Continuous regeneration of the 11-cis-retinal (11cRAL) chromophore of rhodopsin and cone visual pigments is essential for sustaining light-sensitivity and survival of photoreceptors. RPE65 is a key retinoid isomerase in the visual cycle responsible for regenerating 11cRAL. The importance of the RPE65 function in vision and retinal health is reflected by the facts that over 100 different mutations in the RPE65 gene are associated with Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). The long-term goals of the proposed research are 1) to decipher the mechanisms that regulate the expression, stability and activity of normal and disease-causing mutant RPE65s, 2) to identify the molecular pathway leading to photoreceptor death in patients with RPE65 mutations and 3) to develop a new therapeutic intervention to prevent or delay vision loss in the patients. In the recent and preliminary studies, we found that fatty acid transport protein 4 (FATP4), elongation of very long chain fatty acids 1 (ELOVL1) and 26S proteasome non-ATPase regulatory subunit 13 (PSMD13) are negative regulators of RPE65. FATP4 and ELOVL1 inhibited synthesis of 11-cis-retinol catalyzed by RPE65 while PSMD13 promoted degradation of misfolded RPE65 via the ubiquitination- dependent proteasomal pathway in the retinal pigment epithelium (RPE). We observed that the majority of pathogenic missense mutations are mapped on the non-active sites of RPE65 and many of these mutants underwent the PSMD13-mediated proteasomal degradation due to misfolding. Using a living cell-based assay, we discovered that chemical chaperones such as 4-phenylbutyrate (PBA) and glycerol can rescue the stability, membrane-association and the isomerase function of many pathogenic RPE65s with non-active site mutations. Importantly, PBA (a FDA-approved medication) improved cone survival and function in a mouse model of LCA caused by R91W RPE65, the most frequent LCA-associated RPE65 mutant. In addition, our preliminary studies showed that deletion of FATP4 in the R91W knock-in (KI) mouse dramatically improved cone survival and function. To capitalize on these findings, we propose to accomplish two specific aims in the proposed project.
Specific Aim 1 is to identify the molecular mechanisms of how FATP4-deficiency improves cone survival and vision in the KI mouse model of LCA. To this end, we will analyze the visual cycle and the pathogenic pathways leading to cone death in the KI mouse. We will also test if FATP4 suppressor and PBA can exert synergistic effects on long-term preservation of cone vision in the KI mouse.
Specific Aim 2 is to test the hypothesis that RPE-specific knockout of ELOVL1 increases synthesis of the visual chromophores in mouse with wild-type or the mutant RPE65, thereby improving cone survival and function in the KI mouse. We will also analyze the molecular mechanisms by which ELOVL1 inhibits synthesis of 11-cis-retinol catalyzed by RPE65. The results of this innovative project will identify FATP4 and ELOVL1 as new therapeutic targets, providing a knowledge-base for future development of new therapies for patients with RPE65 mutations.
Over 100 different mutations in the RPE65 gene encoding the key retinoid isomerase of the visual cycle are associated with Leber?s congenital amaurosis and retinitis pigmentosa. The goal of the proposed research is to elucidate the molecular mechanisms that regulate expression, stability and the enzymatic function of normal and disease-associated mutant RPE65s. The knowledge gained from this project will promote the development of new therapeutic interventions to prevent or delay retinal degeneration and vision loss in patients with the diseases.
