Role of VDAC Isoforms in Alpha-Synuclein Entry into Mitochondria

Abstract

Parkinson's disease (PD) patients show loss of neurons, mitochondrial dysfunction, and the presence of Lewy bodies. Lewy bodies are composed of misfolded α-synuclein, which is a risk factor in both familial and sporadic PD. Studies have shown that α-synuclein can cause mitochondrial dysfunction and increase reactive oxygen species production. This has been attributed to the interaction of α-synuclein with complexes of the electron transport chain in the inner mitochondrial membrane, but it is not clear how α-synuclein crosses the outer membrane to enter the mitochondria. In neurons, α-synuclein has been shown to bind Voltage-Dependent Anion Channel (VDAC), an outer mitochondrial membrane protein that shuttles metabolites between the cytosol and the mitochondria. Also, single-channel electrophysiology experiments show that monomeric α-synuclein can transiently block and translocate through all VDAC isoforms. However, the three VDAC isoforms in mammals — VDAC1, VDAC2, and VDAC3 that form channels of almost identical conductance and anion selectivity —show distinctly different interaction with α-synuclein in experiments with reconstituted VDACs. To understand the mechanism of α-synuclein translocation into mitochondria in live cells, we performed Förster Resonance Energy Transfer (FRET) microscopy using Alexa488-α-synuclein and VDAC1, 2 or 3 -GFP. The change in FRET signal as α-synuclein enters VDAC pore and translocates through it was used to visualize α-synuclein entry into the mitochondria. The contribution of each isoform in α-synuclein translocation was studied by knocking down individual VDAC isoforms in live cells. Elucidating the mechanism of α-synuclein entry into mitochondria and, in particular, the role of each VDAC isoform in α-synuclein translocation is crucial for understanding of the impact of α-synuclein in mitochondrial dysfunction and the ways to ameliorate mitochondrial function at the early stages of neurodegeneration.