RESUMO
Parkinson's disease (PD) is recognized as the second most common neurodegenerative disorder and has affected approximately one million people in the United States alone. A large body of evidence has suggested that deposition of aggregated alpha-synuclein (α-Syn), a brain protein abundant near presynaptic termini, in intracellular protein inclusions (Lewy bodies) results in neuronal cell damage and ultimately contributes to the progression of PD. However, the exact mechanism is still unclear. One hypothesis is that α-Syn aggregates disrupt the cell membrane's integrity, eventually leading to cell death. We used scanning ion conductance microscopy (SICM) to monitor the morphological changes of SH-SY5Y neuroblastoma cells and observed dramatic disruption of the cell membrane after adding α-Syn aggregates to the culturing media. This work demonstrates that SICM can be applied as a new approach to studying the cytotoxicity of α-Syn aggregates.
Assuntos
Membrana Celular/patologia , Agregação Patológica de Proteínas/patologia , alfa-Sinucleína/metabolismo , Morte Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Microscopia Eletroquímica de Varredura , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/metabolismo , alfa-Sinucleína/análiseRESUMO
Aggregation of the natively unfolded protein α-synuclein (α-Syn) has been widely correlated to the neuronal death associated with Parkinson's disease. Mutations and protein overaccumulation can promote the aggregation of α-Syn into oligomers and fibrils. Recent work has suggested that α-Syn oligomers can permeabilize the neuronal membrane, promoting calcium influx and cell death. However, the mechanism of this permeabilization is still uncertain and has yet to be characterized in live cells. This work uses scanning ion conductance microscopy (SICM) to image, in real time and without using chemical probes, the topographies of live SH-SY5Y neuroblastoma cells after exposure to α-Syn oligomers. Substantial morphological changes were observed, with micrometer-scale hills and troughs observed at lower α-Syn concentrations (1.00 µM) and large, transient pores observed at higher α-Syn concentrations (6.0 µM). These findings suggest that α-Syn oligomers may permeabilize the neuronal membrane by destabilizing the lipid bilayer and opening transient pores.