Project Summary The role of the basal body (BB) and daughter centriole (DC) linkage in photoreceptor biology and pathophysiology has not been explored, while the mother centriole (MC) and DC linkage in cultured mammalian cells is important for cell cycle progression and genome stability. The MC and DC linkage is mediated by ciliary rootlets, which are recruited to the proximal ends of centrioles by CNAP1 (Centrosomal NEK2-Associated Protein 1). NEK2 (NIMA-related kinase 2) phosphorylates CNAP1 and several ciliary rootlet component proteins to regulate the centriole linkage, and CEP78 is known to interact with CNAP1. Genes encoding CNAP1 (CEP250), NEK2 (NEK2), and CEP78 (CEP78) have been identified in patients with retinitis pigmentosa (RP) and combined cone-rod dystrophy and hearing loss (CRDHL). The latter is also considered an atypical type of the most common genetic deaf-blindness disease, Usher syndrome (USH). To understand the disease mechanism underlying the mutations in these genes and the role of the BB and DC linkage, this application focuses on the CEP250 gene. In the preliminary study, new Cep250 mutant mice with both vision and hearing impairments were generated. In the mutant photoreceptors, the BB and DC were disconnected and detached from the ciliary rootlets; DC and axoneme marker proteins were mislocalized; and many proteins in the centriole, connecting cilium, and outer segment were downregulated. The three USH type 2 (USH2) proteins were also reduced, and CNAP1 was found to interact with the USH2 scaffold protein WHRN in vitro, suggesting a mechanistic link between CNAP1 and USH2. Additionally, multiple CNAP1 isoforms were discovered in mouse retinas, which may explain the different phenotypes in our Cep250 mutant mice and the previously reported Cep250R187* mice as well as the different phenotypic manifestations in CEP250 patients. Based on these findings, the following hypothesis will be tested that two functionally important CNAP1 isoforms contribute collectively to the BB and DC linkage and the maintenance of the ciliary structure and USH2 protein complex in photoreceptors and hair cells. In Aim 1, the role of CNAP1 and the BB and DC linkage in the ciliary structure and function will be determined in our Cep250 mutant photoreceptors. In Aim 2, the functional relationship between CNAP1 and the USH2 protein complex will be explored in photoreceptors and hair cells. In Aim 3, the vision and hearing phenotypes of two Cep250 mutant mice, which represent different CEP250 mutation groups and presumably have different CNAP1 isoform disruptions, will be compared. This study will reveal the role of the BB and DC linkage in photoreceptors, understand the vision and hearing pathogenesis caused by different mutations in CEP250 and related genes, uncover the mechanistic connection between CRDHL and USH, and provide new insight and animal models for future therapeutic studies.

Project Narrative CEP250 mutations cause retinal degeneration and sensorineural hearing loss in patients. In this application, we will investigate the CEP250’s protein isoform expression, role in ciliary structure and function, and potential association with other deaf-blindness genes. Our study will provide novel insight into deaf-blindness pathogenesis and establish new animal models for future mechanistic and therapeutic studies.