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

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.