Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 10, 2009). Includes bibliographical references.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

The lens is a crystallin tissue of the anterior part of the eye that focuses light onto the retina. Aged-related cataract, which is the result of loss of lens transparency, is the most common cause of blindness in the world. Being constantly exposed to UV-light, lens is significantly affected by its UVA spectrum. UV-light exposure has been shown to result in apoptosis of lens cells which can lead to cataract formation. This suggests the need for molecular mechanisms to remove apoptotic debris from the lens. In the set of experiments it was proven that integrin αvβ5-mediated pathway is involved in phagocytosis of apoptotic cell debris in the ocular lens, thus contributing to its homeostasis. Additionally, it was shown that exposure to UV-light plays role in cataract formation by influencing integrin αvβ5-mediated phagocytosis function. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Chiu Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 100-116). / Abstract also in Chinese. / TABLE OF CONTENTS --- p.I / ACKNOWLEDGEMENTS --- p.V / LIST OF FIGURES --- p.VI / LIST OF ABBREVIATIONS --- p.VIII / ABSTRACT --- p.1 / Chapter 1. --- INTRODUCTION --- p.5 / Chapter 2. --- LITERATURE REVIEW --- p.7 / Chapter 2.1 --- RETINAL ISCHEMIA --- p.7 / Chapter 2.1.1 --- INDUCTION OF RETINAL ISCHEMIA --- p.7 / Chapter 2.1.2 --- MECHANISMS OF RETINAL ISCHEMIA/REPERFUSION DAMAGE --- p.8 / Chapter 2.1.2.1. --- Free radical --- p.8 / Chapter 2.1.2.2 --- Excitotoxicity --- p.9 / Chapter 2.1.3 --- APOPTOSIS IN RETINAL ISCHEMIA/REPERFUSION INJURY --- p.10 / Chapter 2.2 --- RETINAL DYSTROPHIC ROYAL COLLEGE OF SURGEONS (RCS) RAT --- p.15 / Chapter 2.3 --- BCL-2 FAMILY MEMBERS --- p.16 / Chapter 2.3.1 --- FAMILY MEMBERS AND THEIR INTERACTIONS --- p.16 / Chapter 2.3.2 --- SUBCELLULAR LOCALIZATION --- p.18 / Chapter 2.3.3 --- PHYSICAL STRUCTURE AND PORE FORMATION --- p.19 / Chapter 2.3.4 --- BIOLOGICAL EFFECTS OF BCL-2 --- p.20 / Chapter 3. --- OBJECTIVES --- p.24 / Chapter 4. --- MATERIALS AND METHODS --- p.27 / Chapter 4.1. --- RETINAL ISCHEMIA AND REPERFUSION INDUCED LOSS OF INNER RETINAL ELEMENTS --- p.27 / Chapter 4.1.1. --- TISSUE RESPONSES IN THE RAT RETINAS AFTER TRANSIENT ELEVATED INTRAOCULAR PRESSURE INDUCED RETINAL ISCHEMIA/REPERFUSION INSULT --- p.27 / Chapter 4.1.1.1. --- Induction of retinal ischemia/reperfusion insult with transient elevated intraocular pressure (IOP) --- p.27 / Chapter 4.1.1.2. --- Animal experiments --- p.28 / Chapter 4.1.1.3. --- Histopathology and measurement of inner retinal thickness (IRT) --- p.28 / Chapter 4.1.1.4. --- Flat preparation of the retinas and retinal ganglion cell counts (RGCCs) --- p.29 / Chapter 4.1.2. --- INTERNUCLEOSOMAL DNA FRAGMENTATION AND IN SITU NICKED DNA DETECTIONS AT DIFFERENT TIME AFTER REPERFUSION IN THE RAT RETINAS --- p.30 / Chapter 4.1.2.1. --- Enzyme-linked immunosorbent assay (ELISA) of mono- and oligonucleosomes --- p.30 / Chapter 4.1.2.2. --- In-situ terminal deoxynucleotidyl transferase (TdT)-mediated biotin- dUTP nicked end labelling (TUNEL) --- p.31 / Chapter 4.1.3. --- "IMMUNOHISTOCHEMISTRY OF BCL-2, BAX AND P53" --- p.32 / Chapter 4.1.4. --- "DOUBLE LABELLING OF BCL-2, BAX AND TUNEL" --- p.33 / Chapter 4.1.5. --- IN-SITU REVERSE TRANSCRIPTASE - POLYMERASE CHAIN REACTION OF BCL-2 AND BAX --- p.34 / Chapter 4.1.5.1. --- Primers design and specificity test --- p.34 / Chapter 4.1.5.2. --- In-situ RT-PCR --- p.36 / Chapter 4.2. --- LOSS OF INNER RETINAL ELEMENTS IN THE RETINAL DYSTROPHIC ROYAL COLLEGE OF SURGEONS (RCS) RATS --- p.38 / Chapter 4.2.1. --- HISTOPATHOLOGY --- p.38 / Chapter 4.2.2 --- MORPHOMETRY OF CELLS IN THE RETINAL GANGLION CELL LAYER (RGCL) AND THE INNER NUCLEAR LAYER (INL) --- p.39 / Chapter 4.2.3. --- IMMUNOHISTOCHEMISTRY OF BCL-2 AND BAX --- p.39 / Chapter 5. --- RESULTS --- p.40 / Chapter 5.1. --- RETINAL ISCHEMIA AND REPERFUSION INDUCED LOSS OF INNER RETINAL ELEMENTS --- p.40 / Chapter 5.1.1. --- TISSUE RESPONSES IN THE RAT RETINAS AFTER TRANSIENT ELEVATED INTRAOCULAR PRESSURE INDUCED ISCHEMIA/ REPERFUSION INSULT --- p.40 / Chapter 5.1.1.1. --- Histopathology --- p.40 / Chapter 5.1.1.2. --- Morphometry of inner retinal thickness --- p.40 / Chapter 5.1.1.3. --- Retinal ganglion cell counts (RGCCs) --- p.41 / Chapter 5.1.2. --- INTERNUCLEOSOMAL DNA FRAGMENTATION AND IN SITU NICKED DNA DETECTION AT DIFFERENT TIME AFTER REPERFUSION IN THE RAT RETINAS --- p.41 / Chapter 5.1.2.1. --- Enzyme-linked immunosorbent assay (ELISA) of mono- and oligonucleosomes --- p.42 / Chapter 5.1.2.2. --- In situ TUNEL --- p.42 / Chapter 5.1.3. --- BCL-2 AND RETINAL ISCHEMIA/REPERFUSION INJURY --- p.42 / Chapter 5.1.3.1. --- Immunohistochemistry of Bcl-2 --- p.42 / Chapter 5.1.3.2. --- Double labelling of Bcl-2 and TUNEL --- p.43 / Chapter 5.1.3.3. --- In situ RT-PCR for bcl-2 mRNA --- p.43 / Chapter 5.1.4. --- BAX AND RETINAL ISCHEMIA/REPERFUSION INJURY --- p.44 / Chapter 5.1.4.1. --- Immunohistochemistry of Bax --- p.44 / Chapter 5.1.4.2. --- Double labelling of Bax and TUNEL --- p.45 / Chapter 5.1.4.3. --- In situ RT-PCR for bax mRNA --- p.45 / Chapter 5.1.5. --- P53 IMMUNOREACTIVITY AT VARIOUS TIME AFTER REPERFUSION --- p.46 / Chapter 5.2. --- LOSS OF INNER RETINAL ELEMENTS IN THE RETINAL DYSTROPHIC ROYAL COLLEGE OF SURGEON (RCS) RATS --- p.47 / Chapter 5.2.1. --- HISTOPATHOLOGY --- p.47 / Chapter 5.2.2. --- MORPHOMETRY OF CELLS IN THE RGCL AND INL --- p.47 / Chapter 5.2.3. --- IMMUNOHISTOCHEMISTRY OF BCL-2 AND BAX --- p.47 / Chapter 5.2.3.1. --- Bcl-2 --- p.47 / Chapter 5.2.3.2. --- Bax --- p.48 / Chapter 6. --- DISCUSSION --- p.49 / Chapter 6.1. --- RETINA ISCHEMIA AND REPERFUSION INDUCED LOSS OF RETINAL ELEMENTS --- p.51 / Chapter 6.1.1 --- REPERFUSION TIME DEPENDENT TISSUE RESPONSES IN RAT RETINAS --- p.51 / Chapter 6.1.2 --- ISCHEMIA/REPERFUSION INDUCED APOPTOSIS IN RAT RETINAS --- p.52 / Chapter 6.1.3 --- BCL-2 AND RETINAL ISCHEMIA/REPERFUSION INSULT --- p.53 / Chapter 6.1.4 --- BAX AND RETINAL ISCHEMIA/REPERFUSION INSULT --- p.58 / Chapter 6.1.5 --- P53 AND RETINAL ISCHEMIA/REPERFUSION INJURY --- p.60 / Chapter 6.2. --- LOSS OF INNER RETINAL ELEMENTS IN THE RETINAL DYSTROPHIC ROYAL COLLEGE OF SURGEON (RCS) RAT --- p.61 / Chapter 6.2.1. --- HISTOPATHOLOGY AND MORPHOMETRY --- p.62 / Chapter 6.2.2. --- BCL-2 --- p.63 / Chapter 6.2.3. --- BAX --- p.64 / Chapter 7. --- CONCLUSION --- p.65 / APPENDIX A FIGURES --- p.66 / APPENDIX B REFERENCES --- p.100

Retinal degenerative diseases cause varying degrees of irreversible vision loss in millions of people worldwide. Common to all retinal degenerative diseases is the malfunction or demise of photoreceptor cells or its supportive cells, retinal pigment epithelium cell in the retina. A considerable part of these diseases were resulted from the inherited mutations of essential genes expressed in these retinal cells. The understanding of pathologic mechanism as well as developing of therapeutic treatment for these diseases were discussed in this study. A cutting-edge therapeutic genome editing technology is studied in the first part of study. This technology was invented to treat retinitis pigmentosa via engineered nucleases, which has great clinical potential for autosomal dominant genetic disorders that were previously irreparable by conventional gene therapy interventions. Though customizable gene editing tools can be engineered to target specific mutation sites, however it is too daunting for diseases like retinitis pigmentosa, a progressive retinal degenerative condition associated with more than 150 mutations in the rhodopsin gene alone. Here in this study, we present an “ablate-and-replace” combination strategy that 1) destroys expression of the endogenous gene by CRISPR/Cas9 in a mutation-independent manner, and 2) enables expression of wild-type protein through exogenous cDNA. As proof of concept, we show that our CRISPR-based therapeutic machinery efficiently ablates mRho in vivo, and when combined with gene replacement therapy, ameliorates rod photoreceptor degeneration and improves visual function in two genetically distinct autosomal dominant retinitis pigmentosa animal models. This mutation-independent, ablate-and-replace strategy represents the first electrophysiological recovery by a CRISPR-mediated therapy in an autosomal dominant disorder and it offers a clinically relevant, universal strategy to overcome allelic heterogeneity in debilitating inherited conditions. For the second part of the study, gene editing technology was used to study the pathogenesis of Doyne honey comb dystrophy, another Mendelian disease with extensive similarities to age-related macular degeneration. This monogenic disorder is caused by a unique point mutation on an extracellular matrix protein EFEMP1, expressed by retinal pigment epithelium cell. To precisely gauge the physiological effect resulted from this mutation, CRISPR-mediated gene correction was used to create isogeneic cell pairs from patient donated tissue-derived stem cells. These stem cells were differentiated into retinal pigment epithelium cell before analysis. We found unfolded protein response and immune response were not involved in the pathogenesis, which contradicts existing theories. Via proteomics analysis, we found expression level of a cholesterol catabolic enzyme was affected by the EFEMP1 mutation while those proteins controlling the cholesterol transport remains constant. This result provides supportive evidence to explain the aberrant intracellular accumulation of cholesterol found in patient retinal pigment epithelium cells. This imbalance in lipid homeostasis also suggests Doyne honey comb dystrophy is a retinal pigment epithelium cell-autonomous disease.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Retinal degeneration is characterized by the irreversible loss of photoreceptors. A key research question is the identification and characterization of photoreceptor protective factors that prevent or delay vision loss. The Wnt pathway is a critical cellular communication pathway involved in development and diseases of the central nervous system (CNS). Recently, we discovered that multiple components of the Wnt pathway were differentially expressed in the rd1 mouse model of retinal degeneration. One of the most highly upregulated genes was Dickkopf3 (Dkk3), a secreted Wnt pathway protein of unknown function. Additionally, we demonstrated that Wnt signaling is neuroprotective in primary retinal culture (Yi et al., 2007). These data led to the hypothesis that Dkk3 is a regulator of Wnt-mediated neuroprotection during retinal degeneration. The role of Dkk3 in the retina and its activity in the Wnt pathway was identified in this dissertation project using a series of biochemical, molecular and cell biology methodologies. First, Dkk3 was shown to be expressed and secreted from Muller glia in mouse retinal tissue and primary Muller glia culture. I then demonstrated that Muller glia are a Wnt-responsive cell type and that Dkk3 potentiates Wnt3a-mediated signaling. Interestingly, the latter effect was not observed in other cell types in the retina such as retinal ganglion cells and retinal pigmented epithelial cells. Thus, Dkk3 may act on Muller glia to positively modulate Wnt signaling during retinal degeneration, which could potentially amplify the neuroprotective activity of the Wnt pathway. Next, the role of Dkk3 in cellular viability was explored. HEK293 cells stably expressing Dkk3 were shown to be significantly protected from staurosporine-induced apoptosis compared with vector control. This result suggests that Dkk3 may mediate a direct pro-survival effect onto photoreceptors during retinal degeneration. Protein interaction experiments demonstrated that Dkk3 formed a complex with the single pass transmembrane proteins Krm1 and Krm2 in the membrane, potentially in the endoplasmic reticulum (ER). Furthermore, Wnt signaling luciferase reporter assays demonstrated that Krm2, but not Krm1, abolished Dkk3-mediated Wnt3a potentiation. These data suggest that Dkk3 modulates Wnt signaling by antagonizing Dkk1-Krm dependent Wnt inhibition. Further studies will determine whether this activity is sufficient for the potentiation of Wnt signaling by Dkk3. Lastly, co-immunoprecipitation followed by mass spectrometry analysis was used to identify a novel interacting protein of Dkk3. Dkk3 was shown to interact with glucose response protein 78 (GRP78), an ER-resident chaperone. This suggested that Dkk3 protein is part of the unfolded protein response through GRP78 in the ER. In conclusion, these studies identified two novel functions of Dkk3 in regulating Wnt signaling pathway and cellular viability and suggest a physiological role for Dkk3 and Wnt signaling during retinal degeneration. Future studies will explore the significance of Dkk3-Krm and Dkk3-GRP78 interactions in the retina. Further, elucidation of the regulation of Dkk3 and other Wnt ligands in the ER and the consequence of ER stress on the biological activity of Wnt signaling will provide a better understanding of the role of the Wnt pathway during retinal degeneration.

Retinitis Pigmentosa (RP) refers to a group of inherited retinal dystrophies resulting from progressive photoreceptor degeneration and accumulation of intra-retinal pigment-like deposits. X-linked forms of RP are frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The RPGR transcript undergoes complex alternative splicing to express both constitutive (RPGR^ex1-19) and RPGR^ORF15 variants. Although RPGR is thought to play a role in ciliary function, little is known about the physiological significance of expressing two distinct groups of variants. This study compares Rpgr^ex1-19 and Rpgr^ORF15 expression in developing photoreceptors using immunoblot analysis and immunohistochemistry, assesses ciliary affinity in adult photoreceptors by protein fractionation, examines Rpgr function in transgenic mouse models and identifies a novel Rpgr^ORF15 binding partner using a yeast two-hybrid screen. Our data reveal that Rpgr expression undergoes dynamic temporal regulation during retinal development and indicates variability in ciliary localization of Rpgr variants in adult photoreceptors. Utilization of distinct Rpgr variants during stages of photoreceptor development suggests independent roles. Further examination of Rpgr function using transgenic mouse models over-expressing either the Rpgr^ex1-19 or Rpgr^ORF15 variant reveals that despite normal ciliary localization, an excess of RPGR^ex1-19 results in atypical accumulation of Rpgr in photoreceptor outer segments, abnormal photoreceptor morphology and severe retinal degeneration. The data indicate that the constitutive variant cannot substitute for Rpgr function in photoreceptors and suggest that proper maintenance of the Rpgr isoform ratio is critical to photoreceptor viability. Using mouse retinal cDNA in a yeast two-hybrid screen with the C-terminus of the Rpgr^ORF15 variant, we identified a novel variant of whirlin as an interacting partner. Mutations in whirlin result in Usher syndrome, a disorder characterized by hearing loss and RP. RT-PCR and immunoblot analysis were used to confirm the presence of selected candidate partners in the retina and interaction was confirmed by pull-down assays and co-immunoprecipitation from retinal homogenate. Immunohistochemistry showed co-localization of RPGR and whirlin within photoreceptors and identified isoform specific localization of whirlin. These findings indicate that whirlin binds Rpgr^ORF15 and that this novel isoform may be required for photoreceptor function, thus providing a potential mechanism for the RP phenotype observed in Usher syndrome.

Neurodegeneration is a collective term for the progressive loss of structure, function or even death of neurons. This includes diseases like Alzheimer’s disease, frontotemporal dementia, Parkinson’s disease and motor neuron disease. Recent researches have shown great interest in the role of tau proteins, which have versatile functions including microtubule stabilization and signal relay in the central nervous system. Retina and optic nerve, being part of the central nervous system, can also be affected by similar processes. In neurodegenerative diseases visual disturbances including difficulties in reading and finding object, depth perception, perceiving structure from motion, color recognition and impairment in spatial contrast sensitivity have all been observed. Some of these defects may be attributed to changes at ocular level. The effect of tau mutation was investigated in this study utilizing a transgenic P301L tau mice model. Morphometric analysis has been utilized to quatify the neurodegenerative changes, including the thickness of inner nuclear layer(INL), density of retinal ganglion cells(RGCs) and size of RGCs. Retinal sections stained by hematoxylin and eosin(H&E) were analyzed. Comparisons were made between the P301L tau mice and the control mice in addition to comparisons between different age groups. The study found that there was a significant decrease of thickness of INL of P301L tau mice when compared with control mice. The effect was more pronounced in the peripheral area and the effect increased with age. Regarding density of RGCs, P301L tau mice showed a similar age-related decline as control mice. And regarding the size of RGCs, the RGCs from P301L tau mice increased in size with age and the RGCs from control mice decreased in size with age. / published_or_final_version / Anatomy / Master / Master of Medical Sciences

Choroidal neovascularization (CNV) is a leading threat to severe vision loss, particularly in patients with age-related macular degeneration (AMD). In CNV, newly formed blood vessels sprout from the choroid to the sub-retinal space, where leakage and bleeding of the abnormal vessels lead to photoreceptor death and subsequent vision loss. It is believed that CNV is mediated by growth factors (e.g. vascular endothelial growth factor {VEGF}) produced by the retinal pigment epithelium (RPE) under pathological states (e.g. hypoxia). Current treatments for CNV aiming at countering VEGF only help decrease leakage and inhibit formation of CNV, but none of them is curative and the recurrence rate remains high. In order to find other more powerful potential therapeutic targets, the regulations of VEGF signaling in the pathophysiology of CNV is the focus of numerous translational investigations. Previously, Hypoxia-inducible factor-1 (HIF-1), a crucial transcriptional factor in response to hypoxia, is identified as the master transcriptional factor controlling VEGF expression in the RPE promoting CNV. Alpha-enolase (ENO1), a key glycolytic enzyme, is known to be over expressed in several types of carcinomas also under the regulation of HIF-1. ENO1 has been reported to be closely associated with cancer progression, angiogenesis, and venous invasion. The molecular events of ENO1 in the pathogenesis of promoting angiogenesis are of interest but still barely understood. Recently, the association of ENO1 antibodies with retina has been seen in patients with AMD. We hypothesize that ENO1 expression in the RPE may play a role in the development of CNV, participating in the regulation of VEGF. Hypoxia is an important pathological condition in the formation of CNV. Here, we first determined ENO1 expression and cell death in a human RPE cell line, ARPE-19, under cobalt (II) chloride (CoCl2)-induced hypoxia or anoxia (95% N2, 5% CO2). To further investigate the regulation of ENO1 in CNV, HIF-1α-diminished RPE cells were generated using small interfering RNA (siRNA) and the change of ENO1 expression in response to hypoxic injury was determined. Upon 24 hr of treatment with CoCl2-induced hypoxia or anoxia, the expression of ENO1 and VEGF increased significantly along with HIF-1α in ARPE-19 cells, both of which could in turn be significantly down-regulated by HIF-1α siRNA. Interestingly, cell death remained low in ARPE-19 cells, even after 24 hr of CoCl2-induced hypoxia or anoxia. To further study the role of ENO1 in CNV, we started by investigating the relationship between ENO1 and VEGF. SiRNA was used to knock down the expression of ENO1 in ARPE-19 cells. Upon transfection with the siRNA, ENO1 expression was successfully down-regulated when treated with CoCl2-induced hypoxia. However, VEGF secretions from the ENO1-diminished ARPE-19 cells under CoCl2-induced hypoxia remained unchanged. Double knockdown of ENO1 together with HIF-1α by siRNA also did not help to further suppress VEGF secretion in the hypoxic ARPE-19 cells. Hence, ENO1 was demonstrated to be activated and up-regulated by HIF-1 in RPE cells responding to hypoxia, suggesting a potential role of ENO1 in favoring the formation of CNV, but not through influencing VEGF secretion. / published_or_final_version / Ophthalmology / Master / Master of Philosophy