V63 and N65 of overexpressed α-synuclein are involved in mitochondrial dysfunction

• V63A and N65A mutations restored mitochondrial complex I function and Δψm.
• V63A and N65A mutations inhibited mPTP opening and mitochondrial Ca2+ overload.
• V63A and N65A may modulate the localization of α-Syn in mitochondria.

Parkinson's Disease (PD) is one of the most common neurodegenerative diseases. α-Synuclein (α-Syn)—encoded by SNCA, the first-identified PD-related gene—is the main component of Lewy bodies, which are a pathological hallmark of PD. We previously reported that α-Syn accumulates in mitochondria in PD, causing mitochondrial abnormalities and disrupting mitochondrial membrane potential (Δψm) and mitochondrial potential transition pore (mPTP) opening by interacting with the voltage-dependent anion channel (VDAC) and adenine nucleotide translocator. However, the mechanistic basis of mitochondrial impairment caused by α-Syn has yet to be elucidated. It has been suggested that the amino acid residues Q62, V63, and N65 of α-Syn are important for the interaction of the protein with membranes. To investigate whether this underlies the mitochondrial dysfunction induced by α-Syn overexpression, we mutated these residues to alanine and transfected HEK293T and MN9D cells with the mutated forms of α-Syn protein. The V63A and N65A mutations prevented mitochondrial Ca2+ overload and Δψm dysregulation as well as complex I inactivation and reactive oxygen species production while blocking mPTP opening and caspase 9 activation, possibly by reducing α-Syn accumulation in mitochondria. These results indicate that V63 and N65 are critical residues mediating mitochondrial inactivation. These findings provide novel insight into the molecular events contributing to PD pathogenesis.

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