Mutations in ␣-synuclein have been linked to rare, autosomal dominant forms of Parkinson's disease. Despite its ubiquitous expression, mutant ␣-synuclein primarily leads to the loss of dopamine-producing neurons in the substantia nigra. ␣-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 ␣-synuclein on dopamine homeostasis. In addition to expressing markers of mature dopamine neurons, differentiated MESC2.10 cells are electrically active, produce dopamine, and express wildtype human ␣-synuclein. Lentivirus-induced overexpression of A53T mutant ␣-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 ␣-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 ␣-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 ␣-synuclein might lead to Parkinson's disease by triggering protracted, low grade dopamine toxicity resulting in terminal degeneration and ultimately cell death.

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