A novel microRNA, novel-m009C, regulates methamphetamine rewarding effects

Abstract

Methamphetamine (METH) is a widely abused psychostimulant, whose hyper-rewarding property is believed to underlie its addictive effect, but the molecular mechanism regulating this effect remains unclear. We previously reported that decreased expression of a novel microRNA (miRNA), novel-m009C, is implicated in the regulation of METH hyperlocomotion. Here, we found that novel-m009C may be homologous to hsa-miR-604. Its expression is consistently downregulated in the nucleus accumbens (NAc) of mice when exposed to METH and cocaine, whereas significant alterations in novel-m009C expression were not observed in the NAc of mice subjected to other rewarding and psychiatric stimuli, such as sucrose, morphine and MK-801. We further found the substantial reduction in novel-m009C expression may be regulated by both dopamine receptor D1 (D1R) and D2 (D2R). Increasing novel-m009C levels in the NAc attenuated METH-induced conditioned place preference (CPP) and hyperlocomotion, whereas inhibiting novel-m009C expression in the NAc enhanced these effects but did not change the preference of mice for a natural reward, i.e., sucrose. These effects may involve targeting of genes important for the synaptic transmission, such as Grin1 (NMDAR subunit 1). Our findings demonstrate an important role for NAc novel-m009C in regulating METH reward, reveal a novel molecular regulator of the actions of METH on brain reward circuitries and provide a new strategy for treating METH addiction based on the modulation of small non-coding RNAs.

Fig. 1. Novel-m009C is a miRNA of mice and its expression was substantially reduced by METH.

a The sequence of novel-m009C and its precursor in mice genome. b METH-induced CPP protocol. c METH-induced CPP in mice. Two-way ANOVA. METH: F_{(1, 20)} = 49.36, P < 0.0001; test: F_{(1, 20)} = 50.14, P < 0.0001; METH × test: F_{(1, 20)} = 42.87, P < 0.0001. ****P < 0.0001, different from the saline group in the post-test; ^^^^P < 0.0001, different from the pre-test score of the same group; n = 6. d Significant decrease in the expression of novel-m009C and its precursor in the NAc of mice subjected to the METH-induced CPP test. NAc samples for qPCR were collected after the post-test. Independent t-test. Novel-m009C fold change: t₍₁₀₎ = 2.292; precursor fold change: t₍₁₀₎ = 4.516; *P < 0.05, **P < 0.01; n = 6. e Protocol of the METH-induced locomotor sensitization test. f METH-induced locomotor sensitization of mice. One-way repeated measures ANOVA. METH: F_(1,10) = 173.114, P < 0.0001; day: F_{(7, 70)} = 88.122, P < 0.0001; day × METH: F_{(7, 70)} = 105.414, P < 0.0001. ***P < 0.001, different from the saline group on each day; ^^^P < 0.001, different from the same group on day 3; n = 6. g Substantial reduction in the expression of novel-m009C and its precursor in the NAc of mice after METH sensitization. NAc samples for qPCR were collected 24 h after the last injection. Independent t-test. novel-m009C fold change: t₍₉₎ = 4.363; precursor: t₍₉₎ = 3.77; **P < 0.01; n = 5–6. All values are presented as the mean ± SEM.

Fig. 2. The METH-induced decrease in novel-m009C expression in the NAc of mice was reversed by a D1R or D2R antagonist.

Timeline of SCH (a) or RAC (b) pre-treatment prior to the METH-induced locomotor sensitization test. SCH, RAC or vehicle was given 30 min prior to saline or METH injection. NAc samples for qPCR were collected 24 h after the last injection. SCH (c) or RAC (d) pre-treatment significantly attenuated the hyperlocomotion induced by METH. Mixed-design repeated measures ANOVA with a post hoc multiple comparison test. SCH pre-treatment experiment: METH: F_{(1, 24)} = 178.697, P < 0.001; SCH: F_{(1, 24)} = 100.039, P < 0.001; day: F_{(7, 168)} = 42.688, P < 0.001; day × METH: F_{(7, 168)} = 52.158, P < 0.001; day × SCH: F_{(7, 168)} = 31.872, P < 0.001; METH × SCH: F_(1,24) = 97.336, P < 0.001; day × SCH × METH: F_{(1, 24)} = 97.336, P < 0.001. RAC pre-treatment: METH: F_{(1, 24)} = 310.569, P < 0.001; RAC: F_{(1, 24)} = 82.320, P < 0.001; day: F_{(7, 168)} = 82.759, P < 0.001; day × METH: F_{(7, 168)} = 98.008, P < 0.001; METH × RAC: F_{(1, 24)} = 76.882, P < 0.001; day × RAC: F_{(7, 168)} = 32.951, P < 0.001; day × RAC × METH: F_{(1, 24)} = 31.666, P < 0.001. *P < 0.05, ***P < 0.001, different from the paired saline group mice on each injection day; ^P < 0.05, ^^^P < 0.001, different from the same group on day 3; ^###P < 0.001, different from the vehicle + METH group; n = 6–8. SCH (e) or RAC (f) inhibited the METH-induced decrease in the expression of novel-m009C and its precursor in the NAc of mice. Two-way ANOVA followed by a post hoc multiple comparison test. SCH pre-treatment experiment: novel-m009C fold change: METH: F_{(1, 20)} = 6.778, P = 0.017; SCH: F_{(1, 20)} = 7.529, P = 0.0125; METH × SCH: F_(1,20) = 7.201, P = 0.0143; **P < 0.01; precursor fold change: METH: F_{(1, 20)} = 8.029, P = 0.0103; SCH: F_{(1, 20)} = 1.272, P = 0.2728; METH × SCH: F_(1,20) = 3.268, P = 0.0857. **P < 0.01; n = 6. RAC pre-treatment experiment: novel-m009C fold change: METH: F_{(1, 20)} = 10.84, P = 0.0036; RAC: F_{(1, 20)} = 17.71, P = 0.0004; METH × SCH: F_{(1, 20)} = 0.5452, P = 0.4689; *P < 0.05, **P < 0.01; precursor fold change: METH: F_{(1, 20)} = 9.739, P = 0.0054; RAC: F_{(1, 20)} = 7.17, P = 0.0145; METH × RAC: F_{(1, 20)} = 3.882, P = 0.0628. **P < 0.01; n = 6. All values are presented as the mean ± SEM.

Fig. 3. Overexpression of novel-m009C in the NAc attenuated METH-induced CPP and hyperlocomotion in mice.

a Schematic of the control (AAV-control) and novel-m009C precursor expression AAV vectors. b Timeline of the viral infection experiments performed to validate the location of microinfusion and overexpression efficacy through evaluation of the expression of eGFP and novel-m009C. c eGFP was locally expressed in the NAc of mice, as visualized by fluorescence microscopy. d The expression of novel-m009C and its precursor was substantially heightened in the NAc of AAV-m009C-injected mice. Independent t-test. Novel-m009C fold change: t₍₁₀₎ = 5.313; precursor fold change: t₍₁₀₎ = 4.695; **P < 0.01; n = 6. e Timeline of AAV injection and CPP test. f The regulatory effect of AAV-m009C in the NAc decreased METH-induced CPP. Two-way ANOVA. Virus: F_{(1, 26)} = 3.936, P = 0.0579; test: F_{(1, 26)} = 90.13, P < 0.0001; virus × test: F_{(1, 26)} = 5.54, P = 0.0264; ^^P < 0.01, different from the AAV-control group in the post-test; ****P < 0.0001, different from the pre-test score of the same group; n = 7–8. g Timeline of AAV injection and METH-induced locomotor sensitization test. h The regulatory effect of AAV-m009C in the NAc decreased METH-induced hyperlocomotion. One-way repeated measures ANOVA. Virus: F_{(1, 22)} = 22.595, P < 0.001; day: F_{(7, 154)} = 258.163, P < 0.001; virus × day: F_{(7, 154)} = 5.925, P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001, different from the AAV-control group; ^P < 0.05, ^^P < 0.01, different from the same group on day 3; n = 12. All values are presented as the mean ± SEM.

Fig. 4. Inhibition of novel-m009C in the NAc enhanced METH-induced CPP and hyperlocomotion in mice.

a Schematic of the scrambled control (AAV-scrambled) and anti-m009C TuD RNA expression AAV vectors. Anti-m009C TuD RNA was used to specifically inhibit novel-m009C. b Timeline of the viral infection experiments performed to validate the location of microinfusion and the inhibition efficacy through evaluation of the expression of eGFP and novel-m009C. c eGFP showed local expression in the NAc of mice, as visualized by fluorescence microscopy. d The expression of novel-m009C but not its precursor significantly decreased in the NAc of AAV-anti-m009C-injected mice. Independent t-test. Novel-m009C fold change: t₍₁₂₎ = 3.896; precursor: t₍₁₂₎ = 0.8054; **P < 0.01; n = 6–8. e Timeline of AAV injection and the CPP test. f The regulatory effect of AAV-anti-m009C in the NAc increased METH-induced CPP. Two-way ANOVA. Virus: F_{(1, 32)} = 5.643, P < 0.05; test: F_{(1, 32)} = 76.73, P < 0.0001; virus × test: F_{(1, 32)} = 1.46, P = 0.2357. ****P < 0.0001, different from the pre-test score of the same group; ^P < 0.05, different from the AAV-scrambled group; n = 8–10. g Novel-m009C was significantly decreased in the NAc of AAV-anti-m009C-injected mice following METH-CPP test. Independent t-test. Novel-m009C fold change: t₍₁₄₎ = 2.949; *P < 0.05; n = 8. h Timeline of AAV injection and the METH-induced locomotor sensitization test. i The regulatory effect of AAV-anti-m009C in the NAc increased METH-induced hyperlocomotion. One-way repeated measures ANOVA. Virus: F_{(1, 20)} = 7.861, P < 0.05; day: F_{(7, 14)} = 304.456, P < 0.001; virus × day: F_{(7, 140)} = 1.848, P = 0.155. *P < 0.05, **P < 0.01, different from the AAV-scrambled group; ^P < 0.01, different from the same group on day 3; n = 9–13. j Novel-m009C was significantly decreased in the NAc of AAV-anti-m009C-injected mice following METH-hyperlocomotion test. Independent t-test. Novel-m009C fold change: t₍₁₂₎ = 3.507; **P < 0.01; n = 7. All values are presented as the mean ± SEM.

Fig. 5. Novel-m009C regulates potential target genes involved in neuronal morphology and synaptic activities.

a Procedure used for RIP-Seq and RNA-Seq. The mice were microinjected with AAV-m009C and AAV-control and subjected to the METH-induced locomotor sensitization test. Twenty-four hours after the final injection, NAc tissue was collected and lysed. Half of each NAc homogenate was used for immunoprecipitation (IP) of Ago2, and RNA that cross-linked with Ago2 was then isolated for high-throughput sequencing. RNA extracted from the other half of each homogenate was used for high-throughput sequencing; n = 3 pooled homogenates per group (see Methods). Sequencing data were subjected to bioinformatics analysis to identify the primary targets of novel-m009C, and IPA was performed to analyze the functional networks of the targets. b Venn diagram showing the RISC-associated mRNA targets of novel-m009C (red) and mRNAs with expression levels that were downregulated in the NAc of AAV-m009C-injected mice (green). The 27 overlapping genes were considered direct novel-m009C targets. c The -log10 (P value) of the disease and functional terms of the 27 target genes identified by IPA are shown. The red dotted line indicates P = 0.05. d Interaction network of the 27 direct targets showing the functions and the target genes involved. Correlated connections and networks between the target genes and functional terms were analyzed according to the IPA database. The shape of each gene indicates its functional class, and genes that are green are those with downregulated expression. Functional terms in blue indicate an inhibitory state. The relationships between genes and functional terms are indicated by dotted lines of different colors, which indicate the degree of interaction (blue: inhibition, yellow: inconsistent with the state of the downstream molecule, gray: effect not predicted). e The gene network of these primary target genes. Genes with expression levels that were directly downregulated by AAV-m009C are indicated in green. The high abundance of Grin1 (encoding NMDAR subunit 1) among the targets in red underscores the potential of novel-m009C to control signaling processes associated with neuronal morphology and synaptic activities.

Fig. 6. Grin1 may be a target of novel-m009C in the regulation of METH-induced CPP and hyperlocomotion.

a Procedure for measuring Grin1 expression in the NAc of AAV-injected mice following the METH-induced CPP test. NAc samples were collected after the post-test. b The mRNA level of Grin1 expression was downregulated and upregulated in the NAc of mice in which novel-m009C was overexpression and inhibited, respectively, following the CPP test. Independent t-test. Overexpression: t₍₁₃₎ = 2.745; inhibition: t₍₁₀₎ = 2.35; *P < 0.05, different from the corresponding control group; n = 6–8. Representative western blots (c) and graphs (d) showing the protein level of Grin1 expression was downregulated and upregulated in the NAc of mice in which novel-m009C was overexpressed and inhibited, respectively, following the CPP test. Independent t-test. Overexpression: t₍₁₀₎ = 2.583; inhibition: t₍₁₀₎ = 2.639; *P < 0.05, different from the corresponding control group; n = 6. e Procedure for measuring Grin1 expression in the NAc of AAV-injected mice following the METH-induced hyperlocomotion test. NAc samples were collected 24 h after the final METH injection. f The mRNA level of Grin1 expression was downregulated and upregulated in the NAc of mice in which novel-m009C was overexpression and inhibited, respectively, following the METH-induced hyperlocomotion test. Independent t-test. Overexpression: t₍₁₀₎ = 2.646; inhibition: t₍₁₂₎ = 3.567. *P < 0.05, **P < 0.01, different from the corresponding control group; n = 6–8. Representative western blots (g) and graphs (h) showing the protein level of Grin1 expression was downregulated and upregulated in the NAc of mice in which novel-m009C was overexpressed and inhibited, respectively, following the hyperlocomotion test. Independent t-test. Overexpression: t₍₁₀₎ = 2.667; inhibition: t₍₁₀₎ = 2.35; *P < 0.05, different from the corresponding control group; n = 6. i Procedure for intervening in the AAV-m009C-mediated regulation of METH-induced CPP with SPD. SPD (30 mg/kg, i.p.) was given 30 min before METH injection in the conditioning phase. j SPD treatment reversed the decreasing effect of AAV-m009C on METH-induced CPP. The CPP score of AAV-m009C + SPD-treated mice was compared with that of AAV-m009C- and AAV-control-treated mice. Independent t-test: t_{1 (13)} = 3.192, t_{2 (11.5)} = 2.265, t_{3 (14)} = 0.5665; *P < 0.05, **P < 0.01; n = 7–9. k Procedure for intervening in the AAV-anti-m009C-mediation regulation of METH-induced CPP with Arc. Arc (3.0 mg/kg, i.p.) was given 30 min before METH injection in the conditioning phase. l The effect of AAV-anti-m009C on METH-induced CPP showed an attenuated trend following Arc intervention. The CPP score of AAV-anti-m009C +Arc-treated mice was compared with that of AAV-anti-m009C- and AAV-scrambled-treated mice. Independent t-test: t_{1 (16)} = 2.742, t_{2 (13)} = 1.884, t_{3 (15)} = 1.28; *P < 0.05; n = 7–10. All values are presented as the mean ± SEM.