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 (TSC) 是一种影响多个器官的多系统遗传性疾病。个人有 TSC 患有难治性癫痫、智力障碍和自闭症谱系障碍，以及神经系统疾病对于这些患者来说，症状往往是最严重的。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存在不成熟的畸形神经元。尽管 mTORC1 活性增加显然与 TSC 的大脑表现有关，确定与异常相关的下游分子途径仍然是一个未得到满足的关键医疗需求大脑发育。纤毛病是由影响初级纤毛的基因突变引起的遗传性疾病，是感觉蜂窝天线，在大脑稳态和发育中发挥作用，被认为是关键的调节因子音波刺猬信号传输节点。纤毛病包括一系列共享纤毛的遗传性疾病功能障碍并可能影响多个器官，包括大脑。该提案建立在强大的体外和体内数据表明，TSC 的某些大脑异常概括了纤毛病的表现 Shh 信号通路的改变。特别是，我们发现 Tsc2 敲低海马中的纤毛减少神经元特异性 Tsc1 和 Tsc2 条件敲除小鼠模型以及巨细胞中 TSC 患者的皮质结节。值得注意的是，纤毛发生缺陷与 Shh 信号传导改变有关通路和未成熟神经元的存在。为了深入了解相关的分子机制由于纤毛缺陷，我们对 Tsc2 缺陷神经元进行了表型筛选，并鉴定了热休克 hsp90 蛋白作为药物靶点，可独立于 mTORC1 过度激活而逆转纤毛改变。使用通过高通量细胞检测，我们发现了纤毛恢复的治疗窗口的存在通过hsp90抑制，而不影响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，导致纤毛病样表型 TSC。在这里，我们建议确定 hsp90 抑制恢复 Tsc2 中纤毛的机制基础使用多种药理学和遗传技术并通过识别 hsp90 来识别缺陷神经元 TSC1/2 突变神经元中的相互作用组。存在不成熟的神经元特性、星形胶质细胞增生和异常 Shh 通路的调节是神经元 TSC 表现的一些表现，将对其进行研究以确定恢复纤毛的功能相关性。最后，我们将首先检查皮层神经元中的纤毛 TSC 患者来源的诱导多能干细胞 (iPSC) 及其同基因对照。我们将表演脑渗透和暴露的临床前药代动力学/药效学 (PK/PD) 评估小鼠 hsp90 抑制剂。总而言之，这些实验的结果将有助于阐明下游 TSC 中受 hsp90 影响的途径，为纤毛生物学提供了基本见解，并可能为由热休克反应功能失调引起的其他纤毛病。