Photoreceptor Ciliopathies: RP2, KIF17 and NPHP5

描述（由申请人提供）：对光感受器细胞完整性的挑战涉及一生中蛋白质从内部到外部片段的生物合成后递送。该申请将确定：i) RP2和ARL3在蛋白质转运中的作用；ii)同型二聚体动力蛋白ii （KIF17）介导的棒状和锥状鞭毛内运输的差异；iii)肾囊素-5在光感受器运输和细胞极性中的作用。目的1A将解决

DESCRIPTION (provided by applicant): A challenge to photoreceptor cell integrity concerns the post-biosynthetic delivery of proteins from the inner, to the outer, segments over a lifetime. This application will identify: i) the roles of RP2 and ARL3 in protein transport; ii) differences n rod and cone intraflagellar transport mediated by homodimeric kinesin-II (KIF17); and iii) functions of nephrocystin-5 in photoreceptor trafficking and cell polarity. Aim 1A will address the physiological functions of RP2, ARL3 and the RP2/ARL3/UNC119 complex. We will generate an RP2(G2A) knockin mouse model. The RP2(G2A) knockin construct will incorporate loxP and Frt sites in intron 1 and upstream of ATG to permit, additionally, a germline knockout of RP2 which is expected to mimic XLRP in male mice. In aim 1B, we propose to generate a photoreceptor-specific ARL3 knockout mouse to study the role of ARL3 in rods and cones. Mutant knockin RP2, knockout RP2 and knockout ARL3 mice will elucidate the roles of RP2 and ARL3 in photoreceptor cell biology, and provide important tools for future gene therapy. Aim 2 will focus on molecular motors which move rod phototransduction proteins to the OS by intraflagellar transport (IFT). The role of homodimeric kinesin-II (KIF17) in polarized IMCD3 cells and mouse photoreceptors will be compared and scrutinized. We will generate Kif17flox/flox mice and delete KIF17 specifically in rod photoreceptors by mating with iCre-75 mice. Based on highly conserved IFT pathways in C. elegans, we postulate partial redundancy of KIF3 and KIF17 in rods. Therefore, we will delete both KIF3 and KIF17 in rods by generating a rod-specific KIF3/KIF17 double knockout and study the consequences. Aim 3 will examine the NPHP5 gene that encodes nephrocystin-5, of unknown function. NPHP5 null alleles in human cause nephronophthisis, a medullary cystic kidney disease, and retinitis pigmentosa (Senior-Loken syndrome). We will generate a rod-specific NPHP5 knockout, a mouse model that will elucidate mechanisms causative for the disease phenotype. Further, as nephrocystins are known to be involved in controlling cell polarity, we will use in-vitro renal epithelial cell cultures to probe mechanisms controlling cell polarization. Finally, AAV vectors will be designed to rescue the NPHP5-RP knockout mouse. PUBLIC HEALTH RELEVANCE: We propose to generate mouse models mimicking human retinitis pigmentosa (RP). The first aim focuses on X-linked RP (XLRP) caused by mutations in the RP2 gene, of unknown function. XLRP manifests early in life with symptoms of night blindness, visual field defects and decreased visual function. In aim 3 we focus on the RP component of Senior-Loken syndrome caused by null alleles of nephrocystin-5. NPHP5 null alleles in human cause nephronophthisis (a medullary cystic kidney disease) and retinitis pigmentosa, the combination of which is called Senior-Loken syndrome. Models for XLRP based on mutations in RP2, or models for Senior- Loken Syndrome currently do not exist. Once generated, they will be valuable tools for identification of mechanisms leading to disease and for the development of gene therapy agents. We further propose to study intraflagellar transport (IFT), a pathway that delivers proteins from the inner segment to the outer segment of photoreceptors, passing through a connecting cilium. IFT transports cargo in the anterograde direction (toward microtubule plus ends) and returns products in the retrograde direction (towards the basal body and microtubule minus ends). Our goal is to identify exactly which motor(s) is responsible for ciliary transport of cargo in rods.