Modulation of Poly ADP Ribose Polymerase (PARP) Levels and Activity by Alcohol Binge-Like Drinking in Male Mice

Highlights

• Alcohol consumption in binge-like DID paradigm induces PARP expression and PARP activity in the PFC of C57BL/6J male mouse.

• Virus-induced PARP1 overexpression in the PFC increases voluntary alcohol consumption in C57BL/6J male mice.

• PARP inhibitor ABT-888 decreases voluntary alcohol consumption in C57BL/6J male mice.

• RNA-seq of DID mice PFC samples identified genes upregulated by EtOH involved in addiction behavior and neuronal function.

• Four genes selected based on RNA-seq results were validated via qRT-PCR as upregulated by EtOH and downregulated by ABT-888.

Abstract

Binge drinking is a frequent pattern of ethanol consumption within Alcohol Use Disorders (AUDs). Binge-like ethanol exposure increases Poly(ADP-ribose) polymerase (PARP) expression and activity. PARP enzymes have been implicated in addiction and serve multiple roles in the cell, including gene expression regulation. In this study, we examined the effects of binge-like alcohol consumption in the prefrontal cortex (PFC) of adult C57BL/6J male mice via a 4-day Drinking-in-the-Dark (DID) paradigm. The role of PARP in associated gene expression and behavioral changes was assessed by administering the PARP inhibitor ABT-888 on the last DID day. We then conducted an RNA-seq analysis of the PFC gene expression changes associated with DID-consumed ethanol or ABT-888 treatment. A separate cohort of mice was inoculated with an HSV–PARP1 vector in the PFC and subject to a DID experiment to verify whether overexpressed PARP1 increased ethanol drinking. We confirmed that alcohol increases Parp1 gene expression and PARP activity in the PFC. RNA-seq showed significantly altered expression of 41 genes by DID-consumed ethanol, and of 48 genes by ABT-888. These results were confirmed by qPCR in 7 of the 10 genes validated, 4 of which have been previously associated with addiction. ABT-888 reduced, and overexpression of PFC PARP1 increased DID ethanol consumption. In our model, alcohol binge drinking induced specific alterations in the PFC expression of genes potentially involved in addiction. Pharmacological PARP inhibition proved effective in reversing these changes and preventing further alcohol consumption. Our results suggest an involvement of ethanol-induced PARP1 in reinforcing binge-like addictive behavior.

Introduction

Alcohol Use Disorder (AUD) is among the most common mental health disorders in the US, with a 29.1% lifetime prevalence (Grant et al., 2015). Binge alcohol drinking is defined as the acquisition of a blood ethanol concentration (BEC) ≥ 80 mg/dL, generally reached after 5 drinks in men and 4 drinks in women within 2 hours (“NIAAA Council Approves Definition of Binge Drinking.” NIAAA Newsletter, Department of Health and Human Services – National Institutes of Health, Winter 2004, 3: 3). Binge alcohol drinking contributes to more than half of the deaths associated with excessive alcohol consumption, and can lead to impairments in psychosocial functioning as well as adverse health consequences, including unintentional injuries, alcohol poisoning, gastritis, etc. (Naimi et al., 2003, Stahre et al., 2014). Accumulated data from diverse studies, including human and animal behavioral studies, brain imaging, electrophysiology, and molecular and cellular observations strongly support that the prefrontal cortex (PFC) plays a critical role in addiction (Grusser et al., 2004, Heilig et al., 2017, Wolstenholme et al., 2017). However, the molecular mechanisms underlying binge alcohol drinking as well as its biochemical effects are not fully understood.

Prior studies have focused on the role of PARP activity in cell death pathways following toxic levels of ethanol exposure (Climent et al., 2002, Cherian et al., 2008). The poly (ADP-ribose) polymerase (PARP) family is composed of 18 members. Murai et al (2012) reported that PARP1 is responsible for 90–95% of PARP enzymatic activity while PARP2 contributes the remaining 5–10% (Murai et al., 2012). In mouse primary cortical neuronal cultures, we found that ethanol exposure increased PARP enzymatic activity and led to gene expression changes, and that PARP inhibitors rescued some of the ethanol-induced gene expression changes (Gavin et al., 2016), indicating that PARP enzymes may act as regulators in the biochemical response to ethanol exposure (Gavin et al., 2016).

The accepted addiction cycle model presumes that alcohol drinking induces neuroadaptive changes in the brain that lead to continued alcohol seeking, therefore perpetuating the drinking cycle (Koob, 2003). We hypothesized that ethanol-induced upregulation of PARP expression and activity may lead to changes in the brain involved in increasing the alcohol drinking behavior in an animal model of binge drinking.

In support of the role of PARP enzymes in addiction, cocaine was shown to significantly increase PARP1 expression and enzymatic activity in the nucleus accumbens, and overexpression of PARP1 increased, and PARP inhibition decreased conditioned place preference for cocaine (Scobie et al., 2014). In this study, we show evidence that alcohol drinking increases PARP gene expression and its enzymatic activity in the PFC promoting further alcohol consumption. PARP1 overexpression also increases voluntary alcohol intake; this effect is associated with increased expression of genes involved in binge alcohol drinking. These observed biomolecular effects of alcohol are reduced by PARP inhibition, and PARP inhibition reduces alcohol drinking.