Increased Vesicular Monoamine Transporter 2 (VMAT2; <i>Slc18a2</i>) Protects against Methamphetamine Toxicity

The psychostimulant methamphetamine (METH) is highly addictive and neurotoxic to dopamine terminals. METH toxicity has been suggested to be due to the release and accumulation of dopamine in the cytosol of these terminals. The vesicular monoamine transporter 2 (VMAT2; <i>SLC18A2</i>) is a critical mediator of dopamine handling. Mice overexpressing VMAT2 (VMAT2-HI) have an increased vesicular capacity to store dopamine, thus augmenting striatal dopamine levels and dopamine release in the striatum. Based on the altered compartmentalization of intracellular dopamine in the VMAT2-HI mice, we assessed whether enhanced vesicular function was capable of reducing METH-induced damage to the striatal dopamine system. While wildtype mice show significant losses in striatal levels of the dopamine transporter (65% loss) and tyrosine hydroxylase (46% loss) following a 4 × 10 mg/kg METH dosing regimen, VMAT2-HI mice were protected from this damage. VMAT2-HI mice were also spared from the inflammatory response that follows METH treatment, showing an increase in astroglial markers that was approximately one-third of that of wildtype animals (117% vs 36% increase in GFAP, wildtype vs VMAT2-HI). Further analysis also showed that elevated VMAT2 level does not alter the ability of METH to increase core body temperature, a mechanism integral to the toxicity of the drug. Finally, the VMAT2-HI mice showed no difference from wildtype littermates on both METH-induced conditioned place preference and in METH-induced locomotor activity (1 mg/kg METH). These results demonstrate that elevated VMAT2 protects against METH toxicity without enhancing the rewarding effects of the drug. Since the VMAT2-HI mice are protected from METH despite higher basal dopamine levels, this study suggests that METH toxicity depends more on the proper compartmentalization of synaptic dopamine than on the absolute amount of dopamine in the brain

Dryad-Lohr-etal-excelFSCV

This is an excel spreadsheet that shows all of the raw data collected for the fast scan cyclic voltammetry experiments. Tabs are used to delineate the various experiments

Dryad-Lohr-etal-westerns

These are all of the original images of the western blots in this manuscript. They are in a powerpoint file

In silico and in vitro characterization of nuciferine.

<p>In silico and in vitro characterization of nuciferine.</p

DAT modulation by nuciferine.

<p>(a) Concentration-response curves of nuciferine on vesicular uptake in isolated vesicles, (b) concentration-response curves of nuciferine on vesicular uptake in HEK cells transfected with DAT and VMAT2, (c) concentration response curves of nuciferine on vesicular uptake in HEK cells transfected with DAT. Data are presented as a percentage of the response to vehicle control. The data represent concentration-response curves of normalized data with respect to vehicle performed in triplicate (mean +/- s.e.m.; n = 3).</p

PPI responses to nuciferine in mouse models of hypoglutamatergia and hyperdopaminergia.

<p>Nuciferine rescued PPI in the former, but not in the latter model. (a-c) Null activity (a), startle activity (b), and PPI (c) for C57BL/6J mice treated with vehicle (Veh), 5 or 10 mg/kg nuciferine (Nuc), and/or phencyclidine (PCP). (d-g) Null activity (d), startle activity (e), and PPI for wild-type (WT) (f) and dopamine transporter knockout (DAT-KO) mice (g) given Veh, 2 mg/kg clozapine (CLZ) or 2.5–10 mg/kg Nuc. N = 8–17 mice/group in the C57BL/6J experiment; *<i>p</i><0.05, compared to Veh/Nuc groups; +<i>p</i><0.05, compared to the Veh and PCP groups; †<i>p</i><0.05, compared to all other groups. N = 9–17 mice/genotype/treatment in the DAT experiment; ^<i>p</i><0.05, WT versus KO within dose; #<i>p</i><0.05, dose effect within genotype; &<i>p</i><0.05, overall drug effect regardless of genotype.</p

Comparison of the polypharmacology of nuciferine with atypical and typical antipsychotics.

<p>The empirical affinity values of three antipsychotics are shown (clozapine, haloperidol, aripiprazole). The empirical nuciferine affinity profile from this study is shown in comparison. Values for clozapine, haloperidol, and aripiprazole compiled from data available on the PDSP website accessed 20150428. Only “PDSP verified” data was used for the figure. Data entries of “>10,000” were entered as 10,000 μM.</p

Bioinformatic predictions of lotus phytochemicals.

<p>X axis = number of unique predicted targets, as predicted by SEA. Each unique target included all affinity classes. Y axis = prediction of blood brain barrier penetration using the online blood-brain barrier prediction (BBB) server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150602#pone.0150602.ref017" target="_blank">17</a>] (<a href="http://www.cbligand.org/BBB/" target="_blank">http://www.cbligand.org/BBB/</a>). Higher values are predicted to pass the blood brain barrier. Size of circles = mean of -log10(e-value) for SEA-predicted targets. Larger circles indicate stronger confidence of predicted targets. Color of circles = Mean of max T values. Warmer colors (red) indicate better molecule-molecule matching, a second measure of prediction confidence. Y axis reference line (0.0815) and X axis reference line (17) are the average value for the world’s most widely prescribed psychiatric medications (aripiprazole and quetiapine).[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150602#pone.0150602.ref074" target="_blank">74</a>] These predictions suggest that nuciferine and its metabolites (O-nornuciferine, lirinidine) may be responsible for the psychotropic effects reported in humans. Chemical structures of nuciferine, O-nornuciferine, and lirinidine provided.</p

Nuciferine functional activity at the dopamine D₂ receptor as measured via D₂-mediated G_i signaling in HEKT cells.

<p>(a) Concentration-response curve of nuciferine (red) compared to dopamine (black) and aripiprazole (blue). (b) Concentration-response curve of dopamine (DA) in the presence of 1 μM nuciferine (red) or clozapine (green) showing rightward-shift of DA indicative of competitive antagonist activity. The data represent concentration-response curves of normalized data with respect to dopamine performed in triplicate (mean +/- s.e.m.; n = 3).</p

Locomotor studies of nuciferine.

<p>(a) Nuciferine suppresses the PCP-induced hyperlocomotor response. Data are presented as total distance travelled in 5-minute bins (left) and as cumulative distance travelled between minute 45 and minute 60 (right). N = 18 mice; ^p<0.05, compared to PCP group. (b) Nuciferine (3.0 mg/kg, 15 minute pretreatment) enhances the hyperlocomotor effect of amphetamine (3.0 mg/kg) administration. N = 14 mice; * < 0.001 compared to Amphetamine group. Data are presented as above.</p