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

摘要 帕金森病（Parkinson's disease，PD）是一种以多巴胺能（dopaminergic，DA）神经元通过β-突触核蛋白（β-Synuclein，β-Synuclein）变性为特征的疾病 总计，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症状缓解，没有功能性治愈，这主要是由于 神秘的性质，其中PD是启动。因此，对PD发病机制的更深层次的机制性理解至关重要 进行有效治疗。一个新出现的假设是，PD始于肠道，在那里，PD-S聚集体从 通过迷走神经等途径将肠道输送到大脑。有趣的是，在肠道中检测到这些H2S聚集体，𝛼 在PD诊断之前此外，肠道通透性和功能障碍在PD患者中很常见。虽然这些 肠道病变可能导致肠道细菌和微生物相关分子模式的易位 通过天然免疫受体激活，MAMPs进入宿主组织并随后诱导炎症， 还没有被直接调查。因此，肠道细菌和先天性免疫受体在BRAS聚集中的作用 PD进展尚不清楚。此外，PD样小鼠的哺乳动物模型在生物学上是复杂的， 具有多样的肠道微生物群，并且不能进行无偏诱变筛选以鉴定新的PD因子。 因此，遗传上易处理的并且允许诱变筛选宿主和突变体的最低限度的模型是可行的。 单个微生物将使得能够鉴定对PD发病机制至关重要的新宿主和细菌因子。 为此，我建议使用线虫秀丽隐杆线虫，一种广泛用于疾病的模式生物， 研究和PD研究，以研究肠道细菌如何引发炎症反应， DA神经变性我将使用的特定模型在DA神经元中共表达人类CAPS和GFP，𝛼 导致DA神经元的进行性损失，如GFP信号损失所示。我提议的研究将使用 CRISPR-Cas9基因组编辑技术用于修复对肠道屏障完整性和先天免疫至关重要的基因 受体，然后研究这些基因在PD发病机制中的空间作用，通过监测荧光- 标记的多巴胺能神经元。此外，细菌物种或特异性MAMP将被单独鉴定。 给C.线虫作为细菌的食物来源或治疗，分别，以确定什么细菌的特点， 可以增强或抑制PD。最后，我将对C.线虫和单个细菌 草坪，以确定新的主机和细菌的因素，分别促进或抑制PD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，以阻断或潜在地逆转PD， 利用C. elegans作为一种具有成本效益的筛选工具。该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。这种策略提高了 确定影响PD发病机制的新因素和治疗方法。

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.