Regulation of activity-dependent ProSAp2 synaptic dynamics

描述(申请人提供)：ProSAP2是一种位于兴奋性突触突触后密度(PSD)中的多结构域支架蛋白。它被认为是膜结合的谷氨酸受体、肌动蛋白细胞骨架和第二信使信号通路的功能性偶联[1]。ProSAP2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[见初步数据]刺激存在时是动态的，从而表明ProSAP2的动态特性与其功能是偶联的。这一建议检验了这样的假设，即活性将ProSAP2从不活跃的寡聚形式转化为对NMDAR介导的可塑性做出反应的动态激活状态。活细胞延时共聚焦显微镜和光漂白后荧光恢复(FRAP)将被用来评估ProSAP2在药物操纵下的动态交换特性。实验将在一个专门的温控成像室中使用感染了EGFP-ProSAP2慢病毒结构的分离的海马区培养物进行。本项目旨在明确调节ProSAP2动态的突触活动的特征，然后评估SAM结构域齐聚是否调节ProSAP家族成员的交换特性。综上所述，这里描述的实验将测试ProSAP2的动态是否与突触可塑性有关。相关性：自闭症是一种普遍存在的发育障碍，其特征是社交和交流能力不足。尽管双胞胎研究表明这是一个强大的遗传成分，但图谱研究未能确定这种复杂的社会障碍背后的单个基因，相反，它们涉及几个相互作用的突触蛋白，这些蛋白参与细胞黏附和细胞骨架结构。这个项目探索了调节ProSAP2[3]动态的突触机制，ProSAP2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.