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Abstract
Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron-derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.
Highlights
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by death of neurons in the substantia nigra [1] and atrophy in other brain regions [2, 3]
The sonicated a-synuclein fibrils were similar in appearance to those previously shown to induce PD-like pathology and neurodegeneration in mice [13]
To test whether toxicity was specific to aggregated a-synuclein in our model, recombinant monomeric a-synuclein was added in parallel with a-synuclein preformed fibril (PFF) for 24 h in SN4741 and STHdhQ7/ 7 cells
Summary
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by death of neurons in the substantia nigra [1] and atrophy in other brain regions [2, 3]. Toxin models of PD have been shown to cause cell death via ferroptosis [25], and, apart from iron elevation, additional features of ferroptosis, such as depleted GSH and elevated lipid peroxidation, are observed in PD-affected brains post-mortem [27,28,29]. It is not known whether extracellular a-synuclein causes cell death via ferroptosis, which we explore here. These findings are consistent with growing evidence for a pathogenic role of the lysosome in PD, with variants in genes important for endolysosomal function increasing the risk of sporadic and/or familial disease [30,31,32]
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