AMPA receptor trafficking at cerebellar parallel fiber-stellate cell synapses

描述(申请人提供)：AMPA受体的活性依赖的动员和突触靶向是突触可塑性表达的关键步骤，突触可塑性被认为是大脑中学习和记忆的细胞关联。小脑皮质的平行纤维星状细胞(PF-SC)突触是一种运动控制和协调所必需的结构，表现出一种独特的突触可塑性，其特征是钙渗透的AMPAR被不透钙的AMPAR活性所取代。这两个受体池可以通过简单的生物物理测试相互区分，使其成为探索突触可塑性中亚单位特异性AMPAR靶向的分子基础的理想模型系统。进一步了解钙通透性AMPAR转运的分子机制与人类疾病直接相关。钙通透性AMPAR的调节与多种疾病机制有关，包括中风时的缺血性损伤以及癫痫、肌萎缩侧索硬化症(ALS)和神经病理性疼痛引起的兴奋性毒性损伤。在这个提案中，我将验证我的假设，即在PF-SC突触可塑性过程中，AMPAR辅助亚单位Stargazin是AMPAR亚单位特异性运输所必需的。该提案分为两个具体目标。在特定的目标1中，1将对观星突变小鼠进行电生理记录，以便系统地描述Stargazin在调节AMPAR表面表达和突触中的作用 小脑PF-SC突触的靶向性和可塑性。在具体目标2中，1将研究在PF-SC突触上亚单位特异性AMPAR运输的分子基础。为此，我将在观星小鼠体内进行病毒介导的基因转移，作为一种将完整和缺失突变版本的Stargazin重新引入星状细胞的方式。这样的实验将使我能够集中精力研究在可塑性过程中，观星在调节AMPAR亚单位组成变化中所起的确切作用。这将是第一次证明在突触可塑性过程中，Stargazin介导的靶向性亚基的特异性。与公共健康相关：突触可塑性的一个基本特征，即大脑中神经元存储信息的机制，是对突触供应AMPA受体的精细控制。这一建议旨在了解负责突触AMPA受体调节的分子机制。通过这样做，我希望对大脑的病理状态提供重要的见解，包括中风、癫痫、Lou Gehrig病和慢性疼痛。

DESCRIPTION (provided by applicant): The activity-dependent mobilization and synaptic targeting of AMPA receptors is an essential step in the expression of synaptic plasticity, regarded as the cellular correlate of learning and memory in the brain. The parallel-fiber-stellate cell (PF-SC) synapse of the cerebellar cortex, a structure essential for motor-control and coordination, exhibits a unique form of synaptic plasticity characterized by the activity-dependent replacement of calcium-permeable AMPARs by calcium-impermeable AMPARs. These two pools of receptors can be distinguished from one another by a simple biophysical assay making it an ideal model system for probing the molecular basis for subunit-specific AMPAR targeting during synaptic plasticity. Furthering our understanding of the molecular mechanisms underlying the trafficking of calciumpermeable AMPARs is immediately relevant to human disease. The regulation of calcium-permeable AMPARs is implicated in a wide range of disease mechanisms including ischemic damage during stroke and excitotoxic damage resulting from epilepsy, amyotrophic lateral sclerosis (ALS) and neuropathic pain. In this proposal, I will test my hypothesis that stargazin, an AMPAR auxiliary subunit is required for subunitspecific trafficking of AMPARs during plasticity at PF-SC synapses. The proposal is divided into two Specific Aims. In Specific Aim 1, 1 will carry out electrophysiological recordings from stargazer mutant mice in order to systematically delineate the role of stargazin in regulating AMPAR surface expression, synaptic targeting and plasticity at cerebellar PF-SC synapses. In Specific Aim 2, 1 will examine the molecular basis for subunit-specific AMPAR trafficking at PF-SC synapses. To this end, I will carry out viral-mediated in vivo gene transfer in stargazer mice as a way of reintroducing both a full-length and a deletion mutant version of stargazin into stellate cells. Such experiments will allow me to zero in on the precise role that stargazing plays in mediating changes in AMPAR subunit composition during plasticity. This would be the first demonstration of subunit specificity in stargazin-mediated targeting during synaptic plasticity. PUBLIC HEALTH RELEVANCE: An essential feature of synaptic plasticity, the mechanism through which neurons in the brain store information, is the exquisite control of a synapse's supply of AMPA receptors. This proposal is directed towards understanding the molecular machinery responsible for AMPA receptor regulation at synapses. In so doing, I hope to provide important insights into pathological states of the brain including stroke, epilepsy, Lou Gehrig's Disease and chronic pain.