Membrane Trafficking of the Vesicular Glutamate Transporter, VGLUT1

描述（由申请人提供）：神经传递的一个重要步骤是从突触囊泡中调节神经递质的释放。为了维持对重复刺激的信号反应，突触囊泡必须被回收和再利用。这个过程需要在融合之后，细胞膜和组成囊泡的蛋白质从质膜成分中分离出来，并通过内吞作用回收。已经提出了在不同活动水平下动员的几种内吞途径，尽管通过不同途径回收的后果目前尚不清楚。这些通路以不同的速率运作，这可能会影响突触对持续刺激的反应能力。此外，内吞作用的模式可能通过靶向囊泡来调节未来囊泡释放的倾向。不同的功能池。另外，由于不同的接头蛋白与不同的内吞途径相关，所利用的途径可能调节蛋白质组成，从而调节所产生的囊泡的功能特性。这一观点与许多突触囊泡蛋白含有可能允许独立分类的内化基序的发现是一致的。一种含有这种基序的突触囊泡蛋白是囊泡谷氨酸转运蛋白VGLUT1。VGLUT1还含有两个c端脯氨酸结构域，可以调节转运体使用的内吞作用模式。研究这些结构域可以对这些不同的内吞途径回收的分子机制和功能后果产生重要的见解。由于这些结构域不存在于任何其他泡状神经递质转运体中，VGLUT1的这种额外调节可能解释了VGLUT1突触独特的功能特性，如更高的突触可塑性能力和对突触抑制的抵抗力。因此，我将研究第一聚脯氨酸结构域（PP1）在VGLUT1内吞作用（Aim 1）中的作用，确定VGLUT1是否通过依赖于APS接头蛋白的途径进行循环（Aim 2），并检查Nck1/2支架蛋白是否介导PP1的作用（Aim 3）。我将通过结合分子生物学、生物化学、电子显微镜和活细胞神经元成像来解决这些问题。公共卫生相关性：谷氨酸在神经末梢的释放需要谷氨酸通过VGLUT1-3转运到突触囊泡中。谷氨酸信号与精神分裂症有关，以谷氨酸释放为目标的药物最近成为一种潜在的新型抗精神病药物。此外，由于谷氨酸信号在中枢神经系统中的关键作用，它还涉及其他疾病，如焦虑症、癫痫和神经元变性。

DESCRIPTION (provided by applicant): An essential step in neurotransmission is the regulated release of neurotransmitter from synaptic vesicles. To maintain signaling in response to repeated stimulation, synaptic vesicles must be recycled and reused. This process requires that, following fusion, the membrane and proteins composing a vesicle be sorted from plasma membrane components and retrieved by endocytosis. Several endocytic pathways that are mobilized under different levels of activity have been proposed, although the consequences of recycling via different pathways are presently unknown. These pathways operate at different rates, which may affect the ability of the synapse to respond to continued stimulation. In addition, it is possible that the mode of endocytosis may regulate the propensity for future release of the resulting vesicles by targeting vesicles to. different functional pools. Alternatively, since different adaptor proteins have been associated with different endocytic routes, the pathway utilized may regulate the protein composition, and thus the functional properties, of the resulting vesicles. This idea is consistent with the finding that many synaptic vesicle proteins contain internalization motifs that may allow independent sorting. One synaptic vesicle protein that contains such a motif is the vesicular glutamate transporter VGLUT1. VGLUT1 also contains two Cterminal polyproline domains that may regulate the mode of endocytosis used by the transporter. Studying these domains may yield crucial insight into the molecular mechanisms and functional consequences of recycling by these different endocytic pathways. Since these domains are not present in any other vesicular neurotransmitter transporters, this additional regulation of VGLUT1 may explain the unique functional properties of VGLUT1 synapses such as a higher capacity for synaptic plasticity and an increased resistance to synaptic depression. I will therefore study the role of the first polyproline domain (PP1) in VGLUT1 endocytosis (Aim 1), determine whether VGLUT1 recycles by a pathway dependent on the APS adaptor protein (Aim 2), and examine whether the Nck1/2 scaffolding protein mediates the role of PP1 (Aim 3). I will address these questions through a combination of molecular biology, biochemistry, electron microscopy, and live cell neuronal imaging. PUBLIC HEALTH RELEVANCE: Release of glutamate at the nerve terminal requires transport of glutamate into synaptic vesicles by VGLUT1-3. Glutamate signaling has been implicated in schizophrenia, and drugs targeting glutamate release have recently emerged as a potential new class of antipsychotic. In addition, due to the key role of glutamate signaling in the central nervous system, it has also been implicated in other conditions such as anxiety disorders, epilepsy, and neuronal degeneration.