Molecular mechanisms of neuronal hyperactivity in Tuberous Sclerosis Complex

癫痫和自闭症谱系障碍是影响儿童健康的最常见的神经系统疾病之一。 越来越多的证据表明，雷帕霉素（mTOR）的机制靶点失调是 参与了这两种疾病的发展。多发性硬化综合征（TSC）是一种理想的模型， 研究异常mTOR信号在大脑中的影响，因为这种途径的失调已经被 与动物模型和患者的神经症状有关。尽管TSC-mTOR 信号通路已经被很好地研究，该通路对神经元的失调的下游效应是 不完全理解。我们已经发现，TSC动物模型中Tsc 2的缺失导致TSC- 在锥体神经元的某些亚型中的关键转录因子Egr 1的调节。另外我们 已经观察到从诱导多能干细胞分化的皮质神经元中EGR 1的下调 这与这些神经元中活性增加一致。我们假设 EGFR 1的下调是由于两个单独的信号复合物中mTOR的失调而发生的， 导致TSC中观察到的神经元异常，如兴奋-抑制失衡。证明 这一发现的临床相关性，我们建议证实我们在iPSC来源的神经元中的观察结果， TSC患者和TSC患者的皮质结节。然后我们将研究 TSC 2的缺失改变了iPSC衍生神经元中的EGR 1表达。最后，我们将研究iPSC的兴奋性- 衍生的神经元，并确定使用细胞外基质对该表型的挽救EGR 1表达的影响。 录音. 候选人目前是波士顿儿童医院的神经遗传学研究员，这项提议建立在 他在生物信息学方面的技能，并将他的技能扩展到使用iPSC衍生的神经元来建模遗传疾病， 分子和细胞技术来研究失调的信号通路，单细胞测序，和基本的 电生理学概念和技术。他的建议包括一个全面的指导和说教 计划，使他能够成功地学习新技能，并获得在这些重要领域的专业知识。他 主要导师Mustafa Sahin博士是一位翻译神经科学家，也是临床研究和 TSC分子生物学此外，候选人已成立K 08顾问委员会，成员包括： Lee Rubin博士、Elizabeth Engle博士和亚历山大Rotenberg博士，他们每个人都在各种领域拥有特定的专业知识。 该提案的各个方面，如iPSC衍生神经元的分化和研究，单细胞测序， 和电生理学。候选人致力于翻译研究的职业生涯，重点是神经遗传学 儿童疾病，以及拟议的研究和职业发展计划将使他能够成功地 成为该领域的独立调查员。

Epilepsy and Autism spectrum disorder are among the most common neurological disorders that affect children, and there is increasing evidence that dysregulation of the mechanistic target of rapamycin (mTOR) is involved in the development of both disorders. Tuberous Sclerosis Complex (TSC) is an ideal model in which to study the effects of abnormal mTOR signaling in the brain because dysregulation of this pathway has been implicated in the neurological symptoms of both animal models and patients. Although the TSC-mTOR signaling pathway has been well studied, the downstream effect of dysregulation of this pathway on neurons is not completely understood. We have found that loss of Tsc2 in an animal model of TSC leads to down- regulation of the critical transcription factor, Egr1, in certain sub-types of pyramidal neurons. In addition, we have observed down-regulation of EGR1 in cortical neurons differentiated from induced pluripotent stem cells (iPSCs) from patients with TSC, coincident with increased activity in these neurons. We hypothesize that down-regulation of EGR1 occurs due to dysregulation of mTOR in two separate signaling complexes and contributes to neuronal abnormalities observed in TSC, such excitatory-inhibitory imbalance. To demonstrate the clinical relevance of this finding, we propose to confirm our observation in iPSC-derived neurons from patients with TSC and cortical tubers from individuals with TSC. We will then examine the mechanism by which loss of TSC2 alters EGR1 expression in iPSC-derived neurons. Finally, we will examine excitability in iPSC- derived neurons and determine the effect of rescuing EGR1 expression on this phenotype using extracellular recordings. The candidate is currently a Neurogenetics fellow at Boston Children's Hospital, and this proposal builds upon his skills in bioinformatics and extends his skillset to the use of iPSC-derived neurons to model genetic disease, molecular and cellular techniques to study dysregulated signaling pathways, single cell sequencing, and basic electrophysiological concepts and techniques. His proposal includes a comprehensive mentoring and didactic plan that will allow him to successfully learn new skills and gain expertise in each of these important areas. His primary mentor, Dr. Mustafa Sahin, is a translational neuroscientist and expert in both the clinical research and molecular biology of TSC. In addition, the candidate has assembled a K08 advisory committee consisting of Dr. Lee Rubin, Dr. Elizabeth Engle, and Dr. Alexander Rotenberg, who each have specific expertise in various aspects of this proposal, such as differentiation and study of iPSC-derived neurons, single cell sequencing, and electrophysiology. The candidate is committed to a career in translation research focusing on neurogenetic disorders in children, and the proposed research and career development plans will enable him to successfully transition to become an independent investigator in this field.