Effect of Mutant α-Synuclein on Dopamine Homeostasis in a New Human Mesencephalic Cell Line

Abstract

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.

The phosphorylation state of Ser-129 in human α-synuclein determines neurodegeneration in a rat model of Parkinson disease

“…␣-syn has been implicated in a variety of cellular processes that include regulation of DA homeostasis through regulation of DA transporter activity (8), tyrosine hydroxylase activity (11), or vesicle formation (24). ␣-syn has also been implicated in ER-Golgi trafficking (25), tubulin trafficking (26), interaction with mitochondrial proteins (27), and control of nuclear transcription (12,28).…”

Dopamine and Parkinson's disease: is the killer in the house?

“…11 It is possible that genetic or environmental factors impinge on these processes, putting some individuals at higher risk for Parkinson's disease. The synergy between alpha-synuclein and dopamine to produce neurotoxicity may even be more direct: new evidence 12 indicates that a defective alpha-synuclein can alter the storage of dopamine in vesicle, thus directly increasing its concentration in the cytoplasm, hence its toxic potential, as suggested by the study by Xu et al 1 M-F Chesselet Department of Neurology, The Geffen School of Medicine at UCLA, CA, USA…”

$alpha;-Synuclein regulation of the dopaminergic transporter: a possible role in the pathogenesis of Parkinson's disease

“…2). 4. a-Synuclein and its A53T mutant modulate the amount of the vesicular transporter vesicular monoamine transporter in dopamine nerve terminals A striking aspect of our studies is the absence of any modulation by the A53T mutant of a-synuclein on DAT function. However, recent studies [20,43] suggest that the A53T mutant can modulate the vesicular monoamine transporter (VMAT2) [44]. In MESC2.10 human mesencephalic cells the presence of the A53T mutant decreased expression of VMAT2, accompanied by decreased potassium-induced, and increased amphetamine-induced, dopamine release [43].…”