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

结节性硬化症脑部病理学中纤毛信号传导中断（多样性补充剂）

• 批准号: 10516328
• 负责人: MUSTAFA SAHIN
• 金额: $ 6.6万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10516328
• 关键词: Affect; Award; Biological Assay; Biology; Brain; Brain Pathology; Cells; Cilia; Data; Development; Drug Kinetics; Drug Targeting; Epilepsy; Functional disorder; Genes; Genetic Diseases; Genetic Techniques; Giant Cells; HSP 90 inhibition; Heat shock proteins; Heat-Shock Response; Hippocampus (Brain); Homeostasis; In Vitro; Individual; Intellectual functioning disability; Intractable Epilepsy; Light; Medical; Molecular; Mus; Mutation; Nervous System Physiology; Neuraxis; Neurologic Symptoms; Neurons; Organ; Outcome; Parents; Pathogenesis; Pathway interactions; Patients; Penetration; Pharmacodynamics; Pharmacology; Phenotype; Play; Property; Regulation; Role; SHH gene; Sensory; Signal Pathway; Signal Transduction; TSC1 gene; TSC1/2 gene; Therapeutic; Tuberous Sclerosis; astrogliosis; autism spectrum disorder; base; brain abnormalities; ciliopathy; cilium biogenesis; conditional knockout; cortical tubers; experimental study; in vivo; induced pluripotent stem cell; inhibitor; insight; knock-down; mouse model; mutant; novel therapeutic intervention; pre-clinical; restoration; smoothened signaling pathway

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.

父母奖的项目摘要 结节性硬化症复合物（TSC）是影响多个器官的多系统遗传疾病。有个人 TSC患有难治性癫痫，智力障碍和自闭症谱系障碍以及神经系统 对于这些患者而言，表现通常是最残疾。中枢神经系统（CNS）表现 包括混乱的大脑连通性，增加的星形胶质病以及未成熟的畸形神经元的存在。 尽管事实是，MTORC1活性增加已与大脑表现明显有关 TSC，仍然存在关键的未满足医学需求，以确定与 脑发育异常。纤毛病是由影响的基因突变引起的遗传疾病 原发性纤毛是感觉细胞天线，在大脑稳态和发育中起作用，被认为是 充当声波刺猬信号传导的关键调节节点。纤毛病包括一系列遗传 共享睫状功能障碍并可能影响包括大脑在内的多个器官的疾病。该建议建立 关于强大的体外和体内数据，表明某些脑部的大脑异常概括了 SHH信号通路中有改变的纤毛病的表现。特别是，我们发现减少了 TSC2缩减海马神经元的纤毛，神经元特异性TSC1和TSC2有条件敲除 小鼠模型和TSC患者皮质块茎的巨细胞中。值得注意的是，纤毛发生有缺陷 与SHH信号通路的改变和未成熟神经元的存在有关。获得 对与缺陷纤毛有关的分子机制的见解，我们在 TSC2缺陷型神经元，并确定热休克蛋白HSP90是逆转变化的药物靶标 与MTORC1过度激活独立于纤毛。使用高吞吐量的基于单元的测定，我们 发现了通过HSP90抑制纤毛恢复的治疗窗口的存在，没有 影响TORC1激活。这些发现使我们能够建立我们的中心假设，即HSP90是 MTORC1下游靶标负责TSC中看到的类似纤毛病的表型。在这里，我们建议 确定HSP90抑制使用TSC2缺乏神经元中纤毛的机理基础 多种药理和遗传技术，并通过识别Hsp90相互作用 TSC1/2突变神经元。存在未成熟的神经元特性，星形胶质症和异常调节 SHH途径是一些神经元TSC表现，将研究以建立功能 恢复纤毛的相关性。最后，我们将首先检查由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）及其同源性对照。我们将执行临床前 HSP90的脑渗透和暴露的药代动力学/药效学（PK/PD）评估 小鼠的抑制剂。综上所述，这些实验的结果将有助于阐明下游 TSC中受HSP90影响的途径，提供对纤毛生物学的基本见解，并有可能发明 功能失调的热休克反应引起的其他纤毛病。