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）是第二常见的神经退行性疾病。迄今为止，大多数研究PD发病机制的研究都集中在多巴胺能（DA）神经元上，但最近的结果表明，星形胶质细胞和小胶质细胞在其发育和病理生理中也起着至关重要的作用。患者来源的诱导多能干细胞（iPSC）能够产生携带特定PD相关突变的功能性DA神经元、星形胶质细胞和小胶质细胞，这促进了对PD中改变的不同细胞过程的研究。此外，iPSC衍生的神经胶质细胞共培养物比iPSC衍生的单一培养物或原代培养物更能代表PD的人类模型，因为它们更接近地概括了人脑的细胞环境。因此，本项目的第一部分将是使用混合对照iPSC和PD iPSC衍生的DA神经元和星形胶质细胞共培养物来研究星形胶质细胞在PD发病机制的不同过程中的作用。为了接近这一点，将在星形胶质细胞中研究多种细胞机制，例如自噬体-溶酶体降解途径、线粒体功能和钙活性。此外，还将分析DA神经元和星形胶质细胞之间的α-突触核蛋白转移，以及这种转移如何影响两种细胞类型。最后，将研究神经炎症的不同方面，特别关注星形胶质细胞的反应性或神经保护性表型的获得，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。该项目的第二部分的目标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PD发病机制。为了做到这一点，将使用混合的iPSC衍生的神经元、星形胶质细胞和小胶质细胞三培养物，其中将进行与用神经元和星形胶质细胞共培养物进行的分析类似的分析，以研究不同细胞类型中不同细胞过程可能如何改变。类似地，将研究小胶质细胞在神经炎症中的参与，特别关注小胶质细胞吞噬作用和炎症反应以及由小胶质细胞释放的促炎细胞因子对星形胶质细胞的激活。此外，将使用等基因基因校正的iPSC系作为对照，其中N370 S突变已被校正，以及来自健康个体的iPSC系。使用混合iPSC衍生的共培养物将能够深入研究这种特定突变如何影响不同的细胞类型，以及突变的细胞类型如何对健康细胞类型产生负面影响，后者如何对患病细胞类型产生积极影响。该项目不仅将为星形胶质细胞和小胶质细胞在PD发展中的作用提供新的和更准确的见解，而且还能够优化用于产生和使用iPSC衍生的神经胶质共培养物的方案。所获得的结果还将有助于确定潜在的治疗靶点，从而能够开发和测试新的治疗策略。

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.