Disruption of TRIO signaling through PDE4A5 in neurodevelopmental disorders

在神经发育障碍中通过 PDE4A5 破坏 TRIO 信号传导

• 批准号: 10313299
• 负责人: Ellen Elizabeth Corcoran
• 金额: $ 4.6万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313299
• 关键词: Disruption; TRIO; signaling; through; PDE4A5

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.

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 的人。一系列杂合性功能丧失（LOF）或破坏性变异的等位基因分布在整个基因组中， 整个TRIO基因在SCZ和ASD患者中显著富集，表明TRIO减少 功能导致病理。事实上，我们的实验室已经表明，在小鼠皮层中缺失单个TRIO等位基因， 兴奋性神经元（在NEX-TRIO+/-小鼠中）产生不同的行为缺陷和树突状细胞的严重缺陷。 结构和突触活动。TRIO作用于细胞表面受体下游以控制轴突和树突 寻路和突触发育然而，TRIO变异体损害了信号传导机制， 这些过程中的缺陷如何破坏神经元发育仍然完全不清楚。我们的实验室发现 PDE 4A 5作为新的候选TRIO信号传导伴侣，因为其在TRIO缺陷小鼠皮层中的水平降低 以及其与TRIO共免疫沉淀的能力。我的提议将检验TRIO与 在两个互补的目的中，PDE 4A 5在功能上调节神经元发育和功能。 我的第一个目标是确定TRIO和PDE 4A 5在功能上如何相互作用，以及这些功能是如何相互作用的。 受疾病相关变异的影响。PDE 4A 5与TRIO共免疫沉淀，但这些蛋白质如何 相互作用以及这些相互作用如何影响每个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试验，并确定PDE 4A 5和TRIO是否相互作用 直接和确定最小的TRIO和PDE 4A 5片段足以介导相互作用。我来设计 TRIO：PDE 4A 5相互作用界面内的疾病相关变体，以确定是否以及如何 这些变体影响Trio：PDE 4A 5相互作用。我会用我纯化的蛋白质和一个体外的 基于细胞的测定来测量TRIO和PDE 4A 5之间的相互作用如何影响它们的催化活性。 我的第二个目标是确定TRIO：PDE 4A 5相互作用如何调节神经元发育。 在培养的皮层神经元中破坏单个TRIO等位基因导致树突和树突棘缺陷 与体内观察到的相似。TRIO：PDE 4A 5相互作用的破坏如何有助于 在TRIO缺陷神经元中观察到的病理生理学尚不清楚。我将使用shRNA介导的PDE 4A 5敲低 以确定PDE 4A 5是否是树突和树突棘发育所必需的。我必坚定 敲减/互补和共聚焦显微镜，以确定是否和 TRIO：PDE 4A 5相互作用的中断如何影响PDE 4A 45定位和神经元发育。我会用 小分子PDE 4A 5激活剂，以测试PDE 4A 5的药理学操作是否可以挽救缺陷 在培养物中以及在NEX-TRIO+/-和NEX-TRIO-/-小鼠中TRIO+/-和TRIO-/-皮质神经元的发育中。