Regulation of activity-dependent ProSAp2 synaptic dynamics

描述（由申请人提供）：ProSAP2是一种多结构域支架蛋白，位于兴奋性突触的突触后密度（PSD）。据推测，它在功能上偶联了膜结合谷氨酸受体、肌动蛋白细胞骨架和第二信使信号通路[1]。ProSAP2的交换是活性依赖的，因此它在没有活性的情况下是稳定的，在电[2]或化学刺激的情况下是动态的，从而表明ProSAP2的动态特性与其功能是耦合的。这一提议验证了活性将ProSAP2从非活性寡聚形式转化为动态激活状态的假设，该激活状态响应nmda介导的可塑性。活细胞延时共聚焦显微镜和光漂白后荧光恢复（FRAP）将用于评估ProSAP2在药理学操作下的动态交换特性。实验将在专门的温度控制成像室中使用分离的海马培养物感染EGFP-ProSAP2慢病毒结构进行。本项目旨在明确调控ProSAP2动态的突触活动特征，进而评估SAM结构域寡聚化是否调控ProSAP家族成员的交换特性。综上所述，本实验将测试ProSAP2的动态状态是否与突触可塑性相耦合。相关性：自闭症是一种普遍存在的发育障碍，其特征是缺乏社会交往和沟通。尽管对双胞胎的研究表明了强烈的遗传成分，但图谱研究未能确定这种复杂社会障碍背后的单个基因，而是暗示了涉及细胞粘附和细胞骨架结构的几种相互作用的突触蛋白。该项目探索调节ProSAP2[3]动态的突触机制，ProSAP2[3]是一种与自闭症谱系障碍有关的特别有趣的蛋白质。

DESCRIPTION (provided by applicant): ProSAP2 is a multidomain scaffold protein located in the post-synaptic density (PSD) of excitatory synapses. It is hypothesized to functionally couple membrane-bound glutamate receptors, the actin cytoskeleton and second messenger signaling pathways [1]. ProSAP2 exchange is activity-dependent, such that it is stable in the absence of activity and dynamic in the presence of electrical [2] or chemical [see preliminary data] stimulation, thereby suggesting that the dynamic properties of ProSAP2 are coupled to its function. This proposal tests the hypothesis that activity converts ProSAP2 from an inactive oligomerized form to a dynamic activated state that is responsive to NMDAR-mediated plasticity. Live-cell time-lapse confocal microscopy and Fluorescence Recovery After Photobleaching (FRAP) will be used here to assess the dynamic exchange properties of ProSAP2 in response to pharmacological manipulations. Experiments will be performed using dissociated hippocampal cultures infected with a EGFP-ProSAP2 lentiviral constuct in a specialized temperature controlled imaging chamber. This project specifically aims to define the features of synaptic activity that regulate ProSAP2 dynamics, and then to assess whether SAM domain oligomerization regulates the exchange properties of ProSAP family members. Taken together, the experiments described here will test whether the dynamic state of ProSAP2 is coupled to synaptic plasticity. Relevance: Autism is a prevalent developmental disorder, characterized by deficits in social interaction and communication. Though twin studies suggest a strong genetic component, mapping studies have failed to identify a single gene underling this complex social disorder, rather they have implicated several interacting synaptic proteins that are involved in cell adhesion and cytoskeletal architecture. This project explores the synaptic mechanisms regulating the dynamics of ProSAP2 [3], a particulariy interesting protein implicated in Autism Spectrum Disorders.