Elucidating the origins of cortical tuber cells using human brain organoid models of TSC

使用 TSC 的人脑类器官模型阐明皮质结节细胞的起源

• 批准号: 10574537
• 负责人: Helen S. Bateup
• 金额: $ 38.47万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-15 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10574537
• 关键词: 3-Dimensional; Affect; Astrocytes; Automobile Driving; Autophagocytosis; Behavioral; Binding Proteins; Biological Models; Brain; CRISPR interference; Calcium; Candidate Disease Gene; Caregivers; Cell Line; Cell Lineage; Cells; Complex; Cortical Malformation; Development; Disease; Embryonic Development; Engineering; Epilepsy; FRAP1 gene; Functional disorder; Gene Proteins; Generations; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genome engineering; Goals; Grant; Guide RNA; High Prevalence; Human; Hyperactivity; Image; Impaired cognition; Impairment; Individual; Intellectual functioning disability; Knock-in; Lead; Modeling; Molecular; Mutation; Neurodevelopmental Disorder; Neuroglia; Neurologic; Neurons; Operative Surgical Procedures; Organoids; Outcome; Pathology; Pathway interactions; Patient observation; Phenotype; Production; Role; Sampling; Seizures; Signal Transduction; Slice; System; TSC1 gene; TSC1/2 gene; TSC2 gene; Testing; Tuberous Sclerosis; arm; autism spectrum disorder; body system; brain cell; brain dysfunction; cell cortex; cell type; cellular development; cellular engineering; clinically relevant; cortical tubers; design; developmental disease; disease-in-a-dish; early onset; experimental study; human stem cells; improved; insight; loss of function mutation; multi-electrode arrays; mutant; nerve stem cell; nervous system disorder; neural network; neurogenesis; neuronal survival; newborn neuron; patient variability; pharmacologic; premature; prevent; programs; small hairpin RNA

PROJECT SUMMARY Tuberous Sclerosis Complex (TSC) is a multi-system developmental disorder caused by mutations in the TSC1 or TSC2 genes. The protein products of these genes form a complex that is an essential negative regulator of mTORC1 signaling. In the absence of a functional TSC1/2 complex, mTORC1 signaling is deregulated and constitutively active. While the manifestations of TSC can affect several different organ systems, the neurological and psychiatric aspects of the disease are the most burdensome for caregivers and least well understood. These include early-onset epilepsy, varying degrees of intellectual disability, and a high prevalence of autism spectrum disorder and other behavioral conditions. A hallmark pathology of TSC is the presence of cortical tubers, which are focal regions of enlarged, dysplastic neurons and glia in the cortex that form during embryonic development. Cortical tubers can become epileptic foci and in some cases are surgically removed in individuals with intractable seizures. The size and number of cortical tubers is variable between patients and increased cortical tuber load is associated with worse outcomes including more severe epilepsy and cognitive impairment. The goal of this project is to determine the molecular mechanism(s) by which mutations in TSC1 or TSC2 lead to the formation of cortical tuber cells. To do this we will use our recently established human brain organoid models of TSC in which we have engineered loss of function mutations in TSC1 or TSC2. These human brain organoid models robustly reproduce key cellular features of cortical tubers including dysmorphic neurons, reactive astrocytes, and giant/balloon cells. In addition, we have observed a strong bias towards the production of glial-lineage cells at the expense of neurons in TSC brain organoids, which recapitulates observations from patient tuber samples. Here we will define the molecular basis for altered cortical cell development due to TSC1/2 mutations and investigate how the resulting tuber cells impact the function of the surrounding cortical network. In Aim 1 we will explore two potential hypotheses for altered differentiation of TSC1/2 mutant cells in brain organoids: 1) premature activation of astrogenic transcription programs that interfere with normal neurogenesis and/or 2) impaired survival and development of newborn neurons. To test these hypotheses we will use pharmacological, shRNA, and CRISPRi manipulations to test the contribution of candidate pathways. In Aim 2 we will use different strategies to manipulate mTORC1 signaling and specific downstream arms of the pathway to test whether these can prevent or rescue altered cellular development. In Aim 3, we will perform functional analyses to determine how the presence of cortical tuber cells impacts the activity of the surrounding cortical network. Together the results of these aims will generate new insights into the molecular and cellular mechanisms leading to cortical tuber formation and how these cells ultimately impact cortical function.

项目概要 结节性硬化症 (TSC) 是一种由基因突变引起的多系统发育障碍 TSC1 或 TSC2 基因。这些基因的蛋白质产物形成了一个复合物，这是一个重要的负面因素 mTORC1 信号传导的调节因子。在缺乏功能性 TSC1/2 复合物的情况下，mTORC1 信号传导 放松管制且本质活跃。虽然 TSC 的表现可以影响多个不同的器官 对于护理人员和精神系统而言，该疾病的神经和精神方面的负担最重 最不被理解的。这些包括早发性癫痫、不同程度的智力障碍和高度 自闭症谱系障碍和其他行为状况的患病率。 TSC 的一个标志性病理学是 皮质结节的存在，这是皮质中扩大的、发育不良的神经元和神经胶质细胞的焦点区域， 胚胎发育过程中形成。皮质结节可成为癫痫病灶，在某些情况下 对于顽固性癫痫发作的个体，可通过手术切除。皮质块茎的大小和数量是可变的 患者之间的皮质结节负荷增加与更差的结果相关，包括更严重的结果 癫痫和认知障碍。 该项目的目标是确定 TSC1 或 TSC1 突变的分子机制 TSC2 导致皮质结节细胞的形成。为此，我们将使用最近建立的人脑 TSC 类器官模型，我们在其中设计了 TSC1 或 TSC2 功能缺失突变。这些 人脑类器官模型可以稳健地再现皮质结节的关键细胞特征，包括畸形 神经元、反应性星形胶质细胞和巨细胞/球囊细胞。此外，我们还观察到对 以 TSC 脑类器官中的神经元为代价来产生神经胶质细胞，这概括了 患者块茎样本的观察结果。在这里，我们将定义改变皮质细胞的分子基础 TSC1/2 突变导致的发育，并研究由此产生的块茎细胞如何影响其功能 周围皮质网络。在目标 1 中，我们将探索改变分化的两个潜在假设 脑类器官中的 TSC1/2 突变细胞：1）星形转录程序过早激活， 干扰正常的神经发生和/或 2) 损害新生神经元的存活和发育。测试 这些假设我们将使用药理学、shRNA 和 CRISPRi 操作来测试 候选途径。在目标 2 中，我们将使用不同的策略来操纵 mTORC1 信号传导和特定的 该途径的下游分支，以测试它们是否可以预防或挽救改变的细胞发育。在 目标 3，我们将进行功能分析，以确定皮质结节细胞的存在如何影响 周围皮质网络的活动。这些目标的结果将共同产生新的见解 导致皮质结节形成的分子和细胞机制以及这些细胞最终如何 影响皮质功能。