Disrupted ciliary signaling in the brain pathology of Tuberous Sclerosis Complex (Diversity Supplement)

来自家长奖的项目摘要 多发性硬化症（TSC）是一种多系统遗传性疾病，影响多个器官。人士 TSC患有难治性癫痫、智力残疾和自闭症谱系障碍， 临床表现通常是这些患者最致残的。中枢神经系统（CNS）表现 包括混乱的脑连接、增加的星形胶质细胞增生和未成熟的畸形神经元的存在。 尽管mTORC 1活性的增加明显与脑缺血的表现有关， TSC，一个关键的未满足的医疗需求仍然是确定下游分子途径牵连在 大脑发育异常纤毛病是由基因突变引起的遗传性疾病， 初级纤毛是感觉细胞的触角，在大脑的稳态和发育中起作用，被认为 作为一个关键的调节节点的音刺猬信号。纤毛病包括一系列遗传性 睫状体功能障碍是一种共同的疾病，可以影响包括大脑在内的几个器官。这一建议建立 在强大的体外和体内数据表明，某些大脑异常的TSC重演， 纤毛病变的表现与Shh信号通路的改变。特别是，我们发现， 在Tsc 2敲除海马神经元中，在神经元特异性Tsc 1和Tsc 2条件性敲除中， 小鼠模型和TSC患者皮质结节的巨细胞中。值得注意的是， 与Shh信号通路的改变和未成熟神经元的存在有关。获得 为了深入了解纤毛形成缺陷的分子机制，我们在1999年进行了一项表型筛选， Tsc 2缺陷的神经元，并确定热休克蛋白hsp 90作为药物靶点， 纤毛独立于mTORC 1超活化。使用基于细胞的高通量测定，我们 揭示了通过Hsp 90抑制纤毛恢复的治疗窗口的存在，而不 影响TORC 1的激活。这些发现使我们能够建立我们的中心假设，即hsp 90是 mTORC 1下游靶点负责TSC中观察到的纤毛病样表型。在此，我们建议 确定hsp 90抑制恢复Tsc 2缺陷神经元纤毛的机制基础， 多种药理学和遗传学技术，并通过鉴定热休克蛋白90相互作用组， TSC 1/2突变神经元。存在不成熟的神经元特性、星形胶质细胞增生和对神经元的异常调节。 Shh通路是一些神经元TSC表现，将对其进行研究，以建立功能性TSC。 恢复纤毛的相关性。最后，我们将首先检查TSC患者产生的皮质神经元中的纤毛- 衍生的诱导多能干细胞（iPSC）及其同基因对照。我们将进行临床前 脑渗透和hsp 90暴露的药代动力学/药效学（PK/PD）评估 小鼠体内的抑制剂。总之，这些实验的结果将有助于阐明下游的 热休克蛋白90在TSC中的影响途径，提供了纤毛生物学的基本见解，并可能揭示 其他由热休克反应功能障碍引起的纤毛病。

Project Summary from Parent Award Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder affecting several organs. Individuals with TSC suffer from refractory epilepsy, intellectual disabilities and autism spectrum disorder, and the neurological manifestations are often the most disabling for these patients. Central nervous system (CNS) manifestations include disorganized brain connectivity, increased astrogliosis, and presence of immature dysmorphic neurons. Despite the fact that increased mTORC1 activity has been clearly implicated in the brain manifestations of TSC, a critical unmet medical need remains to identify the downstream molecular pathways implicated in the abnormal brain development. Ciliopathies are genetic disorders caused by mutations in genes affecting primary cilia which are sensory cellular antenna with a role in brain homeostasis and development, thought to act as a key regulatory node for sonic hedgehog signaling. Ciliopathies encompass a range of genetic disorders that share ciliary dysfunction and can affect several organs, including the brain. This proposal builds on robust in vitro and in vivo data indicating that certain brain abnormalities of TSC recapitulate the manifestations of ciliopathies with alteration in the Shh signaling pathway. In particular, we found reduced ciliation in Tsc2-knockdown hippocampal neurons, in neuronal-specific Tsc1 and Tsc2 conditional knockout mouse models and in the giant cells of the cortical tubers of TSC patients. Notably, defective ciliogenesis was associated with an altered Shh signaling pathway and presence of immature neurons. To gain insights into the molecular mechanism implicated in defective ciliation, we performed a phenotypic screen in the Tsc2-deficient neurons and identified the heat shock protein hsp90 as a drug target that reverses altered ciliation independently from mTORC1 hyperactivation. Using a high throughput cell-based assay, we uncovered the existence of a therapeutic window for cilia restoration through hsp90 inhibition, without affecting TORC1 activation. These findings enable us to build our central hypothesis that hsp90 is the mTORC1 downstream target responsible for the ciliopathy-like phenotype seen in TSC. Here, we propose to determine the mechanistic basis by which hsp90 inhibition restores cilia in Tsc2 deficient neurons using multiple pharmacological and genetic techniques and by identifying the hsp90 interactome in the TSC1/2 mutant neurons. Presence of immature neuronal properties, astrogliosis, and aberrant regulation of the Shh pathway are some of the neuronal TSC manifestations that will be investigated to establish the functional relevance of restoring cilia. Finally, we will first examine cilia in cortical neurons generated from TSC patient- derived induced pluripotent stem cells (iPSCs) and their isogenic controls. We will perform preclinical pharmacokinetics/pharmacodynamics (PK/PD) assessment of brain penetration and exposure of hsp90 inhibitors in mice. Taken together, the outcome of these experiments will help elucidate the downstream pathways affected by hsp90 in TSC, provide fundamental insights into cilia biology and potentially shed light for other ciliopathies caused by dysfunctional heat shock response.