Disrupted Ciliary Signaling in the Brain Pathology of Tuberous Sclerosis Complex

结节性硬化症(TSC)是一种影响多个器官的多系统遗传病。具有以下特征的个人 TSC患有难治性癫痫、智力残疾和自闭症谱系障碍，以及神经学 对这些患者来说，症状往往是最令人残疾的。中枢神经系统(CNS)表现 包括大脑连接紊乱，星形胶质细胞增多，以及幼稚畸形神经元的存在。 尽管mTORC1活性增加显然与TSC的脑表现有关， 一个关键的未得到满足的医学需求仍然是识别与异常有关的下游分子通路 大脑发育。纤毛病是由影响原生纤毛的基因突变引起的遗传性疾病 感觉细胞天线在大脑的动态平衡和发育中起作用，被认为是关键的调节 用于音速刺猬信号的节点。纤毛病包括一系列共有纤毛的遗传性疾病。 并可影响包括大脑在内的几个器官。这一建议建立在体外和体内稳健的基础上。 数据显示，TSC的某些脑部异常概括了纤毛疾病的表现 Shh信号通路的改变。特别是，我们发现在TSC2基因敲除的海马区，中介性降低 神经元特异性TSC1和TSC2条件性基因敲除小鼠模型中的神经元，以及 TSC患者的皮质结节。值得注意的是，纤毛发生缺陷与Shh信号改变有关。 未成熟神经元的途径和存在。为了深入了解与之相关的分子机制 缺陷，我们在TSC2缺陷神经元中进行了表型筛选，并确定了热休克 蛋白质HSP90作为药物靶点，独立于mTORC1超激活逆转改变的中介。vbl.使用 这是一项基于细胞的高通量实验，我们发现纤毛修复存在一个治疗窗口。 通过抑制HSP90，而不影响TORC1的激活。这些发现使我们能够建立我们的中央 假设HSP90是mTORC1下游靶基因，导致纤毛病样表型 台糖公司。在这里，我们建议确定HSP90抑制恢复TSC2纤毛的机制基础 利用多种药理学和遗传学技术以及通过鉴定HSP90来鉴定缺陷神经元 TSC1/2突变型神经元中的相互作用组。存在未成熟的神经元特性、星形胶质细胞增生症和异常 Shh通路的调节是一些神经元TSC的表现，将被研究以建立 恢复纤毛的功能相关性。最后，我们将首先检查大脑皮质神经元中的纤毛。 TSC患者来源的诱导多能干细胞(IPSCs)及其同基因对照。我们将表演 临床前药代动力学/药效学(PK/PD)对脑组织渗透和暴露的评估 小鼠体内的HSP90抑制剂。总而言之，这些实验的结果将有助于阐明下游 在TSC中受HSP90影响的通路，提供了对纤毛生物学的基本见解，并可能揭示 其他由热休克反应障碍引起的纤毛病变。

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.