Assessing Vesicular Monoamine Transport and Toxicity Using Fluorescent False Neurotransmitters.

Abstract

Impairments in the vesicular packaging of dopamine result in an accumulation of dopamine in the cytosol. Cytosolic dopamine is vulnerable to two metabolic processes-enzymatic catabolism and enzymatic- or auto-oxidation-that form toxic metabolites and generate reactive oxygen species. Alterations in the expression or activity of the vesicular monoamine transporter 2 (VMAT2), which transports monoamines such as dopamine from the cytosol into the synaptic vesicle, result in dysregulated dopamine packaging. Here, we developed a series of assays using the fluorescent false neurotransmitter 206 (FFN206) to visualize VMAT2-mediated vesicular packaging at baseline and following pharmacological and toxicological manipulations. As a proof of principle, we observed a significant reduction in vesicular FFN206 packaging after treatment with the VMAT2 inhibitors reserpine (IC50: 73.1 nM), tetrabenazine (IC50: 30.4 nM), methamphetamine (IC50: 2.4 μM), and methylphenidate (IC50: 94.3 μM). We then applied the assay to investigate the consequences on vesicular packaging by environmental toxicants including the pesticides paraquat, rotenone, and chlorpyrifos, as well as the halogenated compounds unichlor, perfluorooctanesulfonic acid, Paroil, Aroclor 1260, and hexabromocyclododecane. Several of the environmental toxicants showed minor impairment of the vesicular FFN206 loading, suggesting that the toxicants are weak VMAT2 inhibitors at the concentrations tested. The assay presented here can be applied to investigate the effect of additional pharmacological compounds and environmental toxicants on vesicular function, which will provide insight into how exposures to such factors are involved in the pathogenesis of monoaminergic diseases such as Parkinson's disease, and the assay can be used to identify pharmacological agents that influence VMAT2 activity.