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位置。脊柱可以迅速形成、消除或改变大小/形状，以响应刺激~这种脊柱的可塑性有助于许多生理过程，如突触传递和可塑性。相反，脊柱结构、动力学和功能的异常是许多神经精神疾病的重要因素，包括自闭症谱系障碍(ASD)。ASD是一组神经发育障碍，以社交缺陷、沟通困难和重复行为为特征。AS 作为了解ASD遗传病因的努力的一部分，四项独立的ASD患者基因组研究将染色体区域2q31-32定位为与疾病相关的基因的热点。对这一染色体区域的候选ASD基因的筛查显示，Epac2基因中唯一的单一氨基酸变体仅与自闭症家庭成员分离，但不与未受影响的对照组分离。Epac2是一种鸟嘌呤核苷酸交换因子，它调节小GTP酶Rap，这是一种已知的脊椎结构和功能调节因子。我们的实验室已经证明，几个已识别的Epac2自闭症相关变体(Epac2-AAVs)在体外会导致Rap活性改变和树突棘形态异常。此外，我们发现Epac2基因缺陷的小鼠在体内改变了脊柱动力学。总而言之，这些数据表明，异常的Rap信号，通过功能失调的Epac2活性，可能导致体内脊柱形态发生的异常。然而，由于对Epac2在突触中的作用知之甚少，了解Epac2的S机制在信号转导和调节脊髓下游可塑性中是至关重要的。在这个提案中，我们的目标是从机制上了解Epac2信号复合体以及Epac2-AAVs如何影响复合体的结构和功能。我们实验室的初步数据显示，Epac2与两个突触后支架蛋白Shank3和PSD95形成了一个复合体。因此，我们将进行体外结合分析，以确定这三种蛋白质之间相互作用的性质。然后，我们将通过比较Epac2/Epac2-AAVs在PSD-95/Shank3存在时对下游Rap活性的影响，以及在PSD-95/Shank3存在时Epac2/Epac2-AAVs的定位模式，来确定Shank3/PSD-95对Epac2/Epac2-AAVs功能的影响。我们还打算通过确定Epac2如何在体内调节出生后发育过程中的脊柱动力学，来更深入地了解Epac2的S下游与脊柱可塑性的生理相关性。我们将使用活体双光子显微镜对不同年龄的Epac2基因缺陷小鼠的树突棘进行成像，并将进行药物救援实验以逆转突触变化。总之，我们的工作可能解释了Epac2功能障碍如何导致ASD复杂的神经生物学。 公共卫生相关性： 控制树突结构、动力学和功能的机制--这在大脑发育和功能中很重要--还没有被很好地理解。我们的研究将有助于理解这些过程背后的原因，并可能使我们开始理解几种神经精神疾病的发病机制，如自闭症谱系障碍。

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. PUBLIC HEALTH RELEVANCE: The mechanisms that control dendritic structure, dynamics, and function - which are important in brain development and function - are not well understood. Our studies will contribute to the understanding behind these processes, and perhaps allow us to begin to comprehend the pathogenesis of several neuropsychiatric diseases, such as Autism Spectrum Disorders.