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.

Figure 1.. FFN206 packaging is dependent on VMAT2 function and maintenance of the vesicular proton gradient.

A. Representative 10x image of HEK cells stably transfected with mCherry-tagged VMAT2 (HEK+mCherry-VMAT2, left image) treated with FFN206 (center image). FFN206 fluorescence overlaps with VMAT2 fluorescence (purple, right image). B. Representative 60x TIRF microscope image demonstrates FFN206 fluorescence localized within vesicle-like compartments in a stably transfected HEK cell with VMAT2 (HEK+VMAT2), as denoted by red arrows. Scale bar = 10 μM. C. Representative images of FFN206 fluorescence in control HEK cells with and without VMAT2. In control HEK cells (top), there was no background fluorescence in the absence of FFN206 (left). FFN206 treatment exhibited minimal fluorescence (middle), and FFN206 fluorescence was unchanged following treatment with the VMAT2 inhibitor tetrabenazine (10 μM) (right). Representative images of FFN206 fluorescence in HEK+VMAT2 cells (bottom). In the absence of FFN206, no background fluorescence is observed in HEK+VMAT2 cells (left). HEK+VMAT2 cells treated with FFN206 exhibit robust fluorescence (middle) that was diminished when VMAT2 function is inhibited by treatment with 10 μM tetrabenazine (right). D. Quantification of FFN206 fluorescence from panel C. Data displayed as percent control mean and standard error of the mean, with the control group being HEK+VMAT2 cells incubated with FFN206. Each point represents one well of cells from a 96-well plate. (One-way ANOVA with Dunnett’s multiple comparisons post-hoc test **** p <0.0001 vs. control column HEK+VMAT2+FFN206).

Figure 2.. Real-time uptake of FFN206 in a single cell.

A. Sequential stills taken from a video of FFN206 uptake (blue fluorescence) into the vesicle-like compartments of a single HEK cell stably transfected with mCherry-tagged VMAT2 (red fluorescence). Still photos taken at 0.93s, 3.18s, 3.94s, and 5.94s following FFN206 application. Scale bar = 10 μM. Yellow arrows indicate image progression. B. Comparison of peak FFN206 fluorescence in the final 20 seconds of 1000 total seconds of uptake. HEK+VMAT2 cells display significantly greater fluorescence than HEK cells and HEK+VMAT2 cells treated with tetrabenazine or bafilomycin. Bars are displayed as mean and standard error of the mean. (One-way ANOVA with Dunnett’s multiple comparisons post-hoc test **** p <0.0001 vs. control column HEK+VMAT2).

Figure 3.. Determining optimal parameters for a FFN206 96-well plate assay.

A. Representative images of HEK+VMAT2 cells treated with 0, 0.1, 1, or 10 μM of FFN206. Cells imaged at 10x magnification. B. Representative images of HEK+VMAT2 cells treated with 0, 0.1, 1, and 10 μM of tetrabenazine (TBZ) and 1 μM FFN206. Cells imaged by EVOS system at 10x magnification. Scale bar = 400 μM. C. Quantification of fluorescence in 3A demonstrating FFN206 fluorescence increases as FFN206 dose increases. D. Quantification of fluorescence in 3B demonstrating FFN206 fluorescence decreases as tetrabenazine dose increases. E. Quantification of FFN206 fluorescence in HEK+VMAT2 cells treated with 1 μM FFN206 and 0 μM TBZ compared to cells treated with 1 μM FFN206 and 10 μM TBZ.

Figure 4.. Screening pharmacological inhibitors of VMAT2.

HEK+VMAT2 cells were treated with 0.0001, 0.001, 0.01, 0.1, 1, and 10 μM of tetrabenazine (TBZ); 0.0001, 0.001, 0.01, 0.1, 1, and 10 μM of reserpine; 0.01, 0.1, 1, 10, and 100 μM of methamphetamine; or 0.01, 0.1, 1, 10, and 100 μM of methylphenidate. FFN206 fluorescence represented as percent of control (HEK+VMAT2 cells treated with 0 μM drug). Graphs depict mean and standard error of the mean and curves represent non-linear regressions.

Figure 5.. Screening environmental toxicants for influence on VMAT2 function.

A. HEK+VMAT2 cells were treated with 0.01, 0.1, 1, 10 and 100 μM of the pesticides rotenone, paraquat, or chlorpyrifos. B. HEK+VMAT2 cells were treated with 0.01, 0.1, 1, 10 and 100 μM of the halogenated compounds unichlor, PFOS, Paroil, hexabromocyclododecane, or Aroclor 1260. FFN206 fluorescence represented as percent of control (HEK+VMAT2 cells treated with 0 μM drug). Graphs depict mean and standard error of the mean and curves represent non-linear regressions.