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）的特点是发展充满液体的囊 在双肾中称为囊肿，但导致囊肿形成的关键信号尚不清楚。多个下游蜂窝 在囊肿形成过程中，信号通路失调。然而，靶向这些途径对 ADPKD治疗。ADPKD是由编码多囊蛋白-1（PC 1）和多囊蛋白-2（PC 2）的基因突变引起的 （PC2）.两种多囊蛋白都定位于初级纤毛。初级纤毛指示细胞的决定， 细胞外输入的亚细胞信号。然而，初级纤毛在肾脏中的作用 囊肿形成本身是复杂的。多囊蛋白的缺失导致严重的囊形成，主要是纤毛- 依赖，而纤毛本身的损失导致较小的囊肿。这些结果表明， 纤毛中的反调节正信号和负信号之间抑制正常肾小管中的囊肿形成 促进ADPKD囊肿生长。识别这些信号可能会对小说产生深远影响。 ADPKD的治疗目标和策略。然而，解偶联纤毛信号引起囊肿发生， 下游信号通路在膀胱发生过程中受到影响，纤毛信号难以识别 在缺乏纤毛的情况下阻止了它们的识别。在这里，通过研究纤毛运输适配器， 家族蛋白Tulp 3，我们的目标是确定和目标的关键上游纤毛信号，调节囊肿。 我们先前表明Tulp 3在膜蛋白的纤毛运输中起作用，而不影响相应的 蛋白水平或通过偶联到鞭毛内转运复合物A（IFT-A）破坏纤毛。我们最近展示了 Tulp 3的胚胎肾特异性条件性敲除导致的肾囊肿发生不太严重， 而不是多囊蛋白的丢失与纤毛缺失不同，伴随的Tulp 3缺失并不能抑制PC 1缺失后的囊肿发生。 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 增强纤毛蛋白在囊形成中的多囊蛋白非依赖性抑制作用。其他团体报告说， 在Tulp 3或IFT-A缺失的PKD成年小鼠模型中抑制囊肿形成。因此，我们假设， Tulp 3调节的纤毛货物决定发育过程中纤毛依赖的囊肿抑制和PC 1/2- 抑制成年人纤毛依赖性囊肿激活。在这里，通过利用我们在纤毛贩运和 信号传导和使用新的小鼠模型来阻断潜在货物亚型的运输，我们建议识别 睫状体调节器。在目标1中，我们将确定新的Tulp 3运输的纤毛货物和信号传导。 纤毛的输出与囊形成有关。在目的2中，我们将确定Tulp 3调节的囊肿抑制性纤毛货物， 在小鼠肾脏发育过程中与PC 1基因协同作用的亚型。在目标3中，我们将测试Tulp 3 货物亚型作为PKD体内成年小鼠模型中囊肿形成的启动子，使用遗传上位性。我们 一种方法将确定在成人发病ADPKD期间影响体内膀胱生成的双纤毛驱动因素， 胚胎性囊性疾病解开纤毛产生的信号在任何设置的分子性质。

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.