3 (ZnT3) with pTau remaining at increased levels in the chronic stages of seizures [4]. Interestingly, ablation of tau in mice does not appear to induce either neurological, learning or memory deficits or cell death; animals are more resistant to chemically induced seizures [105]. For example, tau knockouts showed a reduction in seizure severity following pharmacological induction with kainic acid [94]. The effects of tau depletion in epilepsy models have been investigated: The Kcna1 knockout mouse exhibits spontaneous seizures with megencephaly; when tau was knocked down, this resulted in reduced seizures, reduced hippocampal network hyperexcitability and normalisation of brain size [52]. In mice overexpressing wild-type tau, this was associated with a reduction in cortical neuronal activity [15], whereas animal models overexpressing mutant tau show spontaneous epileptic activity [39]. These data have led to the hypothesis that tau modulates sensitivity to excitotoxins and neuronal activity and that tau reduction strategies could be beneficial. How tau exerts beneficial effect on experimentally induced seizures is not fully understood.

4  The Spectrum of ‘Tauopathies’ ‘Tauopathy’ is a collective term for a group of disorders in which accumulation of abnormal tau is considered central to the cellular degeneration and clinical signs and symptoms (Table  2). These are briefly discussed here as they constitute in

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Table 2  Human neurological conditions associated with tau accumulation Primary Tauopathies (Tau Deposition is the Primary Pathological Process) Frontotemporal lobe dementia MAPT (FTDP-17) Pick disease Corticobasal degeneration Progressive supranuclear palsy Globular glial tauopathy Argyrophilic grain disease PART (primary age-related tauopathy) ARTAG (age-related tau astrogliopathy)

Secondary Tauopathies (Widespread Accumulation) Alzheimer’s disease Chronic traumatic encephalopathy Down syndrome Prion diseases Familial British and Danish dementia Postencephalitic parkinsonism Subacute sclerosing panencephalitis Parkinsonism-dementia complex of Guam Amyotrophic lateral sclerosis of Guam Niemann-Pick type C Diffuse neurofibrillary tangles with calcification Myotonic dystrophy

Secondary Tauopathies (Localised Accumulation) Hemimegalencephaly Focal cortical dysplasia II Tuberous sclerosis Ganglioglioma Meningioangiomatosis TLE/HS

diagnostic practice the primary differential diagnosis when encountering pTau accumulation in surgical or post-mortem tissue from a patient with epilepsy. Comparisons of the key features of AD, CTE and epilepsy are also outlined in Table 3.

4.1  Alzheimer’s Disease (AD): Staging and Progression AD is globally the most common and the most extensively studied cause of dementia; AD is confirmed pathologically by the accumulation of β-amyloid protein in addition to tau (recently reviewed in DeTure and Dickson [27], summarised in Table 3). It has long been established that in AD the pathological accumulation in the brain of pTau follows a predictable sequence, progressing for decades and correlating with cognitive decline. This enables a pathological staging from grades I to VI as outlined by Braak and Braak [13]. The earliest involvement is in mesial temporal lobe structures, including the entorhinal cortex/parahippocampal gyrus and hippocampus (Braak stages I to III), before involvement of neocortical structures (stages IV–VI). This staging utilises AT8 pTau immunohistochemistry and has been shown to have a good agreement between pathologists, particularly for the severe stages [1]. Preclinical stages with earliest pTau accumulation are also recognised

Alzheimer’s Disease Well-defined Braak stages (based on AT8): Pre-tangle stage: LC, axons [12] Stages:  I Transentorhinal region, LC

Cortical patterns

Neurone layers III and V preferentially affected Interneurons relatively spared [12]

 II Hippocampus  III Neocortex (fusiform)  IV Neocortex (MTG, STG)  V Neocortex (Frontal, peristriate)  VI Neocortex (occipital, striate) [13] Common macroscopic Ventricular dilatation (ex vacuo); features in late stages widespread cortical atrophy and sulcal widening, small hippocampi (symmetrical), angular atrophy of the thalamus Neuronal cellular Threads (dendrites and axons), localisation pre-tangles, plaques, neurofibrillary tangles, ghost tangles

Distribution/ progression in the brain

Dependent on the underlying cause of epilepsy; in focal epilepsy, asymmetrical pathology dominates, e.g., unilateral hippocampal sclerosis Axonal (including sprouted axons) Pre-tangles > tangles Granular clusters Subpial band (axons) Superficial cortical neurones

Disproportionate dilatation of the third ventricle Septal abnormalities, mammillary body atrophy, old TBI

Neuritic threads, tangles, axonal; ↑neuritic compared to neuronal pathology in the hippocampus [61] Superficial cortical neurones (layer II/III) Perivascular aggregates in depth of sulci

Epilepsy Neurodegeneration Not established Patterns reported may resemble:  CTE  PART  AD (some tangle predominant)  Persistent developmental pTau  ARTAG Novel patterns in epileptogenic lesions:  FCD/HMG: dysmorphic neurones  HS: excitatory/sprouted axons

Chronic Traumatic Encephalopathy Provisional staging outlined (2013) [81]: recently supported by quantitative analysis [3] CTE I: isolated perivascular pTau (usual frontal) CTE II: multiple cortical sites CTE III: widespread cortical tau, involvement of medial temporal lobe and deep grey structures CTE IV: III with cerebral atrophy and neuronal loss

Table 3  Comparisons of pathological tau distribution and staging in Alzheimer’s disease (AD), CTE and epilepsy

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19 to 75% of AD have TDP-43 neuronal inclusions (limbic → diffuse) [27] APP, PSEN1, PSEN2, Sorl1

TDP-43

Several with varying odds ratio: APOEε4, GSK3β, DYRK1A, TOMM40, CLU, PICALM, TREM2, CR1, CD33, etc. (see http://www.alzgene.org/)

Defining feature

β-Amyloid is prominent (%)

Genetic (hereditary forms) Genetic risk factors/ other drivers

In early stages ([12], [20])

Alzheimer’s Disease Early involvement of CA1/ subiculum border and molecular layer of dentate gyrus. Followed by CA2, CA3/CA4 and mossy fibre axons [70] Not a feature (Mmay indicate co-existing ARTAG or other tauopathy)

Axonal pTau

Astroglial pTau

Hippocampal involvement

Epilepsy Neurodegeneration Asymmetric patterns in HS: CA1/CA4 sparing with preferential involvement of CA2, subiculum and granular cells. Mossy fibre axons (early stage) Reports in PM series: subpial, periventricular, cortical (35%) Not reported in some surgical TLE series [46, 121]

(continued)

Several reports: subpial region, WM and hippocampal axonal tracts Aβ can be found (53% of cases) Infrequent Aβ in surgical cases where evaluated (10–15%) : PM Is not currently part of defining criteria series (35%) Neuronal cytoplasmic inclusions in 85% Not identified in PM cases with HS + tau [124]; cytoplasmic pTDP-43 in dysmorphic neurones in FCD II [53] Clear genetic predispositions to CTE not yet identified None identified for neurodegeneration in epilepsy Candidates include: APOEε4, MAPT, C9ORF72, Germline somatic mTOR pathway GRN; TMEM106B protective [19] mutations in MCD

1. Around small vessels in sulcal depths (all cases) 2. Thorn-shaped astrocytes in subpial region and perivascular location frequently present Prominent feature

Defining feature

Chronic Traumatic Encephalopathy In stage III/IV CA1/CA3, subiculum more severely affected; DG less involved [61]

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CSF: low Aβ42 and elevated tau and pTau

Neuroimaging: MRI, PET (FDG, amyloid, tau (AV1451))

Alzheimer’s Disease Less than 50% are pure AD Common concurrent conditions: CVD/SVD, LBD, CAA, HS [27] White matter lesions in AD recognised (axonal degeneration; not SVD) with posterior gradient [80] Evidence that neuronal loss and NFT are better correlates with cognitive decline better than β-amyloid [44] Traumatic encephalopathy syndrome: supportive features including impulsivity, anxiety, apathy, paranoia, suicidality, headache, motor signs, documented functional decline, and delayed onset of symptoms for at least 2 years after significant head impact exposure [34] Neuroimaging biomarkers: cavum septum pellucidum, positive tau neuroimaging such as tau-PET imaging, negative amyloid imaging, Cortical thinning CSF: normal amyloid-beta; elevated pTau/total Tau ratio [34]

Neuroimaging: PET (FDG): hypometabolism correlates with cognitive impairment [55, 74] MRI: progressive cortical thinning in epilepsy [125, 37] CSF: pTau/total tau ratio correlated with white matter impairment [84] No CSF/neuroimaging correlative studies with pathology

Some evidence for correlation with postoperative memory decline (see Table 4).

White matter gliosis, myelin rarefaction [2] and axonal White matter degeneration common degeneration reported in epilepsy [26] but has not been correlated with tau pathology

Chronic Traumatic Encephalopathy Epilepsy Neurodegeneration Limited data Combined pathologies of CTE + AD/amyloid angiopathy/CBD/DLBD/CVD/ALS/HS reported (e.g., One PM series: CVD (40%) [124] Ling et al. [76])

ALS amyotrophic lateral sclerosis, ARTAG age-related tau astrogliopathy, CSF cerebrospinal fluid, CTE chronic traumatic encephalopathy, CVD cerebrovascular disease, DG dentate gyrus, CBD corticobasal degeneration, DLBD diffuse Lewy body disease, HMG hemimegalencephaly, HS hippocampal sclerosis, FCD focal cortical dysplasia, LBD Lewy body disease, LC Locus ceruleus, MCD malformation of cortical development, MTG middle temporal gyrus, NFT neurofibrillary tangle formation, PART primary age-related tauopathy, PM post-mortem, PSP progressive supranuclear palsy, STG superior temporal gyrus, SVD small vessel disease, TBI traumatic brain injury, WM white matter

In vivo (and pre-­ clinical) diagnostic investigations

Clinical correlations

White matter pathology

Co-existing neurodegenerative pathologies/feature

Table 3 (continued)

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[12]. There is increasing evidence that this stereotypical pattern of disease progression in AD occurs through cell-to-cell propagation (for review see Peng et al. [97]). There may exist additional cell type-specific vulnerability; recent evidence also highlights differential neuronal vulnerability in animal models and human tissue, with excitatory (Tbr1, SATB2) neurones preferentially accumulating tau and degenerating (neuronal loss) in both mesial temporal and cortical regions compared to more resilient interneurons (GAD, parvalbumin, calbindin and somatostatin) [36]. However, in transgenic mice overexpressing mutant tau, a reduction of GAD67positive hippocampal interneurons, including both parvalbumin and somatostatin interneurons, was reported, and a co-localisation of pTau in the remaining interneurons was shown in CA1 and the dentate gyrus [75]. The earliest pathological tau accumulation probably occurs in axons; in AD post-mortem samples, hippocampal phosphorylated tau was noted in mossy fibre axons and CA3-Schaffer collaterals prior to neuronal or dendritic tau [20]. 4.1.1  Insights from Seizures Occurring in Alzheimer’s Disease Neuroscientists have long noted parallels between neurodegenerative diseases, particularly Alzheimer’s disease (AD) and epilepsy, suggesting common pathophysiological pathways or shared risk factors. In fact, the first description of senile plaques in 1892, a hallmark of AD, was in a post-mortem series from patients with epilepsy [14]. Seizures and epilepsy may be the first symptom of an underlying neurodegenerative disease; unprovoked, unexplained seizures in adults older than 55 years are associated with a twofold risk for later dementia [62] with AD underlying 7% of all epilepsies in older individuals [112]. Seizures in AD may be difficult to detect or clinically ‘silent’. Subclinical epileptiform activity has been detected in 22–43% of individuals with AD [72, 125] and seizures may not be detectable on scalp EEG but evident on intracranial EEG [71]. Furthermore, subclinical seizures may underlie the fluctuations of cognitive function; accelerated decline is observed in individuals with mild cognitive impairment or AD who also suffer from seizures. Neuronal hyperexcitability is a common factor in both epilepsy and AD. β-Amyloid, a component of senile plaques, can induce hyperexcitability and trigger progressive epilepsy; increases in intrinsic neuronal excitability are associated with tau mutations [65]. Moreover, both epilepsy and AD are associated with interneuronal dysfunction: Transplantation of modified interneurons improved cognitive and behavioural functions in AD mice [79]. Neurophysiological studies have identified paroxysmal slow cortical activity occurring in both AD and epilepsy associated with blood-brain barrier dysfunction, correlating with cognitive decline [83]. Moreover, inflammation, reactive oxygen species generation and mTOR signalling pathways have all been proposed to contribute to the pathophysiology of epilepsy and AD suggesting commonalities.

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4.2  Chronic Traumatic Encephalopathy (CTE) Dementia pugilistica was the term introduced for a distinct neuropsychiatric syndrome (‘punch-drunk’) occurring in retired boxers early in the last century; over the last century, particularly the last decade it  has drawn much scientific attention through its recognition in other contact sports (reviewed in [16]). In 1973, Corsellis described the pathology in a series of post-mortem cases from professional boxers, noting the presence of neurofibrillary tangles among other brainstem and cerebellar pathology [23]. Geddes subsequently confirmed patterns of cortical, sulcal and perivascular pTau in a small series of patients with histories of repetitive head traumas [40]. It has also been recognised that widespread pTau accumulation can follow a single severe head trauma [130]. Similar patterns of neuropathology based on the presence of neurofibrillary tangles (NFT) and tau pathology in athletes other than boxers or patients with documented episodes of single or repeated head trauma (with or without concussion) have been more widely investigated in recent years. The concept of a traumatic tauopathy has gained ground, and the term CTE (chronic traumatic encephalopathy) is now the favoured terminology. Tentative consensus diagnostic neuropathology criteria for CTE have been defined as ‘the presence of pTau aggregates in neurones, astrocytes and cell processes in irregular patterns at the depth of cortical sulci’ [82]. Tentative staging systems for the progression of CTE (stages I to IV) was proposed in post-mortem studies (see Table 3) and recently validated in a large series of 366 cases with quantitative immunohistochemistry [3] (Table 1). Supportive features for CTE also include (i) preferential pTau in superficial cortex (layers II–III), (ii) tangles in CA2 and proximal dendritic swelling of CA4, (iii) pTau in subcortical structures, (iv) pTau in astrocytes in subpial and periventricular regions and (v) grain and dot-like pTau. In addition, signs of TBI may be present and TDP-43 neuronal cytoplasmic inclusions may be identified [82]. 4.2.1  Controversies Regarding CTE Neuropathological Diagnosis Nevertheless, despite the flurry of scientific publications and widespread media coverage (in view of potential implication for sport participants of all ages and implementation of health and safety legislation), the pathological diagnosis of CTE has provoked controversy. The operational neuropathological characterisation, in particular how the disease is staged, is still regarded as preliminary [117]. Of greater importance, the clinical criteria for CTE, ‘traumatic encephalopathy syndrome’, are not well defined or correlated with pathology. The nature and type of repetitive head injury are variable [6], and ‘typical’ neuropathological features of CTE can be encountered in asymptomatic individuals suggesting the pathological heterogeneity of CTE is broad [117]. Importantly, a progressive disease course for CTE as a neurodegenerative condition has yet to be confirmed and neuropathological staging, which requires longitudinal studies (undertaken over decades) and validation of clinical biomarkers [6]. It is also recognised that features of CTE can co-exist with

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other neurodegenerative pathologies including AD, cerebral amyloid, ALS and TDP-43 proteinopathies, which further complicate neuropathological diagnosis. Some experimental studies of TBI have shown that pTau progressively accumulating at the injury site can be transmitted to naïve mice inducing tau pathology and memory defects in inoculated animals [130]. Further experimental and correlative and longitudinal clinical-pathological studies are clearly required to embed CTE as a distinct and progressive form of neurodegenerative tauopathy.

4.3  Primary Age-Related Tauopathy (PART) Primary age-related tauopathy (PART) was a term introduced in 2014 in recognition of a tangle-only dementia with the absence of β-amyloid deposition, commonly encountered in the ageing brain but with mild or limited cognitive impairment compared to the full-blown dementia of late Alzheimer’s disease [24]. Previous terminologies included ‘tangle-predominant senile dementia’ in recognition of the lack of senile plaques. The degree of macroscopic atrophy can be mild in PART with NFTs and ghost tangles comprising 3R and 4R tau, in the hippocampal formation and adjacent regions, as well as the amygdala and in subcortical regions (locus coeruleus, thalamus, raphe nucleus and medulla) but equivalent to Braak limbic stage IV maximally. There is no progression of tau pathology to neocortical regions in PART and glial tau accumulation is not present. Studies do show that within the context of PART, overall tau burden correlates with cognitive function [54]. Recent series have demonstrated progressive accumulation of pTDP-43 protein in the hippocampus in PART, as in AD [132]. Still, PART does not share ApoE ε4 AD risk factors suggesting aetiological differences [8].

4.4  Age-Related Tau Astrogliopathy (ARTAG) ARTAG is an umbrella term for the spectrum of astrocytic tau accumulation, mainly arising in the ageing brain (>60 years), and includes thorn-shaped astrocytes in the subpial, subependymal and perivascular regions, often involving medial temporal lobe and mainly composed of 4R tau [68]. It can occur in isolation, or in association with other neurodegenerative diseases, including an overlap with CTE.  Further work regarding the clinical significance of ARTAG and the role of astroglial tau in influencing neurodegeneration or neuroprotection across a range of tauopathies is required [67].

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4.5  Hippocampal Sclerosis, Ageing and Dementia This is included here not as a tauopathy but concerning the differential diagnosis that can arise in diagnostic pathology. Hippocampal sclerosis (neuronal loss and gliosis) has been long associated with epilepsy, and although not specific for syndrome or seizure type, it is a common finding in particularly unilateral TLE [123]. Patterns of neuronal loss in hippocampal sclerosis in epilepsy have also been associated with specific memory impairments (as reviewed in Tai et al. [122]). A form of hippocampal sclerosis is also recognised in the elderly, presenting with dementia but not epilepsy, which is more usually bilateral (in 40-60%) and subsequently identified as a neurodegenerative condition (a TDP-43 proteinopathy), with TDP-43 (phosphorylated and non-phosphorylated) inclusions in the nucleus and cytoplasm [28, 92]. In comparison to hippocampal sclerosis in epilepsy, in dementia cases there is loss of neurones in CA1 and the subiculum, whereas CA4 neurones are more often spared with less granule cell dispersion or axonal sprouting [7]. Hippocampal sclerosis of ageing is also sometimes observed with arteriolosclerosis [29]. Various nomenclatures have been proposed to encompass this entity and to distinguish this from hippocampal atrophy in AD; recently the unifying term ‘LATE’ (limbic predominant age-related TDP-43 encephalopathy) has been proposed by a working group [93]. LATE is defined as a TDP-43 proteinopathy, with or without hippocampal sclerosis and associated clinical with an amnestic cognitive syndrome that can evolve into dementia. LATE can also co-exist with AD and other neuropathologies in the elderly.

4.6  Tau Propagation and Progression Neurodegenerative diseases are collectively characterised by the cellular accumulation of misfolded proteins (tau, α-synuclein, amyloid, TDP-43, etc.) and often show very stereotypically progression in the brain. As exemplified in AD, systematic progression of tau accumulation led to the theory that tau propagates along neural networks, implying transcellular spread of tau along neural connections [43]. In the past decade, experimental data has accumulated to support transcellular transmission [97]. ‘Seeds’ of tau or oligomers are transmitted from neurone to neurone at the synapse, possibly via exosomes, inducing the same tau conformational change in the recipient cell. The transmission itself is likely to be dependent on the protein conformation (isoform, phosphorylation, mutation) [29]. Furthermore, as neuronal activity promotes the propagation of proteins [127], seizure activity may augment tau spreading [97], but whether there is an altered distribution, reflecting altered networks in epilepsy, is yet to be explored experimentally.

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5  Tau in Refractory Epilepsy There have been over 15 studies, many in the last few years, investigating tau-related neurodegenerative pathology in epilepsy and its potential contribution to cognitive decline. These, summarised in Table  4, have mainly focused on focal epilepsy pathology in surgical resections, particularly in TLE (Fig. 1).

5.1  Temporal Lobe Epilepsy and pTau Tai and colleagues studied a series of 33 TLE patients with hippocampal sclerosis who were over 50 years at the time of surgery [121]. pTau was detected by AT8 immunohistochemistry; both 3R and 4R isoforms were confirmed in positive cases. In 93% of cases, low to moderate pTau expression was noted in the temporal cortex and/or hippocampal regions. In ten cases, an AD-like distribution was observed with pTau expression primarily in neuropil threads and neurones, conforming with early limbic Braak stages. In eight cases, more CTE-like patterns were noted, such as prominent axonal labelling in subcortical white matter, hippocampal white matter involving groups or single axons and patches of pTau in the cortex with more involvement of superficial cortex (layers I–III) than deep cortical layers (layers IV– VI). Perivascular tau neurites or astrocytic tau were not noted in this series, however. A common finding was a band of pTau in the subpial region reminiscent of the foetal expression pattern. There was a relative lack of β-amyloid and neuritic plaques in this series. Unusual aggregates of tau-positive grains surrounded some vessels and neurones but did not co-localise with GFAP astroglial processes or the dendritic marker MAP 2 (Fig. 1h, i). Involvement of pTau in the sclerotic hippocampus showed a different progression through subfields compared to the typical stages in AD. For example, greater levels of pTau were noted in the subiculum with relative sparing of the depleted CA1–CA4 subfields; preferential involvement of the granule cells and mossy fibre axons, including sprouted mossy fibre axons, was noted (Fig. 1a–d). It was proposed that epileptic pTau pathology had both unique and common features that could indicate mixed underlying pathomechanisms: co-­ incidental AD, age-accelerated PART, cumulative moderate head traumas associated with seizures resulting in CTE-like patterns and persistence of developmental pTau distribution. There are also features that suggested pTau was activity driven, with prominent labelling in excitatory granule cells and sprouted mossy fibre axons. Of note, in this relatively small cohort, a correlation between the amount of pTau and postoperative memory decline was noted at 1 year follow-up. Puvenna and colleagues also directly compared tau pathology in epilepsy patients to CTE post-mortem cases and controls [102]. In a range of patients with focal epilepsies (mainly TLE), they also noted prominent cortical subpial pTau (with epitope CP13 recognising phosphorylation site at Ser202) in epilepsy which was not a finding in CTE cases. Additional labelling of superficial cortical neurones, sulcal,

8

20

108

15

Sheng et al. 1994 [114]

Gouras et al. 1997 [45]

Kakita et al. 2005 [57]

Sen et al. 2007 [109]

Author, Year McKenzie and Miller 1994 [78]

TLE

Normal/HS/ porencephalic cyst

Underlying Focal Pathology in Epilepsy Cases TLE (proportion with HS)

1–81 years FCD type II (surgical and PM cases)

4–61 years MCD (varied types)

41– 61 years

10– 45 years

Case Number Age Range in Study at Surgery 101 30– 61 years

Not done

IHC β-amyloid (Dako)

IHC pTau (AT8), 3R and 4R tau

Aβ IHC

Western blot βAPP and IHC

Method for β-amyloid Detection βA4 (Dako) IHC

IHC pTau (AT8)

Not done

Not done

Method for pTau Detection Silver stain and tau IHC (sigma clone)

Clinical Correlations: Epilepsy No correlations with clinical factors including head trauma Not assessed

Higher levels of neuronal βAPP in epilepsy cases than controls 70% of cases with Aβ Not assessed plaques were ApoE ε4 compared to 27% without Not assessed AT8 neurofibrillary tangle-like inclusions in 27% (10% showed many inclusions) Not assessed pTau in dysmorphic neurones in cases >40 years, 4R>3R tau, confined to region of dysplasia (infrequent amyloid plaques)

Key Findings in Epilepsy Cases Senile plaques in 10%, mainly diffuse type, cortical and correlated with age; NFT in 8%

Table 4  Summary of studies investigating pTau, AD and CTE-like pathology in surgical epilepsy series

Not assessed

Not assessed

Not assessed

Not assessed

Clinical Correlations: Neuropsychometric Tests No correlations with neuropsychological tests

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Glioneuronal 30–35 (mean ages tumours of groups)

Prabowo et al. 2015 [100]

61

18– 51 years

Iyer et al. 2014 36 [53]

Lesion negative/ gliosis/ neuronal loss FCD, TS, non-lesional epilepsy

14– 56 years

IHC pTau (AT8)

IHC pTau (AT8)

Not done

Underlying Focal Pathology in Method for pTau Epilepsy Cases Detection All TLE with IHC total tau HS

36

Sima et al. 2014 [115]

Case Number Age Range in Study at Surgery Author, Year Kandratavicius 47 37–47 et al. 2013 [59] (mean ages of groups)

Clinical Correlations: Epilepsy Increased tau expression correlated with epilepsy duration mRNA and No increase in mRNA Not assessed for βAPP but IHC for increased neuronal βAPP labelling No Tau in threads and IHC for dysmorphic neurones correlations APP and with seizure Aβ (Dako (tangles and pre-­ frequency tangle) (>30 years) clone and co-localised with 6F/3D) pS6; APP in dysmorphic neurones but no Aβ Positive No Aβ plaques; 29% Aβ IHC correlation cases pTau neurones (Dako or neuropil threads in with age and Clone duration of lesion only and 6F/3D) epilepsy co-localisation with pS6 Method for β-amyloid Key Findings in Detection Epilepsy Cases Not done Tau in neuronal cell bodies, axons and granule cell layer

(continued)

Not assessed

Not assessed

Not assessed

Clinical Correlations: Neuropsychometric Tests Significant increase in tau in CA2 associated with poor verbal memory Tau Protein in Drug-Resistant Epilepsy and Cognitive Decline 165

50– 65 years

27.6 years (mean)

33

5

Tai et al. 2016 [121]

Liu et al. 2017 [77]

Author, Year Puvenna et al. 2016 [102]

Case Number Age Range in Study at Surgery 19 4 months to 58 years

Table 4 (continued)

TLE

All TLE with HS

Underlying Focal Pathology in Epilepsy Cases Mainly TLE; control groups CTE and normal PM

Aβ IHC (Dako)

31 cases (93%) pTau; 5 cases (15%) Aβ; patterns of tau in axons and excitatory pathways Decreased total tau/ increased relative pTau; increased APP compared to control group

Method for β-amyloid Key Findings in Detection Epilepsy Cases Not done pTau IHC in superficial cortex, pial region, sulcal and perivascular tau in epilepsy. Ration of pTau/total tau higher in epilepsy. Actual soluble and insoluble low and high MW tau higher in CTE

Western blot: total APP tau, pTau (Ser396 and Thr231)

Method for pTau Detection 1. IHC for pTau (AT8 and CP13) and TAU5 (total tau, non-­ phosphorylated) 2. Sarkosyl fractions for tau oligomers 3. Western blotting for pTau and total tau IHC pTau (AT8) and silver stains

Clinical Correlations: Neuropsychometric Tests Not assessed

Negative correlation with pTau and decline in verbal memory postoperatively Not assessed

Clinical Correlations: Epilepsy Not assessed

Association of pTau and generalised seizures Not assessed

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10– 56 years

Gourmaud 19 et al. 2020 [46]

TLE (HS/FCD/ MCD/DNT); AD and autopsy controls

Frontal and temporal lobe resections

5 TLE; 5 FLE

IHC and Western blots; AT8, AT180, 4R, total tau (Tau5)

IHC pTau (AT8)

IHC pTau (AT8) and silver stains

Underlying Focal Pathology in Method for pTau Epilepsy Cases Detection IHC pTau (AT8) TLE with HS (mixed subtypes) Clinical Correlations: Epilepsy No correlations

Correlation with history of head injury No Not done pTau in 38% (substantial in 7%); no correlations CTE features; glial tau (or with head injury/playing reported contact sports) pAPP and Increased APP and IHC and pTau pAPP, Aβ*56 and Western associated blot (APP Aβ42 in epilepsy; and βAPP) increased total tau and with hippocampal 4R tau in the sclerosis hippocampus in pathology epilepsy; pTau increased; activated stress pathways including JNK, mTOR/p70S6K and GSK-3β

Method for β-amyloid Key Findings in Detection Epilepsy Cases Not done Moderate AT8-­ positive tau load in 1.3% of cases (35% of cases AT8 negative), tau in axons, threads, white matter and pre-tangles Not done CTE pattern noted in one case.

Negative correlation of pAPP and pTau with cognitive measures

Preoperative testing neuropsychometry impairments No correlations with neuropsychology tests (pre-op and post-op)

Clinical Correlations: Neuropsychometric Tests AT8 in the DG and subiculum associated with naming decline 1 year postoperatively

APP amyloid precursor protein, CTE chronic traumatic encephalopathy, DG dentate gyrus, FCD focal cortical dysplasia, FLE frontal lobe epilepsy, HS hippocampal sclerosis, IHC immunohistochemistry, MCD malformation of cortical development, TLE temporal lobe epilepsy, NFT neurofibrillary tangle

18– 45 years

60

Smith et al. 2019 [118]

18– 45 years

10

Jones et al. 2018 [56]

Author, Year Prada Jardim et al. 2018 [101]

Case Number Age Range in Study at Surgery 92 18– 55 years Tau Protein in Drug-Resistant Epilepsy and Cognitive Decline 167

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Fig. 1  ‘Unconventional’ (non-AD or CTE) patterns of pTau accumulation encountered in temporal lobe epilepsy with hippocampal sclerosis (HS). (a) A post-mortem case with type I HS (loss of neurones in CA1 and CA4) and evidence of mossy fibre axonal sprouting shown on dynorphin stain in the dentate gyrus (DG) and diminution of the normal mossy fibre pathway (extending to CA4/3). (b) Corresponding pTau labelling observed in the subiculum and CA2 but not in CA1 or CA4; pTau was also present in the sprouted mossy fibres. More extensive pTau was present in all subfields of the contralateral hippocampus (not shown). (c) Surgical TLE/HS cases with extensive pTau labelling of the granule cells in the dentate gyrus and axonal processes running through. (d) The same case as C, double-labelled with ZnT3 for mossy fibres confirming mossy fibre sprouting and focal co-localisation with pTau. (e) A prominent feature reported in TLE/HS cases is a subpial band of pTau in the temporal cortex, reminiscent of foetal patterns. (f) Double labelling of the subpial layer with neurofilament and pTau confirms co-localisation of tau in axons. (g) CA4 neurones labelled with neurofilament and surrounded by pTau/AT8-positive synaptic-like processes on cell body and axon, likely mossy fibre excitatory projections. (h) A pattern noted in the temporal cortex was granular like aggregates of pTau but without the formation of neuritic plaques. These did not co-localise with GFAP or (i) dendritic marker MAP 2

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perivascular tau deposits and a beaded axonal distribution of pTau were also reported. Further ELISA analysis showed lower total tau in epilepsy than CTE and controls; however, pTau (phosphorylated at Ser396) was not differently expressed between CTE and epilepsy cases. When the authors compared the ratio of pTau to total tau, however, it was higher in epilepsy than controls and CTE, and this was observed throughout the lifespan from 4-month-old patients to old age. They also extracted tau from tissue samples to look at tau species, including tau oligomers using sarkosyl-soluble fractions, and both soluble and insoluble forms were higher in CTE cases. Prada Jardim in a series of 92 TLE/HS patients also included a broad age range to identify multiple pathological features that could correlate with memory impairment including pTau [101]. Moderate pTau load was identified in only 1.3% of cases, but there was minor focal positivity in 35% of cases with patterns of tau in axons, threads, white matter and pre-tangles. Interestingly the presence of AT8 immunostaining in the dentate gyrus and subiculum was significantly associated with a decline in verbal memory testing 1 year postoperatively. More recently, Smith et al. studied 60 adults age 18–45 years at time of epilepsy surgery, and pTau was present in 38% of the frontal and temporal cortex specimens [118]. The tau load was described as substantial in 7%, mainly in the older patients; they also reported glial as well as neuronal tau deposits. There was no correlation with neuropsychological testing in this series however. Gourmaud et al. also reported on Alzheimer-like pathology in an epilepsy surgical series discussing the cellular stress pathways that could drive this phenomenon [46]. In a series of 19 TLE cases ages 10–56 years with mixed focal pathologies, the authors showed increased neuronal APP and neuronal and endothelial Aβ42 compared to controls, particularly in the hippocampus but without the formation of extracellular amyloid plaques. Neprilysin, the main amyloid-degrading enzyme, was also increased and so were BACE1 enzymes in hippocampal neurones (BACE1 cleaves APP in amyloidogenic processing pathways). In addition, increased total tau and 4R tau in hippocampal neurones (CA1–CA4 and granule cells) were shown by immunohistochemistry and Western blots and increased pTau (AT8 and AT180) in both hippocampus and temporal cortex, with AT180 showing hippocampal neuronal labelling, but no glial pTau. They also investigated the activation of cell stress pathways involved in the generation of pathological tau and amylodogenic Aβ forms with evidence of phosphorylation of JNK (c-Jun N-terminal kinase), mTOR/ p70S6K and GSK-3β in the hippocampus and temporal lobe neocortex, with PERK and eIF2α (markers of endoplasmic reticulum stress) present in the temporal cortex. Furthermore, pTau and pAPP negatively correlated with preoperative cognitive scores [46].

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5.2  mTORopathies and pTau Malformations of cortical development are a common cause of focal epilepsy, with focal cortical dysplasia (FCD) type II the most typical lesion encountered in epilepsy surgery series [11]. FCD type II is characterised by dysmorphic neurones, abnormal cortical lamination and undifferentiated balloon cells [10]. In a series of 15 cases spanning a wide age range, Sen et  al. in 2007 described age-dependent accumulation of pTau in dysmorphic neurones of FCD II (with AT8 immunohistochemistry) and confirmation of neurofibrillary tangle formation (using Gallyas silver stain) (Fig. 2). Four-repeat tau isoform appeared to be more prominent compared to 3-repeat tau, but this was not quantified. Of note, the proportion of pTau-positive neurones increased with age of the patient, while overall neuronal density declined with age, indirectly implying a neurotoxic effect of tau. Of interest, even in

Fig. 2  Tau accumulation in mTORopathies. (a) In a post-mortem case from a patient with a history of temporal lobe surgery in the 1950s, a further region of focal cortical dysplasia type II was identified in the right cingulate cortex. (b) The typical features of cortical dysplasia type IIB were identified including dyslamination of the cortex, pS6-positive dysmorphic neurones (inset) and balloon cells. (c) Neurofibrillary tangles were identified in the neurones but not in balloon cells and (d) labelled with pTau immunohistochemistry and (e) highlighted as filamentous inclusions on silver stains

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post-mortem cases with whole brain sampling, pTau and inclusions were primarily restricted to the malformation and not extending into other brain regions; this implies inherent vulnerability of dysmorphic neurones but lack of propagation or tau progression beyond the malformation. Iyer and colleagues evaluated pTau and β-amyloid in a series of FCD as well as in resected cortical tubers from patients with tuberous sclerosis (TS) [53]. The authors noted pTau labelling of dysmorphic neurones, including tangles and threads in patients over 30 years of age, but no evidence of β-amyloid. pTau expression was restricted to the region of the malformation; the authors also noted cellular co-­ localisation with pS6 as evidence of mTOR pathway activation. Co-localisation of neuronal pTau and pS6 was also a finding in lesional neurones in a study of low-­ grade glioneuronal tumours in epilepsy [100]. In a series of paediatric hemimegalencephaly (HME) cases, (age 2.5  months to 2.5  years) who underwent surgical treatment for refractory epilepsy, pTau (pSer202) was observed in neuropil and dysmorphic neurones of the malformation and hippocampus with prominent labelling of axons in the molecular layer and neurones in layer II/III; this was not observed in age-matched controls [107]. Similar pTau expression was reported in TS and FCD IIB childhood cases [106]. Recent studies have identified common germline and somatic mutations in mTOR pathway genes and now propose a mechanistic link to histologically similar lesions of FCD, tubers of TS and HME, also referred to as ‘mTORopathies’ with resulting mutations leading to MTOR pathway over-activation [25]. Tau is a substrate of mTOR kinase, and over-activity of the mTOR pathway shown in AD correlates with increased tau, neurofibrillary tangles and amyloid plaques, decreased autophagy and cognitive impairment; these may be reversed by mTOR and ribosomal S6 kinase inhibitors. The propensity for pTau accumulation in mTORopathies therefore raises the possible interactions of seizures, delayed maturation and mTOR dysfunction driving both tau phosphorylation and neurodegeneration.

5.3  Post-mortem Studies of pTau in Epilepsy A limitation of examining tissue from surgical resections in epilepsy is that only one brain region is available for examination and it is not possible to fully stage the disease process and the extent of involvement of different brain regions. In 2011 we undertook a study of a series of 138 post-mortem specimens from patients with epilepsy [124]. One hundred and three of these patients were residents at a national epilepsy centre with long and detailed histories of their lifetime of refractory and difficult to treat epilepsy, many with learning difficulties and progressive cognitive decline. In many cases, a primary focal epileptogenic pathology was present, including cortical malformation or hippocampal sclerosis, in addition to superimposed acquired co-morbid pathologies as cerebrovascular disease and traumatic brain injuries. The aim of this study was to stage regional pTau pathology following the Braak system for AD [1].

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In the 138 cases spanning a wide age range, pTau was identified in 69% but β-amyloid in only 34%; this is in agreement with typical findings in surgical epilepsy cohorts in amyloid-negative tauopathy in epilepsy. Within the series, 15% were stage I, 21% stage II, 13% stage III, 18% stage IV and 2% stage V; no Braak stage VI cases were identified. The frequency of different Braak stages for under 35 years old was similar to large datasets from non-epilepsy populations. However, in the middle-aged group (40–65 years), we noted increased representation of both low (I/II) and mid (III/IV) Braak stages in the epilepsy series with significant increases noted for Braak stages (III/IV). This suggested age-accelerated pTau accumulation in epilepsy. In this cohort, accurate data regarding maximum seizure frequency and total lifetime number of generalised seizures was available in a many cases, but we found no correlation between cumulative seizure events and Braak stage. We did note, however, a significant difference in the Braak stages between epilepsy syndrome diagnoses, with partial epilepsies (including temporal lobe epilepsy) more often associated with higher Braak stages than genetic or idiopathic generalised epilepsies (Fig. 3). These observations support the hypothesis that focal/symptomatic epilepsies are more closely linked to a tauopathy than simply an expression of the severity of the epilepsy. Furthermore, 77% of patients with a Braak stage of 3 or more had progressive cognitive decline; however, 24 patients with cognitive decline had low Braak stage (0–II), suggesting other pathological causes for dementia symptoms. There was a significant correlation noted between the pathological identification of traumatic brain injury, such as old cortical contusions and higher Braak stages (P or = 80 years of age) humans. Acta Neuropathol. 1994;88(3):212–21. 29. Dujardin S, Begard S, Caillierez R, Lachaud C, Carrier S, Lieger S, Gonzalez JA, Deramecourt V, Deglon N, Maurage CA, Frosch MP, Hyman BT, Colin M, Buee L.  Different tau species lead to heterogeneous tau pathology propagation and misfolding. Acta Neuropathol Commun. 2018;6(1):132. 30. Duka V, Lee JH, Credle J, Wills J, Oaks A, Smolinsky C, Shah K, Mash DC, Masliah E, Sidhu A. Identification of the sites of tau hyperphosphorylation and activation of tau kinases in synucleinopathies and Alzheimer’s diseases. PLoS One. 2013;8(9):e75025. 31. Elverman KH, Resch ZJ, Quasney EE, Sabsevitz DS, Binder JR, Swanson SJ. Temporal lobe epilepsy is associated with distinct cognitive phenotypes. Epilepsy Behav. 2019;96:61–8. 32. Falcon B, Zivanov J, Zhang W, Murzin AG, Garringer HJ, Vidal R, Crowther RA, Newell KL, Ghetti B, Goedert M, Scheres SHW. Novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules. Nature. 2019;568(7752):420–3. 33. Feinstein SC, Wilson L. Inability of tau to properly regulate neuronal microtubule dynamics: a loss-of-function mechanism by which tau might mediate neuronal cell death. Biochim Biophys Acta 2005 Jan 3;1739(2-3):268–79. https://doi.org/10.1016/j.bbadis.2004.07.002 34. Fesharaki-Zadeh A.  Chronic traumatic encephalopathy: a brief overview. Front Neurol. 2019;10:713. 35. Fitzpatrick AWP, Falcon B, He S, Murzin AG, Murshudov G, Garringer HJ, Crowther RA, Ghetti B, Goedert M, Scheres SHW. Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature. 2017;547(7662):185–90. 36. Fu H, Possenti A, Freer R, Nakano Y, Hernandez Villegas NC, Tang M, Cauhy PVM, Lassus BA, Chen S, Fowler SL, Figueroa HY, Huey ED, Johnson GVW, Vendruscolo M, Duff KE. A tau homeostasis signature is linked with the cellular and regional vulnerability of excitatory neurons to tau pathology. Nat Neurosci. 2019;22(1):47–56. 37. Galovic M, van Dooren VQH, Postma T, Vos SB, Caciagli L, Borzi G, Rosillo JC, Vuong KA, de Tisi J, Nachev P, Duncan JS, Koepp MJ. Progressive cortical thinning in patients with focal epilepsy. JAMA Neurol. 2019;76(10):1230–9. 38. Galovic M, de Tisi J, McEvoy AW, Miserocchi A, Vos SB, Borzì G, Cueva Rosillo J, Vuong KA, Nachev P, Duncan JS, Koepp MJ. Resective surgery prevents progressive cortical thinning in temporal lobe epilepsy. Brain J Neurol. 2020;143(11):3262–72. 39. Garcia-Cabrero AM, Guerrero-Lopez R, Giraldez BG, Llorens-Martin M, Avila J, Serratosa JM, Sanchez MP.  Hyperexcitability and epileptic seizures in a model of frontotemporal dementia. Neurobiol Dis. 2013;58:200–8.

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Ketogenic Diet, Inflammation, and Epilepsy Detlev Boison

Abstract  Ketogenic diet is a high-fat, low-carbohydrate metabolic intervention that has been in use for the treatment of epilepsy for almost a hundred years. This metabolic intervention affects epilepsy through multiple mechanisms, which combine to exert potent anti-inflammatory activity. Thereby, ketogenic diet not only affects seizures per se but also has the potential to attenuate or prevent the development of epilepsy and its progression. This chapter will focus on novel disease-­ modifying properties of ketogenic diet therapy and will discuss therapeutic challenges and opportunities. Keywords  Metabolism · Metabolic therapy · Adenosine · Disease modification · Epileptogenesis

1  Introduction Although epilepsy is traditionally considered a condition based on an imbalance of neuronal excitation and inhibition, there is now growing consensus that epilepsy is a metabolic disorder and that maladaptive responses of glial function and inflammatory processes, as well as a dysfunction of the blood-brain barrier, play a major role in the pathogenesis and pathophysiology of the condition [10, 13, 15, 27, 80, 105, 110]. The efficacy of conventional antiepileptic drugs (AEDs) may be limited by the development of multiple drug resistance [76]. Furthermore, AEDs have been designed to affect neuronal excitation and inhibition and therefore fail to affect the processes that lead to epilepsy and its progression (epileptogenesis). Those factors combine to the dire situation that conventional AEDs are not effective in up to 40% of all persons with epilepsy, therefore requiring the need to explore alternative

D. Boison (*) Department of Neurosurgery, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_8

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therapeutic options [115]. In particular, metabolic therapeutic strategies have garnered widespread therapeutic interest recently [7, 70, 92, 108]. Disruption of energy homeostasis can be cause or consequence of the epileptic state. Normally, glucose is the primary source of energy, which is needed to maintain the transmembrane potential of neurons. Astrocytes play a major role for the energy supply of neurons through the astrocyte-to-neuron lactate shuttle [85]. This is a metabolic mechanism, by which glutamate stimulates glycolysis and the release of the glycolytic product lactate from astrocytes. Lactate is taken up by neurons and is metabolized in the tricarboxylic acid cycle (TCA). This shuttle system depends on the glycolytic enzyme lactate dehydrogenase (LDH). Epileptic seizures are associated with excessive levels of extracellular glutamate, whose uptake into astrocytes triggers astrocytic glycolysis [6]. Through the enhanced metabolic activity associated with increased synaptic activity, there is a rapid drop in glucose and a corresponding release of high levels of lactate. Thereby, astrocyte-derived lactate becomes a major energy source of neurons during seizures. This mechanism is supported by clinical data which show increased glucose uptake during seizures, whereas interictal periods are characterized by reduced glucose uptake and hypometabolism [32, 33]. Those metabolic disruptions in epilepsy provide a rationale for the use of metabolic therapies designed to restrict the availability of glucose or to block the astrocyte-to-neuron lactate shuttle [14, 92].

2  Mechanisms of Metabolic Therapies The “water diet” treatment pioneered in 1922 by Dr. Hugh Conklin suggested that metabolic intervention, in particular fasting, could be used for seizure control [24]. Those findings spurred the discovery that a diet composed mostly of fats, i.e., a high-fat low-carbohydrate “ketogenic diet” (KD), could replicate the effects of fasting. Therapeutic benefits were ascribed to the production of ketones, such as β-hydroxybutyrate (BHB), which feed directly into the TCA and increase mitochondrial energy production [43]. Metabolic therapies such as the KD are now well-­ established therapeutic options for difficult-to-treat epilepsies in addition to a variety of neurological disorders [100]. The metabolic mechanisms underlying the therapeutic effects of KD therapy have best been characterized in pediatric metabolic epilepsy syndromes. Dravet syndrome (DS), a catastrophic form of childhood epilepsy, has been linked to glucose and oxidative hypometabolism. In line with the metabolic benefits of KD treatment, an overall positive response rate of 60–70% was found in a recent clinical study of 3- and 12-month-old Dravet patients. A molecular mechanism through which ketone bodies improve mitochondrial function has recently been identified in Kcna1 knockout mice [59]. Ketone bodies suppressed seizures in epileptic Kcna1 null mice, improved hippocampal long-term potentiation, and raised the threshold for calcium-induced mitochondrial permeability transition (mPT). Targeted deletion of the cyclophilin D subunit of the mPT complex uncoupled the effects of ketone bodies on mPT, while mPT was directly related to the anti-seizure effects of

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ketone bodies [59]. In addition to pediatric epilepsies, KD therapy is now widely used in adult epilepsies, including temporal lobe epilepsy (TLE) [60, 67]. The efficacy of KD therapy in TLE can best be explained by the direct correction of metabolic defects in TLE. Mechanistically, it has been demonstrated that KD therapy affects hippocampal function through ATP-sensitive K+ (KATP) channels, vesicular glutamate transporter (VGLUT), pannexin channels, and adenosine receptors [58]. Because a KD bypasses glycolysis and thereby directly enters substrates into the TCA, strategies are underway to replace rigid KD treatment with biochemical interventions that inhibit glycolysis or interfere with lactate formation. Based on promising efficacy studies performed in acute seizure models and in the rat kindling model of TLE [101, 102], the glycolytic inhibitor 2-deoxy-d-glucose is currently under evaluation for antiepileptic therapy [83]. More recently, Tsuyoshi Inoue’s group has demonstrated in a seminal study that blockade of LDH hyperpolarizes neurons and suppresses seizures in kainic acid (KA)-induced TLE [92]. Remarkably, LDH was also found to be a molecular target of stiripentol, a clinically used antiepileptic drug used for the treatment of Dravet syndrome [92]. These findings demonstrate that inhibition of this metabolic pathway can mimic the effects of KD therapy (Fig. 1). The energy state of a cell is determined by the ATP/ADP/AMP/adenosine ratio, which is controlled by the adenosine-regulating enzyme adenosine kinase (ADK). This enzyme is regulated by ATP, ADP, AMP, and adenosine and thereby acts as sensor for the energy state of a cell but also acts as a switch to rapidly adjust energy consumption to energy supply [11]. By restricting glucose utilization, ketogenic diets will cause an energy stress to the system. In line with this biological rationale, it was found that KD therapy suppresses epileptic seizures in rodent models of TLE through reducing ADK expression and augmenting adenosine signaling [81]. Under conditions of low energy levels, the KATP channel, a sensor for the energy state of the cell, acts as a feedback system to restrict neuronal activity. KATP in turn is regulated by Bcl-2-associated death promoter (BAD) protein whose genetic manipulation reduces glucose metabolism and increases the activity of neuronal KATP channels, thereby promoting seizure resistance [47]. Consequently, pharmacological inhibition or genetic manipulation of KATP function reduced ketone-induced neuroprotection and seizure resistance [47, 59]. These findings demonstrate a mechanistic link between metabolism, BAD, KATP channel function, and the control of neuronal excitability.

3  Anti-inflammatory Properties of Ketogenic Diets Inflammatory pathways and oxidative stress play a major role in the pathophysiology of epilepsy. Therefore, anti-inflammatory therapies have recently received increased attention for the purpose of seizure prevention, modification, and/or suppression [44]. Of interest, KD therapies and ketone-inducing therapeutics or interventions promote major anti-inflammatory activities, which have been demonstrated in numerous experimental and clinical studies [41, 66]. KD therapies are therefore

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Fig. 1  Astrocyte-neuron lactate shuttle. If glucose levels are sufficiently high, it is transported into neurons and converted into pyruvate via glycolysis. Alternatively, glucose can be transported into astrocytes. If glucose levels are low, astrocytes mobilize stored glycogen as an endogenous source for glucose. Glucose is then converted into lactate by lactate dehydrogenase (LDH). Lactate is then shuttled into neurons, which restore pyruvate from lactate. This pathway constitutes the astrocyte to neuron lactate shuttle. Ketogenic diet increases glucose and increases ketones, which are transported into neurons. Bypassing glycolysis, they directly enter the neuronal tricarboxylic acid (TCA) cycle

clinically used to treat various inflammatory conditions such as those of the joints or skin [41, 48]. Several pathways activated by KD therapy are thought to contribute to the anti-inflammatory activity of the diet. KD-induced beta-hydroxybutyrate (BHB) inhibits the inflammasome in immune cells to reduce production of inflammatory cytokines and thereby reduce inflammation [1, 41, 48]. It was recently proposed that a KD will increase the NAD+/NADH ratio and thereby activate downstream signaling pathways such as the sirtuins, which are associated with anti-­ inflammatory effects [31]. Peroxisome proliferator-activated receptors (PPAR), which are metabolically regulated transcription factors, are involved in mitochondrial biogenesis and the control of genes involved in anti-inflammatory and antioxidant pathways. PPARα is activated by X-box binding protein 1 (XBP1), which in turn is activated by hepatic serine/threonine-protein kinase/endoribonuclease

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inositol-­requiring enzyme 1 (IRE1). The latter enzyme functions as a nutrient sensor that regulates metabolic adaptation to fasting [95]. PPARγ is activated by decanoic acid and might therefore explain the anti-inflammatory and antioxidant properties of medium-chain triglycerides. In line with this function, a PPARγ antagonist abrogated KD-induced seizure protection in Kv1.1 knockout mice, a spontaneously epileptic mouse strain responsive to KD therapy [97], whereas an PPARγ agonist conferred seizure protection; however, KD therapy was ineffective in preventing seizures in PPARγ knockout mice [99]. Since seizure suppression was associated with a PPARγ-induced increase in PPARγ2 expression, the authors of this study concluded that PPARγ2 contributes to the anti-seizure effects of KD therapy. KD therapy also increases the brain levels of adenosine [see details below [78, 81]], which is an important anti-inflammatory molecule [3, 51, 114]. Due to the anti-­ inflammatory properties of adenosine, the KD-induced adenosine increase has disease-modifying and antiepileptogenic properties as will be discussed in the subsequent sections.

4  Adenosine and Epilepsy Because CSF levels of adenosine rise during a seizure and are thought to be a contributing factor for seizure termination, adenosine is considered to be an endogenous anticonvulsant of the brain [8, 28, 30, 69]. Adenosine is a potent vasodilator, and adenosine release during a seizure might therefore contribute to an increase in cerebral blood flow observed during the ictal phase [86]. Apart from seizure suppression, adenosine has anti-inflammatory and epigenetic functions and thereby has the potential to combine potent anti-seizure effects with disease-modifying and antiepileptogenic properties [12].

4.1  Seizure Suppression by Adenosine Purine ribonucleoside adenosine is an endogenous ligand of P1 receptors, a class of G protein-coupled receptors, comprising the adenosine A1, A2A, A2B, and A3 receptor (AR) subtypes [42]. Binding of endogenous adenosine to the Gi-coupled A1Rs induces presynaptic inhibition (prevention of calcium-dependent glutamate release) and induces postsynaptic hyperpolarization by activating inwardly rectifying K+ channels (GIRKs) [29, 94]. Through those mechanisms adenosine dampens neuronal excitability, a key mechanism for seizure suppression. In line with adenosine’s role as an endogenous anticonvulsant acting via A1Rs, adenosine A1R knockout mice as well as mice with a deficiency of adenosine in the brain (caused by transgenic overexpression of the adenosine removing enzyme adenosine kinase, ADK) have a strikingly similar phenotype of spontaneous recurrent subclinical hippocampal seizures [71, 81]. In contrast to A1Rs, which predominantly have an inhibitory

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role, A2AR are coupled to stimulatory G proteins and play multiple roles related to inflammation and glial activation (see below) but also to the control of learning and memory [111], implicating that dysfunctional adenosine metabolism and signaling may contribute to cognitive comorbidities in epilepsy. Adenosine deficiency, linked to astrogliosis and overexpression of ADK, is now considered a major pathological hallmark of temporal lobe epilepsy [4, 5, 50, 74]. Because adenosine deficiency within an epileptogenic brain area can be a direct cause for seizure generation based on insufficient activation of A1Rs [36, 71], the focal reconstitution of adenosine signaling is a rational approach for seizure control. To avoid systemic side effects of global adenosine augmentation, the intracerebral implantation of therapeutic cell grafts engineered to release adenosine or the use of adenosine-releasing polymeric brain implants is an elegant approach to restrict adenosine delivery to seizure-generating brain areas [9, 34]. The first demonstration that focal adenosine augmentation might effectively control seizures was obtained in rats that were electrically kindled in the hippocampus [16]. Kindling is an experimental procedure to induce epilepsy by repetitive administration of subconvulsive electrical stimulation to the limbic system of the brain. Over time repeated kindling stimulations result in a permanent increase in susceptibility and severity of those electrically induced seizures [90]. This animal model is of high predictive value for the clinical efficacy of AEDs [75]. In a proof-of-concept study, synthetic ethylene vinyl acetate copolymers were engineered to release an amount of around 20–50 ng adenosine per day [16] and were implanted into the lateral brain ventricles of rats that had previously been kindled in the hippocampus. Focal adenosine release from the polymers resulted in a strong reduction of convulsive seizures for at least 7 days, whereas control implants had no effects. This was the first published demonstration that the focal release of adenosine can suppress epileptic seizures in  vivo [16]. Similarly, intraventricular implants of encapsulated fibroblasts engineered to release adenosine provided robust but transient seizure suppression in kindled rats [56], and this seizure suppression was found to be independent of prior seizure frequency [17]. Those studies, which demonstrate that focal adenosine delivery to an area of hyperexcitability can be of therapeutic benefit, were subsequently confirmed by an independent research group using intracranial adenosine injection in a rat seizure model [2]. Subsequently, augmentation of adenosine signaling was shown to be effective in a model of pharmacoresistant epilepsy [49, 50]. Importantly, focal adenosine augmentation was shown to effectively suppress seizures in two different species (mice and rats) and in models of electrically induced kindled seizures, as well as in models of spontaneous recurrent seizures that resulted as a consequence of kainic acid-induced status epilepticus [56, 74]. Likewise, robust seizure suppression was achieved by infrahippocampal implants of adenosine-releasing silk, adenosine-­releasing cells, or by gene therapy targeting adenosine metabolism [56, 106, 107]. We have previously shown that the focal delivery of adenosine to the hippocampal formation is devoid of sedative side effects encountered after equivalent seizure suppression via an adenosine A1R agonist [56]. Because local adenosine augmentation therapies have been designed to directly compensate a local

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deficiency of adenosine  – via maladaptive overexpression of ADK  – it can be expected that those therapies restore normal adenosine function in an area of neuronal hyperexcitability rather than leading to excessive adenosine concentrations.

4.2  Adenosine and Inflammation Adenosine is a potent anti-inflammatory molecule which exerts multiple inflammation-­dampening functions in the periphery that are largely based on activation of the A2B receptor by micromolar levels of adenosine, which arise as a consequence of metabolic or energetic stress [38, 52, 88]. In the brain, adenosine is a major regulator of glial function and inflammatory processes. In particular, the regulation of astrocyte proliferation is under the control of adenosine, which through activation of astroglial high-affinity A1Rs under baseline conditions blocks astrocyte proliferation [91]. However, epileptogenic insults such as trauma, inflammation, or hypoxia trigger upregulation of the A2AR [26], which promotes astrocyte proliferation and activation [19, 55]. Through this shift in the expression levels of the two major high-affinity adenosine receptors, epileptogenic insults, which by themselves are associated with a rise in adenosine [23, 30, 36, 50, 87], promote the development of astrogliosis, a common pathological hallmark in many forms of acquired epilepsies [61]. Astrogliosis as such might be epileptogenic through a variety of mechanisms [25, 103, 104]; however, the associated increase in ADK expression might play a crucial role for ictogenesis within an epileptogenic focus [73]. In addition, the secretory functions of astrocytes are under the control of adenosine. Stimulation of A1Rs induces the release of nerve growth factor (NGF) and thereby supports neuronal survival and growth [21, 22]. A2AR activation inhibits the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) under inflammatory conditions and thereby assumes an important protective role [20]. Stimulation of astroglial A2BRs triggers the production and release of neuroprotective IL-6, which is an important damage-control mechanism during brain injury [40, 93]. Finally, the activation of A3Rs stimulates the synthesis of the neuroprotective chemokine CCL2 [113]. Microglia cells express A1, A2A, and A3 receptors and are therefore under the influence of adenosine regulation. Adenosine stimulates microglial cell proliferation through simultaneous activation of A1 and A2ARs, whereas stimulation of A1Rs alone with a synthetic agonist reduced phorbol 12-myristate 13-acetate-stimulated microglial proliferation [46, 96]. In addition, activation of microglial adenosine receptors can cause apoptosis; however the underlying mechanisms and the receptor subtypes involved remain unclear [84]. The A2AR plays an additional role in the regulation of secretory activities of microglia. Activation of this receptor induces upregulation of cyclooxygenase 2 and the release of prostaglandin E2, a potentially pro-inflammatory role of A2AR stimulation [39], as well as the release of the trophic factor NGF [53]. Microglial A3Rs might contribute to the increased phosphorylation

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of extracellular signal-regulated kinase 1,2 (ERK1,2); however the precise function of microglial A3Rs remains unclear. In sum, adenosine appears to combine both pro-­ inflammatory and anti-inflammatory effects on microglial cells. In addition to the direct activity on glial cells, adenosine receptors in infiltrating immune cells regulate inflammatory processes in the brain. The notion that A1Rs on macrophages initiate anti-inflammatory responses is supported by findings that A1R knockout mice have increased expression of genes that encode for the pro-­ inflammatory factors IL-1β and matrix metalloproteinase-12 [109]. Conversely, stimulation of A2ARs on bone marrow-derived cells aggravates ischemic brain injury, whereas selective inactivation of those receptors in chimeric mice protected against middle cerebral artery occlusion induced brain injury through a reduction of the pro-inflammatory mediators IL-1, IL-6, and IL-12 [116].

4.3  Epigenetic Role of Adenosine The contributing role of epigenetic alterations in the pathogenesis and pathophysiology of epilepsy is a relatively new and emerging research area [45, 54, 63, 77, 89]. One potential unifying factor behind many of the pathological changes observed during the development of epilepsy may be epigenetic modifications, which can further be aggravated by epileptogenesis itself [62, 89]. Epigenetic alterations are chemical modifications of DNA or histones, which alter gene transcription and which can respond rapidly to environmental cues. Changes in histone acetylation and methylation, as well as changes in DNA methylation, have been shown to occur in mature cells in the central nervous system [37, 79]. The methylation hypothesis of epileptogenesis suggests that seizures by themselves can induce epigenetic modifications and thereby aggravate the epileptogenic condition [62]. In support of this hypothesis, increased activity of DNA methyltransferases (DNMTs) as well as dysregulation of DNA methylation including a state of global DNA hypermethylation has been associated with human and experimental epilepsies [64, 65, 82, 112, 117]. Adenosine is a product of the S-adenosylmethionine (SAM)-dependent transmethylation pathway, which determines global DNA methylation status. Through the principles of mass action, ADK therefore drives the flux of methyl groups through this pathway by the removal of adenosine [11, 18]. DNA methylation requires DNMTs, which accept methyl groups from SAM and release S-adenosylhomocysteine (SAH) as a product. The latter is further converted into adenosine and homocysteine (HCY) by SAH hydrolase (SAHH). Because the thermodynamic equilibrium of the SAHH-catalyzed reaction lies in the direction of SAH formation [68], the reaction will only proceed when adenosine and homocysteine are constantly removed [18, 68]. If metabolic clearance of adenosine through ADK is impaired, the levels of SAH, which is a direct inhibitor of DNMTs [57], rise [18]. Because adenosine is an obligatory end product of DNA methylation and thereby determines the flux rate of transmethylation reactions, we conclude that an

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increase in ADK as seen in chronic epilepsy [74, 81] would drive increased global DNA methylation in the brain. This process may be amplified, because adenosine is an inhibitor of ADK [11]. Therapeutic adenosine augmentation may thus effectively reverse pathological DNA hypermethylation and thereby prevent epilepsy progression.

4.4  Disease-Modifying Properties of Adenosine Because adenosine is an endogenous anticonvulsant, because adenosine controls inflammatory processes implicated in epileptogenesis, and because adenosine is a regulator of the epigenetic control of gene transcription, adenosine is a prime candidate for disease-modifying and antiepileptogenic activity. An antiepileptogenic role of adenosine was first documented in genetically engineered mice with a forebrain-selective reduction of ADK [74] and in recipients of adenosine-releasing cell grafts [72, 74]. Both infrahippocampal adenosine-releasing cell grafts and intraventricular adenosine-releasing silk provided robust suppression of kindling development in rats [72, 106]; however the underlying antiepileptogenic mechanism remained unresolved. The underlying mechanism was finally elucidated in a seminal study that linked adenosine-related antiepileptogenesis with the epigenetic activity of adenosine in a rat model of systemic kainic acid (KA)-induced progressive TLE [112]. Status epilepticus (SE) as a trigger for subsequent epileptogenesis was induced in young male rats via an acute dose of kainic acid (KA). Only rats that exhibited at least 3  h of acute convulsive SE were used further and subjected to continuous long-term monitoring to quantify seizure activity. Once rats had been diagnosed with “early epilepsy” (3–4 spontaneous recurrent seizures per week) at 9 weeks post KA, the animals received either bilateral intraventricular adenosine-­ releasing silk-implants, silk-only implants, or a corresponding sham treatment. Adenosine-releasing implants were designed and validated to transiently deliver a stable dose of 250 ng adenosine per brain ventricle per day but restricted to only 10 days of drug delivery [106]. After polymer implantation, seizure monitoring was resumed and continued during a time span of 3  months. In both control groups, seizures continued to increase in number and severity, whereas in recipients of adenosine-­releasing implants, seizures were almost completely suppressed after polymer implantation. Remarkably, reduced seizure activity was maintained far beyond expiration of adenosine release from the polymer for at least 12  weeks. Even at 12 weeks after implantation, seizure incidence was reduced by more than 70%. As an additional experimental readout, mossy fiber sprouting at 21 weeks following KA was significantly attenuated in adenosine-treated rats compared to controls. In line with those profound antiepileptogenic effects, the transient delivery of adenosine restored normal DNA methylation status in the long term. These data demonstrate that the transient delivery of adenosine is sufficient to restore normal DNA methylation status and to prevent epilepsy progression in the long term [112].

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5  Ketogenic Diet Therapy of Epilepsy Metabolic therapies, including the ketogenic diet, are gaining renewed interest in the therapy of epilepsy, because – as discussed above – they combine multiple beneficial mechanisms of action and therefore have potential to not only suppress epileptic seizures but to also affect the disease process itself. In the following I will focus on the adenosine-related activities of ketogenic dietary therapies.

5.1  Seizure Suppression Because adenosine is an endogenous anticonvulsant, metabolic strategies that raise adenosine levels have therapeutic value for seizure suppression. In a seminal study we provided direct evidence that ketogenic diet therapy decreased ADK expression in the brain and that the antiepileptic effect of the diet was dependent on the resulting increased adenosine-dependent activation of A1Rs [81]. This study is the first demonstrating that an external environmental stimulus – in this case triggered by a dietary intervention – can decrease the expression of ADK. The antiepileptic effect of the diet-induced decrease in ADK was dependent on increased A1R activation, because seizure suppression in epileptic mice fed a ketogenic diet was reversed through pharmacological blockade of the A1R. Furthermore, in A1R knockout mice, the ketogenic diet had no anti-seizure effects, whereas the diet was effective in ADK-overexpressing transgenic mice, which have reduced levels of brain adenosine. These findings are in line with earlier studies using pharmacological blockade of ADK to potently prevent seizure activity in vivo in mice [50] and with seizure-­ promoting effects of increased ADK levels in the brain [35, 74].

5.2  Antiepileptogenesis A series of recent studies suggests that KD therapy exerts additional disease-­ modifying effects both in genetic models of metabolic epilepsy as well as in rodent models of TLE through epigenetic mechanisms. Remarkably, KD therapy was found to postpone disease progression, delay the onset of severe seizures, and increase the lifespan of Kcna1-null mice, a model of progressive epilepsy and sudden unexpected death in epilepsy (SUDEP) [98]. An epigenetic mechanism of KD therapy with disease-modifying potential was suggested based on a correlation of a predominant increase of DNA methylation with chronic epilepsy in the rat; importantly KD therapy attenuated seizure progression, and ameliorated DNA methylation mediated changes in gene expression in this model [65]. Subsequently it was

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shown that a transient KD therapy restored normal adenosine levels and global DNA methylation levels in epileptic rats that were otherwise adenosine deficient and hypermethylated; importantly, transient KD therapy reduced seizure activity in the long term, even after diet reversal to control diet [78]. Because KD therapy increases adenosine [78, 81] and because adenosine blocks DNA methylation [112], it is likely that the KD exerts its disease-modifying effects through an adenosine-­ dependent epigenetic mechanism.

6  Conclusions and Outlook Due to their anti-inflammatory and adenosine-augmenting properties, metabolic therapeutic interventions are of high interest as potential disease-modifying and antiepileptogenic treatments. Clinical data indeed suggest that KD therapy has lasting disease-modifying effects in patients with epilepsy. To make therapeutic use of diet-based antiepileptogenic treatments, a key issue would be to identify patients at risk for developing epilepsy, which in the absence of reliable biomarkers might be a challenging endeavor. However, data from our prior study show that transient therapeutic adenosine augmentation might be of therapeutic value for the prevention of epilepsy progression even after the onset or diagnosis of epilepsy [112]. Adenosine metabolism plays a key role in linking energy homeostasis to extracellular functions of adenosine that are mediated by the activation of adenosine receptors. Importantly, adenosine metabolism is also tightly linked to a variety of lifestyle choices and external triggers, which thereby can directly influence adenosine receptor-mediated signaling pathways. The realization that adenosine metabolism links metabolic and bioenergetic functions with adenosine receptor-mediated pathways offers new opportunities for therapeutic intervention. Rather than blocking (or activating) adenosine receptors selectively through specific ligands, the therapeutic manipulation of adenosine metabolism, e.g., through dietary interventions, offers unique opportunities to reset the adenosinergic network on a more holistic level. Thereby, restoration of network homeostasis becomes a unique therapeutic opportunity for a variety of pathological conditions.

7  Acknowledgments The author wishes to thank the following agencies for generous research funding: the National Institute of Neurological Disorders and Stroke (NINDS), the National Institute of Mental Health (NIMH), the US Department of Defense (DoD), the Epilepsy Foundation, and Citizens United for the Research in Epilepsy (CURE).

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Role of Regulatory T cells in Epilepsy Dan Xu, Sookyong Koh, and Stephen D. Miller

Abstract  In the past two decades, there has been increasing interest in the potential contributions of immune activation and neural inflammation to the pathogenesis of seizures and epilepsies. Studies have focused on the contribution of antibody-­­ mediated encephalitis as well as pro-­inflammatory mediators produced by brain-­­ resident glial cells to the pathogenesis of epilepsy and seizure induction. Immune activation that leads to functional rewiring of the brain can be both the cause and the consequence of seizures. The immune response, however, is a process tightly controlled by a system of checks and balances to prevent aberrant inflammatory damage. The role of regulatory immune cells that suppress overt immune activation has been largely unexplored in epileptogenesis and seizure control. In this review, we discuss experimental data generated in our laboratory to examine the role of regulatory immune cells in seizure modulation and epileptogenesis. Specifically, we focus on the emerging knowledge of CD4+FoxP3+ regulatory T cells (Tregs) in neurological disorders, recruitment of thymus-­derived natural Tregs (tTregs) to the central nervous system (CNS), generation of peripherally derived antigen-­specific Tregs (pTregs), Treg markers, and the immunosuppressive mechanisms of Tregs. We discuss the interaction between immune regulatory cells and inflammation-­driven effector cells in the regulation of neural inflammation to suppress excessive immune responses deleterious to the host and maintenance of immune homeostasis. Keywords  Neuroinflammation · Immune response · CD4+FoxP3+ regulatory T cells · Lymphocytes · Seizure disorders

D. Xu (*) · S. D. Miller Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA e-mail: [email protected] S. Koh Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA © Springer Nature Switzerland AG 2021 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_9

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Abbreviations Ab: antibody APC: antigen-­presenting cells BBB: blood-­brain barrier CD127: IL-­7 receptor alpha chain CNS: central nervous system CSF: cerebral spinal fluid CTL: cytotoxic T lymphocyte CTLA-4: cytotoxic T lymphocytes antigen-­4 DC: dendritic cells FoxP3: forkhead box P3 FR4: folate receptor 4 GARP: glycoprotein A repetitions predominant GFP: green fluorescent protein GITR: glucocorticoid-­induced tumor necrosis factor receptor ICOS: inducible co-­stimulator IL-10: interleukin-­10 IMP: immune-­modifying nanoparticle iTreg: induced regulatory T cells IPEX: immune dysregulation, polyendocrinopathy, enteropathy, X-­linked KA: kainic acid LAG-3: lymphocyte activation antigen-­3 LAP: latency-­associated peptide MHC: major histocompatibility complex mTOR: mammalian target of rapamycin NFAT: nuclear factor of activated T cells NF-kB: nuclear factor-­kB NOD-Rag: Non-­obese diabetic–recombination activating gene PD-1: programmed cell death-­1 PI3K: phosphatidylinositol-­3-­kinase PLGA: carboxylated poly(lactic-­co-­glycolic) acid PTEN: phosphatase and tensin homolog pTregs: peripherally derived regulatory T cells S1P1: sphingosine phosphate receptor 1 SCID: severe combined immunodeficiency Tconv: conventional T cells TCR: T cell receptor Teff: effector T cells TGF-β: tumor necrosis factor-­β Tr1: type 1 regulatory T cells Tregs: regulatory T cells tTregs: thymus-­derived regulatory T cells TSDR: Treg-­specific demethylation region

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1  I mmune Homeostasis in the Central Nervous System and the Role of Tregs in Neuroinflammation Effective regulation and maintenance of immune homeostasis is crucial to the overall function and health of the brain and spinal cord. Several neuroprotective mechanisms contribute to the balance between pro-­and anti-­inflammatory status of the central nervous system (CNS). Under healthy condition, the endothelial lining that constitutes the blood-­brain barrier (BBB) and the choroid plexus that acts as a blood-­cerebrospinal fluid (CSF) barrier are highly selective for entry of cells and molecules into the CNS. Perturbation of this balance or of the integrity of the BBB is a factor in the initiation and progression of neurological diseases, including epilepsy [1–3]. It has been hypothesized that an immune activation initiating event such as infection, hemorrhage, neurotrauma, or metabolic abnormality in either the CNS or the periphery contributes to neuroinflammatory disease. Inflammatory mediators, including pro-­inflammatory cytokines, complement components, stress-­­ related danger signals, and activated leukocytes, are potential players. All of these have the potential of breaching the BBB resulting in direct impact on CNS-­resident cells and endothelial cells at the BBB to promote adhesion and infiltration of activated antigen-­presenting cells (APC), lymphocytes, and antibodies (Abs) into the brain. The inflammatory milieu in the brain leads to changes in extracellular potassium, sodium, and calcium concentrations resulting in functional changes in neurons and glia. Alterations increase neuronal excitability and decrease seizure threshold and recurrent seizures which further exacerbate neuroinflammation causing a feedback loop of increasing seizure susceptibility [4]. Production and secretion of cytokines are tightly regulated to control immune responses generated in the CNS.  Neuropeptides, such as somatostatin, and anti-­ inflammatory cytokines, including TGF-­β and IL-­10, negatively regulate overt immune activation. A variety of immune regulatory cell populations with diverse cellular origins and activation mechanisms contribute to the homeostasis of the brain, including the CD4+ regulatory T cells (Tregs) [5], type 1 regulatory T cells (Tr1) [6], regulatory CD8+ T cells [7], innate lymphoid regulatory cells, regulatory B cells [8, 9], and tolerogenic dendritic cells (DCs). In this review, we will focus on the role of Tregs in the CNS under healthy and disease conditions. CNS surveillance by peripheral immune cells is an ongoing process in healthy individuals with approximately 1.5x105 T cells detected in the CSF [10, 11]. Normal brain function in fact requires input from peripherally derived lymphocytes. For instance, hippocampal neurogenesis is impaired in mice that lack an intact adaptive immune system, suggesting that normal development depends on functional T and B cells [12]. Neurogenesis is rescued by the transfer of CD4+, but not CD8+, T cells into SCID (severe combined  immunodeficiency) mice lacking adaptive immune cells [13]. Optimal learning and memory performance as measured by the Morris water maze in a healthy animal require CD4+ T cells and MHC class II, as B cell-­­ deficient mice (μMT) and Rag2−/− mice that only received CD8+ T cells failed to recover from memory deficit [14]. Additionally, increased depressive and anxious

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behaviors in immunocompromised mice can be alleviated by systemic transfer of CD4+ T cells [15]. Remarkably, exogenously supplied CD4+ T cells that are specific for a brain-­specific antigen, myelin basic protein, induced enhanced hippocampal neurogenesis in recipient mice as opposed to T cells specific for foreign antigens. This finding suggests possible contribution of natural thymus-­derived Tregs (tTregs) but not extrathymically derived peripheral Treg cells (pTregs) in the maintenance of CNS homeostasis. Foxp3+ tTregs that are primarily generated in the thymus are polyclonal and have been shown to be specific for self-­antigens yet with much lower activation threshold than conventional T cells. In the periphery, FoxP3− naïve conventional CD4+ T cells (Tconv) can upregulate FoxP3 expression to generate pTregs that mediate tolerance toward non-­self-­antigens, such as commensal bacteria that are not represented in the thymus [16]. In contrast to these endogenous populations of Treg, experimental stimulation of Tconv with IL-­2 and transforming growth factor β (TGF-­β) in cell culture gives rise to in vitro-­induced Tregs (iTregs) [4]. A neuroprotective role of CD3+ T cells, most likely Tregs, has been reported to modulate inflammation to alleviate kainic acid-­induced seizure and lesions in the mouse hippocampus [17]. A similar neuroprotective role for T cells was also detected in humans [18]. Vieira et  al. have reported that there was a higher frequency of IL-­10 producing CD4+CD25+FoxP3+ Tregs in the peripheral blood of temporal lobe epilepsy patients [19]. The frequency of Tregs positively correlated with age at onset of seizures in the patients. There is a negative correlation between CD4+ T cells expressing co-­stimulatory molecules (CD69, CD25, and CTLA-­4) with age at onset of seizures. We have also reported detection of IL-­10 producing Tregs in the resected brain parenchyma of patients diagnosed with focal cortical dysplasia and encephalomalacia using an unbiased flow cytometric approach. Remarkably, patients with more Treg in the epileptogenic center of the brain had lower seizure frequency history than those with fewer Tregs. The total number of Tregs in the brain inversely correlated with seizure severity [20]. The critical contribution of Treg in maintaining homeostasis in the CNS is also highlighted in non-­­ epileptic neurological disorder when disturbance of Treg frequency and dysregulation of Treg function is documented in early stages of diseases, such as multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease [21–24]. Supplementation of Tregs confers protection in animal models of these neurological disorders by reducing glia-­mediated inflammation and brain infiltration of peripherally derived leukocytes [25, 26]. To substantiate a protective role for brain-­infiltrating Tregs in the regulation of epileptogenesis, we showed that transfer of autologous tTregs to recipient mice that have experienced kainic acid (KA)-­ induced status epilepticus significantly decreased seizure susceptibility after re-­­ exposure to KA. The symptomatic control coincided with a decrease of total number and frequency of peripherally derived monocytes, macrophage, conventional DC, plasmacytoid DC, CD8+ T cells and B cells in the brain (Fig. 1). The proportion of these cellular subsets expressing activation markers was also reduced. Inhibition of brain infiltration by peripherally derived antigen presenting cells (APCs) and antibody-­­producing B cells through increased activity of tTregs in the brain effectively alleviated seizure severity, suggesting a therapeutic potential of Tregs in

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Fig. 1  tTreg supplementation effectively restricts trafficking of peripherally derived APCs to the brain. The total number of different subsets of viable leukocytes isolated from the brains of P28 mice infused with tTregs and subjected to intraperitoneal injection of kainic acid (KA) was enumerated by flow cytometric analyses. Data is from five brains pooled per group. GD T cells, 𝛾𝛿 T cells; NK cells, natural killer cells; inf monocytes, inflammatory monocytes; pDC, plasmacytoid dendritic cells; mDC, myeloid dendritic cells

seizure control. Conversely, tTreg depletion/inactivation with anti-­CD25 augmented seizure severity supporting the inverse correlation of Treg numbers with seizure frequency in epileptogenic human brains [20]. We have recently developed a novel biodegradable carboxylated poly(lactic-­ co-­ glycolic) acid (PLGA) immune-­­ modifying nanoparticle (IMP)-­based therapy that can be employed to regulate activation of both innate [27] and adaptive [28–30] immune responses. Our results indicate that immunomodulatory IMP treatment markedly reduces seizure severity and alters the epileptogenic process in multiple murine models, including a genetic Dravet model with deficiency in Na  +  channels (Scn1a+/−), an acquired model induced by intrahippocampal injection of kainic acid (KA), and a status epilepticus model using intraperitoneally injected KA (manuscript in preparation). Most importantly, disease modification is accompanied by a significant increase of Treg frequency, from 28% of total CD4+ brain-­infiltrating cells in controls to 54% in IMP-­treated mice. Collectively, these results showed that brain infiltration of Treg in epilepsy may be protective and promote regeneration.

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2  Markers for Identification of Treg in Human and Mouse Molecular signatures and phenotypic markers used to define Tregs differ between mouse and human. Treg markers in mice are well understood and widely accepted by the field, while definitive Treg markers in human remain elusive. In the early days when suppressor cells were originally described, regulatory or “suppressor” T cells were defined at CD8+ T cells which mediated “infectious” immunological tolerance [31, 32]. In a seminal paper by Gershon and Kondo in 1971, negative regulation by a subset of T cells led to the notion that suppressor T cells were a specialized cell type whose main function is to limit immune responses [33]. Subsequently, a previously unrecognized segment within the major histocompatibility complex (MHC) region, the I–J subregion, was reported to be the restricting element which controlled suppressor T-­cell activation [34, 35]. However, with the emergence of molecular cloning and monoclonal antibody, the suppressor T cells theory fell out of favor mainly due to lack of supporting evidence for the I-­J region by genomic sequencing and mRNA transcript detection [36, 37]. The field of suppressor T cells research suffered an abrupt and rapid decline and met its demise as a result of what seems to have been several dubious flaws. The scientific resurgence of suppressor T cells came from Sakaguchi and colleagues in the 1990s reporting an immunoinhibitory effect, critical for restraining the effector function by conventional T cells, by CD4+ T cells that expressed high levels of CD25, the alpha chain of the IL-­2 receptor, which was considered a specific surface marker for murine T suppressors [38]. The “come back” for T suppressor also featured a new nomenclature, the “regulatory T cell” (Treg), to distinguish itself from the abandoned “suppressor T-­cell” predecessor. The Treg theory was strengthened by several studies reporting that transfer of purified T cells lacking markers of recent activation or memory into immune-­deficient hosts led to inflammatory bowel disease, which could be prevented by co-­transfer of CD25hi expressing regulatory T cells with memory markers [39, 40]. Additional cell culture studies demonstrated inhibition of proliferation of conventional CD4+ and CD8+ T cells by CD25hi Tregs [41]. Although Tregs constitutively express high levels of CD25 on the cell surface, which is required for Treg proliferation and survival, the lack of a clear demarcation between CD25hi Tregs and CD25int activated T effector cells (Teff) confounded Treg phenotype and isolation in both human and mouse. FoxP3, the master transcription regulator critical for Treg development and function, was subsequently characterized as another defining marker because non-­synonymous mutations in the FoxP3 gene cause IPEX (immune dysregulation, polyendocrinopathy, enteropathy, and X-­linked)-­like syndrome in human and rodent (scurfy mouse), similar to CD25 deficiency [42–50]. FoxP3 interferes with normal functions of Teff through inhibition of pro-­inflammatory cytokine gene expression by blocking activation of nuclear factor-­kB (NF-­kB) and nuclear factor of activated T cells (NFAT) [51, 52]. FoxP3 is a marker for bona fide Tregs in mice, but activated conventional CD4+ Teff in humans can transiently upregulate both CD25 and FoxP3 [53]. A variety of additional markers subsequently used to define Tregs include GITR

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(glucocorticoid-­induced tumor necrosis factor receptor), CTLA-­4, neuropilin-­­1, PD-­1 (programmed death-­1), LAG-­3 (lymphocyte activation antigen-­3), CD127 (IL-­7 receptor alpha chain), GARP (glycoprotein A repetitions predominant), LAP, FR4, and ICOS (inducible co-­stimulator). These markers are critical for the effector function of Tregs, and differential levels of expression are often used to distinguish thymus-­derived tTregs from extrathymically derived pTregs. We have extensively characterized the role of Tregs in modulating a two-­hit model of KA-­induced status epilepticus mouse model using FoxP3-­GFP (green fluorescent protein) transgenic mice, in which intracellular GFP production is controlled by the FoxP3 promoter to ensure FoxP3-­expressing cells are labeled green (manuscript in preparation). After the first hit of KA in post-­natal day 14 (P14 FoxP3-­GFP mice), Treg frequency in the brain increased from baseline to 15% as a result of inflammation-­driven Treg infiltration. A subsequent second hit of KA at P28 to the same mice resulted in enhanced seizure severity measured by Racine scale, which coincided with a reduction in Treg frequency to 10.8% in the brain. Functional phenotyping showed that single KA-­injected mice had increased surface expression of GITR (60%), GARP (80%), LAP (60%), ICOS (60%), FR4 (70%), CD39 (80%), and PD-­1 (80%). However, after a second hit of KA, all the functional markers were reduced except GITR and FR4. GITR is a functional marker for Tregs in human and mouse and is highly specific for identifying and isolating Tregs that actively regulate Teff to reduce autoimmune disease [54]. Co-­expression of GITR with CTLA-­4, IL-­10, and TGF-­β signifies the association between GITR expression and Treg function. Mature tTregs express high levels of GITR, which is needed to lower the threshold for IL-­2 during development [55]. Signaling through GITR enhances Treg expansion and IL-­10 production [56]. GITR expression, however, is not restricted to tTregs, yet can be detected in many subsets of T cells with regulatory capacity, including Tr1 cells, CD8+ Tregs, and CD4−CD8− double negative Tregs [57–59]. Another functional marker for Tregs is LAG-­3, a CD4 homolog that binds to MHC class II. Similar to GITR, GARP, and CTLA-­4, the expression of LAG-­3 is not specific to Tregs but is required for effective regulatory activity. Blockade of LAG-­ 3 expression inhibits Treg suppressive function. Moreover, LAG-­3 expression is correlated with IL-­10 mRNA production and is robust in CD4+CD25−FoxP3− Tr1 cells. Nevertheless, LAG-­3+FoxP3+ Tregs exert their regulatory function differently from those of Tr1 cells in that LAG-­3+ Tregs are cell contact-­dependent in the absence of IL-­10 and TGF-­β [60, 61]. Ectopic expression of LAG-­3 confers suppressive activity to CD4+ non-­Tregs [62]. Of all the functional markers, ICOS and PD-­1 are the ones approved for clinical use as checkpoint inhibitors in cancer treatment. ICOS is a member of the CD28 superfamily generally expressed on T cells to ligate ICOS-­L expressed on APCs [63]. It is upregulated after TCR (T cell receptor)-­stimulation and T cell activation and thus is expressed at steady-­state in memory T cells and Tregs [64–66]. Signaling through ICOS activates Akt/mTOR and MAPK/ERK pathways providing T cells with proliferative and survival support in Teff, as well as IL-­10 secretion in Tregs [64–66]. Unlike GITR, LAG-­3, GARP, and other functional markers of Treg, ICOS signaling is more important for maintenance of existing Tregs rather than de novo

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Treg activation and differentiation. ICOS blockade diminishes the function and maintenance of Tregs. Blockade of the PD-­1 pathway is more efficacious clinically in cancer immunotherapy than inhibition of the ICOS pathway. The PD-­1 pathway is critical for the maintenance of the suppressive activities of Tregs. PD-­1 and its ligand PD-­L1 are both expressed on Tregs. PD-­L1 induces pTreg differentiation and function through inhibition of PI3K (phosphatidylinositol-­3-­kinase) and mTOR (mammalian target of rapamycin), while promoting PTEN (phosphatase and tensin homolog). Manipulation of PD-­1/PD-­L1 expression using knockout mice or with blocking antibodies significantly diminishes Treg development and function resulting in enhanced autoimmune diseases [67]. FR4 (folate receptor 4) is another functional marker for Tregs with intense expression on CD25hi Tregs. High levels of FR4 expression are crucial for Treg proliferation. In the two-­hit KA mouse model, although the functional phenotype and cytokine production profile suggest that the Tregs identified in the brain were tTreg, we were not able to definitively distinguish them from pTreg. tTregs possess a unique and epigenetically defined marker that reliably identifies this population in humans and mice. It is a completely demethylated region that lies upstream of exon 1 of the FoxP3 locus, termed the Treg-­specific demethylation region  (TSDR) [68]. The methylation status of TSDR is critical for sustained FoxP3 expression and maintenance of Treg phenotype and function, though not an easily accessible marker useful for phenotyping and isolation of live cells. The functional outcome of activated polyclonal tTregs differs markedly from that of pTregs specific for foreign antigens. While tTregs modulate trafficking patterns of Teff through regulation of CXCR4 and S1P1 (sphingosine phosphate receptor 1) expression on Teff to prevent them from migrating out of the lymph nodes to reach the peripheral organs, pTregs inhibit Teff priming [69]. tTregs have the ability to confer immunomodulatory function to pTregs by regulating the GARP expression level to facilitate an increase of TGF-­β concentration in the microenvironment, in which the pTregs reside. Resting mouse tTregs express low levels of GARP. Activation of tTregs, most likely through TCR stimulation by self-­antigens, induces release of GARP from its intracellular storage depot and was followed by detection of the GARP/latent TGF-­β complex on the cellular surface. The latent TGF-­β complex that is bound to LAP does not have biologic activity until it is released from LAP to become secreted TGF-­β. tTreg activation leads to release of biologically active TGF-­β from the latent complex resulting in conversion of the antigen-­specific Foxp3− T cells to pTregs. Therefore, surface expression of GARP is a marker of functional Tregs in the mouse [70–73].

3  The Immunosuppressive Mechanisms of Tregs Tregs have the capacity to actively block immune responses, inflammation, and tissue destruction by suppressing the functions of an array of cell types including classical CD4+ helper T cells, B-­cell antibody production and affinity maturation, CD8+ cytotoxic T-­lymphocyte (CTL) granule release, and antigen-­presenting cell (APC)

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function and maturation state [74–76]. Tregs use surface expression of LFA-­1, CTLA-­4, and LAG-­3 to downregulate expression of co-­stimulatory molecules, such as CD80 and CD86, and stimulate production of IDO (indoleamine 2,3-­­dioxygenase) on APC [61, 77–79]. In the presence of cognate antigen, Tregs use LFA-­1 to form aggregates around APCs in order to create spatial barriers between APCs and Teff before mobilizing CTLA-­4 to downregulate co-­stimulatory molecules on APCs. The amount of antigen required for Treg activation is at least 1 to 2 logs lower than the concentration needed to activate Teff [80]. Teff cell activation is abrogated in the absence of strong co-­stimulatory molecules on APCs, whereas IDO catabolizes tryptophan, an essential amino acid for Teff survival, to kynurenine, which is toxic to Teff cells. The inhibitory effect on APCs in the lymph nodes results in failure of expansion of Teff cells. CTLA-­4 blockade in vivo leads to autoimmune diseases in otherwise healthy mouse and reduces Treg suppressor function when co-­cultured in vitro with Teff [81, 82]. We have examined the potential mechanism(s) of suppression by polyclonal tTregs in immunologically intact mice with systemic kainic acid-­induced status epilepticus or intrahippocampal KA-­induced epilepsy. We carefully monitored the fate and differentiation of Teff and Tregs on a single cell basis. Transfer of polyclonal tTreg cells into wild-­type C57Bl/6 after the first hit of KA induced partial, but highly significant, protection from seizures induced by a subsequent exposure to KA and ameliorated the onset and duration of status epilepticus [20]. Seizure suppression strongly correlated with reduced numbers of Teff in the brain. Mice that had received Treg cells had a 50% reduction in the percentage of CNS-­infiltrating CD4+ T cells, but on a per cell basis, the cytokine production profile of these cells was identical to cells in controls (manuscript in preparation). The therapeutic effects of the tTregs indicated that while the percentage of Teff that infiltrated the CNS was significantly reduced, tTreg-­mediated Teff differentiation was not totally abrogated but partially inhibited. A decrease in brain-­infiltrating Teff cells is accompanied by an increase of the same Teff cell population in the spleen in the presence of an excess of tTregs (manuscript in preparation). However, the resulting cellularity in the spleen is influenced not only by in situ proliferation but also by the rate of egress and ingress. Similar experiments performed in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, showed fewer Teff in the circulation of tTreg-­infused mice compared with controls [26]. Taken together, these observations are consistent with the hypothesis that, in the presence of polyclonal tTregs, priming and expansion of Teff are partially affected and trafficking of Teff to the circulation and ultimately to the brain is inhibited; thus fewer Teff are available to exert function at the inflammatory site. Thangada and colleagues showed that Teff recovered from draining lymph nodes expressed reduced level of CXCR4, syndecan, and S1P1 (sphingosine phosphate receptor 1) [69]. Upon entering lymph nodes, T cells downregulate S1P1 to be retained in the lymph nodes allowing for extensive interaction with APC. Properly primed Teff upregulate S1P1 to facilitate their egress from the lymph nodes into the circulation in response to the high levels of S1P in the blood. By altering S1P1 expression on Teff, Tregs modulate the ability of Teff to migrate from the lymph nodes into the circulation to reach

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inflamed peripheral organ or the brain. Thus, immune modulation by Treg is not only restricted to inhibition of Teff priming, activation, and differentiation but also perturbation of Teff trafficking and homing. This knowledge provides insights into therapeutic development of Treg-­mediated seizure control in that Treg supplementation or augmentation would not necessarily lead to global immunosuppression or unresponsiveness to all antigens like the effect of steroids. Instead, polyclonal tTregs, or better yet antigen-­specific pTregs, have the potential to specifically target autoreactive Teff by altering their trafficking pattern but still allow intact immunity to develop in the secondary lymphoid organs by sparing pathogen-­specific Teff. Whether tTregs need to be activated before being transferred to the recipient remains elusive. One major challenge is that the cognate epitope or the source of antigen that the TCR recognizes for effective tTreg activation is unknown. tTregs have a polyclonal TCR repertoire that are selected on self-­peptides. However, unlike Teff cells, tTregs have more stringent requirements for the diversity of self-­antigens and strength of the signal required for development as tTregs generally do not develop in mice that express a single TCR [83]; Treg development can be restored if enhanced TCR signaling is provided to overcome the limited diversity of TCR. Early TCR sequencing studies by Pacholczyk et al. showed that tTreg TCRs were extremely diverse, but the overlap between TCR sequences of conventional T cells and tTregs was limited [84]. It has been postulated that tTregs are continuously activated by ubiquitous autoantigens at a level orders of magnitude lower than what is required to activate conventional Teff specific for foreign antigens. In a follow-­up study by the same group, a contradictory conclusion was drawn in that tTregs do not preferably recognize self-­antigens but instead express a polyclonal TCR repertoire that is comparable to conventional T cells [85]. Nevertheless, the clonality of tTregs is remarkably diverse irrespective of the antigen source. Limited studies have compared the effectiveness of immune modulation by polyclonal and monoclonal populations of tTregs, and the results are controversial and model dependent. Tang and colleagues showed that a monoclonal tTreg population from a TCR transgenic mouse strain was more effective than polyclonal tTreg to inhibit the incidence of diabetes in a NOD Rag−/− model of type 1 diabetes [86]. However, Hori et al. demonstrated comparable effectiveness of polyclonal and monoclonal tTregs in preventing EAE in a transgenic mouse expressing TCR specific for a myelin basic protein antigen on a RAG−/− background [87]. The benefit of using monoclonal tTregs specific for an organ-­specific antigen to modulate immune response is that they would preferentially home to the target organ to exert suppressive function, while sparing the pathogen-­specific Teff. In contrast to tTregs, pTregs have little effect on Teff migration but mostly inhibit priming of Teff and production of inflammatory cytokines [88, 89]. Secretory immunosuppressive mediators, such as IL-­10, TGF-­β, and galectins, have been reported to mediate Treg suppression [26, 27, 38]. Teff function can be inhibited by Tregs through contact-­dependent (e.g., direct ligation and cytolysis of Teff) and contact-­independent mechanisms (e.g., including secretion of anti-­ inflammatory cytokines such as IL-­10 and TGF-­β, utilization of IL-­2 that is critical for T-­cell survival, and production of inhibitory metabolites) [90, 91].

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4  Conclusion and Perspectives Tregs plays a critical role in maintaining homeostasis in the CNS inflammation. While there are no definitive CD4+ T-­cell subsets that are solely beneficial or entirely detrimental, it is the balance of pro-­inflammatory versus anti-­inflammatory cells and the ratio of Teff to Tregs that would determine disease outcome. Alterations in the subsets of blood-­derived inflammatory cells will affect infiltrates present in the CSF and CNS of patients with epilepsy. Compared to brain-­resident immune cells, such as microglia and astrocytes, the numbers of peripherally derived immune cells in the epileptic brain is small, but the pro-­inflammatory capacity of these infiltrating cells is orders of magnitude greater than glia. The frequency and function of Tregs in peripheral blood and brain lesions appear to correlate with seizure severity. Decreased suppressive capacity of Tregs could be a potential biomarker for drug-­­ resistant epilepsy. The important contribution of Tregs to seizure control and epileptogenesis modulation cannot be overemphasized although the number of studies focusing on the role of Tregs in seizure and epilepsy is limited. Further characterization of Treg populations and more in-­depth studies of regulatory immune subsets are required for the development of more specific and effective immunomodulatory therapies that can avoid general immune suppression. Identification of T-­cell autoantigenic epitopes specific to seizure or epilepsy may provide new insights in precision medicine and therapeutic development.

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Inflammation at the Neurovascular Unit in Post-traumatic Epilepsy Xavier Ekolle Ndode-Ekane, Jenni Kyyriäinen, and Asla Pitkänen

Abstract  Post-traumatic epilepsy (PTE) is one of the clinical outcomes in about 16% of patients with traumatic brain injury (TBI). Patients develop PTE after several months to years following the initial brain injury. Over the years, there have been an increasing number of reports suggesting that post-traumatic inflammation, which is a major component of the pathophysiology of TBI, may be a key player in the development of PTE. The inflammatory process begins within minutes to hours after the primary injury and may proceed for several years after initial insult. Evidence suggest that the neurovascular unit (NVU) play a key role in the post-­traumatic inflammatory cascade. Also, there is growing number of data supporting the hypothesis that antiinflammatory treatments may improve the post-TBI clinical outcome. In this chapter we will give insights into how the different members of the NVU, including the bloodbrain barrier, microglial cells, NG2-glial cells, pericytes, and astrocytes, contribute to the post-traumatic inflammation and the development of PTE. Keywords  Activated microglial · Astrogliosis · Blood-brain barrier · Neurovascular unit · NG2-glial · Post-traumatic epilepsy · Post-traumatic inflammation · Pericytes · Traumatic brain injury

Abbreviations Blood-brain barrier Center for Disease Control and Prevention Central nervous system Cerebrospinal fluid Chemokine ligand Danger-associated molecular patterns

BBB CDC CNS CSF CCL DAMPs

X. E. Ndode-Ekane (*) · J. Kyyriäinen · A. Pitkänen A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_10

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Glial fibrillary acidic protein Interferon gamma Interleukin Magnetic resonance imaging Neurovascular unit Nitric oxide Nitric oxide synthase Platelet-derived growth factor receptor α Platelet-derived growth factor receptor β Post-traumatic epilepsy Post-traumatic seizure Repetitive mild traumatic brain injury Transforming growth factor β Traumatic brain injury Tumor necrosis factor alpha

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GFAP IF-γ IL MRI NVU NO iNOS PDGFR-α PDGFR-β PTE PTS rmTBI TGF-β TBI TNF-α

1  Traumatic Brain Injury Traumatic brain injury (TBI) is defined as an injury to the brain caused by an external force, resulting in the alteration of brain function or brain pathology [69]. The Center for Disease Control and Prevention (CDC) estimated that the incidence of TBI in the United States between 2002 and 2006 was 579 per 100,000 persons or approximately 1.7  million cases per year [34, 66]. In Europe, the incidence was estimated to be at about 235 per 100,000 persons [95]. The risk of TBI is highest among young children (0–4  years of age), older adolescence (15–19  years), and older adults (75 year and above) [48]. TBI often leaves the affected individual with long-term or lifelong disabilities, including physical, psychological, cognitive, and social dysfunction that leads to a decrease in quality of life. The economic burden of TBI, as estimated by the CDC, is about $56 billion in the year 2000 [27]. Post-traumatic epilepsy (PTE) is one of the major long-term clinical outcomes of TBI. In Europe, it is estimated that about 2–16% of TBI patients subsequently develop PTE [35]. The risk of developing PTE increases with the severity of head injury [17]. Among the several risk factors, acute subdural hematoma is by far the intracranial lesion with the highest risk for early or late post-traumatic seizure (PTS) [52, 106]. Early PTS are seizures that occur within 1 week after injury, and late PTS are those that occur more than 1 week after injury. TBI causes several structural, physiological, and biochemical alterations in the brain that are potentially epileptogenic. However, the mechanism that underlies the development of PTE is still unclear. Studies in several animal models of TBI suggest that oxidative stress, excitotoxicity, and neuronal hyperexcitability are some of the mechanisms involved in PTE [13]. Furthermore, there is growing evidence suggesting that the post-injury pathophysiology of the blood-brain barrier (BBB) and the neurovascular unit (NVU), which comprises the brain endothelial cells (together

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with the basal lamina matrix, pericytes, and vascular smooth muscle cells), neuron, microglia cells, astrocytes, and oligodendrocytes, may be involved in PTE.  This chapter will focus on the role of cells of the NVU in post-traumatic inflammation and the pathophysiology of PTE.

2  Traumatic Brain Injury: Primary and Secondary Injury Primary Injury  The primary injury is due to the initial mechanical impact to the brain. The mechanical force tears apart tissue and neurons, shears their axons, and damages the vasculature [70]. Disruption of the blood-brain barrier (BBB) permits blood-borne factors and cells to enter the brain parenchyma [86]. Most notably, especially in moderate and severe TBI, there is intracerebral bleeding and hematomas resulting from damaged blood vessels and consequent hemorrhage [33]. As mentioned above, diffused axonal injury caused by the initial force to the brain is also a principal characteristic of the primary injury. However, subsequent axonal damage is, in most part, due to the secondary injury mechanisms [11]. Secondary Injury  The secondary injury begins within minutes to hours after the primary insult. It comprises several cellular and molecular responses that can go on for several years after the primary insult [82, 103]. These responses include continuous BBB dysfunction, the release of inflammatory mediators by infiltrating cells and resident glial cells, excessive release of free radicals and the neurotransmitter glutamate [31], influx of calcium and sodium ions, mitochondria dysfunction [61], as well as genetic alterations. Hypoperfusion is also a common feature of the secondary injury process and usually occurs during the first few days after the injury. Reports suggest that hypoperfusion may occur due to impaired vasodilation, reduced blood pressure, and increased intracranial pressure [92]. However, these cerebral vasculature changes may occur only acutely after TBI, but the structural vascular changes may persist for months to years after injury.

3  T  he Blood-Brain Barrier and Post-traumatic Epileptogenesis and Epilepsy The BBB, a major component of the NVU, separates contents of the central nervous system (CNS) and the systemic circulation; it regulates homeostasis and blood flow in the brain. The BBB is not only a physical barrier but also a “transport barrier,” regulating transcellular traffic, and a “metabolic barrier,” metabolizing toxic and neuroactive substances [1]. The BBB is composed of a single layer of cerebrovascular endothelial cells linked together by an intercellular tight junction complex [28] and covered by a basement membrane, where the pericytes are located [1]. In

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close contact with the BBB are the astrocyte end feet, microglial cells, and adjacent neurons [1]. Pronounced disruption of the BBB following TBI contributes to edema formation, inflammation, and hyperexcitability. BBB disruption-induced post-traumatic brain edema is a common feature in humans and preclinical models of TBI [18]. Edema occurs during the first few hours after TBI and persists over the next few days and up to 2 weeks after the initial insult [5, 41]. Generally, edema causes increased intracranial pressure, impairs cerebral perfusion, and impairs oxygenation. BBB disruption typically contributes to vasogenic edema, which is the accumulation of extracellular water, but not cytotoxic/ cellular edema, which is a result of intracellular water collection [53]. However, the initial vasogenic edema (first few hours after TBI) is followed by cytotoxic edema. Even though the BBB gradually closes to large plasma protein over the first few days, smaller vascular components remain permeable for up to 7 days, to even several weeks after injury [6, 39, 75]. The mechanisms behind the persistent permeability are not clear, though it is being hypothesized that injury-mediated rearrangement of the endothelial cell cytoskeleton may promote barrier opening [65]. Despite the widely reported occurrence of edema following TBI, there are no reports demonstrating a direct link between edema and PTE. Nonetheless, edema is considered a risk factor for early PTS and PTE. Reports suggest that about 53% of children with moderate to severe cerebral edema are likely to develop PTS and about 20% of pediatric patients with cerebral edema develop early PTE [3, 14]. The mechanisms through which cerebral edema contributes to the appearance of PTS or PTE are not known. Since PTS occur earlier after the injury and PTE occur later after epileptogenesis is established, it is possible that the mechanisms behind PTS and PTE are not the same. It is also likely that edema, together with other factors such as inflammation, may underlie this process. Breakdown of the BBB facilitates the infiltration of blood-borne cells into the brain parenchyma, which subsequently elicits a sequence of inflammatory reactions. Infiltration of peripheral immune cells, such as macrophages and leukocytes, has been described in human and experimental models of TBI [43, 73]. Indeed, we showed that the infiltration of T lymphocytes following the fluid percussion brain injury in rats peaked at 2 days and then decreased by 7 days post-injury (Fig. 1) [73]. Increased infiltration of peripheral immune cells has been shown to be associated with poor functional outcomes. For example, changes in markers for T lymphocyte activation were linked with an unfavorable outcome following TBI in patients [74]. Increased T-cell infiltration in the brain parenchyma was recently shown to be associated with increased somatomotor deficits following TBI in rats [73]. Furthermore, blockage of T-lymphocyte infiltration after TBI in rodents improved functional outcomes [105]. Infiltrating neutrophils were shown to contribute to poor outcomes by promoting edema, oxidative stress, production of inflammatory cytokines, and neurotoxic proteases [45, 76]. Increased production of pro-inflammatory cytokines and the recruitment and activation of glial cells by the infiltrating cells has been suggested to worsen the brain damage, thus leading to the poor outcome [107]. The activated microglial cells, in turn, produced further pro-­ inflammatory cytokines and chemokines, thereby attracting more cells into the

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Fig. 1  CD3 (panels A and B) and CD68 (panels C and D) immunoreactive cells in the cortex (panels A and C) and thalamus (panels B and D) at 4 d and 3 months post-traumatic brain injury (TBI). (a) CD3 immunoreactive cells in the cortex at 4 d post-TBI. CD3 is a marker for T lymphocytes (T cells). Note the ruptured tissue filled with clusters of T cells at the external capsule (ec) (closed arrow). Also, note T cells moving into the brain parenchyma (open arrow). The insert is a high magnification taken from the area around the open arrow. (b) CD3 immunoreactive T cells in the thalamus. Observe the cluster of T cells around a blood vessel (closed arrow) and also some of them moving further from the vessel (open arrow). The insert is a high magnification of T cells around the area with the open arrow. (c) CD68 immunoreactive cells (open arrows) in the cortex at 3 months post-TBI. CD68 is a marker for phagocytic macrophage/monocyte and microglial cells. The asterisk indicates the lesion core. The insert is a high magnification taken from the CD68 cells at layer 6 (open arrow). (d) CD68 immunoreactivity is still very strong in the thalamus at 3 months post-TBI. Note the cluster of cells next to the blood vessel (open arrow). Scale bar in A, B, C, and D equals 100um and scale bar in inserts equals 20 um

brain parenchyma, propagating the inflammatory cascade and oxidative stress in the brain, subsequently leading to cell death [2]. Altered levels of several cytokines including interleukin (IL) 1, IL-1β, IL-6, IL-8, IL-10, IL-12, transforming growth factor β (TGF-β), and tumor necrosis factor (TNF) have been reported in patients and experimental models of TBI [43, 47, 50, 71, 89]. There are no studies linking an increased infiltration of immune cells into the brain parenchyma and the occurrence of PTE. However, evidence suggests that cytokine production contributes to epileptogenesis [8, 85, 96]. Recently it was reported that high levels of CSF/serum IL-1β are associated with increased risk for PTE [20, 21]. It is still a matter of debate how

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the infiltrating immune cells and their production of pro-inflammatory cytokine lead to hyperexcitability and, subsequently, PTE. Post-traumatic BBB breakdown is also suggested to facilitate the development of PTE through the release of blood-borne components such as albumin. Indeed, magnetic resonance imaging (MRI) of patients with PTE has revealed co-localization of brain areas with increased BBB dysfunction, as revealed with gadolinium within the epileptogenic region [97, 98]. Albumin is proposed to trigger epileptogenesis through the albumin/TGF-β-mediated signaling pathway. Binding of albumin to TGF-β receptors, two serine-threonine kinase receptors, on activated astrocytes leads to the phosphorylation of the Smad protein complex and the p38 MAPK pathway [94]. The blockage of albumin binding to the TGF-β receptor has been shown in experimental models of BBB dysfunction to prevent epileptiform activity [50]. It has been demonstrated that TGF-β is involved in excitatory synaptogenesis in experimental TBI epilepsy models [104]. This suggests that TGF-β may also be involved in PTE.

4  Microglial Activation and Traumatic Brain Injury Microglia cells, often described as the macrophages of the CNS, play a significant role in the CNS’s innate immunity. Microglia play an important role in the NVU, including communicating with other cells of the NVU through cytokines, chemokines, and growth factor-mediated signaling. They display a plethora of functions which include immune surveillance, phagocytosis, and secretion of several signaling molecules that affect other cells of the NVU and other immune cells. The specific function displayed by the microglial at any particular moment usually depends on the state of the brain, as during development or pathology. In the normal brain, microglia are often defined as resting cells; however, reports show that they are actually constantly surveying their immediate vicinity by extending thin processes in all directions, interacting with other cells, including members of the NVU [40, 42]. In the presence of a brain injury, like TBI, microglia cells become activated, migrate to the site of damage, proliferate, become phagocytic, and secrete several signaling molecules. Nonetheless, the main functions of microglia are phagocytosis of damaged or apoptotic cells, inactive synapses, infectious agents and tissue debris, regulation of inflammatory response of other brain cells, and peripheral immune cells [37, 56, 99]. Microglial activation is a characteristic feature in human and animal models of TBI [37]. In the activated state, the morphology of the microglia cell changes from their typical ramified state to an amoeboid state with their processes appearing retracted, fewer, and thicker. They also produce a lot of inflammatory mediators that affect the function of other cells. Microglial activation following a TBI occurs early on following the primary injury and has been shown to persist for several months to years in experimental models and postmortem tissue [15, 54, 84]. The role of microglia in the primary and secondary injury process following TBI is still widely

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discussed. Within minutes following the primary insult, microglial cells become activated as a result of a combination of factors including BBB breakdown and release of danger-associated molecular patterns (DAMPs) from damaged cells that signal to other infiltrating and resident cells, thereby causing them to release pro-­ inflammatory cytokines and chemokines [24, 59, 63]. In addition, activated microglia cells upregulate several cell surface receptors that are involved in phagocytosis. These potent inflammatory mediators can also cause the recruitment of other peripheral immune cells to the injury site. It is currently known that activated microglial cells can be divided into two subtypes, M1 and M2, based on the specific cytokine produced. The M1 is considered the pro-inflammatory phenotype, producing interferon-­gamma (INF-γ), tumor necrosis factor alpha (TNF-α), IL-1β, IL-6, iNOS, and others that can exacerbate neural injury. In contrast, the M2 phenotype has increased phagocytotic properties and is considered anti-inflammatory, releasing neurotrophic factors and anti-inflammatory cytokines, including TGF-β and IL-10. It is likely that the specific role of the microglial cells post-TBI is dependent on its phenotypic state; however, this hypothesis is still strongly debated and remains to be proven [63]. Activated microglia affect other cells in the neurovascular unit, following TBI, in a variety of ways. They have been shown to be involved in neurodegeneration by the release of nitric oxide (NO), which inhibits neuronal respiration, causing glutamate release and excitotoxicity [4, 9]. It was shown that chronic microglial activation is associated with progressive neurodegeneration in the controlled cortical impact model of TBI [64]. Besides the mechanical disruption of the vascular walls following TBI, evidence suggests activated microglial cells may be implicated in BBB dysfunction through the release of NO and several pro-inflammatory cytokines such as TNF-α, IL-6, and IL-12 [16, 93]. However, it is still unclear whether (and how) the long-term activation of microglia cells in chronic TBI contributes to chronic BBB dysfunction. Inflammation is a prominent feature following TBI, such that it is no doubt that several preclinical post-TBI treatment strategies target the inflammatory mechanisms to prevent or reduce the post-TBI clinical outcome, including PTE.  For a concise review of anti-inflammatory drug trials in both preclinical and clinical TBI study, see Bergold [7]. In fact, studies on other epilepsy models have implicated inflammation in seizure pathologies. Experimental models of status epilepticus (SE) have broadly been used to demonstrate an increase in inflammation after seizures. For example, using the limbic SE model, De Simoni and colleagues showed that IL-1β, IL-6, and TNFα expression peaked 6  hours after SE initiation and stayed elevated for up to 24 hours afterward. Importantly, the rats that developed spontaneous seizures, later on, showed higher expression of IL-1β. Expression of IL-1β, IL-6, and TNFα was mainly observed in microglia, suggesting their involvement. Interestingly, they showed that the administration of IL-1 receptor antagonist (IL-1Ra) reduced the severity of behavioral seizures [19]. Similarly, in clinical studies, upregulation of IL-6 and IL-1R has been reported in patients following tonic-­ clonic seizures [79, 80]. Several clinical and experimental TBI studies have also demonstrated rapid upregulation of IL-1β during the acute post-TBI period. Using

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the fluid percussion TBI model, it was shown that IL-1β is rapidly upregulated in the brain as early as 1 h post-TBI [32, 57]. Frugier and colleagues show upregulation of IL-1β and other pro-inflammatory cytokines within 6–122 hrs of TBI in postmortem human brain tissues [36]. This data suggests that IL-1β may be involved in PTE. Additionally, in a genetic study performed by Diamond and colleagues, they investigated PTE in 256 Caucasian adults with moderate-to-severe TBI and showed that high levels of cerebrospinal fluid (CSF)/serum IL-1β ratio was associated with increased risk for PTE.  The exact mechanism by which IL-1β is involved in the development of PTE is still unclear [20, 21].

5  NG2-Glial Cells and Pericytes in Traumatic Brain Injury NG2 cells have previously been described as oligodendrocyte progenitors. This is particularly true for the developing brain where they do not only generate oligodendrocytes but also astrocytes and neurons [55, 87, 109]. However, in the adult CNS, very few NG2-glial cells generate oligodendrocytes [12]. NG2-glial cells can be identified based on their unique expression of PDGFR-α, even though some studies suggest they also express PDGFR-β [55, 60, 87, 109]. There are other cell types that express NG2, including pericytes and sometimes microglia and astrocytes [100]. Here, we are referring to NG2-glial cells, which are NG2 and PDGFR-α positive. NG2-glial cells have been described as having a heterogeneous nature, which includes not only their role as progenitor but also their role in neuroprotection, surveillance of the extracellular environment, and their unique interaction with neurons through synapses. There are several excellent review articles that have covered NG2 heterogeneous properties [22, 29, 101]. Here, we will focus on their role after acute brain injury or TBI. The most immediate response of NG2-glial cell during the primary injury process are morphological changes, where the cells around the lesion become hypertrophic, with an enlarged cell body and shorter and thicker processes (Ong and Levine [110]: PMID: 10392832). They also become proliferative [10, 23]. Together with other reports, we recently showed that NG2-glial cells proliferate, migrate, and accumulate around the lesion during the first week post-TBI, suggesting a possible role in scar formation [46, 60] (Fig. 2). Generally, NG2-glial cells have been strongly linked to repair, by generating new oligodendrocytes, as revealed in the cortical stab wound model [58]. There are very few reports showing any role of NG2-glial cells in the post-TBI inflammatory cascade. However, the role of NG2-glial cells in the post-TBI tissue remodeling has been revealed using NG2 knockout mice. In an elegant study by Huang and colleagues, they showed that NG2 deficiency resulted in an upregulation of the Cxcl13 gene that regulates the expression of the chemokine CXCl13 known to attract immune cells into the inflamed area. They further demonstrated that at the chronic phase, post-TBI, the NG2 knockout mice continue to demonstrate neurological deficits and upregulation of Czcl13 gene, which was associated with enhanced CD45+ leukocyte infiltration [44].

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Fig. 2  Expression of NG2 and PDGFR-β at acute and chronic time-points in different cell types post-TBI. (a) A thionin-stained section of the auditory cortex at 2 d post-traumatic brain injury (TBI). Note the neuronal loss and blood leakage (arrows) into the brain. (b) NG2-immunostaining corresponding location in thionin stained section in (A). Note clustering of the NG2+ cells with strong immunoreactivity at the lesion core. (c) PDGFR-β-immunostaining from an adjacent section to that in panels A and B. Note the blood vessel staining (arrow) similar to panel A. (d) A thionin stained section of the auditory cortex at 3 months post-TBI. Note the highly visible lesion core surrounded by glial scar (arrow). (e) NG2-immunostaining corresponding location in panel D from an adjacent section. Increased NG2-immunoreactivity was seen around the lesion core and inside the glial scar. (f) PDGFR-β+ staining was noticeable around the lesion core and glial scar (arrow). (g) Morphology of the NG2+ parenchymal cell at 2 d post-TBI (from panel B). (h) Morphology of the PDGFR-β+ parenchymal cells at 2 d post-TBI (from panel C). Note similar structure with parenchymal NG2+ cell in panel G. (i) Morphology of the structural pericyte (arrow) attached to the blood vessel at 2 d post-TBI (from panel C). (J) Morphology of the reactive migratory pericyte at 2 d post-TBI (from panel C). Scale bar in A–C equals 500 μm, D–F equals 200 μm, and G–J equals 20 μm

Pericytes are also part of the NVU and are located at the abluminal aspect of endothelial cells [62, 88]. Morphologically, they are considered to be polymorphic cells. However, most of them appear to have a spherical or oval cell body with a prominent round nucleus. They can also be identified with the surface markers NG2 and PDGFR-β [25]. Pericytes can be found distributed along the walls of

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precapillary arterioles, capillaries, and post-capillary venules. Traditionally pericytes have been thought of as mainly involved in the development and maintenance of the BBB, vascular stability, angiogenesis, and regulation of blood flow. However, a growing number of reports, principally from cell culture studies, suggest that they can be a source of adult pluripotent stem cells and possess immune, phagocytic, and migratory functions (for detailed review, see [62]); Sa-Pereira et al. [88]). Here, we will focus on the role of pericytes on the post-TBI inflammatory insult. As mentioned above, the traditional role of pericytes has been the maintenance and stabilization of the BBB [88]. In fact, perturbation of the BBB, like in TBI, was shown to cause pericyte migration, which was concomitant with thinning of the basal lamina [26]. We also recently showed that there is a cluster of PDGFR-β+ structural (attached to the blood vessel) and reactive (migratory) pericytes within the lesion cortex and thalamus following TBI in rats, suggesting their role in post-­ TBI scar formation and restoration of vascular integrity [60] (Fig. 2). Evidence of the role of pericytes in post-TBI immune modulation is very limited. Nonetheless, evidence suggests that pericytes respond to interferon-gamma (IF-γ) by the upregulation of the MHC class II molecule and antigen presentation to primed lymphocytes. Furthermore, reports also suggest that pericytes can produce immunoregulatory cytokines like IL-1β and IL-6 [30]. It still remains to be shown whether pericytes play an immune modulatory role following TBI.

6  Astrocyte Activation in Traumatic Brain Injury Astrocytes perform a plethora of functions within the NVU, including neurotrophic support required for neuronal viability; maturation and maintenance of synapses through the release of a variety of molecules; synaptic pruning; and controlling blood flow by the release of molecules that signal vasodilation and vasoconstriction. They also provide additional support to neurons by taking up, metabolizing, and secreting a host of molecules. For example, they produce lactate, which is shuttled to neurons for energy; they take up glutamate and GABA, thereby limiting their actions; they take up water through aquaporin 4 (AQP4) transporter and K+ ions released by the neuron. These aspects, and more about astrocyte function, have been reviewed in Pekny and Pekna [78], Sofroniew and Vinters [91], and [100]). In this section, we will focus on the role of astrocytes – as inflammatory mediators – within the NVU, following traumatic brain injury and in the development of PTE. Most of the post-TBI inflammatory response is being credited to microglia cells; however, there is evidence that astrocytes can also contribute to the post-injury inflammatory cascade. Receptors for several cytokines and growth factors including IL-1β, IL-6, IFN-γ, TNF-α, TGF-β, and CCL-12 have been described in astrocytes [91]. Furthermore, astrocytes can also express tool-like receptors (TLRs), which are receptors of DAMP, produced by dying or damaged cells [90]. Following a brain injury, astrocytes respond by changing their morphology, extending their processes and swelling of cell bodies, becoming proliferative, increasing their expression of

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glial fibrillary acidic protein (GFAP) and vimentin, and secreting inflammatory mediators and growth factors. This transformation process is generally referred to as astrogliosis [38, 77, 108]. Reactive astrocytes have been described in the brain as early as 3 days post-TBI and can be maintained for up to 60 days to several months after the initial insult [68, 102]. Astrogliosis may have several deleterious and beneficial impacts on the primary and secondary injury process following TBI and consequently on the development of PTE.  Blockage of astrocyte nuclear factor-kB (NF-kB) activation, using the NK-kB inhibitor BAY 11-7082, reduced brain edema following fluid percussion brain injury in rats [51]. As discussed earlier, post-traumatic edema is considered a risk factor for PTE [3, 14]. Mannix and colleagues reported that chronic astrogliosis was associated with long-term behavioral deficits in an experimental model of repetitive mild traumatic brain injury (rmTBI) [68]. It was shown that overexpression of insulin-like growth factor-1 (IGF-1) using astrocyte-specific IGF-1 in transgenic mice protected hippocampal neurons and reduced behavioral deficit following controlled cortical impact brain injury in mice [67]. On the other hand, ablating astrocyte proliferation, using an antiviral agent, has been shown to result in substantial neuronal degeneration and inflammation following experimental CCI in mice [72]. Data also points to the fact that an increase in astrocyte activation correlates with peak BBB breakdown and decreased activation correlates with BBB recovery [49]. Also, astrocytes overexpressing IL-6 during the acute post-TBI period are suggested to be neuroprotective [81, 83]. These findings not only indicate the deleterious effect of astrocyte activation after a TBI but also suggest that they play an essential role in preserving neural tissue and restricting inflammation.

7  Concluding Remarks The NVU is closely connected to the pathophysiology of TBI, especially the post-­ traumatic inflammatory process. Through different mechanisms, it is clear that the NVU have a significant role in the development of post-traumatic epileptogenesis and epilepsy. TBI-induced BBB disruption and the ensuing post-traumatic edema are strong risk factors for early PTS and the PTE that occurs later on. Microglial cells, when activated following TBI, exhibit potent inflammatory properties that affect other brain cells, and their effects can potentially lead to PTE. Other cell types of the NVU, such as astrocytes, NG2-glial cells, and pericytes, also exhibit a limited range of inflammatory reactions after TBI that can contribute to post-traumatic epileptogenesis and epilepsy. Quite noticeably is the fact that a huge amount of effort is being made in clinical and experimental studies to target the post-traumatic inflammation to treatment TBI. However, clear answers are still needed to the question of whether and how inflammation contributes to post-traumatic epileptogenesis and epilepsy. Despite these unresolved questions, the results delivered by ongoing research are promising.

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Correction to: Inflammation and Epilepsy: New Vistas Damir Janigro, Astrid Nehlig, and Nicola Marchi

Correction to: D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-­3-­030-­67403-­8 This book was initially published with incorrect ISSN numbers. They were corrected as follows in the imprint: ISSN 1422-7746, eISSN 2296-4525.

The updated version of the book can be found at https://doi.org/10.1007/978-­3-­030-­67403-­8 © Springer Nature Switzerland AG 2022 D. Janigro et al. (eds.), Inflammation and Epilepsy: New Vistas, Progress in Inflammation Research 88, https://doi.org/10.1007/978-3-030-67403-8_11

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