Retinal Degenerative Diseases XIX: Mechanisms and Experimental Therapy (Advances in Experimental Medicine and Biology, 1415) [1st ed. 2023] 3031276809, 9783031276804

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Advances in Experimental Medicine and Biology  1415

John D. Ash · Eric Pierce · Robert E. Anderson · Catherine Bowes Rickman · Joe G. Hollyfield · Christian Grimm   Editors

Retinal Degenerative Diseases XIX Mechanisms and Experimental Therapy

Advances in Experimental Medicine and Biology Volume 1415 Series Editors Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, CNRS and University of Bordeaux, Pessac Cedex, France Haidong Dong, Departments of Urology and Immunology, Mayo Clinic Rochester, MN, USA Heinfried H. Radeke, Institute of Pharmacology and Toxicology, Clinic of the Goethe University Frankfurt Main, Frankfurt am Main, Hessen, Germany Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran Ortrud Steinlein, Institute of Human Genetics, LMU University Hospital Munich, Germany Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of Cardiovascular Sciences, School of Life Science, Shanghai University Shanghai, China

Advances in Experimental Medicine and Biology provides a platform for scientific contributions in the main disciplines of the biomedicine and the life sciences. This series publishes thematic volumes on contemporary research in the areas of microbiology, immunology, neurosciences, biochemistry, biomedical engineering, genetics, physiology, and cancer research. Covering emerging topics and techniques in basic and clinical science, it brings together clinicians and researchers from various fields. Advances in Experimental Medicine and Biology has been publishing exceptional works in the field for over 40 years, and is indexed in SCOPUS, Medline (PubMed), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical Abstracts Service (CAS), and Pathway Studio. 2021 Impact Factor: 3.650 (no longer indexed in SCIE as of 2022)

John D. Ash  •  Eric Pierce Robert E. Anderson Catherine Bowes Rickman Joe G. Hollyfield  •  Christian Grimm Editors

Retinal Degenerative Diseases XIX Mechanisms and Experimental Therapy

Editors John D. Ash Department of Ophthalmology University of Pittsburgh School of Medicine Pittsburgh, PA, USA Robert E. Anderson Health Sciences Center University of Oklahoma Health Sciences Center Oklahoma City, OK, USA Joe G. Hollyfield Department of Ophthalmology Cleveland Clinic Lerner College of Medicine Cleveland, OH, USA

Eric Pierce Ocular Genomics Institute Department of Ophthalmology Massachusetts Eye and Ear Infirmary Harvard Medical School Boston, MA, USA Catherine Bowes Rickman Department of Ophthalmology Duke Medical Center Durham, NC, USA Christian Grimm Laboratory for Retinal Cell Biology Department of Ophthalmology University Hospital Zurich University of Zurich Schlieren, Switzerland

ISSN 0065-2598     ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-3-031-27680-4    ISBN 978-3-031-27681-1 (eBook) https://doi.org/10.1007/978-3-031-27681-1 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

The editors are pleased dedicate this publication to the memory of our long-time friend and colleague, Alan M. Laties. Except for the most recent years, Alan attended each of these biennial retinal degeneration meetings since they began in 1984. Early on Alan recognized the importance of our attempt to provide a continuing international platform for discussions and scientific exchange to take place among investigators focused on retinal degeneration research. Through his scientific leadership at the Foundation Fighting Blindness (formerly the Retinitis Pigmentosa Foundation), we received the first meeting grant to partially cover some of the expenses of the RD meeting held in San Francisco in 1988. The Foundation has provided continuing support for each of the subsequent meetings in the form of travel grant support for young investigators. Born in Beverly, Massachusetts, the son of Russian immigrants, he attended Harvard College (BA, 1954) and completed medical school at Baylor College of Medicine (MD, 1959), followed by a residency in ophthalmology in the Hospital of the University of Pennsylvania (1961–63). A United States Public Health Service Special Research Fellowship supported his

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research training in the Institute of Neurological Sciences at the University of Pennsylvania (1963–64). He joined the faculty at the University of Pennsylvania in 1965 where he moved through the academic ranks until retiring as Emeritus Professor of Ophthalmology at the Perelman School of Medicine in 2020. He held joint appointments in Ophthalmology and Neurology where he was the Irene Heinz Given and John LaPorte Given Research Professor and the Harold G. Scheie Research Professor in Ophthalmology. He served as neuro-ophthalmologist at the Hospital of the University of Pennsylvania while pursuing basic research on the autonomic innervation of the eye, eye growth, and therapeutic approaches to eye diseases. He has published 140 original research papers, 30 review articles, and presented numerous invited lectures at major university medical centers around the world on a variety of topics critical to the treatment of diseases of the eye. He was an inventor holding multiple patents in the area of ophthalmology. In the early 1970s, Alan was approached by the Retinitis Pigmentosa Foundation to help them develop a scientific plan to support targeted research that would lead to an understanding of the causes of retinitis pigmentosa. At the time, it was recognized that these diseases were inherited, but only in a very limited way (autosomal dominant, recessive or X-linked). At the time, no mutations causing RP had been identified and the Human Genome Project would not be initiated for another 20 years. Alan agreed and organized the first Scientific Advisory Board for this Foundation and served as Chairman. In this leadership role, Alan helped identify and direct funding to the first laboratory focused on degenerative retinal disease research, the Berman-Gund Laboratory at the Massachusetts Eye and Ear Infirmary, Harvard University. Research Centers focused on retinal degeneration would later be expanded to many medical centers in North America, England, and Europe. Alan recognized the importance and need for animal models with these inherited retinal diseases and directed funds from the Foundation to support the development of the dog models with RP identified by Dr. Gustavo Aguirre at the College of Veterinary Medicine. In the early 1980s, Alan initiated a scientific plan for the Foundation to identify the major genes responsible for RP. This led in 1989 to the discovery of a mutation in the rhodopsin gene





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responsible for an autosomal dominant form of retinitis pigmentosa. Discovery of mutations in other genes causing retinitis pigmentosa quickly followed. With the discovery of RP-65, a gene that causes a recessive form of RP, gene therapy in a dog model with this recessive disorder could be quickly initiated because of Dr. Laties’ early support from the Foundation of these dog model lines. Dr. Laties’ early leadership was hugely important to gene therapy clinical trials and a number of other therapies related to these inherited retinal diseases. To honor Dr. Laties, the Foundation Fighting Blindness named their physicians’ and physician-scientists’ career development award the Alan Laties Career Development Program and honored him with the inaugural Llura Liggett Gund Lifetime Achievement Award. Dr. Laties was a gifted scientist, outstanding leader, and compassionate human who enriched the lives of his contemporaries. He played a key role in nurturing and expanding research in inherited retinal diseases. He is survived by his wife Deena Gu, a distinguished artist, daughter Jane Laties, sons Alex P. Laties and Nicholas P. Robinson, and a brother, David.

Preface

The XIX International Symposium on Retinal Degeneration was held from September 26 to October 2, 2021. The symposium was initially planned for October of 2020 in Mendoza, Argentina. However, the global pandemic made this meeting impossible. With the availability of vaccines, we decided in March of 2021 that it would be possible to organize the meeting for late September of 2021. From the beginning, we planned the meeting as an in-­ person meeting with the capability of switching to a hybrid or fully online meeting depending on the state of the pandemic, and we moved the in-person meeting to the United States to reduce travel complications for most attendees. As the delta variant began to surge in the weeks leading up to the meeting, we had to activate the hybrid meeting. The meeting platform we established allowed both in-person and virtual platform talks as well as both in-person and virtual attendance. The platform was organized so that all presentations were live and all participants were able to ask questions. All presentations, including posters, were recorded and made available 4  months after the meeting. The in-person sessions were held in the Sonesta Nashville Airport Hotel in Nashville, TN. Because of COVID concerns, the in-person attendance was small (118 scientists) compared to previous meetings (~250 scientists), but the overall attendance increased to 344 attendees. The virtual option was the main driver for the increase in attendance. The meeting program included four outstanding keynote presentations from Michael Chiang, Director of the National Eye Institute on Artificial intelligence for clinical care and research; Douglas Wallace, National academy of Science member and Professor at the University of Pennsylvania on Mitochondria and the etiology of disease; David Gamm, Professor at the University of Wisconsin-­ Madison on Ultrathin micromolded 3D scaffolds for outer retina reconstruction; Valeria Canto-Soler, Professor at the University of Colorado on Human iPSC-derived 3D retinal tissue for stem cell-based therapies for retinal degenerative diseases. Drs Chiang and Wallace presented via the virtual platform, while Drs Gamm and Canto-Soler presented from the podium. The program also included 41 platform talks, with 28 presented in person from the podium and another 13 presented virtually. In addition, 143 posters were presented as short talks on the virtual platform. Seventy-three of the posters were also presented in person during two well-attended poster sessions. New and important data were presented at the meeting, and we were mentioned in a written article published on NPR, and several attendees were interviewed by reporters from Science and other journals. ix

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The RD2021 Travel award competition was highly successful at attracting qualified applicants. We received a 35% increase in TA applications for a total of 196. The applications were reviewed by a panel of 14 expert reviewers, including 6 women, 8 men, and sceintists from a recognized underrepresented minority (URM). Since funding from European sources is dedicated to European early career scientists, we included three reviewers from Europe. Many of the panel members have been prior travel awardees. Each application was assigned four reviewers, and reviewers independently scored applications on a 1–9 scale. Based on scores, the applications are ranked and slotted into funding sources based on funding agency criteria. We were able to support full travel awards for 60 in-person early career scientists and another 41 virtual early-career scientists. This is the largest pool of awardees at an RD meeting. The awards were balanced between men and women. In addition, we implemented a new diversity and inclusion policy and dedicated a minimum of six awards to underrepresented minorities (URM). In the end, we were able to fund 11 URMs to attend the RD meeting. Although the pandemic made the RD2021 meeting more complex and more challenging to organize, the RD2021 meeting was, by all accounts, a terrific success. Pittsburgh, PA, USA Boston, MA, USA Oklahoma City, OK, USA Durham, NC, USA Cleveland, OH, USA Schlieren, Switzerland

John D. Ash Eric Pierce Robert E. Anderson Catherine Bowes Rickman Joe G. Hollyfield Christian Grimm

Contents

Part I Age-related Macular Degeneration High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal Laminar Deposit of Age-­Related Macular Degeneration������������������������������������   3 David M. G. Anderson, Ankita Kotnala, Jeffrey D. Messinger, Nathan Heath Patterson, Jeffrey M. Spraggins, Christine A. Curcio, Richard M. Caprioli, and Kevin L. Schey The Noncanonical Role of Complement Factor H in Retinal Pigment Epithelium (RPE) Cells and Implications for Age-Related Macular Degeneration (AMD)������������������������������������   9 Angela Armento, David Adrian Merle, and Marius Ueffing  acular Pigment Carotenoids and Bisretinoid A2E����������������������������  15 M Ranganathan Arunkumar and Paul S. Bernstein Disturbed Matrix Metalloproteinases Activity in Age-Related Macular Degeneration ����������������������������������������������������������������������������  21 Beatriz Martins and Rosa Fernandes Current Views on Chr10q26 Contribution to Age-Related Macular Degeneration ����������������������������������������������������������������������������  27 Navdeep Gogna, Lillian F. Hyde, Gayle B. Collin, Lisa Stone, Jurgen K. Naggert, and Patsy M. Nishina Untargeted Lipidomic Profiling of Aged Human Retina With and Without Age-Related Macular Degeneration (AMD)����������  37 Ankita Kotnala, David M. G. Anderson, Jeffrey D. Messinger, Christine A. Curcio, and Kevin L. Schey Decoding Race and Age-Related Macular Degeneration: GPR 143 Activity Is the Key��������������������������������������������������������������������  43 Dorothy Tung and Brian S. McKay  eroxisome Proliferator-Activated Receptor Gamma P Coactivator-­1Alpha (PGC-1α): A Transcriptional Regulator at the Interface of Aging and Age-Related Macular Degeneration? ������  49 Freya M. Mowat

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Regulation of ABCA1 by miR-33 and miR-34a in the Aging Eye ��������������������������������������������������������������������������������������  55 Florian Peters and Christian Grimm  he Role of Gene Expression Regulation on Genetic Risk T of Age-­Related Macular Degeneration��������������������������������������������������  61 Rinki Ratnapriya Elastin Layer in Bruch’s Membrane as a Target for Immunization or Tolerization to Modulate Pathology in the Mouse Model of Smoke-Induced Ocular Injury������������������������  67 Bärbel Rohrer, Nathaniel Parsons, Balasubramaniam Annamalai, Crystal Nicholson, Elisabeth Obert, Bryan Jones, and Andrew D. Dick Repurposing Drugs for Treatment of Age-Related Macular Degeneration ����������������������������������������������������������������������������  73 Sarah G. Francisco and Sheldon Rowan Part II Extracellular Vesicles Extracellular Vesicle RNA Contents as Biomarkers for Ocular Diseases����������������������������������������������������������������������������������  81 Heran Getachew and Eric Pierce Proteomics of Retinal Extracellular Vesicles: A Review into an Unexplored Mechanism in Retinal Health and AMD Pathogenesis����������������������������������������������������������������������������  87 Adrian V. Cioanca, Riccardo Natoli, and Yvette Wooff Part III Gene Editing Prime Editing Strategy to Install the PRPH2 c.828+1G>A Mutation ����������������������������������������������������������������������������  97 Salvatore Marco Caruso, Yi-Ting Tsai, Bruna Lopes da Costa, Masha Kolesnikova, Laura A. Jenny, Stephen H. Tsang, and Peter M. J. Quinn Analysis of CRB1 Pathogenic Variants Correctable with CRISPR Base and Prime Editing�������������������������������������������������� 103 Bruna Lopes da Costa, Laura A. Jenny, Irene H. Maumenee, Stephen H. Tsang, and Peter M. J. Quinn Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor������������������������������������������������������ 109 Yi-Ting Tsai, Bruna Lopes da Costa, Salvatore Marco Caruso, Nicolas D. Nolan, Sarah R. Levi, Stephen H. Tsang, and Peter M. J. Quinn

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Part IV Gene Therapy Preexisting Neutralizing Antibodies against Different Adeno-Associated Virus Serotypes in Humans and Large Animal Models for Gene Therapy���������������������������������������� 117 Divya Ail and Deniz Dalkara Optimization of Capillary-Based Western Blotting for MYO7A ���������������������������������������������������������������������������������������������� 125 Kaitlyn R. Calabro, Sanford L. Boye, and Shannon E. Boye AAV Serotypes and Their Suitability for Retinal Gene Therapy ������������������������������������������������������������������������������������������ 131 Lynn J. A. Ebner and Christian Grimm Gene Augmentation for Autosomal Dominant CRX-Associated Retinopathies�������������������������������������������������������������������������������������������� 135 Chi Sun and Shiming Chen  xnip Gene Therapy of Retinitis Pigmentosa Improves T Cone Health���������������������������������������������������������������������������������������������� 143 Yunlu Xue Part V Human Retinal Degeneration Factors Affecting Readthrough of Natural Versus Premature Termination Codons ������������������������������������������������������������ 149 Avigail Beryozkin, Kerstin Nagel-Wolfum, Eyal Banin, and Dror Sharon Integrating Computational Approaches to Predict the Effect of Genetic Variants on Protein Stability in Retinal Degenerative Disease�������������������������������������������������������������� 157 Michelle Grunin, Ellen Palmer, Sarah de Jong, Bowen Jin, David Rinker, Christopher Moth, John A. Capra, Jonathan L. Haines, William S. Bush, and Anneke I. den Hollander  etwork Biology and Medicine to Rescue: Applications N for Retinal Disease Mechanisms and Therapy�������������������������������������� 165 Anupam K. Mondal and Anand Swaroop Non-syndromic Retinal Degeneration Caused by Pathogenic Variants in Joubert Syndrome Genes���������������������������������������������������� 173 Riccardo Sangermano, Egle Galdikaité-Braziené, and Kinga M. Bujakowska Exonic Variants that Affect Splicing – An Opportunity for “Hidden” Mutations Causing Inherited Retinal Diseases�������������� 183 Yogapriya Sundaresan, Eyal Banin, and Dror Sharon  nhanced S-cone Syndrome, a Mini-review������������������������������������������ 189 E Yiyi Wang, Jessica Wong, Jacque L. Duncan, Austin Roorda, and William S. Tuten

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Part VI Inflammation  he Role of Microglia in Inherited Retinal Diseases���������������������������� 197 T Asha Kumari and Shyamanga Borooah CD68: Potential Contributor to Inflammation and RPE Cell Dystrophy������������������������������������������������������������������������������������������ 207 Mayur Choudhary and Goldis Malek  ene Expression of Clusterin, Tissue Inhibitor G of Metalloproteinase-1, and Their Receptors in Retinal Pigment Epithelial Cells and Müller Glial Cells Is Modulated by Inflammatory Stresses���������������������������������������������������� 215 Mengmei Zheng, Eun-Jin Lee, Shinwu Jeong, and Cheryl Mae Craft Part VII Mechanisms of Degeneration  xonal Transport Defects in Retinal Ganglion Cell Diseases�������������� 223 A Iskalen Cansu Topcu Okan, Fatma Ozdemir, and Cavit Agca  onnexins Biology in the Pathophysiology of Retinal Diseases���������� 229 C Alejandro Ponce-Mora, Andrea Yuste, Giuliana Perini-Villanueva, María Miranda, and Eloy Bejarano  ole of Nuclear NAD+ in Retinal Homeostasis�������������������������������������� 235 R Emily E. Brown, Michael J. Scandura, and Eric Pierce Retinal Pigmented Epithelium-­Derived Ectopic Norrin Does Not Promote Intraretinal Angiogenesis in Transgenic Mice���������������� 241 Andrea E. Dillinger and Ernst R. Tamm Caveolin-1 in Müller Glia Exists as Heat-Resistant, High Molecular Weight Complexes ���������������������������������������������������������������� 249 Eric N. Enyong, Jami Gurley, Virginie Sjoelung, and Michael H. Elliott  ole of VLC-PUFAs in Retinal and Macular Degeneration���������������� 257 R Aruna Gorusupudi, Uzoamaka Nwagbo, and Paul S. Bernstein Ocular Amyloid, Condensates, and Aggregates – Higher-Order Protein Assemblies Participate in Both Retinal Degeneration and Function�������������������������������������������������������������������������������������������� 263 Michael H. Hayes, DaNae R. Woodard, and John D. Hulleman  hotoreceptor Ion Channels in Signaling and Disease������������������������ 269 P Shivangi M. Inamdar, Colten K. Lankford, and Sheila A. Baker The Role of Peripherin-2/ROM1 Complexes in Photoreceptor Outer Segment Disc Morphogenesis������������������������������������������������������ 277 Tylor R. Lewis, Muayyad R. Al-Ubaidi, Muna I. Naash, and Vadim Y. Arshavsky

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Human Mutations in Arl3, a Small GTPase Involved in Lipidated Cargo Delivery to the Cilia, Cause Retinal Dystrophy������������������������������������������������������������������������������������ 283 Amanda M. Travis and Jillian N. Pearring Genotype–Phenotype Association in ABCA4-Associated Retinopathy���������������������������������������������������������������������������������������������� 289 Maximilian Pfau, Wadih M. Zein, Laryssa A. Huryn, Catherine A. Cukras, Brett G. Jeffrey, Robert B. Hufnagel, and Brian P. Brooks Retinal Pathoconnectomics: A Window into Neurodegeneration���������������������������������������������������������������������������������� 297 Rebecca L. Pfeiffer and Bryan W. Jones The Role of Ceramide in Inherited Retinal Disease Pathology������������������������������������������������������������������������������������ 303 Xinye Qian, Tanmay Srinivasan, Jessica He, and Rui Chen  xtracellular Matrix: The Unexplored Aspects E of Retinal Pathologies and Regeneration���������������������������������������������� 309 Dmitri Serjanov and David R. Hyde Role of TFEB in Diseases Associated with Lysosomal Dysfunction���������������������������������������������������������������������������� 319 Hsuan-Yeh Pan and Mallika Valapala Retinoic Acid Receptor-Related Orphan Receptors (RORs) in Eye Development and Disease���������������������������������������������� 327 Felix Yemanyi, Kiran Bora, Alexandra K. Blomfield, and Jing Chen Part VIII Mechanisms of Degeneration – Animal Models A Novel Mouse Model for Late-­Onset Retinal Degeneration (L-ORD) Develops RPE Abnormalities Due to the Loss of C1qtnf5/Ctrp5�������������������������������������������������������������������� 335 Shyamanga Borooah, Anil Chekuri, Shikha Pachauri, Bhubananda Sahu, Marina Vorochikhina, John J. Suk, Dirk-­Uwe Bartsch, Venkata R. M. Chavali, Monica M. Jablonski, and Radha Ayyagari  omparison of Mouse Models of Autosomal Dominant Retinitis C Pigmentosa Due to the P23H Mutation of Rhodopsin�������������������������� 341 Shannon R. Barwick and Sylvia B. Smith Compensatory Cone-Mediated Mechanisms in Inherited Retinal Degeneration Mouse Models: A Functional and Gene Expression Analysis���������������������������������������������������������������� 347 Alicia A. Brunet, David M. Hunt, Carla Mellough, Alan R. Harvey, and Livia S. Carvalho

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Inhibition of Ryanodine Receptor 1 Reduces Endoplasmic Reticulum (ER) Stress and Promotes ER Protein Degradation in Cyclic Nucleotide-Gated Channel Deficiency���������������������������������������������������������������������������������� 353 Fan Yang, Hongwei Ma, Rekha Garg, Alfred Lewin, and Xi-Qin Ding  ouse Choroid Proteome Revisited: Focus on Aging�������������������������� 359 M Donita Garland, James Harnly, and Radha Ayyagari Morphological and Functional Comparison of Mice Models for Retinitis Pigmentosa ���������������������������������������������� 365 Prakadeeswari Gopalakrishnan, Avigail Beryozkin, Eyal Banin, and Dror Sharon  urrent Advancements in Mouse Models of Retinal Disease�������������� 371 C T. J. Hollingsworth, Xiangdi Wang, Raven N. Simpson, William A. White, Robert W. Williams, and Monica M. Jablonski Single-Cell Transcriptomic Profiling of Müller Glia in the rd10 Retina���������������������������������������������������������������������������� 377 Duygu Sigurdsson and Christian Grimm  Methods for In Vivo Characterization of Proteostasis in the Mouse Retina �������������������������������������������������������������������������������� 383 Yixiao Wang and Ekaterina S. Lobanova Absence of PRCD Leads to Dysregulation in Lipid Homeostasis Resulting in Disorganization of Photoreceptor Outer Segment Structure������������������������������������������ 389 Sree I. Motipally and Saravanan Kolandaivelu  Expansion Microscopy of Mouse Photoreceptor Cilia ������������������������ 395 Abigail R. Moyel, Michael A. Robichaux, and Theodore Wensel Rod Photoreceptor-Specific Ablation of Metformin Target, AMPK, in a Preclinical Model of Autosomal Recessive Retinitis Pigmentosa �������������������������������������������������������������� 403 Nicholas D. Nolan, Laura A. Jenny, Stephen H. Tsang, and Xuan Cui TLR2 Is Highly Overexpressed in Retinal Myeloid Cells in the rd10 Mouse Model of Retinitis Pigmentosa ���������������������� 409 Alonso Sánchez-Cruz, Enrique J. de la Rosa, and Catalina Hernández-Sánchez Environmental Light Has an Essential Effect on the Disease Expression in a Dominant RPE65 Mutation���������������� 415 Wenjing Wu, Yusuke Takahashi, Xiang Ma, Gennadiy Moiseyev, and Jian-Xing Ma

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Microglia Preserve Visual Function in a Mouse Model of Retinitis Pigmentosa with Rhodopsin-P23H Mutant ���������������������� 421 Chen Yu and Daniel R. Saban Part IX Mechanisms of Degeneration – Metabolism Measuring the Release of Lactate from Wild-Type and rd1 Mouse Retina������������������������������������������������������������������������������ 429 Yiyi Chen, Laimdota Zizmare, Christoph Trautwein, and François Paquet-Durand Aerobic Glycolysis in Photoreceptors Supports Energy Demand in the Absence of Mitochondrial Coupling���������������������������� 435 Daniel T. Hass, Celia M. Bisbach, Martin Sadilek, Ian R. Sweet, and James B. Hurley  Redox Status in Retinitis Pigmentosa���������������������������������������������������� 443 L. Olivares-González, S. Velasco, I. Campillo, J. M. Millán, and R. Rodrigo Perspectives on Retinal Dolichol Metabolism, and Visual Deficits in Dolichol Metabolism-Associated Inherited Disorders���������������������������������������������������������������������������������� 449 Sriganesh Ramachandra Rao, Steven J. Pittler, and Steven J. Fliesler Retinal Metabolic Profile on IMPG2 Deficiency Mice with Subretinal Lesions �������������������������������������������������������������������������� 457 Rong Xu, Yekai Wang, Jianhai Du, and Ezequiel M. Salido Part X Neuroprotection Glutathione Coating of Liposomes Enhances the Delivery of Hydrophilic Cargo to the Inner Nuclear Layer in Retinal Cultures ���������������������������������������������������������������������������������� 467 Gustav Christensen and François Paquet-Durand Modification of Müller Glial Cell Fate and Proliferation with the Use of Small Molecules ������������������������������������������������������������ 473 Marcus J. Hooper A Potential Neuroprotective Role for Pyruvate Kinase 2 in Retinal Degeneration������������������������������������������������������������������������ 479 Jiaming Zhou, Michel Rasmussen, and Per Ekström Part XI Photoreceptors Critical Role of VEGF as a Direct Regulator of Photoreceptor Function���������������������������������������������������������������������� 487 Jianyan Hu, Meili Zhu, Dai Li, Qiang Wu, and Yun-Zheng Le

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 Lysine Ubiquitylation Drives Rhodopsin Protein Turnover���������������� 493 Allen P. F. Chen, Leon Chea, Eun-Jin Lee, and Jonathan H. Lin  Silico Prediction of MYO1C-­Rhodopsin Interactions In and Its Significance in Protein Localization and Visual Function �������������������������������������������������������������������������������� 499 Glenn P. Lobo, Rakesh Radhakrishnan, Matthias Leung, Andrew Gruesen, Hans-­Joachim Knölker, Frederik J. van Kuijk, and Sandra R. Montezuma A Ciliary Branched Actin Network Drives Photoreceptor Disc Morphogenesis�������������������������������������������������������� 507 William J. Spencer and Vadim Y. Arshavsky Part XII RPE  Revisiting the Daily Timing of POS Phagocytosis�������������������������������� 515 Antonio E. Paniagua, Harjas S. Sabharwal, Kausalya Kethu, Andrew W. Chang, and David S. Williams Inhibition of Bacterial Peptidoglycan Cytopathy by Retina Pigment Epithelial PGRP2 Amidase������������������������������������ 521 Marlyn P. Langford, Laura A. Perilloux-Lyons, and A. Scott Kavanaugh Understanding Ischemic Retinopathies: The Role of Succinate and Its Receptor in Retinal Pigment Epithelium �������������������������������������������������������������������������������� 527 Bilge Esin Ozturk  The Amphipathic Helix in Visual Cycle Proteins: A Review���������������� 533 Sheetal Uppal, Eugenia Poliakov, Susan Gentleman, and T. Michael Redmond  The Retinal Pigment Epithelium: Cells That Know the Beat!������������ 539 Elora M. Vanoni and Emeline F. Nandrot Part XIII Stem Cell Models and Therapies Retinal Organoids: A Human Model System for Development, Diseases, and Therapies�������������������������������������������� 549 Sangeetha Kandoi and Deepak A. Lamba  Modeling Retinitis Pigmentosa with Patient-Derived iPSCs �������������� 555 Yeh Chwan Leong and Jane C. Sowden  Primary Retinal Cell Cultures as a Model to Study Retina Biology������������������������������������������������������������������������������������������ 565 Germán A. Michelis, Luis E. Politi, and S. Patricia Becerra

Contents

Contents

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Generation of CRB1 RP Patient-­Derived iPSCs and a CRISPR/Cas9-­Mediated Homology-Directed Repair Strategy for the CRB1 c.2480G>T Mutation���������������������������� 571 Bruna Lopes da Costa, Yao Li, Sarah R. Levi, Stephen H. Tsang, and Peter M. J. Quinn Inducing Neural Regeneration from Glia Using Proneural bHLH Transcription Factors������������������������������������������������������������������ 577 Levi Todd Index��������������������������������������������������������������������������������������������������������  583

Part I Age-related Macular Degeneration

High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal Laminar Deposit of Age-­Related Macular Degeneration David M. G. Anderson, Ankita Kotnala, Jeffrey D. Messinger, Nathan Heath Patterson, Jeffrey M. Spraggins, Christine A. Curcio, Richard M. Caprioli, and Kevin L. Schey

Abstract

Pathologies of the retina are clinically visualized in vivo with OCT and ex vivo with immuD. M. G. Anderson · N. H. Patterson · R. M. Caprioli · K. L. Schey (*) Department of Biochemistry, Vanderbilt University, Nashville, TN, USA Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN, USA e-mail: [email protected] A. Kotnala Department of Biochemistry, Vanderbilt University, Nashville, TN, USA Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham, Birmingham, AL, USA J. D. Messinger · C. A. Curcio Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham, Birmingham, AL, USA J. M. Spraggins Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN, USA Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham, Birmingham, AL, USA

nohistochemistry. Although both techniques provide valuable information on prognosis and disease state, a comprehensive method for fully elucidating molecular constituents present in  locations of interest is desirable. The purpose of this work was to use multimodal imaging technologies to localize the vast number of molecular species observed with matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) in aged and diseased retinal tissues. Herein, MALDI IMS was utilized to observe molecular species that reside in photoreceptor cells and also a basal laminar deposit from two human donor eyes. The molecular species observed to accumulate in these discrete regions can be further identified and studied to attempt to gain a greater understanding of biological processes occurring in debilitating eye diseases such as age-related macular degeneration (AMD). Keywords

Age-related macular degeneration · Macula · Retinal pigment epithelium · Photoreceptors · Basal lamina deposit · MALDI IMS

Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_1

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1 Introduction Matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) can localize and display the tissue distributions of hundreds to thousands of molecules, at cellular resolution, without the need for antibodies or radioisotopes [1]. With effective co-registration to multimodal optical imaging and optical coherence topography (OCT) microscopy, these distributions can be accurately correlated to very small histological features of the neural retina and retinal pigment epithelium (RPE). MALDI IMS methods have been used to examine eye tissues including the retina [2–4], optic nerve [4–6], lens [7, 8], and cornea [9]. Distinct cell and synaptic layers of the retina have unique layer-specific lipid and metabolite signatures distinguished by IMS [4, 10, 11]. By applying multimodal optical imaging technologies with accurate registration and incorporating data-rich IMS images [12], cellular and subcellular localization of specific molecules informative to cellspecific biochemistry can be observed. Human retinal lipid composition studies have been performed in the past. The results, while valuable, provide limited information on cellular origin, as experiments require dissections followed by solvent extractions. MALDI IMS offers a “molecular microscope” that localizes tissue components in situ by molecular weights [11], simultaneously providing hundreds to thousands of spatially resolved signals. In this study, we used a newly developed method of highaccuracy registration [12] to co-­ register high spatial resolution IMS images with OCT autofluorescence and histological images of the same tissue to examine subcellular localizations and molecular features of photoreceptors and AMD pathology.

2 Methods This section has been summarized from Anderson et  al. [3]; for detailed explanations, please see this reference.

D. M. G. Anderson et al.

2.1 Tissue Acquisition and Characterization Whole eyes were obtained from deceased human donors via Advancing Sight Network (formerly the Alabama Eye Bank) by the UAB authors.

2.2 Tissue Handling and Ex Vivo Imaging Methods were optimized for multimodal ex vivo clinical imaging of the ocular fundus [13]. Globes with lens and iris in place were immersed in buffered 4% paraformaldehyde overnight. Iris and lens were removed before imaging. For imaging with OCT and scanning laser ophthalmoscopy, globes were immersed in buffer facing frontward within a custom-built chamber with a 60-diopter lens [13]. Spectral domain OCT images were captured with a Spectralis (HRA&OCT, HRA2; Heidelberg Engineering). Tissues were embedded in 2.5% carboxymethyl cellulose (Sigma C9481), and serial 10 μm cryosections were collected on Superfrost glass slides and on large, 45 × 45 mm in-house, polylysine-coated indium-tin-oxide (ITO) slides (Delta Technologies Loveland, CO, USA).

2.3 MALDI IMS Analysis The matrices 2,5-dihydroxyacetophenone (DHA) and 1,5-diaminonaphthalene (DAN) (Sigma Aldrich, St. Louis, MO, USA) were applied to tissue sections by sublimation [14]. MALDI IMS data were acquired with a laser spot size of 10–15  μm in full scan mode using a Bruker SolariX 9.4T FTICR mass spectrometer (Bruker Daltonics Billerica, MA, USA). Following data acquisition, an advanced image registration workflow [12] was performed. More detailed information of the image registration process can be found in publications by Patterson et al. [12] and Anderson et al. [3]. Molecular identifications were made using LC-MS of chloroform-­methanol extracts from adjacent tissue sections.

High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal…

3 Results 3.1 Signals Specific to Photoreceptors and RPE Figure 1a shows MALDI IMS and optical microscopy focusing on photoreceptors and their ­support cells. The RPE sends delicate processes in the apical direction to contact photoreceptor outer segments, near the RPE cell body for rods and 10–15  μm above the cell body, to contact cone outer segments, which are shorter. Figure 1a is color-coded depiction of photoreceptor and RPE compartments associated with IMS signals in Fig. 1b (blue ONL, red inner segments, yellow outer segments, green RPE). Figure 1b shows MALDI IMS images overlaid with H&E images from this donor. The photoreceptors on the left side of the image are attached to the RPE and detached from the RPE on the right side, a common artifact which can occur during sample preparation. In Fig. 1a, the signal at m/z 818.575 was observed with high abundance in the ONL and was identified as PE(20:0_22:6) (blue). This region is comprised of the photoreceptor cell bodies and processes of Müller (radial) glia. A highly localized signal can be observed with high abundance along a narrow band aligned with photoreceptor inner segments at m/z 1426.0 (red). This signal was identified as

Fig. 1  MALDI IMS signals consistent with localization to photoreceptor and RPE compartments. (a) Schematic diagram of outer retina, excerpted from Fig.  1a. Blue, pink, yellow, and green bands indicate layers formed by highly compartmentalized and vertically aligned photoreceptors and RPE cells in panels b, c. Layers: OPL outer plexiform layer, ONL outer nuclear layer, ELM external limiting membrane, RPE retinal pigment epithelium, BrM Bruch’s membrane, R rod, C cone photoreceptors. (b–f)

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a cardiolipin CL(70:5). Cardiolipins are highly abundant in mitochondria, which are abundant in the ellipsoid portion of photoreceptor inner segments. At the distal part of the photoreceptor cells, outer segments are highly interleaved with apical processes of RPE cells. A DHA-containing PE(18:0_22:6) is observed at m/z 790.539 (yellow) in panel D which can be observed with high abundance in the outer segments, while a signal observed at m/z 728.596 (green) is localized above and within the RPE.

3.2 Signals Specific to Basal Laminar Deposit Figure 2 shows multimodal imaging of a 93-year-­ old donor tissue with the imaging modalities separated into panels. Figure 2a displays ex vivo OCT hyperreflective foci (yellow arrowhead) and an RPE elevation (green arrowhead) near the fovea. Figure 2b shows that retinal layers are visible in H&E-stained sections after IMS data acquisition. The inset magnifies BLamD (PASH staining of an alternate section), clearly indicating thickened extracellular matrix between the RPE plasma membrane and its native basal lamina. Figure 2c shows autofluorescence of the elevated RPE layer and anteriorly migrated RPE cells, which account for high-risk-indicating

MALDI IMS images and H&E-stained tissue images overlaid in perifoveal retina displaying signals from multiple lipid classes that localize to subcellular compartments of the photoreceptor cells. (b) Overlay showing four separate signals. (c) Localized to ONL. (d) Localized to photoreceptor inner and outer segments. (e) Localized to mitochondria-rich photoreceptor inner segments. (f) Localized to RPE apical processes

D. M. G. Anderson et al.

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Fig. 2  Imaging mass spectrometry (IMS) for molecularly informed optical coherence tomography (OCT) and tissue-­level target discovery. Asterisk, foveal pit; RPE, retinal pigment epithelium. Color-coded arrowheads represent corresponding structures across modalities, in a 93-year-old human donor eye. (a) OCT B-scan shows subretinal hyperreflective material (yellow) and an RPE elevation (green). (b) H&E stained cryosection shows

pigmented debris (yellow) and dysmorphic RPE overlying BLamD. Insert, BLamD with basal mound (arrow). Basal mounds contain soft druse material. Layers: GCL ganglion cell, INL inner nuclear, HFL Henle fiber, ONL outer nuclear. (c) Autofluorescent, pigmented debris (yellow) and dysmorphic RPE (green). (d) IMS reveals an m/z signal at 799.673, restricted to BLamD and not detected in the RPE

hyperreflective foci of clinical OCT.  Figure  2d shows that a sphingomyelin-related lipid (PE-Cer-NMe2(42:1)) at m/z 799.671 [15] is highly abundant and localizes to BLamD and RPE, building on previous histochemical and chromatography findings of lipids in this deposit [16, 17].

4 Conclusions MALDI IMS combined with multimodal imaging methods and ex vivo OCT provides a powerful tool to elucidate the molecular composition and localization of molecular species in key regions and pathology associated with ocular dis-

High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal…

ease. Understanding molecular processes occurring in BLamD in early AMD is important as they are early-identified histologic risk factors for AMD progression [18] and are just now being recognized clinically [19, 20]. Acknowledgments This project was supported by the National Institutes of Health P41 GM103391 (R.M.C.) and R01 EY027948 (C.A.C.). Support was also received by a Research to Prevent Blindness Catalyst Award for Innovative Research Approaches for Age-Related Macular Degeneration to K.L.S.

References 1. Caprioli RM, Farmer TB, Gile J.  Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS.  Anal Chem. 1997;69(23):4751–60. 2. Bowrey HE, Anderson DM, Pallitto P, Gutierrez DB, Fan J, Crouch RK, et al. Imaging mass spectrometry of the visual system: advancing the molecular understanding of retina degenerations. Proteomics Clin Appl. 2016;10(4):391–402. 3. Anderson DMG, Messinger JD, Patterson NH, Rivera ES, Kotnala A, Spraggins JM, et al. Lipid landscape of the human retina and supporting tissues revealed by high resolution imaging mass spectrometry. bioR xiv:2020:2020.04.08.029538. 4. Zemski Berry KA, Gordon WC, Murphy RC, Bazan NG. Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry. J Lipid Res. 2014;55(3):504–15. 5. Anderson DM, Spraggins JM, Rose KL, Schey KL. High spatial resolution imaging mass spectrometry of human optic nerve lipids and proteins. J Am Soc Mass Spectrom. 2015;26(6):940–7. 6. Stark DT, Anderson DMG, Kwong JMK, Patterson NH, Schey KL, Caprioli RM, et al. Optic nerve regeneration after crush remodels the injury site: molecular insights from imaging mass spectrometryoptic nerve regeneration imaging mass spectrometry. Invest Ophthalmol Vis Sci. 2018;59(1):212–22. 7. Grey AC, Schey KL. Age-related changes in the spatial distribution of human lens alpha-crystallin products by MALDI imaging mass spectrometry. Invest Ophthalmol Vis Sci. 2009;50(9):4319–29. 8. Stella DR, Floyd KA, Grey AC, Renfrow MB, Schey KL, Barnes S.  Tissue localization and solubilities of alphaA-crystallin and its numerous C-terminal truncation products in pre- and postcataractous ICR/f rat lenses. Invest Ophthalmol Vis Sci. 2010;51(10):5153–61.

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9. Chen Y, Jester JV, Anderson DM, Marchitti SA, Schey KL, Thompson DC, et  al. Corneal haze phenotype in Aldh3a1-null mice: in  vivo confocal microscopy and tissue imaging mass spectrometry. Chem Biol Interact. 2017;276:9–14. 10. Anderson DM, Ablonczy Z, Koutalos Y, Spraggins J, Crouch RK, Caprioli RM, et  al. High resolution MALDI imaging mass spectrometry of retinal tissue lipids. J Am Soc Mass Spectrom. 2014;25(8):1394–403. 11. Anderson DMG, Ablonczy Z, Koutalos Y, Hanneken AM, Spraggins JM, Calcutt MW, et  al. Bis(monoacylglycero)phosphate lipids in the retinal pigment epithelium implicate lysosomal/endosomal dysfunction in a model of Stargardt disease and human retinas. Sci Rep. 2017;7(1):17352. 12. Patterson NH, Tuck M, Van de Plas R, Caprioli RM.  Advanced registration and analysis of MALDI imaging mass spectrometry measurements through autofluorescence microscopy. Anal Chem. 2018;90(21):12395–403. 13. Pang CE, Messinger JD, Zanzottera EC, Freund KB, Curcio CA. The onion sign in neovascular age-related macular degeneration represents cholesterol crystals. Ophthalmology. 2015;122(11):2316–26. 14. Hankin J, Barkley R, Murphy R.  Sublimation as a method of matrix application for mass spectrometric imaging. J Am Soc Mass Spectrom. 2007;18(9):1646–52. 15. Liu A, Chang J, Lin Y, Shen Z, Bernstein PS. Long-­ chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration. J Lipid Res. 2011;51:3217–29. 16. Curcio CA, Presley JB, Malek G, Medeiros NE, Avery DV, Kruth HS.  Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy. Exp Eye Res. 2005;81(6):731–41. 17. Wang L, Li C-M, Rudolf M, Belyaeva OV, Chung BH, Messinger JD, et  al. Lipoprotein particles of intraocular origin in human Bruch membrane: an unusual lipid profile. Invest Ophthalmol Vis Sci. 2009;50(2):870–7. 18. Sarks SH.  Ageing and degeneration in the macular region: a clinico-pathological study. Br J Ophthalmol. 1976;60(5):324–41. 19. Tan ACS, Astroz P, Dansingani KK, Slakter JS, Yannuzzi LA, Curcio CA, et  al. The plateau, an optical coherence tomographic signature of geographic atrophy: evolution, multimodal imaging, and candidate histology. Invest Ophthalmol Vis Sci. 2017;58(4):2349–58. 20. Sura AA, Chen L, Messinger JD, Swain TA, McGwin G Jr, Freund KB, et al. Measuring the contributions of basal laminar deposit and Bruch’s membrane in age-­ related macular degeneration. Invest Ophthalmol Vis Sci. 2020;61(13):19.

The Noncanonical Role of Complement Factor H in Retinal Pigment Epithelium (RPE) Cells and Implications for Age-Related Macular Degeneration (AMD) Angela Armento, David Adrian Merle, and Marius Ueffing

Abstract

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina. Dysfunction of the retinal pigment epithelium (RPE) occurs in early stages of AMD, and progressive RPE atrophy leads to photoreceptor death and visual impairments that ultimately manifest as geographic atrophy (GA), one of the late-stage forms of AMD. AMD is caused by a combination of risk factors including aging, lifestyle choices, and genetic predisposition. A gene variant in the complement factor H gene (CFH) that leads to the Y402H polymorphism in the factor H protein (FH) confers the second highest risk for the development and progression of AMD.  FH is a major negative regulator of the alternative A. Armento (*) · M. Ueffing Institute for Ophthalmic Research, Department for Ophthalmology, Eberhard Karls University of Tübingen, Tübingen, Germany e-mail: [email protected]; [email protected] D. A. Merle Institute for Ophthalmic Research, Department for Ophthalmology, Eberhard Karls University of Tübingen, Tübingen, Germany Department of Ophthalmology, Medical University of Graz, Graz, Austria e-mail: [email protected]

pathway of the complement system, and the FH Y402H variant leads to increased complement activation, which is detectable in AMD patients. For this reason, various therapeutic approaches targeting the complement system have been developed, however, so far with very limited or no efficacy. Interestingly, recent studies suggest roles for FH beyond complement regulation. Here, we will discuss the noncanonical functions of FH in RPE cells and highlight the potential implications of those functions for future therapeutic approaches. Keywords

Age-related macular degeneration (AMD) · Retinal pigment epithelium (RPE) cells · Intracellular complement factor H (CFH)

1 Introduction Age-related macular degeneration (AMD) is a complex and progressive disease of the macula, which affects mainly the elderly population, especially in the developed countries [1, 2]. Due to demographic changes in western societies, AMD incidence is constantly rising, and the concomitant visual impairment threatens not only

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_2

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the individual’s quality of life but also poses a significant burden on healthcare systems worldwide [3]. The early stages of the disease are characterized by the presence of enlarging drusen in the retina. With disease progression, early AMD advance to late stages: wet or dry AMD. Wet AMD is defined by the presence of neovascularization in the retina, while dry AMD is limited to progressive RPE and retinal atrophy, called geographic atrophy (GA), without newly formed vessels. Around 10% of the cases constitute wet AMD, and intravitreal anti-VEGF is effective in treating most cases. However, only recently, one approved treatment has become available for dry AMD in the US [2, 8]. The main pathogenetic driver for AMD is advanced age. Specific genetic factors can predispose for AMD or protect from it. Unhealthy lifestyle habits, like smoking or malnutrition, can further increase the risk for AMD. The combination of those risk factors leads to pathological changes in the retina, involving a plethora of cellular mechanisms promoting disease progression [4].

2 Complement System in AMD Among the processes involved in AMD, the complement system is associated to AMD at different levels. First of all, many single nucleotide polymorphisms (SNPs) associated with AMD cluster into genes of the alternative pathway of the complement system with the Complement Factor H (CFH) gene carrying the second highest risk for AMD [5]. The most common risk haplotype causes an amino acid substitution from tyrosine to histidine at position 402 (called Y402H) in proteins coded by CFH, which are full length FH and a truncated version, FHL-1 [6, 7]. The complement system is a part of the innate immune system designed to recognize and mediate the removal of pathogens or waste material [8]. The eye is an immune-privileged organ, meaning that inflammation needs to be strictly limited, and therefore uncontrolled complement activation is deleterious [8]. FH is a major negative regulator of the complement system, and the AMD variant

A. Armento et al.

402H of FH causes uncontrolled complement activation in  vitro and in  vivo [9]. Moreover, complement factors accumulate in drusen [10], and increased complement system activation has been detected in AMD patients, independent of their genotype [11]. Due to the strong implications of complement system activation in AMD pathology, various complement inhibitors have been tested in clinical trials. However, most agents that entered phase II were discontinued due to a reported lack of efficacy. Interestingly, in some clinical trials, a group of subjects showed improvements, while others did not [12]. Apparently, one of the suspected reasons for the observed lack of efficacy is a lack of patient’s stratification and identification of those patients that are likely to benefit from complement inhibition. Moreover, several studies point to a lack of understanding of the role of FH in AMD pathology, especially in RPE cells, which are degenerating in AMD.

3 Noncanonical Role of Complement Factor H in RPE Cells RPE cells exert several functions vital for retinal homeostasis, which are impaired in AMD [13]. First of all, RPE cells are responsible for phagocytosis of shed photoreceptor outer segments (POS) and recycling of visual pigments, needed for a proper visual cycle. This process is energy intensive, requiring constant mitochondrial respiration and glycolysis within RPE cells. Due to a lack of the inner retina vasculature in the macula, RPE cells are vital to ascertain nutrient supply from the choriocapillaris. Any pathological disruption or perturbance of metabolic activity of RPE cells would affect the amount of nutrients that can reach the neuroretina. Indeed, the so-­called bioenergetics crisis of RPE cells has been described for AMD pathology [14]. Moreover, RPE cells quench short UV light as well as the continuous influx of peroxidized lipids from phagocytosed photoreceptor outer segments and thus need a high degree of antioxidant capacity as well as perfect functioning of their

The Noncanonical Role of Complement Factor H in Retinal Pigment Epithelium (RPE) Cells and…

mitochondria to protect the retina from excessive oxidative stress. Our recent work demonstrates that FH is endogenously produced by RPE cells. Within RPE cells, FH is acting locally supporting retinal homeostasis [15, 16]. This function of FH is context dependent, and we suggest that locally produced FH exerts specific functions at the RPE/retina interface. Most of these newly discovered functions (summarized in Fig. 1) affect a balanced regulation of immune competence and surveillance as well as regulating oxidative stress response and energy metabolism, mechanisms which are part of AMD pathology [8]. FH plays a protective role against oxidative stress in RPE cells. Exogenously applied recombinant FH protects RPE cells from damage induced by the exposure to lipid oxidation products [17, 18]. In parallel, loss of endogenous FH in RPE cells, via CFH silencing, also led to increased vulnerability to oxidative stress, increased levels of oxidized lipids, and reduced viability [15]. Interestingly, addition of recombinant FH in CFH-silenced RPE cells did not cause any rescuing effects [15]. This finding indicates that, although exogenous FH is protective, a proper intracellular FH function must be present to ascertain physiological balance and resilience to stress. This phenomenon may be explained by impaired mitochondrial function seen in iPSC-­ RPE cells carrying the CFH Y402H polymorphism [19] and CFH-silenced hTERT-RPE1 cells [15]. In both models, major processes involved in energy production, glycolysis, and mitochondrial respiration are impaired. In addition, a misbalance in the degree of mitochondria turnover seems to be involved, with an exaggerated increase in mitophagy [15, 19]. Moreover, it has been shown in iPSC-RPE carrying CFH 402H variant that mitochondria are enlarged and accumulating [20]. Mitochondria are essential for a correct response to oxidative stress; therefore, damage or dysfunction of these organelles is deleterious in such an oxidative milieu like the retina. Accumulation of oxidized lipids has been reported in RPE cells under FH dysregulation and induced oxidative stress, when FH function was impaired as a consequence of CFH silencing

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[15] or in the presence of the CFH 402H variant [20]. Moreover, accumulation of lipids was shown also in cfh Y402H mouse models of AMD, especially when subjected to high cholesterol diet [21]. The mechanism of action of intracellular FH is not fully elucidated. Based on the data generated in iPSC-RPE cells carrying the CFH 402H variant, it has been speculated that more than one signaling pathway mediates the effects of FH [19]. Specifically, it has been postulated that the NF-kB pathway and mTOR pathway are likely involved, in concomitance with autophagy and proteasome activity dysregulation, which ultimately lead to mitochondrial damage [19]. In our model of CFH-silenced hTERT-RPE1 cells, we could verify these hypotheses. Indeed, we found that in absence of endogenous FH, both the NF-kB and mTOR pathway are upregulated, with NF-kB triggering pro-inflammatory cytokine expression [22] and the mTOR pathway modulating mitochondrial respiration, but not glycolysis [23]. The mTOR pathway is a major regulator of autophagy [24]. Dysfunction in the autophagy-lysosomal axis in iPSC-RPE cells with CFH high risk was reported but is not regulated by this pathway. Its dysfunction rather depends on the accumulation of complement activation products [25]. Based on our analyses of the intracellular FH protein interactions that form a functional interactome in RPE cells, we suggest a regulatory role of FH in interaction with the ubiquitin proteasome system (UPS) and RB1/E2F signaling, adding a new level of complexity in the understanding of FH mechanism of action [23]. Interestingly, and based on studies in other cell types than RPE, it has already been suggested that FH has a noncanonical intracellular function, independent from complement activation. In clear renal cell carcinoma cells and kidney endothelial cells, FH knockdown impairs cellular homeostasis, at least partially via NF-kB activation [26, 27]. The synopsis of these findings indicates that understanding the function of FH in a specific cell and tissue context is key. In the context of AMD, it is important to advance our understanding on how RPE cells interact with the neuroret-

A. Armento et al.

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Fig. 1  Schematic representation of the noncanonical functions of FH in RPE cells

ina once intracellular FH dysregulation or a CFH 402H high risk variant perturbs intracellular homeostasis within these cells. In this regard, we have recently established a novel coculture model of RPE and neuroretina. Using this system, we show that CFH-silenced RPE cells promote retinal degeneration, which could not be rescued by the addition of exogenous FH.  Moreover, we showed that the damage occurred at the level of mitochondrial activity and lipid oxidation in the photoreceptors, rather than a canonical inflammation or complement activation [16].

4 Future Perspective Recent works of several groups, including ours, emphasize a noncanonical and intracellular role of FH in RPE cells. Besides their reduced capacity to dampen alternative complement activity, CFH risk polymorphisms such as Y402H impair RPE homeostasis and are likely contributing to promote AMD. In consequence, a simple comple-

ment pathway inhibitor strategy to treat AMD progression may not suffice. Rational re-­balancing strategies in a form of combinatorial therapies may be needed to target several drivers of AMD molecular pathology in a systems-­oriented fashion simultaneously. More specifically, the increase of alternative complement pathway activity at the Bruch’s membrane/RPE interface may have to be considered concomitantly with pro-inflammatory, oxidative stress-­ related perturbation and metabolic disbalance of RPE and neuroretina to achieve effective future therapeutic regimes for dry AMD. Last but not least, a very thorough stratification of patient groups may be needed to move to an individualized and rational therapy for dry AMD of the future.

References 1. Ferris FL 3rd, Wilkinson CP, Bird A, Chakravarthy U, Chew E, Csaky K, et al. Clinical classification of age-related macular degeneration. Ophthalmology. 2013;120(4):844–51.

The Noncanonical Role of Complement Factor H in Retinal Pigment Epithelium (RPE) Cells and… 2. van Lookeren Campagne M, LeCouter J, Yaspan BL, Ye W.  Mechanisms of age-related macular degeneration and therapeutic opportunities. J Pathol. 2014;232(2):151–64. 3. Wong WL, Su X, Li X, Cheung CM, Klein R, Cheng CY, et  al. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob Health. 2014;2(2):e106–16. 4. Armstrong RA, Mousavi M. Overview of risk factors for age-related macular degeneration (AMD). J Stem Cells. 2015;10(3):171–91. 5. Fritsche LG, Fariss RN, Stambolian D, Abecasis GR, Curcio CA, Swaroop A. Age-related macular degeneration: genetics and biology coming together. Annu Rev Genomics Hum Genet. 2014;15:151–71. 6. Klein RJ, Zeiss C, Chew EY, Tsai JY, Sackler RS, Haynes C, et  al. Complement factor H polymorphism in age-related macular degeneration. Science. 2005;308(5720):385–9. 7. Day AJ, Willis AC, Ripoche J, Sim RB.  Sequence polymorphism of human complement factor H. Immunogenetics. 1988;27(3):211–4. 8. Armento A, Ueffing M, Clark SJ.  The complement system in age-related macular degeneration. Cell Mol Life Sci. 2021;78(10):4487–505. 9. Skerka C, Lauer N, Weinberger AA, Keilhauer CN, Suhnel J, Smith R, et  al. Defective complement control of factor H (Y402H) and FHL-1  in age-related macular degeneration. Mol Immunol. 2007;44(13):3398–406. 10. Mullins RF, Russell SR, Anderson DH, Hageman GS.  Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease. FASEB J. 2000;14(7):835–46. 11. Heesterbeek TJ, Lechanteur YTE, Lores-Motta L, Schick T, Daha MR, Altay L, et  al. Complement activation levels are related to disease stage in AMD. Invest Ophthalmol Vis Sci. 2020;61(3):18. 12. Park DH, Connor KM, Lambris JD.  The challenges and promise of complement therapeutics for ocular diseases. Front Immunol. 2019;10:1007. 13. Strauss O.  The retinal pigment epithelium in visual function. Physiol Rev. 2005;85(3):845–81. 14. Fisher CR, Ferrington DA.  Perspective on AMD pathobiology: a bioenergetic crisis in the RPE. Invest Ophthalmol Vis Sci. 2018;59(4):AMD41–AMD7. 15. Armento A, Honisch S, Panagiotakopoulou V, Sonntag I, Jacob A, Bolz S, et al. Loss of complement factor H impairs antioxidant capacity and energy metabolism of human RPE cells. Sci Rep. 2020;10(1):10320. 16. Armento A, Murali A, Marzi J, Arrango-Gonzalez B, Kilger E, Clark SJ, et  al. FH loss in RPE cells causes retinal degeneration in a human RPE-­

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porcine retinal explant co-culture model. bioR xiv:2021:2021.07.26.453778. 17. Borras C, Canonica J, Jorieux S, Abache T, El Sanharawi M, Klein C, et al. CFH exerts anti-oxidant effects on retinal pigment epithelial cells independently from protecting against membrane attack complex. Sci Rep. 2019;9(1):13873. 18. Krilis M, Qi M, Qi J, Wong JWH, Guymer R, Liew G, et  al. Dual roles of different redox forms of complement factor H in protecting against age related macular degeneration. Free Radic Biol Med. 2018;129:237–46. 19. Ebeling MC, Geng Z, Kapphahn RJ, Roehrich H, Montezuma SR, Dutton JR, et al. Impaired mitochondrial function in iPSC-retinal pigment epithelium with the complement factor H polymorphism for age-­ related macular degeneration. Cell. 2021;10(4):789. 20. Hallam D, Collin J, Bojic S, Chichagova V, Buskin A, Xu Y, et al. An induced pluripotent stem cell patient specific model of complement factor H (Y402H) polymorphism displays characteristic features of age-related macular degeneration and indicates a beneficial role for UV light exposure. Stem Cells. 2017;35(11):2305–20. 21. Ding JD, Kelly U, Groelle M, Christenbury JG, Zhang W, Bowes Rickman C. The role of complement dysregulation in AMD mouse models. Adv Exp Med Biol. 2014;801:213–9. 22. Armento A, Schmidt TL, Sonntag I, Merle DA, Jarboui MA, Kilger E, et al. CFH loss in human RPE cells leads to inflammation and complement system dysregulation via the NF-kappaB pathway. Int J Mol Sci. 2021;22(16):8727. 23. Merle D, Provenzano F, Jarboui MA, Kilger E, Clark S, Deleidi M, et al. mTOR inhibition via Rapamycin treatment partially reverts the deficit in energy metabolism caused by FH loss in RPE cells. bioR xiv:2021:2021.10.29.466270. 24. Wang Y, Zhang H. Regulation of autophagy by mTOR signaling pathway. In: Qin Z-H, editor. Autophagy: biology and diseases: basic science. Singapore: Springer; 2019. p. 67–83. 25. Cerniauskas E, Kurzawa-Akanbi M, Xie L, Hallam D, Moya-Molina M, White K, et al. Complement modulation reverses pathology in Y402H-retinal pigment epithelium cell model of age-related macular degeneration by restoring lysosomal function. Stem Cells Transl Med. 2020;9(12):1585–603. 26. Daugan MV, Revel M, Thouenon R, Dragon-Durey MA, Robe-Rybkine T, Torset C, et  al. Intracellular factor H drives tumor progression independently of the complement cascade. Cancer Immunol Res. 2021;9:909–25. 27. Mahajan S, Jacob A, Kelkar A, Chang A, McSkimming D, Neelamegham S, et al. Local complement factor H protects kidney endothelial cell structure and function. Kidney Int. 2021;100(4):824–36.

Macular Pigment Carotenoids and Bisretinoid A2E Ranganathan Arunkumar and Paul S. Bernstein

Abstract

Keywords

Lutein (L), zeaxanthin (Z), and meso-­ Abca4 · A2E · Antioxidant · Bisretinoids · zeaxanthin (MZ) are the three macular pigCarotenoids · Lipofuscin · Macular pigments ments (MP) carotenoids that uniquely · Photo-oxidation · Retina · Retinal pigment accumulate in the macula lutea region of the epithelium human retina. L and Z are obtained by humans through dietary intake. The third MP, MZ, is rarely present in diet, and its abundance in the 1 Introduction human fovea is due to the metabolic conversion of dietary L by the retinal pigment epithe- Carotenoids are natural pigments synthesized by lium’s RPE65 enzyme. The major functions of photosynthetic bacteria, algae, yeast, and plants. MP in ocular health are to filter high-intensity, L, Z, and MZ are oxygenated carotenoids (xanphototoxic blue light and to act as effective thophylls) obtained by humans through dietary antioxidants for scavenging free radicals. The intake of leafy greens, fruits, and vegetables. pyridinium bisretinoid, N-retinylidene-N-­ Among the 15 major dietary carotenoids detected retinylethanolamine (A2E), contributes to in human serum, these three xanthophyll carotdrusen formation in dry age-related macular enoids selectively accumulate with a unique disdegeneration (AMD) and to the autofluores- tribution in the macular region of human retina cent flecks in autosomal recessive Stargardt [1]. The MP’s total concentration is about 1 mM disease (STGD1). Retinal carotenoids attenu- at the fovea, and its concentration declines to less ate A2E formation and can directly and indi- than 10 μM in the peripheral retina [2]. The ratio rectly alleviate A2E-mediated oxidative of L:Z:MZ in the peripheral retina is about 3:1:0, damage. In this chapter, we review these more and the concentration of total carotenoids rises recently recognized interconnections between 100-fold in the macula lutea with a change in the MP carotenoids and A2E bisretinoids. ratio of L:Z:MZ of about 1:1:1, as measured by high-performance liquid chromatography (HPLC) [3]. More recently, high-resolution confocal resonance Raman microscopy imaging of R. Arunkumar · P. S. Bernstein (*) the human retina from our laboratory has showed Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah that the ratio of Z + MZ:L can be greater than 9:1 School of Medicine, Salt Lake City, UT, USA at the foveal center, while L is more diffusely dise-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_3

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R. Arunkumar and P. S. Bernstein

tributed across the macula at a relatively lower aided by conjugated double-bond structure with concentration [4]. Retinal carotenoids mainly the hydrophilic group on the ionone ring [2]. The localize to the outer plexiform (Henle fiber) layer absorption maximum of L is around 445 nm, and and the inner plexiform layer, with axial exten- Z is around 450 nm, corresponding with the hazsion from the inner limiting membrane to the ardous blue light range of 430–500  nm. outer limiting membrane. The specificity in the Depending on the retinal carotenoid concentradistribution of retinal carotenoids in the human tion, they are estimated to absorb 40–90% of retina may be due to the selective uptake of reti- incident short-wavelength, high-energy photonal carotenoids by carotenoid binding proteins toxic blue light [8]. StARD3 (L binding protein) and GSTP1 (Z and Retinal carotenoids with conjugated double MZ binding protein). The other major factors that bond (C=C) structures are associated with greater influence the MP carotenoid distributions and singlet oxygen quenching. Z has 11 conjugated concentrations between individuals are the double bonds and is 50% better at quenching sinamount of oral intake of carotenoids and their glet oxygen relative to L with its 10-conjugated bioavailability [5]. L is present in higher concen- double bonds [9]. MZ, a stereoisomer of Z, with trations in the diet, but it is converted to MZ by an identical 11-conjugated double-bond structure the RPE65 enzyme, and the preferential accumu- would be expected to possess the same antioxilation of Z and MZ at the foveal center may be dant properties as Z, but it exhibited better singlet due to their higher antioxidant potential relative oxygen quenching properties relative to L and Z to L. The fovea is prone to photo-damage caused [9]. Furthermore, the antioxidant activity of all by light-induced oxidative stress from reactive three retinal carotenoids as a mixture showed betoxygen species, and singlet oxygen can be gener- ter singlet oxygen quenching ability than any of ated by photo-oxidation of A2E and other bisreti- the individual MP carotenoids. Retinal carotnoid components of lipofuscin [6]. A2E acts as a enoids not only quench singlet oxygen, but they photosensitizer in the presence of short-­ also quench superoxide anion radicals and wavelength blue light and oxygen that generate hydroxyl radicals, the major causative agents of reactive oxygen caused by photo-oxidation and lipid peroxidation and light-induced damage [10, photo-degradation, resulting in retinal degenera- 11]. Generally, retinal carotenoids are better tion and apoptosis of photoreceptor and RPE hydroxyl radical scavengers than superoxide cells [7]. Supplementation with retinal carot- anion scavengers, and Z exhibits better hydroxyl enoids potentially attenuates harmful blue light radical scavenging activity than lutein [10]. MP and effectively scavenges singlet oxygen and carotenoids protect the retina, which is vulnerareactive free radicals in ocular tissues and in bio- ble to oxidative damage caused by exposure to chemical assays. light, high concentration of oxygen, abundant photosensitizers, and high concentrations of polyunsaturated fatty acids (PUFAs).

2 Structure and Function of the Retinal Carotenoids

Retinal carotenoids are characterized by the presence of hydroxyl (O-H) functional groups attached at the 3 and 3′ positions of terminal ionone rings connected by an isoprenoid backbone structure (Fig.  1). The presence of hydroxyl groups and the conjugated double bonds structure determine the light-absorbing properties and antioxidant activities of retinal carotenoids. The light filtering function of retinal carotenoids is

3 A2E Bisretinoids In the visual cycle, the activation of rhodopsin by a photon of light during phototransduction results in the isomerization of 11-cis-retinal to all-trans-­ retinal in photoreceptor outer segments and the release of all-trans-retinal, which is subsequently reduced to all-trans-retinol and transported to the RPE cells. In the RPE, the all-trans-retinol is either stored as a fatty acid ester or is converted

Macular Pigment Carotenoids and Bisretinoid A2E

17 OH

HO

Lutein OH

HO

Zeaxanthin

N

OH

OH

A2E

HO

Meso-Zeaxanthin

Fig. 1  Structures of the macular carotenoids: lutein (L), zeaxanthin (Z), meso-zeaxanthin (MZ), and N-retinylidene-N-­ retinylethanolamine (A2E)

back to an 11-cis-retinoid that is transported back to the photoreceptor outer segment, which can bind to opsin to produce a regenerated visual pigment. Some all-trans-retinal in the photoreceptor outer segments reacts with phosphatidylethanolamine (PE) to form N-retinylidene-PE (NRPE), which is transported or “flipped” by the ATP-­ binding cassette, subfamily A, member 4 (ABCA4) protein present in photoreceptor outer segments. ABCA4 translocates NRPE across the lipid bilayer from the luminal side to the cytoplasmic side of the disc membrane. NRPE released on the cytoplasmic side is hydrolyzed into all-trans-retinal and reduced to all-trans-­ retinol by retinol dehydrogenases (RDHs) [12]. If ABCA4 does not effectively translocate NRPE, it reacts with one more molecule of all-trans-­retinal to form a toxic bisretinoid, dihydropyridinium-­ A2PE.  Dihydropyridinium-A2PE is further oxidized either to A2-dihydropyridine-PE ­ (A2-DHP-PE) by eliminating one hydrogen or to phosphatidylethanolamine-pyridinium bisretinoid (A2PE) by eliminating two hydrogens in the acidic and oxidizing environment (Fig. 2). During phagocytosis of photoreceptor outer segments by

RPE, A2PE is further hydrolyzed to A2E by phospholipase D inside RPE phagosomes. Mutation or dysfunction in the ABCA4 gene leads to excessive accumulation of A2E and other bisretinoids in RPE lipofuscin, resulting in retinal cell death and vision loss. A2E is reported to accumulate with aging and dry AMD and in STGD1 [13]. A2E (C42H58ON; molecular weight, 592) is a well-characterized component of lipofuscin. It exhibits absorbance in both the UV and visible regions of the spectrum [A2E: absorbance maxima (λmax) are 440 and 340  nm, and iso-A2E’s λmax are 430 and 340  nm]. A2E’s hydrophobic side arms and positively charged hydrophilic head group confer an amphiphilic structure (Fig.  1). A2E has a detergent-like structure that may influence membrane properties and inhibit the lysosomal degradation of lipids. A2E induces loss of membrane integrity and perturbs membrane stability, and it inhibits cytochrome C oxygenase and the ATP-driven proton pump. A2E photo-oxidation products can activate the complement system and cause inflammation and DNA damage [12, 13].

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Fig. 2  The visual cycle and bisretinoid formation in photoreceptor outer segments and RPE. When a photon of light is captured by the visual chromophore, its 11-cis-­ retinal (11-cis-RAL) Schiff base chromophore photoisomerizes to all-trans-retinal (all-trans-RAL). The released all-trans-RAL from opsin is reduced to all-trans-­ retinol (all-trans-ROL) by retinol dehydrogenases (RDHs). Alternatively, some all-trans-RAL reacts with phosphatidylethanolamine (PE) in the photoreceptor outer segments to form N-retinylidene-PE (NRPE), which is transported by ABCA4. The bisretinoid synthesis pathway (red) is initiated when NRPE, rather than hydrolyzing to all-trans-ROL and PE, reacts with a second molecule of retinaldehyde. A multistep pathway leads to the formation of the intermediate dihydropyridinium-A2PE.  Auto-­ oxidation of dihydropyridinium-A2PE with loss of two

hydrogens generates A2PE, and loss of one hydrogen generates A2-DHP-PE; phosphate hydrolysis of the latter produces A2-DHP-E.  A2E, lysoA2PE, and A2-GPE are produced from A2PE.  Reaction of the all-trans-RAL dimer with PE with the formation of a Schiff base linkage generates all-trans-retinal dimer-PE (atRALdi-PE), and phosphate hydrolysis of the latter yields all-trans-retinal dimer-ethanolamine (atRALdi-E). All-trans-ROL is transported into RPE cells where all-trans-ROL is esterified to fatty acids to make all-trans retinyl esters (all-­ trans-­RE) by the enzyme lecithin retinol acyl transferase (LRAT). All-trans-RE is converted to 11-cis retinol (11-cis-ROL) by the RPE65 isomerohydrolase. 11-cis retinol dehydrogenase (11cRDH) oxidizes 11-cis-ROL to 11-cis-RAL which enters the photoreceptor outer segments for the regeneration of opsin

4 Retinal Carotenoids Attenuate Bisretinoids in Various Systems

showed additional peaks at m/z 608, 624, 640, 656, 672, and 688, as well as 1239, 1255, 1271, and 1286. The additional higher m/z peaks are photo-oxidation products of A2E and A2PE formed by the insertion of oxygen atoms at the C=C bonds along the side arms of A2E and A2PE.  When A2E and A2PE are irradiated at 430 nm in the presence of L and Z, the additional photo-oxidized peaks are absent [14]. Singlet oxygen quenching abilities of retinal carotenoids were studied in the presence of endoperoxide 1,4-dimethylnapthalene (1  mM), which releases

4.1 In Vitro A2E and its precursor A2PE can be irradiated with 430 nm blue light and analyzed by fast atom bombardment mass spectroscopy (FAB-MS). The FAB-MS spectra after irradiation not only showed a molecular ion peak at m/z 592 and 1223 attributable to A2E and A2PE, but they also

Macular Pigment Carotenoids and Bisretinoid A2E

singlet oxygen with a half-life time of 5  h. @ 25  °C.  The endoperoxide was incubated with A2E (500  μM) with and without retinal carotenoids (500  μM to 4  mM). A2E oxidation was measured using HPLC, and it was found that retinal carotenoids effectively quench singlet oxygen products in a concentration-dependent manner. Furthermore, Z was observed to be a better singlet oxygen quencher than L [14].

4.2 Cell Culture

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3.1-fold in the zeaxanthin-supplemented group. A2E levels increased sixfold in the unsupplemented control group, whereas there was minimal increase in A2E in the MP carotenoid supplemented group, and their A2E levels were significantly lower than the control group [16].

4.5 Rodent Model

Until recently, the protective effects of macular carotenoids against bisretinoids had not been The protective effects of retinal carotenoids on studied in small laboratory mammals because photo-oxidation of A2E were studied in ARPE-­ non-primates do not accumulate substantial 19 cells grown in Dulbecco’s Modified Eagle amounts of carotenoids in ocular tissues due to Medium (DMEM) medium with 10 μM A2E for the presence of very active carotenoid cleavage 10  days. The cells accumulated A2E and were enzymes (Bco1 and Bco2). This led us to cross incubated with L or Z (10  μM), and they were our Bco2−/− “macular pigment mice” that accuthen exposed to blue light (430 nm). Cells incu- mulate dietary MP carotenoids in their retinas bated with retinal carotenoids completely attenu- with a mouse model of STGD that accumulates ated A2E photo-oxidation and inhibited A2E with increasing age. Abca4−/−/Bco2−/− mice proteasome inactivation [15]. fed with L or Z have lower levels of A2E and iso-­ A2E compared to the control group with no carotenoid supplementation, and there was a sta4.3 Human Donor Eyes tistically significant inverse correlation between retinal carotenoids and A2E and iso-A2E levels A2E concentrations in human cadaver eyes in RPE/choroid (Fig.  3). Furthermore, L and Z increase with age in both the macula and in the supplementation improved visual performance in peripheral retina, and A2E levels are threefold Abca4−/−/Bco2−/− mice compared to the control lower in the macula region compared to the group [13]. peripheral retina despite excess light exposure and high metabolic activity. Moreover, A2E concentrations are inversely related to retinal carot- 5 Summary enoid concentrations [16]. Lipofuscin and A2E levels are significantly lower in the fovea region The macular pigment carotenoids are distinct where retinal carotenoids are present in abun- from other nutrients due to their selective accudance [17]. mulation and unique spatial distribution in the fovea, the region most vulnerable to excess light and high metabolic oxygen. While it is widely 4.4 Avian Model appreciated that the beneficial properties of MP carotenoids are in part mediated by their blue The association between retinal carotenoids light-absorbing and antioxidant properties, it is and A2E was studied in Japanese quail, whose now becoming increasingly understood that retinal carotenoids are present as fatty acid attenuation of A2E formation and oxidation by L, esters in distinct photoreceptor oil drops. After Z, and MZ are important protective effects as 16  weeks of feeding, total carotenoids rose well, further emphasizing their valuable role as 1.6-fold in the lutein-supplemented group and safe and cost-effective nutritional interventions

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Fig. 3  Distribution of RPE/choroid A2E + iso-A2E levels in relation to retinal carotenoids in Abca4−/−/Bco2−/− mice. There was a statistically significant inverse correlation between retinal carotenoids and A2E and iso-A2E levels in RPE/choroid (p values: *p