Abstract
ABSTRACTAggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca2+ signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca2+ in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca2+ signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca2+, which prevented the α-synuclein-induced Ca2+ dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca2+ signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca2+ dysregulation and Ca2+-dependent cell death.
Highlights
Neurodegenerative diseases share a common pathological process of misfolding of a native monomeric protein into a range of intermediate oligomeric structures, and polymerising into insoluble amyloid fibrils and depositing in the brain as inclusions
Aggregation proceeds, oligomers consisting of both the Alexa Fluor 488 (AF488)– α-synuclein and the Alexa Fluor 594 (AF594)–α-synuclein will form, and, as the AF488–α-synuclein acts as a FRET donor, and the AF594–αsynuclein as a FRET acceptor, we can use single-molecule confocal FRET analysis to differentiate the oligomeric αsynuclein from the rest of the monomeric protein
Ca2+ signalling and toxicity Given that Ca2+ dysregulation is known to be a key player in cell death induction, we investigated whether the α-synuclein-induced Ca2+ signal leads to neurotoxicity and cell death
Summary
Neurodegenerative diseases share a common pathological process of misfolding of a native monomeric protein into a range of intermediate oligomeric structures, and polymerising into insoluble amyloid fibrils and depositing in the brain as inclusions. In Parkinson’s disease, it is the native protein α-synuclein that undergoes self-aggregation and deposition in Lewy bodies (Cookson, 2009). Extensive evidence suggests that the smaller and soluble intermediate products of α-synuclein aggregation (termed oligomers) are likely to be the pathogenic culprits of disease (Kalia et al, 2013). Monomeric α-synuclein initially forms oligomers (2mer– 50mers) that are unstructured, degradable and non-toxic to cells. These undergo a conversion into highly compact and β-sheet structured oligomers that are stable and toxic to cells (Cremades et al, 2012).
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