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神经递质从突触囊泡中的调节释放。 为了维持信号对重复刺激的响应，突触囊泡必须被回收和重复使用。 这一过程要求，融合后，膜和蛋白质组成的囊泡从质膜成分和回收的内吞作用。已经提出了几种在不同活性水平下动员的内吞途径，尽管目前尚不清楚通过不同途径再循环的后果。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所得囊泡未来释放的倾向。 不同的功能池。或者，由于不同的衔接蛋白与不同的内吞途径相关，所利用的途径可以调节蛋白质组成，从而调节所得囊泡的功能特性。这一想法与许多突触囊泡蛋白含有允许独立分选的内化基序的发现一致。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VGLUT 1。VGLUT 1还含有两个C末端聚脯氨酸结构域，其可调节转运蛋白所使用的内吞模式。研究这些结构域可能会产生至关重要的深入了解这些不同的内吞途径的分子机制和回收功能的后果。由于这些结构域不存在于任何其他囊泡神经递质转运蛋白中，VGLUT 1的这种额外调节可以解释VGLUT 1突触的独特功能特性，例如更高的突触可塑性和对突触抑制的抵抗力。因此，我将研究第一个多聚脯氨酸结构域（PP 1）在VGLUT 1内吞作用中的作用（Aim 1），确定VGLUT 1是否通过依赖于APS衔接蛋白的途径进行转录（Aim 2），并检查Nck 1/2支架蛋白是否介导PP 1的作用（Aim 3）。我将结合分子生物学、生物化学、电子显微镜和活细胞神经成像来解决这些问题。 公共卫生相关性：神经末梢释放谷氨酸需要VGLUT 1 -3将谷氨酸转运到突触囊泡中。谷氨酸信号转导与精神分裂症有关，靶向谷氨酸释放的药物最近已成为一类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，它也与其他疾病如焦虑症、癫痫和神经元变性有关。

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.