Vesicular modulation of dopamine neuron toxicity

描述（由申请人提供）：帕金森病（PD）的一个主要特征是黑质致密部产生多巴胺的神经元的缺失，以及纹状体中多巴胺的缺失。虽然多种过程可能导致多巴胺神经元的丧失，但我们认为，从囊泡中破坏多巴胺的适当储存和释放起着关键作用。这一理论得到了文献的支持，这些文献表明，多巴胺的不当储存会导致氧化多巴胺副产物和有毒醛的形成，从而损害神经元。PD的遗传和毒理学研究表明，许多损伤集中在多巴胺囊泡上发挥其有害作用。我们假设囊泡功能介导多巴胺神经元毒性，因此囊泡功能的增加将增强多巴胺的储存和释放，并赋予对神经毒物的抗性，而囊泡功能的降低将产生相反的效果。我们的实验室已经产生并鉴定了两种独特的小鼠系，使我们能够验证这一假设：一种基于细菌人工染色体的转基因小鼠，其过表达VMAT2 (VMAT2- hi)，以及突触囊泡糖蛋白2C （SV2C-KO）的基因敲除，后者最近与帕金森病的发病机制有关。在这里，我们提供的初步数据清楚地表明，囊泡功能的增加对多巴胺系统有益，并且SV2C是多巴胺摄取和释放的一种新的调节剂。总之，这些发现为以下目标提供了基础。目的1：确定可变VMAT2或SV2C表达对囊泡多巴胺储存和释放动力学的影响。我们将确定这些基因操作如何改变囊泡动力学，包括容易释放的池。目的2：确定PD模型中囊泡储存和释放的改变是否会改变多巴胺神经元的易感性。我们确定减少SV2C或增加VMAT2对老年小鼠MPTP毒性的影响，这是一个经典且成熟的模型。目的3：确定多巴胺囊泡储存和释放的改变是否会影响多氯联苯（PCBs）对pd相关毒性的易感性。我们期望我们的小鼠在囊泡功能和多氯联苯诱导之间表现出负相关。除了在我们实验室产生的新型小鼠系外，我们还将在我们的研究中使用一套尖端技术，包括光遗传刺激，快速扫描循环伏安法，体内微透析和CLARITY。这些目标的完成将建立神经保护的关键概念：增加囊泡功能改善神经递质信号传导和反对多巴胺神经元毒性。这表明突触囊泡功能的调节代表了解决多巴胺神经元功能障碍的创新和未开发的机会，并为新的治疗方法提供了基础。

DESCRIPTION (provided by applicant): A major feature of Parkinson's disease (PD) is the loss of dopamine-producing neurons in the substantia nigra pars compacta and the concomitant loss of dopamine in the striatum. While multiple processes likely contribute to the loss of dopamine neurons, we argue that disruption of the proper storage and release of dopamine from vesicles plays a key role. This theory is supported by the literature, which demonstrates that improper storage of dopamine causes the formation of oxidative dopamine byproducts and toxic aldehydes that damages neurons. Evidence from genetic and toxicological studies of PD demonstrates that many insults converge on the dopamine vesicle to exert their deleterious effects. We hypothesize that vesicular function mediates dopamine neuron toxicity, such that increasing vesicle function will enhance dopamine storage and release and confer resistance to neurotoxicants, while decreasing vesicle function will have the opposite effect. Our laboratory has generated and characterized two unique mouse lines that allow us to test this hypothesis: a bacterial artificial chromosome-based transgenic mouse that overexpresses VMAT2 (VMAT2-HI) and a genetic knockout of the synaptic vesicle glycoprotein 2C (SV2C-KO), which was recently implicated in Parkinson's disease pathogenesis. Here, we provide preliminary data that clearly demonstrate that increased vesicular function is beneficial to the dopamine system and that SV2C is a novel regulator of dopamine uptake and release. Together, these findings provide the basis for the following aims. Aim 1: To determine the effects of variable VMAT2 or SV2C expression on vesicular dopamine storage and release dynamics. We will determine how these genetic manipulations alter vesicular dynamics, including the readily releasable pool. Aim 2: To determine if altered vesicular storage and release alters vulnerability of dopamine neurons in a model of PD. We determine the effect of reduced SV2C or increased VMAT2 on MPTP toxicity in aged mice, a classical and well-established model. Aim 3: To determine if altered vesicular storage and release of dopamine influences vulnerability to the PD-related toxicity of polychlorinated biphenyls (PCBs). We expect that our mice will show an inverse correlation between vesicle function and PCB-induced. In addition to the novel mouse lines generated in our laboratory we will use a suite of cutting-edge techniques in our studies, including optogenetic stimulation, fast-scan cyclic voltammetry, in vivo microdialysis, and CLARITY. Completion of these aims will establish a pivotal concept in neuroprotection: increasing vesicle function improves neurotransmitter signaling and opposes dopamine neuron toxicity. This suggests that modulation of synaptic vesicle function represents an innovative and unexplored opportunity for addressing dopamine neuron dysfunction and provides a foundation for new therapeutic approaches.