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Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral tegmental area

Nature Neuroscience volume 18, pages 415–422 (2015)Cite this article

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Abstract

Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.

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Figure 1: Opiate-induced downregulation of Bdnf expression in human, rat and mouse VTA.
Figure 2: Effect of chronic morphine on expression of Bdnf exons and on binding of Pol II to the Bdnf gene in rat VTA.
Figure 3: Morphine-induced histone modifications at Bdnf promoters in rat VTA.
Figure 4: Chronic morphine regulation of CREB binding to Bdnf promoters in VTA.
Figure 5: Epigenetic regulation of Bdnf by NURR1 and its concomitant behavioral effects.

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Acknowledgements

We thank T. Abel (University of Pennsylvania) for helpful discussions. This work was supported by grants from the National Institute on Drug Abuse (E.J.N. and M.S.M.-R.).

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Author notes

  1. eric.nestler@mssm.edu

Authors and Affiliations

  1. Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Ja Wook Koo, Michelle S Mazei-Robison, Quincey LaPlant, Gabor Egervari, Deveroux Ferguson, Jian Feng, Haosheng Sun, Kimberly N Scobie, Diane M Damez-Werno, Efrain Ribeiro, Catherine Jensen Peña, Deena Walker, Rosemary C Bagot, Michael E Cahill, Sarah Ann R Anderson, Benoit Labonté, Georgia E Hodes, Heidi Browne, Benjamin Chadwick, Alfred J Robison, Vincent F Vialou, Caroline Dias, Zachary Lorsch, Ezekiell Mouzon, Scott J Russo, Yasmin L Hurd & Eric J Nestler

  2. Department of Physiology, Michigan State University, East Lansing, Michigan, USA

    Michelle S Mazei-Robison & Alfred J Robison

  3. Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Gabor Egervari, Sarah Ann R Anderson, Benjamin Chadwick & Yasmin L Hurd

  4. Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Gabor Egervari, Sarah Ann R Anderson, Benjamin Chadwick & Yasmin L Hurd

  5. Department of Pharmacology and Toxicology, The State University of New York at Buffalo, Buffalo, New York, USA

    Kevin M Braunscheidel & Danielle N Adank

  6. Institut National de la Santé et de la Recherche Médicale, U952, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7224, UPMC, Paris, France

    Vincent F Vialou & David M Dietz

  7. Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, USA

    Mary Kay Lobo

  8. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Rachael L Neve

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Contributions

J.W.K., M.S.M.-R., S.J.R., Y.L.H. and E.J.N. designed the study. J.W.K., M.S.M.-R., Q.L., G.E., K.M.B., D.N.A., D.F., J.F., H.S., K.N.S., D.M.D.-W., E.R., C.J.P., D.W., R.C.B., M.E.C., S.A.R.A., B.L., G.E.H., H.B., B.C., A.J.R., V.F.V., C.D., Z.L., E.M., M.K.L. and D.M.D. performed the experiments. J.W.K. and R.L.N. generated viral vectors. J.W.K. and Q.L. analyzed data. J.W.K. and E.J.N. wrote the paper.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of opiate treatment regimens used in this study.

(a) Scatter plot of Bdnf exon IX mRNA levels in VTA of human heroin addicts compared to control subjects. Student’s t-test, *p < 0.05 compared to control subjects, n = 5,9. (b) Acquisition of heroin self administration. Rats robustly self-administered heroin (30 μg/kg/infusion on a designated “active” lever), but not saline. After 10 days of chronic heroin self-administration, animals were killed 24 hrs after last drug use. Repeated measures two-way ANOVA (drug effect: p < 0.001; day effect: p < 0.001; drug×day effect: p < 0.001), Fisher’s post hoc tests, *p < 0.05, **p < 0.01, ***p < 0.001 compared with heroin inactive group; #p < 0.05, ###p < 0.001 compared with saline active group, n = 9,14. (c) Locomotor activity in a standard morphine treatment regimen where rats are injected daily with saline or morphine (5 mg/kg, IP) for 14 days and, two weeks later, are given a challenge dose of saline or morphine. S/S, saline for 14 days, saline challenge; S/M, saline for 14 days, morphine challenge; M/S, morphine for 14 days, saline challenge; and M/M, morphine for 14 days, morphine challenge. Locomotor assays confirmed that this morphine treatment regimen produced the expected behavioral sensitization18. For day 0 to 14, repeated measures two-way ANOVA (drug effect: p = n.s., day effect: p < 0.001; drug×day effect: p < 0.001), Fisher’s post hoc tests, ***p < 0.001 compared with S/S control group, n = 18. On day 28, morphine challenge 14 days after chronic morphine (M/M) elicited hyperactivity compared to S/S, S/M, and M/S groups. Acute morphine exposure 14 days after chronic saline (S/M) showed lower levels of locomotor activity compared to saline controls (S/S) as observed in morphine injected rats on day 1. One-way ANOVA (p < 0.001), Fisher’s post hoc tests, **p < 0.01, ***p < 0.001 compared with S/S group; ###p < 0.001 compared with M/M group, n = 9. (d) Subchronic morphine exposure (15 mg/kg, IP) during 3 day-CPP training induced high levels of CPP scores compared to saline-injected controls. t-test, ***p < 0.001, n = 12. (e) Effect of chronic morphine on gene expression of Bdnf exons III, V, VII, and VIII in rat VTA. qPCR showed that Bdnf transcript levels of exons III (Ct = 33.393 ± 0.295, n = 8), V (Ct = not detectable, n = 8), VII (Ct = 34.776 ± 0.226, n = 8), and VIII (Ct = 34.363 ± 0.392, n = 7,8) are rarely, if ever, detected, and when detected are not significantly altered by morphine. Student's t-tests, t14= 0.573 for exons III; t14= 0.0163 for exons VII, all p’s = n.s.; Mann-Whitney U test, U = 26 for exons VIII, p = n.s. Individual n, p values and degrees of freedom are available in the Supplementary Methods Checklist.

Supplementary Figure 2 Chronic morphine-induced modifications of histones and histone-regulatory proteins at Bdnf gene promoters in rat VTA.

(a-g) Two-way ANOVA followed by Fisher’s post hoc tests showed that chronic morphine (as in Fig. 1c) alters levels of acH4 at Bdnf promoter 4 (-p4) (b) and of H3K27me3 at Bdnf-p2 (f) in VTA. However, there was no morphine-induced alteration in acH3 (a, drug effect: p = n.s.; region effect: p = n.s.; drug×region effect: p = n.s., n = 3,4), H3K4me3 (c, drug effect: p = n.s.; region effect: p = n.s.; drug×region effect: p = n.s., n = 4,5), H3K9me2 (d, drug effect: p = n.s.; region effect: p = n.s.; drug×region effect: p = n.s., n = 4), H3K9me3 (e, drug effect: p = n.s.; region effect: p = n.s.; drug×region effect: p = n.s., n = 5), and H3K36me3 (g, drug effect: p = n.s.; region effect: p = n.s.; drug×region effect: p = n.s., n = 6,5). Additional post hoc analyses with Student’s t-tests showed that chronic morphine changes levels of acH3 (a) and of H3K4me3 (c) at Bdnf-p2. #p < 0.05 compared to S/S controls. (h-p) Chronic morphine also changes occupancy of proteins such as mSIN3a (h), ING2 (i), MLL1 (j), SUZ12 (l), EZH2 (m), RING1A (n), and BMI1 (p) primarily at Bdnf-p2 in VTA (see Fig. 3). However, there was no morphine-induced alteration in G9a (k) and RING1B (o). Fisher’s post hoc tests, *p < 0.05, **p < 0.01, ***p < 0.001 compared with S/S control group. (q) Summary of epigenetic alterations at the Bdnf genes in VTA by chronic morphine. Green thick lines indicate the relative position of amplicons generated by primers used to quantify immunoprecipitated chromatin-DNA. Exons are represented as boxes and the introns as lines. Numbers of the exons are indicated in roman numerals. The positions of the CREB binding sites at Bdnf promoter regions are indicated as red circles. Filled red arrows indicate significant increases; filled blue arrows indicate significant decreases in response to chronic morphine. Individual n, p values and degrees of freedom are available in the Supplementary Methods Checklist.

Supplementary Figure 3 Validation of HSV-EZH2 and of LV-shRNA-EZH2.

(a) Infusion of HSV-EZH2 into VTA of wildtype c57BL/6 mice increased Ezh2, but not Th and Gria1 mRNA expression compared to HSV-GFP controls. Student’s t-tests, t16= 3.044 for Ezh2, ** p < 0.01, n = 9. Mann-Whitney U tests, U = 25 for Th, U = 30 for Gria1, all p’s = n.s., all n’s = 9. (b-d) Validation of LV-shRNA-EZH2. (b) Representative images (scale bar, 25 μm) demonstrating localized LV-mediated shRNA-EZH2 expression (green) in TH-positive (red) cells (white arrows) in rat VTA. (c) Infusion of LV-shRNA-EZH2 into VTA of Sprague Dawley rats decreased Ezh2 mRNA levels compared to LV-GFP-scramble controls. Student’s t-test, t20= 2.193, *p < 0.05, n = 11. (d) Morphine-treated rats that were injected with LV-shRNA-scramble lower Bdnf mRNA levels of compared to saline-treated rats injected with LV-shRNA-scramble. LV-mediated repression of EZH2 in VTA blocked the morphine-induced Bdnf mRNA reduction (one-way ANOVA, F2,13 = 5.003, p < 0.05, n = 5,4,5). Fisher’s post hoc tests, *p < 0.05 compared to saline+LV-shRNA-scramble; #p < 0.05 compared to Morphine+LV-shRNA-scramble.

Supplementary Figure 4 Western blot analysis for validation of HSV-EZH2, HSV-CREB, and HSV-NURR1 in rat VTA.

The viruses were infused into VTA, and protein levels of EZH2 (a), CREB (b), and NURR1(c) were measured in VTA and in anatomical controls, substantia nigra (SN) and red nucleus (RN). The immunoblots show viral-mediated overexpression of the respective protein in VTA (a, HSV-EZH2, Student’s t-test, *p < 0.05 compared to HSV-GFP, n = 7; b, HSV-CREB, one-way ANOVA, F2,14 = 3.764, p < 0.05, n = 5,6,6; c, HSV-NURR1, one-way ANOVA, F2,14 = 5.092, p < 0.05, n = 5,6,6), but not in other brain areas. Fisher’s post hoc tests, *p < 0.05, ** p < 0.01 compared to HSV-TMT controls. Each protein’s expression was normalized to β-tubulin levels, which were not affected by the treatments. Individual n, p values, and degrees of freedom are available in the Supplementary Methods Checklist. For full-length western blots, see in Supplementary Figure 10.

Supplementary Figure 5 Validation of HSV-CREB and HSV-Cre.

(a) Infusion of HSV-CREB into VTA of wildtype c57BL/6 mice increased Creb1 mRNA expression compared to HSV-TMT (tdTomato) controls. Student’s t-test, t16= 2.208, *p < 0.05, n = 9. (b) In contrast, infusion of HSV-Cre into the VTA of floxed CREB mice decreased Creb1 mRNA expression. t-test, t18= 4.344, ***p < 0.001, n = 10. (c,d) Intra-VTA infusion of HSV-CREB up-regulated both total- (c, t-test, t8= 3.813, **p < 0.01, n = 5) and phospho-CREB binding to Nurr1 promoter (d, Mann-Whitney U test, U = 2, *p < 0.05, n = 5) compared to HSV-TMT controls.

Supplementary Figure 6 Effect of opiates on levels of phospho-/total-CREB binding and H3K27me3 at Bdnf promoters or at promoters of other CREB targets, Th and Gria1 in rat VTA.

(a) In contrast to phospho-CREB binding to Bdnf promoters, phospho-CREB binding to Th and Gria1 promoters was increased in morphine-treated rats (Student’s t-tests, for Th, t14= 2.165, n = 9,7; for Gria1, t7= 2.489, n = 5,4, *p < 0.05). (b,c) However, chronic morphine exposure had no effect on levels of total-CREB binding (for Th, t-test, t7= 0.380; for Gria1, Mann-Whitney U test, U = 9; all p’s = n.s., all n’s = 5,4) and H3K27me3 (for Th, t-test, t8= 0.0297; for Gria1, Mann-Whitney U test, U = 7; all p’s = n.s., all n’s = 5) at Th and Gria1 promoters. (d) Self-administered heroin significantly reduced phospho-CREB binding to all Bdnf promoters (two-way ANOVA, drug effect: F1,32 = 32.004, p < 0.001; region effect: F3,32 = 0.132, p = n.s.; drug×region effect: F3,32 = 0.132, p = n.s., n = 5). (e,f) In contrast, heroin self-administration increased total-CREB binding to Bdnf-p2 and -p6 (e, two-way ANOVA, drug effect: F1,20 = 5.683, p < 0.05; region effect: F3,20 = 2.175, p = n.s.; drug×region effect: F3,20 = 2.175, p = n.s., n = 3,4) and H3K27me3 levels (f, two-way ANOVA, drug effect: F1,36 = 19.593, p < 0.001; region effect: F3,36 = 0.542, p = n.s.; drug×region effect: F3,36 = 0.542, p = n.s., n = 5,6) at Bdnf-p2 and -p4. Fisher’s post hoc tests, *p < 0.05, **p < 0.01 compared with each control group. Additional post hoc analyses with Student’s t-tests showed that self-administered heroin induced H3K27me3 at Bdnf-p1,-p2, and -p4 (Bdnf-p1, t9 = 2.789; Bdnf-p2, t9 = 2.495; Bdnf-p4, t9 = 2.549; Bdnf-p6, t9 = 1.262, #p < 0.05).

Supplementary Figure 7 Validation of HSV-NURR1 and NURR1 ChIP antibody.

(a) Infusion of HSV-NURR1 into rat VTA increases Nurr1 mRNA expression in this region. Student’s t-test, t17= 2.166, *p < 0.05, n = 9,10. (b) Effect of Nurr1 overexpression on morphine re-exposure induced hyper-locomotor activity. Repeated injection of morphine (5 mg/kg, IP) for 14 days induced high levels of locomotion (repeated measures two-way ANOVA, drug effect: F1,36 = 7.087, p < 0.05; day effect: F1,36 = 54.272, p < 0.001; drug×day effect: F1,36 = 13.080, p < 0.001, n = 11,27). Fisher’s post hoc tests, ***p < 0.001 compared with saline group. 10 days later, HSV-NURR1 or HSV-TMT were infused into VTA. Four days after the intra-VTA infusion of HSVs (day 28), re-exposure of HSV-TMT infused group (MM-TMT) to morphine challenge dose (5 mg/kg, IP) induced hyper-locomotion compared to saline exposed HSV-TMT infused group (MS-TMT). Importantly, intra-VTA infusion of HSV-NURR1 blocked this morphine re-exposure induced hyper-locomotion (see Fig. 5g,h). Nurr1 overexpression in saline-treated group (i.e., without morphine) had no effect on locomotor activity (Inset). One-way ANOVA, F4,37 = 3.031, p < 0.05, n = 5-10. (c-e) In order to verify the specificity of NURR1 antibody used for ChIP experiments, western blotting was performed with (c) anti-NURR1, (d) anti-NOR1, or (e) anti-NUR77. The analysis shows an enrichment of NURR1 in the anti-NURR1-ChIP’ed samples but not in the corresponding input samples. Student’s t-test, t4= 12.297, ***p < 0.001 compared to input, n = 3 (10 rat VTA punches pooled per sample). No immunoreactivity was detected by anti-NOR1, anti-NUR77, and anti-β-tubulin in the immunoprecipitated samples with anti-NURR1 (all n’s = 3). For full-length western blots, see in Supplementary Figure 10.

Supplementary Figure 8 Schematic model of the complex epigenetic mechanisms underlying suppression of the Bdnf gene in VTA by chronic morphine.

Chronic morphine stalls transcriptional activity of polymerase II (Pol II) at Bdnf by increasing phospho-Ser5-Pol II (Ser5-p) at promoter regions, while reducing phospho-Ser2-Pol II (Ser2-p) at Bdnf exons. Chronic morphine also decreases permissive histone acetylation (e.g., AcH3), and increases repressive histone methylation (H3K27me3), at Bdnf-p2, the latter part of a repressive complex (PRC2, Polycomb repressive complex 2) which includes increased binding of SUZ12 and EZH2 (an H3K27 methyltransferase). As well, chronic morphine paradoxically increases levels of a permissive histone mark (H3K4me3) and its methytransferase (MLL1) at Bdnf-p2. However, these paradoxical increases are associated with morphine induction of ING2 and mSIN3a binding to Bdnf-p2. ING2, a subunit of the repressive mSIN3a-HDAC complex, binds with high specificity to H3K4me3 at particular genes and thereby represses their transcription. In addition, the morphine-induced repression of Bdnf involves reduced binding of two key transcription factors, phospho-CREB (cAMP response element binding protein) and NURR1 (nuclear receptor related 1), which normally induce Bdnf gene expression. In particular, phospho-CREB binding, which is increased at other target genes in VTA by chronic morphine (not shown), is antagonized at Bdnf promoters by morphine-induced increases in H3K27me3. These studies, which provide one of the most comprehensive analyses of epigenetic regulation of a target gene in the brain yet available, reveal novel epigenetic mechanisms for Bdnf down-regulation in VTA in response to chronic morphine, adaptations that contribute to enhanced behavioral responses to the drug.

Supplementary Figure 9 Validation of antibodies used for qChIP experiments.

qPCR amplification was performed with primer sets that were designed based on previous studies validating ChIP antibodies in other systems or on validation information provided by manufacturers (for details, see Supplementary Table 4). Levels of immunoprecipitated DNA are presented as a percentage of input DNA. (a-c) Pol II antibodies. One-way ANOVA, p < 0.05 (a, Total Pol II; c, Ser2-p-Pol II); p < 0.01 (b, Ser5-p-Pol II). Fisher’s post hoc tests, *p < 0.05, **p < 0.01, ***p < 0.001 compared to Gapdh; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to Actb-1, n = 3. (d-j) Antibodies for histone markers. One-way ANOVA, all p’s < 0.001 (d, acH3; f, H3K4me3; g, H3K9me2; h, H3K9me3; i, H3K27me3; j, H3K36me3); p < 0.05 (e, acH4). Fisher’s post hoc tests, *p < 0.05, **p < 0.01, ***p < 0.001 compared to Gapdh; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to Rpl30, n = 2-6. (k,l) HDAC-related proteins. One-way ANOVA, all p’s < 0.05. Fisher’s post hoc tests, *p < 0.05, **p < 0.01 compared to Esr1; #p < 0.05 compared to Rpl30, n = 3-4. (m) MLL1. One-way ANOVA, p < 0.05. Fisher’s post hoc tests, *p < 0.05, **p < 0.01 compared to Esr1, n = 4. (n) G9a. Student’s t-test, *p < 0.05 compared to Rpl30, n = 4,3. (o-s) Polycomb group proteins. t-tests, *p < 0.05 compared to Gapdh (o, SUZ12; p, EZH2); one-way ANOVA, p < 0.05 (q, RING1A); p < 0.001 (r, RING1B); p < 0.01 (s, BMI1). Fisher’s post hoc tests, *p < 0.05, **p < 0.01, ***p < 0.001 compared to Gapdh; ###p < 0.001, compared to Myod1, n = 3-4. (t,u) CREB. t-test, *p < 0.05 compared to Nr4a3. n = 5-6. (v) NURR1. One-way ANOVA, p < 0.01. Fisher’s post hoc tests, *p < 0.05, **p < 0.01, ***p < 0.001 compared to Afm; #p < 0.05, ##p < 0.01 compared to Myod1, n = 2-3. Individual n, p values and degrees of freedom from all ChIP data analyses are available in the Supplementary Methods Checklist.

Supplementary Figure 10 Full-length images of blots from the Supplementary Figures.

(a-c) Immunoblots of all individuals from Supplementary Figure 4. and (d,e) from Supplementary Figure 7. #One blot is excluded from analysis due to highly variable expression of the internal control (c, β-tubulin).

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Koo, J., Mazei-Robison, M., LaPlant, Q. et al. Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral tegmental area. Nat Neurosci 18, 415–422 (2015). https://doi.org/10.1038/nn.3932

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