Neurodevelopmental Function of TBC1D7: A Core Component of the TSC Complex

TBC1D7 的神经发育功能：TSC 复合体的核心组成部分

• 批准号: 10590134
• 负责人: BRENDAN D. MANNING
• 金额: $ 43.86万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10590134
• 关键词: AGFG1 gene; Address; Affect; Architecture; Attention deficit hyperactivity disorder; Autophagocytosis; Axon; Binding; Brain; CRISPR/Cas technology; Cell physiology; Cerebrum; Cilia; Complex; Cortical Dysplasia; Defect; Development; Disease; Dose; Epilepsy; Exhibits; FRAP1 gene; Family; Foundations; GABA-A Receptor; GTPase-Activating Proteins; Gait; Generations; Genes; Genetic Models; Genetic study; Gilles de la Tourette syndrome; Growth; Growth and Development function; Guanosine Triphosphate Phosphohydrolases; Human; Human Genetics; Intellectual functioning disability; Length; Mammalian Cell; Measures; Megalencephaly; Migraine; Molecular; Mus; Mutation; Neurocognitive Deficit; Neurons; Pathway interactions; Pentylenetetrazole; Phenotype; Physiology; Predisposition; Process; Proteins; Role; SHH gene; Seizures; Signal Transduction; Sirolimus; Stimulus; Structure; Syndrome; TSC1 gene; TSC1/2 gene; TSC2 gene; Thick; Tissues; Tuberous Sclerosis; Tuberous sclerosis protein complex; antagonist; autism spectrum disorder; brain overgrowth; cilium biogenesis; genome wide association study; inhibitor; mind control; mouse model; mutant; nervous system disorder; neuronal cell body; neuronal growth; neuropsychiatric disorder; neuropsychiatry; neurotransmission; novel; protein complex; response; scaffold

Abstract Genetic alterations directly or indirectly affecting the function of the tuberous sclerosis complex (TSC) protein complex or its downstream target the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) underlie a myriad of neurological disorders, including, but not limited to, epilepsy, autism spectrum disorder, brain overgrowth (megalencephaly) and focal cortical dysplasia, and neurocognitive deficits. We identified the protein TBC1D7 as a third stochiometric component of the TSC complex that is scaffold together with TSC2 via direct binding to TSC1. TBC1D7 is a component of the TSC complex in all mammalian cells and tissues examined, but also exists as a free, more unstable pool outside of the complex. While the sequence of TBC1D7 predicts that it serves as a GTPase-activating protein (or GAP) for Rab family GTPases, the molecular and cellular functions of TBC1D7, both within the TSC complex and outside of it, are unknown. In addition to its undefined role in the TSC complex, which is functionally disrupted in the severe neurological disorder TSC, independent human genetic studies have now found that TBC1D7 is genetically disrupted in a rare autosomal recessive megalencephaly syndrome associated with intellectual disabilities and neuropsychiatric manifestations. Genome-wide association studies have also implicated the TBC1D7 locus in Tourette’s syndrome, attention deficit/hyperactivity disorder, and migraines. Thus, it is important to understand the role of TBC1D7 in both normal brain development and physiology and in neurological disorders, a problem best addressed through the generation of novel genetic models. We have recently used CRISPR/Cas9 gene editing to generate a line of mice lacking TBC1D7. Initial characterization reveals that these mice exhibit megalencephaly and wide-gait phenotypes and that cultured Tbc1d7-/- primary neurons have elevated mTORC1 signaling, increased soma size, defective axon specification, and longer primary cilia relative to Tbc1d7+/+ neurons. In the two aims of this R21, we will develop a detail characterization of the cerebral and neuronal alterations caused by TBC1D7 loss. In Aim 1, we will focus on signaling, brain growth, cortical layering, cerebellar architecture, seizure susceptibility, and the effects of mTOR inhibitors on these phenotypes. In Aim 2, we use primary neuronal cultures to characterize neuron-intrinsic changes in neuronal signaling, growth, polarity, and the formation and function of primary cilia. We will use these phenotypic endpoints to mechanistically determine the role of both the TSC complex-associated and free pools of TBC1D7, its putative Rab-GAP activity, specific candidate Rab protein targets, and mTORC1 signaling. This new mouse model and study under this proposal will lay an essential foundation for defining the molecular function of TBC1D7 in brain development and the myriad of neurological disorders in which it and the processes and pathway that it regulates have been implicated.

摘要 直接或间接影响结节性硬化症复合体（TSC）蛋白功能的遗传改变 复合物或其下游靶点雷帕霉素（mTOR）复合物1（mTORC 1）的机制靶点 无数的神经系统疾病，包括但不限于癫痫、自闭症谱系障碍、脑 过度生长（巨脑畸形）和局灶性皮质发育不良，以及神经认知缺陷。我们确定了 蛋白质TBC 1D 7作为TSC复合物的第三化学计量组分，所述TSC复合物通过 直接与TSC 1结合。TBC 1D 7是所有哺乳动物细胞和组织中TSC复合物的组分 检查，但也存在作为一个自由的，更不稳定的池外面的复杂。虽然序列 TBC 1D 7预测其作为Rab家族GTP酶的GTP酶激活蛋白（或GAP）， TBC 1D 7在TSC复合物内和其外的分子和细胞功能是未知的。在 除了其在TSC复合体中的不确定作用外，TSC复合体在严重的神经系统疾病中功能被破坏， 独立的人类遗传学研究现在发现，TBC 1D 7在一种遗传学上被破坏， 与智力残疾相关的罕见常染色体隐性巨脑综合征， 神经精神症状全基因组关联研究还表明，TBC 1D 7基因座在 图雷特综合征、注意力缺陷/多动症和偏头痛。因此，重要的是要了解 TBC 1D 7在正常大脑发育和生理学以及神经系统疾病中的作用， 最好通过新的遗传模型来解决。我们最近使用CRISPR/Cas9基因 编辑以产生缺乏TBC 1D 7的小鼠系。最初的特征表明，这些小鼠表现出 巨脑畸形和宽步态表型，培养的Tbc 1d 7-/-原代神经元具有升高的 mTORC 1信号传导、索马大小增加、轴突特化缺陷和初级纤毛相对于 Tbc 1d 7 +/+神经元。在本R21的两个目标中，我们将详细描述大脑和 由TBC 1D 7损失引起的神经元改变。在目标1中，我们将重点关注信号，大脑生长，皮质 分层、小脑结构、癫痫发作易感性以及mTOR抑制剂对这些的影响 表型在目标2中，我们使用原代神经元培养物来表征神经元中的神经元内在变化， 信号传导、生长、极性以及初级纤毛的形成和功能。我们将使用这些表型 终点，以机械地确定TSC复合物相关的和游离的 TBC 1D 7、其推定的Rab-GAP活性、特异性候选Rab蛋白靶标和mTORC 1信号传导。这 新的小鼠模型和在此建议下的研究将为定义分子奠定必要的基础 TBC 1D 7在大脑发育中的功能以及它和神经系统疾病中的无数神经系统疾病， 它所调控的过程和途径都有牵连。