Effect of mutant α-synuclein on dopamine homeostasis in a new human mesencephalic cell line

Abstract

Mutations in α-synuclein have been linked to rare, autosomal dominant forms of Parkinson's disease. Despite its ubiquitous expression, mutant α-synuclein primarily leads to the loss of dopamine-producing neurons in the substantia nigra. α-Synuclein is a presynaptic nerve terminal protein of unknown function, although several studies suggest it is important for synaptic plasticity and maintenance. The present study utilized a new human mesencephalic cell line, MESC2.10, to study the effect of A53T mutant α-synuclein on dopamine homeostasis. In addition to expressing markers of mature dopamine neurons, differentiated MESC2.10 cells are electrically active, produce dopamine, and express wild-type human α-synuclein. Lentivirus-induced overexpression of A53T mutant α-synuclein in differentiated MESC2.10 cells resulted in down-regulation of the vesicular dopamine transporter (VMAT2), decreased potassium-induced and increased amphetamine-induced dopamine release, enhanced cytoplasmic dopamine immunofluorescence, and increased intracellular levels of superoxide. These results suggest that mutant α-synuclein leads to an impairment in vesicular dopamine storage and consequent accumulation of dopamine in the cytosol, a pathogenic mechanism that underlies the toxicity of the psychostimulant amphetamine and the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium. Interestingly, cells expressing A53T mutant α-synuclein were resistant to amphetamine-induced toxicity. Because extravesicular, cytoplasmic dopamine can be easily oxidized into reactive oxygen species and other toxic metabolites, mutations in α-synuclein might lead to Parkinson's disease by triggering protracted, low grade dopamine toxicity resulting in terminal degeneration and ultimately cell death.