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Regulation of dopamine-dependent transcription and cocaine action by Gadd45b

Abstract

Exposure to drugs of abuse produces robust transcriptional and epigenetic reorganization within brain reward circuits that outlives the direct effects of the drug and may contribute to addiction. DNA methylation is a covalent epigenetic modification that is altered following stimulant exposure and is critical for behavioral and physiological adaptations to drugs of abuse. Although activity-related loss of DNA methylation requires the Gadd45 (Growth arrest and DNA-damage-inducible) gene family, very little is known about how this family regulates activity within the nucleus accumbens or behavioral responses to drugs of abuse. Here, we combined genome-wide transcriptional profiling, pharmacological manipulations, electrophysiological measurements, and CRISPR tools with traditional knockout and behavioral approaches in rodent model systems to dissect the role of Gadd45b in dopamine-dependent epigenetic regulation and cocaine reward. We show that acute cocaine administration induces rapid upregulation of Gadd45b mRNA in the rat nucleus accumbens, and that knockout or site-specific CRISPR/Cas9 gene knockdown of Gadd45b blocks cocaine conditioned place preference. In vitro, dopamine treatment in primary striatal neurons increases Gadd45b mRNA expression through a dopamine receptor type 1 (DRD1)-dependent mechanism. Moreover, shRNA-induced Gadd45b knockdown decreases expression of genes involved in psychostimulant addiction, blocks induction of immediate early genes by DRD1 stimulation, and prevents DRD1-mediated changes in DNA methylation. Finally, we demonstrate that Gadd45b knockdown decreases striatal neuron action potential burst duration in vitro, without altering other electrophysiological characteristics. These results suggest that striatal Gadd45b functions as a dopamine-induced gene that is necessary for cocaine reward memory and DRD1-mediated transcriptional activity.

a Adult male rats were administered a single i.p. injection of saline or 10 mg/kg cocaine and returned to their home cage. NAc tissue was collected bilaterally at either 1 hr or 24 hr post injection. RT-qPCR reveals significant increases in immediate early gene (IEG) expression at 1 hr in cocaine-treated rats compared with saline controls. No changes were observed at 24 hr. b Of genes involved in activity-dependent DNA methylation and demethylation processes, only Gadd45b mRNA was significantly increased 1 hr following acute exposure to cocaine. There were no significant changes observed at 24 hr. c To examine cocaine locomotor sensitization, all rats were administered saline on days 1 and 2. On days 3 and 10, half received a 10 mg/kg cocaine injection. This yielded four groups. d Distance traveled was significantly increased in animals that received cocaine either on day 3 or on day 10. Animals who received two consecutive cocaine injections on day 3 and day 10 exhibited further increases in locomotor activity. e Locomotor sensitization was only observed in animals that received cocaine on both day 3 and day 10. Distance traveled on days 3 and 10 was normalized within group to mean locomotion of baseline days 1 and 2. f NAc tissue was collected bilaterally 1 hr post injection on day 10 for RT-qPCR analysis. Gadd45b mRNA was significantly increased in all groups with cocaine experience. All data are expressed as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 for indicated comparisons.

a Male wild-type and mutant Gadd45b knockout mice between 2 and 6 months of age were used for behavioral testing. b Conditioned place preference (CPP) apparatus and experimental timeline for CPP testing. c Cocaine-paired place preference was significantly attenuated in Gadd45b mutant mice. d Targeting strategy to induce insertion/deletion mutations in Gadd45b. CRISPR guide RNAs (gRNAs) were engineered to target exon 2 of Gadd45b. e Illustration of lentivirus construct designed to express gRNA (driven by the U6 promoter) and the Cas9 nuclease (driven by the Ef1α promoter), with bicistronic expression of EGFP. f Sanger sequencing of individual genomic DNA clones reveals CRISPR-mediated mutation in six of eight alleles (75% efficiency) following lentiviral transduction. Primary striatal neuron cultures were transduced with lentivirus at DIV7 and genomic DNA was extracted at DIV11. g Genomic DNA restriction digest at AlwNI site (which is mutated by Cas9 editing) reveals almost complete digestion in lacZ gRNA control, but loss of digestion when AlwNI site has been mutated in Gadd45b gRNA group. Data from primary striatal neuron cultures. h RT-qPCR validation demonstrates robust knockdown of Gadd45b mRNA following Cas9 targeting in striatal neuron cultures. Remaining mRNA contains insertions/deletions resulting in premature translation termination or dysfunctional protein. i Experimental timeline for CRISPR/Cas9 viral transduction and cocaine CPP testing. Lentiviral constructs targeting lacZ or Gadd45b were bilaterally injected into the NAc core 2 weeks prior to behavioral testing. Inset image shows EGFP expressed from SpCas9-2A-EGFP cassette in a representative NAc section. Scale bar = 50 µm. j Rats that received the Gadd45b-targeted CRISPR/Cas9 construct failed to form a place preference to cocaine at both 10 mg/kg and 20 mg/kg. All data are expressed as mean ± s.e.m. *p < 0.05, **p < 0.01, and ***p < 0.001 for indicated comparisons.

a Illustration of experimental design using primary rat striatal culture system. b Unbiased identification of DA-induced DEGs with RNA-seq reveals Gadd45b as one of 103 DA-induced IEGs. c Cells were treated with DA (1 µM) for 1 hr prior to RNA isolation and RNA-seq. c RT-qPCR validation of DA-induced increase in Gadd45b mRNA. d DA-induced increases in Gadd45b mRNA are blocked by co-treatment with the DRD1 receptor antagonist SCH-23390 (1 µM). e Gadd45b mRNA induction is mimicked by DRD1 receptor agonist SKF-38393 (1 µM), but not the D2/D3 receptor agonist Quinpirole (1 µM). f, Gadd45b mRNA is induced by the adenylyl cyclase activator Forskolin (20 µM). g CREB inhibition with 666-15 (1 µM) blocks baseline and DA-induced increases in Gadd45b mRNA. h MEK inhibition with U0126 (1 µM) prevents DA-induced increases in Gadd45b mRNA. U0124 (1 µM) is an inactive analog of U0126 and was included as a negative control. ***p < 0.001 and ****p < 0.0001 for indicated comparisons.

a Illustration of experimental design for shRNA-mediated Gadd45b knockdown and transcriptional profiling. b Volcano plot of 7325 differentially expressed genes (DEGs) in Gadd45b shRNA group as compared with scrambled shRNA control group. c Heatmap showing replicate values of all DEGs (3798 upregulated, 3527 downregulated) after Gadd45b knockdown. d KEGG network analysis reveals differential upregulation of genes implicated in oxidative phosphorylation and neurodegenerative disease categories, and downregulation of genes implicated in dopaminergic synapse function and addiction categories. e Heatmap of DEGs altered by 2 hr stimulation with the DRD1 receptor agonist SKF-38393 (1 µM), stratified by shRNA treatment group. fg Genes upregulated by SKF-38393 stimulation are significantly less induced under conditions of Gadd45b knockdown. ****p < 0.0001 for indicated comparisons.

a Experimental timeline for viral transduction and extracellular single-unit recordings. Primary striatal neurons were grown on multielectrode arrays (MEAs) and transduced with lentiviral shRNA constructs on DIV5. b Live cell imaging of transduced neurons (expressing mCherry) on DIV13 after collecting electrophysiological measures. Scale bar = 100 μm. c Representative traces of four units transduced with either scrambled shRNA or Gadd45b shRNA. d Representative raster plots (one neuron per row). Orange horizontal lines denote action potential bursts. e Gadd45b knockdown does not affect the number of spontaneously active units per well. fg Action potential frequency and burst frequency are not different between neurons transduced with Gadd45b shRNA and scrambled controls. h For all neurons with at least one burst, Gadd45b knockdown does not alter % of spikes occurring in a burst. i shRNA knockdown of Gadd45b significantly decreases burst duration compared with scrambled controls. jk Mean Z score (top) and individual neuron heatmap (bottom) following application of vehicle or 1 µM SKF-38393. l,SKF-38393 increases neuronal firing rate as compared with vehicle control. Effects of SKF-38393 did not differ based on Gadd45b knockdown status. m Left, SKF-38393 response as a function of baseline (pre-SKF-38393) action potential frequency. Right, SKF-38393 response did not differ in Gadd45b shRNA treatment group. All bar graphs expressed as mean ± s.e.m. All violin plots expressed as median ± quartiles. *p < 0.05 and **p < 0.01 for indicated comparisons.

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Acknowledgements

We thank Allison Bauman for generating our primary neuronal cell cultures. We thank Katherine Savell and Jasmin Revanna for their assistance with lentiviral production. We thank the Genomics Core Lab at the UAB Heflin Center for Genomic Sciences for assistance with RNA-seq and RRBS, and the Civitan International Research Center Cellular Imaging Facility for access to the Zeiss Airyscan LSM800 confocal microscope. We thank all current and former Day Lab members for assistance and support.

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JJD performed RT-qPCR analysis of IEGs and DNA methylation machinery. ACB completed cocaine locomotor sensitization assays and RT-qPCR analysis of Gadd45b expression after exposure to cocaine. FAS cloned, designed, and validated CRISPR/Cas9 and shRNA constructs for in vivo and in vitro work, with assistance from GG, JJD, and FAS conceived of and performed CCP assays with transgenic mice. JJD, FAS, and MEZ conceived of and performed CCP assays with CRISPR/Cas9 in rats, with assistance from GG, MEZ, and FAS completed all stereotaxic surgeries for in vivo testing, with assistance from GG and NAS. Microscopy was performed by NVNC and MEZ with assistance from FAS. FAS performed and analyzed in vitro DA, DRD1 agonist, and DRD2 agonist experiments with assistance from GG. MEZ performed and analyzed in vitro DRD1 antagonist, Forskolin, MEK inhibitor, and CREB inhibitor experiments. JJD, FAS, and LI performed statistical and graphical analysis of bulk RNA-seq data sets. MEZ performed all electrophysiological assays and analyzed single-unit data, with assistance from JJD and NAS All projects were supervised by JJD, MEZ, and JJD wrote the main text of the manuscript. All authors have approved the final version of the manuscript.

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Correspondence to Jeremy J. Day.

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Zipperly, M.E., Sultan, F.A., Graham, G. et al. Regulation of dopamine-dependent transcription and cocaine action by Gadd45b. Neuropsychopharmacol. (2020). https://doi.org/10.1038/s41386-020-00828-z

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