Alpha-synuclein aggregation disrupts motility, synaptic transmission, and calcium signaling in the myenteric plexus of the rat colon

项目摘要/摘要 帕金森病(PD)通常被认为是一种躯体运动障碍。然而，胃肠运动 导致便秘的功能障碍通常被描述为对生活质量有更大的负面影响 而不是躯体运动功能障碍。此外，胃肠道功能障碍使帕金森病的临床治疗复杂化，因为它 干扰左旋多巴的吸收，左旋多巴是治疗帕金森病运动症状的标准药物。蛋白质阿尔法- 突触核蛋白(α-SYN)与中枢神经系统退行性变有关，并以聚集的形式存在于神经元中。 肠道神经系统(ENS)。α-SYN在中枢多巴胺能神经元中的过度表达 结果导致α-SYN聚集、神经传递改变和神经退行性变；提供了一种高度 警局的表面效力。然而，到目前为止，还没有人尝试将病理性肠道α-SYN与GI联系起来 功能障碍。为此，我们开发了一种新的基因传递方法，通过这种方法，我们可以引导转基因 特异性地表达于肠神经细胞。重要的是，这种方法将所有转基因表达限制在ens上。 在中枢神经元、肌肉或其他器官中没有表达。换句话说，我们开发了一种 研究ENS功能的新平台，而不增加使用系统应用的试剂的混乱 例如有毒物质或使用普遍存在的转基因表达的转基因动物。使用这种方法 我们将表达α-SYN的腺相关病毒输送到降结肠。在一个月内 病媒动物的粪便排泄量明显减少，抑制率降低 神经肌肉传递，降低神经元和肠神经胶质细胞的基础钙水平，而不是 治疗后ENS中的神经元丢失。α-SYN在突触囊泡的胞吐、循环和对接中起作用，并且 α-SYN的聚集导致神经传递受损。因此，我们假设α-SYN有助于 GI功能障碍，这种作用是通过受损的突触传递介导的，导致 ENS的抑制音调。在此，我们建议(1)确定是否需要α-SYN聚合 观察到的结肠动力障碍。(2)确定过表达α-SYN是否会导致 ENS神经传递的抑制音，以及这是否损害推进运动。(3)。测定 α-SYN过度表达是否损害肠神经节神经传递。我们将执行这些分析 通过使用编码不同α-SYN亚型(野生型、超聚集和不可聚集)的载体。 我们将比较在治疗大鼠和转基因α-SYN过表达动物的小鼠中观察到的数据。我们 也将确定α-SYN在抑制性运动神经元(ENS神经元的群体)中是否过度表达 最常与帕金森病的α-SYN病理有关)本身就足以引起胃肠道功能障碍。团结在一起 本提案旨在证明ENS中聚集的α-SYN导致胃肠功能障碍，并 明确帕金森病患者胃肠功能障碍的分子病因。这项工作将为今后的工作奠定基础 研究工作的重点是研究帕金森病这种破坏性共病的新治疗方法。

Project Summary/Abstract Parkinson's disease (PD) is typically recognized as a somatic motor disorder. However, gastrointestinal motor dysfunction resulting in constipation is often described as having a greater negative impact on the quality of life than somatic motor dysfunction. Moreover, GI dysfunction complicates the clinical management of PD as it interferes with the absorption of levodopa, the standard treatment for PD motor symptoms. The protein alpha- synuclein (α-syn) is linked to CNS degeneration and is present in aggregated forms in neurons throughout the enteric nervous system (ENS). Overexpression of α-syn in specific populations of DA neurons in the CNS results in α-syn aggregation, altered neurotransmission, and neurodegeneration; providing a model with high face validity for PD. However, to date, no attempts have been made linking pathological enteric α-syn to GI dysfunction. To that end, we developed a novel gene delivery method whereby we can direct transgene expression specifically to enteric neurons. Importantly, this approach limits all transgene expression to the ENS with no expression seen in central neurons, muscle, or other organs. In other words, we have developed a novel platform by which to study ENS function without adding confound of using systemically applied agents such as toxicants or the use of transgenic animals with ubiquitous transgene expression. Using this approach we delivered adeno-associated virus (AAV) expressing α-syn to the descending colon. Within one month of vector delivery the animals displayed a significant decrease in fecal output, decreased inhibitory neuromuscular transmission, and decreased basal levels of calcium in neurons and enteric glia, without neuronal loss in the treated ENS. α-syn has a role in synaptic vesicle exocytosis, recycling, and docking, and aggregation of α-syn results in impaired neurotransmission. We therefore hypothesize that α-syn contributes to GI dysfunction, and that this effect is mediated via impaired synaptic transmission, resulting in a net increase of the inhibitory tone of the ENS. Herein we propose to (1) Determine whether α-syn aggregation is required for the observed colonic dysmotility. (2) Determine whether overexpression of α-syn results in a net increase in the inhibitory tone of ENS neurotransmission and whether this impairs propulsive motility. (3). Determine whether α-syn overexpression impairs enteric ganglionic neurotransmission. We will perform these analyses by using vectors that encode different isoforms of α-syn (wildtype, hyper-aggregatable, and non-aggregatable). We will compare the data observed in treated rats and mice with transgenic α-syn overexpressing animals. We will also determine whether α-syn overexpression in inhibitory motor neurons (the population of ENS neurons most often associated with α-syn pathology in PD) per se is sufficient to elicit GI dysfunction. Together the aims in this proposal intend to demonstrate that aggregated α-syn in the ENS causes GI dysfunction, and to define the molecular etiology underlying GI dysfunction in PD. This work will thus provide a basis for future research efforts focused on investigating new treatments of this devastating comorbidity in PD.