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Ubiquitin-proteasomal regulation of chromatin remodeler INO80 in the nucleus accumbens mediates persistent cocaine craving

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Science Advances  09 Oct 2019:
Vol. 5, no. 10, eaay0351
DOI: 10.1126/sciadv.aay0351
  • Fig. 1 INO80 in the NAc is dysregulated during prolonged abstinence following extended-access cocaine self-administration and mediates cocaine craving.

    (A) Schematic of experimental timeline for short- and extended-access self-administration and tissue collection during abstinence. (B) Self-administration training behavior for extended-access [AD1: two-way repeated-measures analysis of variance (ANOVA), treatment × session: F9,190 = 4.724, P < 0.0001, n = 10 to 11 rats per group; AD30: two-way repeated-measures ANOVA, treatment × session: F9,170 = 2.734, P = 0.005, n = 9 to 10 rats per group] and short-access (two-way repeated-measures ANOVA, treatment: F1,90 = 198.8, P < 0.0001, n = 5 to 6 rats per group) paradigms. (C) Cocaine intake for short- and extended-access self-administration (g/kg per hour: t test, t14 = 4.059, P = 0.001; g/kg per session: t test, t14 = 10.87, P < 0.0001). (D) INO80 protein expression in NAc P1 fractions (AD1 after extended-access: t test, t10 = 0.296, P = 0.773; AD30 after extended-access: t test, t14 = 2.213, P = 0.044; AD30 after short-access: t test, t9 = 0.792, P = 0.449; n = 5 to 8 rats per group). (E) Schematic of experimental timeline for extended-access self-administration, viral-mediated gene transfer, and cue-induced seeking test. (F) Extended-access self-administration training behavior before viral-mediated gene transfer for cocaine- (two-way repeated-measures ANOVA, treatment: F2,26 = 0.274, P = 0.763, n = 9 to 10 rats per group) and saline-treated rats (F1,9 = 0.139, P = 0.718, n = 4 to 7 rats per group). (G) Total active and inactive responses during a 60-min cue-induced seeking test on AD30 after extended-access self-administration for cocaine-treated rats [one-way ANOVA, lever × treatment F2,26 = 7.361; Fisher protected least significant difference (PLSD) test for total active lever: GFP versus INO80, P = 0.018; GFP versus ΔNC-EQ, P = 0.018; n = 9 to 10 rats per group] and saline-treated rats (F1,9 = 0.025; Fisher PLSD test for total active lever: GFP versus INO80, P = 0.92; n = 4 to 7 rats per group). Data are means ± SEM. *P < 0.05. s, saline; c, cocaine; EA, extended-access self-administration; SA, short-access self-administration; P1, nuclear fraction; ΔNC-EQ, catalytically inactive mutant INO80; a, active lever; i, inactive lever.

  • Fig. 2 TRIM3 regulates the expression of INO80 during prolonged abstinence and mediates cocaine craving.

    (A) TRIM3 protein expression in NAc P1 fractions (extended-access AD1: t test, t16 = 0.009, P = 0.992; extended-access AD30: t test, t13 = 2.479, P = 0.028; short-access AD30: t test, t10 = 0.177, P = 0.863; n = 5 to 9 rats per group). (B) Co-immunoprecipitation of INO80 with TRIM3 on AD30 (t test, t13 = 1.878, P = 0.042; n = 7 to 8 rats per group). (C) Polyubiquitinated levels of INO80 on AD30 measured by K48-TUBEs (t test, t18 = 2.451, P = 0.012; n = 10 rats per group) and schematic of anti–K48-TUBEs. (D) Trim3 mRNA expression in NAc on AD30 (t test, t8 = 4.245, P = 0.003; n = 5 rats per group). (E) EGR1 protein expression in NAc P1 fractions on AD30 (t test, t15 = 1.915, P < 0.05; n = 8 to 9 rats per group). (F) EGR1 binding along the TRIM3 promoter on AD30 (t test, t14 = 2.365, P < 0.05; n = 3 samples per group, 7 rats per sample). (G) Self-administration training behavior before viral-mediated gene transfer (two-way repeated-measures ANOVA, treatment group: F1,70 = 0.849, P = 0.849, n = 6 to 11 rats per group; saline treatment: F1,70 = 0.036, P = 0.849, n = 5 rats per group). (H) Total active and inactive responses during a 60-min cue-induced seeking test on AD30 for rats that self-administered cocaine (one-way ANOVA, lever × treatment F2,44 = 5.588; Fisher PLSD test for total active lever: GFP versus TRIM3/NLS-TRIM3, P = 0.034; GFP versus ΔRBCC, P = 0.003; n = 6 to 11 rats per group) and saline (F1,16 = 0.168; Fisher PLSD test for total active lever: GFP versus TRIM3, P = 0.227; n = 4 to 7 rats per group). Data are means ± SEM. *P < 0.05 and **P < 0.01. K48, lysine-48; NLS, nuclear localizing signal; ΔRBCC, catalytically inactive mutant TRIM3; IP, immunoprecipitation; IB, immunoblotting; IgG, immunoglobulin G.

  • Fig. 3 INO80 genomic enrichment during prolonged abstinence.

    (A) Genomic distribution of INO80 differential enrichment events in NAc tissue from rats that self-administered cocaine. (B) KEGG 2019 pathway enrichment analysis of INO80 differential enrichment events in rats that self-administered cocaine. (C) Genome browser track sections for selected genes that displayed significant differential enrichment for INO80 comparing rats that self-administered cocaine versus saline. (D) qPCR analysis of Yy1 (one-way ANOVA, treatment: F3,28 = 5.962, P = 0.003; Fisher PLSD test: saline GFP versus cocaine GFP, P = 0.08; cocaine GFP versus cocaine INO80, P = 0.037), Jmjd6 (F3,28 = 24.31, P < 0.0001; saline GFP versus cocaine GFP, P = 0.004; cocaine GFP versus cocaine INO80, P < 0.0001; cocaine GFP versus cocaine ΔNC-EQ, P < 0.0001), and Ddx39b (F3,29 = 7.351, P = 0.0008; saline GFP versus cocaine GFP, P = 0.025; cocaine GFP versus cocaine INO80, P = 0.006; cocaine GFP versus cocaine ΔNC-EQ, P < 0.0001) in the NAc 24 hours after cue-induced seeking test on AD30 (n = 6 to 10 rats per group). Data are means ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaay0351/DC1

    Fig. S1. Incubated cocaine craving during prolonged abstinence following extended-access cocaine self-administration.

    Fig. S2. Experimental timeline and tissue collection.

    Fig. S3. Custom HSV vectors made to manipulate INO80.

    Fig. S4. Cue-induced food seeking and locomotor activity following food self-administration.

    Fig. S5. Custom HSV vectors made to manipulate TRIM3.

    Fig. S6. Expression of TRIM3 and synaptic substrates during abstinence following extended-access self-administration.

    Fig. S7. Similar cocaine-seeking behavior in rats that received intra-NAc injections of HSV-TRIM3 or HSV-NLS-TRIM3.

    Fig. S8. Schematic of INO80 signaling in the NAc.

    Table S1. Primer list for qPCR.

    Table S2. Primer list for ChIP.

    Data S1. INO80 ChIP-seq data tables.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Incubated cocaine craving during prolonged abstinence following extended-access cocaine self-administration.
    • Fig. S2. Experimental timeline and tissue collection.
    • Fig. S3. Custom HSV vectors made to manipulate INO80.
    • Fig. S4. Cue-induced food seeking and locomotor activity following food self-administration.
    • Fig. S5. Custom HSV vectors made to manipulate TRIM3.
    • Fig. S6. Expression of TRIM3 and synaptic substrates during abstinence following extended-access self-administration.
    • Fig. S7. Similar cocaine-seeking behavior in rats that received intra-NAc injections of HSV-TRIM3 or HSV-NLS-TRIM3.
    • Fig. S8. Schematic of INO80 signaling in the NAc.
    • Table S1. Primer list for qPCR.
    • Table S2. Primer list for ChIP.
    • Legend for data S1

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    Other Supplementary Material for this manuscript includes the following:

    • Data S1 (Microsoft Excel format). INO80 ChIP-seq data tables.

    Files in this Data Supplement:

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