Parkinson's and Prokaryotes: roles of bacteria, gut permeability, innate immunity, and genetics in C. elegans dopaminergic neurodegeneration

摘要 帕金森病(PD)，特征是通过𝛼-突触核蛋白使多巴胺(DA)神经元退化(𝛼S) Aggregate，在美国每年花费519亿美元，预计到2030年将影响120万美国人。 目前的治疗方法只能提供有限的和有症状的缓解，没有功能性治愈，主要是由于 帕金森病是在神秘的本质中启动的。因此，对帕金森病发病机制的更深层次的理解是至关重要的。 进行有效的治疗。一种新的假设是帕金森病始于肠道，𝛼S的聚集体从肠道扩散而来 肠道通过迷走神经等途径进入大脑。有趣的是，这些𝛼S聚集体是在肠道年份检测到的 在帕金森病诊断之前。此外，肠道通透性和功能障碍在PD患者中也很常见。尽管这些 肠道病理可能导致肠道细菌和微生物相关分子模式的易位 (MAMPs)进入宿主组织，随后通过天然免疫受体激活诱导炎症，这 还没有受到直接调查。因此，肠道细菌和先天免疫受体在𝛼S聚集中的作用 帕金森病的进展尚不清楚。此外，类似帕金森病小鼠的哺乳动物模型在生物学上是复杂的， 拥有多样化的肠道微生物区系，不能通过无偏见的突变筛选来识别新的PD因素。 因此，一种在基因上易于处理并允许对宿主和 单个微生物将有助于识别对帕金森病发病至关重要的新宿主和细菌因素。 为此，我建议使用线虫秀丽线虫，这是一种广泛用于疾病的模式生物 研究和PD研究，以调查肠道细菌如何触发炎症反应，从而加剧 达神经退行性变。我将使用的特定模型在DA神经元中共同表达人𝛼S和绿色荧光蛋白， 导致GFP信号丢失所示的DA神经元进行性丢失。我提议的研究将使用 CRISPR-Cas9基因组编辑技术失活对肠道屏障完整性和先天免疫至关重要的基因 然后通过荧光监测这些基因在帕金森病发病中的空间作用。 𝛼标记的S和绿色荧光蛋白标记的DA神经元。此外，细菌种类或特定的MAMP将单独 分别给予线虫作为细菌食物来源或治疗，以确定哪些细菌特征 可增强或抑制帕金森病。最后，我将对线虫和单个细菌进行诱变筛选 草坪，以确定新的寄主和细菌因素，分别促进或抑制帕金森病的进展。 我提出的研究将有助于确定新的治疗靶点和治疗方法来阻断或潜在逆转帕金森病， 使用线虫作为一种经济高效的筛查工具。这个项目是高度跨学科的，结合 免疫学、神经生物学、微生物学、肠道生物学和遗传学。这一策略提高了 找出影响帕金森病发病机制的新因素和新疗法。

Abstract Parkinson’s disease (PD), characterized by the degeneration of dopaminergic (DA) neurons via 𝛼-Synuclein (𝛼S) aggregation, costs $51.9 billion annually in the US and is predicted to affect 1.2 million Americans by 2030. Current treatments only provide limited and symptomatic relief, with no functional cure, largely due to the mysterious nature in which PD is initiated. Thus, a deeper, mechanistic understanding of PD pathogenesis is vital for effective treatment. An emerging hypothesis is that PD begins in the gut, where 𝛼S aggregates spread from the gut to the brain via routes like the vagus nerve. Interestingly, these 𝛼S aggregates are detected in the gut years before PD diagnosis. In addition, gut permeability and dysfunction are common in PD patients. Although these intestinal pathologies likely lead to in the translocation of gut bacteria and microbe-associated molecular patterns (MAMPs) into host tissues and subsequent induction of inflammation via innate immune receptor activation, this has not been directly investigated. Thus, the role of gut bacteria and innate immune receptors in 𝛼S aggregation and PD progression is unclear. Furthermore, mammalian models for PD like mice are biologically complex, harbor a diverse gut microbiota, and cannot undergo unbiased mutagenesis screens to identify novel PD factors. Thus, a minimalist model which is genetically tractable and permits mutagenesis screens for both the host and individual microbes would empower identification of novel host and bacterial factors crucial to PD pathogenesis. To this end, I propose to use the nematode Caenorhabditis elegans, a model organism widely used in disease study and PD research, to investigate how gut bacteria may trigger inflammatory responses that exacerbate DA neurodegeneration. The particular model that I will use co-expresses human 𝛼S and GFP in DA neurons, causing a progressive loss of DA neurons as indicated by GFP signal loss. My proposed studies will use the CRISPR-Cas9 genome editing technique to inactivate genes crucial for gut barrier integrity and innate immune receptors and then investigate the spatial role of these genes in PD pathogenesis by monitoring fluorescently- labeled 𝛼S and GFP-labeled DA neurons. Furthermore, bacterial species or specific MAMPs will be individually given to C. elegans as bacterial food sources or treatments, respectively, to identify what bacterial characteristics may enhance or suppress PD. Lastly, I will conduct mutagenesis screens on C. elegans and individual bacterial lawns to identify novel host and bacterial factors, respectively, which either promote or inhibit PD progression. My proposed study will help identify novel therapeutic targets and treatments to block or potentially reverse PD, using C. elegans as a cost-efficient screening tool. This project is highly interdisciplinary, combining immunology, neurobiology, microbiology, enteric biology, and genetics. This strategy improves the possibility of identifying novel factors and treatments which affect PD pathogenesis.