title: Vesicular monoamine transporter 2 regulates the sensitivity of rat dopaminergic neurons to disturbed cytosolic dopamine levels.
abstract: An abnormal accumulation of cytosolic dopamine resulting in reactive oxygen species and dopamine-quinone products may play an important role in the rather selective degeneration of substantia nigra pars compacta (SNc) dopaminergic neurons in Parkinson's disease. The neuronal-specific vesicular monoamine transporter (VMAT2), responsible for uptake of dopamine into vesicles, has been shown to play a central role both in intracellular dopamine homeostasis and sequestration of dopaminergic neurotoxins. Direct or indirect enhancement of VMAT2 activity could therefore have neuroprotective effects by decreasing cytosolic dopamine levels. Here, we demonstrate that transfection of VMAT2 in the dopaminergic cell line, PC12, increases intracellular dopamine content, augments potassium-induced dopamine release and attenuates cell death induced by the cytosolic dopamine enhancer, methamphetamine, suggesting an enhancement in vesicular dopamine storage. In rat ventral mesencephalic cultures highly enriched for dopaminergic neurons, lentiviral delivery of recombinant VMAT2 using a neuronal-specific promoter also resulted in elevated intracellular dopamine content and neurotransmitter release after depolarization. The opposite was seen after downregulation of VMAT2 using virally delivered shRNAs. Furthermore, using this VMAT2 knockdown model, we are the first to report a direct link between enhanced cytoplasmic dopamine levels, measured following mild permeabilization of the plasma membrane using digitonin, and neurite degeneration in primary dopaminergic neurons. In conclusion, our data support the hypothesis that an increase in vesicular sequestration of dopamine by modulation of VMAT2 activity could restore neuronal function and enhance dopaminergic cell survival in conditions of dysregulated dopamine homeostasis such as Parkinson's disease.
authors.affiliation: Department of Molecular Biology, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark.
authors.forename: Sandra
authors.initials: S
authors.lastname: Vergo
authors.forename: Jens Leander
authors.initials: JL
authors.lastname: Johansen
authors.forename: Marcel
authors.initials: M
authors.lastname: Leist
authors.forename: Julie
authors.initials: J
authors.lastname: Lotharius
mesh_terms: D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D049109:Cell Proliferation; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D004347:Drug Interactions; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008694:Methamphetamine / Q000494:pharmacology; D016716:PC12 Cells / Q000166:cytology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D012110:Reserpine / Q000494:pharmacology; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000502:physiology*
chemical_list: D018759:Adrenergic Uptake Inhibitors; C493454:Slc18a2 protein, rat; D013778:Tetrazolium Salts; D013844:Thiazoles; D050493:Vesicular Monoamine Transport Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008694:Methamphetamine; D011189:Potassium Chloride; D012110:Reserpine; D007770:L-Lactate Dehydrogenase; D014446:Tyrosine 3-Monooxygenase; C022616:thiazolyl blue; D004298:Dopamine