Cilia Assembly and Transport in Photoreceptor Cells

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Project Summary Mutations in CEP290 result a number of genetic diseases termed ciliopathies, which manifest with a variety of clinical symptoms, including retinal degeneration. While it is well-established that photoreceptor survival requires Cep290 function, the causes of photoreceptor death remain largely unknown. In vertebrate photoreceptors, Cep290 localizes to the connecting cilium, which is analogous to the transition zone of primary cilia. Work from cell culture and invertebrates suggest that Cep290 organizes the assembly of protein complexes that form a “ciliary gate” within the transition zone. However, whether loss of Cep290 impacts such a ciliary gate have not been demonstrated in photoreceptors. Furthermore, the highly variable nature of CEP290-associated disease phenotypes cannot be explained by traditional genotype-phenotype correlations. Two models have been proposed to explain this variability. One possibility is that second-site genetic modifiers enhance disease severity in some patients. The second possibility is that exons harboring nonsense mutations and that also begin and end in the same reading frame can be preferentially skipped. In such a case the resulting mRNA transcript eludes nonsense-mediated decay and can produce a near-full-length protein. Disease severity therefore correlates with the total amount of full- length and near-full-length protein produced. This proposal seeks to address fundamental questions related to photoreceptor cell biology and the role of Cep290 in photoreceptor degeneration. In Aim 1, we will utilize two distinct zebrafish cep290 mutants to determine if defects in ciliary gating play a role in degeneration. In Aim 2, we will determine if other genes associated with Joubert Syndrome, namely arl13b, ahi1 or cc2d2a act as genetic modifiers to cep290-associated retinal degeneration in zebrafish. Finally, in Aim 3, we will take fibroblasts from patients with CEP290 mutations and generate human induced pluripotent stem cells (hiPSCs) and subsequently differentiated into 3D retinal cups. These hiPSC-derived retinal cups (hiPSC-DRCs) will be used determine if basal exon skipping occurs in from humans carrying CEP290 mutations and whether total protein levels correlate with disease severity. In addition, how disease-causing mutations lead to alterations in cilia architecture and protein trafficking will be investigated. These experiments will establish the molecular and genetic mechanisms that determine the severity of disease progression. Understanding the basis for phenotypic variability will provide much-needed clarification on the mechanisms of pathogenesis and lead to better treatment of ciliary disease.