Proteomics of neuroinflammatory signalling: illuminating cellular mechanisms driving neurodegenerative diseases

Alzheimer"s (AD) and Parkinson"s disease (PD) are prevalent neurodegenerative diseases, but their underlying mechanisms remain poorly understood. Emerging evidence suggests that neuroinflammation, characterized by the release of pro-inflammatory cytokines like IL-1β, TNF-α, and INF-γ, plays a pivotal role in these diseases, challenging the conventional focus on protein aggregates (Aβ, αSyn). In AD, it has been shown that elevated IL-1β levels induce the translocation of NEDD8 from the nucleus to the cytoplasm, where it co-localizes with the E3-ligase Parkin. Together with the kinase PINK1, Parkin is crucial for the removal of defective mitochondria, with loss-of-function mutations in these genes being the primary cause of early-onset PD. Intriguingly, phosphorylated NEDD8 can activate Parkin, implicating a more complex interconnection between neuroinflammation, NEDD8-and Parkin-regulated phosphorylation-dependent signaling in brain cells. Yet, a mechanistic understanding of cellular processes triggered by neuroinflammation remain elusive.

This study aims to investigate spatiotemporal (phospho-)proteome changes in response to neuroinflammatory stress elicited by pro-inflammatory cytokines and αSyn fibrils. To achieve this, spatial-proteomics using subcellular fractionation is combined with high-sensitivity narrow-window data-independent acquisition (nDIA) using the Orbitrap Astral mass spectrometer. This workflow resulted in identification and quantification of ⁓8000 protein groups and between ⁓6000 and 15,000 localized phosphorylation sites per subcellular fraction. IL-1β-dependent subcellular relocation of hundreds of (phospho)proteins was determined in initial analyses of neuronal cell line models. This strategy will be extended to a more physiologically-relevant model system of hPSC derived neurons, astrocytes, microglia, and co-cultures thereof. This system enhances the translational relevance by mimicking neuroinflammation in the complex tissue architecture of the brain. The data obtained reveals the cellular protein abundance changes and phosphorylation site regulation induced under neuroinflammatory stress. This study aspires to unravel the cellular mechanisms underlying neuroinflammation, potentially unlocking new avenues for therapeutic strategies against neurodegenerative diseases.