THM1 modulation of GLI2 activation in cystic kidney disease

描述（由申请人提供）：囊性肾病（CKD）是肾功能衰竭的主要原因，被认为起源于睫状体生理学的潜在缺陷，尽管其分子机制尚不清楚。该拨款提案的目标是阐明 Thm1aln/aln 小鼠模型中 CKD 的分子病因学，该模型表现出纤毛蛋白运输缺陷，导致 Hedgehog (Hh) 信号传导增强。有趣的是，最近在肾结核、Bardet-Biedl 综合征和 Meckel-Gruber 综合征患者中发现了 THM1 突变，其中 CKD 是其主要临床组成部分（N. Katsanis，个人通讯）。重要的是，GLI2（Hh 信号传导的主要转录激活剂）的缺失挽救了 Thm1aln/aln、Gli2-/- 双突变体中的大部分肾脏表型，这表明 Hh 信号传导过度活跃与 Thm1aln/aln 肾囊肿的病因有关。第一个目标是通过使用小分子 Hh 拮抗剂来挽救培养的突变肾脏中的囊肿发生，从而研究过度的 GLI2 活性在 Thm1aln/aln 肾囊肿发生中的作用。在第二个目标中，将通过使用这些相同的小分子 Hh 拮抗剂来探索 GLI2 激活及其受 THM1 调节的分子机制，通过活细胞成像检查 GLI2 纤毛运输与 Thm1 缺陷的内髓集合管 (IMCD) 细胞活性之间的关系，并通过评估 GLI2 蛋白稳定性和 Thm1 突变体提取物中 GLI 蛋白负调节因子 Fused 抑制剂的状态。这些分析将阐明将纤毛缺陷与肾囊肿发生联系起来的分子事件，以及纤毛生理学和 GLI2 激活之间的相互关系。该提案主张 Hh 信号传导在 CKD 病因学中的作用，而该作用在很大程度上尚未被探索，并且与小分子的使用相结合，可能会带来新的治疗方法。 公共健康相关性：囊性肾病 (CKD) 缺乏经过验证的治疗方法，囊性肾病是肾衰竭的主要原因，据认为，囊性肾病源于睫状体生理学的潜在缺陷，但其分子机制尚不清楚。最近在肾结核、Bardet-Biedl 综合征和 Meckel-Gruber 综合征患者中发现了 THM1 突变，其中 CKD 是其主要临床表现（N. Katsanis，个人通讯）。该提案旨在阐明 Thm1aln/aln 突变小鼠 CKD 的分子病因学，该小鼠表现出独特的纤毛缺陷，导致 Hedgehog (Hh) 信号传导增强，利用小分子 Hh 抑制剂来挽救培养的突变肾脏中的囊肿发生，并使用活细胞成像可视化它们对 Thm1 缺陷的肾源性哺乳动物细胞的纤毛生理学的影响。

DESCRIPTION (provided by applicant): Cystic kidney disease (CKD), a leading cause of renal failure, is proposed to originate from an underlying defect in ciliary physiology, though the molecular mechanisms are unclear. The goal of this grant proposal is to elucidate the molecular etiology of CKD in the Thm1aln/aln mouse model, which exhibits a defect in ciliary protein trafficking that leads to increased Hedgehog (Hh) signaling. Interestingly, mutations in THM1 have recently been identified in patients with nephronophthisis, Bardet-Biedl Syndrome and Meckel-Gruber Syndrome, which feature CKD as a major clinical component (N. Katsanis, personal communication). Importantly, the loss of GLI2, the primary transcriptional activator of Hh signaling, rescues most of the kidney phenotype in Thm1aln/aln,Gli2-/- double mutants, implicating overactive Hh signaling in the etiology of Thm1aln/aln renal cysts. In the first aim, the role of excessive GLI2 activity in Thm1aln/aln renal cystogenesis will be investigated by using small molecule Hh antagonists to rescue cystogenesis in cultured mutant kidneys. In the second aim, the molecular mechanism of GLI2 activation and its modulation by THM1 will be explored by using these same small molecule Hh antagonists to examine the relationship between GLI2 ciliary trafficking and activity in Thm1-deficient inner medullary collecting duct (IMCD) cells using live-cell imaging, and by assessing GLI2 protein stability and the status of Suppressor of Fused, a negative regulator of the GLI proteins, in Thm1-mutant extracts. These analyses will elucidate the molecular events connecting a ciliary defect to renal cystogenesis, as well as the interconnection between ciliary physiology and GLI2 activation. This proposal advocates a role for Hh signaling in the etiology of CKD, which has been largely unexplored, and combined with the use of small molecules, can potentially lead to novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: Proven therapies lack for cystic kidney disease (CKD), a leading cause of renal failure, which is proposed to originate from an underlying defect in ciliary physiology, though the molecular mechanisms are unclear. Recently mutations in THM1 have been identified in patients with nephronophthisis, Bardet-Biedl Syndrome and Meckel-Gruber Syndrome, which feature CKD as a major clinical component (N. Katsanis, personal communication). This proposal aims to elucidate the molecular etiology of CKD in the Thm1aln/aln mutant mouse, which exhibits a unique ciliary defect leading to increased Hedgehog (Hh) signaling, by utilizing small molecule Hh inhibitors to rescue cystogenesis in cultured mutant kidneys and to visualize their effects on ciliary physiology of Thm1-deficient, kidney-derived mammalian cells using live-cell imaging.