Opposing Role for Egr3 in Nucleus Accumbens Cell Subtypes in Cocaine Action

Abstract

An imbalance in molecular signaling cascades and transcriptional regulation in nucleus accumbens (NAc) medium spiny neuron (MSN) subtypes, those enriched in dopamine D1 versus D2 receptors, is implicated in the behavioral responses to psychostimulants. To provide further insight into the molecular mechanisms occurring in MSN subtypes by cocaine, we examined the transcription factor early growth response 3 (Egr3). We evaluated Egr3 because it is a target of critical cocaine-mediated signaling pathways and because Egr3-binding sites are found on promoters of key cocaine-associated molecules. We first used a RiboTag approach to obtain ribosome-associated transcriptomes from each MSN subtype and found that repeated cocaine administration induced Egr3 ribosome-associated mRNA in NAc D1-MSNs while reducing Egr3 in D2-MSNs. Using Cre-inducible adeno-associated viruses combined with D1-Cre and D2-Cre mouse lines, we observed that Egr3 overexpression in D1-MSNs enhances rewarding and locomotor responses to cocaine, whereas overexpression in D2-MSNs blunts these behaviors. miRNA knock-down of Egr3 in MSN subtypes produced opposite behavioral responses from those observed with overexpression. Finally, we found that repeated cocaine administration altered Egr3 binding to promoters of genes that are important for cocaine-mediated cellular and behavioral plasticity. Genes with increased Egr3 binding to promoters, Camk2␣, CREB, FosB, Nr4a2, and Sirt1, displayed increased mRNA in D1-MSNs and, in some cases, a reduction in D2-MSNs. Histone and the DNA methylation enzymes G9a and Dnmt3a displayed reduced Egr3 binding to their promoters and reduced mRNA in D1-MSNs. Our study provides novel insight into an opposing role of Egr3 in select NAc MSN subtypes in cocaine action.

Molecular basis of dendritic atrophy and activity in stress susceptibility

“…The NAc (from Bregma, anterior/posterior:+1.6, medial/lateral:+/− 1.5, dorsal/ventral: − 4.4) of D1-Cre mice was bilaterally injected with a cre-inducible, double inverted open (DIO)-reading frame adeno-associated virus (AAV) AAV2.5-Ef1a-DIO-Egr3-EYFP (UNC viral vector core) 22 or AAV2.5-Ef1a-DIO-EYFP and AAV2.5-hsyn-DIO-Egr3-miR-mCitrine or AAV2.5-hsyn-DIO-SS-miR-mCitrine. 22 For calcium imaging experiments, mice were co-injected with AAV2.9-flex-GCaMP6f (UPenn Vector Core) and AAV2.2-DIO-Egr3-miR-LacZ or AAV2.2-DIO-SS-miR-LacZ.…”

E3 Ubiquitin-Protein Ligase SMURF1 in the Nucleus Accumbens Mediates Cocaine Seeking

“…We then identified several putative RUNX2 gene targets based on DNA consensus binding sites (Supplemental Table S1). Following self-administration (two-way repeated-measures ANOVA, drug × session interaction effect, F 9,180 = 3.36, p < .001) (Figure 6B), ChIP assays revealed increased RUNX2 binding in cocaine-treated rats at promoter regions of several target genes that we and others have previously implicated in cocaine-induced plasticity (one-way ANOVA, F 7,29 = 3.087, p = .015) (33,43,62,63) (Figure 6C), including Egr3 ( t test, t 8 = 2.196, p = .029), Dnml1 ( t test, t 7 = 2.419, p = .046), and Smarca4 (gene for Brg1; t test, t 8 = 2.169, p = .031) but not Tgfbr1 ( t test, t 6 = 0.9585, p = .374). Importantly, HSV- wtSmurf1 reduced expression of RUNX2-mediated genes affected by cocaine ( Egr3 : t test, t 13 = 1.872, p = .042; Dnml1: t test, t 14 = 0.746, p = .468; Tgfbr1: t test, t 14 = 0.746, p = .468; Smarca4: t test, t 11 = 2.316, p = .041) (Figure 6D).…”

“…We focused on RUNX2 because of its known interactions with c-Fos and c-Jun, immediate early genes involved in cocaine plasticity (58,59), to cooperatively bind AP-1 binding sites (57), which are essential components of transcriptional events following cocaine exposure (55,60,61). We found that SMAD1/5 and RUNX2 are upregulated in the NAc during withdrawal following cocaine self-administration, which prompted us to examine RUNX2 binding to target genes previously implicated in cocaine plasticity [i.e., Egr3 , Dnm1 , and Smarca4 (33,62,63)]. We found that cocaine self-administration induces enhanced RUNX2 binding at AP-1 sites along the promoters of these genes, suggesting that RUNX2 may be a master regulator of multiple pathways that govern cocaine-induced plasticity.…”

“…We found that cocaine self-administration induces enhanced RUNX2 binding at AP-1 sites along the promoters of these genes, suggesting that RUNX2 may be a master regulator of multiple pathways that govern cocaine-induced plasticity. Interestingly, both EGR3 and DNML1 affect NAc D1-type medium spiny neurons in cocaine plasticity, highlighting the importance of medium spiny neuron subtypes in substance use disorder (62,63). Cocaine-specific RUNX2 binding along Egr3 and Dnml1 promoters suggests that SMURF1-regulated pathways may regulate cocaine-induced plasticity in a subtype-specific manner.…”

RiboTag: Not Lost in Translation

“…More specifically, these methods allow analysis of the 'translatome'-ribosome-associated mRNA-which may be particularly sensitive to eventdependent regulation of protein translation. For example, RiboTag-expressing transgenic mice were recently used to compare differential gene expression responses to cocaine in striatal neurons expressing D 1 and D 2 dopamine receptors (Chandra et al, 2015).…”