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

α-突触核蛋白聚集破坏大鼠结肠肌间神经丛的运动、突触传递和钙信号传导

• 批准号: 9414029
• 负责人: Fredric Manfredsson
• 金额: $ 34.88万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9414029
• 关键词: Address; Animals; Biological Assay; CNS degeneration; Calcium; Calcium Signaling; Cells; Clinical Management; Colon; Comorbidity; Constipation; Data; Dependovirus; Descending colon; Development; Disease; Docking; Electrophysiology (science); Enteral; Enteric Nervous System; Etiology; Exhibits; Exocytosis; Face; Frequencies; Functional disorder; Ganglia; Gene Delivery; Goals; Homeostasis; Impairment; Individual; Injections; Knowledge; Levodopa; Link; Mediating; Mediator of activation protein; Methods; Modeling; Molecular; Monitor; Motor Activity; Motor Neurons; Movement; Mus; Muscle; Myenteric Plexus; Nerve Degeneration; Nervous System Physiology; Neuraxis; Neuroglia; Neurons; Neurotransmitters; Organ; Output; Parkinson Disease; Pathologic; Pathology; Patients; Pattern; Phenotype; Plant Roots; Population; Protein Isoforms; Proteins; Quality of life; Rattus; Recombinant adeno-associated virus (rAAV); Recycling; Regulation; Relaxation; Reporting; Research; Role; Signal Transduction; Smooth Muscle; Stomach; Synaptic Transmission; Synaptic Vesicles; Testing; Tissues; Transgenic Animals; Transgenic Organisms; Work; absorption; alpha synuclein; associated symptom; cell motility; combination gene therapy; dopaminergic neuron; gastrointestinal; improved; in vivo; motility disorder; motor disorder; motor symptom; mutant; neuromuscular transmission; neuron loss; neurotransmission; neurotransmitter release; new therapeutic target; novel; overexpression; prevent; standard care; therapeutic development; toxicant; trafficking; transgene expression; vector

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.

项目总结/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具有更大的负面影响 而不是躯体运动功能障碍此外，GI功能障碍使PD的临床管理复杂化，因为它 干扰左旋多巴的吸收，左旋多巴是PD运动症状的标准治疗。蛋白质阿尔法- 突触核蛋白（α-syn）与CNS变性有关，并以聚集形式存在于整个中枢神经系统的神经元中。 肠神经系统（ENS）。α-syn在中枢神经系统特定DA能神经元群中的过表达 导致α-syn聚集，改变神经传递和神经变性;提供了一个高 PD的表面效度。然而，迄今为止，尚未尝试将病理性肠道α-syn与GI 功能障碍为此，我们开发了一种新的基因传递方法， 肠神经元特异性表达。重要的是，这种方法将所有转基因表达限制在ENS中， 在中枢神经元、肌肉或其他器官中未见表达。换句话说，我们开发了一个 一种新的平台，通过该平台研究ENS功能，而不会增加使用系统应用代理的混淆 例如毒物或使用具有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（AAV）递送至降结肠。后一个月内 载体递送动物显示粪便排出量显著减少，抑制性降低， 神经肌肉传递，神经元和肠神经胶质细胞的基础钙水平降低， α-syn在突触囊泡胞吐、再循环和对接中起作用， α-syn的聚集导致神经传递受损。因此，我们假设α-syn有助于 胃肠道功能障碍，这种作用是通过受损的突触传递介导的，导致 在此，我们建议（1）确定是否需要α-syn聚集 观察到的结肠运动障碍。(2)确定α-syn的过表达是否导致 ENS神经传递的抑制性张力以及这是否损害推进运动。（三）、确定 α-syn过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α-syn的不同同种型（野生型、超聚集型和非聚集型）的载体。 我们将比较给药大鼠和小鼠与转基因α-syn过表达动物中观察到的数据。我们 还将确定抑制性运动神经元（ENS神经元群体）中α-syn过表达是否 最常与PD中的α-syn病理学相关）本身足以引起GI功能障碍。一起 本提案旨在证明ENS中聚集的α-syn导致GI功能障碍，并 明确PD患者胃肠道功能障碍的分子病因。这项工作将为今后的工作奠定基础。 研究工作集中于研究PD中这种毁灭性合并症的新治疗方法。