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”，是 一种将信息存储在神经网络中的机制，从而产生更高阶的认知技能 比如学习和记忆。在长时程增强（LTP）过程中，一种广泛研究的突触可塑性形式， 突触外 AMPAR 是从附近的储备库中招募的，包括细胞上的突触周围区域 表面和细胞内区室，然后用突触后密度（PSD）锚定。一个大 经过数十年调查的大量证据已经建立了 AMPAR 的机制 锚定在 PSD 内。相比之下，控制 AMPAR 突触靶向的分子机制 建立突触外受体储备池在很大程度上是未知的。鉴于储备池的招募 突触外 AMPAR 的增加是 LTP 期间发生的突触快速强化的基础，LTP 是分子 建立这种储备池的机制对于我们理解突触可塑性和 代表了我们知识上的一个重大差距。 SynDIG（突触分化诱导基因）定义了四个基因家族（SynDIG1-4），它们编码大脑 特异性跨膜蛋白。这里我们来确定SynDIG4（SD4）的功能，也称为Prrt1 （富含脯氨酸的跨膜蛋白 1）在 AMPAR 储备池的调节中。蛋白质组学研究 表明 SD4 是 AMPAR 复合物的组成部分；然而，SD4并没有在PSD上进行丰富，而是 与包含 GluA1 的 AMPAR 在非突触位点共定位。值得注意的是，破伤风诱发的 LTP 依赖于 GluA1，在 SD4 敲除 (KO) 的急性海马切片中被废除，同时 theta 爆发 刺激 LTP (TBS-LTP) 独立于 GluA1，不会受到损害。此外，SD4 KO 小鼠表现出 两项独立的认知测试（莫里斯水迷宫、新物体识别）存在严重缺陷， 证明 SD4 在海马依赖性学习和记忆中发挥关键作用。此外，突触外 与野生型 (WT) 神经元相比，SD4 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 SD4 维持着这样的储备池 突触外含有 GluA1 的 AMPAR 在破伤风诱导的 LTP 期间部署。 在这个协作双 PI 应用中，我们提出了一种全面的多学科方法来研究 SD4 依赖性突触外含 GluA1 AMPAR 的分子、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 GluA1- 储备池的突触目标是否为 在塑性过程中含有 AMPAR 需要 SD4。在目标 3 中，我们将测试同聚 GluA1 是否依赖 突触可塑性机制在多个年龄段都需要离体 SD4。这些研究的结果将提供 对管理储备建立和维护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重大空白。

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.