IschemiRs: MicroRNAs in Ischemic Stroke: From Basics to Clinics [1st ed.] 9789811547973, 9789811547980

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Rajanikant G. K. · Pierre Gressens  Sreekala S. Nampoothiri  Gokul Surendran · Cindy Bokobza

IschemiRs: MicroRNAs in Ischemic Stroke From Basics to Clinics

IschemiRs: MicroRNAs in Ischemic Stroke

Rajanikant G. K. • Pierre Gressens • Sreekala S. Nampoothiri • Gokul Surendran • Cindy Bokobza

IschemiRs: MicroRNAs in Ischemic Stroke From Basics to Clinics

Rajanikant G. K. School of Biotechnology National Institute of Technology Calicut Calicut, India

Pierre Gressens NeuroDiderot, Inserm Université de Paris Paris, France

Sreekala S. Nampoothiri Development and Plasticity of the Neuroendocrine Brain Lille Neuroscience & Cognition INSERM 1172 Lille, France

Gokul Surendran School of Biotechnology National Institute of Technology Calicut Calicut, India

Cindy Bokobza NeuroDiderot, Inserm Université de Paris Paris, France

ISBN 978-981-15-4797-3 ISBN 978-981-15-4798-0 https://doi.org/10.1007/978-981-15-4798-0

(eBook)

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Preface

MicroRNAs are small endogenous noncoding RNA molecules that function as a regulator of gene expression at the posttranscriptional level. In recent years, the scientific community has witnessed a massive increase in the wealth of knowledge that bridges the microRNA and its strategic role as a biomarker and therapeutic in a wide range of diseases, including ischemic stroke. However, there is still a lack of comprehensive details linking this small RNA molecule to an ischemic stroke, the more preponderant among the two main types of stroke. This monograph is intended as a medium to examine the emerging role of microRNAs in cerebral ischemia. The book addresses topics related to the activity of microRNAs in multiple pathological manifestations of strokes such as oxidative stress, excitotoxicity, cell death trends, their use as a biomarker, diagnostic agents, and currently practiced therapeutic interventions. It also highlights the latest research on how microRNAs help in neuroregeneration during poststroke events, the myriad of computational tools and databases used in microRNA research, and how they modulate other cerebrovascular diseases. The book concludes with a special insight into the potentials of developing microRNA-based therapies for ischemic stroke. This book is the first of its kind to bring together pieces of puzzle concerning the effect of the microRNAs on the ischemic stroke. Given the vast literature available in public libraries, our book seeks to provide readers with cutting-edge knowledge on microRNA covering various aspects of stroke. This alone makes our book, one that researchers in this area would be pleased to have in their bookshelves. This book can serve as a comprehensive tool for researchers, doctoral and graduate students, and clinicians in the field of miRNA and ischemic stroke and aid in the development of reliable biomarkers and neurotherapeutics by identifying suitable microRNAs. We would like to thank the Springer team, especially Madhurima Kahali and Selvakumar Rajendran, for their patient, professional, and constant support throughout the production of this book. Calicut, India Paris, France Lille, France Calicut, India Paris, France

Rajanikant G. K. Pierre Gressens Sreekala S. Nampoothiri Gokul Surendran Cindy Bokobza

v

Contents

Part I

microRNAs and Ischemic Stroke: Back to Basics

1

microRNAs in Normal Brain Physiology . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 microRNA Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 miRNA in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 miRNA in Neurons . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2 miRNA in Oligodendrocytes . . . . . . . . . . . . . . . . . . . 1.3.3 miRNA in Astrocytes . . . . . . . . . . . . . . . . . . . . . . . . 1.3.4 miRNA in Microglia . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

3 3 4 6 6 7 8 8 9 10

2

Ischemic Stroke: An Imperative Need for Effective Therapy . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Current Treatment Choices for Ischemic Stroke Victims . . . . . 2.2.1 IV Pharmacological Thrombolysis . . . . . . . . . . . . . . . 2.2.2 Endovascular Recanalization Strategies . . . . . . . . . . . 2.3 Other Reperfusion Strategies . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Post-Ischemic Neuroprotective Agents . . . . . . . . . . . . . . . . . . 2.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .

15 15 16 16 18 23 24 25 25

MicroRNAs in Ischemic Stroke Pathophysiology: Special Emphasis on Early Molecular Events . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Post-Ischemic Glutamate Excitotoxicity and Ionic Imbalance . . . 3.2.1 miRNAs Targeting Glutamate Receptors . . . . . . . . . . . 3.2.2 miRNAs and Glutamate Transporters . . . . . . . . . . . . . . 3.2.3 miRNA Regulation of Post-Ischemic Calcium Overload . 3.3 miRNAs in Post-Ischemic Oxidative Stress Regulation . . . . . . .

33 33 34 36 37 37 39

Part II 3

microRNAs in Ischemic Stroke Pathophysiology: A Bird’s Eye View

vii

viii

Contents

3.3.1 Endogenous Antioxidant Defense Mechanisms . . . . . . 3.3.2 Oxidative Stress-Induced Mitochondrial Dysfunction . 3.3.3 miRNAs and the Master Regulator Nrf2 . . . . . . . . . . 3.3.4 Endothelial Integrity and Blood–Brain Barrier . . . . . . 3.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

39 41 41 42 43 43

microRNA Regulation of Ischemic Stroke Inflammatory and Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Stroke Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 microRNAs Associated with Ischemic Neuro-Inflammation . . . 4.4 microRNAs and Innate Immune Cells in Ischemic Stroke . . . . 4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . .

49 49 50 51 53 54 54

5

Regulatory Role of microRNAs in Ischemic Cell Death . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 Intrinsic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2 Extrinsic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 MicroRNAs and Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 MicroRNAs and Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .

59 59 60 61 61 61 62 63 65

6

The Emerging Role of microRNAs in Post-ischemic Angiogenesis and Neurogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Post-Stroke Induced Neurogenesis . . . . . . . . . . . . . . . . . . . . . 6.2.1 miRNA Regulation of Early Stages of Neurogenesis . . 6.2.2 Neuronal Maturation and Integration . . . . . . . . . . . . . 6.2.3 miRNAs Regulating Neurotrophic Factors . . . . . . . . . 6.2.4 miRNAs and Neurogenesis Signaling Pathways . . . . . 6.3 miRNA Regulation of Post-Ischemic Angiogenesis . . . . . . . . . 6.3.1 EC-Specific miRNAs in Angiogenesis . . . . . . . . . . . . 6.3.2 Post-Ischemic miRNA Regulation of Angiogenic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Coupling Angiogenesis and Neurogenesis Post-Ischemia . . . . . 6.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .

67 67 68 68 71 73 74 75 76

. . . .

76 79 80 80

. . . .

87 87 89 89

4

7

MicroRNAs as Potential Diagnostic, Prognostic, and Therapeutic Biomarkers in Ischemic Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Important Circulating microRNAs in Ischemic Stroke . . . . . . . 7.2.1 Let 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Contents

8

9

7.2.2 MiRNA-125a-5p/miR-125b-5p/miR-125b-2
. . . . . . . 7.3 Embolic Stroke Versus Thrombotic Stroke . . . . . . . . . . . . . . . 7.4 Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Neurogenesis and Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

Interplay Between microRNAs and Other Cerebrovascular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Hemorrhagic Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Intracerebral Hemorrhage . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1 microRNAs as ICH Biomarkers . . . . . . . . . . . . . . . . 8.3.2 Regulatory Role of microRNAs in ICH . . . . . . . . . . . 8.4 Subarachnoid Hemorrhage . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1 microRNAs as SAH Biomarkers . . . . . . . . . . . . . . . . 8.4.2 Disease Modifying Role of microRNAs in SAH . . . . . 8.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. 95 . 95 . 96 . 96 . 96 . 97 . 99 . 99 . 101 . 101 . 103

New Insights into the Regulatory Role of lncRNA, circRNA, piRNAs, and ceRNAs in Ischemic Stroke . . . . . . . . . . . . . . . . . . . 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.1 Non-coding RNAs at a Glance . . . . . . . . . . . . . . . . . 9.2 New Players in the Game . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1 Long Non-coding RNA . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Circular RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.3 PIWI RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

107 107 107 109 109 110 111 112 112 112

. . . . . . . . .

117 117 118 118 120 120 121 121 122

Part III 10

ix

90 90 90 91 91 92

IschemiRs: The Bench and Beyond

Computational Resources for microRNA Research . . . . . . . . . . . . 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 MiRNA Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 MicroRNA Prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 MicroRNA Disease Interaction . . . . . . . . . . . . . . . . . . . . . . . 10.5 MicroRNA-Target Interaction . . . . . . . . . . . . . . . . . . . . . . . . 10.6 MicroRNA Interaction with Transcription Factors . . . . . . . . . . 10.7 Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

x

11

Contents

MicroRNA-Targeted Therapeutics for Ischemic Stroke: Status, Gaps and the Way Forward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Restoration of miRNA Function as a Potential Ischemic Stroke Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Development of Anti-miRNA-Based Ischemic Stroke Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.1 Antisense Oligonucleotides . . . . . . . . . . . . . . . . . . . . 11.3.2 Chemically Modified Antisense Oligonucleotides/ Antagomirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.3 Locked Nucleic Acid (LNA) . . . . . . . . . . . . . . . . . . . 11.3.4 miRNA Sponge . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Delivery of miRNA Modulators for Ischemic Stroke Therapy . 11.4.1 Viral-Based miRNA Delivery System . . . . . . . . . . . . 11.4.2 Non-Viral miRNA Delivery Systems . . . . . . . . . . . . . 11.4.3 Exosomal Mediated Delivery of miRNAs . . . . . . . . . 11.5 Pharmacological Agents and Small-Molecules as Therapeutic Modulators of miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 Challenges, Perspectives, and Way-Forward . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. 125 . 125 . 126 . 127 . 127 . . . . . . .

128 128 129 130 130 131 133

. 133 . 134 . 135

About the Authors

Rajanikant G. K. is currently working as a Professor at the School of Biotechnology and served as the Coordinator of DBT—Bioinformatics Infrastructure Facility at National Institute of Technology Calicut, Calicut, India. Having done his Ph.D. in Radiation Biology and Toxicology from Division of Radiobiology and Toxicology, Manipal University, Manipal, India, he was also awarded a Senior Research Fellowship from Indian Council of Medical Research, Government of India, New Delhi, for the year 2001–2004. Till date, he has worked as a Principal Investigator in various government-funded research projects and has further guided and supervised several Doctorate and Post-Doctorate students and researchers. He has also successfully applied for an Indian Patent in the year 2017 for using a pharmaceutical composition consisting of 1,4-DI(1H-benzimidazol-2-YL)-1,2,3,4-butanetetrol (BTT) as an ischemic neuroprotectant. He has also been invited as a speaker to various prestigious conferences and has conducted many conferences, workshops, and seminars successfully. He has several articles in renowned national and international journals, as well as book chapters and edited volumes, to his credit. Pierre Gressens is presently holding several prominent positions, including a Research Officer (DR1 Inserm) and Director, UMR 1141 Inserm-Paris University, Robert Debré Hospital, Paris, France; Professor of Fetal and Neonatal Neurology, KCL, London, UK; Consultant, Service of Pediatric Neurology, Robert Debré Hospital, Paris, France; Director, PremUP Foundation, Paris; Coordinator of the University-Hospital Department PROTECT, Robert Debré Hospital, Paris; and Vice-Dean for Research, Paris-Diderot Medical School, Paris, France. He obtained his M.D. and Ph.D. degrees from University of Louvain Medical School at Brussels, Belgium. He has received various honors, such as Roger de Spoelberch award for neurosciences (Switzerland) in 2010, FRM Fondation Guillaumat-Piel award (France) in 2013, and the Elsie Widdowson Lecture at the Neonatal Society, London, UK in 2017. He has been invited as a speaker to various prestigious conferences. He has published his research/review articles in many international journals of great repute. Sreekala S. Nampoothiri is currently a postdoctoral researcher at INSERM 1172, Development and plasticity of the neuroendocrine brain, Lille Neuroscience & xi

xii

About the Authors

Cognition, Université de Lille, Lille, France. She has a PhD in Biotechnology from National Institute of Technology Calicut, Kerala, on the thesis entitled “microRNA9 Upregulation Integrates Postischemic Neuronal Survival and Regeneration: A Systems Biology Approach.” She is a recipient of Raman-Charpak Fellowship 2015 and performed a part of her doctoral thesis research at INSERM 1141, Université Paris Diderot, Hôpital Robert Debré, Paris. She was the University topper for her Masters (M.Tech) program in Biotechnology and Biochemical Engineering (specialization in Molecular Medicine) at Sree Buddha College of Engineering, University of Kerala, Thiruvananthapuram, India, as well as for her Bachelors (B.E.) in Biomedical Engineering, from Anna University, Tamil Nadu, India. She was also selected by Indian Academy of Sciences as Summer Junior Research Fellow (SJRF) in May 2012. She has published articles in journals of good repute and has also presented her research at various conferences and seminars. Gokul Surendran is currently pursuing his PhD. from National Institute of Technology Calicut, Kerala, India. He did his M.S. (Pharm.) Biotechnology from National Institute of Pharmaceutical Education and Research, Hajipur, India. He was also awarded a Fellowship during his M.S. (Pharm.) from Ministry of Chemicals and Fertilizers (Government of India) and has further qualified the National Institute of Pharmaceutical Education and Research (NIPER) Joint Entrance Examination (NIPER-JEE-2014) with All India Rank of 400 and Graduate Pharmacy Aptitude Test (GPAT) Examination (GPAT 2014) with All India Rank of 204. He has also presented his research at various conferences and seminars, and published articles in good journals. Cindy Bokobza holds a Master’s degree from the European Master’s in Genetics (Magistère) Program at the Université de Paris and from the University of Trieste, Italy. She worked as an intern at the Center for Human Disease Modeling (CHDM) at Duke University, USA. She obtained her PhD in Neuroscience from the Université de Paris (Ecole Doctorale BioSPC) for her studies on the impact of perinatal inflammation on neurodevelopment in the team of Pierre Gressens at the Hôpital Robert Debré, Paris. She was a G.E.N.E. Fellow for postdoctoral research, funded by the EUR G.E.N.E. Graduate School (#ANR-17-EURE-0013). She also served as a Student Representative at PhD School Bio Sorbonne Paris Cité.

Abbreviations

20 -OMe AAV AGO2 AMPA aNPCs APAF-1 ARE aSAH ATP AuNPs BBB BCL2 Bcl-2 Bcl2l11 BDNF bFGF BIM Ca2+ CALM1 CaM CaMKIIδ CaMKK CaMKs CAT CEBPb circRNA DLGAP4 CMV CNS CypD DAMP DGCR8 DISC DRG

20 -O-methyl Adeno-associated viruses Argonaute 2 α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Amplifying neural progenitor cells Apoptotic protease-activating factor 1 Antioxidant response element Aneurysmal subarachnoid hemorrhage Adenosine triphosphate Gold nanoparticles Blood–brain barrier B-cell lymphoma 2 B-cell lymphoma 2 BCL2 like 11 Brain-derived neurotrophic factor Basic fibroblast growth factor BCL2 interacting mediator of cell death Calcium ion Calmodulin 1 Calmodulin Calcium/calmodulin-dependent protein kinase II δ Calcium/calmodulin-dependent protein kinase kinase Calcium/calmodulin-dependent protein kinases Catalase CCAAT enhancer binding protein beta circDLGAP4 Cytomegalovirus Central nervous system Cyclophilin D Damage-associated molecular pattern DiGeorge syndrome chromosome region 8 Death-inducing signaling complex Dorsal root ganglion xiii

xiv

Drp-1 EAAT1–5 EC eEF2 EGFL7 EGR2 eNOS ePAM-R EPAR ER FADD FasIII FDA GDNF GLAST/EAAT1 GLT-1/EAAT2 GluR2 GSHPx H2O2 HI HO-1 HSPs I/R IA IA IAT ICAM-1 ICH ICV IGF-1 IL IMS IV JAG1 Keap1 LimK1 LNA lncRNA LSD1 Map1b MAPK/ERK1/2 Mash1 MCA MDA

Abbreviations

Dynamin-related protein-1 Excitatory amino acid transporters 1–5 Endothelial cell Eukaryotic elongation factor-2 kinase Epidermal growth factor-like-domain 7 gene Early growth response 2 Endothelial nitric oxide synthase Polyamidoamine ester Endovascular photoacoustic recanalization Endoplasmic reticulum Fas-associated death domain protein Fasciclin III Food and Drug Administration Glial-derived neurotrophic factor Glutamate-aspartate transporter Glutamate transporter-1 Glutamate receptor-2 Glutathione peroxidase Hydrogen peroxide Hypoxic-ischemic Heme oxygenase-1 Heat shock proteins Ischemia/reperfusion Intra-arterial Intracranial aneurysm Intra-arterial thrombolysis Intercellular adhesion molecule-1 Intracerebral hemorrhage Intracerebroventricular Insulin-like growth factor 1 Interleukin Interventional management of stroke Intravenous Jagged-1 Kelch-like erythroid cell-derived protein LIM domain kinase 1 Locked nucleic acid Long noncoding RNA Lysine demethylase 1A Microtubule-associated protein 1b Mitogen-activated protein kinase/extracellular regulated kinase 1/2 Achaete-scute homolog 1 Middle cerebral artery Methane dicarboxylic aldehyde

Abbreviations

MeCP2 Mib1 miRNA miRNAs miRs MMP MPTP MSC Na+ ncRNAs NCX NeuroD1 NF-kB NGF NIHSS NINDS NMDA NO NOX NPCs Nrf2/NFE2L2 Nrg NSC/NSPC NSCs Numbl O2 OGD OLs OPCs PAMAM PAMAM-Arg PAMP PDGF PEI PI3K/mTOR PLGA pMCAO Pol II PRDXs PS PTA PTEN PUMA qHTS

xv

Methyl CpG binding protein 2 Mind bomb one microRNA microRNAs MicroRNAs Matrix metalloproteinase Mitochondrial permeability transition pore Mesenchymal stem cell Sodium Noncoding RNAs Sodium–calcium exchanger Neuronal differentiation 1 Nuclear factor kappa b Nerve growth factor National Institutes of Health Stroke Scale National Institute of Neurological Disorders and Stroke N-methyl-D-aspartate Nitric oxide Nicotinamide adenine dinucleotide phosphate-oxidase Neural progenitor cells Nuclear factor erythroid 2-related factor 2 Neuroglian Neural stem/progenitor cell Neural stem cells NUMB like endocytic adaptor protein Oxygen Oxygen–glucose deprivation Oligodendrocytes Oligodendrocytes progenitor cells Polyamidoamine PAMAM conjugated with arginine Pathogen-associated molecular pattern molecule Platelet-derived growth factor Polyethylenimine Phosphatidylinositol-3-kinase/mammalian target of rapamycin Polylactic-co-glycolic acid Permanent middle cerebral artery occlusion RNA polymerase II Peroxiredoxins Phosphorothioate Percutaneous transluminal angioplasty Phosphatase and tensin homolog p53 upregulated modulator of apoptosis Quantitative high-throughput screening

xvi

REST RGCs Rheb RISC RNA RNS ROS RTN rt-PA SAH SAINT Sema3A SESs SGZ SHIP-1 siRNA SOCS-1 SODs SOX9 Spred-1 SPRY2 STAIR SUMO SVZ TAp73 TF TGF TJs TLR TLR4 TNF t-PA Usp14 UTR VCAM VEGF VEGFR WHO XPO-5 ZO-1

Abbreviations

RE1 silencing transcription factor Retinal ganglion cells Ras homolog, MTORC1 binding RNA-induced silencing complex Ribonucleic acid Reactive nitrogen species Reactive oxygen species Retrograde transvenous neuroperfusion Recombinant tissue-plasminogen activator Subarachnoid hemorrhage Stroke-acute ischemic NXY treatment Semaphorin 3A Self-expanding stents Subgranular zone Inositol polyphosphate-5-phosphatase D Small interfering RNA Suppressor of cytokine signaling 1 Superoxide dismutases SRY-box transcription factor Sprouty-related EVH1 domain containing 1 Sprouty 2 Stroke Treatment Academic Industry Roundtable Small ubiquitin-like modifier Subventricular zone Tumor protein P73 Tissue factor Transforming growth factor Tight junctions Toll-like receptor Toll-like receptor 4 Tumor necrosis factor Tissue-plasminogen activator Ubiquitin specific peptidase 14 Untranslated Vascular cell adhesion molecule Vascular endothelial growth factor Vascular endothelial growth factor receptor World Health Organization Exportin-5 Tight junction protein

Part I microRNAs and Ischemic Stroke: Back to Basics

1

microRNAs in Normal Brain Physiology

Abstract

MicroRNAs (miRs) are short single-stranded non-coding RNAs that regulate protein synthesis by translational repression or degradation by targeting mRNAs at the post-transcriptional level. Highly conserved across species, they are known to regulate many genes in critical pathways during the lifetime. Any dysregulation at the cellular level, but also in the entire organism, is therefore harmful. The brain is a privileged organ in many ways, and interestingly, the majority of miRs are expressed in the brain. We describe miRs biogenesis in this chapter and focus on the role of cell-specific miRs in normal brain physiology and homeostasis. Keywords

miRNAs · Brain · Biogenesis · Physiology · Neurons oligodendrocytes · Astrocytes · Microglia

1.1

Introduction

In general, gene expression passes through several processes, including transcription of the genomic DNA sequence in mRNA that then translates to a protein. To modulate which gene is expressed and at which level (in terms of quantity and localization), evolutionarily integrated several checkpoints and/or keylocks are required to fine-tune the regulation of gene expression. All of them are mostly grouped in epigenetics, which is the study of gene expression modulation without affecting the nucleotide sequence of the genome. This regroups DNA methylation, histone modifications, and non-coding RNA. A non-coding RNAs (ncRNAs) are RNA molecules that are not translated into a protein. Many of the newly identified ncRNAs have not been validated for their function. Many ncRNAs are also likely to be non-functional. A microRNA # Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_1

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(abbreviated miRNA) is a small non-coding RNA molecule (containing approximately 22 nucleotides) found in plants, animals, and some viruses, that functions in silencing RNA and post-transcriptional regulation of gene expression [1] (Box 1.1). The expression of aberrant miRNAs is implicated in various diseases. miRNAs appear to control the development and function of the nervous system [2]. Box 1.1: miRNAs Discovery • Historically, the first miRNA was described in 1993 in Caenorhabditis elegans (C. elegans). • This non-coding RNA is a sequence of 22 nucleotides that partially base-to-base matched to multiple sequences in the 3’UTR of lin-14 mRNA sequence; this double-strand demonstrated a reduction of LIN-14 protein. • It was only several years after that a second ncRNA was characterized not only in C. elegans, but also in Drosophila and humans. Historically, miRs have been linked to numerous cellular processes, including differentiation, cell proliferation, embryonic development, metabolism, and stress response [3]. Given the fundamental role of miR in several systems, several studies indicate that any aberration in miR biogenesis may lead to the ontogeny of human pathologies such as cancer, diabetes, cardiovascular disease, HIV, obesity, and nervous system disorders [4]. Besides, it is also proposed that miRs be quantifiable biomarkers of disease progression and especially in stroke [5, 6]. The advancement of high-throughput sequencing has allowed an exponential number of miR discovery since 1993. Most of them are grouped in miRBase catalog (version 22, 2019), comprising around 49,000 mature miR sequences from 271 organisms [7]. The sequence of most miRs are conserved, suggesting a preserved role in the regulation of gene expression at the cellular level. In mammals, miRs interfere with 3’-UTR of their target mRNAs by partial complementary, leading to a translation inhibition [8]. Mounting evidence demonstrates that a single mRNA can be targeted by several miRs, whereas a single miR can target several mRNAs. In this chapter, we will describe miRs biogenesis and its role in maintaining normal brain physiology. Primarily, we will describe miRs function in all brain cells (neurons, astrocytes, oligodendrocytes, and microglia) under normal conditions.

1.2

microRNA Biogenesis

MiRs have a unique biogenesis pathway but also a variety of expression patterns that enable their specific functions in various biological processes. We generally consider two pathways for miR biogenesis: (1) the canonical pathway, with RNA polymerase II (Pol II) transcription, leading to the generation of a long transcript (several

1.2 microRNA Biogenesis

5

Nucleus

Genomic DNA E1

Canonical pathway

E2

miRtron pathway E1

Pri-miRNA AAA 3’

5’

E2

DROSHA

Pre-miRNA EXPORTIN

AGO proteins

Pre-miRNA RISC complex

DiCER

GW182 proteins 5’

3’

Ribosome

3’

5’

Fig. 1.1 Schematic representation of mature microRNA biogenesis

kilobases) with a hairpin structure, so-called the pri-miR; and (2) the miRtron pathway, non-canonical pri-miRNAs are encoded in introns of coding genes (miRtrons) [9–11] (Fig. 1.1). Canonical miRNA biogenesis begins with post-transcriptional or co-transcriptional processing of primary miRNA transcripts (pri-miRNAs) from the genome. The canonical pri-miRNAs are transcribed by RNA Polymerase II (POL II), possess a 50 -cap, and do not usually have a poly-A tail. Pri-miRNAs

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microRNAs in Normal Brain Physiology

form a hairpin structure in the nucleus and are cleaved into premature miRNAs (pre-miRNAs) by the RNase III enzyme DROSHA, which is in complex with RNA-binding protein (RBP) DiGeorge syndrome chromosome region 8 (DGCR8). The pre-miRNA is released into the cytoplasm from the nucleus by Exportin-5 (XPO-5) and further processed by the RNase III enzyme DICER, bound to transactivation-responsive RNA-binding protein (TRBP) [12]. TRBP binds to the pre-miRNA and DICER, and cleaves the loop of the hairpin, resulting in a miRNA duplex. The miRNA duplex interacts with components of the RNA-induced silencing complex (RISC) loading complex (RLC). The Argonaute 2 (AGO2) destabilizes the miRNA duplex, which is unwinded by RNA helicases. The miRNA loaded RISC (miRISC) binds to reverse complementary sequences within the 30 -untranslated region (UTR) of target mRNAs. Mirtrons are intronsencoded non-canonical microRNAs, the biogenesis of which begins with splicing. They are not being processed by Drosha and join the canonical pathway at the Exportin-5 level. The critical functional outcome of miRNA activity is a decreased abundance of translated protein from a target mRNA. The guide miRNA leads the RISC complex to a corresponding sequence on the target mRNA, typically located on the 3’UTR, resulting in translational inhibition and mRNA destabilization.

1.3

miRNA in the Brain

Literature reveals that 70% of miR are expressed in the brain; however, only a shortlist of them is enriched in the brain or are brain-specific [13, 14]. In 2013, Jovičić et al. described miR expression in four dcell types in culture, and determine specifically enriched miRs in each [15]. We will explain in the following sections brain cell-specific microRNAs and their functions in brain physiology.

1.3.1

miRNA in Neurons

In the brain, we generally discriminate neurons from other glial cells (astrocytes, oligodendrocytes, and microglia). However, astrocytes and oligodendrocytes are differentiated from the same neural stem cells (NSCs) that neurons are differentiated [9, 16, 17]. During neurogenesis, microRNAs contribute to the cell fate decision of astrocytes versus neural progenitor cells (NPCs) that will become adult neurons. Similar mechanisms occur in adult neurogenesis [17]. Figure 1.2 groups some of the miRs associated with neuronal differentiation, proliferation, axonal outgrowth, and synaptogenesis. miR-9 enhances differentiation of neuronal stem cells, promotes neuronal migration, and inhibits neural proliferation [18–20]. miR-124 also promotes neuronal differentiation by stimulation of neurite branching; it also modulates synaptic activity [21–23]. Let-7 is a primary miR that acts in many levels, such as proliferation, auto-renovation, and differentiation of NSCs [24, 25]. miR-106-25 works with

1.3 miRNA in the Brain

Differenaon/proliferaon • • • • • • • • •

Let-7 miR-9 miR-34 miR-124 miR-134 miR-137 miR-184 miR-17-92 miR-106-25

7

Axonal/dendrite outgrowth • • • • • • •

miR-9 miR-124 miR-132 miR-134 miR-218 miR-430 family miR-17-92 cluster

Synaptogenesis & plascity • • • • • • • •

miR-132 miR-134 miR-135 miR-138 miR-219 miR-284 miR-485 miR-125b

Fig. 1.2 Major functional role miRNAs in neuronal cells

miR-25 to enhance the proliferation of NSCs and NPCs [26]. miR-134 acts during development on differentiation and synaptogenesis, and during adulthood, it regulates synaptic plasticity [27]. miR-137 inhibits proliferation, induce neuronal maturation, regulates differentiation of neural stem cells [28, 29].

1.3.2

miRNA in Oligodendrocytes

Oligodendrocytes (OLs) represent 20–40% of brain cells. OLs are myelin-producing cells that surround neuronal axons for effective saltatory conduction of electrophysiological signals. miRNAs play critical roles in normal cellular physiology and development of OL lineage cells. During development, OLs pass through specific stages of proliferation and differentiation. Recent evidence demonstrates that these stages are fine-tuned and regulated by miRs [30, 31]. During the first stage, NSCs differentiate into oligodendrocytes progenitor cells (OPCs) by inhibition of neurons differentiation promoters via miR-219, miR-145, miR-7a, and miR-19b [32, 33]. OPCs are highly proliferative cells due to their sensitivity to the plateletderived growth factor (PDGF). Data demonstrated that PDGFRα promotes OPCs proliferation and inhibition of their differentiation. In a specific time window of development, OPCs overexpress miR-219, miR-138, miR-297c-5p, and miR-338 that will target PDGFRα, Sox6, Hes5, and other proteins to inhibit proliferation [32, 34–36]. At the same time, a decreased expression of miR-7a and miR-9 allows SRF, PMP22, CNPase, and Sp1 expression, pre-myelinating OLs (pre-OLs) differentiation promoters [33, 37, 38]. The final step of OLs differentiation is myelin production. Pre-OLs increase the expression of miR-138 and miR-23a to inhibit UHRF1 and LaminB1 [39–41]. The overexpression of miR-23a, miR-125-3p, and miR146a activate signaling pathways leading to myelin proteins production [36, 42– 45]. In normal brain physiology, miRs also modulate metabolism and gene expression in OLs, and more specifically, lipid metabolism via the expression of miR-219 and miR-32 [44, 46] (Fig. 1.3).

miR-9 miR-27a miR-138 miR-145 miR-205 miR-214 miR-338 miR-715 miR-125-3p miR-297c-5p

• • • • •

miR-23a miR-138 miR-219 miR-146a miR-125-3p

microRNAs in Normal Brain Physiology

• miR-17-92 OPCs expansion

• • • • • • • • • •

Myelinaon

1

Differentiation/maturation

8

Fig. 1.3 miRNAs involved in oligodendrocyte differentiation, maturation and myelination

1.3.3

miRNA in Astrocytes

Astrocytes represent the largest population of glial cells in the CNS. They are traditionally viewed as structural cells supporting the brain structure in terms of homeostasis by regulation of ion, pH, neurotransmitter, and blood flow. Astrocytes are also essential for proper neuronal functioning during development via their roles in synaptogenesis, and in the adult brain for synaptic transmission and information processing [47]. Moreover, due to the presence of damage-associated molecular pattern (DAMP) and pathogen-associated molecular pattern molecule (PAMP) receptors, astrocytes also contribute to neuroinflammation in the case of infection via the expression of specific markers [48]. As described before, miRs contribute to the cell specification of neurons. Interestingly, miRs that are enriched in neurons are diminished in astrocytes, demonstrating that the regulation of differentiation from one cell type to another at the stem cell level is miRs dependent (Table 1.1).

1.3.4

miRNA in Microglia

The last major component of the brain cell population is microglia. Microglia are CNS-resident macrophages that have essential physiological roles in the brain in addition to their inflammatory activity [62]. They are derived from erythromyeloid precursors from the yolk sac that migrate to the neuroepithelium during early embryonic development [63]. As macrophages, in healthy conditions, they continually scan their environment and participate in synaptic plasticity. However, during stress conditions such as bacterial and/or infection, trauma or ischemia, microglia will activate and adopt a pro-inflammatory phenotype [62, 64, 65]. This phenotype is

1.4 Conclusion

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Table 1.1 Astrocyte specific microRNAs miRNAs miR-124a miR-125b miR-143 miR-145 miR-146a miR-181a miR-21 miR-210 miR-221 miR-223

Targets GLT-1 Fyn, Nrg1, Smad4, RhoA HK2, ADAR1, PUMA, PDGFRA, PRKCE, MAPK7, DSSP, DMP-1, KRAS, and BCL-2 GFAP, AQP-4 IRAK1, TRAF6 MeCP2 PTEN, PDCD4 AKT CX43, ICAM1 SDF-1

References [49, 50] [51] [52] [53] [54, 55] [56] [57, 58] [59] [60] [61]

harmful to the brain, especially during ischemia, and will further be described in Chap. 4. All of these processes and phenotypes have been recently linked to miRs. Some miRs are enriched in microglia and inhibit neuronal phenotypes. Interestingly, miR-124 that is particularly enriched in the brain and has a major function in neurons and astrocytes, is also present in microglia; and seems to be a microglia-miR in comparison to periphery macrophages [65]. Let-7a reduces neurite production and limits pro-inflammatory cytokines production; it also increases the production of neuroprotective molecules [66]. Data suggest that miR-204 and miR-424 may regulate microglial proliferation and apoptosis [67, 68]. Microglia specific miR-155 has also been linked to angiogenesis by targeting CCN1 [69]. Many miRs such as miR-146a/b, miR-155, miR-124, miR-689 have been correlated with the microglia inflammatory polarization [65, 70].

1.4

Conclusion

In many regards, the brain is a privileged organ, with a fine-tune regulation of all processes during the lifetime. One of the actors of this regulation are nc-RNAs, and more specifically miRs, that are post-transcriptionally modulators of gene expression. We have described the four major cell types of the brains and how miRs participate in brain physiology. In an interesting manner, many miRs are present in different cell types, and their functions are cell-context specific during time and place. As an example, miR-146 has been associated with both microglia and oligodendrocytes, respectively, for the regulation of neuroinflammation and myelin production. In light of these observations, we should consider the use of miRs as therapeutic candidates due to their ability to target several pathways in different cell types to protect the brain from internal and external aggressions.

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2

Ischemic Stroke: An Imperative Need for Effective Therapy

Abstract

While it is currently estimated that stroke causes eight million annual deaths worldwide, the death rate only gives a first account of the overall burden of disease. Ischemic stroke is caused by the blockade or occlusion of the cerebral artery compromising blood supply and energy flow to the brain. The likelihood of restoration of blood flow depends on the extent of thrombosis within the cerebral artery. Thrombolysis is currently coupled with endovascular approaches for the removal and dissolution of occlusive thrombus. This chapter provides an overview of the current thrombolytic and endovascular treatment approaches for poststroke care along with the emerging neuroprotective agents that are lost in translation. Keywords

Ischemic stroke · Thrombolysis · Endovascular recanalization · Neuroprotection

2.1

Introduction

Therapeutic skepticism and inadequate treatment opportunities make ischemic stroke the third most common cause of death and disability worldwide [1, 2]. Ischemic stroke induces a broad spectrum of sensory, motor, and cognitive impairments resulting in a massive personal and financial burden on society [2]. The amplitude of brain damage and the outcome depends on the tolerance threshold of the brain region and neuronal population exposed to ischemic insult (Box 2.1). Though thrombolytic therapy with recombinant tissue-plasminogen activator (rt-PA) remains the first and the only Food and Drug Administration (FDA) approved treatment [3], intravenous (IV) rt-PA thrombolysis is no longer the only panacea for acute ischemic stroke. Currently, thrombolytic treatment is coupled with endovascular approaches for the removal and dissolution of occlusive thrombus. Besides, preclinical research # Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_2

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2 Ischemic Stroke: An Imperative Need for Effective Therapy

continues to discover and improve appropriate neuroprotective agents with little progress in clinical trials, primarily due to the overestimation of therapeutic efficacy in rodent models. This chapter describes the current landscape surrounding the thrombolytic and endovascular methods used for ischemic stroke treatment, the role of emerging neuroprotectants, their successes and failures, and recommendations essential for the improvement of the quality of research at both preclinical and translational levels. Box 2.1: Stroke Incidence and Prevalence • Stroke incidence refers to the number of individuals experiencing stroke for the first time. • Stroke incidence varies around the world depending on age, sex, susceptibility to vascular diseases and other risk factors in a population. • Between 2009 and 2016, ischemic stroke incidence is reserved at approximately 795,000 victims in the USA • Stroke incidence is lower in France compared to East European countries • In the developing nations such as China and India, stroke rate is highly precarious with more than one billion population, diverse socio-economic and cultural backgrounds, and varying vascular risk factors. • Stroke prevalence is the total number of individuals who have had a stroke at any time within a population. • 1.5–2 people per 1000 population experience stroke each year. • Overall prevalence rate is estimated to be about 10 per 1000 population, which corresponds to 50 per 1000 for those aged above 65 years.

2.2

Current Treatment Choices for Ischemic Stroke Victims

This section enumerates currently accepted and utilized treatments for ischemic stroke that can be broadly categorized into (1) IV pharmacological thrombolysis and (2) endovascular procedures (thrombectomy or embolectomy) with or without the administration of thrombolytic agents (Table 2.1).

2.2.1

IV Pharmacological Thrombolysis

The first generation thrombolytic agents involve streptokinase, a protein secreted by streptococci, and urokinase, a human serine protease [4]. Their use in stroke thrombolysis has been hampered by the high risk of intracerebral hemorrhage (ICH), non-specificity to fibrin clots, low clinical efficiency, and allergic reactions, particularly with streptokinase [5]. The second-generation recombinant tissue-plasminogen activator thrombolytic therapy is based on the adsorption of endogenous tissueplasminogen activator (t-PA, a serine protease enzyme in the plasma) to the surface

2.2 Current Treatment Choices for Ischemic Stroke Victims

17

Table 2.1 Reperfusion strategies to restore blood flow following acute ischemic stroke Endovascular recanalization strategies

Pharmacological IV and/or IA thrombolysis

Endovascular thrombectomy Endovascular thromboaspiration Mechanical thrombus disruption

Augmented fibrinolysis Thrombus entrapment

Other reperfusion approaches

Temporary endovascular bypass Global reperfusion or trans-arterial retrograde reperfusion Flow reversal or transvenous retrograde reperfusion

Thrombolytic agents: Plasminogen activators, direct fibrinolytics, fibrinogenolytic agents Adjunctive therapy: Heparin, direct thrombin inhibitors, GP IIb/IIIa antagonists MERCI Retrievera, catch device, Phenox Clot Retriever, Attracter-18 Alligator, in-time retriever Possis AngioJet system, NeuroJet, penumbra stroke systema Micro-guidewire, snares, percutaneous transluminal angioplasty, endovascular photoacoustic recanalization (EPAR), and LaTIS laser device EKOS MicroLys US infusion catheter and transcranial Doppler ultrasonography Self-expanding and balloon-expandable stents Resheathable (closed-cell) stents, solitaire, Trevo stentrievers Pharmacological treatment (IV administration of phenylephrine infusion) and mechanical methods (NeuroFlow device) Retrograde transvenous neuroperfusion (RTN) for partial venous flow reversal. ReviveFlow system for complete arteriovenous flow reversal

a

The MERCI Retriever and Penumbra stroke system have been FDA approved for the removal of clots obstructing the brain artery in ischemic stroke patients

of fibrin clots that converts plasminogen into plasmin for the lysis of clots occluding the cerebral blood vessel [3]. In contrast with the first generation thrombolytic drugs, rt-PA is fibrin-specific and remains relatively inactive in the absence of fibrin. Other advantages include less allergic reactions, the short plasma half-life of 4–5 min, and rapid clearance from the circulating blood [6]. In 1995, the National Institute of Neurological Disorders and Stroke (NINDS) conducted a clinical trial of IV administration of rt-PA. The investigators found that the administration of rt-PA within three-hour stroke onset increased the prospects of a favorable outcome [7]. Later in 1996, the FDA approved the use of second-generation thrombolytic, rt-PA (Alteplase™) for stroke treatment, which became a broadly accepted standard of ischemic stroke care [8]. Despite being the only FDA approved treatment for ischemic stroke, the daunting side effects and other pitfalls revolving around the strict treatment protocol have precluded the extensive clinical use of rt-PA. One of the most severe consequences includes the symptomatic and fatal hemorrhagic transformation of the infarcted cerebral area and reperfusion damage. The routine use of IV thrombolysis is also

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2 Ischemic Stroke: An Imperative Need for Effective Therapy

limited by the narrow therapeutic window as the brain damage progressively increases from the time of stroke onset. There is also a considerable concern regarding the potential occurrence of arterial reocclusion because of its short halflife and limited penetration into the clot matrix. Strict adherence to the guidelines for IV thrombolysis lowers the rate of symptomatic hemorrhage, but at the cost of excluding a majority of patients who could benefit from acute stroke therapy [9]. Given the overall low rates of clinical usage and therapeutic response of IV rt-PA, endovascular treatment strategies have been devised for the effective removal of thrombus that has expanded the number of patients treated within a targeted population.

2.2.2

Endovascular Recanalization Strategies

Recanalization refers to the removal and dissolution of the occlusive thrombus to restore antegrade flow. Based on the primary mechanism of action, the recanalization strategies may be categorized into (1) pharmacological intra-arterial thrombolysis (IAT) and (2) intra-arterial (IA) mechanical thrombolysis, including endovascular thrombectomy, thromboaspiration, mechanical thrombus disruption, augmented fibrinolysis, thrombus entrapment, and temporary endovascular bypass (Box 2.2).

2.2.2.1 Pharmacological IAT in Ischemic Stroke IAT uses coaxial microcatheters for the infusion of a fibrinolytic agent specifically into the thrombus in the intracranial vessel [10]. In contrast to the systemic infusion, IAT requires a low dose of a fibrinolytic agent to reach a higher concentration at the localized region and allows better recanalization, while reducing the intracerebral hemorrhagic (ICH) complications. Ideally, the treatment window for IAT can be drawn-out beyond the typical window of 3 h for IV thrombolysis [10, 11]. Although IAT has several theoretical advantages over IV thrombolysis, it is a relatively complicated procedure with many limitations, including delays in the initiation of the treatment, requirement of high-level technical expertise, and risks and expense of an invasive procedure. Box 2.2: Types of Stroke • All ischemic strokes are caused by disruption of blood supply to the brain. • However, ischemic strokes can be initiated in different parts of the body and may be initiated by various kinds of blockages such as an embolic or a thrombotic stroke. • An embolic stroke occurs when an embolus (which could be a blood clot, plaque sections, or other debris) formed elsewhere in the body traverses to the blood vessels in the brain. • A thrombotic stroke is the result of thrombus or clot formed in the blood vessels within the brain.

2.2 Current Treatment Choices for Ischemic Stroke Victims

19

2.2.2.2 IA Mechanical Thrombolysis IA mechanical thrombolysis for the treatment of ischemic stroke has gained attention over pharmacological thrombolysis and can be employed as a primary or adjunctive strategy for four main reasons. First, IA mechanical thrombolysis minimizes or eliminates the need for pharmacological thrombolytics. Second, it has strong prospects for the extension of the therapeutic window beyond 6 h. Third, mechanical fragmentation of clot provides a larger surface area for the action of fibrinolytic agents, enabling rapid thrombolysis with the inflow of fresh plasminogen. Finally, clot retrieval devices may offer faster recanalization and may be more efficient at managing resistance to enzymatic degradation, while dealing with mature embolic clots/emboli formed from calcium, cholesterol, or other debris from atherosclerotic lesions [12, 13]. IA mechanical approaches with little or no fibrinolytic agent have been prevalent as a vital choice of treatment for stroke patients who either have a contraindication to pharmacological thrombolysis or present late after the onset of stroke. In addition, adjunctive endovascular therapy may be crucial to achieving successful thrombolysis via recanalization of a proximal occlusive lesion. However, the shortcomings of IA mechanical thrombolysis include (1) the challenge of navigating mechanical devices into the intracranial circulation, (2) massive trauma to the vasculature leading to vasospasm, perforation, vessel dissection, or rupture, and (3) distal embolization of fragmented thrombus in previously unaffected areas [14]. Despite these challenges, the benefits of IA mechanical thrombolysis significantly outweigh its drawbacks and risks. Endovascular Thrombectomy Endovascular thrombectomy or thrombus retrieval technique restores rapid blood flow with a potentially lower possibility of clot fragmentation and distal embolism compared to other endovascular approaches. The devices vary based on the place of application of force on the thrombus, which could take either a proximal approach with aspiration or “grasper” devices or a distal approach with basket-like or snarelike devices. The technique has been tested in a swine stroke model, where the proximal device allowed fast repeated application with a minor risk of thromboembolic events and vasospasm but conferred low success in thrombus retrieval compared to the distal device. The rate of embolic events with the distal device could be counterbalanced with the use of proximal balloon occlusion [15]. The MERCI Retriever (Concentric Medical, Inc., Mountain View, CA) and The Catch device (Balt Extrusion, Montmorency, France) have been used for thrombectomy with promising results. The MERCI Retriever has a flexible nitinol wire with coil loops, which is used along with a microcatheter and a French balloon guide catheter of 8–9 in. in size. This system was approved by the US FDA for intracranial clot retrieval in individuals who underwent an acute ischemic stroke [16, 17]. To date, more than 9000 patients have been treated using this device. The Catch device (Balt Extrusion, Montmorency, France) is also used for thrombectomy and has a self-expanding distally closed nitinol cage design [18]. When comparing the MERCI and the Catch devices in vivo, it was found that the MERCI Retriever

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2 Ischemic Stroke: An Imperative Need for Effective Therapy

performed better, with higher rates and chances of recanalization at the first attempt, and a lower chance of thrombus fragmentation [19]. The Phenox Clot Retriever (pCR, Phenox, Bochum, Germany) has an extremely flexible nitinol or platinum alloy compound core wire bordered by a dense palisade of perpendicularly oriented stiff polyamide microfilaments that are trimmed in a conical shape, with increasing distal diameter. The device is molded to the body of a 0.010-in. micro-guidewire (3–1 mm proximally and 5–2 mm distally). It is inserted into the target vessel via a 0.021- or 0.027-in. microcatheter, which is positioned distally to the thrombus, and gradually pulled back under constant aspiration through the guiding catheter. In October 2006, this device was made available for the treatment of acute ischemic stroke patients in Europe [20]. A few other endovascular recanalization devices include the Attracter-18 device (Target Therapeutics, Freemont, CA), The Alligator Retrieval Device (ARD, ev3 Neurovascular, Irvine, CA), and In-Time Retriever (Boston Scientific, Natick, MA). The Attracter-18 device has been effective in the recanalization of an obstructed superior division branch of the left middle cerebral artery (MCA), which was otherwise intractable to IAT [21]. The Alligator Retrieval Device with four small grasping jaws attached to the tip of the flexible wire and juxtaposed with a 0.21-in. microcatheter [22]. It was used to treat six patients with mainly MCA intracranial clots, where two out of six patients previously underwent treatment failure with another clot retrieval device, and three patients did not need systemic thrombolytics [23]. The In-Time Retriever with 4–6 wire loops was efficiently used to remove basilar occlusion as well as MCA occlusion previously resilient to thrombolytics and balloon angioplasty [24, 25]. An in vitro pulsatile flow model compared the performance of five thrombectomy systems. While the MERCI, Catch, and Phenox Clot Retrievers mobilize and remove thrombi equally, the In-Time and Attracter devices provide only partial thrombus removal with extensive effort during initial thrombus penetration and deployment of the device. Nevertheless, all devices generate micro- and macro-fragments during thrombus penetration and retrieval. Among other thrombectomy devices, the Phenox retriever captures most of the clot fragments and delivers superior performance in terms of foiling distal embolization [26]. The study, however, mainly focused on the interaction between the thrombi and the devices and marginalized several other factors relevant to mechanical thrombectomy in humans (such as the interactions between the retriever devices and the vessel wall/the thrombus and the vessel wall, and the occurrence of endogenous thrombolysis and vasospasm). Endovascular Thromboaspiration Thromboaspiration or suction thrombectomy with either a guiding catheter or a microcatheter can be used for a fresh non-adhesive clot [27–29]. Though aspiration devices induce relatively less vasospasm and embolic events, their use is often limited by the difficulty of navigating through intracranial circulation due to the intricate design. The Possis AngioJet system (Possis Medical Inc., Minneapolis, MN) uses high-pressure saline jets to produce a distal Venturi suction that gently agitates the clot face and sucks the generated clot fragments into the access catheter.

2.2 Current Treatment Choices for Ischemic Stroke Victims

21

Three acute stroke patients with ICA occlusion were successfully treated using a five French Possis AngioJet catheter [30]. The NeuroJet (Possis Medical Inc.) is a small, single-channel device explicitly designed for its use in the intracranial circulation. However, the trial was discontinued due to the complications with vessel dissection and difficulty in navigating through the carotid siphon, as observed in a pilot study for acute ischemic stroke. The risk of hemolysis also limits the usage of these devices for a longer duration [31, 32]. The Penumbra stroke system (Penumbra Inc., Alameda, CA) offers two different revascularization techniques: (1) debulking of thrombus and aspiration of the clot using reperfusion catheter, while a separator device facilitates clot fragmentation and prevents catheter blockade or (2) direct extraction of the thrombus by a ring retriever, while balloon guide catheter temporarily arrests the flow. This system was first tested in a pilot trial in Europe involving 23 subjects enrolled with 21 vessel occlusions, where three patients were excluded from the trial due to vessel tortuosity. Only 20 patients were treated at a time-window of 8 h following the onset of symptoms. Recanalization before IAT was accomplished in all treated cases. Six out of 20 patients were non-compliant to IV thrombolytic therapy, whereas nine patients were responsive to post-device IA rt-PA. At 30 days, 45% of the patients demonstrated a good outcome or National Institutes of Health Stroke Scale (NIHSS) four-point improvement. It was also detected that adjunctive IA thrombolytic therapy was linked with a higher incidence of hemorrhage [33]. In January 2008, another prospective, single-arm, multicenter Penumbra Stroke Trial carried out in the USA and Europe routed FDA approval of the thrombus aspiration device to remove clots in acute stroke patients [34].

Mechanical Thrombus Disruption Probing the thrombus with a micro-guidewire or passing a snare (e.g., Amplatz Goose-Neck Microsnare, Microvena, White Bear Lake, MN) through the occlusion multiple times forms the standard methods for mechanical thrombus disruption [25, 35]. A snare may also be used for clot retrieval when the clot is firm or solid [36]. Many studies have demonstrated the efficiency and feasibility of percutaneous transluminal angioplasty (PTA) as one of the techniques for mechanical thrombus disruption following acute stroke [37–40]. PTA is particularly useful in atherothrombotic disease, where residual stenosis sufficiently reduces the blood flow leading to re-thrombosis [35]. Owing to the risks associated with the procedure, this technique is generally reserved as a salvage treatment for patients, where the flow cannot be restored by other conservative methods [37, 41]. The EPAR (Endovascular Photoacoustic Recanalization; Endovasix Inc., Belmont, CA) and the LaTIS laser device (LaTIS, Minneapolis, MN) use different laser technologies to disrupt intracranial clots. The EPAR converts photonic energy from the laser to acoustic energy at the fiberoptic tip for the mechanical fragmentation of the thrombus [42]. On the other hand, the LaTIS laser device generates a “light pipe” with the slow injection of contrast material to carry the energy from the

22

2 Ischemic Stroke: An Imperative Need for Effective Therapy

catheter to the embolus [31, 43]. These devices have been assessed in small safety and feasibility trials; however, their efficacy trials have not been performed. More recently, a randomized trial of thrombectomy was performed in patients after 6 h of stroke onset. The authors established that thrombectomy was more efficient compared to standard medical care alone, especially between 6 and 24 h of stroke symptom onset in patients who presented stroke deficits greater than the infarct volume. The latest acute stroke guidelines also recommend mechanical thrombectomy in selected patients with large vessel occlusion acute ischemic stroke within 6–16 h of last known normal who have large vessel occlusion and meet DAWN or DEFUSE three eligibility criteria. Mechanical thrombectomy is listed as a reasonable method to treat acute ischemic stroke in selected patients within 6–24 h of onset of stroke symptoms [43]. Augmented Fibrinolysis The EKOS MicroLys US infusion catheter (EKOS Corporation, Bothell, WA) was designed to generate a microenvironment of ultrasonic vibration using a microcatheter with a piezoelectric ultrasound element (2-mm, 2.1-MHz) at its distal tip facilitating thrombolysis. This occurs with a combination of non-cavitating ultrasound that reversibly separates fibrin strands as well as acoustic streaming that elevates fluid permeation leading to increased drug-thrombus surface interaction [44]. This technique enhances clot dissolution without the fragmentation of emboli, while its utility has been tested in Interventional Management of Stroke (IMS) II trial with further investigations in the current randomized IMS III trial [45, 46]. Similarly, the CLOTBUST trial demonstrated that fibrinolysis with IV rt-PA could be enhanced with the use of continuous 2-MHz transcranial Doppler ultrasonography [47]. Thrombus Entrapment Using Stents Stent, a small wire mesh tube, is placed in an acutely occluded intracranial vessel for faster recanalization via the thrombus entrapment between the stent and the vessel wall. This may be ensured by the dissolution of thrombus by endogenous and/or pharmacological thrombolysis. Generally, self-expanding stents (SESs) are advantageous over expandable balloon stents as SESs are more flexible and relatively easier to navigate within the intracranial circulation. Besides, SES has been shown to provide higher rates of recanalization and lower degrees of vasospasm as opposed to balloon-mounted stents in a canine model with acutely occluded vessels [48, 49]. There are five intracranial SES currently available (1) Neuroform stent (Boston Scientific, Natick, MA), (2) Leo stent (Balt Extrusion, Montmorency, France), (3) Enterprise stent (Cordis Neurovascular Inc., Miami Lakes, FL), (4) Wingspan stent (Boston Scientific Corp., Natick, MA), (5) Solitaire stent (ev3 Neurovascular, Irvine, CA). Wingspan stent may be used for the treatment of intracranial atherosclerosis, while other devices find application in stent-assisted coil embolization of wide-necked aneurysms [50–52]. These devices vary based on their design, where Wingspan and the Neuroform stents have an open-cell design, while the Enterprise, the Leo, and the Solitaire stents have a closed-cell design. The

2.3 Other Reperfusion Strategies

23

closed-cell design of Enterprise and Leo facilitates the resheathing of the stent after partial or even full placement in the case of Solitaire [52–54]. Despite the successful recanalization and resheathing of the stent in closed-cell design, the use of stents in patients has been associated with severe complications such as acute in-stent thrombosis and fatal ICH. However, Enterprise stent has been shown to aid in immediate recanalization of an occluded vessel insensitive to other conventional thrombolytic procedures such as IV and IA administration of rt-PA, IA abciximab, and mechanical thrombus disruption. The free end of the stent expands and operates as a temporary bypass to displace and disrupt the clot with the administration of additional abciximab through the guiding catheter. After 20 min, the partially expanded stent is constrained and removed. The technique can be applied to other stent-like devices, which then also function as clot retrievers and have been termed stentrievers [55]. The Solitaire FR (ev3 Neurovascular) and the Trevo (Concentric Medical, Inc.) devices are commercially available stentrievers in Europe. Preliminary clinical data indicates that these stentrievers may result in higher recanalization rates than earlier devices. The utility of these devices has been tested in swine stroke models and the histological evaluation revealed that Solitaire device parallels well with the MERCI Retriever, whereas Trevo treated arteries result in severe disruption of the intima [56, 57]. Despite the popularity of post-stroke recanalization, it is often associated with immediate reocclusion of the artery due to the presence of either residual emboli in the event of partial recanalization, or an underlying atheromatic lesion. In addition, reperfusion failure leads to the slow-down of blood flow within the ischemic territory, thereby contributing to reocclusion (no-reflow phenomenon) [58, 59]. Therefore, a significant understanding of the mechanisms of recanalization and reocclusion is fundamental to set the principles for endovascular treatment of ischemic stroke.

2.3

Other Reperfusion Strategies

The reperfusion strategies also include non-recanalization modalities that are categorized into global reperfusion (flow augmentation or trans-arterial retrograde reperfusion) and transvenous retrograde reperfusion (flow reversal) based on the flow manipulation and the involvement of collateral vasculature. Global reperfusion augments the cerebral blood flow via leptomeningeal and/or Willisian collaterals, which perfuse the tissue regions distal to the occluding thrombus. Flow augmentation can be attained by both pharmacological treatment (IV administration of phenylephrine infusion) and mechanical methods (NeuroFlow device) [60, 61]. Transvenous retrograde reperfusion or flow reversal treatment modality reverses the normal direction of cerebral flow from arteries-capillaries-veins to veins-capillaries-arteries. Retrograde transvenous neuroperfusion (RTN) was first used for partial venous flow reversal. It comprised of an external pump to reap arterial blood from the patient’s femoral artery and to carry the blood via two catheters positioned into the transverse sinuses near the torcula. However, larger

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2 Ischemic Stroke: An Imperative Need for Effective Therapy

clinical trials using RTN have not been reported [62]. The ReviveFlow system (ReviveFlow, Inc., Quincy, MA) is a complete arteriovenous cerebral flow reversal method in which a balloon guide catheter is positioned in the cervical internal carotid arteries and jugular veins on one or both sides of the neck. With the inflation of balloons, the blood is aspirated from the proximal ICA into the distal jugular vein through an external pump system. The device is presently undergoing preclinical studies [63].

2.4

Post-Ischemic Neuroprotective Agents

Both endovascular procedures and thrombolysis effectively target the thrombus blocking the cerebral artery, but neither of these therapies essentially targets a specific mechanism directly involved in the ischemic cascade, a multifaceted series of temporally regulated events leading to differential death of cellular populations in the brain. The term neuroprotection has been in use for more than five decades, and barbiturate drugs were among the first pharmacological neuroprotective agents found to inhibit neuronal death [64]. Currently, “neuroprotection” is synonymous with the term “cytoprotection” because the entire neurovascular unit containing neurons, glial cells, and vascular connectivity must be protected following an episode of ischemic stroke [65]. Hence, post-ischemic neuroprotection refers to the implementation of therapies that alleviate brain injury via direct actions on the stroke pathophysiology rather than the pharmacological thrombolysis or recanalization for the improvement of cerebral blood flow. Over the course of last 20 years, more than 1000 neuroprotective agents have been tested in experimental stroke models that directly or indirectly target the molecular events associated with ischemic stroke such as energy failure, excitotoxicity, excessive free radical generation, inflammation, and neuronal apoptosis (each event has been discussed in detail in Chapter-3). An exhaustive list of compounds have been tested preclinically based on their neuroprotection targets, which can be antioxidants or free radical scavengers, NO inhibitors, GABA agonists, glutamate antagonists, calcium and sodium channel blockers, potassium channel openers, calcium chelators, opioid antagonists, phosphatidylcholine precursors, leukocyte adhesion inhibitors, growth factors, and serotonin agonists [66]. A few clinical trials such as Stroke-Acute Ischemic NXY Treatment (SAINT) I–II trials to test a free radical scavenger NXY-059 [67], Radicut (Edaravone; no organized randomized double-blind trial) [68], Transcranial laser therapy (NEST 1–3 trials) [69], Albumin (ALIAS trials) [70], Magnesium (FAST-mag trials) [71] were expected to yield positive outcome. Despite promising results in preclinical studies, many neuroprotective agents failed at different phases of clinical trials for not being safe and efficient. The plausible reasons for these “translational roadblocks” include (1) underestimation of the complexity of ischemic pathophysiology, (2) poor quality of preclinical studies with results that have low internal validity, (3) experimental stroke animal models do not match the patient characteristics such as age, sex, and comorbidities, (4) undesirable publication bias

References

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mainly in preclinical research, (5) heterogeneity of ischemic stroke patients, where the therapies do not match the individual pathophysiology, (6) clinically irrelevant timing of therapies that do not correspond to preclinical findings, and finally (7) species-specific differences with respect to preclinical rodent models and clinical trials in humans. In an attempt to make preclinical results perceptive for clinical trials, the Stroke Treatment Academic Industry Roundtable (STAIR) criteria offered specific guidelines that emphasize the careful design of the clinical trial analogous to animal experiments [72]. SAINT trials to test NXY 059 were reported to have satisfied the STAIR criteria, yet failed to show beneficial effect during SAINT II trial. The subsequent analysis of SAINT trials revealed that STAIR criteria were not met during preclinical studies. As a result, recent STAIR recommendations highlight the equal importance of the quality of preclinical studies [73]. Therefore, the development of novel neuroprotective therapies is inextricably related to the regulation of translational research practices, transparency of data, and strict adherence to guidelines. International multicenter randomized animal trials may also aid in overcoming the discrepancies in preclinical experimental studies [74].

2.5

Conclusion

Despite years of meticulous research, the paucity of efficient stroke treatment highlights the necessity to delineate the disease mechanism further as well as develop clinically viable therapeutic interventions. However, the path to successful stroke therapy remains elusive due to the complexity and plasticity of the neuronal network in the brain, where the functional specialization of neurons and glia hinge on highly organized and coordinated gene expression. Numerous studies have already demonstrated how ischemic stroke alters the expression of genes that function as pro-survival or pro-apoptotic mediators in different brain regions and cell types. More recently, it has been estimated that around 30% of these proteincoding genes are regulated by non-coding microRNAs (miRNAs) playing a significant role in the pathophysiology of neurological disorders, including ischemic stroke. In this setting, there has been a rising interest in deciphering the role miRNAs in response to acute ischemic stress, how they can be modulated to enhance the neurological outcome, and the significance of the development of miRNA-based therapeutic interventions.

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25. Bergui M, Stura G, Daniele D, Cerrato P, Berardino M, Bradac GB (2006) Mechanical thrombolysis in ischemic stroke attributable to basilar artery occlusion as first-line treatment. Stroke 37:145–150 26. Liebig T, Reinartz J, Hannes R, Miloslavski E, Henkes H (2008) Comparative in vitro study of five mechanical embolectomy systems: effectiveness of clot removal and risk of distal embolization. Neuroradiology 50:43–52 27. Chapot R, Houdart E, Rogopoulos A, Mounayer C, Saint-Maurice JP, Merland JJ (2002) Thromboaspiration in the basilar artery: report of two cases. AJNR Am J Neuroradiol 23:282–284 28. Brekenfeld C, Remonda L, Nedeltchev K, von Bredow F, Ozdoba C, Wiest R et al (2005) Endovascular neuroradiological treatment of acute ischemic stroke: techniques and results in 350 patients. Neurol Res 27:S29–S35 29. Lutsep HL, Clark WM, Nesbit GM, Kuether TA, Barnwell SL (2002) Intraarterial suction thrombectomy in acute stroke. AJNR Am J Neuroradiol 23:783–786 30. Bellon RJ, Putman CM, Budzik RF, Pergolizzi RS, Reinking GF, Norbash AM (2001) Rheolytic thrombectomy of the occluded internal carotid artery in the setting of acute ischemic stroke. AJNR Am J Neuroradiol 22:526–530 31. Nesbit GM, Luh G, Tien R, Barnwell SL (2004) New and future endovascular treatment strategies for acute ischemic stroke. J Vasc Interv Radiol 15:S103–S110 32. Molina CA, Saver JL (2005) Extending reperfusion therapy for acute ischemic stroke: emerging pharmacological, mechanical, and imaging strategies. Stroke 36:2311–2320 33. Bose A, Henkes H, Alfke K, Reith W, Mayer TE, Berlis A, Branca V, Sit SP, Penumbra Phase 1 Stroke Trial Investigators (2008) The penumbra system: a mechanical device for the treatment of acute stroke due to thromboembolism. AJNR Am J Neuroradiol 29:1409–1413 34. Penumbra Pivotal Stroke Trial Investigators (2009) The penumbra pivotal stroke trial: safety and effectiveness of a new generation of mechanical devices for clot removal in intracranial large vessel occlusive disease. Stroke 40:2761–2768 35. Qureshi AI, Siddiqui AM, Suri MF, Kim SH, Ali Z, Yahia AM et al (2002) Aggressive mechanical clot disruption and low-dose intra-arterial third-generation thrombolytic agent for ischemic stroke: a prospective study. Neurosurgery 51:1319–27; discussion 1327–9 36. Kerber CW, Barr JD, Berger RM, Chopko BW (2002) Snare retrieval of intracranial thrombus in patients with acute stroke. J Vasc Interv Radiol 13:1269–1274 37. Mangiafico S, Cellerini M, Nencini P, Gensini G, Inzitari D (2005) Intravenous glycoprotein IIb/IIIa inhibitor (tirofiban) followed by intraarterial urokinase and mechanical thrombolysis in stroke. AJNR Am J Neuroradiol 26:2595–2601 38. Ueda T, Sakaki S, Nochide I, Kumon Y, Kohno K, Ohta S (1998) Angioplasty after intraarterial thrombolysis for acute occlusion of intracranial arteries. Stroke 29:2568–2574 39. Nakano S, Iseda T, Yoneyama T, Kawano H, Wakisaka S (2002) Direct percutaneous transluminal angioplasty for acute middle cerebral artery trunk occlusion: an alternative option to intra-arterial thrombolysis. Stroke 33:2872–2876 40. Abou-Chebl A, Bajzer CT, Krieger DW, Furlan AJ, Yadav JS (2005) Multimodal therapy for the treatment of severe ischemic stroke combining GPIIb/IIIa antagonists and angioplasty after failure of thrombolysis. Stroke 36:2286–2288 41. Nogueira RG, Schwamm LH, Buonanno FS, Koroshetz WJ, Yoo AJ, Rabinov JD et al (2008) Low pressure balloon angioplasty with adjuvant pharmacological therapy in patients with acute ischemic stroke caused by intracranial arterial occlusions. Neuroradiology 50:331–340 42. Berlis A, Lutsep H, Barnwell S, Norbash A, Wechsler L, Jungreis CA et al (2004) Mechanical thrombolysis in acute ischemic stroke with endovascular photoacoustic recanalization. Stroke 35:1112–1116 43. Lutsep H. Mechanical thrombolysis in acute stroke. Emedicine. Updated: July 19, 2018 44. Mahon BR, Nesbit GM, Barnwell SL, Clark W, Marotta TR, Weill A et al (2003) North American clinical experience with the EKOS MicroLysUS infusion catheter for the treatment of embolic stroke. AJNR Am J Neuroradiol 24:534–538

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45. IMS II Trial Investigators (2007) The interventional management of stroke (IMS) II study. Stroke 38:2127–2135 46. Khatri P, Hill MD, Palesch YY, Spilker J, Jauch EC, Carrozzella JA et al (2008) Methodology of the interventional management of stroke III trial. Int J Stroke 3:130–137 47. Alexandrov AV, Molina CA, Grotta JC, Garami Z, Ford SR, Alvarez-Sabin J et al (2004) Ultrasound enhanced systemic thrombolysis for acute ischemic stroke. N Engl J Med 351:2170–2178 48. Levy EI, Ecker RD, Horowitz MB, Gupta R, Hanel RA (2006) Stent-assisted intracranial recanalization for acute stroke: early results. Neurosurgery 58:458–63; discussion 458–63 49. Levy EI, Sauvageau E, Hanel RA, Parikh R, Hopkins LN (2006) Self-expanding versus balloon-mounted stents for vessel recanalization following embolic occlusion in the canine model: technical feasibility study. AJNR Am J Neuroradiol 27:2069–2072 50. Akpek S, Arat A, Morsi H, Klucznick RP, Strother CM, Mawad ME (2005) Self-expandable stent-assisted coiling of wide-necked intracranial aneurysms: a single-center experience. AJNR Am J Neuroradiol 26:1223–1231 51. Yu SC, Leung TW, Lee KT, Wong LK (2013) Angioplasty and stenting of intracranial atherosclerosis with the Wingspan system: 1-year clinical and radiological outcome in a single Asian center. J Neurointerv Surg 6:96–102 52. Lubicz B, Leclerc X, Levivier M, Brotchi J, Pruvo JP, Lejeune JP et al (2006) Retractable selfexpandable stent for endovascular treatment of wide-necked intracranial aneurysms: preliminary experience. Neurosurgery 58:451–7; discussion 451–7 53. Peluso JP, van Rooij WJ, Sluzewski M, Beute GN (2008) A new self-expandable nitinol stent for the treatment of wide-neck aneurysms: initial clinical experience. AJNR Am J Neuroradiol 29:1405–1408 54. Yavuz K, Geyik S, Pamuk AG, Koc O, Saatci I, Cekirge HS (2007) Immediate and midterm follow-up results of using an electrodetachable, fully retrievable SOLO stent system in the endovascular coil occlusion of wide- necked cerebral aneurysms. J Neurosurg 107:49–55 55. Kelly ME, Furlan AJ, Fiorella D (2008) Recanalization of an acute middle cerebral artery occlusion using a self-expanding, reconstrainable, intracranial microstent as a temporary endovascular bypass. Stroke 39:1770–1773 56. Jahan R (2009) A novel, self-expanding, fully retrievable flow restoration device for treatment of acute ischemic stroke. Stroke 40:146 57. Nogueira RG, Levy EI, Gounis M, Siddiqui AH (2012) The Trevo device: preclinical data of a novel stroke thrombectomy device in two different animal models of arterial thrombo-occlusive disease. J Neurointerv Surg 4:295–300 58. del Zoppo GJ, Mabuchi T (2003) Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab 23:879–894 59. Adhami F, Liao G, Morozov YM, Schloemer A, Schmithorst VJ, Lorenz JN, Dunn RS, Vorhees CV, Wills-Karp M, Degen JL, Davis RJ, Mizushima N, Rakic P, Dardzinski BJ, Holland SK, Sharp FR, Kuan CY (2006) Cerebral ischemia-hypoxia induces intravascular coagulation and autophagy. Am J Pathol 169:566–583 60. Mistri AK, Robinson TG, Potter JF (2006) Pressor therapy in acute ischemic stroke: systematic review. Stroke 37:1565–1571 61. Lylyk P, Vila JF, Miranda C, Ferrario A, Romero R, Cohen JE (2005) Partial aortic obstruction improves cerebral perfusion and clinical symptoms in patients with symptomatic vasospasm. Neurol Res 27:S129–S135 62. Frazee JG, Luo X, Luan G, Hinton DS, Hovda DA, Shiroishi MS et al (1998) Retrograde transvenous neuroperfusion: a back door treatment for stroke. Stroke 29:1912–1916 63. Nogueira RG, Schwamm LH, Hirsch JA (2009) Endovascular approaches to acute stroke, part 1: drugs, devices, and data. AJNR Am J Neuroradiol 30:649–661 64. Hall R, Murdoch J (1990) Brain protection: physiological and pharmacological considerations. Part II: the pharmacology of brain protection. Can J Anaesth 37:762–777

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Part II microRNAs in Ischemic Stroke Pathophysiology: A Bird’s Eye View

3

MicroRNAs in Ischemic Stroke Pathophysiology: Special Emphasis on Early Molecular Events

Abstract

It is not surprising, given the complexity of the central nervous system, that a vast number of unique brain-enriched microRNAs (miRNAs) are expressed differentially after an ischemic stroke. MicroRNAs control multifaceted pathophysiological events such as excitotoxicity, calcium overload, and oxidative stress within minutes of an inflammatory response and neuronal apoptosis within hours and days of the onset of the stroke. This chapter brings together the functional roles of various miRNAs reported to regulate post-ischemic events, particularly in the acute phase. Keywords

microRNA · Acute ischemic stroke · Excitotoxicity · Calcium overload · Mitochondria · Oxidative stress

3.1

Introduction

Ischemic stroke orchestrates a myriad of interrelated pathophysiological events such as neuronal depolarization, loss of energy, ionic imbalance, glutamate excitotoxicity, and inflammation leading to neuronal death (Fig. 3.1). Traditional therapeutic strategies for an ischemic stroke either attempted to restore cerebral blood flow using endovascular recanalization techniques or by targeting specific genes or proteins involved in stroke pathophysiology to minimize ischemic brain damage. More recently, microRNAs (miRNAs), the non-coding regions of the genome, have revolutionized our knowledge of gene regulation and its translation into a functional protein. With the onset of ischemic stroke, miRNAs exhibit distinct expression patterns that suggest their role in stroke-related pathophysiological processes. The ability of miRNAs to regulate several target genes

# Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_3

33

34

3

MicroRNAs in Ischemic Stroke Pathophysiology: Special Emphasis on Early. . .

Brain restoration

Brain damage

ATP depletion Ionic imbalance Glutamate & Ca2+ overload Oxidative stress

Neuroinflammation Neuronal death Minutes

Hours

Post-ischemic neuroprotection

Days

Weeks

Neuro-restoration /regeneration

Fig. 3.1 Representation of complex cascade of pathophysiological events involved in ischemic stroke progression

underscores their unique significance in ischemic stroke pathophysiology (Table 3.1). Nevertheless, innovative miRNA-based therapeutic approaches have opened novel clinical avenues to support early diagnosis and ischemic stroke care, where the primary therapeutic target is the brain. This chapter highlights the potential role that miRNAs play in a series of molecular events that occur in the acute ischemic stroke phase, such as glutamate excitotoxicity, calcium overload, and oxidative stress.

3.2

Post-Ischemic Glutamate Excitotoxicity and Ionic Imbalance

Excitotoxicity is a focal point of ischemic stroke research to investigate molecular mechanisms that can be aimed at neuroprotection. A large number of unequivocal evidence suggests glutamate excitotoxicity as one of the major causes of ischemic neuronal death. After an ischemic episode, neurons and glia undergo depolarization due to the loss of adenosine triphosphate (ATP) and energy failure. This is also accompanied by an abundant release of neurotransmitter glutamate into the extracellular spaces, which activates α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate and N-methyl-D-aspartate receptor (NMDA)-type glutamate receptors. These receptors permit an excessive influx of sodium and calcium ions (Ca2+) via ion-gated channels causing metabolic imbalance, generation of excess free radicals, and eventually cell death. The glutamate release triggers multifaceted death signaling cascades depending on the type and severity of ischemic stroke. Increasing evidence suggests that miRNAs modulate glutamate release and promote the blockade of downstream post-ischemic events to confer neuroprotection

3.2 Post-Ischemic Glutamate Excitotoxicity and Ionic Imbalance

35

Table 3.1 miRNAs and their validated targets in ischemic stroke pathophysiology Post-ischemic exogenous miRNAs regulation Glutamate excitotoxicity miR-223 Overexpression miR125b

Overexpression

miR-107

Inhibition

miR-29a

Overexpression

miR181a

Inhibition

miR-124

Overexpression

miROverexpression 1000 Calcium overload miRInhibition 103-1 miR-335 Overexpression miR-219

Overexpression

miROverexpression 148a Oxidative stress miR-145 Inhibition miRInhibition 146a miR106b-5p

Inhibition

miR-424

Overexpression

miR23a-3p

Overexpression

miR-210

Overexpression

miR-410

Overexpression

Biological role

Validated gene targets

References

Attenuates post-ischemic NMDAinduced calcium influx Reduces miniature excitatory post-synaptic currents in hippocampal neurons Decreases glutamate release following cerebral I/R injury Preserves astrocyte GLT-1 to promote neuronal survival Increases BCL2 expression and GLT-1 levels to confer neuroprotection Reverses the pathological loss of GLT-1 during cerebral ischemia Regulate glutamate release at the synapse

GluR2 ", NR2B " NR2A #

[1]

GLT-1 "

[3]

PUMA #

[4]

BCL2 "

[5]

GLT1 "

[6]

Vglut #

[7]

Enhances NCX-1 expression to abate post-ischemic brain damage Regulates CaM protein expression during acute ischemic injury Decreases glutamate-induced neuronal excitotoxicity Mitigates Toll Like Receptor 4 (TLR4)-mediated inflammation

NCX-1 "

[8]

CALM1 #

[9]

CaMKIIγ #

[10]

CaMKIIα #

[11]

SOD2 " SOD2 "

[12] [13]

SOD2 "

[14]

SOD2 ", ECSOD ", NFE2L2 " SOD2 "

[15]

a7nAchR "

[17]

SOD ", GSHPx "

[18]

Reduces infarction Reverses the loss of SOD2 and augments cell viability following oxidative stress Restores SOD2 activity and MDA content to counteract postischemic oxidative damage Protection against oxidative stress-induced cerebral ischemia and reperfusion injury Reduces NO and 3NT while increasing SOD2 levels to confer neuroprotection Post-ischemic neuroprotection and maintenance of antioxidant stress response Increases neuronal survival and reduces infarct volume

[2]

[16]

(continued)

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MicroRNAs in Ischemic Stroke Pathophysiology: Special Emphasis on Early. . .

Table 3.1 (continued)

miRNAs miR-25 miR-34a miR-30 miR-499

Post-ischemic exogenous regulation Overexpression Inhibition Overexpression

miR743a miR181a

Overexpression

miR-29b

Overexpression

miR-34b

Overexpression

miR-27b

Inhibition

miR-93

Inhibition

Inhibition

Biological role Mitigates oxidative/nitrative stress Enhances cell viability Functions in regulating oxidative stress and neuronal apoptosis through mitochondrial fission machinery Oxidative stress regulation in the brain Decreases infarct size in ischemic stroke Protects astrocytes and mitochondrial function Ameliorates ischemic strokeinduced oxidative stress Protects against oxidative brain damage

Defense against oxidative stress triggered by ischemia/reperfusion

Validated gene targets NOX-4 # NOX-2 "

References [19] [20]

Drp1 #

[21–23]

MDH2 #

[24]

GRP78/ HSP70 ", BCL2 " PUMA #

[25, 26]

Keap1 #

[27]

Nrf2 ", Hmox1 ", SOD1 ", Nqo1 " Nrf2 "

[28]

[26]

[29]

[30, 31]. This section probes into the salient role of miRNAs in regulating postischemic glutamate excitotoxicity and ionic imbalance.

3.2.1

miRNAs Targeting Glutamate Receptors

During cerebral ischemia, the change in miRNA transcriptome was reported to regulate glutamate release and excitotoxicity of the neurons [32]. Reducing the release of synaptic glutamate, either by mitigating presynaptic activity or by inhibiting receptors of ionotropic glutamate, decreases neuronal damage after ischemia. miR-223 upregulation attenuates post-ischemic NMDA-induced calcium influx in hippocampal neurons. The resultant suppression of AMPA subunit glutamate receptor-2 (GluR2) and NMDA subunit NR2B controls neuronal excitatory response and protects the ischemic brain from excitotoxic neuronal death. The study was further corroborated by the inhibition of miR-223, resulting in an increased expression of glutamate receptors (GluR2 and NR2B) with the subsequent increase in the influx of calcium ions and excitatory post-synaptic currents [1]. miR-125b targets NMDA receptor subunit NR2A and reduces miniature excitatory post-synaptic currents in hippocampal neurons [2]. Exogenous administration of miR-132 induces the upregulation of glutamate receptors (NR2A, NR2B, and GluR1) via mitogen-

3.2 Post-Ischemic Glutamate Excitotoxicity and Ionic Imbalance

37

activated protein kinase/extracellular regulated kinase 1/2 (MAPK/ERK1/2) pathway [33]. Several other miRNAs such as miR-219, miR-34a, miR-539, and miR-19a also target glutamate receptors, but their involvement in glutamate excitotoxicity caused by ischemic stroke warrants further research [34].

3.2.2

miRNAs and Glutamate Transporters

Glutamate transporters [excitatory amino acid transporters 1–5 (EAAT1–5)] facilitate glutamate reuptake and clearance to maintain low levels of extracellular glutamate. Glutamate uptake is primarily dependent on astrocytic glutamate transporters, including glutamate transporter-1 (GLT-1)/EAAT2, and glutamate-aspartate transporter (GLAST/EAAT1) [35]. Cerebral ischemia downregulates GLT-1, which hoists glutamate release causing glutamate excitotoxicity [36]. Various studies have identified the close association of GLT-1 with the aberrant expression of specific miRNAs post-ischemia. miR-107 directly targets GLT-1 inducing glutamate accumulation and neuronal apoptosis. The inhibition of miR-107 enhances GLT-1 expression with the consequent decrease in glutamate release following cerebral ischemia/reperfusion (I/R) injury [3]. Another study identified the use of magnesium lithospermate B (a component extracted from Chinese herbal medicine (Danshen) to modulate miR-107/GLT-1 pathway against glutamate excitotoxicity. Magnesium lithospermate B inhibits miR-107 and upregulates GLT-1 to promote post-ischemic neuroprotection [37]. Astrocyte impairment determines the extent of neuronal death in ischemia, which is associated with the reduced GLT-1 expression [38]. Astrocyte-enriched miRNAs (miR-29a and miR-181a) regulate GLT-1, one of the potential neuroprotective strategies to reduce the vulnerability of neurons to ischemic stroke. Overexpression of miR-29a suppresses p53 upregulated modulator of apoptosis (PUMA) and preserves astrocyte GLT-1 to promote neuronal survival [4]. Conversely, inhibition of miR-181a increases B-cell lymphoma 2 (BCL2) expression with the concomitant increase in GLT-1 levels [5]. Morel et al. reported that the exosome-mediated transfer of miR-124 from neuron to astrocyte increases GLT-1 expression, an efficient strategy likely to reverse the pathological loss of GLT-1 during cerebral ischemia [6]. Besides, miR-1000 targets vesicular glutamate transporter (Vglut) to regulate glutamate release at the synapse. The genetic deletion of miR-1000 elevates glutamatergic excitotoxicity and induces neuronal apoptosis in Drosophila melanogaster [7]. Thus, miRNAs potentially help to elucidate the concurrent mechanisms involved in over-activation of NMDA-receptors and may help to develop novel NMDAR antagonists.

3.2.3

miRNA Regulation of Post-Ischemic Calcium Overload

Ca2+ ion acts as a pervasive intracellular messenger during and immediately after the onset of ischemic stroke and influences the cascade of events that lead to neuronal

38

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MicroRNAs in Ischemic Stroke Pathophysiology: Special Emphasis on Early. . .

death. Post-ischemic over-activation of NMDA receptor triggers the translocation of extracellular Ca2+ to the intracellular spaces within the cerebral tissues [39]. A few early reports suggested that accumulation of intracellular Ca2+ contributes to postischemic neurological impairment, and mortality and those calcium antagonists may keep the neuronal damage under control [40, 41]. A recent meta-analysis and systematic studies, however, suggest that the administration of calcium antagonists in patients following ischemic stroke may not be helpful [42, 43]. Nevertheless, the uptake and release of intracellular Ca2+ is regulated by mitochondrial calcium uniporter, sodium (Na+)-calcium (Ca2+) exchanger (NCX), and mitochondrial permeability transition pore (MPTP), along with the Na+-independent Ca2+ efflux pathway and H+/Ca2+ antiporter pathways. Many miRNAs tightly regulate these Ca2+ signaling proteins and affect calcium ion homeostasis within the brain [44]. NCX is a bi-directional membrane ion transporter comprising of three isoforms (NCX-1, NCX-2, NCX-3) that facilitates the exit of one Ca2+ ion in exchange of three Na+ ions into the cell under normal physiological conditions. During ischemia, NCX reverses the ion exchange with an overwhelming influx of Ca2+ ions resulting in the dysregulation of intracellular calcium and sodium homeostasis [45]. The downregulation, knock-out or pharmacological inhibition of isoforms NCX-1 and NCX-3 aggravates post-ischemic brain damage [46–49]. Identifying miRNAs targeting NCX-1 is one of the rational strategies to prevent the reduction of postischemic NCX-1. miR-103-1 selectively regulates NCX-1, where the inhibition of miR-103-1 enhances NCX-1 expression to abate post-ischemic brain damage [8]. Contrary to this, Aurora et al. found that miR-214 suppresses the expression of NCX-1 mRNA to control the influx of Ca2+ in response to I/R injury. The study shows that miR-214 inhibits calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ), cyclophilin D (CypD), and BCL2 interacting mediator of cell death (BIM), the downstream effectors of Ca2+ signaling pathways, to attenuate calcium overload and ischemic cell death. Also, the genetic deletion of miR-214 results in impaired calcium handling and increased sensitivity to I/R apoptosis [50]. However, the reported mechanism is cardiac-tissue specific and has no direct correlation with the expression of miR-214 and NCX-1 in the brain. Calcium overload due to the increase in calmodulin (CaM) activity also engenders brain damage in the acute phase of ischemic stroke. CaM is a Ca2+binding protein that controls trans-membrane transportation, absorption, and secretion of calcium, which in turn is regulated by miRNAs. miR-335 targets calmodulin 1 gene (CALM1) and influences CaM protein expression during acute ischemic injury. The study suggests that miR-335 is downregulated in the plasma of patients with acute cerebral infarction and the miR-335 overexpression negatively regulates CALM1 in vitro [9]. Besides CaM, calcium/calmodulin-dependent protein kinases (CaMKs) critically regulate Ca2+ signaling. It consists of a large family of Ser/Thr protein kinases such as CaMKI, CaMKII, and CaMKIV and is activated when Ca2+/CaM binds to their regulatory region. Though the activity of CaMKIII (also known as eukaryotic elongation factor-2 kinase, eEF2) is also triggered by Ca2+/CaM, it is not strictly related to other CaMKs and grouped into the family of atypical protein kinases [51].

3.3 miRNAs in Post-Ischemic Oxidative Stress Regulation

39

CaMKII is an essential mediator of excitatory glutamate signals with implications in learning and memory. It consists of four isoforms (α, β, γ, and δ) encoded by different genes and potentially regulated by specific miRNAs [52, 53]. miR-219 targets CaMKIIγ to assuage glutamate-induced neuronal excitotoxicity in hippocampal neurons, while miR-148a represses CaMKIIα to mitigate toll-like receptor 4 (TLR4)-mediated inflammation in hepatic I/R injury [10, 11]. Other miRNAs such as miR-145 and miR-30b-5p repress CaMKIIδ expression and prevent Ca2+ overload in cardiomyocytes [54, 55]. It is noteworthy that not many miRNAs have been identified to target CaMKII, especially in the brain, despite the well-established role of CaMKII in ischemic stroke pathophysiology. Emerging data also reflects that an increase in intracellular calcium concentration is not always detrimental and might trigger endogenous neuroprotective pathways following ischemic stroke. For instance, the inhibition of calcium/calmodulindependent protein kinase kinase (CaMKK) and CaMKIV disrupt the blood–brain barrier and exacerbate stroke injury [56]. It also explains the failure of several clinical trials that block calcium signaling pathways to promote post-stroke recovery. Therefore, further studies on miRNA mediated regulation of post-ischemic Ca2+ homeostasis are essential to circumvent the existing approaches that involve non-specific targeting of Ca2+ signaling.

3.3

miRNAs in Post-Ischemic Oxidative Stress Regulation

Ischemic stroke elicits the excessive release of free radicals in the form of superoxide anions, hydrogen peroxide, and hydroxyl radicals causing irreversible neuronal damage. The surplus generation of free radicals in the form of reactive oxygen/ nitrogen species (ROS/RNS) results in oxidative stress [57]. miRNAs are differentially expressed in response to oxidative stress and modulate redox homeostasis to either promote neuroprotection or aggravate neuronal damage [58, 59].

3.3.1

Endogenous Antioxidant Defense Mechanisms

miRNAs regulate the activity of enzymatic and non-enzymatic endogenous antioxidants that are critical for the scavenging of excessive free radicals after ischemic brain damage. Superoxide dismutases (SODs), glutathione peroxidase (GSHPx), catalase (CAT), and peroxiredoxins (PRDXs) belong to the group of enzymatic antioxidants, while glutathione, NAD(P)H, vitamin C, vitamin E, uric acid, and bilirubin form the non-enzymatic antioxidants [60]. The enzymatic antioxidant SOD encompasses a family of proteins categorized into Copper/Zinc SOD (SOD1), Manganese SOD (SOD2), and extracellular SOD (ECSOD) based on their metal-ion constitution and localization within the brain cells. SODs catalyze the dismutation of oxygen free radicals to generate hydrogen peroxide (H2O2) and oxygen (O2) [61]. Other enzymatic antioxidants such as GSHPx, CATs, or PRDXs convert the harmful H2O2 to the end-product water [62].

40

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MicroRNAs in Ischemic Stroke Pathophysiology: Special Emphasis on Early. . .

According to several reports, the transgenic overexpression of SODs mitigates oxidative stress-induced neuronal death [63–69]. These findings intersect with the inhibition of specific miRNAs targeting SODs and the concomitant activation of the antioxidant defense system following cerebral ischemia. For instance, miR-145 directly targets SOD2 mRNA and prevents the corresponding protein translation after ischemic stroke. The administration of antagomir-145 in ischemic rats increases the expression of SOD2 in the cortical neurons and reduces infarction due to the plausible attenuation of oxidative stress [12]. Similarly, miR-146a interferes with the post-transcriptional expression of SOD2 in response to H2O2 induced oxidative stress. The inhibition of miR-146a reverses the loss of SOD2 and augments cell viability following oxidative stress [13]. Blocking the activity of miR-106b-5p also restores SOD2 activity and lowers methane dicarboxylic aldehyde (MDA) content to counteract post-ischemic oxidative damage [14]. Other miRNAs and their targets affected by ROS generation include miR-181a/GSHPx-1, miR-30b/CAT, and miR-874-3p/caspase 8 [70]. In addition to the inhibition of miRNAs targeting SODs, overexpression of miR-424, miR-23a-3p, miR-210, and miR-410 indirectly enhances SOD levels to diminish post-ischemic oxidative stress. miR-424 and miR-23a-3p abrogate H2O2induced generation of free radicals and enhance SOD activity to ameliorate cerebral I/R injury [15, 16]. miR-210 mediates post-ischemic neuroprotection and maintains antioxidant stress response through the reduction in MDA as well as an increase in SOD and glutathione levels [17, 71]. Furthermore, a recent study indicates that the miR-410 overexpression results in enhanced neuronal survival and reduced infarct size, while increasing SOD and GSHPx activities [18]. In addition to enhancing the antioxidant defense system, restricting the enzymatic source of free radical generation is one of the most distinctive approaches to alleviate post-ischemic oxidative stress. Nicotinamide adenine dinucleotide phosphateoxidase (NADPH-oxidase/NOX) is a pro-oxidant enzyme that forms superoxide ions and functions exclusively towards the production of ROS. NOX consists of multiple protein subunits with an active catalytic site that promotes electron transfer from NADPH to oxygen, facilitating ROS formation. There are five NOX isoforms out of which NOX-1, NOX-2, and NOX-4 are expressed in the central nervous system [72]. miRNAs target these NOX isoforms to balance the amount of ROS produced. miR-29a, -29c, -126a, -132, -136, -138, -139, -153, -337, and -376a were downregulated following MCAO and potentially target NOX-2 and NOX-4 genes. Moreover, the intravenous administration of NOX inhibitor VAS2870 elevates the expression levels of miR-29a, -29c, -126a, and -132 [73]. Another study revealed that miR-25 mimic downregulates NOX-4 and decreases superoxide production to mitigate oxidative/nitrative stress [19]. Overexpression of miR-34a exerts pro-apoptotic activity with enhanced NOX-2 expression and ROS production in the human glioma cell line [20]. Despite the crucial role of NOX-2 and NOX-4 in post-ischemic oxidative stress regulation, substantial research on miRNAs targeting these NOX isoforms is lacking. Therefore, identifying novel miRNAs that target NOX and their mechanism of action would exalt the existing strategies for the regulation of ROS generation and prevention of post-ischemic oxidative stress.

3.3 miRNAs in Post-Ischemic Oxidative Stress Regulation

3.3.2

41

Oxidative Stress-Induced Mitochondrial Dysfunction

Ischemic stroke-induced oxidative stress causes mitochondrial dysfunction and impairs mitochondrial ATP generation [21]. miRNAs govern post-stroke mitochondrial function by targeting specific mitochondrial-associated proteins. miR-30 and miR-499 target dynamin-related protein-1 (Drp-1) and regulate mitochondrial fission machinery during oxidative stress [22, 23]. Oxidative stress downregulates miR-30 and induces mitochondrial fission via p53 transcriptional activation of Drp-1 [22]. Further, Shi and Gibson have reported miR-743a-mediated posttranscriptional upregulation of the mitochondrial enzyme malate dehydrogenase 2 (MDH2) by oxidative stress [24], rendering insight into the potential role of miRNAs in oxidative stress-induced mitochondrial dysfunction. The excessive release of superoxide anions in the mitochondria stimulates an inflammatory response after cerebral ischemia [74]. The exogenous administration of miR-145 antagomir promotes SOD2 expression to confer neuroprotection [12]. Other miRNAs such as miR-141, miR-338, and miR-15b, target genes (solute carrier family 25 member 3, COX-IV, and ADP-ribosylation factor-like protein 2) that are necessary for ATP production [75–77]. Besides regulating mitochondrial function, miRNAs target mitochondrial protective proteins such as heat shock proteins (HSPs), to restore brain function following ischemic stroke. miR-181a inhibition increases the level of glucose-regulated protein, 78 KD (GRP78/HSP70) and decreases the infarct size in an experimental ischemic stroke model. GRP78 belongs to HSP70 family that regulates protein folding in the endoplasmic reticulum, responds to unfolded proteins, functions in cytoprotection and inhibition of apoptosis [25]. Varying single miRNA levels in astrocytic mitochondria have reaped promising results with high clinical translational value. Astrocytes are essential regulators of neuronal function and a valid target for in-vivo stroke therapy. Astrocyte localized miRNAs, miR-181a and miR-29a, coordinate mitochondrial homeostasis through independent pathways. miR-181a overexpression in glucose-deprived astrocytes reduces mitochondrial membrane potential and increases ROS formation leading to cell death. The targeted decrease in miR-181a raises BCL2 levels and enhances the survival of astrocytes. This action of miR-181a is astrocyte-specific, with no significant effect on primary neurons. Conversely, miR-29b upregulation protects astrocytes and mitochondrial function by targeting PUMA [26]. Therefore, it is speculated that combining mitochondrial and miRNA-based modalities into a single intervention might help in overcoming the current multi-dimensional approach towards the development of ischemic stroke therapy.

3.3.3

miRNAs and the Master Regulator Nrf2

The redox-sensitive nuclear factor erythroid 2-related factor 2 (Nrf2/NFE2L2) serves as a “master regulator” of cell survival. Several miRNAs influence Nrf2 expression and have been reported to restore endogenous antioxidant enzyme levels, promoting

42

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MicroRNAs in Ischemic Stroke Pathophysiology: Special Emphasis on Early. . .

redox homeostasis [78]. A previous study suggested that 85 miRNAs can bind to cytoplasmic Nrf2 mRNA to inhibit its translation [79]. miR-153, miR-27a, miR-1425p, and miR-144 directly target Nrf2 and affect Nrf2 mRNA abundance as well as its nucleo-cytoplasmic concentration. Moreover, the ectopic expression of these miRNAs deregulates glutathione and ROS levels and disrupts cellular redox potential, suggesting their likely role in sensitizing neuronal cells to oxidative stress [80]. Besides the direct binding of miRNAs to Nrf2, other proteins in the Nrf2 “regulome” also alter ARE-mediated redox signaling. Nrf2 is suppressed by Kelch-like erythroid cell-derived protein (Keap1) comprising of more than 25 cysteine residues [78]. Under oxidative stress, these cysteine residues are occluded, and Nrf2 translocates from the cytoplasm into the nucleus, where it dimerizes with the antioxidant response element (ARE) in the promoter region of its downstream target genes [81, 82]. miR-34b targets Keap1 to ameliorate ischemic stroke-induced oxidative stress. The overexpression of miR-34b suppresses Keap1, while enhancing Nrf2 and heme oxygenase-1 (HO-1) expression. This resulted in reduced infarction volume, lowered nitric oxide (NO), as well as 3-nitrotyrosine levels and increased total SOD and SOD2 [27]. On the other hand, the upregulation of miR-27b and miR-93 aggravates oxidative stress-induced neuronal damage. miR-27b represses the expression of Nrf2, Hmox1, SOD1, and Nqo1, whereas the inhibition of miR-27b protects against intracerebral-hemorrhage (ICH)-induced brain damage [28]. This effect is partly attributed to the role of miR-27b in Nrf2/ARE regulation. miR-93 directly binds to the predicted 30 -UTR target sites of the Nrf2 to attenuate the expression of Nrf2 and HO-1 [29]. The Nrf2/HO-1 pathway is a critical cellular defense mechanism against oxidative stress triggered by ischemia/reperfusion [83]. Thus, miRNAs targeting Nrf2 might be valuable therapeutic agents to antagonize post-ischemic oxidative stress.

3.3.4

Endothelial Integrity and Blood–Brain Barrier

The blood–brain barrier (BBB) is shaped by brain endothelial cells lining the cerebral microvasculature. It is an essential mechanism to protect the brain from changes of plasma fluid chemistry and from circulating substances such as neurotransmitters and xenobiotics capable of upsetting neuronal activity. Mounting evidence indicates that ischemic stroke causes rapid alterations of endothelial integrity and function [84, 85]. Several mechanisms, including ROS development and MMP activation or inflammatory cytokines, contribute to the vascular disturbance that ultimately leads to blood–brain barrier (BBB) disruption. The majority of miRNAs adversely affect BBB permeability, such as miR-15a/16-1, miR-130a, miR-143, miR-150, miR-155, and miR-210 [86–91]. They augment ischemiainduced BBB disruption by either directly or indirectly targeting TJs including ZO-1, claudin-5, occludin, cadherin, and β-catenin. Alternatively miR-21, miR-132, miR-149-5p, and miR-539 have been shown to maintain BBB integrity through different mechanisms, including improving the expression of TJ, MMP-9 regulations, and blocking the signaling trajectory of MAPK [92, 93].

References

3.4

43

Conclusion

Over the past decade, miRNAs have been the frontiers of ischemic stroke research to comprehend the known as well as unknown molecular mechanisms. Modulating miRNA expression, either using specific miRNA mimic or inhibitor, holds a promising therapeutic value in ischemic stroke and is expected to shun the “translational roadblock” in the development of alternative stroke therapies. MiRNAs may also improve the process of drug development as it interferes with the pharmacological mechanisms at the post-translational level. However, it has been estimated that over a 1000 miRNAs with more than 30% of human genes regulated by these miRNAs have been identified so far. With the increasing number of miRNAs, any given miRNA may have multiple genes targets and vice versa. Several computational network models have been implemented to check the complex miRNA–gene interactions, integrating mathematics with state-of-the-art quantitative experimental data. It could be used to predict the precise role of miRNAs in signaling cell death or survival pathways and their cross-talking in various cellular modalities before laboratory testing. This approach would, therefore, help optimize the prognostic and predictive parameters of new strategies to rationalize existing treatments and identify new targets for therapeutic modulation of neuronal death and restoration pathways.

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4

microRNA Regulation of Ischemic Stroke Inflammatory and Immune Response

Abstract

Ischemic stroke is one leading cause of death worldwide, and inflammation has been extensively associated with its ontogeny and subsequent brain lesions. MicroRNAs (miRs) are non-coding RNA molecules that specifically repress gene expression post-transcriptionally. Several miRs have been correlated with innate immune response, and evidence accumulating suggests miRs regulatory role during stroke-related neuroinflammation. The use of miRs as a potential neuroprotective target for ischemic stroke is increasingly being considered as a result of this build-up knowledge. In this chapter, our communication focuses on the identification of miRs linked to neuroinflammatory ontogeny associated with ischemic stroke. Keywords

Ischemic stroke · Inflammation · Microglia · Immune response · Astrocytes · microRNAs

4.1

Introduction

In 2016, World Health Organization (WHO) identified ischemic stroke as the second leading cause of death and the world’s third cause of disability [1]. Stroke is defined as a lack of oxygen supply to brain cells after cerebral artery breach or occlusion. In fact, with poor treatment options, ischemic stroke is associated with 87% of strokes in the USA (see Chap. 2). Nevertheless, researchers have made significant progress in identifying the underlying cellular processes in ischemic pathophysiology, opening new stroke therapeutic targets such as microRNAs (miRs) (see Chap. 1) [1]. Short and long-term cerebral ischemic effects are associated with the disruption of several molecular and cellular processes such as blood flow, oxygen and nutrient delivery. In this context, neurons are more vulnerable to hypoxia-ischemia than any # Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_4

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microRNA Regulation of Ischemic Stroke Inflammatory and Immune Response

Table 4.1 miRNAs associated with immune response to ischemia Pathogenic process Coagulation Oxidative stress Inflammation Immune system

microRNA miR-19a, miR-let-7i, miR-146, miR-148, miR-122, miR-17, miR-126, miR-223 miR-424, miR-155, miR-410

References [8–12]

miR-181c, miR-155, miR-146a, miR-98, miR-22, miR-93, miR-195, miR-182-5p, miR-122, miR-544, miR-320 miR-340-5p, miR-424, miR-210, miR-99a-5p

[14, 16–25]

[13–15]

[26–29]

other type of brain cell, and will become dysfunctional and/or more likely to die. Cellular events following cerebral ischemia include coagulation, oxidative stress, inflammation, and apoptosis. All of these events are seriously detrimental to neurons and glial cells, leading to subsequent neuronal death and brain damage. During cerebral ischemia, several immune responses are triggered, which aggravate adverse neuronal reactions. Various immune cells are associated with subsequent lesions, including lymphocytes, astrocytes, and microglia [2]. In addition to cerebral lesions, evidence has shown a correlation between ischemic stroke and a significant reduction in the functioning of the global immune system, leading to a higher susceptibility to infection. The specific function of each immune cell will be further described in the following sections. As described before (see Chap. 1), miRs are a class of non-coding RNAs that post-transcriptionally regulate specific mRNA translation. Mounting evidence demonstrated the active role of miRs in stroke-related molecular events and reperfusion-associated neuroinflammation. It is, therefore, important to understand the regulatory role of microRNAs in the immune system following an ischemic stroke to find appropriate therapy. Given the complexity of the CNS, the brain-specific miRs appeared to be regulators of both the homeostatic and pathological functions of stroke [3–7]. During ischemic stroke progression, a cascade of molecular and cellular events occurred, including blood–brain barrier (BBB) disruption, oxidative stress, neuroinflammation, and cell death. Interestingly, several studies demonstrated that specific miRs target these deleterious processes listed in Table 4.1.

4.2

Stroke Immunology

Before vessel occlusion, a non-homeostatic immune response can induce local inflammation at vascular levels, which impairs its normal functioning. Indeed, leukocyte cells recruitment triggers inflammation, leading to the formation of atherosclerotic plaques and thrombosis. At stroke onset, immune cells, such as lymphocytes, participate in ischemic stroke pathogenesis by releasing proteins that are described as key actors of cell death and brain lesions. This immune response

4.3 microRNAs Associated with Ischemic Neuro-Inflammation

51

within brain tissue and its vasculature induces brain lesions associated with local excitotoxicity, mitochondrial dysfunction, causing neuronal death. The leader of the immune response following vessel occlusion is microglia and infiltrating leukocytes through disrupted BBB. The neuronal death process induces the release of chemokines that initiate immune cell recruitment such as neutrophils, natural killer cells, macrophages, and dendritic cells. Activated immune cells release inflammatory proteins such as cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS), exacerbating BBB disruption [3]. At the vascular level, there is a significant production of reactive oxygen species (ROS), leading to platelet and endothelial cells activation, followed by coagulation. This leads to the containment of platelets and immune cells within the local brain microvessels. One process linked to ischemic events is thrombosis, which also triggers the immune response. Indeed, thrombin activated nuclear factor kappa B (NF-kB) pathway is a chemotactic for peripheral immune cells. Moreover, thrombin activates the complement system and worsens the BBB disruption and subsequent brain lesions. In summary, inflammation can trigger stroke occurrence and is highly harmful. The targeting of inflammation processes could, therefore, be successful in reducing the stroke of subsequent lesions.

4.3

microRNAs Associated with Ischemic Neuro-Inflammation

During ischemia initiation, miRs regulate platelets, thrombus formation, and coagulation process (Fig. 4.1). The first event initiating the coagulation cascade is the tissue factor (TF), produced mainly by monocytes. Data demonstrated that under LPS exposure, TF production is increased and is associated with a reduction of miR-19a; on the contrary, miR-19a overexpression is associated with a decrease of TF production [12]. In addition to TF, miRs also regulate thrombin formation through miR-let-7 and miR-146, while coagulation factor III and VIII are targeted by miRs-148/19a and miR-122 [8, 9]. Plasminogen activator inhibitor-1 (Serpine-1) is the primary coagulation activator by acting on a tissue plasminogen activator (tPA) and inhibiting fibrin degradation. During a stroke, underexpression of miR-148 and miR-19a lead to an overexpression of Serpine-1 [9]. In normal conditions, several miRs are expressed by endothelial cells and regulate cellular functions such as cell growth, survival, and angiogenesis [30, 31]. Rapidly after ischemia, proinflammatory signals are produced, including the tumor necrosis factor (TNF), leading to the expression of several cell surface markers by endothelial cells that bind to recruited leukocytes (P/E-selectins, ICAM-1, and VCAM-1). This agglomeration of peripheral cells contributes to the vessel obstruction and to subsequent lesions. Interestingly, this recruitment of cells by endothelial cells through TNF production is regulated by miR-126, miR-31, and miR-17-3p that target, respectively, VCAM-1, E-selectin, and ICAM-1 expression [16, 32]. In normal conditions, nitric oxide (NO) has been associated with beneficial outcomes within the brain [33]. Moreover, as a potent vasodilator and an inhibitor

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Platelets BBB disrupon Cell death: • miRs-181a/c • miR-21 • miR-30d

Coagulaon: • miR-19a • • miR-146a • • miR-let7 • • miR-148 •

Endothelial cells: • miR-126 • miR-155 • miR-31 • miR-320 • miR-17-3p• miR-225 miR-122 miR-17 miR-126 miR-223

Reacve astrocytes

Dying neurons

Acvated Microglia

Oligodendrocytes

Microglia: • miR-126 • miR-223 • miR-181c • miRs-146a/b • miR-124

Astrocytes: • miR-7

Fig. 4.1 MicroRNAs and their involvement in various cellular components of the brain

of cell adhesion to endothelial cells, it helps to maintain brain homeostasis. Thus, NO can inhibit the platelet formation and subsequent vessel occlusion. During ischemia, there is a modification of NO bioavailability at the occlusion site associated with an overexpression of ROS leading to oxidative stress [34]. Through inflammation, miR-155 is upregulated and has been associated with a reduction of endothelial nitric oxide synthase (eNOS) expression and NO production. Targeting miR-155 could be a potential target to promote NO bioavailability and reduced vessel occlusion [35]. miR-424 expression during ischemia can reduce ROS production and thus improve neuron survival [36]. Matrix metallopeptidases (MMPs) are a class of modulators involved in endothelial disruption during inflammatory stress [37]. Several miRs have been associated with MMPs regulation in stroke. Recent evidence in a mouse model of stroke demonstrated that overexpression of miR-320 is deleterious and inhibits IGF-1 production [38]. One target of miR-320, ETS-2, is overexpressed in blood samples from stroke patients [39]. ETS-2 regulates several MMPs and TNF expression

4.4 microRNAs and Innate Immune Cells in Ischemic Stroke

53

[40]. miR-885 and miR-491 have been described as modulators of MMP-9 expression, and thus limited the barrier disruption [41]. Several miRs have been associated with ischemic stroke initiation at different levels. It indicates the need to understand the role of miRs in stroke better to develop a larger panel of potential targets. Zhao et al. have shown that lentiviral overexpression of miR-424 significantly reduces post-ischemic brain injury by suppressing microglial activity [36]. MiR-let-7c-5p has also been reported to confer ischemic neuroprotection from inflammation by inhibiting the activation of microglial and the translational repression of caspase-3 [42]. Ischemic stroke caused the upregulation of miR-124, which was shown to specifically inhibit CCAAT/ enhancer-binding protein alpha (C/EBP-α) and its downstream factor PU.1, promoting microglia quiescence [43, 44]. MiR-181c suppressed the expression of TLR4 by binding to its 3’UTR, therefore reducing the level of NF-κB and the production of downstream proinflammatory factors [45]. MiR126, known to be endothelialspecific, is a potent vascular inflammatory regulator. miR126 modulates the expression of vascular cell adhesion molecule (VCAM), and controls leukocyte extravasation into the brain [46]. miR155 and let-7 have anti-inflammatory effects and regulate expression of molecules important in stroke recovery, including IL4, IL10, and BDNF in microglia and downregulate iNOS and IL6 [47–49]. miR155 has proinflammatory effects in the brain after stroke. It controls macrophage signaling, differentiation, and proinflammatory phenotypes by decreasing the equilibrium between suppressor of cytokine signaling 1 (SOCS-1) protein, and the upregulation of inflammatory molecules such as iNOS [50, 51]. The administration of miR155 antagomir lowered post-stroke inflammation, diminished neuronal apoptosis, and improved neurological deficits [17]. Compared to the action of miR155, the antagomir for miR210 decreased the expression of proinflammatory cytokines IL6, TNF-a, IL1b, CCL2, and CCL3, decreasing neurological impairment, rendering miR210 a powerful inhibitor of inflammation during stroke rehabilitation [28]. The inflammasome is a cytosolic complex that plays a role in ischemic stroke by fostering inflammatory and cell death processes. MiR223 has a role to play in suppressing NLRP3 inflammasome by binding to its 30 UTR sites and therefore inhibiting IL1b and caspase-1 activity, decreasing brain edema and increasing neurological performance [52]. miR31 and miR17-3p regulate the expression of E-selectins and Intercellular Adhesion Molecule-1 (ICAM-1), reducing neutrophil adhesion and post-stroke leukocyte infiltration [53]. While miR98 and let-7g decrease leukocyte adhesion and migration across the BBB [47].

4.4

microRNAs and Innate Immune Cells in Ischemic Stroke

As described before, microglia is a highly induced immune cell activated by stroke and the following reperfusion. In this condition, they produce a large panel of molecules that promote inflammation and subsequent neuronal death. After ischemia, neuronal death occurs within and at the distance of the occlusion site. Dying cells are key actors in inflammation post-ischemia by expressing

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specifics signals. These signals are called danger-associated molecular pattern molecules (DAMPs) and can be recognized by specific receptors at the membrane of microglia, macrophage, endothelial cells, and astrocytes. Response to those DAMPs contributes to microglial activation and the worst inflammation following ischemia. DAMPs are not always proteins; one of the non-protein DAMPs signals is ATP. After an ischemic event, ATP is abundant in cerebral parenchyma. Microglia present at its cell surface purinergic receptor, such as P2Y12 receptor. When activated, P2Y12R induced microglial migration toward ATP source at the ischemic site. Thus, ATP and P2Y12R contribute to microglial activation and neurotoxicity [54]. During ischemia, microglia overexpress toll-like receptor 4 (TLR-4), which passes through the upregulation of TNF-a and IL-1B expression [55]. In the opposite, miR-181c represses expression of TLR-4 in microglia and limits TNF-a and IL-1B production [45]. miR-146 family is highly associated with neuroinflammation in several adult brain diseases and described as overexpressed in microglial cell cultures [56]. Unpublished data from our team demonstrate that overexpression of miR-146b can prevent microglial activation and limited subsequent lesions (Bokobza et al., in preparation). Brain-specific miR-124 expression is decreased under neurological disorder; however, its overexpression could reestablish microglia quiescence through overexpression of neuroprotective molecules [57]. miR-181a has been demonstrated to be upregulated by OGD by downregulation of apoptosis inhibitor [58]. On the contrary, mir-181c have been reported to be downregulated by microglia in OGD in vitro analysis, and that overexpression of mir-181c can protect neuron from cell death [59]. In the same way, overexpression of miR-21 could protect neurons from cell death by targeting apoptosis factors [60]. miR-30d have been demonstrated as an active modulator of apoptosis in astrocytes [61].

4.5

Conclusion

In conclusion, cerebral ischemia is strongly associated with inflammation, which can lead to severe brain lesions. Many miRs have been demonstrated to be modulators of neuroinflammation following cerebral ischemia. The prospect of cerebral ischemia treatment could be reached by controlling any of these inflammatoryassociated miRs.

References 1. Johnson W, Onuma O, Owolabi M, Sachdev S (2016) Stroke: a global response is needed. Bull World Health Organ 94(9):634–634A 2. Ao LY, Yan YY, Zhou L, Li CY, Li WT, Fang WR, Li YM (2018) Immune cells after ischemic stroke onset: roles, migration, and target intervention. J Mol Neurosci 66(3):342–355 3. Thounaojam MC, Kaushik DK, Basu A (2013) MicroRNAs in the brain: it’s regulatory role in neuroinflammation. Mol Neurobiol 47(3):1034–1044

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5

Regulatory Role of microRNAs in Ischemic Cell Death

Abstract

Cell death is a biological process that results in old cells dying and being replaced by new ones or maybe a result of pathological disorders, localized injury, or trauma resulting in the death of cells. There are many cell death pathways so far reported in various kinds of literature, and many are continuously being discovered and reported having regulatory functions. Even though there are many cell death pathways activated in various diseases here in this chapter, we focus on only those cell death pathways that are activated during cerebral ischemia and are regulated by microRNAs. Keywords

Apoptosis · Autophagy · Cell death pathways · Necroptosis

5.1

Introduction

Cell death may be categorized by morphological appearance as apoptotic, necrotic, autophagic, enzymological, or immunological in nature [1]. All of these have specific characteristic features as well as functional importance. The most wellestablished and studied cell death pathway is apoptosis due to its immense importance in various developmental processes [2]. A general idea of the so-called programmed cell death “apoptosis” and other pathways are briefed in the first section of the chapter. In contrast, the latter part describes various microRNAs that are well known to regulate these cell death pathways during stroke onset.

# Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_5

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5.2

5

Regulatory Role of microRNAs in Ischemic Cell Death

Apoptosis

Apoptosis is a cellular mechanism that is essential to the development and homeostasis of an orderly occurring multicellular organism. Unchecked apoptosis can result in severe diseases that can threaten the organism’s existence. Biologically, apoptosis is strongly associated with inflammation and host protection against intracellular pathogens such as viruses [2]. Apoptosis takes place via characteristic morphological changes which rely on caspase activities. Caspases are cysteine proteases that precisely cleave after aspartic acid residues. Because a great deal of human diseases are related to apoptosis as well as inflammation, research in these areas is extremely important biologically. Apoptosis is not only limited to the developmental process but also plays an imminent role in the nervous system processes and the associated neurological disorders. Neurons follow the same basic apoptosis system with all other forms of cells. However, various types of neurons and neurons at different stages of development express the different combinations of Bcl-2 and caspase family members [3]. Bcl-2 expression is elevated in the central nervous system throughout development and is downregulated after birth, while Bcl-2 expression in the peripheral nervous system is retained throughout life. Neuronal apoptosis crafts the brain that is growing and has a potential effect on neurodegenerative diseases. Apaf-1, Bcl-2 proteins, and caspase families are the main molecular components of the neuron apoptosis system. Neurotrophins are molecules that control neuronal apoptosis via protein kinase cascades, such as phosphoinositide 3-kinase/Akt and mitogenactivated protein kinase pathways [4]. The protein family Bcl-2 plays a key role in transducing intracellular apoptotic signal. This family of genes contains both anti-apoptotic and pro-apoptotic proteins, which contain one or more domains of Bcl-2 homology. The Bcl-2 family’s key antiapoptotic members include Bcl-2 and Bcl-xL, respectively, which are found on the outer membrane of the mitochondrial and endoplasmic reticulum and perinuclear membranes. A transgenic mouse expressing Bcl-2 in the nervous system has been shown to be safe during development against neuronal cell death, comparable to models of neuronal damage such as occlusion of the middle cerebral artery. Explicit control over apoptosis is highly beneficial in preventing harmful effects on tissues and organs. Bcl-2 is crucial for the maintenance of neuronal survival. For example, the growth of the nervous system in Bcl-2-knockout mice is stable, but there is a corresponding loss of motor, sensory, and sympathetic neurons after birth. Like Bcl-2, Bcl-xL is an apoptosis regulator expressed during brain development; however, Bcl-xL expression tends to increase into adult life, unlike Bcl-2 expression. Bcl-2 and Bcl-xL function by the inhibition by heterodimerization of pro-apoptotic members of the Bcl-2 family. Bax is a pro-apoptotic member of the Bcl-2 family that expresses itself extensively in the nervous system. There are two different pathways by which the apoptosis act; intrinsic and extrinsic pathway [2, 5].

5.3 Autophagy

5.2.1

61

Intrinsic Pathway

The intrinsic pathway of apoptotic cell death is induced in a mitochondria-dependent manner in response to intracellular insults. A central platform of a molecular complex known as apoptosome is formed via CARD-containing protein Apaf-1 resulting in caspase activation in this pathway. Cytochrome c, which typically remains in the intermembrane space of the mitochondria, is released into the cytosol after cell damage occurs through mitochondrial leakage. Then, the apoptosome recruits caspase-9 through the interaction of CARD between Apaf-1 and caspase-9.

5.2.2

Extrinsic Pathway

The extrinsic mechanism of apoptosis requires the presence of death receptors found on the plasma membrane. They are often referred to as the “death receptor pathway.” Cell surface death receptors belong to the superfamily of tumor necrosis factor receptors, namely TNFR-1, Fas, and p75NTR. FasL triggers apoptosis by binding to its receptor (Fas receptor) which triggers the recruitment of the cytoplasmic adaptor protein Fas-associated death domain protein (FADD). FADD contains a “death effector domain” at the N terminus, which interacts with the death effector domain by binding to procaspase-8. This complex (FasL–Fas–FADD–procaspase-8) is referred to as the Death Inducing Signaling Complex (DISC) and is formed within seconds of the Fas receptor presence. The signal complex catalyzes Procaspase-8 to induce Caspase-8 proteolytic cleavage and transactivation. Once activated, Caspase8 is released from DISC into the cytoplasm and initiates downstream cleavage of Caspase-3 either directly or by mitochondrial dependent pathways [1].

5.3

Autophagy

Autophagy is the process of partial auto-digestion in cells, which prolongs the cell survival in conditions of starvation associated with nutritional shortages such as amino acids for a brief period of time. They provide nutrients essential for cell viability. It is recently been shown that autophagy also protects cells from ingested bacteria by killing them. The molecular mechanism causing autophagy has been extensively researched, primarily in yeast (by testing autophageal deficient mutants to classify the responsible genes). The autophagy cycle includes the creation of autophagosomes, which are double-membrane cytoplasmic vesicles engineered to engulf different cellular constituents, including cytoplasmic organelles [6]. Such autophagosomes combine into lysosomes to form autolysosomes, where the cellular components are digested. Autophagy is still a morphological concept, although there is also no definitive proof that a particular cause of autophagic death currently exists. Autophagy is regulated by type I and III PI3 kinases as well as the mTOR pathway [6].

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It has been proposed that autophagic death plays a crucial role in both physiological and pathological cell death. In addition, autophagic death is normal during tissue remodeling processes (metamorphosis) in insects and organ morphogenesis throughout growth. Because autophagy can provide the necessary nutrients required for cells to survive, it is possible that autophagy can be used to provide nutrients to other cells in multicellular organisms [7]. Numerous studies indicate the role of autophageal cell death in different disorders, as the autophageal symptoms are present in the lesions in some neurodegenerative conditions, such as Parkinson’s disease and Alzheimer’s disease, as well as in certain types in myopathy [8]. MicroRNAs are involved in all of the cellular death mechanisms: necroptosis, mitophagy, etc. besides the above-mentioned cell death pathways. This chapter details specifically with only those which are activated during ischemic or hypoxic insults, emphasizing on ischemic stroke.

5.4

MicroRNAs and Autophagy

There is a lot of microRNAs that are differentially regulated during ischemic insult. Some of them might act through specific cell death pathways, while others act via targeting multiple pathways. The ischemic onset triggers a wide variety of cellular and molecular phenomenon resulting in the death of neuronal cells [6]. Autophagy is one of the mechanisms by which it occurs to have plenty of regulatory players, especially microRNAs. Autophagy has shown to be activated in neurons of neonatal or adult rodents upon cerebral ischemia [9]. Several microRNAs regulating key players of autophagy have been specifically targeted by many labs to evaluate its potential impact on post-ischemic neuroprotection. A detailed entry of all the microRNAs and its target genes involved in ischemic cell death is given in Table 5.1. The most widely targeted gene in autophagic pathway is Beclin-1; being one of the key players in triggering autophagy, several microRNAs have been reported to act on Beclin-1. MicorRNA-30d-5p, miRNA-30a, miR-215 are some of the miRs that work on Beclin-1. The interaction studies were predicted using bioinformatics tools and validated by the experimental approach. MicorRNA-30d-5p has been Table 5.1 The major genes and their miRNA target during the ischemia-induced cell death process Cell death pathways Autophagy

Apoptosis

Target genes Beclin-1 Atg5 Act1 iASPP IGF-1 Caspase7 and Bclaf1 Bcl-xL Bcl-2

microRNAs miR-30d-5p, miR-30a, miR-215 miR-30d-5p, miR-9a-5p miR-298 miR-124, miR-182 miR-186-5p miR-146a miR-24 miR-181

References [9, 10] [11, 12] [13] [14] [15] [16] [17] [18]

5.5 MicroRNAs and Apoptosis

63

shown to be one of the most downregulated miRNAs in brains with hypoxiaischemia [9]. Aside from Beclin-1, MiR-30d-5p was found to target 3’UTR of Atg5 [11]. MiRNA-30a, a differentially expressing microRNA in the hippocampus of mice with an ischemic attack, supports ischemic neuroprotection by improving Beclin-1-mediated autophagy [10]. MiR-9a-5p is one of the well-studied miRs and has been shown to be decreased in the ischemic region of rats following stroke. One of the study reported its role in regulating the process of autophagy via targeting ATG5 expression, which was confirmed using western blotting, immunofluorescence staining, and luciferase assays [12]. Some microrRNAs regulate complex pathways bringing in multiple players at a time such as miR-215. It has been stated that MiR-215 mimic decreases ischemic infarct and inhibits autophagy through a negative modulation of Act 1/IL-17RA and an inhibition of JNK/pBcl-2/Beclin pathway [19]. In this set the second one contains miR-298. The Act1/JNK/NF-kB pathway is shown to be specifically controlled miR-298 and inhibits the production of Act1 protein in vitro and in vivo. In addition, Act 1 is known to be a primary target of miR-298 by luciferase assay studies [13]. There have been several miRs that control several cell death pathways, especially apoptosis and autophagy. For starters, miR-30d-5p has been shown to be involved in both apoptosis and autophagy in neonatal rat brains subjected to hypoxic injury. MiR-215 is the second in line to inhibit both apoptosis and autophagy in vitro. In certain cases apart from targeting key autophagic players, miRs may target the formation of lysosomes and autolysosome formation, necessary for the process of autophagy. After an ischemic stroke, MiR-207 was shown to be downregulated, besides miR-207 agonists could inhibit the number of lysosomes and autolysosomes, and increase the number of autophagic vacuoles. Thus, the latter part of lysosomalautophagy flux may be affected [20].

5.5

MicroRNAs and Apoptosis

Apoptosis is one of the well-established mechanisms to cell death in the mammalian system and has a large spectrum to functions in physiology and pathophysiology. Its presence in various pathological conditions, especially neurodegenerative diseases, has sparked much interest in dissecting the molecular and regulatory mechanisms underlying it. MicroRNAs have been reported to be involved in apoptotic gene regulation, and play a crucial disease-modifying function after cerebral ischemia. The most widely targeted apoptotic genes include caspase, Bcl-2, and Bcl-xL. Besides these, some inflammatory regulators, and other anti-apoptotic proteins are also targeted. MiR-124 is one of the critical miRs in ischemic settings and has been linked to regulating cell apoptosis by targeting anti-apoptotic protein iASPP in the early stages of cerebral ischemia [21]. The anti-apoptotic protein iASPP is a target of not only a single miRNA but is regulated by other miRs; miR-182, reported to bind 3’-UTR of iASPP [14]. MiR-146a is another miR that has been reported to show anti-apoptotic properties upon downregulation. This is due to its interaction with 3’-UTR of Caspase7 and Bclaf1 (bioinformatics analysis) [16]. IGF-1 is one of the

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most active anti-apoptotic growth factors associated with the modulation of various processes, such as cell development, proliferation, and neuron survival, which have been shown to be deregulated during ischemic insults. MiR-186 was found to control IGF-1 by specifically suppressing its expression and by inducing neuronal apoptosis [15]. There are several miRs that regulate apoptosis without directly targeting the key players of apoptosis. For example, the lowering of miR-224 decreases OGD-induced ischemic injury by preventing neuronal apoptosis by targeting SPG7 [22]. Another example of this is miR-181c, which was shown to suppress TNF and partially prevents neuronal apoptosis following OGD [23]. The key players of the apoptotic pathway targeting miRs were numerous. miRNA-24 was highly expressed, and inhibition of miRNA-24 prevents hippocampus or cortex apoptosis and increases cell viability, leading to a protective effect on ischemic stroke. Further, miR-24 was also showed to have pro-apoptotic function via targeting Bcl-xL [17]. A similar finding was made in a study in which miR-99a blocked all pro-caspase-3 and active caspase-3 signals as well as the prevention of neural apoptosis. Numerous cases of miRs targeting Bcl-2 have been published. For example, the miR-15 cluster was found to be upregulated after focal ischemia and target Bcl-2, and thus inhibition of miR-15 raises Bcl-2 protein levels [24]. Another significant miRs in cerebral ischemia is miR-497 which also targets Bcl-2 and antagomiR-497 therapy with elevated rates of Bcl-2 followed by decreased amount of infarction. Following global cerebral ischemia, it has been shown that miR-181a suppression raises Bcl-2 expression and decreases neuronal loss of CA1 in hippocampus [25]. In cortical neurons, miR-124 agomiR improved the anti-apoptotic expression of Bcl-2 and Bcl-xl without altering pro-apoptotic Bax or Bad expression and thereby protecting neurons against OGD [26]. Mir-29b suppresses a variety of pro-survival members of the Bcl-2 family, including Bcl-2, Mcl-1, and Bcl-w (Bcl2L2). MiR-25 overexpression in neural cell lines has been shown to inhibit OGD-apoptosis by reducing Fas levels. It has also been shown that MiR-25 overexpression prevents Bax and Caspase-3 expression and facilitates Bcl-2. Antagomir MiR-15a/161 has enhanced anti-apoptotic protein production in ischemic brain regions and reduced production of the proinflammatory molecules [25]. For example, some of the miRs family, members of the miR-181 family, are specifically involved in controlling both apoptosis regulatory proteins (BCL2 and XIAP family) and inflammatory mediators. MiR-378 levels have decreased dramatically in vitro following OGD. It was confirmed by laboratory studies (luciferase reporter assay) that miR-378 could bind to the 3’UTR of Caspase-3 mRNA and repress its translation. Another miRNA binding to caspase includes miR-1247-3p. OGD/R mediated caspase expression is blocked by the miR-1247-3p mimic [27]. Bcl2l11 has been involved in controlling intrinsic death of cells triggered by different forms of stimuli. MiR-9 has been reported to target and control Bcl2l11 [28]. There were several studies relating to miRs in astrocytes where Bcl-2 was shown as a direct target of miR-181, and miR-181a inhibition in primary astrocytes increased Bcl-2 protein and decreased oxidative stress during glucose deprivation

References

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[18]. In addition, the amount of miR-496 in brain tissues has decreased after cerebral ischemia, affecting a pro-apoptotic member of the Bcl-2 family; BCL2L14 (BCL-G) and negatively regulating them to display a defensive role in cerebral I/R injury by suppressing cell apoptosis [29].

References 1. Kroemer G et al (2009) Classification of cell death: recommendations of the nomenclature committee on cell death 2009. Cell Death Differ 16(1):3–11 2. Park HH et al (2007) The death domain superfamily in intracellular signaling of apoptosis and inflammation. Annu Rev Immunol 25:561–586 3. Yuan J, Yankner BA (2000) Apoptosis in the nervous system. Nature 407(6805):802–809 4. Nafis S et al (2015) Apoptosis regulatory protein-protein interaction demonstrates hierarchical scale-free fractal network. Brief Bioinform 16(4):675–699 5. Yip KW, Reed JC (2008) Bcl-2 family proteins and cancer. Oncogene 27(50):6398–6406 6. Tsujimoto Y, Shimizu S (2005) Another way to die: autophagic programmed cell death. Cell Death Differ 12(Suppl 2):1528–1534 7. Tait SW, Ichim G, Green DR (2014) Die another way--non-apoptotic mechanisms of cell death. J Cell Sci 127(Pt 10):2135–2144 8. Green DR, Levine B (2014) To be or not to be? How selective autophagy and cell death govern cell fate. Cell 157(1):65–75 9. Zhao F et al (2017) miR-30d-5p plays an important role in autophagy and apoptosis in developing rat brains after hypoxic-ischemic injury. J Neuropathol Exp Neurol 76(8):709–719 10. Wang P et al (2014) Down-regulation of miRNA-30a alleviates cerebral ischemic injury through enhancing beclin 1-mediated autophagy. Neurochem Res 39(7):1279–1291 11. Jiang M et al (2018) Exosomes from MiR-30d-5p-ADSCs reverse acute ischemic strokeinduced, autophagy-mediated brain injury by promoting M2 microglial/macrophage polarization. Cell Physiol Biochem 47(2):864–878 12. Wang N et al (2018) MicroRNA-9a-5p alleviates ischemia injury after focal cerebral ischemia of the rat by targeting ATG5-mediated autophagy. Cell Physiol Biochem 45(1):78–87 13. Sun H et al (2018) MiR-298 exacerbates ischemia/reperfusion injury following ischemic stroke by targeting act1. Cell Physiol Biochem 48(2):528–539 14. Yi H et al (2017) MicroRNA-182 aggravates cerebral ischemia injury by targeting inhibitory member of the ASPP family (iASPP). Arch Biochem Biophys 620:52–58 15. Wang R et al (2018) miR-186-5p promotes apoptosis by targeting IGF-1 in SH-SY5Y OGD/R model. Int J Biol Sci 14(13):1791–1799 16. Zhou X et al (2016) MicroRNA-146a down-regulation correlates with neuroprotection and targets pro-apoptotic genes in cerebral ischemic injury in vitro. Brain Res 1648(Pt A):136–143 17. Liu W, Chen X, Zhang Y (2016) Effects of microRNA-21 and microRNA-24 inhibitors on neuronal apoptosis in ischemic stroke. Am J Transl Res 8(7):3179–3187 18. Xu LJ et al (2015) Post-stroke treatment with miR-181 antagomir reduces injury and improves long-term behavioral recovery in mice after focal cerebral ischemia. Exp Neurol 264:1–7 19. Sun H et al (2018) Upregulation of miR-215 exerts neuroprotection effects against ischemic injury via negative regulation of Act1/IL-17RA signaling. Neurosci Lett 662:233–241 20. Tao J et al (2015) MiR-207/352 regulate lysosomal-associated membrane proteins and enzymes following ischemic stroke. Neuroscience 305:1–14 21. Liu X et al (2013) MicroRNA-124-mediated regulation of inhibitory member of apoptosisstimulating protein of p53 family in experimental stroke. Stroke 44(7):1973–1980 22. Fu F, Wu D, Qian C (2016) The MicroRNA-224 inhibitor prevents neuronal apoptosis via targeting spastic paraplegia 7 after cerebral ischemia. J Mol Neurosci 59(3):421–429

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23. Moon JM, Xu L, Giffard RG (2013) Inhibition of microRNA-181 reduces forebrain ischemiainduced neuronal loss. J Cereb Blood Flow Metab 33(12):1976–1982 24. Yang X et al (2017) MicroRNA-15a/16-1 antagomir ameliorates ischemic brain injury in experimental stroke. Stroke 48(7):1941–1947 25. Ouyang YB, Giffard RG (2014) MicroRNAs affect BCL-2 family proteins in the setting of cerebral ischemia. Neurochem Int 77:2–8 26. Sun Y et al (2013) MicroRNA-124 protects neurons against apoptosis in cerebral ischemic stroke. CNS Neurosci Ther 19(10):813–819 27. Zhang R et al (2019) miR-1247-3p mediates apoptosis of cerebral neurons by targeting caspase2 in stroke. Brain Res 1714:18–26 28. Wei N et al (2016) MicroRNA-9 mediates the cell apoptosis by targeting Bcl2l11 in ischemic stroke. Mol Neurobiol 53(10):6809–6817 29. Li G et al (2018) Impact of microRNAs on ischemic stroke: from pre- to post-disease. Prog Neurobiol 163–164:59–78

6

The Emerging Role of microRNAs in Post-ischemic Angiogenesis and Neurogenesis

Abstract

Post-stroke angiogenic and neurogenic processes have become one of the highly studied domains to identify multifaceted therapeutic strategies bridging neurodevelopment and neuropathology. microRNAs (miRNAs) regulate the generation of new neurons in the stroke-damaged adult brain to propagate postischemic self-repair. Similarly, a large number of individual miRNAs control endothelial cell (EC) proliferation and migration and promote vascular-network formation pertinent to angiogenesis. The expression of these miRNAs can be modulated to achieve structural and functional re-establishment of neurovascular networks after ischemic stroke. This chapter highlights the role of multiple miRNAs in different stages of post-ischemic neurogenesis and angiogenesis that potentially favor long-term recovery against ischemic stroke. Keywords

Neurogenesis · Angiogenesis · miRNA · Neuronal regeneration · Neuronal repair

6.1

Introduction

Post-ischemic angiogenesis and neurogenesis form two major endogenous adaptive responses with the potential to limit brain damage. Emerging preclinical data indicates that microRNAs (miRNAs) regulate these interwoven endogenous restorative events, particularly in the sub-acute phase of ischemic stroke, enabling functional recovery after stroke [1, 2]. The specific role of miRNAs in post-ischemic angiogenesis and neurogenesis has been established by studying the effect of (1) the mutation of Dicer, an enzyme necessary for the maturation of miRNAs and (2) the modulation of individual miRNAs that fine-tune the post-transcriptional expression of angiogenic and neurotrophic factors. This chapter elaborates on the miRNA# Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_6

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mediated regulation of different stages of post-ischemic neuro- and angiogenesis that potentially favor long-term recovery against ischemic stroke.

6.2

Post-Stroke Induced Neurogenesis

Neurogenesis refers to the generation of new neurons that mature to form an intricate neuronal circuitry in an embryonic as well as in an adult brain. Neurogenesis encompasses multiple processes, including neural stem/progenitor cell (NSC/NSPC) proliferation, neuronal differentiation, and cell fate determination [3]. In the recent past, adult neurogenesis has become one of the highly studied domains to identify multifaceted mechanisms bridging neurodevelopment and neuropathology. It has been well-established that de-novo neurogenesis predominantly occurs at two distinct regions in the adult brain: the subgranular zone (SGZ) of the dentate gyrus of the hippocampus and the subventricular zone (SVZ) adjacent to the lateral ventricle [4, 5]. The stroke-damaged adult brain induces the formation of new neurons from NSPCs localized in these regions to propagate post-ischemic selfrepair. Besides, ischemia-induced neurogenesis may also occur in the non-neurogenic region, such as the striatum in the intact brain, where the proliferating NSCs migrate to the injured striatum and cortex. However, the effect is temporary, and most NSCs fail to survive. To have a significant impact on the post-stroke recovery, the potential self-repair mechanism must be enhanced, predominantly by regulating the survival and differentiation of the generated neuroblasts [6]. Every step in neurogenesis has been identified to be governed explicitly by different signaling pathways, growth factors, coding, and non-coding RNAs [7, 8]. Therefore, this section puts forth the critical role of miRNAs in the optimization of neurogenesis concurrent with other endogenous neuroregenerative responses vital for the efficient restoration of the brain against ischemic damage.

6.2.1

miRNA Regulation of Early Stages of Neurogenesis

Neurogenesis can be divided into several distinct stages, each of which is under the precise control of cell-intrinsic miRNA–target interactions (Table 6.1). miRNAs permit tight temporal and spatial regulation of the neurogenic processes within a confined microenvironment [33]. After cerebral ischemia, multiple miRNAs potentiate the activation of quiescent NSCs in the SGZ to re-enter the cell-cycle and turn into proliferative NSCs. The activated NSCs undergo self-renewal giving rise to early amplifying neural progenitors (aNPCs) to maintain and expand NSC pool [34]. However, the final fate of NSCs continues to be controversial, as activated NSCs may differentiate into astrocytes terminally [35] or may re-enter quiescence [36], after several rounds of asymmetric division.

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Table 6.1 An overview of miRNAs and its targets involved in neurogenic processes miRNAs involved in neurogenic Validated processes target NSC proliferation and differentiation miR-9 TLX

miR-137

let-7b miR-17-92 cluster

TLX and its co-repressor LSD1 TLX PTEN

miR-124

SOX9, JAG1

Migration of newborn neurons miR-379-410 N-cadherin cluster miR-9 Stathmin

miR-19

Rapgef2

Neuronal maturation and integration miR-9 Map1b

miR-21 miR-17-92

SPRY2 PTEN

miR-431

Kremen-1

miR-124 miR-132

miR-134

Sema3A p250GAP, MeCP2, MMP9 LimK1

miR-9

REST

miR-137

Mib1

miR-34a miR-541

TAp73 Synapsin-I

Biological function

References

Form a regulatory feedback loop to maintain a balance between NSC proliferation and differentiation Maintain NSC proliferation and differentiation

[9]

Maintains NSC transition states Overexpression of miR-17-92 cluster amplifies NSC proliferation in ischemic SVZ neural progenitor cells miR-124 upregulation reduces strokeinduced NSC proliferation and promotes neural differentiation

[11] [12]

Controls neuronal differentiation and migration Loss of miR-9 promotes migration of human embryonic stem cell-derived neural progenitors Migration of neurons during adult neurogenesis

[15]

Overexpression of miR-stimulates axonal branching and decreases axonal outgrowth Augments peripheral nerve regeneration Overexpression of miR-17-92 cluster enhances axonal outgrowth via PI3K/ mTOR signaling pathway Promotes axonal growth or elongation without affecting axonal branching through Wnt/β-catenin signaling pathway Axonal guidance Augments dendritic growth and remodeling

[18]

Negatively regulates dendritic spine size and modulates synaptic plasticity Loss of miR-9 leads to reduced dendritic length and complexity Overexpression of miR-137 in later stages of neurogenesis reduces dendritic complexity Synaptogenesis miR-541 upregulation reduces the neurite extensions

[10]

[13, 14]

[16]

[17]

[19, 20] [21]

[22]

[23] [24–26]

[27] [28] [29]

[30, 31] [32]

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6.2.1.1 NSC Proliferation and Differentiation NSCs undergo several rounds of proliferation to expand the NSC pool before differentiating into new neurons or astrocytes. Several miRNAs, along with their targets, strictly regulate this shift from proliferation to differentiation state. The nuclear receptor TLX works as a hub in miRNA-mediated regulation of NSC proliferation and differentiation. Under normal physiological conditions, TLX forms a regulatory loop with miR-9 and miR-137 to control neurogenesis in the brain. Zhao et al. showed that miR-9 targets TLX to impede NSC proliferation and enhance the differentiation, whereas TLX accelerates NSC proliferation by repressing miR-9 pri-miRNA expression. This regulatory feedback loop between TLX and miR-9 ensures a balance between NSC proliferation and differentiation in embryonic as well as in the adult brain [9]. Recently, miR-9 upregulation has been shown to positively affect neuronal regeneration in an in vitro model of ischemic stroke [37]. miR-137 forms a closed regulatory loop containing TLX and its co-repressor lysine demethylase 1A (LSD1) to maintain NSC proliferation and differentiation [10]. It is also noteworthy that miR-9 and miR-137 are differentially expressed post-ischemia, and their exogenous modulation favors neuronal survival [37–39]. Another miRNA, let-7b targets TLX receptor directly or via cyclin D1, an effector downstream of TLX, to maintain NSC transition states. Cyclin D1 is crucial for cell-cycle progression, and its downregulation by let-7b lengthens the G1 phase of cell-cycle, deters the cell-cycle progression into the S phase, and initiates the differentiation of NSCs [11]. Although let-7b has been remotely identified as a biomarker for ischemic stroke, its post-ischemic mechanism of action is not wellcharacterized [40]. Nevertheless, the complex regulation of TLX and the associated miRNAs by a feedback loop may act as a perfect mechanism to drive the transient switch of post-ischemic active NSCs from their proliferative state into differentiation. miR-17-92 cluster impacts NSC proliferation and differentiation in an adult brain. The members of the miR-17-92 cluster are upregulated in ischemic SVZ neural progenitor cells, suggesting their potential role in post-stroke induced neurogenesis [12]. The miR-17-92 cluster consists of six miRNAs on chromosome 13 that are transcribed as a single polycistronic unit. Overexpression of miR-17-92 cluster amplifies NSC proliferation, whereas the knockdown of its members (miR-18a and miR-19a) limits proliferation and exacerbates cell death. The effect of miR-18a and miR-19a in NSC proliferation can be attributed to their ability to target phosphatase and tensin homolog (PTEN), which has been previously identified as one of the negative regulators of NSC proliferation and survival [41, 42]. Emerging evidence suggests the unique role of miR-124 in post-stroke induced neurogenesis. In a non-ischemic brain, miR-124 regulates the temporal progression of neurogenesis by targeting SRY-box transcription factor (SOX9) in the SVZ region of an adult brain. miR-124 expression increases during the switch between transit-amplifying cells and neuroblasts, which continues to increase as neuroblasts exit the cell-cycle. Inhibition of miR-124 retains SVZ cells as dividing precursors, whereas its ectopic overexpression leads to precocious neuronal differentiation [13]. Further, it has been demonstrated that cerebral ischemia downregulates

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miR-124a in SVZ neural progenitor cells. The administration of miR-124a mimic dramatically reduces stroke-induced NSC proliferation and promotes neural differentiation by targeting jagged-1 (JAG1). Intriguingly, miR-124 mediates neural differentiation by distinctly targeting JAG1/SOX9 based on the ischemic/nonischemic condition in the brain [14]. The exosome-mediated delivery of miR-124 further supports its role in promoting neurogenesis after cortical ischemia [43].

6.2.1.2 Migration of Newborn Neurons The migration of newborn neurons in the dentate gyrus is essential for the development of accurate synaptic contacts with the pre-existing neuronal network. This migration is under the control of several chemo-attractants, such as Reelin, and adhesion proteins that are targeted by multiple miRNAs in the dentate gyrus. N-cadherin belongs to the superfamily of cadherins that must be precisely regulated to ensure complete neurogenesis and neuronal migration. miR-379-410 cluster finetunes the expression of N-cadherin in neural progenitors in the ventricular zone as well as in migrating neurons, controlling neuronal differentiation and migration [15]. Loss of miR-9 reportedly promotes migration of human embryonic stem cellderived neural progenitors perhaps due to its inverse correlation with Stathmin, the regulator of microtubule dynamics and cellular cytoskeleton [16]. The gain-offunction of miR-19 directly targets Rapgef2, contributing to an efficient migration of neurons during adult neurogenesis in addition to its role in NSC proliferation [17].

6.2.2

Neuronal Maturation and Integration

Neuronal maturation and integration encompass a set of individual processes such as axonal outgrowth, dendritogenesis, synaptogenesis, and neuronal guidance, to form a neuronal circuit. The newborn neurons migrate horizontally, and develop axonal processes as well as dendritic spines, facilitating neuron-neuron communication [44]. Strong synaptic connection with afferent neurons is critical to prevent the selective apoptosis of immature neurons [45–47]. The role of miRNAs in neuronal maturation and integration has been well-characterized, where each process is regulated by a specific miRNA or a subset of miRNAs.

6.2.2.1 Axonal Outgrowth and Neurite Outgrowth The role of the Dicer-dependent-miRNA pathway in nerve regeneration is one of the early studies that established the potential function of miRNAs in axonal outgrowth or regeneration [48]. Multiple reports suggest that loss of Dicer impaired axonal extension and implied that the dorsal root ganglion (DRG) axonal development relies on miRNAs [49–52]. Dajas-Bailador et al. demonstrated that miR-9 overexpression stimulates axonal branching and decreases axonal outgrowth by targeting microtubule-associated protein 1b (Map 1b) [18]. miR-9 also influences the activity of brain-derived neurotrophic factor (BDNF) on axonal growth and branching in a bi-phasic manner. Short or long-term stimulation with BDNF varies miR-9 expression to promote axonal growth and branching, respectively [18].

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miR-21 augments peripheral nerve regeneration by regulating the mRNA expression of Sprouty 2 (SPRY2) [19, 20]. It was first revealed by quantifying the expression levels of miR-21, miR-223, miR-455-5p, miR-431 miR-18, miR-138, miR-483, and miR-383 after sciatic nerve transection. Among the tested set of eight miRNAs, overexpression of miR-21 displayed maximum neurite outgrowth in adult rat DRG neurons [19]. The function of miR-21 in neuronal regeneration is independent of PTEN, one of the previously reported targets of miR-21 [53, 54]. However, inverse regulatory action between PTEN and miR-17-92 affects axonal outgrowth in embryonic cortical neurons. Overexpression of miR-17-92 cluster suppresses PTEN concomitant with the activation of the phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathway to enhance axonal outgrowth following spinal cord injury [21]. miR-431 promotes injury-induced axonal regeneration by targeting Kremen-1 through Wnt/β-catenin signaling pathway. miR-431 explicitly promotes axonal growth or elongation without affecting axonal branching [22]. This observation is in congruence with the fact that axonal outgrowth enhances regeneration process, whereas axonal branching can be detrimental; hence the axon growth and branching mechanisms are tightly regulated in mature neurons [55]. Following axonal and neurite outgrowth, the neurons undergo a definite alignment pattern. Differential expression of miR-124, miR-221, and miR-222 has been shown to affect the alignment of neurons after neurite outgrowth (referred to as neuron guidance) [56].

6.2.2.2 Dendritic Development and Synaptogenesis Synaptogenesis refers to the process of formation of synapses between neurons as the axons and dendrites continue to develop. Axon guidance, dendritic growth, and assembly of large synaptic protein complexes form the major events of synaptogenesis. Several miRNAs including the components of the miRNA machinery (Dicer and Argonaute homologue eIF2c), are expressed within dendrites and post-synaptic densities to regulate synaptogenesis [57]. One of the primary events of synaptogenesis is axon guidance including long-range guidance, fasciculation, and identifying correct targets. Axon guidance is modulated by molecules such as Ephrins, Netrins, Semaphorins, Slits, growth factors, morphogens, and cell adhesion molecules [58, 59]. Though miRNA regulation of axon guidance was rarely studied in the past, increasing evidence shows that miRNAs might play a pivotal role in the process of axon guidance. One of the early studies has shown that the depletion of Dicer leads to aberrant guidance of retinal ganglion cells (RGCs) axons [60]. Further, Dicer mutation in zebrafish exhibits defasciculated axons disrupting the axonal trajectory, and members of the miR-430 family partially rescue the aberrant axonal phenotype [61]. Interestingly, knockdown of miR-124 in RGCs causes inappropriate stalling of axons within the optic tectum due to incorrect axonal response to Semaphorin 3A (Sema3A) that wrongly targeted regions in the ventral border [23]. Hence, miRNAs are important regulatory molecules in axons; however, their specific roles in axonal guidance post-stroke remain mostly unknown. While axons transmit signals away from the cell body to the post-synaptic cell, dendrites receive signals from the pre-synaptic cell. miRNAs can control dendritic

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development and maturation. To date, numerous studies have enumerated the regulatory function of miR-132 in dendritic morphology, synaptic plasticity, and pathophysiology of neurological disorders like stroke, epilepsy, Alzheimer’s, and Parkinson’s disease [62–64]. miR-132 augments dendritic growth and remodeling through the repression of small Rho GTPase p250GAP in cortical and hippocampal neurons. miR-132 targets methyl CpG binding protein 2 (MeCP2), marking its role in epigenetics [24, 25]. Jasinska et al. showed that miR-132 also regulates dendritic spine structure in association with matrix metalloproteinase (MMP)-9 [26]. Another miRNA, miR-134, negatively regulates dendritic spine size and modulates synaptic plasticity in association with LIM Domain Kinase 1 (LimK1) mRNA [27]. miR-9 also influences dendritic development. Loss of miR-9 leads to reduced dendritic length and complexity via its downstream target RE1 silencing transcription factor (REST) [28]. Intriguingly, miR-137 exhibits distinct regulatory effects during the course of early and late stages of adult neurogenesis. Though miR-137 promotes proliferation and differentiation of adult NSCs, overexpression of miR-137 in later stages of neurogenesis reduces dendritic complexity by targeting Mind bomb one (Mib1), a ubiquitin ligase [29]. Another study established the functional role of two MIR137 gene variants in downregulation of matured miR-137 that adversely affects synaptogenesis and neural transmission in patients with schizophrenia and bipolar disorder [65]. miRNA precursors and mature miRNAs are expressed in different regions of the synapse, including post-synaptic densities and soluble components of synaptic fractions [66, 67]. miR-8 has been deemed necessary for the development of neuromuscular synapse in Drosophila at advanced stages of development [68, 69]. miR-8 mediates the localization of synaptic cell adhesion molecules such as Fasciclin III (FasIII) and Neuroglian (Nrg) and promotes synaptogenesis [69]. The miRNA-target combination of miR-34a/tumor protein p73 (TAp73) modulates postnatal brain development and neuronal differentiation with implications in synaptogenesis [30, 31]. The transcription factor TAp73 drives the expression of miR-137, which in turn regulates the expression of synaptotagmin-1 and syntaxin1A, the synaptic specific targets [31]. This miRNA-transcription factor machinery works in parallel with synaptogenesis reinforcing its role in neuronal development. Overexpression of miR-541 represses synapsin-I and reduces the neurite extensions drastically, whereas the knockdown of miR-541 displays converse effects [32]. Other synaptically enriched miRNAs include miR-134, miR-138, miR-132, and miR-124 [70].

6.2.3

miRNAs Regulating Neurotrophic Factors

Neurotrophic factors are small polypeptide molecules that mediate neuronal proliferation, migration, differentiation, and development of the nervous system [71]. The lack of neurotrophic factors (nerve growth factor (NGF), BDNF, ciliary neurotrophic factor, glial-derived neurotrophic factor (GDNF), vascular endothelial growth factor

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(VEGF), and insulin-like growth factor 1 (IGF-1)) in the penumbra zone of an ischemic brain can trigger neuronal apoptosis. In preclinical studies, these neurotrophic factors are modulated by different miRNAs and have all been shown to decrease infarct size in animal models [72]. For instance, let-7f antagomir acts on the IGF-1 signaling pathway for translation activation of IGF-1 to promote neuroprotection in the experimental ischemic stroke model [73]. Inhibition of miR-134 enhances BDNF and B-cell lymphoma-2 (Bcl-2) expression in neurons following oxygen-glucose deprivation, an in vitro model for ischemic stroke [74]. Other miRNAs, miR-107 and miR-30-5p, are also BDNF regulators. However, their precise mechanism in post-ischemic neuronal survival needs further investigation [75].

6.2.4

miRNAs and Neurogenesis Signaling Pathways

The initiation of neurogenesis in an ischemic brain occurs in association with the signaling pathways, namely, Hedgehog, Notch, Wnt, and transforming growth factor-beta (TGF)-β signaling pathways. miRNAs fine-tune these signaling pathways to effectuate proliferation, migration, and differentiation of NSCs to promote neuronal repair post-ischemia [76–79]. miR-21, which is upregulated post-ischemia, acts as an NSC regulator via Wnt and TGF-β signaling pathways. Following ischemic stroke, miR-34a negatively regulates the NSC proliferation through the inhibition of Notch, Wnt, Hedgehog, and TGF-β signaling pathways [80]. It has been already discussed earlier in this chapter that increased miR-124 levels promote neural differentiation with the post-transcriptional downregulation of SOX9. The effect has been ascribed to the ability of miR-124a to target the JAG1/ Notch signaling pathway, where miR-124a in NPCs reduces JAG1 mRNA and protein levels concurrent with the inactivation of Notch signaling [14]. Another study suggests a possible link between the Wnt signaling pathway, loss of function of Kremen-1 and gain of function of miR-431 in axonal regeneration [22]. Further, the Notch signaling pathway specifically drives proliferation, via its repressor protein NUMB Like, Endocytic Adaptor Protein (Numbl). Lack of Numbl in NSCs causes increased proliferation and symmetric division, taxing the NSC pool. Numbl expression has been reported to be regulated by both miR-184 and miR-34a, thus driving symmetric division [81, 82]. miR-34a also indirectly regulates Notch signaling by targeting neuronal differentiation 1 (NeuroD1) and Achaete-Scute Homolog 1 (Mash1), two proteins downstream of Notch [82]. Insulin/IGF pathway also aids in stem cell maintenance throughout aging. FoxO3, a component of the Insulin/IGF pathway, directly affects the expression of the miR106b-25 cluster. Expression of miR-25, a chief miRNA in the miR106b-25 cluster, increases NSC proliferation [83]. Besides NSC pool maintenance by regulating the rate of proliferation and cell division, miRNAs also control NSC quiescence. This phenomenon has not been demonstrated in adult hippocampal neurogenesis yet, and studies involving other stem cell niches are scarce. However, certain significant findings indicate the function of miRNAs in the regulation of NSC quiescence state. The conditional

6.3 miRNA Regulation of Post-Ischemic Angiogenesis

75

knock out of Dicer in muscle stem cells exhibited a rise in the rate of cellular proliferation, suggesting a transition from quiescent to an activated state. Advanced analysis of differences in miRNA expression between quiescence and activation state of cells provided various miRNA candidates, out of which miRNA-489 formed a critical regulator of stem cell quiescence [84].

6.3

miRNA Regulation of Post-Ischemic Angiogenesis

Angiogenesis refers to the formation of new blood vessels from the existing vasculature, which is typically active in developing tissues. Angiogenic activation involves endothelial cell (EC) proliferation, migration, and formation of tube-like structures that eventually mature into new blood vessels. These processes are orchestrated by maintaining a dynamic balance between pro- and anti-angiogenic factors transducing stimulatory or inhibitory signals for an adequate angiogenic response [85]. Regardless of the quiescent angiogenic processes and the stable vasculature in an adult brain, ischemic stroke induces angiogenesis, especially in the ischemic boundary zone, in an attempt to restore oxygen and nutrient supply to the affected regions of the brain. Extensive experimental research suggests that the ECs around the infarct area begin to proliferate as early as 12–24 h following ischemic stroke with a considerable increase in microvessel density at 3 days post-ischemia. Moreover, neovascularization in an ischemic brain can occur for a longer time as evidenced by the continued vessel proliferation for more than 21 days after cerebral ischemia [86]. Interestingly, stroke patients with higher cerebral microvascular density have been reported to recover faster and survive longer compared to patients with lower vascular density, indicating the role of post-ischemic angiogenesis in promoting neurological functional recovery [87]. There has been a recent upsurge in the studies linking the role of miRNAs in postischemic angiogenesis [1]. The early experimental evidence on the significance of miRNAs in angiogenesis involves the genetic manipulation of Dicer, an enzyme necessary for the maturation of miRNAs. Vascular-selective Dicer deletion in mice disrupts vascular phenotype and impairs angiogenic ability, which in turn is associated with reduced endothelial tube formation and slowed EC migration. These pathophysiological changes are further correlated with the ability of miRNAs to regulate key angiogenesis-related genes such as VEGF, its receptors KDR (VEGFR2) and FLT-1 (VEGFR1), as well as the putative angiopoietin-2 receptor Tie-1. Thereafter, a large number of individual miRNAs have been identified to control EC proliferation, migration, and formation of vascular-network relevant to angiogenesis [88].

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EC-Specific miRNAs in Angiogenesis

Several miRNAs exhibit prominent EC-specific expression patterns, and their dysregulation leads to vascular and neurological disorders [89–91]. The first comprehensive analysis involved the identification of 15 highly expressed miRNAs in ECs targeting receptors of angiogenic factors [92]. miR-126 is one of the wellcharacterized EC-specific miRNA located in the seventh intron of epidermal growth factor-like-domain 7 gene (EGFL7) [93–95]. Overexpression of EGFL7 directly influences the role of miR-126 in neovascularization following vascular injury. miR-126 regulates angiogenic processes such as (1) the migration and survival of ECs, (2) organization of the cytoskeleton, and (3) the formation of the capillary network. It has been reported that loss of miR-126 in mice causes vascular leakage, hemorrhaging, and embryonic lethality. Targeted deletion of miR-126 is also associated with defective vascularization and early death of animals subjected to ischemic insult [94], portraying the plausible role of miR-126 in post-ischemic angiogenesis. miR-155 is a multi-functional miRNA that potentially regulates endothelial morphogenesis [96]. Inhibition of miR-155 at 48 h following the experimental ischemia maintains cerebral vasculature and promotes blood supply to the periinfarct area by preserving the tight endothelial junctions (TJs) as well as BBB integrity. This has been attributed to the stabilization of tight junction protein (ZO-1), a scaffolding protein essential for the TJ assembly, parallel with the downregulation of miR-155. However, miR-155 mediated regulation of ZO-1 is associated with claudin-1, the direct target of miR-155. miR-155 mediated stroke recovery process is interpreted to be the outcome of (1) maintenance of vascular integrity, which inhibits the spread of ischemic damage into the peri-infarct area, (2) the stimulation of interleukin (IL)-10 mediated neuroprotective mechanisms, (3) switch from damaging microglia/macrophage phenotype to the neuroprotective and reparative phenotype. Other miR-155 targets include Ras homolog, MTORC1 binding (Rheb), SMAD5, Rictor, endothelial nitric oxide synthase (eNOS), suppressor of cytokine signaling 1 (SOCS-1), Inositol Polyphosphate-5-Phosphatase D (SHIP-1), and CCAAT Enhancer Binding Protein Beta (CEBPb) [97]. miRNAs such as let-7e and miR-27b, are also expressed in ECs and exert proangiogenic effects [98, 99].

6.3.2

Post-Ischemic miRNA Regulation of Angiogenic Factors

The gene regulation by miRNA typically follows three main principles, (1) coordinate principle, where miRNAs may act cooperatively through multiple target sites in one gene, (2) co-regulatory principle, where miRNA with the ability to target multiple genes could potentially regulate a group of functionally related genes, and (3) principle of differential regulation, where a gene with multiple binding sites for a number of miRNAs can be regulated by discrete miRNAs in cells under different conditions. All these principles apply to miRNA regulation of angiogenesis

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owing to the range of pro- and anti-angiogenic genes targeted by different miRNAs. Under ischemic stress, miRNAs modulate the angiogenic activation with the release of polypeptide growth factors and upregulation of the specific proangiogenic factors such as VEGF, TGF-β, platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF)-2, and MMPs [100].

6.3.2.1 Maneuvring the Proangiogenic VEGF Among a variety of proangiogenic factors, VEGF is ubiquitously expressed in the brain and acts as the central mediator of post-ischemic angiogenesis. VEGF binds to its receptors on the endothelial cell surface and activates intracellular tyrosine kinases. It triggers multiple downstream signals that favor a wide range of responses in ECs, including migration, proliferation, survival, differentiation, and increased permeability. Though there are multiple variants of VEGF ligand and its receptor (VEGFR), angiogenesis is predominantly mediated through the interaction of VEGF-A (often referred to as VEGF) with VEGFR-2 (Xk-1) [101–103]. A detailed understanding of how VEGF ligands and receptors elicit angiogenesis has been reviewed elsewhere [104, 105]. VEGF mRNA and protein expression increase in the salvageable penumbra region as early as 3 h, extending up to 7 days following ischemic stroke [105]. Besides, the transgenic overexpression of VEGF gene decreases infarct volume and promotes neurological recovery in a rodent model of focal cerebral ischemia [106]. Given the dynamic significance of VEGF in adult angiogenesis and ischemic stroke, it is not surprising that miRNAs can regulate VEGF signaling pathways by targeting transcripts encoding VEGF ligands and its receptors as well as other proteins that cross-talk with VEGF. For instance, miRNAs such as miR-107 and miR-126 augment the expression of VEGF, establishing their roles in post-ischemic EC function and angiogenesis. Permanent middle cerebral artery occlusion (pMCAO) in rats induces a strong miR-107 expression in the ischemic boundary zone with implications in post-stroke angiogenesis. The study reveals that miR-107 upregulation enhances tube formation and migration of ECs, while its inhibition reduces capillary density. The angiogenic activity of miR-107 has been attributed to the increased expression of endogenous VEGF165 via direct downregulation of its target, Dicer-1 gene [107]. Nonetheless, miR-126 overexpression enhances the proangiogenic activity of VEGF and maintains vascular integrity by repressing sprouty related EVH1 domain containing 1 (Spred-1), an intracellular inhibitor of angiogenic signaling [93]. Additionally, miR-210 promotes angiogenesis through VEGF signaling pathway in a renal ischemia/perfusion model [108]. A few miRNAs downregulate VEGF mRNA levels and potentially block postischemic angiogenesis. For instance, miR-377 inhibition restores VEGF and early growth response 2 (EGR2) levels to promote angiogenesis with the consequent reduction in ischemic brain injury and cerebral inflammation [109]. Interestingly, VEGFA and histone deacetylase 9 (HDAC9) form a positive feedback loop, such that the silencing of HDAC9 inhibits VEGFA expression in ECs. Being inversely correlated with HDAC9, miR-17-20 inhibition protects the sprouting defects impelled by the silencing of HDAC9 and the potential downregulation of VEGFA

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[110]. Recently, miR-93 and miR-200b are downregulated by hyperglycemic conditions. Both the miRNAs target the 30 UTR of VEGFR, and their inhibition increases VEGFA expression [111, 112]. miR-15a directly suppresses VEGF and FGF2 in ECs to control angiogenesis, while miR-20b represses VEGF levels in tumor cells to regulate cell survival in hypoxic conditions [113, 114]. VEGF also induces the expression of specific microRNAs that act downstream of VEGF signaling pathway. For instance, VEGF induces the expression of miR-16 and miR-424 that share a typical seed sequence. These microRNAs jointly target VEGFA, VEGFR-2, and FGFR1, and function in a negative feedback loop to control angiogenesis [115]. Another miRNA, miR-296, indirectly regulates VEGFR-2expression through hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), which defines sorting of ligand/receptor complexes to lysosomes for degradation [116]. Though multiple miRNAs influence VEGF expression to promote angiogenesis, it is imperative to consider that post-ischemic VEGF activity is time-dependent. Early post-ischemic administration of VEGF may cause increased blood–brain barrier (BBB) leakage, hemorrhagic transformation, and ischemic lesions. In contrast, late administration may enhance angiogenesis in the ischemic penumbra to promote neurological recovery [117]. Thus, miRNA regulation of VEGF during stroke recovery may diminish the neurological deficits after cerebral ischemia.

6.3.2.2 MMPs and Post-Ischemic Angiogenesis? MMPs are a family of endopeptidases that selectively degrade components of the extracellular matrix. Based on their domain organization, sequence similarities, and substrate specificity, MMPs can be of 24 different types. Activation of MMPs immediately after ischemic stroke is known to cause degradation of neurovascular matrix followed by increased BBB permeability, edema, and hemorrhagic transformation [118]. However, increased MMP activity may not always be confined to the declining neurological outcome, necessitating a re-evaluation of the development of MMP inhibitors to protect the brain against ischemic stroke. While MMPs seem to contribute towards brain damage in the early phase of ischemic stroke, it may aid in post-ischemic vascular remodeling and neurovascular repair as the days progress [119, 120]. Blocking MMP-9 7–14 days following ischemic stroke aggravates brain injury, suppresses neurovascular remodeling, and impairs functional recovery. Late inhibition of MMP-9 may also attenuate endogenous VEGF signals necessary for post-ischemic vascular repair [120]. Hence, it is imperative to tightly regulate MMP expression and re-assess the approach towards targeting MMPs throughout poststroke recovery. miRNAs can strictly regulate the proteolytic activity of MMPs at the transcriptional and protein level. Anti-miR-320 increases MMP-9, whereas anti-miR-181a-5p raises MMP-14 levels, influencing cell migration, invasion, and activation of angiogenesis [121, 122]. This is in line with the observation that the downregulation of miR-320 as well as miR-181a reduces infarct volume and improves neurological outcomes to protect the brain against ischemic stroke [123, 124]. Other miRNAs such as miR-191, miR-491-5p, and miR-497 target MMP-9 [125–127], while

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miR-29b and miR-15b suppress MMP-2 and MMP-3, respectively [128, 129], emphasizing the regulatory role of miRNAs in MMP induced angiogenesis. However, most research currently focuses on miRNA regulation of MMPs in tumor angiogenesis warranting more studies dealing with post-ischemic angiogenesis and vascular repair.

6.4

Coupling Angiogenesis and Neurogenesis Post-Ischemia

The processes of angiogenesis and neurogenesis following ischemic stroke are highly synchronized and interrelated. For instance, neuroblasts in the SVZ region migrate to the infarct boundary zone where post-stroke angiogenesis occurs. The migrating neuroblasts localize around cerebral blood vessels in areas of active vascular sprouting and remodeling. Blocking post-ischemic angiogenesis causes a tenfold reduction in neuroblasts in the peri-infarct cortex 7 days post-stroke, signifying the causal link between angiogenesis and neurogenesis within the poststroke neurovascular niche [130]. Specific miRNAs such as miR-9, miR-124, miR-210 promote the coupling of angiogenic and neurogenic processes in the normal as well as in the ischemic brain. In a developing brain, miR-9 connects neurogenesis and angiogenesis by the formation of neurons expressing VEGFA. miR-9 regulation of VEGFA is arbitrated by directly targeting the expression of the transcription factors, TLX and ONECUT. Moreover, MiR-9-mediated untimely rise in neuronal VEGFA has been reported to cause thickening of blood vessels outlying the normal formation of the neurovascular network in the brain and retina [131]. Thus, this dual role of miR-9 on NSC proliferation and angiogenesis can be exploited to develop regenerative therapies against ischemic stroke. miR-210 is often referred to as a pleiotropic hypoxamir, which is upregulated under hypoxic conditions in vitro. miR-210 is neuroprotective following cerebral ischemia, and the effects have been plausibly ascribed to the activation of brain angiogenesis and neurogenesis via VEGF [132, 133]. The study demonstrated that the overexpression of miR-210 increases the number of microvessels and the ECs in conjunction with the rise in the number of neural precursor cells and their proliferation in the SVZ upto 28 days [133]. Similarly, exogenous administration of miR-124 also enhances neurovascular remodeling and promotes angio-neurogenesis 8 weeks following ischemic stroke. A feedback loop between miR-124 and REST through the deubiquitinating enzyme ubiquitin specific peptidase 14 (Usp14) has been shown to mediate angio-neurogenesis and subsequent recovery following stroke [134]. Therefore, miRNA-mediated coupling of angio- and neurogenic processes is one of the radical neuronal restoration strategies for long-term improvement against ischemic stroke.

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Conclusion

The post-ischemic activation of angiogenesis and neurogenesis has stirred immense interest in the scientific community renewing our knowledge of the time window for stroke recovery. It also challenges the current concept of “the earlier, the better” for the administration of therapeutic interventions against ischemic damage. The poststroke structural and functional re-establishment of neurovascular networks is governed by an array of miRNAs that fine-tune the expression of various angioneurogenic targets. Though multiple miRNAs have been identified to regulate different stages of angio- and neurogenic processes, their effects on functional consequences and the level of post-ischemic brain repair remain elusive. Future steps may encompass the application of findings from rodents to primate models of stroke to ascertain the indispensable role of miRNAs in post-ischemic neuronal regeneration and vascular modeling.

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7

MicroRNAs as Potential Diagnostic, Prognostic, and Therapeutic Biomarkers in Ischemic Stroke

Abstract

Biomarker represents an unavoidable tool, having immense clinical utility serving the purpose of ease and accuracy in diagnosing a disease. Its clinical value extends beyond diagnosis to therapeutic applications for better treatment outcomes. The gold standard of protein biomarkers has outlasted over decades, but a slow progression of microRNAs (miRs) as a novel biomarker has risen as of late. The unique nature of miRs has enabled them to be more efficient than proteins and hence is considered over the latter in many diseases, including stroke. This chapter looks into some of the critical miRs reported to have clinical application; emphasizing circulating RNA in the context of ischemic stroke. Keywords

Biomarker · Circulating microRNA · Diagnostic marker · Prognostic marker

7.1

Introduction

Ischemic stroke, due to its complex pathophysiological nature, needs a holistic approach for diagnosis and pharmacological intervention. This intricacy can be attributed to several of the underlying risk factors such as hypertension, diabetes, atherosclerosis, atrial fibrillation, etc. Stroke, according to Trial of Org 10,172 in Acute Stroke Treatment diagnostic criteria can be classified into five subtypes: largevessel disease, small vessel disease, cardioembolic stroke, other determined etiology, and cryptogenic ischemia stroke [1, 2]. The clinical side of different subtype’s calls for several imaging techniques such as CT & MRI, electrocardiogram among others to delineate one from the other. However, efficient diagnosis and management of stroke demand better tools and approaches which needs to be promising and reliable like biomarker. A biomarker can be any biomolecule such as a protein or nucleic acids (DNA or RNA) used as an agent to assess the state, severity, # Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_7

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7 MicroRNAs as Potential Diagnostic, Prognostic, and Therapeutic Biomarkers. . .

occurrence, and prognosis of a disease compared to control subjects [2, 3]. Thus on the basis of their application they can either act as diagnostic or prognostic marker. The clinical use of a biomarker can be even extended towards the therapeutic side, where impeding its activity can result in positive outcomes (Box 7.1). Box 7.1 • Diagnostic Biomarker They are biological entities that aid in the diagnosis of diseases. These are mostly used as a preliminary test to confirm the presence of a pathological state. Some examples include the presence of particular genetic variants and the levels of specific proteins in body fluids. • Prognostic Biomarker Similar to a diagnostic marker, the use of prognostic markers is necessary for predicting the likely outcome of a disease. These are bio-molecules that are measured to evaluate the course of a disease or a response to a therapeutic intervention in patients. Examples include the presence of a particular gene variant, patterns of gene expression or levels of a specific protein in body fluids. The recent years have seen a significant improvement in the field of biomarker research considering the wealth of information accumulated over the years. They are essential markers in many diseases such as cancer, neurodegenerative disorders, diabetes, and ischemic stroke [3]. Protein biomarkers were more prominent in the past decade; however, RNA, especially microRNAs (miRs) are gaining more attention over proteins [3, 4]. This is credited for their remarkable stability in whole blood, plasma, serum, and in circulation; thus evading endonuclease cleavage and loss of function. Moreover, miRs play a predominant role at several stage of stroke, not limited to inflammation, immune response, cell death, neuroprotection, etc. [1, 2]. Thus miRs as a peripheral biomarker would be a powerful tool for monitoring the progression of the disease as well as a measure of patient’s response to treatment. Peripheral microRNA biomarkers can be detected in plasma, serum, or as circulating miRs in blood. However, besides, cerebrospinal fluid (CSF) reflects a complete profile of miRs during any disease associated with CNS, thereby making them a better option to consider than blood or plasma. This is, in fact, true considering blood cells as a significant source of plasma miRs (58%), questioning the reliability of circulating biomarkers [5]. In a microRNA profile from patient samples (CSF), there were several miRs (miR-9-5p, miR-9-3p, miR-124-3p, and miR-1283p), brain enriched that were significantly elevated, correlating to those with severe brain damage caused by ischemia [6]. However, one major limitation to this is that it is invasive, and there are ethical issues when performing a lumbar puncture on healthy subjects [6]. Circulating miRs, on the other hand, takes advantage of the ease of detection in blood plasma or serum. Circulating miRs has emerged as a promising biomarker for

7.2 Important Circulating microRNAs in Ischemic Stroke

89

Table 7.1 Circulating microRNAs associated with ischemic stroke Circulating miRs miR-125b-2, miR-27a, miR-422a, miR-488, and miR-627 miR-30a and miR-126 Let-7b (LA)/Let-7b (others) miR-15a, miR-19b, miR-32 miR-136, and miR-199a3p miR-126, miR-130a, and miR-378 miR-222, miR-218, and miR-185 miR-21-5p and miR-30a-5p miR-9-5p and miR-128-3p miR-148b-3p/miR-151b and miR-27b-3p Let-7e-5p miR-223 miR-134

Expression Upregulated

Reference [10]

Down-regulated Down-regulated/ upregulated Upregulated

[11] [11]

Down-regulated Upregulated Upregulated Upregulated Down-regulated/ upregulated Upregulated Upregulated Upregulated

[12] [13] [13] [14] [6] [15] [16] [17] [18]

ischemic stroke recently, evidenced by several literature studies, where its diagnostic and prognostic values are highlighted [7–9]. Some potential circulating miRs for ischemic stroke observed in various studies are listed in Table 7.1.

7.2

Important Circulating microRNAs in Ischemic Stroke

7.2.1

Let 7

Let-7 is a large family of miRNA comprising of 12 members in humans, and are involved abundantly as regulators of gene expression in the CNS [19]. Let-7 family miRNA alterations were found in several studies recently stressing its importance as a diagnostic biomarker during cerebral ischemia. A clinical study involving 197 ischemic stroke patients were utilized to analyze the biomarker potential of one of the family members of let-7; let-7b (circulating miRNA) [11]. Similar studies were conducted on a different family member; let-7e/let-7e-5p [16, 20]. In all the cases, the let-7 family members were expressed highly in relative to control subjects except that circulating let-7b from a sub-group of patients (largevessel atherosclerosis) showed a lowered expression level, indicating a role in stroke pathology [11]. This was further confirmed via bioinformatics analysis, revealing some critical stroke-related genes as their targets.

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7.2.2

7 MicroRNAs as Potential Diagnostic, Prognostic, and Therapeutic Biomarkers. . .

MiRNA-125a-5p/miR-125b-5p/miR-125b-2

MicroRNA-125a and miR-125b both have been previously implicated in several pathways related to cerebral ischemia, such as inhibition of angiogenesis [21]. Besides, miR-125a-5p has been reported to support endothelial barrier in the blood-brain barrier and also aid in the differentiation of inflammatory cells [22, 23]. In the case of miR-125b-5p, they were shown to regulate synaptic morphology and function in the brain [24]. In a case-control based study to evaluate the potentiality of circulating miRs as biomarker, showed that both miR-125a-5p/125b5p level is upregulated and while the former stays elevated up to 90 days while the latter returned to normal. Therefore, the biological effects of miR-125a-5p might have more sustained relevance after acute stroke [25]. In a second study where patient samples, as well as in-vivo rat model of stroke, were utilized to show the importance of miR-125b-2 in acute stroke [10].

7.3

Embolic Stroke Versus Thrombotic Stroke

The main difference between embolic and thrombotic stroke is that the former is caused by a blood clot or plaque that develops elsewhere and then travels through the bloodstream to one of the blood vessels in the brain. In the latter, the blood clot develops in the blood vessels inside the brain. For efficient treatment, the two subtypes of stroke need to be identified and differentiate separately. To solve this issue, a group of researchers compiled a set of miRs via bioinformatics analysis, potentially discriminating ES to TS. These miRs include miR-15a-5p, miR-17-5p, miR-19b-3p, and miR-20a-5p. Besides, network analysis of the above miRs provides the possible gene targets, and subsequent pathway analysis revealed that PI3K-Akt signaling pathway was significantly enriched for the miR-17-5p and miR-15a-5p [26].

7.4

Cell Death

Apoptosis is the leading cause of neuronal death in the penumbra region following acute ischemic stroke [27]. Increasing evidences support the involvement of miRNAs in the regulation of these apoptotic processes before and after IS [28]. MiR-21 was first reported to have a protective role in ischemia reperfusioninduced cardiocyte apoptosis by inhibiting the phosphatase and tensin homolog (PTEN)/Akt dependent pathway [29]. In a study Plasma miR-21 and miR-24 were found to be significantly lower in acute cerebral infarction patients compared to the healthy subjects. Utilizing the National Institutes of Health Scales Score, the authors concluded that plasma miR-21 and miR-24 could act as potential early stage markers of acute cerebral infarction [30]. Moreover, further analysis on N2A neuroblastoma cells following oxygen-glucose deprivation (OGD) and reoxygenation indicates that

7.6 Neurogenesis and Angiogenesis

91

miR-21 may have an anti-apoptotic effect, while miR-24 may have a proapoptotic effect [30]. MiR-16 belongs to cluster miR-15/miR-16 that has been well documented as an apoptosis-related miRNA and also involved in p53 signaling pathway. However, its relevance in hyperacute cerebral infarction proved the diagnostic, prognostic, and disease management properties of miR-16 [31, 32]. miR-15a/16-1 cluster dysregulation in plasma has been reported in patients with stroke acting as a potential biomarker for diagnostic and prognostic use. miR-15a/16-1 cluster upon inhibition via antagomir significantly reduced cerebral infarct size and improved neurological outcomes in stroke mice. This reduced infarct size is mainly attributed to their effect on both antiapoptotic proteins (upregulation) and suppression of proinflammatory molecules [33].

7.5

Inflammation

The inflammatory response is one of the important factors in IS, playing a pivotal role in the early events of ischemic insult, besides its role in long-term neuronal recovery. It has been reported that inhibition of inflammatory cells reduces ischemic brain injury [34]. Studies on IS patients have revealed several miRNAs significantly altered by targeting inflammatory factors. MiR-145 is another circulating miR that was found to be significantly upregulated within 24 h after stroke onset [35]. On the other hand, targeting miR-145 with an antagomir was found to be neuroprotective in vivo, indicating the potential use of miR-145 as a candidate biomarker or therapeutic target for stroke [36]. MiR-124 is the most abundant miRNA of the CNS and there is documented evidence of an association between high plasma levels of miR-124-3p and severity of stroke. The circulating miR-124-3p is increased in patients who died but it was most predictive within 6 h after onset of symptoms. This suggests the usefulness of miR-124-3p for prognosis, stratification, and predicting mortality [32]. Furthermore, there was significant correlation between ischemic lesion size and plasma miR-1243p.

7.6

Neurogenesis and Angiogenesis

Angiogenesis and neurogenesis are crucial for tissue repair and remodeling after brain injury. MiR-210 is a pleiotropic hypoxia-miR, which is the only miRNA that is upregulated under hypoxia [37]. miR-210 upon intracerebral injection can promote endothelial cell proliferation, new microvessel formation, and increase the number of neural progenitor cells in the subventricular zone via the vascular endothelial growth factor (VEGF) pathway [38]. However, miR-210 was found to be significantly upregulated in the adult rat ischemic cerebral cortex, thereby enhancing Notch1 signaling pathway [39]. But in acute IS patient’s miR-210 level was found to be significantly decreased upon stroke onset, and patients with higher circulating

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blood miR-210 provide a better clinical outcome, indicating the importance of blood miR-210 as a sensitive biomarker for clinical prognosis in acute cerebral ischemia.

References 1. Chen W et al (2019) Monogenic, polygenic, and microRNA markers for ischemic stroke. Mol Neurobiol 56(2):1330–1343 2. Georgios K, Gerasimos S, Konstantinos S (2013) The emerging role of microRNA in stroke. Curr Top Med Chem 13(13):1573–1588 3. Vijayan M, Reddy PH (2016) Peripheral biomarkers of stroke: focus on circulatory microRNAs. Biochim Biophys Acta 1862(10):1984–1993 4. Jickling GC, Sharp FR (2011) Blood biomarkers of ischemic stroke. Neurotherapeutics 8 (3):349–360 5. Pritchard CC et al (2012) Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies. Cancer Prev Res (Phila) 5(3):492–497 6. Sorensen SS et al (2017) Elevation of brain-enriched miRNAs in cerebrospinal fluid of patients with acute ischemic stroke. Biomark Res 5:24 7. Eyileten C et al (2018) MicroRNAs as diagnostic and prognostic biomarkers in ischemic strokea comprehensive review and bioinformatic analysis. Cell 7(12):249 8. Yi-Bing O et al (2013) microRNAs: innovative targets for cerebral ischemia and stroke. Curr Drug Targets 14(1):90–101 9. Gandolfi M et al (2017) Assessed and emerging biomarkers in stroke and training-mediated stroke recovery: state of the art. Neural Plast 2017:1389475 10. Sepramaniam S et al (2014) Circulating microRNAs as biomarkers of acute stroke. Int J Mol Sci 15(1):1418–1432 11. Long G et al (2013) Circulating miR-30a, miR-126 and let-7b as biomarker for ischemic stroke in humans. BMC Neurol 13(1):178 12. Selvamani A et al (2014) Circulating miRNA profiles provide a biomarker for severity of stroke outcomes associated with age and sex in a rat model. Clin Sci (Lond) 127(2):77–89 13. Jin F, Xing J (2017) Circulating pro-angiogenic and anti-angiogenic microRNA expressions in patients with acute ischemic stroke and their association with disease severity. Neurol Sci 38 (11):2015–2023 14. Wang W et al (2018) Diagnosis of hyperacute and acute ischaemic stroke: the potential utility of exosomal microRNA-21-5p and microRNA-30a-5p. Cerebrovasc Dis 45(5–6):204–212 15. Cheng X et al (2018) Exploring the potential value of miR-148b-3p, miR-151b and miR-27b-3p as biomarkers in acute ischemic stroke. Biosci Rep 38(6):BSR20181033 16. Huang S et al (2016) Identification of blood let-7e-5p as a biomarker for ischemic stroke. PLoS One 11(10):e0163951 17. Chen Y et al (2017) Increased circulating exosomal miRNA-223 is associated with acute ischemic stroke. Front Neurol 8:57 18. Zhou J et al (2018) Increased serum exosomal miR-134 expression in the acute ischemic stroke patients. BMC Neurol 18(1):198 19. Lee H et al (2016) Biogenesis and regulation of the let-7 miRNAs and their functional implications. Protein Cell 7(2):100–113 20. Peng G et al (2015) MicroRNA let-7e is a potential circulating biomarker of acute stage ischemic stroke. Transl Stroke Res 6(6):437–445 21. Che P et al (2014) miR-125a-5p impairs endothelial cell angiogenesis in aging mice via RTEF-1 downregulation. Aging Cell 13(5):926–934 22. Banerjee S et al (2013) miR-125a-5p regulates differential activation of macrophages and inflammation. J Biol Chem 288(49):35428–35436

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8

Interplay Between microRNAs and Other Cerebrovascular Diseases

Abstract

Cerebrovascular disorders constitute the world’s leading cause of morbidity and mortality. The role of miRNAs in initiating and advancing cerebrovascular diseases has recently become an important focus of attention. Through this chapter, we provide a brief overview of the ever-expanding role of miRNAs as a diagnostic and therapeutic tool for cerebrovascular diseases. Keywords

Intracerebral hemorrhage · Subarachnoid hemorrhage · Cerebral aneurysm · MicroRNAs · Biomarkers

8.1

Introduction

Cerebrovascular disease refers to a group of pathological conditions that affect the blood vessels of the brain and the cerebral circulation. Extended roles of miRNAs increase the complexity of gene-regulatory processes in cerebrovascular diseases. Recent studies have indeed shown that miRNAs are closely linked to cerebrovascular diseases such as intracerebral hemorrhage, subarachnoid hemorrhage, and cerebral aneurysms (Box 8.1). Such findings indicate a new miRNA-based therapeutic point cut for cerebrovascular diseases. Moreover, circulating extracellular miRNAs are stable in bodily fluids and emerging as potential biomarkers for non-invasive disease diagnosis and prognosis. This chapter focuses on the potential for blood or cerebrospinal fluid miRNAs to be used as novel biomarkers in the early diagnosis of cerebrovascular diseases. The findings discussed here can provide new insights into the physiological significance of microRNAs that promote the initiation and progression of various cerebrovascular diseases.

# Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_8

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Box 8.1: Cerebrovascular Diseases • Cerebrovascular diseases are a heterogeneous group of medical conditions such as stroke (hemorrhagic and ischemic), transient ischemic attacks, intracranial stenosis, cerebral aneurysms, moyamoya disease, and vascular malformations. • It is estimated that there are 31 million stroke survivors globally, although about six million deaths have been due to cerebrovascular disease (second most common cause of death in the world and sixth most common cause of disability). • Cerebrovascular disease occurs primarily at an advanced age; the risk of developing it increases significantly after 65 years of age.

8.2

Hemorrhagic Stroke

A hemorrhagic stroke refers to bleeding (hemorrhage) in the brain that unexpectedly interferes with its function. This bleeding may occur either inside the brain or between the skull and the brain. Hemorrhagic strokes make up for about 13% of all strokes and are divided into categories depending on the site and the cause of the bleeding: Intracerebral hemorrhage (ICH) and Subarachnoid hemorrhage (SAH). miRNAs have been reported to play key roles in the regulation of molecular processes following hemorrhagic stroke. Hemorrhagic stroke diagnosis and treatment may benefit from information about the stroke–miRNA system.

8.3

Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a subtype of stroke that occurs for 20–30% of all strokes, contributes to a severe injury, and has a 6-month mortality rate of more than 50%.

8.3.1

microRNAs as ICH Biomarkers

Hematoma enlargement (HE) is one of the most devastating and independent determinant of mortality and the functional outcome following IHC [1]. Since miRNAs are the most stable bioactive molecules that are expressed promptly during disease progression, the identification of specific differentially expressed miRNA signatures in HE may help in the diagnosis and risk stratification of ICH. Zheng and colleagues were the first to show that variations in miRNA expression occur after the onset of ICH [2]. Genome-wide miRNA expression levels in peripheral blood of ICH patients with or without HE indicated that a total of 30 independent miRNAs had differential expressions. The differentially expressed miRNAs were closely related to biological process such as inducing apoptosis (miRNA-29b/miRNA-29c/

8.3 Intracerebral Hemorrhage

97

miRNA-29b-1
, miRNA-574-5p, miRNA-22, and miRNA-122) and inflammatory mechanisms (miR-let-7f, miR-19a, miR-122, miR-29b, miR-29b-1
, and miR-29c), underlining an early hematoma expansion. The perihematomal edema (PHE) formed immediately after ICH deteriorates the hematoma’s space-occupying effect, resulting in intracranial hypertension, herniation, or even death [3]. The development and/or progression of PHE vary substantially and hematoma size alone is therefore inadequate to estimate the severity of brain edema at an early stage. The choice of therapeutic intervention(s) requires a novel, more sensitive indicator to evaluate PHE volume. Low expression of miR-126 was positively correlated with the extent of PHE, suggesting it may have a pathogenic role in the development of PHE after ICH [4]. Serum miR-130a has been found to be a non-invasive marker of brain edema and a location-dependent prognosis predictor in acute ICH [5]. MiR-130a originates mainly from thrombin-stimulated brain microvascular endothelial cells (BMECs) and aggravates the brain edema by increasing the permeability of blood–brain barrier (BBB) through the cav-1/MMP-2 and MMP-9 signaling pathway. miRNAs are expressed in a tissue- and/or cell-specific manner and their pattern of expression reflects underlying pathophysiologic processes. miRNAs can be detected in a remarkably stable form in serum and plasma, making them desired biomarkers for human diseases. To date, function and clinical implications of microRNAs are not well understood in ICH. Wang et al. discovered that miRNA, miR-124, was synchronously expressed in brain tissue and plasma in the ICH stroke model of the rat. Further, a similar miR-124 expression pattern was seen in human ICH patient plasma specimens [6].

8.3.2

Regulatory Role of microRNAs in ICH

Inflammatory mechanisms play a critical role in the pathophysiology of intracerebral hemorrhage [7]. Inflammasome, multi-protein complexes of the innate immune system, serves as a platform for caspase-1 activation and interleukin-1 (IL-1) maturation as well as pyroptosis [8]. The evidence demonstrates that NLRP3 inflammasome contributes to inflammation after intracerebral hemorrhage [9]. It has been demonstrated that miR-223 acts as crucial regulator of microglial activation, inflammation, and neuron injury after ICH by directly targeting NLRP3 [9]. The TLR/IL-1R superfamily mediates the innate immune response mainly by upregulating the expression of inflammatory genes [10]. A member of the IL-1 receptor (IL-1R)-associated kinase (IRAK) family, IRAK4, has been shown to play an essential role in toll-like receptor (TLR)-mediated signaling [11]. Yuan et al. identified an inverse relationship between miR-367 and IRAK4 expression [12]. The 30 UTR of IRAK4 mRNA contained conserved miR-367 binding sites, and miR-367 directly regulated IRAK4 expression through these 30 UTR sites. The results indicated that miR-367 downregulation leads to increased IRAK4 expression of microglia after ICH. The upregulated miR-367 attenuated NF-κB and inflammatory mediator expression of erythrocyte lysate-treated microglia. These results demonstrated that miR-367 attenuated inflammatory response of microglia via IRAK4. Following tissue injury, miRNAs are released into the extracellular space.

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Comparison of miRNAs from the same patient’s cerebrospinal fluid (CSF) and blood is unique as CSF is likely to reflect local events in damaged brain tissue compared to miRNAs in the bloodstream. Iwuchukwu et al. identified a significantly greater number of miRNAs in the CSF than plasma of ICH patients [13]. The large fold decrease in the plasma miRNAs that regulates inflammation-associated genes suggests that the systemic regulation of these genes may be affected. Such miRNAs may provide insights into the underlying neuroinflammation in ICH. Microgliamediated inflammation leads to secondary injury caused by ICH. Activated microglia has dual roles in brain injury and recovery as both proinflammatory (M1) and anti-inflammatory (M2) factors. MiR-124 was considered to be the most abundant miRNAs in the brain and reduced in ischemic stroke. Yu et al. showed that miR-124 plays a key role in inhibiting activation of microglia and promoting polarization of microglia M2 in ICH [14]. Furthermore, in vivo administration of miR-124 attenuates inflammatory injury and offers promising therapeutic strategy against ICH. The two major factors related to the severity of ICH are hematoma and inflammation. miRNA-132 has a well-known ability to potentiate the cholinergic anti-inflammatory pathway by targeting acetylcholinesterase (AChE), a hydrolytic enzyme for acetylcholine (ACh), thereby increasing ACh content. However, the role of miR-132 in intracerebral hemorrhage (ICH) remains unexplored. The mice overexpressing miR-132 in the brain responded with reduced neurological deficits and brain edema. The counts of activated microglia and the expression of pro-inflammatory cytokines were also reduced in these mice. In addition, BBB integrity improved, and the extent of neuronal death decreased in ICH mice injected with lentivirus encoding miR-132. On the contrary, a decrease in miR-132 expression elevated inflammation rate and increased cellular apoptosis. Overall, these results support neuroprotective role of miR-132 in an ICH mouse model, providing new therapeutic intervention opportunities [15]. The pathophysiologic process following ICH can be divided into two phases: the primary injury triggered by the mass effect of intraparenchymal hematoma and the secondary brain damage arising from oxidative stress (OS) and perihematomal neuroinflammation. miR-27b is an abundant neuronal miR implicated in numerous OS-related neurological disorders [16]. MiR-27b expression was decreased in the striatum after ICH, while Nrf2 mRNA and protein expression were increased. Intracerebroventricular injection of miR-27b antagomir and transfection of miR-27b inhibitor inhibited endogenous miR-27b in rats and PC12 cells, respectively. The activation of the ICH-induced Nrf2/ARE pathway was promoted by MiR-27b antagomir and reduced lipid peroxidation, neuroinflammation, cell death, and neurological deficits otherwise seen after ICH. The miR-27b inhibitor in PC12 cells reduced oxidative stress, inflammation, and apoptosis caused by iron, and these effects were blocked by the knockdown of Nrf2. MiR-27b inhibition reduced oxidative and inflammatory injury caused by ICH, raising the prospect of using miR-27b inhibition as a therapeutic strategy for ICH [17]. miR-126 is involved in the regulation of vascular inflammation, blood vessel integrity, and angiogenesis. In ICH patients, there is a reverse correlation between serum miR-126 and perihematomic edema. However, intracerebroventricular administration of miR-126-3p mimic reduced cerebral edema, inhibited neuron loss, and alleviated

8.4 Subarachnoid Hemorrhage

99

neurological impairment following ICH by targeting PIK3R2 and the Akt signaling pathway in brain vascular endothelium [18]. During the innate immune response to ICH, microglia are among the first non-neuronal cells on the scene. These are extremely important for hematoma removal and debris clearance, but they are also a source of persistent inflammation. Expression of microRNAs could modulate inflammatory response and brain damage in ICH. For instance, miR-590-5p expression was downregulated following ICH in vivo and LPS-induced microglia cells in vitro. miR-590-5p overexpression alleviated ICH-induced brain injury and inflammation through targeting Peli1 gene expression [19]. Peli1 was expressed abundantly in microglia and was also associated with neuroinflammation and brain injury following subarachnoid hemorrhage [20]. Similarly, miR-27a-3p was downregulated in the serum, hematoma, and perihematomal tissues in vivo, and intracerebroventricular administration of miR-27a-3p mimic reduced ICH-induced brain edema, leukocyte infiltration, microglia activation, BBB disruption, neuronal apoptosis, and neurologic deficits following ICH. miR-27a-3p overexpression inhibited the expression of AQP11 in BMECs by directly targeting the 3’-UTR of AQP11 [21]. These observations indicate that miR-27a-3p is critical in maintaining post-ICH brain homeostasis. Microglia polarization plays a key role in triggering ICH mediated inflammatory brain injury. Casein Kinase 2 Interacting Protein 1(CKIP-1) was established as a molecular transcription to control polarization of microglia. MiRNAs control the expression of genes and polarization of microglia. CKIP-1 was a target gene of let-7a and that let-7a regulated microglia M2 polarization by targeting CKIP-1 following ICH. Let-7a overexpression reduced CKIP-1 protein levels and inhibited proinflammatory cytokine expression, reduced brain edema, and improved neurological functions in the mouse model of ICH [22]. Recently, miR-181c overexpression has been shown to have an anti-apoptotic effect on neurons and improve neurological function in ICH rats, which is likely mediated by activation of the PI3K/Akt pathway by PTEN targeting [23].

8.4

Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a life-threatening form of stroke triggered by bleeding into the brain’s surrounding space. A ruptured aneurysm, arteriovenous malformation, or head injury may induce SAH.

8.4.1

microRNAs as SAH Biomarkers

Recent studies have shown that circulating serum miRNAs may potentially be used as biomarkers to indicate tissue damage. It was hypothesized that SAH contributes to changes in the expression of miRNAs in the brain and that these miRNAs are secreted into serum where they may serve as biomarkers for SAH. Serum samples on day three after the onset of SAH were subjected to microarray and quantitative PCR analysis. The results indicated that miR-502-5p and miR-1297 were potentially

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valuable indicators of the diagnosis, severity, and prognosis of SAH, and miR-4320 was a potentially valuable indicator of the diagnosis of SAH [24]. A pilot study was conducted to evaluate the feasibility of extraction and subsequent profiling of miRNA from CSF specimens in an aSAH patient population and to establish whether there is a distinct signature of CSF miRNAs between patients who experience cerebral vasospasm and patients who do not. The results showed that temporal miRNA profiling could detect differences between CSF from aSAH and non-SAH patients. Furthermore, the miRNA profile of CSF samples from patients who developed cerebral vasospasm was distinguishable from those who do not. Such findings provide the basis for future research into the discovery of novel CSF microRNA biomarkers that may predispose the development of cerebral vasospasm after SAH and therefore influence subsequent clinical management [25]. Delayed cerebral infarction (DCI) is a clinically relevant surrogate marker for neurological outcome following SAH and is a major cause of morbidity post-aSAH. Lu et al. demonstrated that a combination of four miRNAs (miR-4463, miR-4532, miR-4793, and miR-1290) could produce two non-overlapping clusters to clearly distinguish SAH with and without DCI [26]. Further, the classifier could also differentiate healthy controls from each SAH subtype with or without DCI, demonstrating its disease specificity. In an explorative study, miRNA profiles in cerebrospinal fluid (CSF) were compared between neurologically healthy patients and SAH patients with and without subsequent development of DCI. miR-21 and miR-221 showed a relative increase in SAH patients with DCI compared with those without, indicating that SAH is associated with marked changes in the CSF miRNA profile and these changes may be associated with the development of DCI [27]. In another study, a global miRNA expression analysis profile of patients following aSAH was carried out using small RNA deep-sequencing. Eight miRNAs were found to be expressed differently in patients with aSAH, three being upregulated: hsa-miR-146a-5p, hsa-miR-589-5p, and hsa-miR-941; and five being downregulated: let-7f-5p, hsa-miR-126-5p, hsa-miR-17-5p, hsa-miR-451a, and hsa-miR-486-5p. The MYC gene was found to be correlated with its regulation with the highest number of closely related differentially expressed miRNAs, indicating it as an important target involved in SAH. In addition, as novel players in this complex disease, 15 potential novel miRNAs were found occurring only in samples from aSAH patients. Such findings provide tools for potential studies to identify novel biomarkers to help in the clinical management of patients with a predisposition to develop an aSAH [28]. Arterial vasospasm is a well-known delayed complication of aSAH. However, no validated biomarker exists to help clinicians discriminate aSAH patients who may develop vasospasm (VSP+) and recognize those who merit intensive preventive therapy afterwards. Whole-blood miRNAs may be a source of candidate biomarkers for vasospasm. In a prospective cohort study, whole-blood miRNA profiling between VSP+ patients with aSAH and non-vasospasm patients (VSP) showed significantly lower levels of hsa-miR-3177-3p in VSP. There is evidence that the decrease in levels of hsa-miR-3177-3p after aSAH has been associated with a rise in levels of LDHA mRNA in VSP patients. Therefore, whole-blood levels of hsa-miR-3177-3p can be used as a valid biomarker for post-aSAH vasospasm [29].

8.5 Conclusions

101

Early brain injury (EBI) is the leading potential cause of SAH mortality and morbidity. Apoptosis is one of the main pathologies of SAH-induced EBI. The mesenchymal stem cells (hucMSCs) derived from human umbilical cord exert neuroprotective effect through exosomes. Additionally, miR-206 targets BDNF and plays a critical role in brain injury diseases. Zhao et al. found that treatment with hucMSCs-derived miR-206-knockdown exosomes has a greater neuroprotective effect on SAH-induced EBI compared to treatment with simple exosomes [30]. The miR-206-knockdown exosomes significantly improved neurological deficit and brain edema and reversed neuronal apoptosis by targeting BDNF/ TrkB/CREB signaling. Likewise, glycine, a non-essential amino acid, was neuroprotective post-SAH by upregulating miRNA-26b, leading to downregulation of PTEN accompanied by activation of AKT. Further, glycine treatment suppressed SAH-induced M1 microglial polarization and thereby inflammation [31].

8.4.2

Disease Modifying Role of microRNAs in SAH

After the discovery of miRNAs and their significance in control of posttranscriptional gene expression, genetic profiling has shifted from DNA level to RNA level over the past decade. Su et al. compared the circulating microRNA profiles of SAH patients and healthy individuals, and the circulating microRNA profiles of SAH patients with and without delayed cerebral infarction (DCI) [32]. KEGG pathway analysis showed the involvement of miRNAs and target genes for axon guidance and TGF-β signaling, suggesting that the resulting differential pattern of miRNA expression represents the effects of SAH rather than disease etiology. In contrast to healthy controls, miR-132 and miR-324 displayed upregulation in both SAH DCI and non-DCI cohorts. In another study, microRNA profiling of human cerebrospinal fluid from eight patients after aneurysmal SAH (aSAH) was performed daily for 10 days with the aim of identifying changes in microRNA abundance [33]. Two unique clusters of differentially regulated microRNAs over time were identified. The first cluster contained miRs (miR-92a and let-7b) known to be present in blood and decreased in abundance over time. The second cluster contained several poorly characterized miRs (most notably, miR-491) that increased in abundance over time. The study assumed that these miRs may have pleiotropic effects on brain injury and the brain’s response to injury. Further research into CSF miRs may contribute to a better understanding of brain injury after aSAH and lead to the development of novel therapeutics [33].

8.5

Conclusions

Cerebrovascular disorders represent a set of severe and complex neurological illnesses, and the exact etiologies of which are unknown. MicroRNAs are as important mediators of post-transcriptional gene silencing in both the physiology of brain development and pathology of cerebrovascular diseases (Table 8.1). Given their multifaceted functions, microRNAs may serve as a novel and promising theranostic target for the early diagnosis, prevention, and treatment of various cerebrovascular diseases.

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Table 8.1 Summary of reported miRNAs involved in various cerebrovascular diseases microRNA miR-21-5p

Disease Intracerebral hemorrhage

Treatment/ biomarker Antagomir

miR-93

Antagomir

miR-23b

Exogenous overexpression by transfection with lentivirus or miR-23b mimics

miR-24

Subarachnoid hemorrhage

Antagomir or mimic

Effect • Reduced the neurological defects • Repaired cognitive impairment • Alleviated BBB permeability • Inhibited neuronal apoptosis posthemorrhage • Accelerated hematoma absorption • Suppressed TLR4/ NF-κB signaling pathway • Improved neurological function • Suppressed inflammation • Attenuated cerebral edema • Repressed neuronal apoptosis • Suppressed inflammation in vitro by targeting inositol polyphosphate multikinase (IPMK) • Autophagy regulation through the Akt/mTOR pathway • miR-24 expression levels were increased in SAH patients with vasospasm • miR-24 inhibition increased NOS3 expression • Negative regulatory association exists between miR-24 and NOS3 • Downregulation of NOS3 may induce vasospasm following SAH, which may be due to the upregulation of miR-24

Reference [34]

[35]

[36]

[37]

(continued)

References

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Table 8.1 (continued) microRNA miR-1297

Disease Aneurysmal subarachnoid hemorrhage (aSAH)

Circulating exosomal miRNAs

Intracranial aneurysm (IA)

Treatment/ biomarker Prognostic biomarker

Diagnostic biomarkers

miR-3313p

Clinical and preclinical profiling

miR-4735

Overexpression and downregulation in vitro

Effect • Poor outcome at 1 year was associated with significantly higher levels of serum miR-1297 value in aSAH patients • miR-145-5p and miR-29a-3p were upregulated in plasma exosomes IA patients. • To distinguish IA patients from controls, the area under the receiver operating characteristic curve was 0.791 for miR-29a-3p • The area under the receiver operating characteristic curve for miRNA-145-5p was 0.773 in terms of discriminating whether the aneurysm was ruptured. • Maintains the contractile type of vascular smooth muscle cells, to inhibit the progression of IA. • Inhibits the formation of IA by downregulating TNF-α and CD14. • Plays an important role in phenotypic modulation in IA by regulating autophagypromoted smooth muscle cells proliferation and migration

Reference [38]

[39]

[40]

[41]

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9

New Insights into the Regulatory Role of lncRNA, circRNA, piRNAs, and ceRNAs in Ischemic Stroke

Abstract

The past decade witnessed a paradigm shift of attention from a protein-centric approach to RNA centric approach in understanding the complexity of multifactorial diseases like ischemic stroke. Thus, marking the beginning of a new class of non-protein-coding genes, exemplified by the intense research on microRNA, in human physiology and disease. However, microRNAs are just the beginning of a whole new world of non-coding RNAs (ncRNAs), with unexplored limitless functionalities. These ncRNAs are emerging as prominent regulators of gene expression and as a potential therapeutic modality in various disease pathologies including but not limited to cancer, neurological, and cardiovascular diseases. Here, we look upon some relevant ncRNAs: long non-coding RNAs (lncRNAs), PIWI-interacting RNAs (piRNAs), circular RNAs (circRNAs), and others, their molecular interaction and therapeutic potential in the context of stroke pathology. Keywords

lncRNA · piRNA · circRNA · Non-coding RNA · Ischemic stroke

9.1

Introduction

9.1.1

Non-coding RNAs at a Glance

At the beginning of the genomic period, there was a great deal of interest in discovering the unlimited potential of protein-coding genes in normal physiology and development, as well as in various human diseases. Nevertheless, coding genes are only a small part of the genome (1–2%), most of which are considered junk genes without physiological significance [1]. The notion of junk gene shifted when it was revealed that a class of small RNA molecules called microRNAs (miRNAs) play a # Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_9

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New Insights into the Regulatory Role of lncRNA, circRNA, piRNAs, and. . .

Table 9.1 Different classes of ncRNAs and their approximate sizes

S.No. Short ncRNAs 1. 2. 3. 4. 5. 6. 7. 8. Long ncRNAs 9. 10. 11. 12.

Classes of ncRNAsa

Size

miRNA tiRNA piRNA TSSa-RNAs PASRs snRNA snoRNA PROMPTs

19–24 bp 17–18 bp 26–31 bp 20–90 bp 22–200 bp ~150 bp 60–300 bp 200 bp >200 bp >200 bp >200 bp

snoRNA small nucleolar RNA, snRNA small nuclear RNA, XIST X-inactivation specific transcript, miRNA microRNA, piRNA PIWIinteracting RNA, lncRNA long non-coding RNA, tiRNA transcription initiation RNA, PROMPTs promoter upstream transcripts, PASRs promoter-associated small RNAs, TSSa-RNAs TSS-associated RNAs a Indicates that the different classes of ncRNAs are not necessarily clear and distinct as some may be part of a larger class like lncRNA (H19 and XIST)

significant role in a wide variety of cellular and molecular processes [2–4]. These RNA species along with others were collectively referred to as non-coding RNAs (ncRNAs). The functional aspect of ncRNAs, especially miRNAs, became more prominent considering their role in numerous pathological disorders including but not limited to cancer, neurological, and cardiovascular diseases [5, 6]. In recent years, a plethora of ncRNAs has been discovered (Table 9.1), with miRNAs being the highly worked out RNA, while others such as long non-coding RNAs (lncRNAs) are slowly gaining importance [7]. These intriguing molecules regulate many cellular processes like apoptosis, differentiation, development, and, most importantly, transcriptional and post-transcriptional modulation resulting in gene silencing effect [7–9]. A tissue-specific expression are often observed for certain class of RNA molecules like circular RNAs (circRNAs), enriched in the brain, especially in neuronal cells, points to their specific demands in orchestrating complex molecular interactions [10, 11]. As of late, reports have emerged as to the role of ncRNAs in the brain such as neuronal development and maintenance, synaptic plasticity, and synaptogenesis [12–14]. Their dysregulation is often linked to many pathological conditions: Alzheimer’s diseases, glioblastoma, ischemic stroke, etc. and thus emerged as a potential therapeutic target having clinical implications [15– 18]. Here, we will look at different species of ncRNAs, their biogenesis in general,

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regulatory cross-talk during cerebral ischemia, and where the field of non-coding RNA will lead us in the future of translational research. There is no bottom to the ocean of information that could be extracted from different ncRNAs and translate it into clinical applications.

9.2

New Players in the Game

9.2.1

Long Non-coding RNA

Long non-coding RNAs are a diverse class of ncRNAs that are grouped based on the length of their transcript (Table 9.1); covering a large proportion of non-protein coding genes. Despite other members of the parent class, the transcriptome of lncRNAs outreaches that of the protein-coding genes [19]. The expression pattern of lncRNAs reveals a tendency towards specific cell/tissue types and are involved in different stages of development and disease. A look at the functional aspects shows the importance of lncRNAs in various biological processes: genomic imprinting, dosage compensation, X chromosome inactivation, etc. The importance of lncRNAs in organ development, especially brain and CNS, can be attributed to their crucial role in numerous processes like synapse formation, neuronal development, and even neurogenesis [7, 19]. This factor alone can be considered for its implication in several neurological diseases ranging from Alzheimer’s, Parkinson’s to glioma, and cerebral ischemia [18, 20]. The expression profile of lncRNAs was much like that of miRNAs upon ischemic insult, and many studies have arrived at different lncRNAs, for example, linc-DHFRL1-4, linc-SNHG15, and linc-FAM98A-3. There are many lncRNAs differentially expressed during cerebral ischemia, which are involved in many important pathways like angiogenesis, inflammation, cell death, etc. (Table 9.2). Besides the above-mentioned roles, lncRNA-N1LR was found to promote neuroprotection via acting through p53 pathway [26, 28, 30–32]. The concept of competing endogenous RNAs (ceRNAs) has recently emerged, wherein different ncRNAs like lncRNA, cirRNAs compete towards mRNA binding Table 9.2 Different lncRNAs, their molecular target, and expression level during cerebral ischemia lncRNAs lncRNA SNGH12 lncRNA MEG3

Expression Upregulated Upregulated

lncRNA MALAT1 lncRNA SNHG14

Upregulated Upregulated

lncRNA-N1LR lncRNA SNHG1

Upregulated Upregulated

Target miR-199a miR-181b p53 protein miR-21 miR-30a miR-145-5p miR-136–5p p53 protein miR-338

Reference [21] [22–24]

[25] [26, 27] [28] [29]

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site on miRNA, thereby altering its function [22, 32]. In a study by Yan et al., lncRNA MEG3 acts as ceRNA and aids in ischemic neuronal cell death by competing with programmed cell death 4 (PDCD4) mRNA in binding directly to miR-21 [23]. Thus, lncRNAs associate with different aspects of stroke pathology, including its role as neuroprotectant, making them a viable target to explore further.

9.2.2

Circular RNA

Circular RNAs are an intriguing and exciting class of ncRNAs with unique biological and structural properties, conserved, and expressed across diverse species: humans, flies, mouse, etc. [33]. It is one of the emerging classes of RNA molecules reported to have functional relevance (Box 9.1), not only physiological but also pathological relevance in the mammalian system, but still in its early stages [8, 34]. The widespread interest in understanding the mechanistic behavior of circRNAs in various diseases owing to its role as gene expression regulators often via circRNA-miRNA-gene axis [35]. Circular RNAs abundant expression in brain and its functional play in numerous cellular processes: neuronal differentiation and development, synaptogenesis, and neuronal plasticity have made it a prominent target to explore in various neurological diseases, especially stroke [36]. Box 9.1 Circular RNA Circular RNA are widely known for its involvement in several important functions such as neuroplasticity, synaptogenesis, nervous system development, neurogenesis, and differentiation, etc. besides they are considered as a crucial target in various pathological conditions like glioma, Parkinson’s disease, multiple sclerosis, etc. The expression profile of circRNAs analyzed in an in vitro and in vivo stroke model reveals a significant alteration in the level of several circRNAs: mmu_circRNA_40001, mmu_circRNA_013120, mmu_circRNA_40806 to name a few, following cerebral ischemia [37]. A similar study in oxygen-glucose deprivation/reoxygenation (OGD/R) model revealed mmu-circRNA-015947 to be involved in the pathogenesis of cerebral ischemia/reperfusion injury [38]. Besides expression profile studies, the miRNA sponge function of circRNAs (circDLGAP4) was first demonstrated in a study by Bai et al., where circDLGAP4 acts as an endogenous sponge to inhibit miR-143 activity (Table 9.3). Furthermore, increasing the expression of circDLGAP4 resulted in a positive outcome: decreased infarct areas and blood–brain barrier damage in an in vivo stroke model [39]. In addition to the above study, circ_008018 and circHectd1 were validated experimentally to interact with miR-99a and miR-142, further confirming its role as endogenous miRNA sponges. To complement this, knockdown of the two above-

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Table 9.3 Circular RNAs differentially expressed during ischemic stroke circRNA circDLGAP4 mmu_circRNA_40001 mmu_circRNA_013120 mmu_circRNA-015947 circ_008018 circHectd1 a

Expression Down-regulated Upregulated

Targeta miR-143 –

Reference [39] [37]

Upregulated Upregulated Upregulated

– miR-99a miR-142

[38] [40] [41]

Indicates only experimentally verified target were included

mentioned circRNAs attenuated neurological deficits, reduced cerebral I/R induced injury, and ameliorated astrocyte activation [40, 41]. As evidenced from the above studies circRNAs represent a class of potential RNA molecule that could serve as a novel biomarker and therapeutic target for ischemic stroke. Despite all the progress to date, the broader picture regarding its biology remains unanswered and unexplored.

9.2.3

PIWI RNA

The new entrant into the class of ncRNAs is called PIWI (P-element induced wimpy testis interacting)-RNAs (piRNAs), which were initially observed in germ-line cells in flies (Drosophila melanogaster) and mouse [9]. They are regarded as the “guardian of genome” due to their ability to suppress transposons mediated genetic mutations [42]. The unique feature of piRNA that stands out from the rest of its allies lies in its biogenetic pathway: primary processing and ping-pong amplification pathway [43, 44]. Recently, piRNAs were identified in the mouse hippocampus, where they were enriched in dendritic spines. Besides, piRNA pathways present in neurons were shown to contribute to axon growth, neural repair, and regeneration [45]. There were reports of piRNAs role in various cancers, but of recently, they were identified to play a significant role during cerebral ischemia. In the first study, a substantial alteration in piRNAs expression level could be seen upon transient focal ischemia. Moreover, through a bio-informatics approach, the authors have identified transposons as targets of piRNAs and that TFs play a considerable role in the regulation of stroke responsive piRNAs during ischemia [46]. This is the first report on the effect of piRNAs during a stroke, and further extensive study at the molecular level is required to untangle the functional significance of brain enriched piRNAs. Even though the available data relating piRNAs role in cellular homeostasis and various disease is limited and inconclusive, yet it holds enormous potential in regulating gene expression at different tissue levels.

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9.3

Conclusion

The discovery of ncRNAs and their prominent interaction at multiple levels of gene expression; transcriptional and post-transcriptional level, has changed the landscape of research from the outlook of protein-centric to RNA centric studies. The technological advancement has enabled researchers to identify new transcripts, initially thought of as transcriptional noises, with functional relevance. The small world era began with a single player (microRNAs) in the game, but to date, it has been expanded to different classes of RNAs, each with a complex nature and functionality. These studies represent just the tip of the iceberg with an incomplete picture, and further, more in-depth analysis is required to understand their biology.

9.4

Future Perspective

In the past two decades, we have only scratched the surface of RNA world, which itself has provided a glimpse of its immense importance in the development of an organism to its complex interplay in various pathological states. The depth of the pot is still untouched, and we are far from understanding and discovering the complete map of different ncRNAs. These little transcripts offer a full potential in deciphering the intricate balance of cellular homeostasis. However, from the standpoint of translational research, it is questionable in a way as to whether targeting a single or multi-ncRNAs will outsmart the conventional method of treatment (proteincentric). Due to the overcomplicated nature of all the RNA molecules so far discovered, only time can answer whether a transition is possible for ncRNAs from bench to bedside.

References 1. Wright MW, Bruford EAJHG (2011) Naming ‘junk’: human non-protein coding RNA (ncRNA) gene nomenclature. Hum Genomics 5(2):90 2. Miska EA (2005) How microRNAs control cell division, differentiation and death. Curr Opin Genet Dev 15(5):563–568 3. Carthew RW (2006) Gene regulation by microRNAs. Curr Opin Genet Dev 16(2):203–208 4. Kosik KS (2006) The neuronal microRNA system. Nat Rev Neurosci 7(12):911–920 5. Khoshnam SE et al (2017) Emerging roles of microRNAs in ischemic stroke: as possible therapeutic agents. J Stroke 19(2):166–187 6. Hayes J, Peruzzi PP, Lawler S (2014) MicroRNAs in cancer: biomarkers, functions and therapy. Trends Mol Med 20(8):460–469 7. Fatica A, Bozzoni I (2014) Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet 15(1):7–21 8. Haque S, Harries LW (2017) Circular RNAs (circRNAs) in health and disease. Genes (Basel) 8 (12):353 9. Ozata DM et al (2019) PIWI-interacting RNAs: small RNAs with big functions. Nat Rev Genet 20(2):89–108 10. Rybak-Wolf A et al (2015) Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed. Mol Cell 58(5):870–885

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35. Rong D et al (2017) An emerging function of circRNA-miRNAs-mRNA axis in human diseases. Oncotarget 8(42):73271–73281 36. Hanan M, Soreq H, Kadener S (2017) CircRNAs in the brain. RNA Biol 14(8):1028–1034 37. Liu C et al (2017) Screening circular RNA expression patterns following focal cerebral ischemia in mice. Oncotarget 8(49):86535–86547 38. Lin SP et al (2016) Circular RNA expression alterations are involved in OGD/R-induced neuron injury. Biochem Biophys Res Commun 471(1):52–56 39. Bai Y et al (2018) Circular RNA DLGAP4 ameliorates ischemic stroke outcomes by targeting miR-143 to regulate endothelial-mesenchymal transition associated with blood-brain barrier integrity. J Neurosci 38(1):32–50 40. Yang X et al (2018) Downregulation of circ_008018 protects against cerebral ischemiareperfusion injury by targeting miR-99a. Biochem Biophys Res Commun 499(4):758–764 41. Han B et al (2018) Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke. Autophagy 14(7):1164–1184 42. Halic M, Moazed D (2009) Transposon silencing by piRNAs. Cell 138(6):1058–1060 43. Beyret E, Liu N, Lin H (2012) piRNA biogenesis during adult spermatogenesis in mice is independent of the ping-pong mechanism. Cell Res 22(10):1429–1439 44. Luteijn MJ, Ketting RF (2013) PIWI-interacting RNAs: from generation to transgenerational epigenetics. Nat Rev Genet 14(8):523–534 45. Phay M, Kim HH, Yoo S (2018) Analysis of piRNA-like small non-coding RNAs present in axons of adult sensory neurons. Mol Neurobiol 55(1):483–494 46. Dharap A, Nakka VP, Vemuganti R (2011) Altered expression of PIWI RNA in the rat brain after transient focal ischemia. Stroke 42(4):1105–1109

Part III IschemiRs: The Bench and Beyond

Computational Resources for microRNA Research

10

Abstract

MicroRNAs being one of the most diversely conserved regulatory non-coding RNAs with an immense role in various physiological and pathological processes, have found its ways into the spotlight. The exponential flow of data related to their discovery, target prediction, function, and interaction with other non-coding RNAs, etc. can be seen over the years. Several bioinformatics tools and databases can be seen recently, that were devised to manage this growing body of data. Some of them are widely relied upon by researchers globally, and more such databases and web servers are arising with sophisticated functionality. This chapter briefly touches the highly important computational tools (databases, webservers) in general and specific to microRNAs and ischemic stroke. Keywords

Bioinformatics tools · Databases · Web server · Computational methods

10.1

Introduction

Over the past few years, a large amount of data has been generated related to microRNAs using high throughput techniques such as next-generation sequencing. These data are associated with sequence identification, novel genes, target prediction, disease interactions, etc. Based on computational predictions, 60% of human protein-coding genes are targeted by miRNAs through conserved base-pairing between the 3’ UTR of mRNA and the 5’ region of miRNA, called the seed region [1]. Given the enormous involvement of microRNAs in gene regulation as well as disease processes, several bioinformatics tools are available to manage the mounting data flow. Both basic and applied miRNA research can be enhanced by computational tools and databases. Most of these tools are accessible through an online interface; researchers globally can utilize these cutting-edge tools and databases, and # Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_10

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even laboratories with poor computational infrastructure can apply this through free online interfaces. Here we look into some of the bioinformatics tools and accessories that can be applied for microRNA research. Each section describes specific categories of miRNA research, and various computational tools widely relied upon are detailed.

10.2

MiRNA Identification

MiRNA identification is complicated and requires an interdisciplinary strategy [2]. In recent years, biological and bioinformatics approaches have enabled discovery of thousands of miRNAs in plants, animals, unicellular eukaryotes, and viruses [3, 4]. They are now collected in the miRBase, the leading online repository of miRNA sequences and annotation. The latest release of miRBase (v22) contains microRNA sequences from 271 organisms: 38,589 hairpin precursors and 48,860 mature microRNAs [5]. A variety of algorithms are applied to discover new miRNAs from NGS data. These tools consider some major miRNA features, like sequence conservation among species, and structural features like a hairpin and minimal folding free energy [6]. MiRscan and miRseeker are the primary tools that target conserved intragenic sequences that can form hairpin structures based on RNAfold and Mfold, respectively [7, 8]. A newer approach, known as machine learning, has subsequently been devised to enhance and improve the prediction of novel microRNAs. BayesmiRNAfind, MiRFinder, MatureBayes are some of the databases which use machine learning algorithms to predict novel miRNAs [9–11]. In miRNA identification, cleavage site, binding and target discovery tools are also an important tool. Since the mechanism of Dicer cleavage site selection is not completely understood, tools such as LBSizeClea and PHDcleav are used to predict these site (utilizing a Support Vector Machine (SVM) model) [12, 13]. There have been many tools developed base on AGO protein family to decipher the miRNA binding sites. This is due to the central role played by AGO protein family an essential component of the RISC. Thus some of the tools that were developed include miRBShunter, Antar, and miRTar2GO [14–16].

10.3

MicroRNA Prediction

MicroRNA prediction involves initial identification of genes that code for particular miRNA and many different techniques have been utilized, such as cloning followed by sequencing, blotting, microarray, etc., however, the major drawback of these techniques were that they are tedious and time-consuming. Moreover, some of the miRNAs whose expression levels are low may not be detected via the traditional approaches. The emergence of Next Generation Sequencing technology has bypassed the above drawbacks and has become a reliable and sensitive method to identify miRNAs. Besides these experimental approaches, several bioinformatics tools have been developed to complement them to validate novel miRNAs [17].

10.3

MicroRNA Prediction

Table 10.1 Some of the commonly referred databases for miRNA research

119 Purpose MiRNA identification

MicroRNA prediction

MicroRNA-disease interaction

MicroRNA-target interaction

MicroRNA-TF interaction

Database • miRBase • MiRscan • miRseeker • BayesmiRNAfind • MiRFinder • MatureBayes • Mfold • RNAmicro • ProMir, • HHMMiR • Triplet-SVM • PalGrade • MiR2Disease • PhenomiR • HMDD • NSDNA • TargetScan • RNAhybrid • DIANA-microT-CDS • DIANA-TarBase • miRTarBase • TransmiR • ChIPBase • CircuitsDB

The bioinformatics tools developed to identify miRNAs have run on several algorithms, which are mostly based on sequence conservation. For instance, Mfold and RNAfold tools are based on minimum free energy concept, thereby predict putative secondary structure [18, 19]. In a nutshell, the computational algorithms used for miRNAs structure prediction can be classified into two categories; comparative and non-comparative methods. Some of the tools that are included in comparative methods are MiRscan, miRseeker, RNAmicro, MiRFinder ProMir, HHMMiR, etc. (Table 10.1). The comparative tools also include machine learning approaches that use different strategies to predict novel miRNAs such as Hidden Markov Model (HMM), Support Vector Machine (SVM), and Naïve Bayes Classifier. RNAmicro and MiRFinder are some of the examples of SVM based tools, while MiRRim, SSCprofiler, ProMir, HHMMiR are HMM based tools [20]. The non-comparative method does not rely on the concept of sequence conservation and has thus the advantage of identifying species-specific or non-conserved miRNAs. These non-comparative methods includes tools such as Triplet-SVM, HHMMiR, miPred, miR-abela, and PalGrade [15].

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Computational Resources for microRNA Research

MicroRNA Disease Interaction

It is an established fact that miRNA deregulation is associated with several human diseases [21]. There have been a lot of studies aimed at understanding the complex regulation of microRNA during any pathological condition. Thus to organize, retrieve, and to keep these data up to date, an online knowledge base is crucial. In the past, a lot of computational tools have been established (Table 10.1). MiR2Disease is a manually curated database detailing on miRNA deregulation in various human diseases. It contains miRNA-disease interaction data, miRNA expression pattern in the disease experimentally verified miRNA target gene(s), and literature references [22]. PhenomiR is one of the highly cited databases providing information about miRNAs differentially regulated in disease and other biological processes [23]. Human microRNA Disease Database (HMDD) is another most widely referred database that provides experimentally supported human miRNA-disease interaction data from various sources such as genetics, epigenetics, circulating miRNAs and miRNA-target interactions [24]. The Nervous System Disease NcRNAome Atlas (NSDNA) is a manually curated database that provides comprehensive experimentally supported associations between Nervous System Diseases (NSDs) and ncRNAs. The current version of NSDNA documents 26,128 associations between 144 NSDs and 8736 ncRNAs in 11 species, curated from 1410 articles [25]. Besides, there are several online resources linking microRNA to disease, which are specific to certain diseases like cancer.

10.5

MicroRNA-Target Interaction

It is a fact that miRNAs down-regulate their target gene expression by targeting 3’ UTRs of mRNAs through sequence-specific binding. Moreover, a single miRNA can target multiple genes and several miRNAs can target a single gene [26]. Thus knowing the target mRNA of a microRNA could be beneficial for microRNA therapeutics. MicroRNA-target interaction prediction tools as well as experimentally validated targets are available via databases and webservers. Validating a possible miRNA target in the laboratory is expensive and time-consuming, since a single miRNA can have a large number of potential target sites. Computational approaches can help reduce the number of experimental validation. The prediction algorithm takes into account of a number of features to accurately predict the target gene and site. An in-built feature of most tools is that prediction thresholds can be entered, to manage prediction sensitivity or accuracy level. Some of the widely used and cited web tools include TargetScan, RNAhybrid, DIANA-microT-CDS, to name a few [27–29]. Even with the most sophisticated computational resources in hand, experimental validation is well needed. There are numerous techniques for experimental validation, which includes qRT–PCR, luciferase reporter assays, and western blotting. The technological advancement has given rise to high-throughput sequencing (HITS) techniques such as microarrays,

10.7

Future Prospects

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proteomics, and sequencing-based methodologies such as RNA-Seq, HITS-CLIP, PARCLIP, and Degradome-Seq to establish the interaction [26] experimentally. DIANA-TarBase is one of the most cited manually curated target databases. The latest version contains more than half a million miRNA-target interactions (MTIs), curated from published experiments. It incorporates data from high throughput experiments to support the interaction data. The miRTarBase is second in the line of the widely relied database to understand the target interaction. It has accumulated more than 50,000 MTIs from over 18 species that are collected using a data mining approach. All the MTIs are validated experimentally by using reporter assay, western blotting, microarray, and NGS experiments [30].

10.6

MicroRNA Interaction with Transcription Factors

MiRNAs and transcription factors (TFs) are two important classes of regulatory molecules in gene regulatory networks. MiRNAs play a regulatory role at the posttranscriptional level. It has become clear that they do not act independently, but cooperate with other molecules like TFs to regulate target genes, or execute specific functions indirectly. Thus a lot of studies have come up with establishing the link between transcription factors and microRNA. These data are compiled and organized in an easily accessible way. TransmiR is one such database, manually curated that uses TF-miRNA regulatory relationships from the literature. It provides experimentally validated TF-miRNA regulations for multiple species and miRNA associated diseases, where available [31]. ChIPBase is the mostly updated database, which integrates chromatin immunoprecipitation with next generation DNA sequencing (ChIP-Seq) data to facilitate annotation and discovery of TF binding maps and transcriptional regulatory relationships of miRNAs from ChIP-Seq data [32]. CircuitsDB is another database that provides miRNA-TF regulatory circuits in the human and mouse genomes. Mainly the miRNA-TF feed-forward regulatory loop (FFL), data can be retrieved from the database; such that a master regulator is targeting a microRNA or TF and gene [33].

10.7

Future Prospects

Even with the highly equipped technology and use of high-throughput data, a lot of web servers and databases have emerged recently. These tools can come in hand for the researchers in reducing a considerable amount of time. But because computational tools are not a fool-proof resource for microRNA research, and that more sophisticated and accurate tools are needed to prevent false-positives. Moreover, the data has to be experimentally validated to finalize and conclude any hypothesis.

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MicroRNA-Targeted Therapeutics for Ischemic Stroke: Status, Gaps and the Way Forward

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Abstract

Post-stroke miRNA modulation is anticipated to revolutionize ischemic stroke therapeutic landscape despite the challenges and the future efforts necessary for the successful translation of preclinical observations to clinical practice. This chapter summarizes the currently employed strategies for the restoration or inhibition of microRNA (miRNA) function and the methods used for the delivery of miRNAs. The rising interest in the use of pharmacological agents and smallmolecules to regulate miRNA expression, with special emphasis on ischemic stroke, has also been discussed. Keywords

miRNA · Mimics · Inhibitors · miRNA delivery · Exosomes · Small-molecule

11.1

Introduction

An increasing inventory of studies demonstrating post-stroke gain- or loss-of-function of miRNAs or a family of miRNA suggests the decisive role of microRNAtargeted therapeutics for the treatment of ischemic stroke [1–3]. The short mature miRNA sequences are conserved across different vertebrate species making it relatively simple to target while allowing their use in preclinical studies and clinical trials. Currently, there are two primary methods to modulate miRNA activity: (1) restoration of miRNA function using synthetic RNA duplexes or vector-based overexpression and (2) inhibition of miRNA activity using chemically modified antimiR oligonucleotides, locked nucleic acids (LNAs), or miRNA sponges. This chapter provides useful insight into the current landscape of miRNA-targeted therapeutics in ischemic stroke, challenges, and future efforts necessary for the successful translation of miRNA-based bench discoveries into therapeutic modalities.

# Springer Nature Singapore Pte Ltd. 2020 R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke, https://doi.org/10.1007/978-981-15-4798-0_11

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Restoration of miRNA Function as a Potential Ischemic Stroke Therapy

The most common strategy for the restoration of miRNA activity is to introduce miRNA mimics, a small RNA molecule that resembles endogenous miRNA. Typically, miRNA mimics are synthetic RNA duplexes, where one strand is identical to the miRNA of interest (guide or antisense strand), and the opposite (passenger or sense) strand harbors chemical modifications [4–6]. These modifications include a cholesterol molecule link to enhance stability and cellular uptake, asymmetric 50 modification of small interfering RNA (siRNA) strands to check RISC loading [7], or 20-fluoro (20-F) modification to protect against exonucleases in addition to enhancing the stability of guide strand without interfering with RNA-induced silencing complex (RISC) loading [7, 8]. However, the application of these chemical modifications is limited because the guide strand needs to function as a miRNA, and the cell must identify it per se. miRNA mimics are designed to contain a sequence motif on its 50 -end partially complementary to the 30 untranslated (UTR) sequence of its target gene. Once introduced into the cells, miRNA mimics load the active strand into the RISC and then bind specifically to its target gene inducing post-transcriptional repression or translational inhibition of the gene [9]. Numerous reports have highlighted the utility of miRNA mimics in the downregulation of target genes associated with ischemic stroke pathology, eventually leading to neuroprotection. For instance, the transfection of miR-9 mimic downregulates Bcl2l11 and suppresses neuronal apoptosis in an experimental model of ischemic stroke [10]. Recently, miR-9 upregulation has been reported to augment neuronal survival as well as neuronal regeneration following oxygen-glucose deprivation, an in vitro model for ischemic stroke [11]. miR-124 upregulation promotes stroke-induced neurogenesis by suppressing jagged 1 (JAG1) in neural progenitor cells [12]. The exogenous upregulation of miRNAs such as miR-17-92 cluster, miR-122, miR-21, and miR-210 also enhances neuronal survival and functional outcome following experimental ischemic stroke [13–16]. Although systemic miRNA-mimic transfection restores the post-stroke miRNA levels, it might cause potential off-target effects due to the undesirable cellular uptake into tissues that do not generally express the transfected miRNA. To prevent these unwanted side effects, targeted delivery of miRNA to the appropriate cell/ tissue type is of utmost importance. Apart from these chemically modified miRNA mimics, many studies have also reported the use of lenti-, adeno- or adenoassociated viruses (AAV) to steer the expression of a given miRNA and to restore its activity within the brain [17–19]. These delivery strategies are discussed later in the chapter.

11.3

11.3

Development of Anti-miRNA-Based Ischemic Stroke Therapeutics

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Development of Anti-miRNA-Based Ischemic Stroke Therapeutics

One of the most widely used approaches to regulate miRNA levels in an experimental ischemic stroke model involves the use of an anti-miRNA molecule to block the action of a specific miRNA known to aggravate the post-ischemic neuronal injury. Anti-miRNAs are generally modified antisense oligonucleotides containing the full/ partial complementary reverse sequence of a mature miRNA (Fig. 11.1). Ideally, an efficient anti-miR must be cell-permeable, stable in vivo, highly specific, and non-toxic.

11.3.1 Antisense Oligonucleotides Antisense-oligonucleotides can bind to a target miRNA and stop the subsequent translational arrest [20, 21]. However, these oligonucleotides have been rendered

Anti-sense oligonucleotides

Locked Nucleic Acid

Anti-miRNA therapeutic strategies

Chemically modified Antagomirs

Fig. 11.1 Current anti-miRNA therapeutic strategies in ischemic stroke

miRNA sponge

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impractical due to their (1) susceptibility to serum nucleases, (2) negatively charged backbone causing poor cell-permeability, and (3) high off-target effects as they hybridize with other similar oligonucleotide sequences or stimulate immune responses. Also, the overall use of antisense oligonucleotides for therapeutic purposes has been limited by their pharmacokinetic profile [22, 23].

11.3.2 Chemically Modified Antisense Oligonucleotides/Antagomirs Since simple antisense oligonucleotides were deemed unsuitable for therapeutic investigation, chemically modified antisense oligonucleotides were designed with superior features for the inhibition of specific miRNAs. One of the most common chemical modifications for improved nuclease resistance and rapid, but stable hybridization includes 20 -O-methyl (20 -O-Me)-modification [20, 24]. Although 20 -O-Me-modified RNAs effectively inhibited the slicing action of miRISC, they are still disposed to degradation by serum exonucleases, making them unsuitable for in vivo studies [20, 23, 24]. Another most viable modification has been the substitution of non-bridging oxygen in the phosphate backbone with a sulfur atom to form a phosphorothioate (PS) bond. As a result, an improvement in the half-life time of oligonucleotides in the bloodstream was detected that reportedly ranges between 1 and 4 weeks [25]. However, the enhanced stability of PS-bond containing oligonucleotides comes at the expense of low hybridization affinity to the corresponding target miRNAs [26]. Nevertheless, PS-bond containing oligonucleotides can bind to plasma proteins facilitating more uniform distribution and cellular uptake via bloodstream compared to other modified oligonucleotides. Other modifications include the introduction of a methoxyethyl group at the 20 -sugar position (20 -MOE) or a fluorine atom substituent at the C20 -ribose position (20 -F). As opposed to 20 -O-Me, 20 -MOE-modified oligonucleotides are nuclease resistant, more lipophilic, and demonstrate a higher affinity to their target sequences. Though 20 -F modification does not affect serum nuclease resistance, the fluorine atom in the 20 -position induces the ribose ring into a high C30 -endo conformation, which is distinctive for A-form duplexes and contributes to an excellent affinity for target RNAs [27].

11.3.3 Locked Nucleic Acid (LNA) With the advancement of miRNA research, it has been challenging to develop and improve tools for miRNA detection and functional analysis at low expression and concentration levels. One of the most successful nucleotide derivatives and essential inclusion in the miRNA toolbox is LNA [28]. In LNAs, the ribose moiety consists of an additional methylene bridge linking the 20 oxygen and 40 carbon that “locks” the ribose in the C30 -endo conformation. This arrangement improves base stacking and backbone pre-organization in A-form duplexes and shields from degradation.

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LNA-modified oligonucleotides have high thermal stability when hybridized with their corresponding mRNA target molecules, making it an ideal nucleotide derivative to target small RNAs, such as miRNAs [29, 30]. LNA-modified oligonucleotides are of great importance to study the effects of miRNA inhibition following ischemic stroke. For instance, LNA-modified antisense miR-92a has been reported to induce cell-protective, pro-angiogenic, and anti-inflammatory effects in a porcine model of ischemia and reperfusion [31]. The inhibition of miR-210 by its complementary LNA post-hypoxic-ischemic (HI) insult significantly reduces miR-210 levels in the brain, decreases brain infarct size, and enhances long-term neurological recovery in HI neonatal rat model [32]. Presently, LNAs have been used in clinical trials to manipulate the miRNA activity in specific disease settings and one of the most potential druggable candidates.

11.3.4 miRNA Sponge The miRNA “sponge” method was introduced as an approach to produce continuous miRNA loss-of-function in cell lines and transgenic animal models. Sponge RNAs consist of complementary binding sites specific to the seed sequence of miRNA of interest that allows them to inhibit the activity of an entire family of related miRNAs. A sponge construct typically consists of four to ten binding sites that are set apart by a few nucleotides each. The efficacy of a miRNA sponge rests on two main factors (1) affinity and avidity of binding sites and (2) concentration of sponge RNAs concerning the concentration of the miRNA of interest. The expression of a sponge could be maximized by using the most potent available promoter based on the cell type, such as cytomegalovirus (CMV) promoter for mammalian cell lines. Although deletion of the miRNA encoding gene is the sole way to ensure complete loss of the miRNA’s activity, the sponge method offers many advantages such as (1) the ease of creating dominant-negative transgenics over knockouts, and their utility in broader range of cell lines and animal models, (2) supports the individual knockout of miRNAs and the animals are cross-bred to produce a strain with complete knockout, where the seed family members of these miRNAs are encoded at multiple distant loci, and present functional redundancy (3) the sponges act on the mature miRNA and their efficiency is unaltered by the proximity of miRNAs within a cluster [33]. Lately, miRNA sponges have been put to use for ischemic stroke translational research. A recent study demonstrated that circRNA DLGAP4 (circDLGAP4) acts as an endogenous miR-143 sponge that blocks miR-143 activity, and the level of circDLGAP4 reduces following acute ischemic stroke. Furthermore, the overexpression of circDLGAP4 decreases the infarct area and improves neurological function in an ischemic stroke model [34]. Another study showed how long non-coding RNA (lncRNA) TUG1 sponges miRNA-9 and increases post-ischemic neuronal apoptosis [35]. Although sponge technology has advantages in experimental settings, further research is warranted to convert it into a therapeutically viable strategy.

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Delivery of miRNA Modulators for Ischemic Stroke Therapy

The development of miRNA-based therapeutics remains divisive in terms of efficient and safe delivery methods, especially for the treatment of ischemic stroke, where the introduced miRNA should pass through the blood–brain barrier (BBB) without inducing neurotoxicity and/or off-target effects. Thus, different miRNA delivery systems have been tested to surpass the challenge of crossing BBB, including viral and non-viral vector-based delivery systems.

11.4.1 Viral-Based miRNA Delivery System The commonly used viral-based miRNA delivery systems include retroviruses, adenoviruses, AAVs, and lentiviruses that are modified to check self-replication and improve safety [36]. These delivery systems provide high transfection efficiency and facilitate a constant expression of miRNA mimics or antagomirs in an in vitro or in vivo experimental setting (Box 11.1). While retroviral vectors are generally used to deliver miRNAs into germline and somatic cells, lenti- and adenoviruses can infect mitotic as well as post-mitotic cells (for example, glia and neurons) [37, 38]. Lentiviruses, a subclass of retrovirus, translocate across the nuclear membrane and stably integrate into chromosomes offering multiple advantages over other viral delivery systems. First, lentiviral vectors can mediate long-term miRNA expression making it feasible to evaluate the therapeutic efficiency of the transferred miRNA without multiple injections [39]. This could be especially favorable in monitoring long-term post-ischemic neuronal repair mechanisms such as angiogenesis and neurogenesis. Second, the lentiviral vectors are reported to have higher compliance with experimental brain tissue and may transduce to astrocytes, neurons, or endothelial cells. Third, lentiviral vectors have relatively low adverse side effects and produce minimal immune response compared to an adenovirus vector [39]. As a result, lentiviral vectors have gained immense attraction as one of the main miRNA delivery methods for ischemic stroke research. Several studies have employed lentiviral-based miRNA expression system (for miRNAs such as miR-210, miR-424, miR-107, miR-134, miR-17-92, miR-124, miR-29b) to reinforce the understanding of the associated molecular mechanisms and their therapeutic potential following ischemic stroke [13, 16, 40–44]. For instance, lentiviral overexpression of miR-210 increases microvessel density and the number of neural progenitor cells with the resultant improvement in neurobehavioral outcomes in an in vivo model for ischemic stroke [16]. Similarly, lentivirus-mediated miR-424 overexpression inhibits neuronal apoptosis and microglia activation to confer neuroprotection following permanent focal cerebral ischemia [40]. However, the approach of locally delivering a miRNA to the brain using viral vectors is still far from practice because of the complications associated with direct injection into the human brain. The presence of endogenous neutralizing antibodies in human sera also limits the direct use of viral vectors. Further, the intravenous or intramuscular administration of viral vectors may drive their entry into an undesired cell or tissue

11.4

Delivery of miRNA Modulators for Ischemic Stroke Therapy

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types. Therefore, the choice of a reliable delivery method is critical to examining the therapeutic efficiency of miRNA. Box 11.1: Viral-Based Delivery System • In a viral-based delivery system, the virus binds to the host and introduces genetic material into the host cell during the course of its replication cycle. • Lytic and lysogenic are the two main types of virus infections. • Viruses of the lytic cycle are released from the cell infecting more cells soon after insertion of their DNA. • Lysogenic viruses integrate their DNA into the host cell and do not inflict any harm unless they are triggered to release the DNA.

11.4.2 Non-Viral miRNA Delivery Systems Unlike viral vectors, non-viral miRNA delivery systems are less toxic and generate a low immunogenic reaction. An efficient non-viral delivery system should carry the exogenous miRNAs or miRNA-expressing vectors, and protect the sequences from nuclease-mediated degradation within cells [36, 45]. Typically, non-viral delivery systems include physical and chemical approaches. Physical approaches apply external forces using gene gun, electroporation, hydrodynamic, ultrasound, and laser-based energy techniques to make the cell membrane transiently permeable for gene delivery. However, these methods impair cellular integrity, causing apoptosis and nuclease cleavage [46]. Physical methods are usually applied in in vitro studies and occasionally used for the miRNA delivery. Non-viral chemical methods involve carriers that are lipid-based, polymer-based, or inorganic [47, 48]. Lipid-based carriers, often known as lipoplexes or liposomes, encapsulate nucleic acids within a charged membrane-like surface that can be cationic, anionic, or neutral. Cationic liposome forms the most commonly used non-viral delivery system as they possess unique characteristics such as the ease of production, low pathogenicity, low immunogenic response, high affinity with the cell membrane and allows blood–brain barrier penetration [49, 50]. Currently, there are various commercially available cationic lipoplex for miRNA delivery, such as SilentFect™ (Bio-Rad) [51], Lipofectamine® RNAi-MAX (Invitrogen) [11, 40, 52], DharmaFECT® (Dharmacon) [53], and SiPORT™ (Invitrogen) [54], which have been widely reported to provide favorable results in ischemic stroke research. However, cationic liposomes may induce cytotoxic effects due to the presence of excessive positive charges, which may eventually interfere with the negatively charged proteins in the cells. This signifies that carriers ought to have low charge density to decrease cytotoxicity. Low charge density refers to the unstable carrier/ RNA complexes due to the weak charge interactions between the carrier and RNA. One key to this problem could be biodegradable carriers.

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Polymer-based non-viral systems include polyethylenimine (PEI), polyamidoamine (PAMAMs) dendrimers, polylactic-co-glycolic acid (PLGA), or cell-penetrating peptide (CPP) as delivery carriers. PEI has been mainly implemented for the delivery of miRNA mimics. Reportedly, PEI promotes endosomal escape through the proton sponge effect and increases the entry of hydrogen ions into acidic endosomes causing swelling and disruption of the intracellular vesicles. However, PEI has a very low transfection efficiency and poor biodegradability. PLGA has been primarily employed for the delivery of antisense oligonucleotides and siRNAs. Though PLGA is hydrophobic and must be coated/ functionalized for efficiency before oligonucleotide delivery, it mediates long-term dissociation from the carrier, leaving a prolonged effect [55]. Recently, PLGA nanoparticles were complexed with miR-124 to investigate the effect of miR-124 on survival and differentiation of neural stem cells in vitro, and stroke outcome in vivo [56]. PAMAMs are positive-charged polymers with relatively high transfection efficiency but may result in the accumulation of the polymers in the liver. Another variant of PAMAM conjugated with arginine (PAMAM-Arg) has been reported to exhibit remarkable transfection efficiency to primary neuronal and glial cultures compared to any other liposome or polymer [57, 58]. To overcome the degradability aspect of PAMAMs, polyamidoamine ester (ePAM-R), a degradable copolymer variety of PAMAM-Arg has been used for gene delivery to the ischemic brain [59]. Besides the aforementioned synthetic polymers, cell-penetration peptides (CPPs) are naturally derived polymers that are used as miRNA carriers [60]. As opposed to synthetic polymers, CPPs are less toxic but are susceptible to degradation in sera. In addition to lipids and polymers, a few studies have employed inorganic materials for miRNA delivery. Some of the current inorganic miRNA vectors involve carbon-nanotubes or nanoparticles (NPs) composed of gold (AuNPs), iron (II, III) oxide (Fe3O4), and silica, out of which AuNPs are the most frequently used miRNA carriers. AuNPs functionalized with a single layer of alkylthiol-modified, double-stranded RNA molecules can enter cells (without the help of cationic co-carriers), pass through the endocytic pathway into late endosomes, settle there for 24 h following transfection, and eventually mimic the function of endogenous miRNAs. These inorganic carriers are stable in vivo and generally free of microbial contamination, but they generate a weak interaction with nucleic acids. Nonetheless, the conjugation of inorganic carriers with organic hybrid materials improves transfection efficiency and half-life [47]. Therefore, both viral and non-viral miRNA delivery systems have advantages as well as disadvantages. While viral vectors provide higher transfection efficiency, they are more toxic and immunogenic. On the other hand, non-viral carriers have low delivery efficiency but are relatively safer. An efficient delivery method should combine the benefits of both the systems that would ensure the translation of miRNA therapies from bench to bedside.

11.5

Pharmacological Agents and Small-Molecules as Therapeutic Modulators. . .

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11.4.3 Exosomal Mediated Delivery of miRNAs Exosomes are extracellular lipid vesicles of endocytic origin (30–100 nm size) and secreted by nearly every cell type in humans. These vesicles establish inter-cellular communication by carrying macromolecules, including proteins, lipids, and nucleic acids (particularly RNA). Unlike other vectors, exosomes form an endogenous cellfree system for the RNA transport between cells, protect RNA from digestion, selectively target cells and rapidly transferred to target cells. Exosomes serve as an efficient vehicle for the delivery of miRNAs [61–63], and the transferred miRNAs can modulate the characteristics of the recipient cell. For instance, exosomemediated delivery of miR-124 enhances cortical neurogenesis following stroke. Moreover, the nano-size of exosomes possibly permits the entry into the brain increasing the likelihood of therapeutic and neuroplasticity effect of systemic administration of exosomes [64]. Recently, mesenchymal stem cell (MSC) derived exosomes have been shown to regulate recipient cell protein expression as well as cell characteristics through miRNA transfer. The miRNA within MSC-derived exosomes could be manipulated to enhance neurovascular plasticity and functional recovery following stroke [65].

11.5

Pharmacological Agents and Small-Molecules as Therapeutic Modulators of miRNAs

Besides miRNA mimics and inhibitors, pharmacological agents and smallmolecules act as therapeutic modulators of miRNAs to promote neuroprotection. These pharmacological agents may either block the expression of miRNAs that induce ischemic stroke damage or upregulate miRNAs that promote neuronal survival. For instance, acetylbritannilactone suppresses miR-155 expression to alleviate ischemia-induced cerebral inflammation [66]. On the other hand, VELCADE in combination with tissue-plasminogen activator (tPA) increases miR-146a levels, inactivates ischemia-induced toll-like receptor (TLR) signaling pathway, and leads to a reduction in infarct volume [67]. Glucosamine enhances miR-9 expression to promote neuronal survival and regeneration, as demonstrated in an in vitro model of ischemic stroke [11]. Pretreatment with nicorandil upregulates miR-7 and assuages oxygen-glucose deprivation (OGD)-induced endoplasmic reticulum stress as well as an inflammatory response in astrocytes [68]. Further, the emerging body of evidence points to the significance of smallmolecule-mediated regulation of miRNAs in ischemic stroke treatment. A luciferase-based cellular assay for the screening of small-molecules to target miRNA was among the first studies involving the small-molecule mediated modulation of miRNAs [69]. Recently, a quantitative high-throughput screening (qHTS) system has been aimed at identifying small-molecules capable of targeting miR-182 family and increasing SUMOylation as a means to develop novel ischemic stroke therapeutics [70]. Small-molecules have low-molecular weight and are mostly hydrophobic, which enables them to bind to miRNAs and modulate their function.

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The success of small-molecules in drug discovery depends on their ability to cross BBB, resistance to degradation in the blood-stream, and bioavailability. Thus, the use of small-molecules to target miRNAs may overcome the challenges associated with poor cell-permeability and pharmacokinetics.

11.6

Challenges, Perspectives, and Way-Forward

One of the common difficulties in miRNA-based therapeutics involves the rapid degradation of miRNAs by endogenous RNases. Though chemical modifications helped resolve this issue, their half-life can be very short, necessitating frequent injections or infusions. Secondly, the potential off-target effects remain a primary concern in developing miRNA therapeutics. While some of these off-target effects may be induced by the “passenger-strand” of commercially available mimics, some may be the result of the action of miRNA on other target genes producing unwanted side effects or activating pathways that offset neuroprotection. Moreover, it is imperative to carefully consider the time course of action of miRNA on its targets for the efficient functioning of miRNA therapeutics. For instance, early administration of miR-124 mimic following ischemic stroke increases neuronal survival, while late administration has no effect [71]. The need to bypass or penetrate the BBB following miRNA delivery remains the next primary concern. Nearly two-thirds of published research has employed intracerebroventricular (ICV) administration or intrathecal injection to circumvent the BBB. While such studies provide proof-principle, it is generally not possible to translate to humans. An intravenous (IV) injection of chemically modified miRNA mimics and antagomirs is one way to achieve adequate cell and BBB penetration, but with the challenge of systemic off-target effects. Viral-vector mediated miRNA delivery systems consume more time, making them unsuitable for the treatment of acute ischemic stroke. However, they could be applied for long-term recovery if immunological risks are thoroughly assessed and controlled. In the face of these difficulties, AAV delivery has provided favorable ways for tissue-specific delivery of miRNAs and presently tested in several preclinical models as well as clinical trials for cancer gene therapy. More recently, exosomes or engineered nanoparticlesmiRNA-based drugs have been presented to augment the BBB passage avoiding delivery to undesirable sites. miRNA-based therapeutics is still in its infancy and has not advanced to clinical trials for the treatment of ischemic stroke. Combining specific miRNA-based therapeutics with endovascular recanalization strategies may offer novel insights for the treatment of ischemic stroke. Moreover, understanding the activity of miRNAs in specific brain cell types, such as neurons, oligodendrocytes, astrocytes, and microglia along with their downstream targets, under normal as well as ischemic stroke conditions may aid in the improvement of specificity and effectiveness of post-stroke miRNA therapy. In spite of these challenges, there has been a rising interest surrounding miRNAs as therapeutic entities. Pioneering miRNA-based therapeutic strategies have unleashed novel clinical avenues to support early

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