基于帕金森病患者特异性iPSC来源的中脑星形胶质细胞研究疾病发生的非细胞自主性因素

Parkinson's disease(PD) is the second most common neurodegenerative disorder. Tremendous studies had been done with this disease, but the mechanism of pathogenesis is still not clear, and none of clinical treatments is working well. Astrocytes could be one of the non-cell autonomous components which play an important role in the pathogenesis of PD. More attention have been paid to how astrocytes are involved in PD, and most of these studies are using animal models, which cannot fully recapitulate the clinical symptoms of the human diseases. In order to understand the role of astrocytes in the pathogenesis of PD in a more accurate way, we are planning to take the patient-specific induced pluripotent stem cells (iPSCs) as the model in this study. To generate PD patient-specific isogenic iPSCs, the PINK1 mutation in PD patient-specific iPSCs was corrected using TALEN-based gene editing method. Mesencephalic astrocytes will be differentiated from PD patient-specific isogenic iPSCs or normal human PINK1 knock-out isogenic iPSCs. How PINK1 mutant affects the physiological and pathological function of mesencephalic astrocytes will be addressed. Using co-culture system, the study will also focus on how mesencephalic astrocytes affect the viability of PD patient-specific isogenic iPSCs-derived dopaminergic neurons treated with cell stress factors. Through the studies we propose here, we are trying to elucidate how important the non-cell autonomous component is with regard to the initiation and pathogenesis of Parkinson's disease, and to supply the new target for drug screening, which may benefit the PD clinical treatment in the near future.

帕金森病是临床上发病率第二的神经退行性疾病，其发病机制仍不明确且缺乏有效的治疗措施。星形胶质细胞介导的非细胞自主因素参与了帕金森病的发生、发展，但具体的作用机制尚不清晰。为了更为客观全面地反映星形胶质细胞在帕金森病疾病发生中的作用，本研究拟建立PINK1点突变帕金森病人的诱导多能干细胞(iPSCs)，应用TALEN技术获得患者个性化的纠正突变的正常细胞，体外分化成中脑区域星形胶质细胞，研究PINK1点突变对星形胶质细胞功能的影响。同时，通过星形胶质细胞与多巴胺能神经元共培养，试图寻找来源于星形胶质细胞的，能影响神经元在细胞压力因子作用下的存活能力的因素，探讨非细胞自主因素在帕金森病理性改变的启动与发展过程中的重要性，为临床新药的筛选提供新的靶点。

帕金森病是临床上发病率第二的神经退行性疾病，炎症反应参与了帕金森病的发生、发展，但具体的作用机制尚不清晰。利用病人来源的诱导多能干细胞（iPSC）分化成疾病相关的细胞类型如多巴胺神经元、胶质细胞、巨噬细胞等可在体外模拟疾病发生，实现在人类基因背景下探索致病的发生机理。本项目围绕诱导多能干细胞模拟帕金森病的中心，以原有研究计划为导向并根据研究结果适时调整，建立了PINK1突变、PINK1和LRRK2分别敲除的多株iPSC，同时，也建立了多巴胺神经元、星形胶质细胞和巨噬细胞分化平台。利用这些技术平台，我们发现了LRRK2在巨噬细胞中可通过蛋白质翻译和自噬途径调控巨噬细胞和炎症反应。在基于iPSCs的疾病模型研究中，目前认为通过基因编辑产生具有相同遗传背景（isogenic）的不同基因型iPSC亚克隆是解决克隆间异质性的最佳方法。但是，关于这种相同遗传背景iPSCs疾病模型是否存在亚克隆间异质性、其异质性产生机制以及对疾病模拟的影响等问题，需进行科学系统的分析。本项目为了建立能够更客观准确再现人帕金森病病理、生物化学改变的星形胶质细胞模型，采用相对星形胶质细胞分化而言，分化时程较短的神经元为研究对象，通过转录组分析和生理功能分析，来回答了相同遗传背景iPSCs疾病模型是否存在亚克隆间异质性及其对疾病模型建立的影响这些科学问题。研究结果表明，同基因型的iPSC亚克隆存在异质性，这种异质性不能通过提高亚克隆来源神经元的分化效率而消除。而对不同遗传背景和重编程方法所得的iPSC克隆的多株亚克隆进行研究，验证了这种基因表达异质性的普遍存在性，并可部分体现在单细胞水平。进一步实验结果表明，相同基因背景iPSC亚克隆在基因表达、分化能力存在显著异质性。另外，通过CAT对氧化应激条件下细胞毒性实验结果的干扰例证了其中基因表达异质性对疾病模拟的干扰。上述isogenic iPSC亚克隆异质性的研究结果将有利于完善帕金森病星形胶质细胞模型的建立，推动非细胞自主性因素对帕金森病发生影响的研究。