Abstract
BACKGROUND: A key factor in the propagation of α-synuclein pathology is the compromised protein quality control system. Variations in membrane association and astrocytic uptake between different α-synuclein forms suggest differences in exocytosis or membrane cleavage, potentially impacting the secretome's influence on dopaminergic neurons. We aimed to understand differences in protein degradation mechanisms of astrocytes for both wild-type (WT) and mutant forms of α-synuclein, specifically during periods of reduced degradation efficiency. We also investigated α-synuclein release into the secretome and its effects on healthy dopaminergic neurons. METHODS: Cellular models used were rat primary astrocytes alongside hiPSC-derived astrocytes, whose impact on rat primary dopaminergic neurons and the human SH-SY5Y cell line was investigated. We examined the release and accumulation of α-synuclein resulting from impaired degradatory pathways, including matrix metalloprotease-MMP9, the ubiquitin proteasomal pathway-UPS, and the autophagy-lysosomal pathway-ALP, using immunocytochemical analysis and flow cytometry. Additionally, we explored the effect of astrocytic secretome on dopaminergic-neuronal survival, neurite collapse and function. RESULTS: At early stages, astrocytes were able to deal efficiently with monomeric α-synuclein (via UPS), and larger aggregates (through MMP9 and autophagy), clearing extracellular α-synuclein and maintaining neuronal health. However, extended exposure to extracellular monomeric and aggregated α-synuclein compromised their proteasomal activity, inhibiting MMP9 and destabilizing autophagy, transforming astrocytes from protectors to promoters of neurodegeneration. This study is the first to elucidate the astrocytes' preferred degradation pathways for both monomeric and aggregated forms of α-synuclein, along with the subsequent effects of these payloads on the cellular degradation machinery. The astrocytic transformation is characterized by α-synuclein expulsion, increased release of inflammatory cytokines, and diminished secretion of growth factors leading to dopaminergic neuronal apoptosis and dysfunction, particularly neurite collapse, intracellular Ca(2+) response and vesicular dopamine release. The presence of phosphorylated and nitrated α-synuclein species in astrocytes also suggests their potential involvement in modifying both forms of the protein. CONCLUSION: The initial protective action of astrocytes in clearing and degrading extracellular α-synuclein is severely compromised at latter stages, leading to astrocytic dysfunction and impairing neuron-glia cross-talk. This study underscores the criticality of integrating astrocytes into treatment paradigms in synucleinopathies.