SynDIG1/Prrt1 regulation of extrasynaptic GluA1-containing AMPARs during plasticity

突触AMPA受体（AMPAR）含量的活动依赖性变化，称为“突触可塑性”， 一种信息存储在神经网络中的机制，可以产生更高级的认知技能 比如学习和记忆。在长时程增强（LTP）过程中，一种被广泛研究的突触可塑性形式， 突触外AMPAR从附近的储备池（包括细胞上的突触周围区域）募集 表面和细胞内区室，并随后与突触后密度（PSD）锚定。大 经过几十年的研究，大量证据已经建立了AMPAR的机制， 锚定在PSD内。相反，控制AMPAR突触靶向的分子机制， 建立突触外受体储备库在很大程度上是未知的。鉴于准备金池的征聘 突触外AMPAR的表达是LTP过程中突触快速增强的基础， 建立这种储备池的机制对我们理解突触可塑性至关重要， 代表了我们知识上的一个重大缺口。 SynDIG（Synapse Differentiation Induced Gene，突触分化诱导基因）定义了一个由四个基因（SynDIG 1 -4）组成的家族，其编码脑- 特异性跨膜蛋白。在这里，我们将确定SynDIG 4（SD 4）的功能，也称为Prrt 1 （富含脯氨酸的跨膜蛋白1）在AMPAR储备库的调节中的作用。蛋白质组学研究 表明SD 4是AMPAR复合物的组分;然而，SD 4并不富集在PSD中，而是 在非突触位点与含GluA 1的AMPAR共定位。值得注意的是，破伤风诱导的LTP， 依赖于GluA 1，在SD 4敲除（KO）的急性海马切片中被消除，而θ-爆发 刺激LTP（TBS-LTP），这是独立的GluA 1，不受损害。此外，SD 4 KO小鼠表现出 在两个独立的认知试验（莫里斯水迷宫，新物体识别）中存在严重缺陷， 证明了SD 4在依赖于大脑的学习和记忆中的关键作用。此外，突触外 与野生型（WT）神经元相比，SD 4 KO中的AMPAR减少。鉴于储备池 突触外AMPAR对突触可塑性至关重要，我们假设SD 4维持这样的储备池 突触外含有GluA 1的AMPAR在破伤风诱导的LTP期间部署。 在这个协作的双PI应用程序中，我们提出了一种全面的多学科方法来调查 突触外含GluA 1的AMPAR的SD 4依赖性调节与分子、细胞和 电生理学方法。在目标1中，我们将定义SD 4维持突触外突触的机制。 含GluA 1的AMPAR。在目标2中，我们将确定是否突触靶向储备库的GluA 1- 在可塑性过程中含有AMPAR需要SD 4。在目标3中，我们将测试是否同源GluA 1依赖 突触可塑性机制需要SD 4在多个年龄离体。这些研究的结果将提供 从分子上深入了解控制保护区建立和维持的基本机制 突触外AMPAR池对突触可塑性至关重要，并解决了我们知识中的一个主要空白。

Activity-dependent variation in synaptic AMPA receptor (AMPAR) content, referred to as ‘synaptic plasticity’, is a mechanism whereby information is stored in neural networks that give rise to higher order cognitive skills such as learning and memory. During long-term potentiation (LTP), a widely studied form of synaptic plasticity, extrasynaptic AMPARs are recruited from nearby reserve pools, including perisynaptic regions on the cell surface and intracellular compartments, and subsequent anchored with the postsynaptic density (PSD). A large body of evidence spanning decades of investigation has established mechanisms by which AMPARs are anchored within the PSD. In contrast, the molecular mechanisms that govern AMPAR synaptic targeting to establish reserve pools of extrasynaptic receptors are largely unknown. Given that recruitment of reserve pools of extrasynaptic AMPARs underlies the rapid strengthening of synapses that occurs during LTP, the molecular mechanisms that establish such reserve pools are critical to our understanding of synaptic plasticity and represent a major gap in our knowledge. SynDIG (Synapse Differentiation Induced Gene) defines a family of four genes (SynDIG1-4) that encode brain- specific transmembrane proteins. Here we will determine the function of SynDIG4 (SD4), also known as Prrt1 (Proline-rich transmembrane protein 1) in the regulation of the reserve pool of AMPARs. Proteomic studies indicate that SD4 is a component of AMPAR complexes; however, SD4 is not enriched in the PSD, but instead colocalizes with GluA1-containing AMPARs at non-synaptic sites. Remarkably, tetanus-induced LTP, which is dependent on GluA1, is abolished in acute hippocampal slices from SD4 knockout (KO) while theta-burst stimulation LTP (TBS-LTP), which is independent of GluA1, is not impaired. Furthermore, SD4 KO mice exhibit profound deficits in two independent cognitive assays (Morris water maze, novel object recognition), demonstrating a critical role for SD4 in hippocampal-dependent learning and memory. Moreover, extrasynaptic AMPARs are reduced in SD4 KO compared with wild-type (WT) neurons. Given that reserve pools of extrasynaptic AMPARs are critical for synaptic plasticity, we hypothesize that SD4 maintains such reserve pools of extrasynaptic GluA1-containing AMPARs that are deployed during tetanus-induced LTP. In this collaborative dual-PI application we propose a comprehensive multidisciplinary approach to investigate SD4-dependent regulation of extrasynaptic GluA1-containing AMPARs with molecular, cellular, and electrophysiological methods. In Aim 1 we will define the mechanism by which SD4 maintains extrasynaptic GluA1-containing AMPARs. In Aim 2 we will determine whether synaptic targeting of reserve pools of GluA1- containing AMPARs during plasticity requires SD4. In Aim 3 we will test whether homomeric GluA1-dependent synapse plasticity mechanisms require SD4 ex vivo at multiple ages. The results of these studies will provide molecular insight into fundamental mechanisms that govern establishment and maintenance of the reserve pools of extrasynaptic AMPARs critical for synaptic plasticity and address a major gap in our knowledge.