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

来自家长奖的项目摘要 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活性的增加显然与脑部疾病的表现有关 TSC，一个关键的未得到满足的医学需求仍然是确定与TSC有关的下游分子通路 大脑发育异常。纤毛病是由基因突变引起的遗传性疾病 初级纤毛是一种感觉细胞触角，在大脑的动态平衡和发育中发挥作用，人们认为 作为Sonic Hedgehog信号的关键调节节点。纤毛病包括一系列遗传因素 具有睫毛功能障碍的疾病，可能会影响包括大脑在内的几个器官。这项建议建立了 关于稳健的体外和体内数据表明，TSC的某些脑异常概括了 伴有Shh信号通路改变的纤毛病的表现。特别是，我们发现减少了 神经元特异性TSC1和TSC2条件性基因敲除在海马神经元中的调节作用 在小鼠模型和TSC患者皮质结节的巨细胞中。值得注意的是，纤毛发生缺陷 与Shh信号通路的改变和未成熟神经元的存在有关。为了获得 对缺陷调节所涉及的分子机制的洞察，我们在 并确定热休克蛋白HSP90是逆转改变的药物靶点 独立于mTORC1超激活的调节。使用高通量的基于细胞的分析，我们 发现了通过抑制HSP90进行纤毛修复的治疗窗口的存在，而没有 影响TORC1的激活。这些发现使我们能够建立我们的中心假设，即HSP90是 MTORC1下游靶基因与TSC中见到的纤毛病样表型有关。在此，我们建议 HSP90抑制恢复TSC2缺陷性神经元纤毛的机制 多种药理和遗传技术，并通过鉴定HSP90相互作用组在 TSC1/2突变神经元。存在未成熟的神经元特性、星形胶质细胞增生症和异常调节 Shh通路是一些神经元TSC的表现，将被研究以建立功能性的 恢复纤毛的相关性。最后，我们将首先检查TSC患者大脑皮质神经元中的纤毛- 衍生诱导多能干细胞(IPSCs)及其同基因对照。我们将进行临床前研究 热休克蛋白90脑内渗透和暴露的药代动力学/药效学评价 小鼠体内的抑制剂。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。

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.