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 (IC 50 : 73.1 nM), tetrabenazine (IC 50 : 30.4 nM), methamphetamine (IC 50 : 2.4 μM), and methylphenidate (IC 50 : 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.

Inhibition of VMAT2 by β2-adrenergic agonists, antagonists, and the atypical antipsychotic ziprasidone

“…Two hit compounds, SMT and ZPS had IC 50 values of 53 ± 28 and 39 ± 17 nM, respectively, a potency comparable to that of the specific VMAT2 inhibitor TBZ, i.e., IC 50 = 37 ± 28 nM in our assay. The IC 50 measured for TBZ, and the SSRI fluoxetine (1.98 ± 0.25 μM) is comparable to previously determined IC50 values 32 , 37 , 39 , 40 . Formoterol, a β 2 -AR antagonist, had an IC 50 of 0.56 ± 0.31 μM and several other stabilizing and destabilizing compounds showed inhibition in the range 1–4 μM (Table 1 ).…”

The dopamine membrane transporter plays an active modulatory role in synaptic dopamine homeostasis

“…Direct measurement of synaptic dopamine levels is not feasible with currently available technology. Advances in synthetic compounds such as false fluorescent neurotransmitters that can mimic dopamine and can be packaged into vesicles by the vesicular monoamine transporter (Black et al., 2020; Pereira et al., 2016) hold potential for direct measurements of vesicular contents in the future. Surrogate tools such as genetically encoded or chemical indicators for calcium to monitor Ca 2+ influx (Tian et al., 2009) have the potential to bolster conclusions derived from the FRAP and behavioral approaches reported in the current study.…”

Vitamin D: A potent regulator of dopaminergic neuron differentiation and function

“…Molecules such as FFN200 are fluorescent DA derivatives that are taken up into cells and sequestered by any cell containing the vesicular monoamine transporter VMAT2. To date, FFN200 has been used to visualise DA release in striatal slices, cultured mesencephalic DA neurons, human kidneys and PC12 cells (Black et al, 2021; Hu et al, 2019; Pereira et al, 2016). To the best of our knowledge, our group is the 1st to report the use of this agent in differentiated SH‐SY5Y neuroblastoma cells.…”