Functions of alpha-Synuclein in Neurotransmitter Release

描述(申请人提供)：帕金森病(PD)是第二常见的神经退行性疾病，影响1.8%的65岁以上的人。帕金森病的一个临床特征是在脑神经元中形成细胞内蛋白质内包涵体，称为路易小体。路易体的主要成分是α-突触核蛋白，在家族性帕金森病和散发性帕金森病的神经元中都有蓄积。帕金森病患者的治疗策略并不是很成功，因为临床症状发生在神经元功能受损到难以挽救的程度的阶段。治疗策略的改进依赖于对帕金森病的病理生理发展的更好的了解，包括由α-突触核蛋白积聚引起的症状前神经元功能障碍。先前发现，阿尔法-突触核蛋白水平的升高在症状性帕金森病之前会损害神经递质的释放。然而，导致这种损伤的机制却知之甚少。由于α-突触核蛋白主要表达在神经末梢，而神经末梢通常太小，不能用高灵敏度的电生理学方法进行研究，因此以往的研究不能直接评估许多重要的突触前因子的作用，如动作电位、电压依赖性钙内流和通过内吞作用进行囊泡循环等。为了确定神经末梢中升高的α-突触蛋白的主要细胞和分子靶点，我们的研究将利用Hold的花瓣，这是一种可通过膜片钳技术获得的大型哺乳动物突触。在初步实验中，我们发现，升高的α-突触核蛋白抑制了花萼突触的囊泡胞吐。这一观察结果为进一步研究α-突触核蛋白升高抑制胞吐作用的机制奠定了基础。在目标1中，我们将确定升高的α-突触核蛋白与钙信号之间的相互作用。在目标2中，我们将确定升高的α-突触核蛋白是否通过影响囊泡池、内吞和动员来减少胞吐作用。在目标3中，我们将确定在花萼识别的机制是否适用于中脑多巴胺能神经元，这些神经元与神经退行性变的发展有关。实现这些目标可以提供在症状性神经退行性疾病之前α-突触核蛋白的积累如何导致突触传递障碍的关键信息。该项目可以阐明帕金森病的病理生理变化过程，并有助于针对这些变化开发新的治疗方法。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is the second most common neurodegenerative disease, which affects 1.8% of people over the age of 65 years. One clinic hallmark of PD is formation of intracellular proteinaceous inclusions, termed Lewy Bodies, in the brain neurons. The main constituent of the Lewy Bodies is alpha-synuclein, which is found to accumulate in neurons in both familial forms of PD and sporadic PD. Therapeutic strategies for PD patients have not been very successful, because clinical symptoms occur at a stage when the neuronal functions have been damaged to an extent hard to rescue. Improvement of the therapeutic strategies depends on better understanding of the pathophysiological development of PD, including presymptomatic neuronal dysfunctions induced by alpha-synuclein accumulation. Increased level of alpha-synuclein has previously been found to impair neurotransmitter release prior to the symptomatic PD. However, mechanisms causing such impairment are poorly understood. Because alpha-synuclein is mainly expressed in nerve terminals, which are usually too small for using the highly sensitive electrophysiology methods, previous studies could not directly evaluate contributions of many essential presynaptic factors, such as action potential, voltage dependent calcium entry, and vesicle recycling through endocytosis. In order to identify the primary cellular and molecular targets of elevated alpha-synuclein in nerve terminals, our studies will take advantage of the calyx of Held, a large mammalian synapse which is accessible to patch-clamp techniques. In preliminary experiments, we found that elevated alpha-synuclein inhibited vesicle exocytosis at the calyx synapse. This observation provides the foundation to further investigate the mechanisms underlying the inhibited exocytosis by elevated alpha-synuclein. In Aim 1, we will determine the interaction between elevated alpha-synuclein and calcium signaling. In Aim 2, we will determine whether elevated alpha-synuclein reduces exocytosis by affecting vesicle pool, endocytosis and mobilization. In Aim 3 we will determine whether the mechanisms identified at the calyx apply to midbrain dopaminergic neurons, which are related to the development of neurodegeneration. Achieving these aims can provide crucial information on how accumulation of alpha-synuclein causes the impairment of synaptic transmission prior to the symptomatic neurodegenerative disorders. This project can shed light on the process of pathophysiological changes in PD, and help the development of new therapeutic approaches targeting to these changes.