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Lens Adaptation to Glutathione Deficiency

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Lens Adaptation to Glutathione Deficiency LENS ADAPTATION TO GLUTATHIONE DEFICIENCY: IMPLICATIONS FOR CATARACT by JEREMY A. WHITSON Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology CASE WESTERN RESERVE UNIVERSITY May, 2017 1 Table of Contents List of Tables 6 List of Figures 7 Acknowledgements 11 List of Abbreviations 12 Abstract 17 1. Introduction 19 1.1. The Crystalline Lens - Biological Glass 20 1.1.1. Anatomy of the Lens 20 1.1.2. Functions of the Lens 21 1.1.3. Lens Cell Biology 22 1.1.4. Transport and Homeostasis in the Lens 23 1.1.5. Conclusions 25 1.2. Age-Related Cataract - A Disease of Protein Aggregation 26 1.2.1. Pathology of Age-Related Cataract 26 1.2.2. Risk Factors for and Impact of Age-Related Cataract 27 1.2.3. Clinical Treatment of Cataract 28 1.2.4. Posterior Capsular Opacification 29 1.2.5. Conclusions 30 1.3. Glutathione - The Guardian of Lens Redox Homeostasis 30 1.3.1. Structure and Biosynthesis of Glutathione 30 1.3.2. Regulation of Cellular Glutathione Content 32 1.3.3. The Essential Role of Glutathione in the Lens 32 1.3.4. Lenticular Glutathione Declines with Age 34 1.3.5. A Loss of Glutathione Activity Leads to Cataract 34 1.3.6. The LEGSKO Mouse Model 35 1.3.7. A Salvage Pathway for Glutathione Synthesis 36 1.3.8. Conclusions 37 1.4. Glutathione Transport – Known Mechanisms 38 1.4.1. Glutathione Transport in the Lens 38 1.4.2. Active Transport Systems 39 1.4.3. Passive Transport 41 1.4.4. Conclusions 42 1.5. Thesis Statement 44 2. Evidence of Dual Mechanisms of Glutathione Uptake 45 in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis 2.1. Abstract 46 2.1.1. Purpose 46 2 2.1.2. Methods 46 2.1.3. Results 46 2.1.4. Conclusions 47 2.2. Introduction 47 2.2.1. Antioxidant Functions of Glutathione 47 2.2.2. Lenticular Glutathione Homeostasis 48 2.2.3. The LEGSKO Mouse Model of Cataract 48 2.2.4. Glutathione Transport 49 2.2.5. Purpose of Study 50 2.3. Materials and Methods 50 2.3.1. Chemicals 50 2.3.2. Animal Work 51 2.3.3. LC-MS/MS Analysis 51 2.3.4. Cultured Lens Uptake Experiments 52 2.3.5. Lens Wash Test 53 2.3.6. Cultured Lens GSH Efflux Assay 53 2.3.7. GSH Uptake Imaging 54 2.3.8. In Vivo Uptake Experiments 55 2.3.9. Vascular Eye Perfusion 56 2.3.10. Lens Surface Permeability Assay 56 2.3.11. Tissue Procurement and Dissection 57 2.3.12. Statistical Analysis 58 2.4. Results 58 2.4.1. Lenticular LEGSKO GSH Is Supplied by Circulating 58 GSH 2.4.2. Salvage Pathway Is Not the Source of LEGSKO Mouse 60 Lens GSH 2.4.3. Cultured Lens GSH Uptake Occurs by Passive Diffusion 62 2.4.4. Lens GSH Uptake From Aqueous Humor Is 72 Carrier-Mediated 2.4.5. Mouse Vitreous Contains High Levels of GSH 75 2.4.6. Vitreous but Not Aqueous Contributes Highly to Lens 77 GSH In Vivo 2.4.7. High Vitreous GSH Content Is Unique to Small Animals 81 2.5. Discussion 84 2.5.1. Evidence of Vitreous GSH Transport From Other Studies 84 2.5.2. Uptake From Vitreous but Not Aqueous Is Sufficient to 85 Maintain Steady State GSH Concentration in LEGSKO Lenses 2.5.3. Vitreous GSH Flows Into Lenses but Not Vice Versa 86 2.5.4. Vitreous GSH Varies Greatly Among Species 87 3 2.5.5. Role of Vitreous GSH in Health and Disease 88 2.5.6. Conclusions 89 2.6. Acknowledgements 90 3. Transcriptome of the GSH-Depleted Lens Reveals 91 Changes in Detoxification and EMT Signaling Genes, Transport Systems, and Lipid Homeostasis 3.1. Abstract 92 3.1.1. Purpose 92 3.1.2. Methods 92 3.1.3. Results 92 3.1.4. Conclusions 93 3.2. Introduction 93 3.2.1. The Role of Glutathione In Age-Related Cataract 93 3.2.2. Analysis of the LEGSKO Mouse 94 3.2.3. Purpose of Study 95 3.3. Materials and Methods 95 3.3.1. Animal Work 95 3.3.2. Sample Preparation 96 3.3.3. RNA-Seq 96 3.3.4. Cluster Generation and HiSeq Sequencing 97 3.3.5. Bioinformatic Analysis 98 3.3.6. Validation of RNA-Seq Data by Real Time PCR (qPCR) 98 3.3.7. Statistical Analysis 100 3.4. Results 100 3.4.1. Overview of Significant Gene Expression Changes 100 Resulting from GSH Depletion in the Lens 3.4.2. RNA-Seq Data Shows Strong Differential Expression of 103 Lens Epithelia and Fiber Cell Marker Genes and Excellent Agreement with qPCR Results 3.4.3. GSH Depletion Results in a Partial Downregulation of β- 106 and γ-Crystallins 3.4.4. Trends Identified in Upstream Regulators and 108 Molecular/Cellular Functions of Genes with Modulated Expression 3.4.5. Expression of Several Detoxification Genes Increases as a 111 Function of Decreasing Lenticular GSH Content 3.4.6. Several Transport Systems Are Modulated as a Function of 113 Decreasing Lenticular GSH Content 3.4.7. An Array of Lipid Metabolism Genes Show Modulated 116 Expression as a Function of Decreasing Lenticular GSH 4 Content 3.4.8. Lens GSH Depletion Induces Gene Expression Changes 118 Related to Epithelial-Mesenchymal Transition (EMT) Pathways. 3.5. Discussion 130 3.5.1. Interpretation of Data 130 3.5.2. The GSH-Deficient Lens Transcriptome Shows an Unusual 131 Oxidative Stress Response and Indicates Protective Detoxification Genes 3.5.3. The GSH-Deficient Lens Shows Numerous Changes in 134 Transport Systems 3.5.4. GSH-Deficient Lenses Have Altered Lipid Metabolism 136 3.5.5. GSH-Deficient Lenses Show Activation of EMT signaling 138 and a Loss of Differentiation 3.5.6. Analogies and Differences from Other Models of 139 Oxidative Stress and GSH-Depletion 3.5.7. Limitations of this Study 142 3.5.8. Conclusions 142 4. Analysis of Downstream Metabolic Effects of GSH-Dependent 143 Transcriptomic Changes in the Lens 4.1. Introduction 144 4.2. Materials and Methods 144 4.2.1. Animal Work 144 4.2.2. Lens Total Iron Assay 144 4.2.3. Urea Assay 145 4.2.4. BCA Normalization 145 4.2.5. Statistical Analysis 146 4.3. Results 146 4.3.1. Lens Total Iron Content 146 4.3.2. Urea Within and Surrounding the Lens 147 4.4. Discussion 148 4.4.1. Can Lens Fiber Cells Undergo Ferroptosis? 148 4.4.2. Urea Homeostasis in the Lens 149 4.4.3. Conclusions 150 5. Discussion 151 5.1. Summary of Findings 152 5.1.1. The Source and Mechanism of Residual GSH Levels in 152 the LEGSKO Lens 5.1.2. The Major Consequences of Glutathione Deficiency in 153 the Lens 5 5.1.3. Conclusions 153 5.2. Future Directions and Open Questions 153 5.2.1. Glutathione as a Potential Therapeutic Agent 153 5.2.2. The Source of Vitreous Glutathione 155 5.2.3. Identity of the Lens Epithelia Glutathione Transporter 156 5.2.4. The Glutathione-Depleted Lens Proteome 157 5.2.5. The Potential Protective Role of GSTK1 in the Lens 157 5.2.6. Conclusions 159 5.3. Concluding Remarks 159 6. Appendix 160 References 191 6 List of Tables Table 1.1 Risk factors for age-related cataract development. 27 Table 2.1. LC-MS/MS MRM Settings. 52 Table 3.1. Top Upstream Regulators and Molecular and Cellular 109 Functions of Gene Expression Changes in GSH-Deficient Lenses. Table 3.2. Comparison of Overlapping Gene Expression Changes in 128 GSH-Deficient and EMT Lens (Medvedovic et al., 2006) Transcriptomes. Table 6.1. All significant (P < 0.05; FDR < 0.1) gene expression changes 178 in LEGSKO and BSO-treated LEGSKO lenses compared to WT. 7 List of Figures Figure 1.1. Anatomy of the lens and its local environment. 21 Figure 1.2. Chemical structure of glutathione. 31 Figure 1.3. Diagram of glutathione transport, function, and metabolism. 43 Figure 2.1. Purity of synthesized GS-B tested by LC-MS/MS analysis. 54 Figure 2.2. Analysis of the source of LEGSKO lens GSH. 59 Figure 2.3. Analysis of the mechanism of LEGSKO lens GSH uptake. 61 Figure 2.4. Comparison of GSH uptake in cultured lenses. 62 Figure 2.5. Time course of uptake in cultured lenses. 63 Figure 2.6. Initial rate curves of uptake in cultured lenses. 65 Figure 2.7. Temperature dependence of transport in cultured lenses. 66 Figure 2.8. Test for competitive inhibition of uptake. 67 Figure 2.9. Wash test to assess cellular localization of 68 13 15 GSH-(glycine- C2, N) Figure 2.10. Measurement of cultured lens GSH efflux. 69 13 15 Figure 2.11. Comparison of GS-B and GSH-(glycine- C2, N) 71 uptake rate. Figure 2.12. Visualization of GS-B uptake in cultured lenses. 72 Figure 2.13. Comparison of initial uptake rates from anterior chamber 73 in WT and LEGSKO. lenses. Figure 2.14. Test for competitive inhibition of uptake from the anterior 74 chamber. Figure 2.15. Permeability of lens surfaces. 75 8 Figure 2.16. Comparison of concentrations of aqueous, vitreous, and lens 76 GSH in LEGSKO and WT eyes. Figure 2.17. Comparison of in vivo GSH transport and homeostasis in 78 mouse eyes.
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