Dysregulation of TRIO GEF1 activity in neurodevelopmental disorders

项目摘要 TRIO基因的遗传变异增加了神经发育障碍（NDD）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质 Rho家族GTP酶的交换因子（GEF）结构域：GEF 1激活Rac 1和RhoG，GEF 2激活 Rhoa.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 GEF 1活性。虽然我们的研究结果强调了这种酶活性对适当的 大脑发育，TRIO GEF 1活性调节的分子机制，下游 TRIO GEF 1信号传导的靶点，以及这些过程如何被GEF 1靶向变体破坏， 基本的，尚未解决的问题。对它们的分析将揭示TRIO中的变体如何导致NDD， 为新的治疗干预提供信息。我们的建议将从三个方面解决这些问题： 目标1.阐明TRIO GEF 1的激活机制。我们发现，血影蛋白重复6-9， TRIO结合并自身抑制其GEF 1活性，血影蛋白重复序列8中的NDD相关变体缓解了这一点 自我抑制一个简短的受体和激酶清单已被鉴定为已知或可能的TRIO GEF 1调节剂， 但这些激活剂与TRIO结合以激活GEF 1活性的机制尚不清楚。我们将使用 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 GEF 1 活动我们还将使用基于FRET的活性生物传感器和形态学测量来揭示这些细胞是如何被激活的。 Rac 1/RhoG激活和受体激活诱导的神经元发育。 目标二。鉴定和表征TRIO GEF 1活性调节的神经元信号传导事件。我们 产生了携带TRIO变体等位基因的小鼠，其具有降低的（K1431 M）或升高的（R1078 Q）TRIO GEF 1 活动我们将使用比较蛋白质组学和磷酸化蛋白质组学，从野生型小鼠和 那些携带TRIO GEF 1抑制或激活等位基因以鉴定蛋白质、信号传导事件和TRIO- 受TRIO GEF 1活动变化影响的互动伙伴。我们将系统地测试如何操纵 这些GEF 1介导的事件影响神经元发育和突触连接。 目标3。为了测量TRIO GEF 1活性的选择性变化如何影响神经元发育， 突触传递GEF 1缺陷型TRIOK 1431 M等位基因的杂合性导致脑体积减小 和行为缺陷，这与我们的假设一致，即TRIO GEF 1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， 在携带K1431 M和R1078 Q变体的小鼠中进行电子显微镜检查，以揭示TRIO GEF 1活性如何改变 影响轴突、树突乔木和突触发育。全细胞电生理学和光遗传学 操作将使我们能够确定TRIO GEF 1活性变化对神经元细胞的影响。 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.