The role of astrocytes and microglia in Parkinson's disease pathogenesis in specific GBA N370S patient iPSC-derived neuro-glial co-cultures

帕金森病(PD)是第二种最常见的神经退行性疾病。迄今为止，对帕金森病发病机制的研究大多集中在多巴胺能神经元，但最近的研究结果表明，星形胶质细胞和小胶质细胞在帕金森病的发生和病理生理中也起着至关重要的作用。患者诱导的多能干细胞(IPSCs)能够产生功能性DA神经元、星形胶质细胞和携带PD相关突变的小胶质细胞，这有助于研究PD改变的不同细胞过程。此外，IPSC来源的神经胶质细胞共培养比IPSC来源的单一培养或原代培养更具代表性的人类模型，因为它们更接近人脑的细胞环境。因此，本项目的第一部分将使用混合对照IPSC和PD IPSC来源的DA神经元和星形胶质细胞共同培养来研究星形胶质细胞在PD发病机制中的不同过程中的作用。为了达到这一目的，将研究星形胶质细胞的多种细胞机制，如自噬-溶酶体降解途径、线粒体功能和钙活性。此外，还将分析α-突触核蛋白在DA神经元和星形胶质细胞之间的转移，以及这种转移如何影响这两种细胞类型。最后，将研究神经炎症的不同方面，特别是星形胶质细胞获得反应性或神经保护性表型，以及促炎细胞因子或神经营养因子的释放及其对神经元的影响。项目的第二部分的目标将是研究小胶质细胞如何与星形胶质细胞和DA神经元相互作用并影响帕金森病的发病。为此，将使用IPSC来源的神经元、星形胶质细胞和小胶质细胞的混合三种培养方法，其中将进行类似于神经元和星形胶质细胞共培养的分析，以研究不同细胞类型中不同的细胞过程可能发生的变化。同样，将研究小胶质细胞在神经炎症中的参与，特别是小胶质细胞的吞噬和炎症反应以及小胶质细胞释放的促炎细胞因子对星形胶质细胞的激活。本项目将使用的PD iPSC克隆系来自携带GBA N370S突变的患者。此外，将以N370S突变已被纠正的等基因校正的IPSC系作为对照，以及来自健康个体的IPSC系作为对照。使用IPSC衍生的混合共培养将能够深入研究这种特定的突变如何影响不同的细胞类型，以及突变的细胞类型如何可能对健康的细胞类型产生负面影响，以及后者如何对患病的细胞类型产生积极的影响。该项目不仅将对星形胶质细胞和小胶质细胞在帕金森病发生发展中的作用提供新的、更准确的见解，而且还将能够优化产生和使用IPSC衍生的神经胶质细胞共培养的方案。所获得的结果还将有助于确定潜在的治疗靶点，这将使新的治疗策略得以开发和测试。

Parkinson's disease (PD) is the second most common neurodegenerative disorder. To date, most studies investigating the mechanisms driving PD pathogenesis have focused on dopaminergic (DA) neurons, but recent results have shown that astrocytes and microglia also play crucial roles in its development and pathophysiology. Patient-derived induced pluripotent stem cells (iPSCs) enable the production of functional DA neurons, astrocytes and microglia carrying specific PD- associated mutations, which has facilitated the investigation of the different cellular processes altered in PD. Moreover, iPSC-derived neuro-glial co-cultures are much more representative human models of PD than iPSC-derived monocultures or primary cultures, as they more closely recapitulate the cellular milieu of the human brain.Therefore, the first part of this project will be to study the role of astrocytes in different processes of PD pathogenesis, using mixed control iPSC and PD iPSC-derived DA neuronal and astrocytic co- cultures. To approach this, multiple cellular mechanisms will be studied in astrocytes, such as the autophagosome-lysosome degradation pathways, mitochondrial function, and calcium activity. Furthermore, the transfer of alpha-synuclein between DA neurons and astrocytes will also be analysed, as well as how this transfer affects both cell types. Lastly, different aspects of neuroinflammation will be investigated, with a particular focus on the acquisition of either a reactive or neuroprotective phenotype by astrocytes, and the release of pro-inflammatory cytokines or neurotrophic factors and their effect on neurons.The objective of the second part of the project will be to study how microglial cells interact with astrocytes and DA neurons and affect PD pathogenesis. In order to do so, mixed iPSC-derived neuronal, astrocytic and microglial tri-cultures will be used, where a similar analysis to the one conducted with the neuron and astrocyte co-culture will be done to study how different cellular processes might be altered in the different cell types. Similarly, the involvement of microglia in neuroinflammation will be investigated, particularly focusing on microglial phagocytosis and inflammatory response and the activation of astrocytes by the pro-inflammatory cytokines released by microglia.The PD iPSC clonal lines that will be used in this project are derived from patients carrying the GBA N370S mutation. Furthermore, isogenic gene-corrected iPSC lines will be used as controls, in which the N370S mutation has been corrected, as well as iPSC lines derived from healthy individuals. The use of mixed iPSC-derived co-cultures will enable to study in depth how this particular mutation affects different cell types, as well as how a mutated cell type might negatively affect a healthy cell type and how the latter can positively affect the diseased cell type.This project will not only give new and more accurate insight into the role of astrocytes and microglia in the development of PD, but it will also enable to optimize the protocols for the generation and use of iPSC-derived neuro-glial co-cultures. The results obtained will also help identify potential therapeutic targets, which will enable new treatment strategies to be developed and tested.