Dysregulation of TRIO GEF1 activity in neurodevelopmental disorders

项目总结 这三个基因中的遗传变异会增加患神经发育障碍(NDDS)的风险，包括 精神分裂症、自闭症和相关疾病。TRIO编码一个含有两个鸟嘌呤核苷酸的大蛋白 Rho家族GTP酶的交换因子结构域：GEF1激活rac1和RhoG，GEF2激活 罗亚。我们发现了一组与自闭症和智力残疾相关的变异，它们选择性地激活或 抑制TRIO GEF1活性。虽然我们的发现强调了这种酶活性对适当的 大脑发育，调节TRIO GEF1活性的分子机制，下游 TRIO GEF1信号的靶点，以及这些过程是如何被GEF1靶向变体破坏的 根本而又悬而未决的问题。回答这些问题将揭示TRIO中的变异是如何导致NDD的，并可能 告知新的治疗干预措施。我们的建议将从三个方面解决这些问题： 目的1.阐明TRIO-GEF1的激活机制。我们发现幽灵蛋白在体内重复6-9次 TRIO结合并自动抑制其GEF1活性，而血影蛋白重复序列8中NDD相关变体缓解了这一点 自我抑制。一小部分受体和激酶已被确定为已知或可能的三个GEF1调节因子， 但这些激活剂参与TRIO激活GEF1活性的机制尚不清楚。我们将使用 纯化重组蛋白以测试这些受体的细胞质结构域和激酶对TRIO GEF1的影响 活动。我们还将使用基于FRET的活性生物传感器和形态测量来揭示这些 RhoG/rac1激活和受体激活诱导神经元发育的机制。 目的2.鉴定和表征受TRIO-GEF1活性调控的神经元信号事件。我们 产生了携带三个变异等位基因的小鼠，其中(K1431M)或(R1078Q)三个GEF1降低或升高 活动。我们将在野生型小鼠的样本中使用比较蛋白质组学和磷酸蛋白质组学 那些携带三重GEF1抑制或激活等位基因以识别蛋白质、信号事件和三重GEF1的人。 互动伙伴受到TRIO GEF1活动变化的影响。我们将系统地测试操纵 其中，GEF1介导的事件影响神经元发育和突触连接。 目的3.测量TRIO GEF1活性的选择性变化如何影响神经元发育和 突触传递。GEF1缺陷的TRIOK1431M等位基因杂合性导致脑体积缩小 和行为缺陷，与我们的假设一致，即TRIO GEF1活性的选择性改变 损害正常的神经元发育和突触功能。我们将使用定量组织病理学和 携带K1431M和R1078Q变异的小鼠的电子显微镜显示TRIO GEF1活性是如何改变的 影响轴突、树突和突触的发育。全细胞电生理学与光遗传学 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、突触功能和电路连通性。

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.