Kappa Opioid Receptor Antagonism Restores Phosphorylation, Trafficking and Behavior induced by a Disease Associated Dopamine Transporter Variant

Abstract

Aberrant dopamine (DA) signaling is implicated in schizophrenia, bipolar disorder (BPD), autism spectrum disorder (ASD), substance use disorder, and attention-deficit/hyperactivity disorder (ADHD). Treatment of these disorders remains inadequate, as exemplified by the therapeutic use of d-amphetamine and methylphenidate for the treatment of ADHD, agents with high abuse liability. In search for an improved and non-addictive therapeutic approach for the treatment of DA-linked disorders, we utilized a preclinical mouse model expressing the human DA transporter (DAT) coding variant DAT Val559, previously identified in individuals with ADHD, ASD, or BPD. DAT Val559, like several other disease-associated variants of DAT, exhibits anomalous DA efflux (ADE) that can be blocked by d-amphetamine and methylphenidate. Kappa opioid receptors (KORs) are expressed by DA neurons and modulate DA release and clearance, suggesting that targeting KORs might also provide an alternative approach to normalizing DA-signaling disrupted by perturbed DAT function. Here we demonstrate that KOR stimulation leads to enhanced surface trafficking and phosphorylation of Thr53 in wildtype DAT, effects achieved constitutively by the Val559 mutant. Moreover, these effects can be rescued by KOR antagonism of DAT Val559 in ex vivo preparations. Importantly, KOR antagonism also corrected in vivo DA release as well as sex-dependent behavioral abnormalities observed in DAT Val559 mice. Given their low abuse liability, our studies with a construct valid model of human DA associated disorders reinforce considerations of KOR antagonism as a pharmacological strategy to treat DA associated brain disorders.

Figure 1:. The KOR agonist U69,593 increases DAT-mediated uptake via a Thr53 dependent mechanism.

EM4 cells were transfected with DAT and KOR and uptake of [³H]DA was assessed as described in Methods. A: U69,593 (10 μM) increased WT DAT-mediated uptake in rDAT/rKOR cotransfected EM4 cells in a concentration-dependent manner as compared to vehicle (n= 3 independent observations per condition, one-way ANOVA, followed by Bonferroni post-hoc tests). B: Treatment with 10 μM U69,593 enhanced WT DAT specific uptake in a time-dependent fashion (n= 5 independent observations per condition, one-way ANOVA, followed by Bonferroni post-hoc tests). C: Incubation with 10 μM U69,593 increased WT DAT V_max (two-tailed unpaired t-test) without effect on K_M (n=3 independent observations per condition, two-tailed Student’s unpaired t-test). D: Treatment with U69,593 significantly augmented WT DAT-mediated uptake when compared to vehicle in cells that expressed KOR plus either WT DAT or a truncated version of DAT, lacking the first 22 N-terminal residues (DAT Δ1–22). In contrast, truncation of the first 55 N-terminal residues (DAT Δ1–55 DAT) prevented KOR-agonist induced increases in [³H]DA uptake. (n=5–6 independent observations per condition, one-way ANOVA, followed by Bonferroni post-hoc tests) E: Site-directed mutagenesis of DAT Thr53 to alanine (DAT-Ala53) rendered transporter-mediated DA uptake insensitive to pre-treatment with U69,593 at 5 and 10 μM (n=4 observations per condition, One-way ANOVA, followed by Bonferroni posthoc tests). F: U69,593 did not affect the kinetic parameters K_M and V_max of the DAT-Ala53 mutant (n=3 independent observations per condition, Student’s two-tailed unpaired t-test) Data are shown as the mean with error bars reflecting the SD *=P<0.05; **=P<0.01; ***=P<0.001; ****=P<0.0001.

Figure 2:. Capacity to phosphorylate rDAT at Thr53 is required for rKOR activation to elevate transporter surface expression

EM4 cells were transfected with either WT DAT or DAT Ala53 plus rKOR, subjected to U69,593 and biotinylated as described in Methods. A: Treatment with U69,593 did not affect WT DAT band densities at ~55–60 kDa and ~85–90 kDa, derived from total protein lysates (n=3 independent observations per condition, Student’s two-tailed unpaired t-test). B: Incubation with U69,593 increased the WT DAT band densities at ~55–60 kDa and ~85–90 kDa, derived from biotinylated protein lysates (unpaired, two-tailed t-test). No effect was observed on the DAT Ala53 band densities at ~55–60 kDa (n=3 independent observations per condition, Student’s two-tailed unpaired t-test). C and D: Protein lysates were immunoblotted with p-Thr53 antibody. Bands marked with an asterisk indicate non-specific immunoreactivity. C: In total protein lysates, incubation with U69,593 increased the density of the p-Thr53 immunoreactive band at ~55–60 kDa for WT DAT (Student’s unpaired, two-tailed t-test). No effect was observed on the band densities at ~85–90 kDa. No specific bands were detected for the DAT Ala53 variant (n=3–5 independent observations per condition, Student’s two-tailed unpaired t-test). D: U69,593 augmented band densities at ~55–60 and ~85–90 kDa for biotinylated WT DAT (n=5 independent observations per condition, Student’s two-tailed unpaired t-test). No specific bands were detected for the DAT Ala53 variant. All bars represent the mean with error bars reflecting the SD. Individual quantifications are displayed as individual symbols. *=P<0.05; **=P<0.01; ****=P<0.0001. n.s.=not significant

Figure 3:. KOR-agonism enhances phosphorylation of DAT at Thr53 in native preparations and augments DAT-mediated clearance in the NAc in vivo.

A) Male rat striatal synaptosomes were treated with vehicle or 50 μM U0126 for 15 min at 30°C, followed by treatment with vehicle or 10 μM U69,593 for an additional 15 min 30°C. After treatment, samples were subjected to SDS-PAGE and western blotted for total and p-Thr53 DAT. Upper panel shows representative western blots. Lower panel shows quantification of p-Thr53 DAT staining as a percentage of basal levels. (n=4; one-way ANOVA, Tukey’s multiple comparisons test). B and C) Rats were injected s.c. with vehicle or U69,593 (0.32 mg/kg) and sacrificed at indicated times. Tissue from the dorsal (B) or ventral striatum (C) were western blotted for total and p-Thr53 DAT in duplicate. Signals from the U69,593 treated samples were compared to the time-matched control. The upper panels show representative western blots for the dorsal and ventral striatum with each time-matched control followed by U69,593 treatment. The lower panels show quantification of the p-Thr53 DAT staining as a percentage of the time-matched control (Two-tailed, unpaired t-test). D) Schematic representation of the electrode-micropipette assembly that was lowered into the NAc of anaesthetized rats to allow for in vivo chromatographic measurement of DA clearance rates. E) Representative oxidation currents converted to micromolar values observed upon pressure ejection of DA before (gray traces) and 2 min after intra-NAc injection of vehicle (black trace), U69,593 (890 μM, barrel concentration, red trace). The leftward-shift in the representative trace following U69,593 injection is indicative of increased DA clearance. F) U69,593 decreased the clearance time of exogenously applied DA in the NAc (T₅₀ and T₈₀; n= 7 observations per condition, two-tailed paired t-test) when compared to the pre-drug value. Bars indicate the mean and error reflect SD. Individual values from each animal are reflected by the corresponding symbols. *denotes P<0.05; **denotes P<0.01, ns = not significant.

Figure 4:. mKOR agonism induces enhanced mDAT surface expression and phosphorylation at mDAT Thr53 while mKOR antagonism normalizes enhanced surface expression and Thr53 phosphorylation of mDAT Val559 in acute brain slices containing the DS or VS

A-D) Acute slices containing the DS and/or the VS were exposed to 10 μM of U69,693 or vehicle for 7 min and surface expression and phosphorylation of p-Thr53 DAT was assessed. A and B) In the DS, treatment with U69,593 increased surface DAT levels (A) (n=9; two-way ANOVA, Šídák’s multiple comparisons test) as well as phosphorylation of mDAT at Thr53 (B) (n=10; two-way ANOVA, Šídák’s multiple comparisons test). Higher basal phosphorylation levels at Thr53 were detected for the DAT Val559 when compared to WT DAT in the DS (n=11; two-way ANOVA, Šídák’s multiple comparisons test). C) In the VS, KOR-agonist treatment increased surface mDAT levels independent of genotype as compared to vehicle treatment (n=8; two-way ANOVA, Šídák’s multiple comparisons test). D) Treatment with U69,593 enhanced WT mDAT phosphorylation at Thr53, but remained without effect on DAT Val559 phosphorylation. E-H) Acute coronal slices containing the DS or the VS were treated with the KOR antagonist norBNI (1 μM) for 20 min and DAT surface expression and phosphorylation at Thr53 were determined. E and F) In the DS, antagonism of mKOR reduced the enhanced surface expression (E) (n=8; two-way ANOVA, Šídák’s multiple comparisons test) and Thr-53 phosphorylation (F) (n=6; two-way ANOVA, Šídák’s multiple comparisons test) of tansporters in DAT Val559 mice. No effect of norBNI was detected on WT DAT. G and H) Treatment with norBNI remained without effect on DAT surface expression (G) (n=6, two-way ANOVA, Šídák’s multiple comparisons test) and Thr-53 phosphorylation (H) (n= 7, two-way ANOVA, Šídák’s multiple comparisons test) in the VS. All bars show the mean and SD. Symbols reflect individual observations. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001

Figure 5:. Treatment with norBNI normalizes sex-specific behavioral phenotypes of male and female DAT Val559 mice

A-D) Male homozygous DAT Val559 mice and their WT littermates were injected with saline (vehicle; n= 13 for WT and 12 for DAT Val559, respectively) or norBNI (10 mg / kg, i.p.; n= 13 for WT and 12 for DAT Val559, respectively) 30 min prior to testing, placed into the center of the open Y-maze and the number of alternating arm entries, distance travelled and direct revisits were recorded as described in Methods. A) Representative heat maps showing the explorative behavior of WT and Val559 mice following vehicle or norBNI administration. Time spent in each area is directly correlated to the color gradient ranging from dark blue to dark red, with the latter indicating highest value. B) Systemic administration of norBNI 30 min prior to the test normalized the deficit in the percentage of alternations of DAT Val559 mice when compared to WT control mice. C) No acute effect of norBNI was observed for total distance travelled during the test session. D) Administration of norBNI reduced the number of direct revisits of DAT Val559 mice. E-J) The NOR task was performed with saline treated female WT (n=11) and DAT Val559 littermates (n=10) and compared to norBNI treated (10 mg/kg, i.p., 30 min prior to testing) female WT (n=13) and littermate DAT Val559 (n=10) mice. E) provides representative heat maps representing location in relation to objects (circles) used in NOR sessions. The relative interaction time with the familiar versus the novel object and the discrimination index are shown in (F) and (G), respectively. H) shows the total distance travelled and the total object interaction times on day 1 (two novel objects) and day 2 (one novel and one familiar object) are shown in panels (I) and (J), respectively. Data are given as mean and SD and were analyzed using two-way ANOVA with Šídák’s multiple comparisons test. *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001, n.s.= not significant.

Figure 6. norBNI treatment of male DAT Val559 mice normalizes abnormal locomotor responses to injection stress and locomotor response to systemic cocaine as monitored in the open field test

A-E) Homozygous male DAT Val559 and WT littermates were injected with saline (vehicle) or norBNI (10 mg/kg, i.p.) and placed in open activity chambers one week post injection. A) Representative activity traces of WT and DAT Val559 mice. DAT Val559 mice previously injected with vehicle (saline) displayed significantly less B) forward locomotor activity C) spent less time in the center of the activity chamber and exhibited fewer D) vertical counts and E) stereotypies when compared to WT mice. No differences between genotypes were detected when the mice were pretreated with norBNI. F-G) Homozygous male DAT Val559 were injected with saline or cocaine (10 mg/kg, i.p.) one week post administration of norBNI (10 mg/kg, i.p.) and placed into open activity chambers. F) Representative traces of male DAT Val559 mice injected with the indicated drug combinations. G) Total distance travelled and time spent in the center of the chamber of male DAT Val559 mice injected with the indicated combinations of saline, norBNI and cocaine. All bars show the mean and SD. n=12–13 individual animals per group, data in B, C, D, E and G were analyzed with two-way ANOVA, Šídák’s multiple comparisons test. Symbols reflect individual animals. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, ns = not significant.