Systematic study of extracellular vesicles and their integrative analysis with Parkinson's organoids MAP

项目摘要和摘要 帕金森病(PD)是与运动异常相关的最常见的神经系统疾病。已经25岁了 帕金森病的第一个遗传原因被发现已经有几年了，但仍然没有有效的治疗方法。其中之一 我们认为的障碍是缺乏评估早期帕金森病发病机制和治疗反应的模型 神经生理环境。这给我们在这一疾病上取得进展的能力提供了一个重大瓶颈。 最近的两条证据促使我们在真实的神经生理环境中研究帕金森病的发病机制：(1)人类 神经成像数据和动物模型都表明，突触破坏会导致神经元死亡，这证明了这一点 帕金森病是一种突触。(2)许多新的、调节的、非编码的RNA与帕金森病的发病机制有关。例如, 我们在人死后大脑的多巴胺神经元中发现了超过20,000个候选增强子RNA(或eRNAs) 艾尔自然神经科学，2018年)。它们显著地与帕金森病风险变异体共同定位。另一类新的RNA是 环状RNA(CircRNAs)，主要富含在大脑中，高度特异于突触，并且超稳定 (例如，半衰期比线性RNA长10倍)。我们发现了11,000个在多巴胺神经元中活跃表达的CircRNA， 其中许多与帕金森病的病理密切相关(董等人)。呈交)。更重要的是，CircRNA可以 与lncRNAs和miRNAs形成调控网络，并可包裹到细胞外小泡(EV)中，穿透 血脑屏障。在此基础上，我们假设调控RNA包括。CircRNAs，eRNAs，miRNAs，IncRNAs 可在EV中检测到，并可能在PD发病机制中突触功能障碍中发挥作用。 为了验证这一假说，我们需要一个模型来概括帕金森病发病机制的动态生理微环境。 在这项研究中，我们将结合我们在脑有机体、PD生物学、外显体分析、单细胞组学、 生物信息学和生物医学工程开发新的3D脑有机物质微生理分析平台 (MAP)重述多巴胺神经元的相互连接并研究分子神经退行性变 系统地。我们将(1)首先开发PD类有机化合物并分析转录组(包括。CircRNA，miRNAs，mRNAs， InncRNAs等)分泌性EV和脑器官单细胞转录组，以鉴定PD相关RNA，然后(2) 在一种新型、高通量、迷你脑片上平台中绘制PD有机物的病理生理动力学图，并最终 将(3)整合EV-有机物时间多维数据以推断与PD相关的RNA及其调控 帕金森病发病过程中的动力学变化。 最近在RNA疗法方面的突破导致了多项首例人体试验和临床批准(例如，莫德纳， Alnylam和Ionis PharmPharmticals)。与线性RNA相比，CircRNAs有许多优点，这使它们有可能更好 适合翻译成治疗学和诊断学。由钯类有机物分泌的电动汽车提供了很好的液体替代品 用于研究帕金森病脑神经病理的活检。因此，这种交叉学科(神经学、生物医学工程、 计算基因组学)研究将为理解帕金森病神经病理奠定重要的、高度创新的基础 和外切体治疗。

PROJECT SUMMARY AND ABSTRACT Parkinson's disease (PD) is the most common neurological disease associated with movement abnormality. It has been 25 years since the first genetic cause of PD was identified, and yet there is still no effective treatment for the disease. One of the hinders we think is the lack of models that assess early PD pathogenesis and therapy responses in its real neurophysiological environment. This provides a significant bottleneck in our ability to make progress in this disease. Two lines of recent evidence motivate us to study PD pathogenesis in a real neurophysiological environment: (1) Human neuroimaging data and animal models both showed that synaptic disruption proceeds neuronal death, making the case that PD is a synaptopathy. (2) Many novel, regulatory, non-coding RNAs show linkage to PD pathogenesis. For instance, we found over 20,000 enhancer RNAs (or eRNAs) candidates in dopamine neurons of human post-mortem brains (Dong et al. Nature Neuroscience, 2018). They significantly co-localized with PD risk variants. The other class of novel RNAs is circular RNAs (circRNAs), which are predominantly enriched in the brain, highly specific to the synapse, and ultra-stable (e.g., 10x longer half-life than linear RNAs). We identified >11,000 circRNAs actively expressed in the dopamine neurons, many of which are significantly associated with PD pathology (Dong et al. in submission). More importantly, circRNAs can form a regulatory network with lncRNAs and miRNAs, and can be wrapped into extracellular vesicles (EV), penetrating blood-brain barriers. Based on these, we hypothesize that regulatory RNAs incl. circRNAs, eRNAs, miRNAs, lncRNAs can be detected in EV and might play a role in the synaptic dysfunction in PD pathogenesis. To test this hypothesis, we need a model to recapitulate the dynamic physiological microenvironment of PD pathogenesis. In this study, we will combine our expertise in brain organoids, PD biology, exosome analysis, single-cell omics, bioinformatics, and biomedical engineering to develop a new 3D brain organoids microphysiological analysis platform (MAP) to recapitulate the dopamine neurons' interconnectivity and study molecular neurodegeneration systematically. We will (1) first develop PD organoids and profile the transcriptome (incl. circRNAs, miRNAs, mRNAs, lncRNAs, etc.) of secreted EV and single-cell transcriptome of brain organoids, to identify PD-associated RNAs, then (2) map the pathophysiological dynamics of PD organoids in a novel, high-throughput, mini-brain-on-chip platform, and last will (3) integrate the EV-organoid temporal multi-dimensional data to infer the PD-associated RNAs and their regulatory dynamics during the PD pathogenesis. Recent breakthroughs in RNA therapeutics have led to multiple first-in-human trials and clinical approval (e.g., Moderna, Alnylam, and Ionis pharmaceuticals). circRNAs have many advantages over linear RNAs, making them potentially better suited for translation into therapeutics and diagnostics. EVs secreted from PD organoids provide a good proxy of fluid biopsy for studying PD brain's neuropathology. Thus, this interdisciplinary (neurology, biomedical engineering, computational genomics) study will set an important, highly innovative foundation for understanding PD neuropathology and exosome treatment.