Analysis of ROS-Sensitive HDAC5 PTMs and Identification of HDAC5 Repressor Complexes in Rats Self-Administering Cocaine or Heroin
Christopher Cowan, Medical University of South Carolina
Project background and rationale (related to R01 DA0327089 and P50 DA046373): Repeated daily cocaine or heroin self-administration (SA) produces acute and chronic changes in redox homeostasis in the nucleus accumbens (NAc), and our preliminary findings indicate that oxidative conditions produced histone deacetylase 5 (HDAC5) intramolecular cysteine disulfide bond formation, HDAC5 S-glutathionylation, and Reactive oxygen species (ROS)-dependent retention of HDAC5 in the cytoplasm under conditions that normally promote nuclear accumulation (data not shown). As such, we hypothesize that ROS production by drugs of abuse, and changes in the redox balance, opposes HDAC5’s repression of genes that support drug cue-associated learning and memory, thereby enabling the formation of stable drug-memories that exacerbate relapse vulnerability. To explore this idea, we will use cutting-edge, tandem mass spectrometry approaches to characterize ROS-related HDAC5 cysteine thiol post-translational modifications from NAc tissues isolated from cocaine or heroin SA rats, and we will study the function of those ROS-modified residues (including C696=C698 disulfide bonding) using site-directed mutagenesis and analysis of cell type-specific HDAC5 subcellular localization in vitro and in vivo during and after drug SA.
Core experiment A: Analysis of ROS-sensitive HDAC5 PTMs in Rats that are Self-Administering Cocaine or Heroin. In these experiments, we will use LC-MS/MS to detect ROS-sensitive HDAC5 PTMs in a cell type-selective manner. We will infuse AAV2-DIO-Flag-HDAC5 virus bilaterally into the NA core of D1-cre or D2-cre rats (young adult males and females). Virus-infused rats will be allowed to recover for 2 weeks prior to being trained to self-administer cocaine, heroin or yoked saline. We will extract NAc tissues at two key time points: 1) immediately after a 2-hr SA session, and 2) immediately after the final 2-hr extinction session. To measure redox PTMs on HDAC5, we will rapidly isolate NAc and PL tissue punches from the virus-infused D1- or D2-cre rats and immunoprecipitate Flag-HDAC5 protein under redox buffer conditions that stabilize cysteine PTMs and prevent non-experimentally-induced intramolecular disulfide artifacts (i.e. all exposed cysteines will be irreversibly alkylated with N-ethylmaleimide (NEM) during tissue homogenization). Immunopecipitated (IPed) Flag-HDAC5 will be treated with trypsin, and peptide masses corresponding to predicted tryptic fragments of HDAC5 will be isolated for analysis. The tryptic fragments will be analyzed for evidence of disulfide bond formation or s-glutathionylation following heroin, cocaine or saline SA.
Core experiment B: Identification of HDAC5 repressor complexes in drug SA animals. Overexpression of nuclear HDAC5 (3SA mutant) in the adult rat NAc blocks both cued and drug-primed seeking of heroin or cocaine, but not sucrose. HDAC5’s intrinsic histone deacetylase activity is reported to be ~1% of class I HDAC activity, and HDAC5 is reported to associated with the class I HDAC, HDAC3, and HDAC3 is reported in the literature to influence drug behaviors in animals. To better understand how and why HDAC5 can suppress relapse-like behaviors in rats, we will examine the nature of the associated protein complexes to test the hypothesis that HDAC5 targets the complex to specific genomic loci, but associated repressor proteins provide the key enzymatic activity necessary to regulate gene expression and drug cue memories. Using a similar strategy as described above, we will IP the AAV-expressed Flag-HDAC5 from NAcore tissues using mild IP conditions to preserve protein complexes associated with HDAC5 under saline, heroin or cocaine SA conditions. We will solubilize the associated protein complexes (compared to a GFP-only control virus condition) and separate the proteins by SDS-PAGE and visualize bands using MS/MS-compatible silver-staining conditions. Visualized bands will be gel extracted, in-gel digested with trypsin and subsequently analyzed for peptide identification from LC-MS/MS spectra.
Core experiment A: Analysis of ROS-sensitive HDAC5 PTMs in Rats that are Self-Administering Cocaine or Heroin. In these experiments, we will use LC-MS/MS to detect ROS-sensitive HDAC5 PTMs in a cell type-selective manner. We will infuse AAV2-DIO-Flag-HDAC5 virus bilaterally into the NA core of D1-cre or D2-cre rats (young adult males and females). Virus-infused rats will be allowed to recover for 2 weeks prior to being trained to self-administer cocaine, heroin or yoked saline. We will extract NAc tissues at two key time points: 1) immediately after a 2-hr SA session, and 2) immediately after the final 2-hr extinction session. To measure redox PTMs on HDAC5, we will rapidly isolate NAc and PL tissue punches from the virus-infused D1- or D2-cre rats and immunoprecipitate Flag-HDAC5 protein under redox buffer conditions that stabilize cysteine PTMs and prevent non-experimentally-induced intramolecular disulfide artifacts (i.e. all exposed cysteines will be irreversibly alkylated with N-ethylmaleimide (NEM) during tissue homogenization). Immunopecipitated (IPed) Flag-HDAC5 will be treated with trypsin, and peptide masses corresponding to predicted tryptic fragments of HDAC5 will be isolated for analysis. The tryptic fragments will be analyzed for evidence of disulfide bond formation or s-glutathionylation following heroin, cocaine or saline SA.
Core experiment B: Identification of HDAC5 repressor complexes in drug SA animals. Overexpression of nuclear HDAC5 (3SA mutant) in the adult rat NAc blocks both cued and drug-primed seeking of heroin or cocaine, but not sucrose. HDAC5’s intrinsic histone deacetylase activity is reported to be ~1% of class I HDAC activity, and HDAC5 is reported to associated with the class I HDAC, HDAC3, and HDAC3 is reported in the literature to influence drug behaviors in animals. To better understand how and why HDAC5 can suppress relapse-like behaviors in rats, we will examine the nature of the associated protein complexes to test the hypothesis that HDAC5 targets the complex to specific genomic loci, but associated repressor proteins provide the key enzymatic activity necessary to regulate gene expression and drug cue memories. Using a similar strategy as described above, we will IP the AAV-expressed Flag-HDAC5 from NAcore tissues using mild IP conditions to preserve protein complexes associated with HDAC5 under saline, heroin or cocaine SA conditions. We will solubilize the associated protein complexes (compared to a GFP-only control virus condition) and separate the proteins by SDS-PAGE and visualize bands using MS/MS-compatible silver-staining conditions. Visualized bands will be gel extracted, in-gel digested with trypsin and subsequently analyzed for peptide identification from LC-MS/MS spectra.