Abstract

Multiple system atrophy (MSA) is a fatal neurodegenerative synucleinopathy characterized by the accumulation of α-synuclein in oligodendrocytes, forming glial cytoplasmic inclusions. Although iron dysregulation and ferroptosis, an iron-dependent form of regulated cell death, have been implicated in neurodegeneration, their specific role in MSA oligodendrocytes remains unknown. We investigated ferroptosis pathways in postmortem brain tissues from patients with MSA, Parkinson’s disease (PD), and healthy controls (HCs) by immunofluorescence for GPX4 co-labelled with CNPase (oligodendrocyte marker) or TH (dopaminergic neuron marker). To validate these findings, we employed PLP-hαSyn transgenic mice, an established MSA model, and the human oligodendrocytic cell line MO3.13, subjected to α-synuclein over-expression, preformed fibrils (PFF) exposure, and brain homogenates derived from MSA pons. Mechanistic insights were pursued through immunofluorescence, JC-1, FerroOrange, western blotting, and co-immunoprecipitation. Finally, we developed a novel biomarker assay using nanoscale flow cytometry to quantify FTH1-containing, CNPase-positive (oligodendrocyte-derived) EVs (ODFC-EVs) in plasma samples from 49 MSA patients, 46 PD patients, and 48 HCs. GPX4, the key ferroptosis regulator, was significantly reduced in CNPase + oligodendrocytes of MSA brains versus PD and HCs, while, as expected, GPX4 loss in PD predominated in TH + neurons. PLP-hαSyn mice recapitulated the unique GPX4 suppression in oligodendrocytes. In MO3.13 cells, α-synuclein enhanced erastin-induced GPX4 loss, increased labile Fe 2+ accumulation and aggravated mitochondrial depolarisation. Mechanistically, α-synuclein was found to directly bind and stabilize NCOA4, impairing its ubiquitination-mediated degradation. This enhanced NCOA4 activity drove excessive ferritinophagy, leading to the lysosomal degradation of the iron-storage protein FTH1 and subsequent iron overload. Translationally, plasma levels of