Photoreceptor Ciliopathies: RP2, KIF17 and NPHP5

对光感受器细胞完整性的挑战涉及蛋白质的生物合成后传递 从内到外，一生一世的片段。此应用程序将标识：i)以下角色 RP2和ARL3在蛋白质转运中的差异；II)杆状和锥状鞭毛内转运的差异 由同源二聚体激动素-II(KIF17)介导；以及iii)肾囊蛋白-5在脑内的作用 光感受器运输和细胞极性。 目标1A将阐述RP2、ARL3和RP2/ARL3/UNC119的生理功能 很复杂。我们将产生一个RP2(G2a)敲门小鼠模型。RP2(G2A)敲门 构建将在内含子1和ATG上游整合loxP和FRT位点以允许， 此外，RP2的生殖系敲除有望在雄性小鼠中模拟XLRP。在AIM 1B，我们建议建立一种光感受器特异性的ARL3基因敲除小鼠来研究 Arl3为杆状和圆锥状。突变敲除RP2、敲除RP2和敲除ARL3的小鼠将 阐明RP2和ARL3在光感受器细胞生物学中的作用，并提供重要工具 用于未来的基因治疗。 Aim 2将专注于通过以下方式将杆状光转导蛋白移动到OS的分子马达 鞭毛内转运(IFT)。同源二聚体激动素-II在极化IMCD3细胞中的作用 老鼠的光感受器将被比较和仔细观察。我们将产生Kif17Flox/Flox小鼠 并通过与iCre-75小鼠交配，特异性地删除杆状光感受器中的KIF17。基于高度 在线虫中保守的IFT通路，我们假设KIF3和KIF17在 棒子。因此，我们将通过生成特定于杆的杆来删除杆中的KIF3和KIF17 KIF3/KIF17双基因敲除并研究其后果。 目的3将检测编码肾囊蛋白-5的NPHP5基因，功能未知。NPHP5 人类零等位基因可导致肾小球肾炎、一种髓样囊性肾病和视网膜炎 色素变性(老年L综合征)。我们将生成特定于棒的NPHP5基因敲除，一个 小鼠模型，将阐明疾病表型的致病机制。更进一步，如 已知肾囊素参与控制细胞的极性，我们将使用体外肾脏 上皮细胞培养，以探索控制细胞极化的机制。最后，AAV载体将 旨在拯救NPHP5-RP基因敲除小鼠。

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 in 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 specifcally 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-L¿ken 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.