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

项目总结和摘要 帕金森病（Parkinson's disease，PD）是最常见的伴有运动异常的神经系统疾病。已经25了 自帕金森病的第一个遗传原因被发现以来，已经有多年了，但仍然没有有效的治疗方法。之一 我们认为障碍是缺乏评估早期PD发病机制和真实的治疗反应的模型 神经生理环境这为我们在这一疾病方面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PD的发病机制：（1）人类 神经影像学数据和动物模型都表明，突触破坏会导致神经元死亡， 帕金森病是一种突触病(2)许多新的、调节性的、非编码RNA显示出与PD发病机制的联系。比如说， 我们在人类死后大脑的多巴胺神经元中发现了超过20，000种增强子RNA（或eRNA）候选物（Dong et al. Nature Neuroscience，2018）。它们与PD风险变体显著共定位。另一类新的RNA是 环状RNA（circRNA），主要富集在大脑中，对突触高度特异，并且超稳定 (e.g.,半衰期比线性RNA长10倍）。我们鉴定了> 11，000个在多巴胺神经元中活跃表达的circRNA， 其中许多与PD病理学显著相关（Dong等人提交）。更重要的是，circRNA可以 与lncRNA和miRNAs形成调控网络，并可包裹在细胞外囊泡（EV）中，穿透 血脑屏障基于这些，我们假设调控RNA包括。circRNA、eRNA、miRNA、lncRNA 在EV中可以检测到，并且可能在PD发病机制中的突触功能障碍中起作用。 为了验证这一假设，我们需要一个模型来概括PD发病机制的动态生理微环境。 在这项研究中，我们将联合收割机结合我们在脑类器官，PD生物学，外泌体分析，单细胞组学， 生物信息学和生物医学工程，开发新的3D脑类器官微生理分析平台 (MAP)来重现多巴胺神经元的相互连接并研究分子神经退化 系统地我们将（1）首先开发PD类器官并分析转录组（包括circRNA，miRNAs，mRNAs， lncRNA等）分泌的EV和脑类器官的单细胞转录组，以鉴定PD相关RNA，然后（2） 在一个新的、高通量的、微型脑芯片平台上绘制PD类器官的病理生理动力学， 将（3）整合EV-类器官时间多维数据以推断PD相关RNA及其调控基因。 PD发病过程中的动力学。 RNA疗法的最新突破已经导致多个首次人体试验和临床批准（例如，Moderna， Alnylam和Ionis制药公司）。circRNA与线性RNA相比具有许多优势，使其可能更好地 适用于治疗和诊断。从PD类器官分泌的EV提供了流体的良好代表 为研究帕金森病的脑神经病理学提供了一种新的方法。因此，这个跨学科（神经学，生物医学工程， 计算基因组学）研究将为理解PD神经病理学奠定重要的、高度创新的基础 和外泌体治疗。

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.