AMPA receptor trafficking at cerebellar parallel fiber-stellate cell synapses

描述（由申请人提供）：AMPA受体的活性依赖性动员和突触靶向是突触可塑性表达的重要步骤，突触可塑性被认为是大脑中学习和记忆的细胞相关物。小脑皮层的平行纤维星状细胞（PF-SC）突触是运动控制和协调所必需的结构，具有独特的突触可塑性形式，其特征在于钙渗透性AMPAR被钙不渗透性AMPAR的活动依赖性替代。这两个受体池可以通过简单的生物物理测定彼此区分，使其成为用于探测突触可塑性期间亚基特异性AMPAR靶向的分子基础的理想模型系统。进一步了解钙渗透性AMPAR的分子机制与人类疾病密切相关。钙渗透性AMPAR的调节涉及广泛的疾病机制，包括中风期间的缺血性损伤和由癫痫、肌萎缩性侧索硬化（ALS）和神经性疼痛引起的兴奋性毒性损伤。在这个建议中，我将测试我的假设，stargazin，一个AMPAR辅助亚基是必需的亚基特异性贩运的AMPAR在PF-SC突触的可塑性。该提案分为两个具体目标。在具体目标1中，1将进行stargazer突变小鼠的电生理记录，以系统地描述stargazin在调节AMPAR表面表达，突触后神经元和突触后神经元中的作用。 小脑PF-SC突触的靶向和可塑性。在具体目标2中，1将研究PF-SC突触上亚单位特异性AMPAR运输的分子基础。为此，我将在stargazer小鼠中进行病毒介导的体内基因转移，作为将stargazin的全长和缺失突变版本重新引入星状细胞的一种方式。这些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亚基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介导的靶向亚基特异性。公共卫生相关性：突触可塑性是大脑中神经元储存信息的机制，它的一个基本特征是对突触AMPA受体供应的精确控制。该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，我希望能为大脑的病理状态提供重要的见解，包括中风，癫痫，卢伽雷病和慢性疼痛。

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.