<p>Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh+/- and Cfh-/- mice fed a high fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (RPE) deposit formation, specifically basal laminar deposits, following high fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch’s membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh+/- and Cfh-/- mice, RPE damage accompanied by loss of vision occurred only in old Cfh+/- mice. We demonstrate that such pathology is a function of excess complement activation and C5a production, associated with monocyte recruitment, in Cfh+/- mice versus complement deficiency in Cfh-/- animals. Due to the CFH dependent increase in sub-RPE deposit height we interrogated the potential of CFH as a novel regulator of Bruch’s membrane lipoprotein binding and show, using human Bruch’s membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Interestingly, although the CFH H402 variant shows altered binding to BrM, this does not affect its ability to remove endogenous lipoproteins. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD.</p> / Dissertation

Vision provides a critical interface with the physical world. This work examines visual development and vision loss in mice to glean the influence of the retinal state on visual connections. I first assessed the impact of retinal activity on the eye-specific segregation of retinal afferents in the lateral geniculate nucleus (LGN) of young Gβ5 -/- mice. Gβ5 is the fifth member of the β subfamily of heterotrimeric G proteins. Gβ5 binds and stabilizes the R7 family of regulators of G-protein signaling (RGS), which accelerate Gi/o GTP hydrolysis. Gβ5 -/- mice, which lack R7RGS activity, have malformed synapses in the outer plexiform layer (OPL) and impaired OPL transmission. Altered spontaneous retinal activity in Gβ5-/- mice at P7, P12, P14, and P28 correlates with impaired eye-specific segregation of retinal afferents in the LGN at corresponding timepoints. However, Gβ5-/- mice exhibit a normal transition from cholinergic to glutamatergic drive that corresponds with a temporary recovery of refinement at P10. Thus the abnormal-normal-abnormal pattern of activity in the retina is coupled with abnormal-normal-abnormal segregation. This activity-segregation profile suggests activity may instruct early retinogeniculate development. nob mice, which also exhibit impaired OPL transmission, have aberrant retinal waves that align with loss of segregation. nobxGβ5-/- mice have similar levels of segregation as Gβ5-/- at P21, but activity only similar P14 nobxGβ5-/- and Gβ5-/- RGCs. This suggests that the critical period of eye-specific segregation closes shortly after P14 and that R7RGS activity is critically important to postnatal RGCs. Next, I investigated the aged visual system via the retinofugal projections of mice with retinal remodeling after photoreceptor degeneration (PD). ΔCT mice, with mild remodeling, and TG9N mice, with aggressive remodeling, retain gross anatomical and physiological connectivity in the presence of attenuated visual activity compounded by organic remodeling. However, the magnitude of pupillary light responses in PD mice was diminished. Reduced melanopsin signal in the retina, not downstream anomalies, explains this functional deficiency. These observations suggest that changes to eye-specific segregation are limited once projections are established, regardless of retinal activity or remodeling. These observations bode well for future retina-based treatments of vision loss.

O Corpo Ciliar (CC) é uma fonte de células tronco da retina de animais adultos, mas sua ativação permanece desconhecida. GTPases Rho são proteínas que reorganizam do citoesqueleto de actina, regulam vias de sinalização e transcrição gênica, sobrevivência celular e proliferação. Neste trabalho, investigamos a expressão das GTPases Rho nas células do CC e seu efeito na regulação do ciclo celular. As GTPases RhoA, RhoB e Rac1 foram expressas nas células do CC e sua ativação pelo ácido lisofosfatidico (LPA) aumentou a expressão dos genes progenitores retinianos Pax6 e Chx10. A inibição das proteínas por Toxina A de Clostridium difficile aumentou a proliferação no CC e potencializou o efeito proliferativo dos fatores de crescimento. A inibição especifica destas proteínas diminuiu a expressão dos transcritos de p21cip, p27kip, p16INK4a e p19INK4d e aumentou de Ki67, CiclinaA e D1. O estudo da via de Wnt indicou que Rac1 regulou os genes de componentes da degradação de -catenina e Lef1. Concluímos que a inativação das GTPases Rho induziu a proliferação das células progenitoras retinianas localizadas no CC e regulou a via de Wnt. Sua ativação induziu o perfil de célula progenitora, sugerindo uma nova ferramenta para o mecanismo de reparo retiniano. / Ciliary Body (CB) is a potential source of stem cells in the adult retina, but its activation is still unknown. Rho GTPases play a role in actin-based cytoskeleton reorganization, regulate signaling pathways and gene transcription, cell survival and cell proliferation. In this study we investigated the expression of Rho GTPases in CB cells and their role on cell cycle regulation. The GTPases RhoA, RhoB and Rac1 were present in CB cells and the activation by lysophosphatidic acid (LPA) increased the expression of the progenitor genes Pax6 and Chx10. The inhibition by Clostridium difficile Toxin A increased the proliferation of CB cells and potentiated the proliferative effect of growth factors. The specific inhibition decreased the expression of p21cip, p27kip, p16INK4a and p19INK4d as well as increased Ki67, cyclinA and D1 transcripts. The Wnt pathway study indicated that Rac1 regulated -catenin degradation genes components and Lef1. Taken together, the inactivation of Rho GTPases stimulated the proliferation of progenitor cells located in CB as well as regulate the Wnt signaling pathway. The proteins activation was correlated to progenitor profile induction. These different mechanisms may provide a potential new approach on retinal repair.

A neuroproteção é um dos tópicos mais relevantes aplicados à neurociência. As junções comunicantes (JC), formadas pelas conexinas (Cx) estão envolvidas na neurodegeneração após lesão. Estudos com animais KO apresentam resultados contraditórios sobre papel das JCs. O objetivo deste trabalho foi analisar o papel das Cxs a partir do trauma mecânico na retina, modelo que permite a visualização do foco, penumbra, e áreas adjacentes à lesão. Observamos regulação distinta das Cx36 e Cx43 durante a neurodegeneração. A Cx36 não se alterou e a Cx43 apresentou desorganização e aumento da imunorreatividade após 7 dias, concomitantemente com GFAP. Células amácrinas apoptóticas encontram-se acopladas a células vizinhas por Cx36. O papel funcional das JCs foi avaliado, utilizando bloqueadores, para verificar a viabilidade/morte de células. Carbenoxolone (CBX), reduziu o espalhamento da apoptose, após 4h, enquanto a quinina, teve o mesmo efeito após 1h. A distribuição de núcleos apoptóticos confirmou que a utilização de bloqueadores de JCs reduz a propagação da apoptose. A quinina, mas não o CBX, diminuiu a expressão de caspases iniciais e efetoras. O controle da permeabilidade de canais de JCs pode participar de estratégias de neuroproteção. / The neuroprotection stands out as one of the most pursued hot topics in applied neurosciences. The gap junctions (GJ), formed by connexin (Cx) are involved in neurodegeneration injury. Studies using KO animal models endowed apparently contradictory results in relation to the role of coupling in neuroprotection. The aim of this study was to analyze the role of Cx-mediated communication in focal lesion induced by mechanical trauma in the retina, a model that alow the visualization of the focus, penumbra and adjacent areas. We observed distinct regulation of Cx36 and Cx43 during neurodegeneration. The Cx36 did not change during the lesion progression and Cx43 showed disorganized pattern and upregulated after 7 days, the same as GFAP. Apoptotic amacrine cells are coupled with health neighborhood cells by Cx36. The functional role of GJ was evaluated using blockers to verify the viability/cell death. Carbenoxolone (CBX) reduced the spread of apoptosis after 4h while quinine had the same effect after 1h. The distribution of apoptotic nuclei confirmed that the use of GJ blockers reduced the propagation of apoptosis. Quinine, but not CBX, decreases initial and effector caspases expression. The control of GJ channels permeability can participate in neuroprotection strategies.

Retinose Pigmentar (RP) é uma doença hereditária que conduz progressivamente à cegueira. Mais de 150 mutações da rodopsina associadas à RP foram descritas, e causam a alteração da sua conformação. Esta tese testou a hipótese de que pequenas moléculas auxiliam na formação da rodopsina e/ou reduzem a morte dos fotorreceptores. As mutações da RP, N15S e P23H, revelaram diferenças quanto às características e gravidade devido à má-formação das proteínas mutantes. Ligação de pequenas moléculas (retinóides, íons metálicos, clorofilas e antocianinas) à rodopsina foi demonstrada in vitro. O derivado da clorofila, Ce6, mostrou-se mais efetivo, conferindo maior estabilidade e foi então testado em ratos submetidos à degeneração por luz ou em modelos de RP (P23H e S334ter). Observou-se uma proteção contra a degeneração por luz e uma significante diminuição da degeneração no P23H. Em contraste, Ce6 causou um aumento na degeneração dos fotorreceptores do S334ter. Finalmente, resultados clínicos, bioquímicos e in vivo foram comparados e mostraram estar altamente relacionados. / Retinitis Pigmentosa (RP) is an inherited disease that progressively leads to blindness. More than 150 mutations associated with RP are known in rhodopsin, causing its misfolding. This thesis tested the hypothesis that small molecules can rescue folded rhodopsin and/or reduce photoreceptor cell death. RP mutations, N15S and P23H, revealed differences in characteristics and severity of misfolding of the mutant proteins. Binding of small molecule classes (retinals, metal ions, chlorophylls and anthocyanins) to rhodopsin was demonstrated in vitro. The chlorophyll derivative, Ce6, was most effective in conferring stability and therefore tested in rats subjected to light-damage and RP rat models, P23H and S334ter. Protection against the light-induced retinal degeneration and more importantly a significant slowing of the photoreceptor degeneration rate in the P23H rat were observed. In contrast, Ce6 increased photoreceptor degeneration in the S334ter rat. Finally, clinical, biochemical and in vivo rat data were compared and it was found to be highly correlated.

Tissue inhibitors of metalloproteinases (TIMPs) comprise a family of four proteins in humans that modulate the turnover of the extracellular matrix by regulating the activities of endopeptidases that catalyze its degradation, especially the matrix metalloproteinases (MMP). In general, the four TIMPs are broad-spectrum tight binding inhibitors of MMPs with individual differences in specificity. In this study, we attempted to understand the basis of such variation by using membrane type-1 MMP (MT1-MMP) as a model, since it is inefficiently inhibited by TIMP-1 in contrast with the other TIMPs. We designed and engineered mutations in the N-domain of TIMP-1, based on current knowledge of TIMP interactions. By measuring inhibition levels of each mutant against several MMPs, including MT1-MMP, we were able to obtain a triple mutant with an vii improved affinity for MT1-MMP. / Our results, along with previous data, confirm that multiple residues in the critical interface segments between TIMPs and MMPs, namely at positions 2, 4, 5, 6, and 98, are key in determining the basic interaction between the two molecules. The second part of this work focused on naturally occurring mutations in TIMP-3 which cause an early form of macular degeneration called Sorsby's Fundus Dystrophy (SFD). The TIMP-3 mutants identified so far share certain features but the mechanism by which they result in macular disease is not yet understood. As an initial step, we expressed recombinant TIMP-3 carrying a truncation mutation, glutamic acid 139 to a stop codon (E139X), and assessed its activity towards representative MMPs and tumor necrosis factor-(Sa (Bconverting enzyme, another metalloproteinase normally inhibited by TIMP-3. Our results indicate that this mutation does not impair the inhibitory activity of TIMP-3. / Expression of this mutant in mammalian retinal cells revealed a difference in localization between wild-type and E139X mutant TIMP-3. Therefore, we concluded that the SFD mutations may actually influence the processing and/or binding properties of TIMP-3 in the retina. / by Asmaa Bilal Hamze. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

The term 'Retinitis pigmentosa' (RP) represents a group of inherited, late-onset diseases characterised by progressive retinal degeneration due to photoreceptor death. Mutations in the RP2 gene are found in 7-18% of patients with X-linked RP, one of the most severe forms. The RP2 gene product is a membrane-associated protein which encompasses two distinct domains. The N-terminal domain is well characterised as possessing GTPase-activating protein (GAP) activity towards the small GTPase ARL3 and thus regulate the transport of lipid-modified proteins within the photoreceptor cell. However, it is not known if the loss of this particular function of RP2 is the sole reason that causes the disease, while the role of the protein's C-terminus remains unknown. This thesis focuses on the characterisation of two novel protein-protein interactions of RP2 with the aim to investigate novel roles of the protein. Firstly, evidence is provided that a highly-conserved cluster of RP2 residues that span both the N- and C-terminus participate in direct interaction with Osteoclast-stimulating factor 1 (OSTF1). Two hypotheses are explored about the potential role of the complex in SRC-mediated RP2 phosphorylation and the regulation of cell motility. Secondly, the catalytic subunit of DNA-dependent protein kinase (DNA PK) is identified as a novel interaction partner of RP2 in cultured cells. The two proteins are shown to co-localise in the nuclear and membrane compartments of a retinal-derived cell line and might engage in a kinase-substrate relationship. So far, no evidence was found that RP2 participates in the canonical function of DNA PK which is the regulation of DNA double-stranded breaks. Finally, the CRISPR/Cas9 genome editing method was applied on zebrafish embryos to generate a novel vertebrate animal model for the loss of RP2 function. One out of three different zebrafish lines with rp2 mutations was shown by histology to have mild late-onset thinning of the photoreceptor outer segments. The present thesis reports previously unexplored aspects of RP2's function and will, therefore, contribute to understanding the molecular mechanisms that underlie RP. Moreover, this thesis will contribute to the discussion about the usefulness of zebrafish as an RP model.

Retinal hypoxia and inadequate oxygen delivery have been implicated as causal for the development of several eye diseases, including diabetic retinopathy, glaucoma, and retinopathy of prematurity. The imaging of oxygen tension in the retina, generated from a measure of the phosphorescence lifetimes of bolus-injected palladium-porphyrin probes, has been used successfully to study retinal oxygen dynamics in numerous animal models. However, the specific parameters for applying this technique in the mouse have not been thoroughly investigated. The goals of this project were to calibrate a newly-constructed phosphorescence lifetime imaging instrument and data analysis software against known oxygen concentrations, to determine specific parameters for probe excitation and image collection and analysis in the mouse eye, and to assess any damage caused to the eye by the technique using histological analysis. An in vitro system was developed for calibration of the probe and for estimation of power of excitation light and camera settings necessary to produce acceptable oxygen maps. In vivo experiments were then performed, and plots indicating camera settings necessary for producing varying qualities of oxygen maps were constructed. Trypsin digestion of retinal tissue was used in an attempt to assess any damage to experimental subjects, but this histological technique was deemed inadequate for analyzing the capillary structures of the mouse eye. Alternatively, damage was assessed using the instrument itself to calculate changes in oxygen tension during the experimental process. The results of this work will allow the phosphorescence lifetime imaging system to be used in the mouse to study how changes in retinal oxygen tension correlate with the progression of eye diseases where oxygen is implicated, including diabetic retinopathy.

INTRODUCTION: Retinal Pigment Epithelia (RPE) degeneration is a key event in the development of age-related macular degeneration (AMD). RPE dysfunction in AMD is thought to occur through the accumulation of reactive oxygen species (ROS) and oxidative damage. The transcriptional co-activators, PGC-1α and PGC-1β, are important regulators of mitochondrial biogenesis and anti-oxidant capacity. Our group has previously shown that the PGC-1α protein promotes RPE oxidative metabolism and that overexpression of the PGC-1α gene protects cells from AMD-associated pro-oxidants. On the other hand, PGC-1β gene expression has been found to be upregulated in patients with neovascular AMD, and in-vitro overexpression of PGC-1β damages cells and induces pro-oxidant conditions. OBJECTIVE: Given the divergence of PGC-1α and PGC-1β functions in RPE and their clinical relevance in AMD pathogenesis, this project will seek to investigate the impact of the upregulation of PGC-1α and PGC-1β in RPE metabolism. PGC-1α will be upregulated through treatment with compound ZLN005. A new methodology for PGC-1β expression will be developed to closely modulate in-vitro PGC-1β induction. METHODS: In-vitro experiments were performed on the ARPE-19 cell line. Cells were treated with 10µM of ZLN005 for 24 hours. Oxidative stress was induced by exposure to H2O2 and NaIO3 under serum-free conditions. Lactate dehydrogenase (LDH) levels were used to quantify cell death. Quantitative PCR (qPCR) and Western Blot were performed to measure changes in gene and protein expression respectively. Superoxide production by the mitochondria was measured to evaluate ROS levels within the cell. Intravitreal injections of 20µM ZLN005 were performed on eight-week old male C57BL/6J mice. After 24 and 72 hours of treatment, the mice were euthanized and the enucleated eyes were dissected to obtain the RPE and neural retina layers. Total RNA was extracted from these layers and qPCR was performed to measure gene expression. A tetracycline-inducible PGC-1β plasmid was designed and transfected into ARPE-19 cells. The cells were exposed to 0.01-100µg/ml doxycycline for 48-hours and qPCR was used to measure gene expression. Transfected cells were treated with ZLN005 and cell death upon exposure to oxidative stress was quantified. RESULTS: Gene expression analysis on ARPE-19 cells treated with ZLN005 showed robust upregulation of PGC-1α, PGC-1β and their associated transcription factors and enzymes. Induction of PGC-1α at the protein level was also confirmed. ZLN005 efficiently protected ARPE-19 cells from H2O2 and NaIO3 cytotoxicity and its protection was negated in PGC-1α-silenced cells. Treatment with ZLN005 also decreased mitochondrial superoxide production. ZLN005 intravitreal injections were safely administered to the animals and did not cause cataracts or other damage to the ocular tissues. While statistical significance in gene expression changes was limited due to the small sample size, anti-oxidants GPX1 and TXN2, and electron transport chain gene, ATP50, were found to be potentially induced in the neuro-retina, while FOXO3 was found to be downregulated. Evaluation of our novel tetracycline-inducible PGC-1β adenoviral vector showed that upregulation of PGC-1β was efficiently controlled by the addition of doxycycline to transfected cells. Upon exposure to H2O2, transfected cells treated with doxycycline experienced greater cell death than transfected cells not exposed to doxycycline. ZLN005 treatment was able to decrease cell death in both conditions. CONCLUSION: The present study shows that ZLN005 efficiently protects RPE cells from oxidative damage through selective induction of PGC-1α. While still preliminary, the in-vivo study indicates that ZLN005 is safe to be injected into the eye and may be able to increase the expression of mito-protective and anti-oxidant genes in the neuronal retina. In addition, our design of the tetracycline inducible PGC-1β plasmid allows for tight control over PGC-1β expression through doxycycline addition. Upregulation of PGC-1β at levels similar to those observed in clinical conditions caused increased pro-oxidant induced cell death and treatment with ZLN005 was able to protect against cell death. / 2021-06-30T00:00:00Z

Age-Related Macular Degeneration (AMD) is a retinal eye disease that is the leading cause of blindness in those over 50 years of age throughout the developed world. Oxidative and metabolic dysfunction of the retinal pigment epithelium (RPE) has been shown to play an important role in AMD. However, the mechanism of dysfunction in the RPE is poorly understood. The peroxisome proliferator-activated receptor-gamma coactivator 1α and β (PGC1A and PGC1B) are coactivators that interact with transcription factors to regulate mitochondria metabolism. In a previous study, it was demonstrated that one of the isoforms, PGC1A, protects RPE cells from oxidative stress through the upregulation of transcription factors that regulate important antioxidant enzymes. There is experimental and clinical evidence that demonstrates that PGC1B may play a deleterious role in the RPE cell. The objective of this study is to characterize the pathological effect of PGC1B on the RPE cell. PGC1B was overexpressed in the human retinal pigment epithelium cell line (ARPE-19) and expression of the PGC1 isoforms and their main gene targets was evaluated using quantitative polymerase chain reaction (qPCR). Cell death was evaluated under basal and pro-oxidant conditions by quantification of lactate dehydrogenase (LDH) release from the RPE cell. The effect of PGC1B gain of function on the RPE pro-angiogenic function was evaluated using the choroid explant sprouting assay and by testing the proliferative, migratory, and tube formation potential of RPE-derived conditioned media on the rhesus monkey chorioretinal cell line (RF/6A). Quantitative PCR analysis showed that overexpression of PGC1B in ARPE-19 cells leads to increased mitochondrial metabolism and decreased antioxidant enzyme expression, causing oxidative stress. After treatment with H2O2, PGC1B overexpression caused ARPE-19 cells to become more susceptible to cytotoxicity. The ex vivo choroid sprouting assay demonstrated that PGC1B overexpression in RPE is pro-angiogenic. However, cell proliferation as measured by MTT and the cell migration assay provided conflicting results on the pro-angiogenic effect of PGC1B. Previous research has demonstrated that oxidative stress in the RPE cell plays a role in AMD progression. It has been demonstrated in this study that PGC1B expression leads to increased mitochondrial metabolism and repression of antioxidant enzymes needed to prevent oxidative stress and dysfunction in the RPE cell. While experiments to test the effect of PGC1B on angiogenesis provided conflicting results, a different endothelial cell model may be better suited in demonstrating the pro-angiogenic effect of PGC1B. The hope is that the information provided from this study may be used to further our understanding of AMD and lead to the development of therapeutic targets to combat the effects of AMD.