Photoreceptor cells in the retina use several opsin proteins to detect light and confer visual information. Mutations to genes encoding opsins are associated with varying degrees of color blindness and retinal degeneration. In a previous mouse model for color blindness the retina degenerated, with a significant reduction of healthy cones by 3 months (Ma, et al., 2022, Human Gene Therapy). I am characterizing a new mouse line that was gene edited to lack both blue and green sensitive cone opsins, making it a double knock out. We fix and embed the eyes before freezing them, and then I stain frozen eye sections with fluorescent antibodies.  Using a high-resolution microscope, I am able to examine retina health and cone populations. I am comparing knockout animals with a wild type strain of normal mice and a retinal degeneration strain processed in the same way. I confirmed the absence of both blue and green cone opsin in the knockout mice, and despite being a model for color blindness, the mice maintain a healthy population of ospin-less cones for at least one year. However, at one year the knockout mice have approximately 30% fewer cones in their retinas than normal mice. The populations of dying cells and immune response cells in the knockout retinas match those seen in the wild type retinas, and are significantly lower than the populations seen in the degeneration model. This suggests that the retina is not in an active state of degeneration for at least one year. This model will be useful for future development of cone opsin gene therapies, and can serve as a model for color blindness. It also has implications for the health of cones without the cone opsin protein.

Age-related macular degeneration (AMD) is an acquired degeneration of the retina characterized by the presence of lipid-rich deposits, or drusen, underneath the retinal pigment epithelium (RPE). Development of drusen has been linked to degradation of extracellular matrices and aberrant RPE lipid metabolism. Mutations in tissue inhibitor of metalloproteinase 3 (TIMP3), involved in extracellular matrix (ECM) degradation, have been associated with Sorsby Fundus Dystrophy (SFD), an autosomal dominant inherited disease phenotypically similar to AMD. SFD and AMD share clinical features, such as the presence of drusen, geographic atrophy, and choroidal neovascularization. The aim of this project is to evaluate the hypothesis that increased ECM degradation results in reprograming of SFD RPE metabolism towards increased branched chain amino acid (BCAA) oxidation, resulting in lipid synthesis and deposition. We found that SFD induced pluripotent stem cells (iPSC)-RPE have increased apolipoprotein E deposits, and may have increased lipid metabolism. SFD RPE were found to have decreased levels of FABP7, a lipid binding protein that regulates lipid metabolism by increasing fatty acid oxidation, by proteomics and confirmed by Western blot. SFD RPE cells show increased BCAA consumption and upregulated expression of branched chain amino acid transaminase 1 (BCAT1), an enzyme that catalyzes the animation of BCAAs. Control RPE were treated with BCAT1 inhibitors, BCATc Inhibitor 2 and gabapentin. Treatment of RPE with 50 µM of BCATc Inhibitor 2 resulted in a greater than two-fold decrease in BCAA consumption at 48 hours, indicating effective inhibition of BCAT1. Results from this study will help determine whether enhanced BCAA oxidation results in activation of increased lipid synthesis and lipid deposits in SFD iPSC RPE.

Age-related macular degeneration (AMD), a multifactorial eye disease, is distinguished by drusen formation and thickening of Bruch’s membrane. Early onset macular drusen (EOMD) is a rare inherited retinal degeneration with clinical similarity to AMD. EOMD results from genetic variants that cause decreased protein expression levels of complement factor H (CFH) and factor H-like protein 1 (FHL-1). The precise mechanism of drusen formation is unknown, although there are multiple lines of evidence that complement dysregulation and inflammation play a major role. RPE cells derived from EOMD patient induced pluripotent stem cells (iPSCs) can serve as in vitro models for understanding the effects of altered local complement. The complement system is a cascade of proteins and reactions that modulate inflammatory responses for the removal of pathogens. Specific components, such as C3a, could be involved in inflammatory responses that contribute to drusen formation and AMD. The purpose of this project is to observe how increased C3a impacts inflammatory cytokine secretion in EOMD RPE cells. iPSCs were generated from peripheral blood mononuclear cells collected from two patients in an EOMD family and differentiated into RPE cells. Western blot analysis was performed on EOMD and control RPE cells to quantify C3a, CFH, and FHL-1 levels to determine baseline expression levels. Control and EOMD RPE were treated with C3a, and inflammatory cytokine (IL-6 and IL-8) secretion was measured by ELISA. Decreased CFH/FHL-1 secretion and increased local C3a were observed in EOMD RPE. Preliminary data indicate that increased C3a levels may alter cytokine secretion by RPE, indicating that increased complement may play a role in local inflammatory responses that contribute to the pathophysiology of EOMD and AMD.