Cdc-42 and the Exocyst in Ciliogenesis and Polycystic Kidney Disease

 描述（由申请人提供）： 囊肿形成对于早期肾脏发育至关重要;然而，如果肾脏囊肿形成延迟和/或在不正确的情况下发生，所产生的囊肿会破坏肾脏结构，从而促进多囊肾病（PKD）和肾衰竭。人类患者和动物模型中几乎所有形式的PKD都与肾初级纤毛结构和/或功能的扰动相关。例如，编码纤毛蛋白多囊蛋白-1和-2的基因突变导致常染色体显性PKD（ADPKD），这是人类最常见的潜在致死性单基因疾病，而多种纤毛蛋白的突变导致肾单位结核，这是儿童和年轻人肾衰竭的最常见遗传原因。虽然初级纤毛结构和功能的维持似乎是预防/治疗PKD的关键，但异常纤毛发生如何促进疾病尚不完全清楚。此外，由于没有任何形式的PKD的批准治疗，即使是最有希望的治疗也具有显著的副作用和/或毒性，因此更彻底地了解初级纤毛形成/功能对于确定未来治疗PKD的治疗靶点至关重要。我们实验室的长期目标是确定纤毛发生的机制基础，并确定该过程中的畸变如何导致PKD。我们的实验室是第一个表明，高度保守的八蛋白外囊复合物，促进靶向和对接的囊泡携带的蛋白质从trans-Golgi网络（TGN），本地化，是必要的，初级纤毛的形成。值得注意的是，在一个肾单位营养不良家族中发现的外囊蛋白突变支持外囊在预防PKD中的作用。外囊已被证明是至关重要的靶向Rab 8阳性囊泡轴承纤毛蛋白的新生纤毛。最近，我们证明了小的GTbaseCdc 42和Tuba，Cdc 42纤毛特异性鸟嘌呤核苷酸交换因子（GEF），调节外囊本身对新生纤毛的靶向，其中外囊的Sec 10组分直接与Par复合物相互作用。最值得注意的是，我们最近证明，cdc 42敲除斑马鱼导致纤毛缺陷和cdc 42肾特异性敲除小鼠阻止纤毛发生，这导致PKD肾单位肾萎缩表型，肾功能衰竭和死亡。初步数据显示，在斑马鱼中敲除tuba会导致类似的表型。基于这些数据，我们假设Cdc 42被一个或多个纤毛特异性GEF激活是将外囊募集到新生纤毛所必需的，从而 促进Sec 10/Par 6稳定相互作用，其允许靶向携带纤毛蛋白的Rab 8阳性囊泡以促进纤毛发生（在目的1和2中测试）。我们处于进行这些实验的独特位置，因为我们拥有所有必要的工具，技术和试剂。由于适当的纤毛形成和功能对于预防PKD是至关重要的，因此进一步了解纤毛蛋白的主要递送途径将可能鉴定出许多新的 候选目标和治疗干预点。我们还表明，Cdc 42和Sec 10在体外和体内的破坏导致MAPK激活和磷酸化ERK（pERK）的增加。由于MAPK活化发生在肾单位萎缩pcy小鼠中，并且pERK的药理学正常化防止了囊形成，我们进一步假设，在新生纤毛处抑制囊外定位/稳定化导致异常MAPK活化，反过来， 导致PKD。在目标3中，我们将采用药理学方法在Cdc 42和Sec 10敲除斑马鱼和条件性小鼠敲除模型中测试这一假设，并将利用我们最近描述的新型腺相关病毒（AAV）载体和逆行递送系统，该系统允许在小鼠肾小管细胞中进行基因表达。综上所述，我们的工作为PKD的治疗开辟了新的途径：利用药理学手段抑制MAPK通路，并通过分子生物学方法递送野生型基因来拯救遗传性纤毛病。

 DESCRIPTION (provided by applicant): Cystogenesis is critical for early kidney development; however, if renal cystogenesis is delayed and/or occurs in an incorrect context, the resulting cysts can destroy kidney structure, thereby promoting polycystic kidney disease (PKD) and renal failure. Virtually all forms of PKD in human patients and animal models are associated with perturbations in renal primary cilia structure and/or function. For example, mutations in genes encoding the ciliary proteins polycystins-1 and -2 cause autosomal dominant PKD (ADPKD), the most common potentially lethal monogenic disorder in humans, while mutations in multiple ciliary proteins lead to nephronophthisis, a form of PKD that is the most common genetic cause of renal failure in children and young adults. Although maintenance of primary cilia structure and function appears to be key to preventing/treating PKD, it is not entirely clear how aberrant ciliogenesis promotes disease. Moreover, since there are no approved treatments for any form of PKD, and even the most promising treatments have significant side effects and/or toxicities, a more thorough understanding of primary cilia formation/function is critical for the identification of future therapeutic targets for the treatment of PKD. The long-term goal of our laboratory is to identify the mechanistic basis for ciliogenesis, and determine how aberrations in this process contribute to PKD. Our laboratory was the first to show that the highly-conserved eight-protein exocyst complex, which promotes targeting and docking of vesicles carrying proteins from the trans-Golgi network (TGN), localizes to, and is necessary for formation of, primary cilia. Notably, mutations in an exocyst protein, identified in a family with nephronophthisis, support a role for the exocyst in prevention of PKD. The exocyst has been shown to be critical for targeting Rab8-positive vesicles bearing ciliary proteins to the nascent cilium. More recently, we demonstrated that both the small GTPase Cdc42 and Tuba, a Cdc42 ciliary-specific guanine nucleotide exchange factor (GEF), regulate targeting of the exocyst itself to the nascent cilium, where the Sec10 component of the exocyst directly interacts with the Par complex. Most notably, we recently demonstrated that cdc42 knockdown in zebrafish causes ciliary defects and Cdc42 kidney-specific knockout in mice prevents ciliogenesis, which leads to a PKD nephronophthisis phenotype, renal failure, and death. Preliminary data show that tuba knockdown in zebrafish results in a similar phenotype. Based on these data, we hypothesize that activation of Cdc42 by one or more ciliary-specific GEFs is required to recruit the exocyst to the nascent cilium, thereby promoting Sec10/Par6 stabilizing interactions that allow for the targeting of Rab8-positive vesicles bearing ciliary proteins to promote ciliogenesis (tested in Aims 1 and 2). We are in a unique position to undertake these experiments as we have all the necessary tools, techniques, and reagents. As proper cilia formation and function are critical for preventing PKD, further understanding of a major delivery route for ciliary proteins will likely identify a number of novel candidate targets and points of therapeutic intervention. We also showed that Cdc42 and Sec10 disruption in vitro and in vivo leads to MAPK activation and an increase in phosphorylated ERK (pERK). As MAPK activation occurs in the nephronophthisis pcy mouse, and pharmacologic normalization of pERK prevents cystogenesis, we further hypothesize that inhibition of exocyst localization/stabilization at the nascent cilium results in abnormal MAPK activation that, in turn, leads to PKD. In Aim 3, we will take a pharmacologic approach to test this hypothesis in our Cdc42 and Sec10 knockdown zebrafish and conditional murine knockout models, and will also exploit our recently described novel adeno-associated viral (AAV) vector, and retrograde delivery system, that allows for gene expression in murine kidney tubule cells. Taken together, our work opens up new avenues for the treatment of PKD: utilizing pharmacologic means to inhibit the MAPK pathway, and molecular biology to deliver wild-type genes to rescue inherited ciliopathies.