Disrupted Ciliary Signaling in the Brain Pathology of Tuberous Sclerosis Complex

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TSC的脑表现有关， 一个关键的未满足的医学需求仍然是确定与异常的细胞凋亡有关的下游分子途径。 大脑发育纤毛病是由影响初级纤毛的基因突变引起的遗传性疾病， 是感觉细胞的触角，在大脑的稳态和发育中起作用，被认为是调节大脑活动的关键。 刺猬信号的节点。睫状体病包括一系列遗传性疾病， 功能障碍，并可能影响几个器官，包括大脑。该提案建立在体外和体内稳健的基础上 数据表明TSC的某些脑异常概括了纤毛病的表现， Shh信号通路的改变。特别是，我们发现Tsc 2敲低的海马神经元中纤毛的形成减少， 在神经元特异性Tsc 1和Tsc 2条件性敲除小鼠模型中以及在巨细胞中， TSC患者的皮质结节。值得注意的是，纤毛发生缺陷与Shh信号的改变有关。 通路和未成熟神经元的存在。为了深入了解 我们在Tsc 2缺陷的神经元中进行了表型筛选，并确定了热休克 蛋白hsp 90作为药物靶点，其逆转不依赖于mTORC 1超活化的改变的纤毛形成。使用 一个高通量的基于细胞的检测，我们发现了纤毛恢复的治疗窗口的存在， 通过抑制hsp 90，而不影响TORC 1的激活。这些发现使我们能够建立我们的中央 假设hsp 90是mTORC 1下游靶点，负责在 TSC。在这里，我们建议确定抑制热休克蛋白90恢复Tsc 2纤毛的机制基础。 使用多种药理学和遗传学技术，并通过鉴定HSP 90， TSC 1/2突变神经元中的相互作用体。存在不成熟的神经元特性、星形胶质细胞增生和异常 Shh通路的调节是一些神经元TSC表现，将对其进行研究以建立 恢复纤毛的功能相关性。最后，我们将首先检查由大脑皮层神经元产生的纤毛。 TSC患者来源的诱导多能干细胞（iPSC）及其同基因对照。我们将执行 脑渗透和暴露的临床前药代动力学/药效学（PK/PD）评估 HSP 90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。

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.