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到蛋白质在整个生命周期内从内部到外部的生物合成后传递。这一应用将识别：1)RP2和ARL3在蛋白质转运中的作用；2)同源二聚体激动素-II(KIF17)介导的杆状和锥状鞭毛内转运的差异；3)肾囊藻毒素-5在感光细胞运输和细胞极性中的功能。目标1A将针对 RP2、ARL3和RP2/ARL3/unc119复合体的生理功能。我们将产生一个RP2(G2a)敲门小鼠模型。RP2(G2a)敲打蛋白构建物将在内含子1和ATG上游包含loxP和FRT位点，以允许另外一种胚系敲除RP2，预计它将在雄性小鼠中模拟XLRP。在目标1B中，我们建议建立一个光感受器特异性ARL3基因敲除小鼠来研究ARL3在视杆细胞和视锥细胞中的作用。突变敲除RP2、敲除RP2和敲除ARL3小鼠将阐明RP2和ARL3在光感受器细胞生物学中的作用，并为未来的基因治疗提供重要工具。AIM 2将专注于通过鞭毛内转运(IFT)将杆状光转导蛋白移动到OS的分子马达。同源二聚体激动素-II(KIF17)在极化的IMCD3细胞和小鼠光感受器中的作用将被比较和仔细研究。我们将产生Kif17Flox/FLOX小鼠，并通过与iCre-75小鼠交配来删除杆状光感受器中的KIF17。基于线虫高度保守的IFT通路，我们假设KIF3和KIF17在杆状结构中存在部分冗余。因此，我们将通过生成特定于棒的KIF3/KIF17双敲除来删除棒中的KIF3和KIF17，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。人类的NPHP5零等位基因可导致肾小球肾炎、一种髓性囊性肾病和视网膜色素变性(High-Loken综合征)。我们将产生一个杆状特异的NPHP5敲除，一个将阐明疾病表型致病机制的小鼠模型。此外，由于已知肾囊素参与控制细胞的极性，我们将使用体外肾上皮细胞培养来探索 控制细胞极化的机制。最后，将设计AAV载体来拯救NPHP5-RP基因敲除小鼠。 公共卫生相关性：我们建议建立模拟人类视网膜色素变性(RP)的小鼠模型。第一个目标是研究由未知功能的RP2基因突变引起的X连锁RP(XLRP)。XLRP在生命早期表现为夜盲、视野缺陷和视觉功能下降的症状。在目标3中，我们重点研究由肾囊素-5零等位基因引起的老年Loken综合征的RP成分。人类NPHP5缺失等位基因可引起肾小球肾炎(一种髓样囊性肾病)和视网膜色素变性，这两种疾病的组合称为High-Loken综合征。基于RP2突变的XLRP模型或老年Loken综合征模型目前尚不存在。一旦产生，它们将成为识别导致疾病的机制和开发基因治疗试剂的宝贵工具。我们进一步建议研究鞭毛内转运(IFT)，这是一种通过连接纤毛将蛋白质从光感受器内段输送到外段的途径。IFT沿顺行方向运输货物(朝向微管加末端)，并以逆行方向(朝向基底和微管减去末端)返回产品。我们的目标是准确地确定哪台发动机(S)负责棒状货物的纤毛运输。

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.