Recessive mutations in CRB1 cause Leber Congenital Amaurosis 8 (LCA8) and Retinitis Pigmentosa 12 (RP12). Both diseases are currently untreatable, leading to retinal blindness at birth (LCA8) or at young adulthood (RP12). CRB1 is a single-spanning transmembrane protein homologous to Drosophila Crumbs (Crb). Crb family proteins contain a small intracellular domain and a large extracellular domain, and execute their functions through protein interactions. Crb proteins interact with PDZ- and FERM- domain proteins through its intracellular domain, whereas form homodimer through its extracellular domain. Crb proteins have multiple cellular functions: an apical determinant essential for apicobasal polarity and cell adhesions, and a regulator of supracellular actomyosin cable in morphogenesis. Crb proteins are also involved in cell signaling pathways, including Hippo/YAP/TAZ, Notch1, Wingless (Wnt), and mTOR. None of these functions have been assessed in CRB1-deficient human retinal cells. In mice, Crb1 is found in the subapical region of retinal progenitor cells and in the outer limiting membrane of the adult retina. In contrast to early-onset blinding LCA8, Crb1-knockout mice only display late-onset mild retinal phenotypes, indicating limitations of mouse models in studying the mechanisms of CRB1-associated retinal disease. Collectively, it is still unclear how CRB1 mutations affect human retinal cells. The goal of this study is to determine molecular functions of CRB1 in pluripotent stem cell-derived human retinal cells. We hypothesize that CRB1 regulates apicobasal polarity and cell adhesions in human neuroretinal progenitors; CRB1 mutations affect retinal epithelial structure leading to differentiation defects and degenerations. We will 1) elucidate the functions of CRB1 in the regulation of apicobasal polarity, cell adhesions, and differentiation in human neuroretinal tissue; and 2) determine CRB1 interactome and trafficking in human retinal cells. Accomplishments of the studies will move a step closer toward therapeutic development for CRB1-associated retinal disease.
Vision impairment and loss caused by CRB1-associated retinal disease, progressive retinitis pigmentosa 12 and early-onset Leber congenital amaurosis 8, are currently untreatable, leading to severe burdens for the patients and society. Accomplishments of the proposed studies will decipher molecular functions of CRB1 in near-native condition?differentiated human retinal cells, establish a disease-in-a-dish model for CRB1 associated retinal disease, and thus lead to therapeutic development to prevent and delay the progression of retinitis pigmentosa 12. !