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背侧部多巴胺产生神经元的丢失和纹状体多巴胺的伴随丢失。虽然多个过程可能导致多巴胺神经元的损失，但我们认为，多巴胺从囊泡中的正确储存和释放的破坏起着关键作用。这一理论得到了文献的支持，文献表明，多巴胺的储存不当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。来自PD的遗传学和毒理学研究的证据表明，许多损伤聚集在多巴胺囊泡上以发挥其有害作用。我们假设囊泡功能介导多巴胺神经元毒性，因此增加囊泡功能将增强多巴胺储存和释放，并赋予对神经毒物的抗性，而降低囊泡功能将产生相反的效果。我们的实验室已经产生并表征了两种独特的小鼠品系，使我们能够测试这一假设：一种细菌人工染色体为基础的转基因小鼠，过表达VMAT 2（VMAT 2-HI）和突触囊泡糖蛋白2C（SV 2C-KO）的基因敲除，这是最近参与帕金森病的发病机制。在这里，我们提供了初步的数据，清楚地表明，增加囊泡功能是有益的多巴胺系统和SV 2C是一种新的调节多巴胺的摄取和释放。这些研究结果为实现以下目标提供了基础。目的1：确定可变VMAT 2或SV 2C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：确定在PD模型中改变囊泡储存和释放是否会改变多巴胺神经元的脆弱性。我们确定了老年小鼠中SV 2C降低或VMAT 2增加对MPTP毒性的影响，这是一种经典且完善的模型。目标3：确定多巴胺囊泡储存和释放的改变是否影响多氯联苯（PCB）对PD相关毒性的易感性。我们预计，我们的小鼠将显示囊泡功能和PCB诱导的负相关。除了在我们实验室中产生的新型小鼠品系外，我们还将在我们的研究中使用一套尖端技术，包括光遗传学刺激，快速扫描循环伏安法，体内微透析和微透析。这些目标的完成将建立一个关键的概念，在神经保护：增加囊泡功能，改善神经递质信号传导，并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.