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

突触AMPA受体(AMPAR)含量的活性依赖性变化，称为突触可塑性，是 一种将信息存储在神经网络中的机制，可产生更高层次的认知技能 比如学习和记忆。长时程增强(LTP)是突触可塑性的一种被广泛研究的形式， 突触外AMPAR是从附近的储备池招募的，包括细胞上的突触周围区域 表面室和胞内室，并随后与突触后密度(PSD)锚定。大号 经过数十年的调查，大量证据已经建立了AMPAR被 固定在PSD内部。相反，控制AMPAR突触靶向的分子机制 建立突触外受体的储备库在很大程度上是未知的。鉴于后备库的招募 突触外AMPAR的变化是突触迅速加强的基础，这种突触发生在LTP，分子 建立这种储备池的机制对于我们理解突触可塑性和 代表着我们知识上的一个重大缺口。 SynDIG(突触分化诱导基因)定义了一个由四个基因组成的家族(SynDIG1-4)，这些基因编码大脑- 特定的跨膜蛋白。这里我们将确定SynDIG4(SD4)的功能，也称为Prrt1 (富含脯氨酸的跨膜蛋白1)在AMPAR储备库的调节中。蛋白质组学研究 表明SD4是AMPAR复合体的一个组成部分；然而，SD4不是富含在PSD中，而是在PSD中 与含有GluA1的AMPAR在非突触位置共定位。值得注意的是，破伤风引起的LTP 依赖于GluA1，在SD4基因敲除(KO)的急性海马片中被取消，而theta-Burst 不依赖GluA1的刺激性LTP(TBS-LTP)不受损害。此外，SD4 KO小鼠还表现出 两个独立认知测验(莫里斯水迷宫、新奇物体识别)的严重缺陷， 展示了SD4在海马区依赖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，SD4KO中AMPAR的表达减少。考虑到这些储备库 突触外AMPAR对突触的可塑性至关重要，我们假设SD4维持这样的储备池 在破伤风诱导的LTP期间部署的突触外含有GluA1的AMPAR。 在这种协作的双PI应用中，我们提出了一种综合的多学科方法来研究 SD4依赖的突触外含GluA1的AMPAR与分子、细胞和 电生理学方法。在目标1中，我们将定义SD4维持突触外的机制 含有GluA1的AMPAR。在目标2中，我们将确定突触靶向GluA1储备池- 在可塑性过程中包含AMPAR需要SD4。在目标3中，我们将测试同构体GluA1依赖于 突触的可塑性机制需要在多个年龄段的体外SD4。这些研究的结果将提供 对建立和维持保护区的基本机制的分子洞察 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缺口。

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.