Dysregulation of TRIO GEF1 activity in neurodevelopmental disorders

TRIO GEF1 活性在神经发育障碍中的失调

基本信息

批准号：
10714793
负责人：
Anthony J Koleske
金额：
$ 85.39万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10714793
关键词：
Address Adhesions Alleles Anxiety Axon Behavioral Binding Biochemical Biochemical Process Biophysics Biosensor Brain Cells Characteristics Cytoplasmic Receptors Cytoplasmic Tail Defect Dendrites Development Developmental Delay Disorders Disease Electron Microscopy Electrophysiology (science)Event Family Fluorescence Resonance Energy Transfer Genes Genetic Guanine Nucleotide Exchange Factors Guanosine Triphosphate Phosphohydrolases Heterozygote Histopathology Impairment Individual Intellectual functioning disability Interruption Lead Measurement Measures Mediating Modeling Molecular Morphology Mus Neurodevelopmental Disorder Neurons Pathologic Pathology Phosphorylation Site Phosphotransferases Process Prosencephalon Proteins Proteomics Receptor Activation Recombinant Proteins Regulation Risk Sampling Schizophrenia Signal Transduction Signaling Protein Social Interaction Spectrin Symptoms Synapses Synaptic Transmission Synaptic plasticity TRIO gene Testing Therapeutic Translating Variant Wild Type Mouse Work autism spectrum disorder brain behavior brain size comparative enzyme activity excitatory neuron genetic risk factor genetic variant impaired brain development individuals with autism spectrum disorder light microscopy loss of function model organism motor impairment neuron development neuronal excitability neurotransmission novel therapeutic intervention optogenetics phosphoproteomics receptor rho schizophrenia risk success synaptic function tool

项目摘要

PROJECT SUMMARY Genetic variants in the TRIO gene increase risk for neurodevelopmental disorders (NDDs) including schizophrenia, autism, and related disorders. TRIO encodes a large protein with two guanine nucleotide exchange factor (GEF) domains for Rho family GTPases: GEF1 activates Rac1 and RhoG, and GEF2 activates RhoA. We found a cluster of variants associated with autism and intellectual disability that selectively activate or inhibit TRIO GEF1 activity. While our findings highlight the central importance of this enzyme activity for proper brain development, the molecular mechanisms by which TRIO GEF1 activity is regulated, the downstream targets of TRIO GEF1 signaling, and how these processes are disrupted by GEF1-targeting variants remain fundamental, yet unresolved questions. Answering them will reveal how variants in TRIO lead to NDDs and may inform new therapeutic interventions. Our proposal will address these questions in three Aims: Aim 1. To elucidate the mechanism of TRIO GEF1 activation. We discovered that spectrin repeats 6-9 in TRIO bind and autoinhibit its GEF1 activity and that NDD-associated variants in spectrin repeat 8 relieve this autoinhibition. A short list of receptors and kinases has been identified as known or likely TRIO GEF1 regulators, but the mechanisms by which these activators engage TRIO to activate GEF1 activity are unclear. We will use purified recombinant proteins to test how these receptors’ cytoplasmic domains and kinases impact TRIO GEF1 activity. We will also use a FRET-based activity biosensor and morphological measurements to reveal how these mechanisms contribute to Rac1/RhoG activation and neuronal development induced by receptor activation. Aim 2. To identify and characterize the neuronal signaling events regulated by TRIO GEF1 activity. We have generated mice bearing TRIO variant alleles with reduced (K1431M) or elevated (R1078Q) TRIO GEF1 activity. We will use comparative proteomics and phospho-proteomics in samples from wild-type mice versus those bearing TRIO GEF1-inhibiting or activating alleles to identify proteins, signaling events, and TRIO- interaction partners impacted by changes in TRIO GEF1 activity. We will systematically test how manipulation of these GEF1-mediated events impacts neuronal development and synaptic connectivity. Aim 3. To measure how selective changes in TRIO GEF1 activity impact neuronal development and synaptic transmission. Heterozygosity for the GEF1-defective TRIOK1431M allele causes reduced brain size and behavioral defects, consistent with our hypothesis that selective alterations in TRIO GEF1 activity compromise normal neuronal development and synaptic function. We will use quantitative histopathology and electron microscopy in mice bearing the K1431M and R1078Q variants to reveal how altered TRIO GEF1 activity impacts axon, dendritic arbor, and synapse development. Whole-cell electrophysiology and optogenetic manipulation will enable us to identify the consequences of changes in TRIO GEF1 activity on neuronal excitability, synaptic function, and circuit connectivity.

项目概要 TRIO 基因的遗传变异会增加神经发育障碍 (NDD) 的风险，包括 TRIO 编码具有两个鸟嘌呤核苷酸的大蛋白，用于精神分裂症、自闭症和相关疾病。 Rho 家族 GTPases 的交换因子 (GEF) 结构域：GEF1 激活 Rac1 和 RhoG，GEF2 激活我们发现了一组与自闭症和智力障碍相关的变异，它们选择性地激活或抑制 TRIO GEF1 活性，而我们的研究结果强调了这种酶活性对于正常的重要性。大脑发育、TRIO GEF1 活性调节的分子机制、下游 TRIO GEF1 信号传导的目标，以及这些过程如何被 GEF1 靶向变体破坏仍然存在回答这些基本但尚未解决的问题将揭示 TRIO 中的变异如何导致 NDD 并可能。我们的建议将通过三个目标解决这些问题：目的 1. 阐明 TRIO GEF1 激活机制我们发现血影蛋白在 6-9 中重复。 TRIO 结合并自动抑制其 GEF1 活性，血影蛋白重复序列 8 中的 NDD 相关变体可缓解这种情况一小部分受体和激酶已被确定为已知或可能的 TRIO GEF1 调节剂，但我们将使用这些激活剂与 TRIO 激活 GEF1 活性的机制尚不清楚。纯化的重组蛋白以测试这些受体的胞质结构域和激酶如何影响 TRIO GEF1 我们还将使用基于 FRET 的活性生物传感器和形态学测量来揭示这些活性。机制有助于 Rac1/RhoG 激活和受体激活诱导的神经发育。目标 2. 识别和表征 TRIO GEF1 活性调节的神经信号事件。已产生携带 TRIO 变异等位基因的小鼠，其 TRIO GEF1 降低（K1431M）或升高（R1078Q）我们将使用比较蛋白质组学和磷酸化蛋白质组学来研究野生型小鼠和野生型小鼠的样本。那些带有 TRIO GEF1 抑制或激活等位基因的基因，用于识别蛋白质、信号事件和 TRIO- 受 TRIO GEF1 活动变化影响的互动合作伙伴我们将系统地测试如何操纵。这些 GEF1 介导的事件会影响神经发育和突触连接。目标 3. 测量 TRIO GEF1 活性的选择性变化如何影响神经发育和 GEF1 缺陷 TRIOK1431M 等位基因的杂合性导致大脑尺寸减小。和行为缺陷，与我们的假设一致，即 TRIO GEF1 活性的选择性改变损害正常的神经发育和突触功能我们将使用定量组织病理学和对携带 K1431M 和 R1078Q 变体的小鼠进行电子显微镜观察，揭示 TRIO GEF1 活性如何改变影响轴突、树突轴和突触发育。操纵将使我们能够确定 TRIO GEF1 活性变化对神经元的影响兴奋性、突触功能和电路连接。