Disrupted Ciliary Signaling in the Brain Pathology of Tuberous Sclerosis Complex

结节性硬化症脑病理学中纤毛信号传导中断

基本信息

批准号：
10654265
负责人：
MUSTAFA SAHIN
金额：
$ 14.34万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2022-09-10
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10654265
关键词：
Affect Animal Model Astrocytes Benign Biological Assay Biology Brain Brain Pathology Cells Characteristics Cilia Client Complex Data Defect Development Disinhibition Distal Dose Drug Kinetics Drug Targeting Epilepsy Epileptogenesis FRAP1 gene Functional disorder Genes Genetic Diseases Genetic Techniques Giant Cells Grant HSP 90 inhibition Hamartoma Heat shock proteins Heat-Shock Proteins 90 Heat-Shock Response Hippocampus (Brain)Homeostasis Human Impairment In Vitro Individual Intellectual functioning disability Intractable Epilepsy Knockout Mice Lead Light Measurement Medical Molecular Mus Mutation Nervous System Physiology Neuraxis Neurodevelopmental Disorder Neurologic Symptoms Neurons Organ Outcome Pathogenesis Pathway interactions Patients Penetration Pharmacodynamics Pharmacology Phenotype Play Process Property Proteins Regulation Resected Role SHH gene Seizures Sensory Signal Transduction Sirolimus Sonic Hedgehog Pathway TSC1 gene TSC1/2 gene TSC2 gene Text Therapeutic Tuberous Sclerosis Tubulin astrogliosis autism spectrum disorder base brain abnormalities ciliopathy cilium biogenesis conditional knockout cortical tubers experimental study in vivo in vivo Model induced pluripotent stem cell inhibitor insight knock-down migration mouse model mutant neuron development neuropathology novel therapeutic intervention pre-clinical prevent restoration smoothened signaling pathway tumor

项目摘要

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.

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MTORC1活性增加的事实已与TSC的大脑表现明显有关，但仍然存在关键的未满足医学需求，以识别与异常有关的下游分子途径大脑发育。纤毛病是由影响原发性纤毛的基因突变引起的遗传疾病，是感觉细胞天线，在大脑稳态和发育中起作用，被认为是关键调节 Sonic刺猬信号传导的节点。纤毛病包括一系列共享睫状体的遗传疾病功能障碍，可能会影响包括大脑在内的多个器官。该建议建立在体外和体内的强大的基础上数据表明TSC的某些大脑异常概括了纤毛病的表现 SHH信号通路的变化。特别是，我们发现TSC2-KNOCKDOWN HAMPOCAMPAL的纤毛减少神经元，在神经元特异性TSC1和TSC2中有条件敲除小鼠模型以及在 TSC患者的皮质块茎。值得注意的是，纤毛发生有缺陷与SHH信号改变有关未成熟神经元的途径和存在。洞悉与涉及的分子机制有缺陷的纤毛，我们在TSC2缺陷神经元中进行了表型筛选，并鉴定了热休克蛋白质HSP90是一种药物靶标，可独立于MTORC1过度激活而逆转纤维化。使用一个基于高吞吐量的细胞测定法，我们发现了纤毛恢复的治疗窗口的存在通过HSP90抑制，而不会影响TORC1激活。这些发现使我们能够建立我们的中心假设HSP90是MTORC1下游靶标，负责纤毛病的表型。 TSC。在这里，我们建议确定HSP90抑制恢复TSC2中纤毛的机械基础使用多种药理和遗传技术的神经元缺陷，并通过识别HSP90 TSC1/2突变神经元中的相互作用组。存在未成熟的神经元特性，星形胶质病和异常 SHH途径的调节是一些神经元TSC表现，将研究以建立恢复纤毛的功能相关性。最后，我们将首先检查由从 TSC患者衍生的诱导多能干细胞（IPSC）及其同源性对照。我们将表演临床前药代动力学/药效学（PK/PD）评估脑穿透和暴露小鼠中的HSP90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。