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. 2014 Jul 1;76(1):47-56.
doi: 10.1016/j.biopsych.2013.09.034. Epub 2013 Oct 16.

Methamphetamine downregulates striatal glutamate receptors via diverse epigenetic mechanisms

Subramaniam Jayanthi  1 , Michael T McCoy  1 , Billy Chen  2 , Jonathan P Britt  2 , Saїd Kourrich  2 , Hau-Jie Yau  2 , Bruce Ladenheim  1 , Irina N Krasnova  1 , Antonello Bonci  2 , Jean Lud Cadet  3
Affiliations

Methamphetamine downregulates striatal glutamate receptors via diverse epigenetic mechanisms

Subramaniam Jayanthi et al. Biol Psychiatry. .

Abstract

Background: Chronic methamphetamine (METH) exposure causes neuroadaptations at glutamatergic synapses.

Methods: To identify the METH-induced epigenetic underpinnings of these neuroadaptations, we injected increasing METH doses to rats for 2 weeks and measured striatal glutamate receptor expression. We then quantified the effects of METH exposure on histone acetylation. We also measured METH-induced changes in DNA methylation and DNA hydroxymethylation.

Results: Chronic METH decreased transcript and protein expression of GluA1 and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) and GluN1 N-methyl-D-aspartate receptor subunits. These changes were associated with altered electrophysiological glutamatergic responses in striatal neurons. Chromatin immunoprecipitation-polymerase chain reaction revealed that METH decreased enrichment of acetylated histone H4 on GluA1, GluA2, and GluN1 promoters. Methamphetamine exposure also increased repressor element-1 silencing transcription factor (REST) corepressor 1, methylated CpG binding protein 2, and histone deacetylase 2 enrichment, but not of sirtuin 1 or sirtuin 2, onto GluA1 and GluA2 gene sequences. Moreover, METH caused interactions of REST corepressor 1 and methylated CpG binding protein 2 with histone deacetylase 2 and of REST with histone deacetylase 1. Surprisingly, methylated DNA immunoprecipitation and hydroxymethylated DNA immunoprecipitation-polymerase chain reaction revealed METH-induced decreased enrichment of 5-methylcytosine and 5-hydroxymethylcytosine at GluA1 and GluA2 promoter sequences. Importantly, the histone deacetylase inhibitor, valproic acid, blocked METH-induced decreased expression of AMPAR and N-methyl-D-aspartate receptor subunits. Finally, valproic acid also attenuated METH-induced decrease H4K16Ac recruitment on AMPAR gene sequences.

Conclusions: These observations suggest that histone H4 hypoacetylation may be the main determinant of METH-induced decreased striatal glutamate receptor expression.

Keywords: AMPAR; Addiction; CoREST; HDAC2; MeCP2; NMDAR; REST; valproic acid.

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Figures

Figure 1
Figure 1
Effects of chronic METH treatment on the mRNA and protein expression of AMPAR (GluA1 and GluA2) subtype of glutamate receptors. The rats were treated with saline or METH for two weeks as described in table S1. Total RNA was extracted from the striatum (n=8 rats per group) and quantitative PCR for (A) GluA1 and (B) GluA2 were carried out as described in the text. The relative amounts of mRNA were normalized to OAZ1 (ornithine decarboxylase antizyme 1) and quantified. Western blot analyses (n=6 rats per group) showed significant decreases in the membrane protein levels of (C) GluA1 and (D) GluA2. Representative photomicrographs show results of three samples per group. For quantification, the signal intensity was normalized to 3-tubulin. Values represent means ± SEM of fold changes relative to the controls. Statistical significance was determined by un-paired Student’s t-test. Key to statistics: * p< 0.05; ** p< 0.01; *** p< 0.001 vs. control group.
Figure 2
Figure 2
Glutamate receptor function is decreased following chronic METH administration. Chronic METH administration had no effect on the (A) frequency or (B) amplitude of mEPSCs in medium spiny neurons in the dorsal lateral striatum. (C) A significant decrease in the input-output ratio was observed in the METH group. (D) AMPAR/NMDAR ratio in MSN was significantly increased by chronic METH administration. The increase in the AMPAR/NMDAR ratio is consistent with the observed decreases in (E) GluN1/NR1 mRNA and (F) protein levels.
Figure 3
Figure 3
Chronic exposure to METH promotes hypoacetylation of H4K5, H4K12 and H4K16 on the promoters of AMPA GluA1, GluA2 and NMDA GluN1 subunits. The rats were treated as mentioned in Figure 1. Chronic METH treatment decreases the levels of nuclear (A) H4K5ac; (B) H4K12ac and (C) H4K16ac proteins in the dorsal striatum (n=6 rats per group). Representative photomicrographs show results of three samples per group. For quantification, the signal intensity was normalized to 3-tubulin. ChIP assays (n=6 - 8 rats per group) were carried out using antibodies against histone H4 acetylated at lysine 5 (H4K5ac), at lysine 12 (H4K12ac) and at lysine 16 (H4K16ac) on (D) GluA1, (E) GluA2 and (F) GluN1 DNA sequences. Quantitative PCR was conducted as described in the text using specific ChIP primers directed at GluA1, GluA2 or GluN1 promoters (see Table S2). Values represent means ± SEM of fold changes relative to the controls. Statistical significance was determined by un-paired Student’s t-test. Key to statistics: * p< 0.05; ** p< 0.01; *** p< 0.001 vs. control group.
Figure 4
Figure 4
Chronic METH treatment induced the expression of HDACs (HDAC1, HDAC2, SIRT1 and SIRT2), REST, and CoREST proteins in the dorsal striatum. Chronic METH administration increased the protein levels of (A) HDAC1, (B) HDAC2, (C) SIRT1, (D) SIRT2, (E) REST, and (F) CoREST. Representative photomicrographs show results of three samples per group. For quantification, the signal intensity was normalized to 3-tubulin. Values represent means ± SEM of fold changes relative to the controls. Statistical significance was determined by un-paired Student’s t-test. Key to statistics: * p< 0.05; ** p< 0.01; *** p< 0.001 vs. control group.
Figure 5
Figure 5
Chronic METH increases enrichment of CoREST on GluA1 and GluA2 gene promoters whereas enrichment of REST was observed on GluN1 promoter. ChIP assays (n=8 rats per group) were performed on striata of control and METH-treated rats with (A) anti-REST and (B) anti-CoREST antibodies. Quantitative PCR was conducted as described in the text using specific ChIP primers directed at GluA1 or GluA2 or GluN1 promoter (see Table S2). Values represent means ± SEM of fold changes relative to the controls. Statistical significance was determined by un-paired Student’s t-test. Key to statistics: * p< 0.05; ** p< 0.01; *** p< 0.001 vs. control group. Co-immunoprecipitation assays of (C) REST and HDAC1, and (E) CoREST and HDAC2. Immunoprecipitates were prepared from striatal nuclear extracts of control and METH-treated rats using antibody against anti-REST, anti-CoREST, and recovery of HDAC1 and HDAC2 was determined by western blot assay. The levels of HDAC1 and HDAC2 from non-specific IgG are indicated. Input levels (5 %) of HDAC1 and HDAC2 are shown for comparison. ChIP assays (n=6 - 8 rats per group) were carried out using antibodies against HDAC1 (D), and HDAC2 (F) on GluA1, GluA2 and GluN1 DNA sequences. Quantitative PCR was conducted as described in the text using specific ChIP primers directed at GluA1, GluA2 or GluN1 promoters (see Table S2). Values represent means ± SEM of fold changes relative to the controls. Statistical significance was determined by un-paired Student’s t-test. Key to statistics: * p< 0.05; ** p< 0.01; *** p< 0.001 vs. control group.
Figure 6
Figure 6
Chronic METH exposure causes down-regulation of GluA1 and GluA2 transcription by formation of a MeCP2-HDAC2 complex. Western blot analysis of (A) MeCP2, (D) DNMT1, (E) DNMT3A and (F) DNMT3B Representative photomicrographs show results of 3 samples per group. For quantification, the signal intensity was normalized to 3-tubulin. Co-immunoprecipitation assays of (B) MeCP2 and HDAC2 show METH-induced interactions of MeCP2 with HDAC2. The level of HDAC2 from non-specific IgG is indicated. Input levels (5 %) of HDAC2 are shown for comparison. ChIP assays (n=6 – 8 rats per group) were carried out using antibodies against MeCP2 (C). Quantitative PCR was conducted using specific ChIP primers (see Table S2). Denatured genomic DNA of ~200 – 600 bp (generated by sonication) was incubated with an antibody directed against 5mC (G) or 5hmC (H), in order to isolate methylated or hydroxymethylated DNA by immunoprecipitation. Relative enrichment of 5mC and 5hmC in the bound over input fractions was calculated by real-time PCR. Values represent means ± SEM of fold enrichment relative to the controls. Statistical significance was determined by un-paired Student’s t-test. Key to statistics: * p< 0.05; ** p< 0.01; *** p< 0.001 vs. control group.
Figure 7
Figure 7
Co-treatment with valproic acid (VPA) blocked the METH-induced decreases in (A) GluA1 mRNA, (B) GluA2 mRNA, and (C) GluN1 mRNA. Sodium valproate (300 mg/kg) was injected intraperitoneally twice a day 30 min prior to either saline or to METH injections. Drug administration, RNA extraction and RT-PCR of GluA1, GluA2 and GluN1 are as described in the text. The relative amounts of mRNA were normalized to OAZ1 and quantified. Values represent means ± SEM of fold changes relative to the controls. Statistical significance for the four groups (n=7–8 rats per group) were compared by two-way analysis of variance and Bonferroni correction. Key to statistics: * p< 0.05; ** p< 0.01 (Bonferroni).
Figure 8
Figure 8
Co-treatment with valproic acid (VPA) blocked METH-induced increased enrichment of HDAC2 on (A) GluA1 and (B) GluA2 gene promoters. VPA also blocked decreased enrichment of H4K16ac on (C) GluA1 but not on (D) GluA2 gene promoters. Sodium valproate (300 mg/kg) was injected intraperitoneally twice a day 30 min prior to either saline or to METH injections. Drug administration and ChIP-PCR for GluA1 and GluA2 are as described in the text. ChIP assays (n=4 – 6 rats per group) were carried out using an antibody against HDAC2 and H4K16ac. Values for all experiments represent means ± SEM of fold changes relative to the controls. Statistics are as described in Figure 7. Key to statistics: * p< 0.05 (Bonferroni).
Figure 9
Figure 9
Schematic models showing chronic METH-induced epigenetic modifications in the dorsal striatum. Under control condition, there exists a balance between histone acetylases (HATs) and histone deacetylases (HDACs) that regulate the histone acetylation/deacetylation status that maintain the baseline transcription levels. However, chronic METH exposure leads to formation of protein repressor complexes MeCP2-CoREST-HDAC2 that cause H4K5, H4K12 and H4K16 hypoacetylation at enhancer or promoter sequences of GluA1 and GuA2 genes. This then leads to decreased expression of these receptors. Rats chronically exposed to METH also show formation of a protein repressor complex that contains REST-HDAC1 that produces hypoacetylation of H4K5, H4K12 and H4K16 at the promoter region of GluN1 and subsequent decreased GluN1 (NR1) expression in the dorsal striatum. Co-treatment of METH-treated animals with valproic acid that inhibits HDAC1 and HDAC2 blocked METH-mediated hypoacetylation and METH-induced decreased expression of these glutamate receptors.
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References

    1. Bowers MS, Chen BT, Bonci A. AMPA receptor synaptic plasticity induced by psychostimulants: the past, present, and therapeutic future. Neuron. 2010;67:11–24. - PMC - PubMed
    1. Chen BT, Hopf FW, Bonci A. Synaptic plasticity in the mesolimbic system: therapeutic implications for substance abuse. Ann N Y Acad Sci. 2010;1187:129–139. - PMC - PubMed
    1. Luscher C, Malenka RC. Drug-evoked synaptic plasticity in addiction: from molecular changes to circuit remodeling. Neuron. 2011;69:650–663. - PMC - PubMed
    1. Belin D, Everitt BJ. Cocaine seeking habits depend upon dopamine-dependent serial connectivity linking the ventral with the dorsal striatum. Neuron. 2008;57:432–441. - PubMed
    1. Doyon J, Bellec P, Amsel R, Penhune V, Monchi O, Carrier J, et al. Contributions of the basal ganglia and functionally related brain structures to motor learning. Behav Brain Res. 2009;199:61–75. - PubMed

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