Understanding the Role of Epac2 in Cognitive Function

描述（由申请人提供）： 树突棘是树突上的微小突起，是哺乳动物大脑中大多数兴奋性突触的部位。脊柱可以迅速形成，消除，或改变大小/形状，以响应刺激-这种脊柱可塑性有助于许多生理过程，如突触传递和可塑性。相反，异常的脊柱结构、动力学和功能是许多神经精神疾病（包括自闭症谱系障碍（ASD））发病机制的重要贡献者。ASD是一组神经发育障碍，其特征在于社交缺陷，沟通困难和重复行为。作为 作为理解ASD遗传病因学的努力的一部分，四项独立的ASD患者基因组研究已经将染色体区域2 q31 -32定位为与该疾病相关的基因的热点。在该染色体区域中筛选候选ASD基因揭示了EPAC 2基因中的排他性单氨基酸变体，该变体仅与自闭症家庭成员分离，而不与未受影响的对照分离。Epac 2是一种鸟嘌呤核苷酸交换因子，可调节小GTap Rap，这是一种已知的脊柱结构和功能调节因子。我们的实验室已经表明，几种鉴定的Epac 2自闭症相关变体（Epac 2-AAV）在体外引起改变的Rap活性以及异常的树突棘形态。此外，我们发现EPAC 2缺陷小鼠体内的脊柱动力学发生了改变。总的来说，这些数据表明，通过功能失调的Epac 2活性，异常的Rap信号传导可能导致体内异常的脊柱形态发生。然而，由于对Epac 2在突触中的作用知之甚少，因此了解Epac 2在信号传导和调节下游脊柱可塑性中的机制是至关重要的。在这项提案中，我们的目标是获得Epac 2信号复合物的机制理解，以及Epac 2-AAV如何影响复合物的结构和功能。我们实验室的初步数据显示Epac 2与两个突触后支架蛋白Shank 3和PSD 95形成复合物。因此，我们将进行体外结合试验，以确定这三种蛋白质之间的相互作用的性质。然后，我们将通过比较在PSD-95/Shank 3存在下Epac 2/Epac 2-AAV对下游Rap活性的影响以及通过表征在PSD-95/Shank 3存在下Epac 2/Epac 2-AAV的定位模式来确定Shank 3/PSD-95如何影响Epac 2/Epac 2-AAV的功能。我们还打算通过确定Epac 2如何在体内的整个出生后发育过程中调节脊柱动力学来更深入地了解Epac 2与脊柱可塑性的下游生理相关性。我们将使用活体双光子显微镜对不同年龄的EPAC 2缺陷小鼠的树突棘进行成像，并将进行药理学拯救实验以逆转突触改变。总之，我们的工作可以解释Epac 2功能障碍如何有助于ASD复杂的神经生物学。

DESCRIPTION (provided by applicant): Dendritic spines are tiny protrusions on dendrites and are the sites of most excitatory synapses in the mammalian brain. Spines can be rapidly formed, eliminated, or change size/shape in response to stimuli~ this spine plasticity contributes to numerous physiological processes such as synaptic transmission and plasticity. Conversely, abnormal spine structure, dynamics, and function are important contributors to the pathogenesis of numerous neuropsychiatric disorders, including Autism Spectrum Disorders (ASD). ASD are a group of neurodevelopmental disorders that are characterized by social deficits, communication difficulties, and repetitive behaviors. As part of an effort to understand the genetic etiologies of ASD, four independent genome studies of ASD patients have localized the chromosome region 2q31-32 as a hotspot for genes relevant to the disease. A screen for candidate ASD genes in this chromosomal region revealed exclusive single amino-acid variants in the EPAC2 gene that segregate exclusively with autistic family members but not unaffected controls. Epac2 is a guanine nucleotide exchange factor that modulates the small GTPase Rap, a known regulator of spine structure and function. Our lab has shown that several of the identified Epac2 autism- associated variants (Epac2-AAVs) cause altered Rap activity as well as aberrant dendritic spine morphologies in vitro. In addition, we found that EPAC2-deficient mice have altered spine dynamics in vivo. Collectively, this data suggests that aberrant Rap signaling, via dysfunctional Epac2 activity, may lead to abnormal spine morphogenesis in vivo. However, as little is known about the role of Epac2 at synapses, understanding Epac2's mechanism in signaling and in regulating downstream spine plasticity is essential. In this proposal, we aim to gain a mechanistic understanding of the Epac2 signaling complex and how Epac2-AAVs may affect the complex's structure and function. Preliminary data from our lab show that Epac2 is in a complex with two post- synaptic scaffolding proteins, Shank3 and PSD95. We will thus perform in vitro binding assays to determine the nature of the interactions between these three proteins. We will then determine how Shank3/PSD-95 affects Epac2/Epac2-AAVs function by comparing the effects of Epac2/Epac2-AAVs, in the presence of PSD- 95/Shank3, on downstream Rap activity and by characterizing Epac2/Epac2-AAVs localization patterns in the presence of PSD-95/Shank3. We also intend to gain a deeper insight into Epac2's downstream physiological relevance to spine plasticity by determining how Epac2 modulates spine dynamics throughout postnatal development in vivo. We will use intravital two-photon microscopy to image dendritic spines in EPAC2-deficient mice of various ages and will perform pharmacological rescue experiments to reverse synaptic alterations. In conclusion, our work may explain how Epac2 dysfunction contributes to the complicated neurobiology of ASD.