Disruption of TRIO signaling through PDE4A5 in neurodevelopmental disorders

项目摘要 精神分裂症(SCZ)和自闭症(ASD)是影响数百万人的高度衰弱的神经发育障碍 一群人。一系列杂合性功能丧失(LOF)或破坏性变异的等位基因序列 在患有SCZ和ASD的个体中，整个trio基因显著丰富，这表明trio基因减少 功能导致病理。事实上，我们的实验室已经证明了小鼠大脑皮质中单个三对等位基因的缺失 兴奋性神经元(在NEX-TRIO+/-小鼠中)产生各种行为缺陷和严重的树突状细胞缺陷 结构和突触活性。TRIO作用于细胞表面受体下游，控制轴突和树突 寻路和突触发育。然而，Trio变种和Trio变体破坏的信号机制 这些过程中的缺陷如何扰乱神经元的发育仍然完全不清楚。我们的实验室鉴定出 PDE4A5是一种新的候选TRIO信号伙伴，因为它在TRIO缺陷小鼠皮质中的水平降低 以及它与TRIO共免疫沉淀的能力。我的提议将检验三人组相互作用的假设 在功能上与PDE4A5一起在两个互补的目标中调节神经元的发育和功能。 我的第一个目标是确定TRIO和PDE4A5在功能上如何交互，以及这些功能是如何 受疾病相关变异的影响。PDE4A5与TRIO共免疫沉淀，但这些蛋白是如何 相互作用以及这些相互作用如何影响每种蛋白质的功能还知之甚少。我会用纯净的 重组蛋白进行定量结合分析并确定PDE4A5和TRIO是否相互作用 并定义足以调节相互作用的最小TRIO和PDE4A5片段。我会当工程师 位于Trio：PDE4A5交互界面中的与紊乱相关的变体，以确定是否以及如何 这些变体影响了Trio：PDE4A5的相互作用。我会用我的纯化蛋白质和大量的体外和 以细胞为基础的分析，以衡量三者与PDE4A5之间的相互作用如何影响其催化活性。 我的第二个目标是确定TRIO：PDE4A5相互作用如何调控神经元发育。 破坏培养的皮质神经元中的单个三对等位基因会导致树突和树突棘的缺陷 发育与体内观察到的相似。TRIO：PDE4A5相互作用的中断如何促进 TRIO缺陷神经元的病理生理学尚不清楚。我将使用shRNA介导的PDE4A5基因敲除 以确定PDE4A5是否是树突和树突棘发育所必需的。我会表演的 基因敲除/互补和共聚焦显微镜在培养的皮质神经元中确定是否和 TRIO：PDE4A5相互作用的中断如何影响PDE4A45的定位和神经元发育。我将使用 小分子PDE4A5激活剂测试PDE4A5的药理操作是否可以修复缺陷 在培养和NEX-TRIO+/-和NEX-TRIO-/-小鼠的皮层神经元发育中。

Project Summary Schizophrenia (SCZ) and autism (ASD) are highly debilitating neurodevelopmental disorders that affect millions of people. An allelic series of heterozygous loss-of-function (LOF) or damaging variants spread throughout the entire TRIO gene are significantly enriched in individuals with SCZ and ASD, suggesting that reduced TRIO function causes pathology. Indeed, our lab has shown that deletion of a single TRIO allele in mouse cortical excitatory neurons (in NEX-TRIO+/– mice) yields diverse behavioral deficits and severe defects in dendritic structure and synaptic activity. TRIO acts downstream of cell surface receptors to control axon and dendrite pathfinding, and synapse development. However, the signaling mechanisms compromised by TRIO variants and how defects in these processes disrupt neuronal development remain completely unclear. Our lab identified PDE4A5 as a new candidate TRIO signaling partner due to its reduced levels in TRIO-deficient mouse cortex and its ability to co-immunoprecipitate with TRIO. My proposal will test the hypothesis that TRIO interacts functionally with PDE4A5 to regulate neuron development and function in two complementary aims. My first aim is to determine how TRIO and PDE4A5 interact functionally and how these functions are impacted by disease-associated variants. PDE4A5 coimmunoprecipitates with TRIO, but how these proteins interact and how these interactions impact each protein’s function is poorly understood. I will use purified recombinant proteins to perform quantitative binding assays and determine whether PDE4A5 and TRIO interact directly and define the minimal TRIO and PDE4A5 fragments sufficient to mediate interaction. I will engineer disorder-associated variants that lie within the TRIO:PDE4A5 interaction interface to determine whether and how these variants impact the Trio:PDE4A5 interaction. I will use my purified proteins and an arsenal of in vitro and cell-based assays to measure how interactions between the TRIO and PDE4A5 impact their catalytic activities. My second aim is to determine how TRIO:PDE4A5 interactions regulate neuronal development. Disruption of a single TRIO allele in cultured cortical neurons yields defects in dendrite and dendritic spine development similar to those observed in vivo. How disruption of TRIO:PDE4A5 interactions contributes to the pathophysiology seen in TRIO deficient neurons is unclear. I will use shRNA-mediated knockdown of PDE4A5 to determine whether PDE4A5 is necessary for dendrite and dendritic spine development. I will perform knockdown/complementation and confocal microscopy in cultured cortical neurons to determine whether and how disruptions in TRIO:PDE4A5 interaction affects PDE4A45 localization and neuron development. I will use a small-molecule PDE4A5 activator to test whether pharmacological manipulation of PDE4A5 can rescue deficits in development of TRIO+/– and TRIO–/– cortical neurons in culture and in NEX-TRIO+/– and NEX-TRIO-/– mice.