Retinal Degenerative Diseases XIX: Mechanisms and Experimental Therapy (Advances in Experimental Medicine and Biology, 1415)

Description

This book contains the proceedings of the XVIII International Symposium on Retinal Degeneration (RD2018). A majority of those who spoke  and presented posters at the meeting contributed to this volume. The blinding diseases of inherited retinal degenerations have no treatments, and age-related macular degeneration has no cures, despite the fact that it is an epidemic among the elderly, with 1 in 3-4 affected by the age of 70. The RD Symposium focused on the exciting new developments aimed at understanding these diseases and providing therapies for them. Since most major scientists in the field of retinal degenerations attend the biennial RD Symposia, they are known by most as the “best” and “most important” meetings in the field. The volume presents representative state-of-the-art research in almost all areas of retinal degenerations, ranging from cytopathologic, physiologic, diagnostic and clinical aspects; animal models; mechanisms of cell death; candidate genes, cloning, mapping and other aspects of molecular genetics; and developing potential therapeutic measures such as gene therapy and neuroprotective agents for potential pharmaceutical therapy. While advances in these areas of retinal degenerations were described, there will be many new topics that either are in their infancy or did not exist at the time of the last RD Symposium. These include the role of inflammation and immunity, as well as other basic mechanisms, in age-related macular degeneration, several new aspects of gene therapy, and revolutionary new imaging and functional testing that will have a huge impact on the diagnosis and following the course of retinal degenerations, as well as to provide new quantitative endpoints for clinical trials. The retina is an approachable part of the central nervous system (CNS), and there is a major interest in neuroprotective and gene therapy for CNS diseases and neurodegenerations, in general. It should be noted that with successful and exciting initial clinical trials in neuroprotective and gene therapy, including the restoration of sight in blind children, the retinal degeneration therapies are leading the way towards new therapeutic measures for neurodegenerations of the CNS. Many of the successes recently reported in these areas of retinal degeneration sprang from collaborations established at previous RD Symposia, and many of those were reported at the RD2016 meeting and included in the current volume. We anticipate the excitement of those working in the field and those afflicted with retinal degenerations is reflected in the volume.  Preface 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 1 Introduction 2 Methods 2.1 Tissue Acquisition and Characterization 2.2 Tissue Handling and Ex Vivo Imaging 2.3 MALDI IMS Analysis 3 Results 3.1 Signals Specific to Photoreceptors and RPE 3.2 Signals Specific to Basal Laminar Deposit 4 Conclusions References The Noncanonical Role of Complement Factor H in Retinal Pigment Epithelium (RPE) Cells and Implications for Age-Related Macular Degeneration (AMD) 1 Introduction 2 Complement System in AMD 3 Noncanonical Role of Complement Factor H in RPE Cells 4 Future Perspective References Macular Pigment Carotenoids and Bisretinoid A2E 1 Introduction 2 Structure and Function of the Retinal Carotenoids 3 A2E Bisretinoids 4 Retinal Carotenoids Attenuate Bisretinoids in Various Systems 4.1 In Vitro 4.2 Cell Culture 4.3 Human Donor Eyes 4.4 Avian Model 4.5 Rodent Model 5 Summary References Disturbed Matrix Metalloproteinases Activity in Age-Related Macular Degeneration 1 Introduction 2 MMPs Are Implicated in AMD 2.1 MMPs in Dry AMD 2.2 MMPs in Wet AMD 3 Concluding Remarks References Current Views on Chr10q26 Contribution to Age-Related Macular Degeneration 1 Introduction 2 ARMS2 2.1 Reported Tissue, Cellular and Subcellular Expression of ARMS2/ARMS2 2.2 ARMS2 Risk Variants and Their Putative Role in AMD 3 HTRA1 3.1 Reported Tissue and Cellular Expression of HTRA1/HTRA1 3.2 Contribution of HTRA1 Gene Variants to AMD 4 Conclusions References Untargeted Lipidomic Profiling of Aged Human Retina With and Without Age-Related Macular Degeneration (AMD) 1 Introduction 2 Material and Methods 2.1 Sample Procurement and Preparation for LC-MS/MS Analysis 2.2 High Flow LC-MS/MS Procedure 2.3 NanoLC-MS/MS Procedure 2.4 Data Analysis 3 Results 3.1 Elution Profile of Lipids Identified by High Flow LC-MS/MS 3.2 Untargeted Lipidomic Analysis of Healthy Human Donor RPE-Choroid Using High Flow LC-MS/MS 3.3 Comparison of Lipid Profiling from High Flow LC-MS/MS and nLC-MS/MS 4 Discussion 5 Conclusion References Decoding Race and Age-Related Macular Degeneration: GPR 143 Activity Is the Key 1 Introduction 2 GPR143 3 GPR143 and Trophic Factors 4 L-Dopa Halts Extracellular Vesicle Release 5 GPR 143 Signaling in Photoreceptor Outer Segment Digestion 6 Clinical Studies with L-Dopa and AMD 7 Conclusion References Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1Alpha (PGC-1α): A Transcriptional Regulator at the Interface of Aging and Age-Related Macular Degeneration? 1 Introduction 2 Regulation of Tissue Activity of PGC-1α 3 Retinal Effects of Perturbation of PGC-1α Activity 4 Role of PGC-1α in Human AMD 5 Potential Modulation of the PGC-1α Pathway in AMD Treatment References Regulation of ABCA1 by miR-33 and miR-34a in the Aging Eye 1 Introduction 2 miRNA 3 Regulation of ABCA1 by miR-33 4 Regulation of ABCA1 by miR-34a 5 Concluding Remarks and Future Therapeutic Perspectives References The Role of Gene Expression Regulation on Genetic Risk of Age-Related Macular Degeneration 1 Introduction 2 Connecting AMD-GWAS Discoveries to Biology 3 Variant to Function by Studying eQTLs 4 eQTL Studies in AMD-Relevant Human Tissues 5 Future Experimental Approaches References 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 1 Introduction 2 Cigarette Smoke Exposure (CSE) as a Model for Dry AMD 3 Neoepitopes Trigger Chronic Inflammation in Smoke-Induced Pathology 4 Is Pathology in the CSE Model Preventable with Peptide Immunotherapy? References Repurposing Drugs for Treatment of Age-Related Macular Degeneration 1 Introduction 2 L-DOPA 3 Metformin 4 Fluoxetine 5 Drugs Being Evaluated in Preclinical Models 5.1 Mitochondrial Activation Drugs 5.2 Serotonin Receptor Agonists 5.3 Antidiabetic Drugs 6 Conclusions and Outlook References Part II: Extracellular Vesicles Extracellular Vesicle RNA Contents as Biomarkers for Ocular Diseases 1 Introduction 2 EV RNAs as Biomarkers in Ocular Disorders 3 Concluding Remarks References Proteomics of Retinal Extracellular Vesicles: A Review into an Unexplored Mechanism in Retinal Health and AMD Pathogenesis 1 Introduction 2 Part I: Retinal EV Proteome in Normal Health 2.1 Part I.I: Retinal EV Proteins Have Distinct Roles in the Neural Retina and RPE to Support Host-Cell Function in Retinal Homeostasis 3 Part II: Retinal EV Proteome in Disease 3.1 Part II.I: RPE EV Proteins Modulate Cell Death, Oxidative Stress, and Inflammatory Pathways in Retinal Degeneration 3.2 Part II.II: RPE-EV Contributes to Drusen Formation 4 Conclusion References Part III: Gene Editing Prime Editing Strategy to Install the PRPH2 c.828+1G>A Mutation 1 Introduction 2 Materials and Methods 2.1 Clinical Evaluation 2.2 Cloning and Plasmid Constructs 2.3 Cell Culture and Transfection 2.4 DNA Extraction 2.5 Analysis of Prime Editing Efficiency 3 Results and Discussion References Analysis of CRB1 Pathogenic Variants Correctable with CRISPR Base and Prime Editing 1 Introduction 2 Materials and Methods 3 Results and Discussion References Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor 1 Introduction 2 Materials and Methods 2.1 Cloning and Plasmid Constructs 2.2 Cell Culture and Transfection 2.3 DNA Extraction 2.4 Analysis of Prime Editing Efficiency 3 Results 4 Discussion References Part IV: Gene Therapy Preexisting Neutralizing Antibodies against Different Adeno-Associated Virus Serotypes in Humans and Large Animal Models for Gene Therapy 1 Introduction 2 Preexistence of Antibodies Against Different Serotypes in Humans 3 Preexistence of Antibodies Against Different Serotypes in Large Animal Models 4 Strategies to Deal with the Anti-AAV Antibodies References Optimization of Capillary-Based Western Blotting for MYO7A 1 Introduction 2 Materials and Methods 2.1 AAV Production 2.2 Use of Animals and Eye Collections 2.3 In Vitro Infections and Cell Harvesting 2.4 Jess™ Simple Western Optimization 3 Results 3.1 Optimizing Sample and Antibody Concentrations for MYO7A and VCL 3.2 Determining Final Run Conditions for MYO7A Jess™ Assays 4 Discussion References AAV Serotypes and Their Suitability for Retinal Gene Therapy 1 Introduction – Adeno-Associated Viral Gene Therapy 2 Serotype Tropism Defines Cell- Tissue- and Host-Infection 3 The Eye as a Therapeutic Target 4 Concluding Remarks and Future Directions References Gene Augmentation for Autosomal Dominant CRX-Associated Retinopathies 1 Pathogenic Mechanisms of CRX Mutations 2 Gene Augmentation as a Strategy to Treat CRX-Associated Retinopathies 3 Unanswered Questions and Challenges 4 Methods 4.1 Transgenic Mouse Lines 4.2 Quantitative PCR (qPCR) References Txnip Gene Therapy of Retinitis Pigmentosa Improves Cone Health 1 Introduction 2 RP Glucose Hypothesis 3 Txnip Presents a Robust Therapeutic Effect 4 Future Directions References Part V: Human Retinal Degeneration Factors Affecting Readthrough of Natural Versus Premature Termination Codons 1 Introduction 2 Protein Translation 3 The Process of Translation Termination 4 Nucleotide Sequences Upstream the Stop Codon 5 Nucleotide Sequence Downstream the Stop Codon 6 Nonsense Mutations and TR 7 Decoding of Termination Codons References Integrating Computational Approaches to Predict the Effect of Genetic Variants on Protein Stability in Retinal Degenerative Disease 1 Part I. Protein Stability: Role and Importance 2 Part II. Common Computational Methods for Determining Variant Impact 3 Part III. New Methodologies: PathProx and POKEMON 4 Part IV. Retinal Diseases, AMD, and Protein Stability References Network Biology and Medicine to Rescue: Applications for Retinal Disease Mechanisms and Therapy 1 Introduction 2 Molecular Genetics of IRDs 3 Network Medicine and Healthcare 4 Retinal Degeneration Mechanisms and Therapy 5 Conclusion References Non-syndromic Retinal Degeneration Caused by Pathogenic Variants in Joubert Syndrome Genes 1 Introduction 2 Joubert Syndrome Genes Mutated in Non-syndromic Retinal Degeneration 2.1 CEP290 2.2 OFD1 2.3 ARL3 2.4 AHI1 2.5 INPP5E 3 Conclusions References Exonic Variants that Affect Splicing – An Opportunity for “Hidden” Mutations Causing Inherited Retinal Diseases 1 Introduction 2 Exonic Mutations Affecting the Donor and Acceptor Splice Sites 3 Exonic Variants Affecting Exonic Splicing Enhancer Sequences 4 Conclusion References Enhanced S-cone Syndrome, a Mini-review 1 Introduction 2 NR2E3 Gene Affects Photoreceptor Differentiation 3 Retinal Structure of ESCS 4 Visual Function of ESCS 4.1 Visual Sensitivity 4.2 Color Vision 4.3 Temporal Vision 4.4 Spatial Vision References Part VI: Inflammation The Role of Microglia in Inherited Retinal Diseases 1 Introduction 2 Phosphodiesterase 6B (Pde6b) 3 Membrane Frizzled Related Protein (Mfrp) 4 Crumbs Cell Polarity Complex Component 1 Gene (Crb1) 5 CX3C Chemokine Receptor 1 (Cx3cr1) 6 MER Proto-Oncogene, Tyrosine Kinase (Mertk) 7 Nuclear Receptor Subfamily 2 Group E Member 3 (Nr2e3) 8 Retinoid Isomerohydrolase (Rpe65) 9 Future Perspectives References CD68: Potential Contributor to Inflammation and RPE Cell Dystrophy 1 Introduction 2 CD68 Expression in the Eye 3 CD68 Expression Is Associated with RPE Dystrophy and Inflammation 4 Unresolved Roles of CD68 in AMD Pathogenesis References Gene Expression of Clusterin, Tissue Inhibitor of Metalloproteinase-1, and Their Receptors in Retinal Pigment Epithelial Cells and Müller Glial Cells Is Modulated by Inflammatory Stresses 1 Introduction 2 Methods 2.1 Cytokine Treatments and Quantitative RT-PCR 3 Results 4 Discussion References Part VII: Mechanisms of Degeneration Axonal Transport Defects in Retinal Ganglion Cell Diseases 1 Introduction 2 Axonal Degeneration and Transport Defects 3 Optic Neuropathies 4 Animal Models and Axonal Transport Defects 5 Future Perspectives References Connexins Biology in the Pathophysiology of Retinal Diseases 1 Connexin Biology in Different Retinal Components 2 Connexin Biology in Retinal Pathophysiology 3 Concluding Remarks References Role of Nuclear NAD+ in Retinal Homeostasis 1 Introduction 2 Enzymes Involved in NAD+ Synthesis Are Essential in the Retina 3 Role of NAD+ Consumers and NAD+ Depletion in Retinal Degeneration 4 Conclusions References Retinal Pigmented Epithelium-Derived Ectopic Norrin Does Not Promote Intraretinal Angiogenesis in Transgenic Mice 1 Introduction 2 Material and Methods 2.1 Animals 2.2 Immunohistochemistry and Retinal Flat Mount Preparation 2.3 Fluorescence Microscopy 3 Results 3.1 Canonical Wnt/β-Catenin Signaling Is Activated in NdpKO/Rpe65-Norrin Mice 3.2 RPE-Derived Norrin Does Not Promote Retinal Angiogenesis 3.3 Analysis of Blood-Retinal Barrier Integrity 4 Discussion References Caveolin-1 in Müller Glia Exists as Heat-Resistant, High Molecular Weight Complexes 1 Introduction 2 Materials and Methods 2.1 Cell Lines and Culture Conditions 2.2 Western Blotting 2.3 Immunoprecipitation 2.4 Mass Spectrometry 3 Results 3.1 Cav1 in MIO-M1 Müller Glia Exist as High Molecular Weight Complexes 3.2 Proteins Associated with Cav1 Complexes Are Involved with the Cell Cytoskeleton 4 Discussion References Role of VLC-PUFAs in Retinal and Macular Degeneration 1 Introduction 2 ELOVL4 and STGD3 3 ELOVL4 and AMD 4 Synthesis of VLC-PUFAs 5 Future Directions References Ocular Amyloid, Condensates, and Aggregates – Higher-Order Protein Assemblies Participate in Both Retinal Degeneration and Function 1 Introduction 2 Methods 2.1 Thioflavin T and Immunofluorescence Staining 3 Results 3.1 Aggregates Are Identifiable in Multiple Retinal Layers 4 Discussion 4.1 Do Aggregates Contribute to the Structure and Function of the Basal Body and Permeability Barrier of the Connecting Cilium? 4.2 Do Aggregates Participate in the Structure and Organization of the Photoreceptor Disc? 5 Summary and Future Directions References Photoreceptor Ion Channels in Signaling and Disease 1 Introduction 2 CNG Channels Respond to Light 3 Cav1.4 Channels Respond to CNG-Regulated Membrane Potential 4 HCN1 Channels Shape Light Responses 5 Kv2.1/Kv8.2 Channels 6 Future Directions References The Role of Peripherin-2/ROM1 Complexes in Photoreceptor Outer Segment Disc Morphogenesis 1 Peripherin-2 Is a Tetraspanin Protein Whose Mutations Cause Retinal Degeneration 1.1 Molecular Structure of Peripherin-2 and ROM1 1.2 Mutations in Peripherin-2 Are Associated with Retinal Degeneration 1.3 Supramolecular Organization of Peripherin-2/ROM1 2 The Function of Peripherin-2 2.1 Discs Are Formed through Peripherin-2-Dependent Suppression of Ciliary Ectosome Release 2.2 The Role of Peripherin-2 in Disc Rim Formation and Enclosure 3 Concluding Remarks and Future Avenues of Study References Human Mutations in Arl3, a Small GTPase Involved in Lipidated Cargo Delivery to the Cilia, Cause Retinal Dystrophy 1 Arl3 Unloads Lipidated Cargo onto Ciliary Membranes 2 Mouse Models of Arl3 3 Human Mutations in Arl3 References Genotype–Phenotype Association in ABCA4-Associated Retinopathy 1 Introduction 2 Genotype–Phenotype Correlation Analyses 2.1 Qualitative Genotype–Phenotype Correlation Analyses 2.1.1 Qualitative Descriptions of Mild Variants 2.1.2 Qualitative Descriptions of Severe Variants 2.2 Semi-quantitative Genotype–Phenotype Correlation Analyses 2.3 Quantitative Genotype–Phenotype Correlation Analyses 3 Future Directions 3.1 Interval-Scaled Severity Measures as Input 3.2 Interval-Scaled Reporting of Variant Severity 3.3 Differentiating Age-Dependent Severity from Genuine Phenotypic Differences 3.4 Latent Variable Methods 4 Summary References Retinal Pathoconnectomics: A Window into Neurodegeneration 1 Introduction 2 Remodeling 3 Pathoconnectomics and RPC1 4 Early Findings 5 Future of Pathoconnectomics References The Role of Ceramide in Inherited Retinal Disease Pathology 1 Introduction 2 Ceramide Metabolism 3 Ceramide Depletion by CerSs Deletion Also Has Deleterious Effects on the Retina 4 Conclusion References Extracellular Matrix: The Unexplored Aspects of Retinal Pathologies and Regeneration 1 Introduction 2 ECM in the Retina 3 Inner-Limiting Membrane 4 Bruch’s Membrane 5 Interphotoreceptor Matrix 6 ECM Composition 7 Collagens 8 Laminins 9 ECM Receptors 10 Dynamic Reciprocity 11 Concluding Remarks and Future Perspectives References Role of TFEB in Diseases Associated with Lysosomal Dysfunction 1 Introduction 2 Involvement of Lysosomal Dysfunction in the Pathogenesis of Several Diseases 3 Lysosomes and Its Importance in the Proper Functioning of the RPE 4 Lysosomal Dysregulation in the Pathogenesis of AMD 5 Enhancing TFEB as a Strategy to Induce Lysosomal Function 6 Conclusion References Retinoic Acid Receptor-Related Orphan Receptors (RORs) in Eye Development and Disease 1 Introduction 2 RORα Regulates Eye Development and Vascular Eye Diseases 2.1 RORα Regulates Lens and Cone Photoreceptor Development 2.2 Genetic Variations of RORA Are Linked with Age-Related Macular Degeneration (AMD) 2.3 RORα Modulates Retinal Inflammation and Neurovascular Interaction in a Mouse Model of Oxygen-Induced Retinopathy (OIR) 3 RORβ Regulates Retinal Neuronal Differentiation 4 RORγ in Autoimmune Regulation and Eye Diseases 4.1 RORγt, a Key Regulator of Th17 Cells, Is Implicated in Ocular Autoimmunity 4.2 RORγt/IL17 Signaling Axis in Retinal Vasculature 5 Conclusions References 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 1 Introduction 2 Results 2.1 Exons 2 and 3 of Ctrp5 Are Deleted in Ctrp5−/− Mice 2.2 Loss of Ctrp5 Expression in Ctrp5−/− Mice 2.3 Accumulation of Autofluorescent Spots in Ctrp5−/− Mice 2.4 Early RPE Stress and Accumulation of Sub-RPE Deposits with Age in Ctrp5−/− Mice 3 Materials and Methods 3.1 Generation of Mice 3.2 Evaluation of the Expression of CTRP5 and the Transcript 3.3 Autofluorescence Imaging 3.4 Histology and Ultrastructure Analysis of the Retina 4 Discussion References Comparison of Mouse Models of Autosomal Dominant Retinitis Pigmentosa Due to the P23H Mutation of Rhodopsin 1 Introduction 2 Mouse Models of the Rho-P23H Mutation 3 Pathogenic Mechanism Studies References Compensatory Cone-Mediated Mechanisms in Inherited Retinal Degeneration Mouse Models: A Functional and Gene Expression Analysis 1 Introduction 2 Materials and Methods 2.1 Animals 2.2 Electroretinography (ERG) 2.3 Optomotor Response 2.4 Flow Cytometry 2.5 qRT-PCR Analysis 2.6 Statistical Analysis 3 Results 3.1 Compensatory Gene Expression Changes in Response to Cell Death 3.2 ERG and Behavioral Visual Responses Persist in the Absence of Cone Function 4 Discussion References Inhibition of Ryanodine Receptor 1 Reduces Endoplasmic Reticulum (ER) Stress and Promotes ER Protein Degradation in Cyclic Nucleotide-Gated Channel Deficiency 1 Introduction 2 Materials and Methods 2.1 Experimental Animals 2.2 Construction of AAV2/5-SaCas9/gRNA-Ryr1 Vector 2.3 Subretinal Injection 2.4 Western Blotting and Immunofluorescence Labeling 3 Results 3.1 Treatment with AAV2/5-SaCas9/gRNA-Ryr1 Reduced Expression Level of Ryr1 in Cnga3−/−/Nrl−/− Retinas and Increased Expression Levels of Cone-Specific Proteins 3.2 Treatment with AAV2/5-SaCas9/gRNA-Ryr1 Reduced ER Stress Responses in Cnga3−/−/Nrl−/− Retinas and Increased Expression Levels of ERAD Proteins 4 Discussion References Mouse Choroid Proteome Revisited: Focus on Aging 1 Introduction 2 Methods 3 Results 4 Discussion References Morphological and Functional Comparison of Mice Models for Retinitis Pigmentosa 1 Introduction 2 Morphological Alterations of Retinal Structure 3 Comparison of Retinal Functional Response 4 Comparison of Protein Expression 5 Conclusion References Current Advancements in Mouse Models of Retinal Disease 1 Introduction 2 Inherited Retinal Dystrophies (IRDs): A “Monogenic” Retinal Degeneration 3 Current Mouse Models of RP 4 AMD: A Perfect Storm of Genetic Complexity and Environmental Stressors 5 Current Mouse Models of AMD 6 Systems Genetics and the BXD Family of Mice: The Future of Retinal Disease Modeling? 7 Conclusions References Single-Cell Transcriptomic Profiling of Müller Glia in the rd10 Retina 1 Introduction 2 Materials and Methods 3 Results 4 Discussion References Methods for In Vivo Characterization of Proteostasis in the Mouse Retina 1 Introduction 2 Approaches to Assess Proteostasis in Mouse Retina In Vivo 2.1 Assessments of UPS with Genetic Reporters 2.2 Autophagy Flux Measurements 2.3 Assessments of Protein Translation References Absence of PRCD Leads to Dysregulation in Lipid Homeostasis Resulting in Disorganization of Photoreceptor Outer Segment Structure 1 Introduction 2 PRCD Is Essential for Photoreceptor Outer Segment Maintenance 3 Phenotypic Variations in Different PRCD Mutant Animal Models 4 Retina Lacking PRCD Exhibit Altered Membrane Lipid Composition 5 Conclusions and Future Directions References Expansion Microscopy of Mouse Photoreceptor Cilia 1 Introduction 2 Materials and Methods 2.1 Solutions Needed 2.2 Gelation Solution 2.3 Method 2.4 Sectioning 2.5 Animals 2.6 Antibodies 3 Results 4 Discussion References Rod Photoreceptor-Specific Ablation of Metformin Target, AMPK, in a Preclinical Model of Autosomal Recessive Retinitis Pigmentosa 1 Introduction 2 Materials and Methods 2.1 Animals 2.2 Tamoxifen Injection 2.3 Immunohistochemistry and Morphometry 2.4 Electroretinography (ERG) 3 Results 3.1 Lack of Functional and Structural Rescue in Pde6bH620Q; Pde6gCreERT2; and AMPKfl/fl Mice 4 Discussion References TLR2 Is Highly Overexpressed in Retinal Myeloid Cells in the rd10 Mouse Model of Retinitis Pigmentosa 1 Introduction 2 Materials and Methods 2.1 Animals 2.2 RNA Isolation and Quantitative PCR 2.3 Flow Cytometry 2.4 Statistical Analysis 3 Results 3.1 Expression of Tlr Genes in WT and rd10 Retinas 3.2 TLR2 Levels in Retinal Cells from WT and rd10 Retinas 4 Discussion References Environmental Light Has an Essential Effect on the Disease Expression in a Dominant RPE65 Mutation 1 Introduction 2 Materials and Methods 2.1 Animals 2.1.1 Physiological Day-Light Exposure 2.2 ERG 2.3 Histology 2.3.1 Spectral Domain Optical Coherence Tomography (SD-OCT) 3 Results 3.1 DayL-Exposed RPE65 WT/KI Mice Exhibited Both Retinal Function and Morphological Changes 4 Discussion References Microglia Preserve Visual Function in a Mouse Model of Retinitis Pigmentosa with Rhodopsin-P23H Mutant 1 Introduction 2 Materials and Methods 2.1 Animals 2.2 Microglia-Conditional Depletion 2.3 Electroretinogram (ERG) 2.4 Tissue Processing for Histology and Immunoblotting 2.5 Statistics 3 Results 3.1 Conditional Microglial Depletion in RhoP23H Mice 3.2 Effect of Microglial Depletion on Photoreceptor Cell Numbers 3.3 Effect of Microglial Depletion on Visual Function 4 Discussion References Part IX: Mechanisms of Degeneration – Metabolism Measuring the Release of Lactate from Wild-Type and rd1 Mouse Retina 1 Introduction 2 Materials and Methods 2.1 Animals 2.2 Organotypic Retinal Explant Cultures 2.3 Lactate Measurements 2.4 Lactate Measurements Based on 1H-NMR 2.5 Statistics 3 Results 3.1 Steady Secretion of Lactate from Mouse Retina 3.2 Increased Lactate Release from rd1 Mouse Retina 4 Discussion References Aerobic Glycolysis in Photoreceptors Supports Energy Demand in the Absence of Mitochondrial Coupling 1 Introduction 2 Materials and Methods 2.1 Animals and Euthanasia 2.2 Ex Vivo Incubations 2.3 Glucose Assay 2.4 Lactate Assay 2.5 Photoreceptor Contributions to Retina Metabolism 2.6 Metabolite Extraction and Gas Chromatography-Mass Spectrometry 2.7 Measurement of O2 Consumption Rate (OCR) 2.8 Statistical Analysis 3 Results 4 Discussion References Redox Status in Retinitis Pigmentosa 1 Introduction 2 Oxidative Stress 3 Antioxidant Therapies with Nutraceuticals 4 Oxidative Stress and Inflammation 5 Conclusions and Future Perspectives References Perspectives on Retinal Dolichol Metabolism, and Visual Deficits in Dolichol Metabolism-Associated Inherited Disorders 1 Introduction 2 Dolichol Synthesis and Dolichol-Linked Oligosaccharides in the Neural Retina 3 Inherited Disorders Affecting Dolichol Metabolism 3.1 DHDDS and NUS1-CDG 3.2 SRD5A3-CDG 3.3 DOLPP1-CDG 3.4 DOLK-CDG 4 Conclusions References Retinal Metabolic Profile on IMPG2 Deficiency Mice with Subretinal Lesions 1 Introduction 2 Materials and Methods 2.1 Animals 2.2 Retina Collection 2.3 Mass Spectrometry Sample Preparation and Metabolite Analysis by LC-MS/MS 2.4 Statistical Analysis 3 Results 4 Discussion 4.1 Altered IPMInterphotoreceptor matrix (IPM) and Retina Metabolism 4.2 Experimental Limitations and Future Directions 4.3 Subretinal Lesions, Nutrient Stress, and Macular Degeneration References Part X: Neuroprotection Glutathione Coating of Liposomes Enhances the Delivery of Hydrophilic Cargo to the Inner Nuclear Layer in Retinal Cultures 1 Introduction 2 Materials 3 Methods 3.1 Liposome Preparation and Encapsulation 3.2 Liposome Uptake in Organotypic Retinal Explant Cultures 4 Results and Discussion References Modification of Müller Glial Cell Fate and Proliferation with the Use of Small Molecules 1 Introduction 2 Drugs and Pathways Shown to Affect MG Cell Fate After Injury 3 Emphasis on Pathways Common with Astrocytes 3.1 Effects of Combination Treatments in Astrocytes 3.2 Non-cell Autonomous Effects In Vivo 4 Discussion References A Potential Neuroprotective Role for Pyruvate Kinase 2 in Retinal Degeneration 1 Introduction 2 Materials and Methods 2.1 Experiment with Animals 2.2 Organotypic Retinal Explant Culture 2.3 Cryosectioning 2.4 TUNEL Assay 2.5 Microscopy and Image Processing 2.6 Statistics 3 Results 4 Discussion References Part XI: Photoreceptors Critical Role of VEGF as a Direct Regulator of Photoreceptor Function 1 Introduction 2 Critical Factors in Experimental Design 3 Effect of VEGF on Photoreceptor Function 4 Contribution of VEGF on Reducing Photoreceptor Function in Diabetes and Hypoxia 5 Potential Mechanism of VEGF-Mediated Direct Effect on Photoreceptor Function 6 Clinical Relevance References Lysine Ubiquitylation Drives Rhodopsin Protein Turnover 1 Introduction 2 Materials and Methods 2.1 Cell Culture and Transfection 2.2 Immunoblotting Analysis and Immunoprecipitation 2.3 Cycloheximide (CHX) Chase Assay 2.4 Statistical Analysis 3 Results 3.1 P23H Rhodopsin Is Less Ubiquitylated When Lysine Is Replaced with Arginine 3.2 K-null WT Rhodopsin Is Less Ubiquitylated Than WT Rhodopsin 4 Discussion References In Silico Prediction of MYO1C-Rhodopsin Interactions and Its Significance in Protein Localization and Visual Function 1 Introduction 2 Materials and Methods 2.1 Phylogenetic Sequence Analysis 2.2 Protein–Protein Interaction Bioinformatics Tools 2.3 In Vitro Transfections and Co-localization Studies 3 Results 3.1 Myosin Family Controlling Cytosolic Dynamics 3.2 MYO1C Interaction with Rhodopsin 3.3 Co-Localization of MYO1C with Rhodopsin in COS1 Cells 4 Discussion References A Ciliary Branched Actin Network Drives Photoreceptor Disc Morphogenesis 1 Filamentous Actin Resides Within the Photoreceptor’s Cilium at the Site of Disc Formation 2 Inhibition of Actin Polymerization Prevents Initiation of Disc Formation 3 The Arp2/3 Complex Initiates Actin Polymerization During Disc Formation 4 PCARE Recruits Actin Polymerization Machinery to the Cilium 5 A Cycle of Actin Polymerization and Depolymerization Initiates Disc Formation References Part XII: RPE Revisiting the Daily Timing of POS Phagocytosis 1 Introduction 2 Daily Cycle of Phagosome Number 3 Ingestion and Degradation Rates 4 POS Tip Ingestion 5 Conclusion References Inhibition of Bacterial Peptidoglycan Cytopathy by Retina Pigment Epithelial PGRP2 Amidase 1 Introduction 2 Materials and Methods 2.1 Cell Culture 2.2 Peptidoglycan Reagents 2.3 Immunofluorescent Antibody Assay 2.4 Cleavage of FITCMDP by RPE Lysate 2.5 NOD-Induced Cytopathy 2.6 Statistical Analysis 3 Results 3.1 Detection of RPE PGRP2 and NAMAA Activity 3.2 Concentration and Time-Dependent RPE Lysate NAMAA Cleavage of FITCMDP 3.3 RPE Lysate NAMAA Inhibition of PGN Cytopathy 4 Discussion References Understanding Ischemic Retinopathies: The Role of Succinate and Its Receptor in Retinal Pigment Epithelium 1 Introduction 1.1 Role of the Retinal Pigment Epithelium (RPE) in the Eye 1.2 Molecular Pathways Involved in the Regulation of Vascularization in the Retina 2 Succinate and Its Receptor SUCNR1 3 Conclusion, Therapies, and Prevention References The Amphipathic Helix in Visual Cycle Proteins: A Review 1 Introduction 2 Materials and Methods 2.1 Amino Acid Sequence Retrieval and Structure Visualization 2.2 Prediction for Amphipathic α-Helix Segments in the Protein 3 Results 3.1 Retinal Pigment Epithelium-Specific 65 kDa (RPE65) Protein 3.2 Lecithin:Retinol Acyltransferase 3.3 Cellular Retinaldehyde-Binding Protein 3.4 Retinol Dehydrogenases 4 Discussion References The Retinal Pigment Epithelium: Cells That Know the Beat! 1 Introduction 2 POS Phagocytosis 3 Retinal Adhesion 4 Potential Link with the Intracellular Circadian Clock 5 Phenotypes Linked to Deregulated Circadian Functions 6 Conclusion and Perspectives References Part XIII: Stem Cell Models and Therapies Retinal Organoids: A Human Model System for Development, Diseases, and Therapies 1 Introduction 2 Retinal Organoids: “Mini-retinas” 3 Modeling Inherited Retinal Degenerations Using Retinal Organoids 4 Therapeutic Approaches Using Retinal Organoids 5 Conclusion References Modeling Retinitis Pigmentosa with Patient-Derived iPSCs 1 Introduction 2 Challenges in RP Disease Modeling 3 Modeling RP with Patient-Derived iPSCs 3.1 Benefits of Patient iPSCs for RP Disease Modeling 3.2 Factors Important for the Success of RP Modeling with Patient iPSCs References Primary Retinal Cell Cultures as a Model to Study Retina Biology 1 Introduction 2 Cell Lines 3 Primary Retinal Cell Cultures 4 Dispersed Cell Cultures 5 Future Directions References Generation of CRB1 RP Patient-Derived iPSCs and a CRISPR/Cas9-Mediated Homology-Directed Repair Strategy for the CRB1 c.2480G>T Mutation 1 Introduction 2 Materials and Methods 2.1 Clinical Evaluation 2.2 Cell Culture 2.3 DNA Extraction and PCR Primers 2.4 CRISPR-Mediated Gene Correction 3 Results and Discussion References Inducing Neural Regeneration from Glia Using Proneural bHLH Transcription Factors 1 Introduction 2 Ascl1 3 Neurogenin (Neurog2) 4 Atonal–Homolog 5 NeuroD 6 Outlook References Index