| Summary: | <p>Parkinson’s disease (PD), the second most common neurodegenerative disease, is characterized by the loss of dopaminergic neurons (DAn) in the substantia nigra. However, the research available indicates that other cells found in the midbrain, such as astrocytes, are also involved in the disease pathogenesis, although their role has not been fully elucidated yet. Moreover, it is still to be determined whether the neuroinflammation found in PD patients has a causative effect in disease onset or is in turn a consequence of neurodegeneration. Similarly, the currently available data regarding the effects of α-synuclein in human astrocytes remains inconclusive. Therefore, the aim of this thesis was to investigate the role of astrocytes in PD pathogenesis, focusing on cells carrying the PD-associated <em>GBA-N370S</em> mutation. In addition, the response of astrocytes to a range of inflammatory cytokines and α-synuclein pre- formed fibrils (PFFs) was extensively investigated, as well as the impact of reactive astrocytes on DAn.</p>
<p>To generate a representative in vitro model of midbrain cells, induced pluripotent stem cells (iPSC) obtained from healthy donors and PD patients carrying the <em>GBA-N370S</em> mutation were derived into dopaminergic neurons (iDAn) and midbrain-specific astrocytes (iASTROs). An in- depth characterization of iASTROs demonstrated their regional and astrocytic identity and their functional maturity. Overall, throughout the experiments conducted in this thesis, very few cell-autonomous alterations were observed in <em>GBA-N370S</em> iASTROs, and no significant genotypic differences were observed regarding the response of iASTROs to inflammatory cytokines and PFFs. Nonetheless, <em>GBA-N370S</em> iASTROs displayed differences in calcium signalling and a higher production of reactive oxygen species. Additionally, a direct iDAn- iASTRO co-culture was established, optimized, and used to investigate the effects of the <em>GBA-N370S</em> mutation on iDAn-iASTRO homeostasis. Whereas control and <em>GBA-N370S</em> iASTROs presented similar neurosupportive characteristics, <em>GBA-N370S</em> iDAn elicited a different response in iASTROs compared to control iDAn.</p>
<p>Furthermore, bulk RNAseq data of control iASTROs treated with inflammatory cytokines and PFFs for 24h revealed very extensive transcriptomic changes in response to inflammatory cytokines but not PFFs, a finding that was verified with subsequent immunocytochemistry and RT-qPCR experiments conducted after 24h and 7 days of treatment. Various genes related to calcium signalling and homeostasis, as well as vitamin D metabolism, were upregulated in iASTROs after exposure to TNFα+IFNγ. Further in vitro experiments showed the timescale and magnitude of the increases in calcium signalling in reactive control and <em>GBA-N370S</em> iASTROs, but partial blockage of endoplasmic reticulum calcium release did not result in changes in iASTROs reactivity. Moreover, even if <em>CYP27B1</em> and <em>VDR</em> expression and nuclear translocation were elevated in reactive iASTROs, vitamin D supplementation in iDAn did not result in changes in cell viability.</p>
<p>Lastly, to investigate if iASTROs treated with inflammatory cytokines presented neurotoxic or neuroprotective characteristics, experiments in iDAn-iASTRO co-cultures were conducted. Pre-treatment of iASTROs with TNFα+IFNγ before the co-cultures did not affect their neurosupportive capacity, whereas some of the data suggests that when treating and maintaining the co-cultures with cytokines, control and <em>GBA-N370S</em> iASTROs became neurotoxic and/or lost neurosupportive functions. In the case of co-cultures treated with TNFα+IFNγ, this neurotoxic activity was potentiated with the addition of PFFs, suggesting that PFFs modulate the reactive response of iASTROs to inflammatory cytokines.</p>
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