Molecular mechanisms of neuronal hyperactivity in Tuberous Sclerosis Complex

癫痫和自闭症谱系障碍是最常见的影响 儿童，越来越多的证据表明，雷帕霉素机械性靶点(MTOR)的调节失调 参与了这两种疾病的发展。结节性硬化症(TSC)是一种理想的动物模型 研究大脑中异常的mTOR信号的影响，因为这一通路的失调已经被 与动物模型和患者的神经症状有关。尽管TSC-mTOR 信号通路已经得到了很好的研究，该通路失调对神经元的下游影响是 不能完全理解。我们发现，在TSC的动物模型中，TSC2的缺失导致了- 关键转录因子Egr1在某些锥体神经元亚型中的调节。此外，我们 在诱导的多能干细胞分化的皮质神经元中观察到Egr1的下调 来自TSC患者的(IPSCs)，与这些神经元的活动增加一致。我们假设 Egr1的下调是由于mTOR在两个独立的信号复合体和 导致TSC中观察到的神经元异常，这种兴奋-抑制失衡。为了证明 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。然后我们将研究通过什么机制 TSC2的缺失改变了IPSC来源神经元中Egr1的表达。最后，我们将检查IPSC的兴奋性- 并确定细胞外挽救Egr1表达对这一表型的影响 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衍生的神经元来模拟遗传病， 分子和细胞技术研究失调的信号通路、单细胞测序和基础 电生理学概念和技术。他的建议包括全面的指导和说教 这将使他能够成功地学习新技能，并在这些重要领域获得专业知识。他的 首席导师穆斯塔法·沙欣博士是一位翻译神经学家，也是临床研究和治疗方面的专家。 TSC的分子生物学。此外，候选人还组建了一个K08顾问委员会，由Dr。 李·鲁宾、伊丽莎白·恩格尔博士和亚历山大·罗滕贝格博士，他们各自在各种不同领域拥有特定的专业知识 这一建议的各个方面，如IPSC来源神经元的分化和研究、单细胞测序、 和电生理学。应聘者致力于从事神经遗传学方面的翻译研究 儿童精神障碍和拟议的研究和职业发展计划将使他能够成功地 过渡成为这一领域的独立调查者。

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.