Role of primary cilium-generated signaling in polycystic kidney disease

摘要 常染色体显性遗传性多囊肾病(ADPKD)的特征是发展为充满液体的囊 在两个肾脏中被称为囊性，但导致囊性形成的关键信号尚不清楚。多个下行蜂窝 在膀胱癌的发生过程中，信号转导通路处于失调状态。然而，以这些通路为靶点对 ADPKD治疗。ADPKD是由编码多囊蛋白-1(PC1)和多囊蛋白-2的基因突变引起的 (PC2)。两种多囊蛋白均定位于初生纤毛。初级纤毛指示细胞作出决定以响应 通过将亚细胞信号划分为细胞外输入。然而，初级纤毛在肾脏中的作用 膀胱发生本质上是复杂的。多囊藻毒素的丢失会导致严重的囊变，主要是纤毛- 依赖的，而纤毛的丧失本身会导致较小的包囊。这些结果表明，一个复杂的相互作用 纤毛中反调节正负信号之间的相互作用抑制正常肾小管的囊性形成 和促进ADPKD的囊性生长。识别这些信号可能会对小说产生深远的影响 ADPKD的治疗靶点和策略。然而，解偶联导致囊变的纤毛信号 囊变过程中下游信号通路的影响及纤毛信号识别的困难 在没有纤毛的情况下，阻止了它们的识别。在这里，通过研究纤毛交易适配器，Tubby 家族蛋白Tulp3，我们的目标是识别和靶向调控膀胱发生的关键上游纤毛信号。 我们之前已经证明Tulp3在膜蛋白的纤毛运输中起作用，而不影响相应的 蛋白质水平或通过与鞭毛内运输复合体A(IFT-A)偶联而破坏纤毛。我们最近展示了 胚胎肾脏特异的Tulp3条件性敲除会发展成不那么严重的肾脏囊变 而不是因为多囊藻毒素的丢失。与纤毛不同，伴随的Tulp3丢失不会抑制PC1丢失后的囊变 破坏，但导致早期死亡，表明肾功能加速丧失。这些结果进一步 加强多囊蛋白在成囊过程中对纤毛蛋白的非依赖性抑制作用。其他组织也报告了 Tulp3或IFT-A缺失抑制成年小鼠PKD模型的囊变。因此，我们假设 Tulp3调节的纤毛决定发育过程中纤毛依赖的囊抑制和PC1/2- 抑制成人纤毛依赖的包囊激活。在这里，通过利用我们在纤毛贩运和 发出信号并使用新的鼠标模型来阻止潜在货物亚型的贩运，我们建议识别 成囊的纤毛调节剂。在目标1中，我们将确定新的Tulp3传输的纤毛货物和信号 纤毛输出量与孢子发生有关。在目标2中，我们将确定Tulp3调节的包囊抑制纤毛货物 在小鼠肾脏发育过程中与PC1基因协同的亚型。在目标3中，我们将测试Tulp3 利用遗传上位性研究成年小鼠PKD体内模型中Cargo亚型作为囊变促进剂的作用。我们的 方法将确定在成人发病的ADPKD和ADPKD期间影响体内囊变的双重纤毛驱动因素 胚胎发病的囊性疾病解开了纤毛产生的信号的分子本质，在两种情况下。

Abstract Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of fluid-filled sacs called cysts in both kidneys, but key signals that cause cyst formation are unknown. Multiple downstream cellular pathways are dysregulated during cystogenesis. However, targeting these pathways has limited effects on ADPKD treatment. ADPKD is caused by mutations in genes encoding for polycystin-1 (PC1) and polycystin-2 (PC2). Both polycystins localize to primary cilia. The primary cilium instructs cellular decisions in response to extracellular inputs by compartmentalizing subcellular signaling. However, the role of primary cilium in kidney cystogenesis is inherently complex. Loss of polycystins causes severe cystogenesis, which is mostly cilia- dependent, while loss of cilia by itself causes smaller cysts. These results suggest that a complex interplay between counter-regulatory positive and negative signals in cilia inhibit cyst formation in normal renal tubules and promote cyst growth in ADPKD, respectively. Identifying these signals could have profound impacts on novel therapeutic targets and strategies for ADPKD. However, uncoupling ciliary signals causing cystogenesis from downstream signaling pathways affected during cystogenesis and the difficulty in identification of ciliary signals in absence of cilia have prevented their identification. Here, by studying the ciliary trafficking adapter, tubby family protein Tulp3, we aim to identify and target the key upstream ciliary signals that regulate cystogenesis. We previously showed that Tulp3 functions in ciliary trafficking of membrane proteins without affecting respective protein levels or disrupting cilia by coupling to the intraflagellar transport complex A (IFT-A). We recently showed that embryonic kidney-specific conditional knockouts of Tulp3 developed renal cystogenesis that was less severe than from polycystin loss. Concomitant Tulp3 loss did not inhibit cystogenesis upon PC1 loss, unlike ciliary disruption, but caused early lethality, suggesting accelerated loss of renal function. These results further reinforce the polycystin independent inhibitory role of ciliary proteins in cystogenesis. Other groups have reported suppression of cystogenesis in adult mouse models of PKD from Tulp3 or IFT-A loss. Thus, we hypothesize that Tulp3-regulated ciliary cargoes determine cilia-dependent cyst inhibition during development and PC1/2- inhibited cilia-dependent cyst activation in adults. Here by leveraging our expertise in ciliary trafficking and signaling and using novel mouse models to block trafficking of potential cargo subtypes, we propose to identify ciliary regulators of cystogenesis. In Aim 1, we will determine novel Tulp3 trafficked ciliary cargoes and signaling outputs in cilia relevant to cystogenesis. In Aim 2, we will determine Tulp3 regulated cyst inhibitory ciliary cargo subtypes that genetically synergize with PC1 during murine kidney development. In Aim 3, we will test Tulp3 cargo subtypes as promoters of cystogenesis in adult mouse models of PKD in vivo using genetic epistasis. Our approach will determine dual ciliary drivers impacting cystogenesis in vivo during adult-onset ADPKD and embryonic-onset cystic disease unraveling the molecular nature of cilium-generated signaling in either setting.