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. 2019 May 16;129(8):3407-3419.
doi: 10.1172/JCI127411.

Autism-linked Dopamine Transporter Mutation Alters Striatal Dopamine Neurotransmission and Dopamine-Dependent Behaviors

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Free PMC article

Autism-linked Dopamine Transporter Mutation Alters Striatal Dopamine Neurotransmission and Dopamine-Dependent Behaviors

Gabriella E DiCarlo et al. J Clin Invest. .
Free PMC article

Abstract

The precise regulation of synaptic dopamine (DA) content by the dopamine transporter (DAT) ensures the phasic nature of the DA signal, which underlies the ability of DA to encode reward prediction error, thereby driving motivation, attention, and behavioral learning. Disruptions to the DA system are implicated in a number of neuropsychiatric disorders, including attention deficit hyperactivity disorder (ADHD) and, more recently, Autism Spectrum Disorder (ASD). An ASD-associated de novo mutation in the SLC6A3 gene resulting in a threonine to methionine substitution at site 356 (DAT T356M) was recently identified and has been shown to drive persistent reverse transport of DA (i.e. anomalous DA efflux) in transfected cells and to drive hyperlocomotion in Drosophila melanogaster. A corresponding mutation in the leucine transporter, a DAT-homologous transporter, promotes an outward-facing transporter conformation upon substrate binding, a conformation possibly underlying anomalous dopamine efflux. Here we investigated in vivo the impact of this ASD-associated mutation on DA signaling and ASD-associated behaviors. We found that mice homozygous for this mutation display impaired striatal DA neurotransmission and altered DA-dependent behaviors that correspond with some of the behavioral phenotypes observed in ASD.

Keywords: Molecular biology; Neuroscience; Psychiatric diseases; Transport.

Conflict of interest statement

Conflict of interest: HC is the president and CSO of DRI Biosciences Corporation that is developing treatments for neurological disorders including Fragile X Syndrome and Autism Spectrum Disorder.

Figures

Figure 1
Figure 1. The DAT T356M mutation impairs striatal DA reuptake while maintaining normal DAT expression.
(A) Stimulated dopaminergic current recorded (using carbon fiber amperometry) from acute striatal slices of WT (black) and DAT T356M+/+ (red) mice. (B) Peak dopaminergic current recorded from acute striatal slices of WT (black) and DAT T356M+/+ (red) mice. There was no significant difference in the peak DA released between WT and DAT T356M+/+ mice at baseline (–cocaine [–COC]: WT = 1.921 ± 0.17 μM; DAT T356M+/+ = 1.526 ± 0.18 μM; n = 17; P = 0.1908, 2-way ANOVA). With addition of cocaine, peak DA released decreased significantly in DAT T356M+/+ striatum, but not WT striatum (+COC: WT = 1.766 ± 0.19 μM; DAT T356M+/+ = 0.916 ± 0.13 μM; n = 17; P = 0.23 and P = < 0.001, respectively, 2-way ANOVA). ****P < 0.0001; **P = 0.0013 (C) Decay time (t80–t20) of the dopaminergic signal recorded in acute striatal slices of WT (black) and DAT T356M+/+ (red) mice. The decay time was significantly longer at baseline in DAT T356M+/+ striatum when compared with WT striatum (–COC: WT = 121.5 ± 12.39 ms; DAT T356M+/+ = 244.9 ± 19.83; n = 17; P = 0.001, 2-way ANOVA). Addition of cocaine increased decay time of the dopaminergic signal in both WT and DAT T356M+/+ mice (+COC: WT = 334.1 ± 25.82 ms; DAT T356M+/+ = 384.9 ± 32.21 ms; n = 17; P = <0.001 P = and 0.006, respectively, 2-way ANOVA followed by Šidák’s multiple comparisons test), as expected, and confirming the identity of the current as dopaminergic. ***P = 0.001; ****P < 0.0001 (D) Immunoblotting for DAT showed no difference in DAT expression in the striatum between WT and DAT T356M+/+ mice (n = 6; P = 0.877, 2-tailed t test).
Figure 2
Figure 2. The DAT T356M mutation drives increased striatal DA metabolism and reduced striatal DA synthesis.
(A) The tissue concentration of DA (measured by HPLC) is significantly reduced in the DAT T356M+/+ striatum compared with WT striatum (WT = 153.5 ± 15.38 ng/mg; DAT T356M+/+ = 65.55 ± 6.71 ng/mg; n = 6 WT, 5 DAT T356M+/+; P < 0.0001, 2-way ANOVA followed by Šidák’s multiple comparisons test). There was no difference in the concentration of other biogenic amines (serotonin). ****P < 0.0001. (B) The ratio of the tissue content of DA in striatum to its metabolites is significantly lower in DAT T356M+/+ mice compared with WT mice (see Supplemental Table 2), providing evidence for increased metabolism of DA in the striatum (likely due to reduced reuptake of released DA). ****P < 0.0001. (C) Immunoblotting revealed significantly decreased pTH expression in the striatum of DAT T356M+/+ mice when compared with WT mice (n = 12 from 4 animals; P = 0.0172, Student’s 2-tailed t test). *P = 0.0172. (D and E) Immunoblotting revealed significantly decreased p-ERK1 expression in the striatum of DAT T356M+/+ mice when compared with WT mice (n = 6; P = 0.03, Student’s 2-tailed t test). *P = 0.0279 (D).
Figure 3
Figure 3. DAT T356M+/+ male, but not female, mice exhibit slower weight gain in early life and have lower body weights in adulthood than DAT WT male mice.
(A) Male DAT T356M+/+ mice gain weight significantly more slowly in the period following weaning (P21 – week 5 of life; n = 37 WT, 39 DAT T356M+/+; *P = 0.02, **P < 0.009, 2-way ANOVA followed by Šidák’s multiple comparisons test). In adulthood (at 10 weeks of age), the body weight of male DAT T356M+/+ mice averaged 3.09 g ± 0.92 g lower than that of adult male WT mice. (B) Female DAT T356M+/+ mice never differed statistically in body weight compared with WT female mice.
Figure 4
Figure 4. DAT T356M+/+ mice do not demonstrate deficits in strength, coordination, motor learning, or anxiety.
(A) There was no difference in latency to fall on the inverted screen test (a proxy for strength) between WT and DAT T356M+/+ mice (WT = 120.0 ± 21.79 s, DAT T356M+/+ = 109.6 ± 19.29 s; n = 10 WT, n = 11 DAT T356M+/+; P = 0.72, Student’s 2-tailed t test). (B) There was no difference in latency to reach the platform on the pole climb test (a proxy for coordination) between WT and DAT T356M+/+ mice (WT = 10.11 ± 2.360 s, DAT T356M+/+ = 8.545 ± 1.836 s; n = 9 WT, 11 DAT T356M+/+; P = 0.6, Student’s 2-tailed t test). (C) On days 1 and 2 of the rotarod test of coordination and motor learning, there was no statistically significant difference in performance between WT and DAT T356M+/+ mice. However, on the third day of testing, DAT T356M+/+ took a significantly longer time to fall or rotate than WT mice, indicating improved motor learning and indicating a propensity for the formation of repetitive motor routines in DAT T356M+/+mice (WT days 1, 2, 3 = 129.43 ± 15.89 s, 159.06 ± 21.52 s, 167.03 ± 22.1 s, respectively; DAT T356M+/+ days 1, 2, 3 = 153.73 ± 10.16 s, 207.36 ± 15.28 s, 236.45 ± 15.22 s, respectively; n = 10 WT, n = 11 DAT T356M+/+; P = 0.68 [day 1], P = 0.14 [day 2]), P = 0.02 [day 3], 2-way ANOVA followed by Šidák’s multiple comparisons test). (D) There was no difference in the percentage of time spent in the closed arms of the elevated zero maze between WT and DAT T356M+/+ mice, indicating no anxiety-like phenotype in the DAT T356M+/+ mice (WT = 56.94% ± 3.12%, DAT T356M+/+ = 62.55% ± 1.75%; n = 10 WT, n = 11 DAT T356M+/+; P = 0.13, Student’s 2-tailed t test). *P = 0.0157.
Figure 5
Figure 5. DAT T356M+/+ mice exhibit spontaneous, persistent hyperlocomotion, repetitive rearing behavior, reduced marble burying, and altered social behaviors.
(A) DAT T356M+/+ mice traveled significantly further than WT mice during a 60-minute test period (WT = 3648 ± 312.4 cm; DAT T356M+/+ = 20571 ± 1062 cm; n = 15 WT, 16 DAT T356M+/+; ****P < 0.0001, Student’s 2-tailed t test). (B) DAT T356M+/+ mice exhibited hyperlocomotor activity across all 10-minute intervals of the 60-minute test (for all intervals, ****P < 0.0001 by 2-way ANOVA followed by Šidák’s multiple comparison test). (C) DAT T356M+/+ mice exhibited repetitive rearing behavior (WT = 71 ± 5; DAT T356M+/+ = 123 ± 9; n = 15 WT, n = 16 DAT T356M+/+; ****P < 0.0001, 2-way ANOVA followed by Šidák’s multiple comparisons test). (D) WT animals displayed a statistically significant preference for the social target, while DAT T356M+/+ mice exhibited no preference for either target (WT = 115.41 ± 11.06 s with social target, 66.07 ± 8.12 s with empty chamber; n = 15; P = 0.001, 2-way ANOVA followed by Šidák’s multiple comparisons test; DAT T356M+/+ = 72.51 ± 5.91 s with social target, 66.125 ± 5.97 s with empty chamber; n = 16; P = 0.81, 2-way ANOVA followed by Šidák’s multiple comparisons test). **P = 0.0012. (E) DAT T356M+/+ mice won significantly fewer bouts against both familiar and novel mice than would be expected by chance (dashed line indicates chance-level performance; n = 24 bouts from 12 pairs of mice; *P < 0.05, **P < 0.01 by χ2).
Figure 6
Figure 6. Treatment with DAT inhibitors reduces spontaneous locomotor activity in DAT T356M+/+ mice.
(A) ACT-01 treatment significantly reduced spontaneous locomotor activity in DAT T356M+/+ mice as early as 20 minutes into the observation period compared with vehicle-treated animals (n = 6–9; **P = 0.0032; *P < 0.032, 2-way ANOVA followed by Šidák’s multiple comparisons test). (B) Total locomotor activity in DAT T356M+/+ mice was reduced by acute treatment with ACT-01 (DAT T356M+/+ vehicle = 6847 cm ± 901.2 cm; DAT T356M+/+ ACT-01 = 2361 cm ± 595.2 cm; n = 6–9; **P = 0.0045, Student’s t test). (C) GBR12909 treatment significantly reduced spontaneous locomotor activity in DAT T356M+/+ mice as early as 10 minutes into the observation period compared with vehicle-treated animals (n = 4; *P = 0.045, 2-way ANOVA followed by Šidák’s multiple comparisons test). (D) Total locomotor activity in DAT T356M+/+ mice was rescued by acute treatment with GBR12909 (DAT T356M+/+ vehicle = 5188 cm ± 411.7 cm; DAT T356M+/+ GBR12909 = 2578 cm ± 716.5 cm; n = 4; *P = 0.0196, Student’s 2-tailed t test).

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