GI Endotoxin as an Environmental Trigger in an alpha-Synuclein Transgenic Mouse

描述（由申请人提供）：帕金森病（PD）是一种常见的神经退行性疾病，在美国影响约100万人。虽然黑质多巴胺能神经元的丧失是帕金森运动障碍的主要原因，但非运动障碍，包括痴呆、抑郁、胃肠功能障碍和自主神经不稳定，可由中枢、肠和自主神经系统的其他地方的神经变性引起。尽管经过几十年的研究，大多数帕金森病的病因仍不清楚。帕金森病（PD）的家庭研究、双胞胎研究和遗传关联研究支持这样的观点：PD是一种复杂的全身性神经系统疾病，由一种或多种环境因素作用于遗传易感个体引起。遗传易感性可以很强，如由1-突触核蛋白基因（SNCA）错义或拷贝数突变引起的罕见的家族性PD，也可以很微妙，如更常见的散发性疾病，遗传关联研究表明SNCA位点的多态变异有助于遗传易感性。流行病学和实验观察表明，许多不同的环境因素可能有助于PD的发展。这些包括病毒感染、重金属、细菌毒素、微生物引起的炎症以及包括杀虫剂在内的化学制剂。遗传易感性和环境侮辱之间的平衡可能会因易感性的强弱而有所不同。导致疾病所需的环境暴露强度可能与遗传易感性的程度成反比：遗传易感性越大，所需的环境暴露越少，疾病的遗传性就越明显。暴露于与PD相关的环境因子可能是通过胃肠道（尽管吸入暴露嗅觉神经也有可能），这表明肠神经系统（ENS）可能在疾病早期受到影响，而肠神经系统是暴露于这些环境因子的第一线。事实上，有充分的证据表明，在PD患者出现运动体征之前，胃肠道功能障碍就已经发生了几年到几十年。Braak和他的同事报告说，在他们的大型尸检系列中，最年轻的个体仅在肠神经系统（ENS）中显示PD的病理改变，而中枢神经系统的PD病理出现在年龄较大的个体中，主要影响脑干和中枢神经系统。最终，中脑（包括黑质）和皮层的中枢神经系统病理出现在年龄更大的个体中。我们建议验证这样的假设，即基因易感小鼠通过胃肠道暴露于致病性环境暴露将模拟PD，并且早期ENS功能障碍不仅暂时先于中枢神经系统功能障碍，而且实际上通过增加暴露于这些物质而导致其因果关系。我们已经开发了一系列转基因小鼠，以模仿因SNCA突变而患有家族性PD的遗传易感个体。这些小鼠从含有人类SNCA位点的P1人工染色体上过表达野生型或突变型（A53T和A30P）人类1-突触核蛋白。表达两种突变型（但非野生型）1-突触核蛋白中的任何一种的小鼠早在3月龄时就出现ENS功能障碍，Gl转运时间延长，结肠运动性下降，但中枢神经系统功能障碍和中枢神经系统神经元损失最小。我们将把这些转基因动物暴露于革兰氏阴性内毒素和鱼藤酮中，这两种物质通常被认为是与帕金森病最密切相关的环境毒素。我们将询问，在我们的1Syn转基因模型中，肠道神经系统缺陷是否会(1)导致肠道菌群分布和组成的变化以及肠道细菌暴露，(2)增强内毒素的吸收，导致全身炎症效应和神经缺陷，以及(3)增加鱼藤酮的吸收和神经毒性，鱼藤酮是一种之前在细胞和动物模型中被证明会导致神经变性的农药。

DESCRIPTION (provided by applicant): Parkinson Disease (PD) is a common neurodegenerative disease affecting ~1 million people in the United States. While loss of substantia nigra dopaminergic neurons is primarily responsible for the characteristic parkinsonian movement disorder, non-motor disabilities including dementia, depression, gastrointestinal dysfunction, and autonomic instability arise from neurodegeneration elsewhere in the central, enteric and autonomic nervous systems. The cause of most PD is unknown despite decades of research. Family studies, twin studies, and genetic association studies in Parkinson disease (PD) support the view that PD is a complex systemic nervous system disorder caused by one or more environmental agents acting on genetically vulnerable individuals. The genetic predisposition can be strong, as in the rare, familial forms of PD caused by missense or copy number mutations in the 1-synuclein gene (SNCA), or subtle, as in the more common sporadic disease, where genetic association studies indicate that polymorphic variants at the SNCA locus contribute to genetic predisposition. Epidemiological and experimental observations have suggested many different environmental agents may contribute to the development of PD. These include viral infection, heavy metals, bacterial toxins, microbe-induced inflammation, and chemical agents including pesticides. The balance between genetic predisposition and environmental insult is likely to be different depending on the strength of the predisposition. The intensity of environmental exposure required to cause disease is likely inversely related to the degree of genetic vulnerability: the greater the genetic susceptibility, the less the environmental exposure required and the more apparently heritable the disease will appear to be. Exposure to the environmental agents implicated in PD is probably via the gastrointestinal (GI) tract (although inhalation exposing the olfactory nerves is also possible), suggesting that the enteric nervous system (ENS), which would be on the front lines of exposure to these environmental agents, might be affected early in the disease. Indeed, GI dysfunction is well documented to occur years to decades before the motor signs in PD patients. Braak and his colleagues reported that the youngest individuals in their large autopsy series showed pathological changes of PD only in the enteric nervous system (ENS), while PD pathology in the CNS appeared at older ages, affecting predominantly the brain stem, as well as in the ENS. Ultimately, CNS pathology in the midbrain (including substantia nigra) and cortex was seen in still older individuals. We propose to test the hypothesis that exposure of genetically predisposed mice to pathogenic environmental exposures through the GI tract will model PD and that early ENS dysfunction not only temporally precedes CNS dysfunction but actually contributes causally to it by increasing the exposure to these agents. We have developed a series of transgenic mice engineered to mimic genetically vulnerable individuals with familial PD due to SNCA mutations. These mice overexpress either wildtype or mutant (A53T and A30P) human 1-synuclein from a P1 artificial chromosome containing the human SNCA locus. Mice expressing either of the two mutant, but not wildtype, 1-synucleins have ENS dysfunction as early as 3 months of age, with prolonged Gl transit time and decreased colonic motility, but minimal CNS dysfunction and CNS neuronal loss. We will expose these transgenic animals to gram-negative endotoxin and rotenone, two agents frequently considered as environmental toxins most strongly implicated in PD. We will ask whether the enteric nervous system defect in our 1Syn transgenic models will (1) cause changes in distribution and composition of gut flora and exposure to intestinal bacteria, (2) enhance the absorption of endotoxin leading to systemic inflammatory effects and neurological defects, and (3) increase absorption and neurotoxicity of rotenone, a pesticide previously shown in cellular and animal models to cause neurodegeneration. PUBLIC HEALTH RELEVANCE: Evidence is accumulating that environmental exposures in individuals genetically predisposed to Parkinson disease are critical in the development of the disease. The applicant wants to know if mice, engineered to mimic the genetic make-up of humans with a form of hereditary Parkinson disease, are at significantly increased risk for developing a mouse equivalent of Parkinson disease when exposed through their gastrointestinal tract to two different environmental agents thought to have a role in causing human PD: endotoxin made by normal bacteria in the gut and the commonly used pesticide rotenone. The ultimate goal is to study in detail the role of gastrointestinal exposure to environmental agents in the development of neurodegeneration in a model of the natural human disease. By understanding the disease process, we can identify points at which interventions could slow or stop its progression.