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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Neurochem Int. 2019 Jun 25;129:104497. doi: 10.1016/j.neuint.2019.104497

PARP inhibition in vivo blocks alcohol-induced brain neurodegeneration and neuroinflammatory cytosolic phospholipase A2 elevations

Dimitrios E Kouzoukas a,d,f,*, Jennifer A Schreiber b,d, Nuzhath F Tajuddin a, Simon Kaja a,b,c,d,e,f, Edward J Neafsey a, Hee-Yong Kim g, Michael A Collins a,b,d
PMCID: PMC6760246  NIHMSID: NIHMS1041436  PMID: 31251945

Abstract

Chronic alcoholism promotes brain damage that impairs memory and cognition. High binge alcohol levels in adult rats also cause substantial neurodamage to memory-linked regions, notably, the hippocampus (HC) and entorhinal cortex (ECX). Concurrent with neurodegeneration, alcohol elevates poly(ADP-ribose) polymerase-1 (PARP-1) and cytosolic phospholipase A2 (cPLA2) levels. PARP-1 triggers necrosis when excessively activated, while cPLA2 liberates neuroinflammatory ω−6 arachidonic acid. Inhibitors of PARP exert in vitro neuroprotection while suppressing cPLA2 elevations in alcohol-treated HC-ECX slice cultures. Here, we examined in vivo neuroprotection and cPLA2 suppression by the PARP inhibitor, veliparib, in a recognized adult rat model of alcohol-binging. Adult male rats received Vanilla Ensure containing alcohol (ethanol, 7.1 ± 0.3 g/kg/day), or control (dextrose) ± veliparib (25 mg/kg/day), by gavage 3x daily for 4 days. Rats were sacrificed on the morning after the final binge. HC and ECX neurodegeneration was assessed in fixed sections by Fluoro-Jade B (FJB) staining. Dorsal HC, ventral HC, and ECX cPLA2 levels were quantified by immunoblotting. Like other studies using this model, alcohol binges elevated FJB staining in the HC (dentate gyrus) and ECX, indicating neurodegeneration. Veliparib co-treatment significantly reduced dentate gyrus and ECX neurodegeneration by 79% and 66%, respectively. Alcohol binges increased cPLA2 in the ventral HC by 34% and ECX by 72%, which veliparib co-treatment largely prevented. Dorsal HC cPLA2 levels remained unaffected by alcohol binges, consistent with negligible FJB staining in this brain region. These in vivo results support an emerging key role for PARP in binge alcohol-induced neurodegeneration and cPLA2-related neuroinflammation.

Keywords: alcohol, cytosolic phospholipase A2, neurodegeneration, poly(ADP-ribose) polymerase, veliparib

1. Introduction

A prominent outcome of chronic alcohol misuse and binge alcoholism is an elevated risk of cognitive abnormalities including dementia, and corresponding damage to supporting brain structures (Erdozain et al., 2014; Harper, 2009). Excluding instances of thiamine malnutrition or hepatic dysfunction, chronic alcohol misuse comprises ~10% of early onset dementia cases (Cheng et al., 2017). As in humans (Erdozain et al., 2014; Harper, 2009; Wilson et al., 2017), binge alcohol-related neurodamage in rodent models typically involves structures supporting learning and memory, including the hippocampus (HC) and entorhinal cortex (ECX) (Collins et al., 1996; Obernier et al., 2002; Vetreno et al., 2011). Neuroinflammation is also evident in these brain regions.

Concerning phospholipase-derived neuroinflammation, we documented that repeated alcohol binges in rats elevated levels of cytosolic phospholipase A2 (cPLA2 IVA), which is responsible for liberating the ω−6 proinflammatory fatty acid, arachidonic acid, from membrane phospholipids (Tajuddin et al., 2014). In other neuroinflammatory pathways, alcohol binging raised levels of the neuroinflammatory alarmin, HMGB1, and its receptors in rodent and postmortem alcoholic brains (Crews et al., 2013; Vetreno et al., 2013). Alcohol binging also elevated CD11B, an indicator of primed microglia that exacerbate neuroinflammatory responses (Marshall et al., 2016). Collectively, these findings indicate that binge alcohol exposure drives neuroinflammatory pathways linked to neurodegeneration in vulnerable brain regions.

We previously reported that alcohol-induced neurodamage associated with elevated levels of poly (ADP-ribose) polymerase-1 (PARP-1) in adult rat HC and ECX and in organotypic HC-ECX slice cultures (Collins et al., 2014; Tajuddin et al., 2014). Although PARP is a key DNA repair enzyme, its overactivation can trigger regulated necrosis of neurons (parthanatos) (Fatokun et al., 2014). Indeed, blocking PARP activity with pharmacological inhibitors in the alcohol-binged HC-ECX slice cultures reduced or prevented neurodegeneration (Tajuddin et al., 2018). Furthermore, PARP inhibition in brain slice cultures prevented binge alcohol’s augmentation of cPLA2 levels (Tajuddin et al., 2018), revealing a previously unacknowledged connection between neuronal death by brain PARP overactivation and the cPLA2 neuroinflammatory pathway.

In the current study, we tested whether the brain-penetrable PARP inhibitor, veliparib, counteracted alcohol-induced neurodegeneration and associated neuroinflammatory changes in cPLA2 in a 4-day severe binge intoxication model (Collins et al., 1996; Majchrowicz, 1975; Tajuddin et al., 2014). It differs from the our previous studies (Tajuddin et al., 2013; Tajuddin et al., 2014;Tajuddin et al., 2018) by focusing on PARP inhibition and prevention of cPLA2 changes in vivo. The results indicate that PARP mediates binge alcohol-induced neurodegeneration and neuroinflammatory cPLA2 upregulation in the HC and ECX, consistent with previous in vitro results (Tajuddin et al., 2018).

2. Materials and methods

2.1. Alcohol exposure and pharmacological treatment

Experiments were approved by the Loyola University Chicago Institutional Animal Care and Use Committee and performed according to the guidelines of the National Institutes of Health. Adult male Sprague Dawley rats (321.0 ± 1.9 g) received alcohol (0 to 5 g/kg ethanol) or dextrose caloric supplement (control) in Vanilla Ensure Plus (Abbott Laboratories, Chicago, IL) by gavage every 8 hrs for 4 days (Collins et al., 1996; Faingold, 2008; Majchrowicz, 1975). After a 5 g/kg alcohol priming dose, subsequent alcohol treatments were titrated according to a 6-point scale based on a visual inspection of intoxication behaviors: 0=normal movement (5 g/kg), 1=hypoactive (4 g/kg), 2=ataxia (3 g/kg), 3=delayed righting reflex, ataxia with no abdominal elevation (2 g/kg), 4=loss of righting reflex (1 g/kg), and 5=loss of eye blink reflex (0 g/kg) (Faingold, 2008; Nixon and Crews, 2004). This scale highly correlates with blood alcohol concentrations (BAC). Rats received the PARP inhibitor, veliparib (ApexBio, Boston, MA; 25 mg/kg/day) orally in control or alcohol solutions. Veliparib was chosen due to its high oral bioavailability, long half-life, and efficiency in penetrating the blood-brain-barrier (Donawho et al., 2007; Li et al., 2011; Muscal et al., 2010). Using a commercial kit (Pointe Scientific, Canton, MI), peak BACs were measured in serum of tail blood collected 90 min after the 1st gavage on day 4.

2.2. Tissue collection & sectioning

On the morning after the final gavage, isoflurane-anesthetized rats were transcardially perfused with ice-cold phosphate-buffered saline (PBS). Brains were removed, split along the midsagittal plane, and frozen in isopentane cooled by a methanol/dry ice slurry. Horizontal sections (25 μm) from one hemisphere, taken every 0.5 mm from −8.6 to −5.6 mm below bregma, were thaw-mounted onto positively-charged glass slides (Diamond; Globe Scientific, Paramus, NJ). From the other hemisphere, HC and ECX were microdissected on ice (Chiu et al., 2007). Isolated HC were bisected into dorsal and ventral regions. All samples were stored at −80°C.

2.3. Fluoro-Jade B staining for neurodegeneration and analysis

Fluoro-Jade B (FJB) is a well-accepted fluorescent marker of dead or dying neurons, including in this alcohol binge model (Leasure and Nixon, 2010, Obernier et al., 2002). Sections were fixed (4% paraformaldehyde in PBS, 15 min), air-dried, then processed for FJB (EMD Millipore, Billerica, MA) staining as described (Leasure and Nixon, 2010; Obernier et al., 2002). Slides were immersed in solutions of 1% sodium hydroxide in 80% ethanol for 5 min, 70% ethanol for 2 min, distilled water for 2 min, 0.06% potassium permanganate for 10 min, distilled water for 2 min, 0.01% FJB in 0.1% acetic acid for 20 min, and washed 3x with distilled water for 1 min. Air-dried sections were sealed under coverglass with Cytoseal 60 (Richard-Allan Scientific, San Diego, CA) and dried overnight.

Sections were imaged using a Cytation5 digital imager (BioTek, Winooski, VT). Structures were determined by a stereotaxic atlas (Paxinos and Watson, 2014). HC and ECX regions of interest (ROIs) were drawn on section scans using ImageJ (NIH, Bethesda, MD), and areas (mm2) measured. In the dentate gyrus (DG), FJB+ cells mostly appeared in the granule cell layer, whereas ECX FJB+ cells localized to layer III (Fig. 1A) similar to previous reports (Collins et al., 1996; Obernier et al., 2002). ROIs were created for entire structures. Since ROI area, and consequently FJB+ cell counts, depended on dorsal-ventral brain coordinates, counts were normalized to the ROI surface area and averaged across sections for each rat. Only FJB+ cells that resembled neuronal soma morphologically (see insets, Fig. 1A) were counted in ROIs. Counts were verified by a blinded observer. For each rat, a minimum of three horizontal sections (an average of 5.40 ± 0.22 and 5.50 ± 0.25 sections for each DG and LEnt, respectively) were analyzed.

Fig. 1. PARP inhibitor, veliparib, reduced alcohol-related neurodegeneration in dentate gyrus (DG) and lateral entorhinal cortex (LEnt).

Fig. 1.

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A) Panels show neurodegeneration (FJB labeling) in rat dentate gyrus (DG; above) and lateral entorhinal cortex (LEnt; below) after 4 days of alcohol binges. White arrows identify examples of FJB+ (degenerating) cells. For each viewfield, insets are the boxed areas magnified 300%. ROIs were drawn on horizontal HC and ECX scans using ImageJ (NIH, Bethesda, MD) and areas (mm2) measured. For each ROI, FJB+ cells were counted and then verified by a blinded observer. B) Bar graphs show that veliparib (25 mg/kg/day) reduced alcohol-induced DG and LEnt neurodegeneration by 79.1% and 66.3%, respectively. N = 4 – 5 rats/group. (* different from all, # different from alcohol and no treatment control groups; Tukey’s HSD, p ≤ 0.05).

2.4. Western blotting

Dorsal HC, ventral HC, and ECX were homogenized in cold lysis buffer (1 mg tissue/12 μl RIPA buffer; Sigma, Burlington, MA) containing protease and phosphatase inhibitor cocktail (50X, Sigma, Burlington, MA), gently shaken for 2 hrs (4°C), and then centrifuged to pellet debris. Collected supernatants were aliquoted and frozen at −80°C. Bicinchoninic acid assay (Pierce Biotech, Rockford, IL) determined total proteins concentrations. Reduced samples (40 g protein + 100 μM dithiothreitol) in Laemmli buffer (Bio-Rad, Hercules, CA), were separated by SDS-PAGE in 4–20% gradient Bis-Tris gels (Genscript, Piscataway, NJ) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Cytosolic PLA2 was quantified using a primary antibody (sc-454 at 1:250 dilution; Santa Cruz Biotech, Dallas, TX), a horseradish peroxidase-linked secondary antibody (sc-516102; Santa Cruz Biotech, Dallas, TX), and the SuperSignal West Femto Substrate ECL kit (ThermoFisher Scientific, Waltham, MA). The loading control, GAPDH (probed with sc-365062; Santa Cruz Biotech, Dallas, TX), was measured after stripping (Restore; Thermo Fisher Scientific, Waltham, MA). All blot images were acquired by a Chemidoc XRS imaging system (BioRad, Hercules, CA) and analyzed in ImageJ (NIH, Bethesda, MD) using rolling ball background subtraction. Relative density values were calculated as the target protein band density divided by that of GAPDH, then expressed as a percent of control group levels (mean ± SEM) in each blot. Samples run on multiple blots were normalized to their respective blot’s control group, then averaged.

2.5. Statistical analyses

ANOVAs determined significant interactions between factors and differences between treatment groups. Post-hoc analyses consisted of Tukey’s Honest Significant Difference (HSD) test, or planned comparisons (Student’s t-tests) where indicated. For western blot data, statistical extreme outliers, defined as greater than 3.0 × interquartile range, were removed (one data point). Outliers in the control group (no alcohol or veliparib) were kept, since all values were normalized (100%) to the cPLA2 levels in the control group within each blot.

All data are presented as mean ± SEM, with statistical differences of p ≤ 0.05 considered significant. All statistical analyses were performed using SPSS Statistics (IBM, New York, NY).

3. Results

Weight loss is typical and expected with this binge alcohol intoxication model (Marshall et al., 2016). Significant weight loss (Tukey’s HSD, p ≤ 0.05) over the 4-day binge paradigm occurred in animals receiving either Alcohol (14.5 ± 0.7%) or Alcohol + Veliparib (9.3 ± 2.5%) (Table 1). No differences in peak BAC (Student’s t-test, p > 0.05) were detected in alcohol-binged rats given veliparib (25 mg/kg/day) or not (369.5 ± 36.0 vs. 350.6 ± 31.1 mg/dl, Table 1). Veliparib-treated rats appeared slightly less intoxicated as determined by the 6-point visual intoxication scale (1.8 ± 0.3 vs. 2.4 ± 0.1; Student’s t-test, p ≤ 0.05). This difference necessitated that rats treated with alcohol and veliparib receive more alcohol overall than rats receiving only alcohol (9.0 ± 0.7 vs. 7.1 ± 0.3 g/kg/day; Student’s t-test, p ≤ 0.05).

Table 1.

Weight and alcohol intake across treatment groups.

Group N Initial
Weight
(g)		 Final
Weight
(g)		 Weight
Loss
(%)		 Alcohol
Intake
(g/kg/day)		 Peak BAC
(mg/dl)
Control 5 320 ± 4.8 320 ± 4.0 0.2 ± 1.1 - -
Control + Veliparib 5 322 ± 1.9 327 ± 3.7 −1.3 ± 0.6 - -
Alcohol 5 321 ± 5.0 274 ± 5.6 * 14.5 ± 0.7 * 7.1 ± 0.3 359.0 ± 31.8
Alcohol + Veliparib 4 320 ± 3.7 290 ± 9.6 * 9.3 ± 2.6 * 9.0 ± 0.7 # 378.4 ± 36.9
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Means ± SEM are displayed.

*

(different from control groups, Tukey’s HSD; # different from alcohol alone, Student’s t-test, p ≤ 0.05). Peak BAC in alcohol-treated groups averaged 79.8 ± 4.9 mM (367.7 ± 22.6 mg/dl). Rats in the control groups received no alcohol.

3.1. Veliparib reduced binge alcohol neurodegeneration in dentate gyrus (DG) and lateral ECX (LEnt)

To examine alcohol-induced neurodegeneration, ROIs for HC and ECX were drawn on section images and FJB+ cells were counted. As shown in Table 2, control rat sections showed few FJB+ cells (< 0.75 cells/mm2) in the HC and ECX, and veliparib alone did not raise FJB+ cell counts (Tukey’s HSD, p > 0.05 vs. control) in any examined brain region. Binge alcohol treatment promoted significant DG and LEnt neurodegeneration (Table 2 and Fig. 1; Tukey’s HSD, p ≤ 0.05) as shown by elevated counts of FJB+ cells vs. control in the DG (8.4 ± 2.1 cells/mm2) and LEnt (10.7 ± 1.1 cells/mm2). No significant changes in FJB+ cell counts were seen in other HC regions (cornu ammonis and subiculum) or in the medial entorhinal cortex, agreeing with earlier cupric-silver neurodegeneration stain results (Collins et al., 1996). No FJB+ cells appeared in the dorsal HC (above bregma −4.6 mm). Two-way ANOVAs (2×2) revealed the interaction of veliparib and alcohol exposure on FJB+ counts in the DG and LEnt (p ≤ 0.05, Fig. 1B). Co-treatment with veliparib in alcohol-binged rats promoted significant neuroprotection as evidenced by a 78.6% reduction in FJB+ cell counts (1.8 ± 0.6 cells/mm2) in DG, and a 66.4% reduction in FJB+ counts (3.6 ± 1.2 cells/mm2) in LEnt (Tukey’s HSD, p ≤ 0.05 vs alcohol only; Table 2 and Fig. 1).

Table 2.

The effect of veliparib on alcohol-induced neurodegeneration in the HC and ECX.

Group N Coor.
(mm)		 Neurodegeneration (FJB+ Cells/ mm2)
Hippocampus Entorhinal Cortex
S CA1 CA2 CA3 DG MEnt LEnt
Control 5 −6.96 ± 0.05 0.5 ± 0.2 0.1 ± 0.1 0.5 ± 0.2 0.2 ± 0.1 0.5 ± 0.1 0.7 ± 0.1 0.3 ± 0.1
Control + Veliparib 5 −7.00 ± 0.03 0.2 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 0.5 ± 0.1 0.7 ± 0.2 1.1 ± 0.6 0.8 ± 0.2
Alcohol 5 −7.07 ± 0.14 1.0 ± 0.4 0.6 ± 0.2 0.9 ± 0.3 1.6 ± 0.6 8.4 ± 2.1* 2.1 ± 0.4 10.7 ± 1.1*
Alcohol + Veliparib 4 −7.15 ± 0.03 0.5 ± 0.2 0.6 ± 0.4 0.5 ± 0.2 0.7 ± 0.2 1.8 ± 0.6 1.6 ± 0.4 3.6 ± 1.2
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Degenerating neurons (FJB+ Cells) and ROI areas (mm2) were determined in the HC and ECX sections of rats receiving treatments. Means ~± SEM are displayed. Four days of alcohol binges caused neurodegeneration in the dentate gyrus (DG) and lateral entorhinal cortex (LEnt)

*

( different from all, Tukey’s HSD, p ≤ 0.05), which was prevented by the PARP inhibitor, veliparib (25 mg/kg/day). Other abbreviations: Vertical coordinates (Coor.); Cornu Ammonis (CA); Medial entorhinal cortex (MEnt); Subiculum (S).

3.2. Veliparib blocked the binge alcohol-induced increase of cPLA2 levels in ventral HC and ECX

Since veliparib diminished neurodegeneration (FJB+ cells) in the ventral HC and LEnt (Table 2), we evaluated whether PARP inhibition also affected neuroinflammatory changes in cPLA2 in these regions. Consistent with previous reports (Tajuddin et al., 2014; Tajuddin et al., 2013), binge alcohol treatment significantly elevated cPLA2 in the ventral HC (+34.4 ± 4.4%; Tukey’s HSD, p ≤ 0.05, Fig. 2B) and in the ECX (+71.9 ± 2.3%; Tukey’s HSD, p ≤ 0.05, Fig. 2C). No cPLA2 changes from binge alcohol treatment were observed in the dorsal HC (Fig. 2A), which is consistent with FJB+ cells absent in the dorsal HC (above bregma −4.6 mm). Two-way ANOVAs (2×2) revealed the interaction of veliparib and alcohol exposure on cPLA2 levels in the ventral HC and ECX (p ≤ 0.05, Figs 2B, 2C). PARP inhibition by veliparib co-treatment in alcohol-binged rats abolished the amplification of cPLA2 levels in the ventral HC, suppressing cPLA2 by 44.7%. A similar reduction in cPLA2 levels (34.6%) was observed in the ECX.

Fig. 2. PARP inhibitor, veliparib, reduced alcohol-related changes in cPLA2 levels in the HC and ECX.

Fig. 2.

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Representative bands from each immunoblot are at the top of each panel. Graphs respectively show cPLA2 levels in the dorsal HC (A), ventral HC (B), and ECX (C) normalized to GAPDH, relative to corresponding levels in controls (rats with no exposure to alcohol or veliparib). Bar graphs show that alcohol binges elevated cPLA2 levels in the ventral HC and ECX, but not in the dorsal HC. Two-way ANOVAs indicated veliparib (25 mg/kg/day) reduced alcohol-related changes in cPLA2 in the ventral HC and the ECX (p ≤ 0.05). N = 4 – 5 rats/group. Differences by Tukey’s HSD (one-way ANOVA) are indicated: * (different from all, p ≤ 0.05), # (different from no treatment control group, p ≤ 0.05).

4. Discussion

These results indicate that PARP overactivation plays a key role in binge alcohol-induced neurodegeneration and phospholipid-associated neuroinflammation in vivo. This conclusion is supported by demonstrating the in vivo efficacy of a PARP inhibitor in preventing alcohol neurodegeneration in vulnerable brain regions and neuroinflammatory changes in cPLA2 levels. Furthermore, this study strengthens the link between PARP and changes in phospholipid-mediated neuroinflammatory signaling by being the first in vivo study that links PARP activity to brain cPLA2 elevations.

The current study confirmed previous findings from alcohol-binged adult rats. Specifically, binge alcohol treatment caused HC and ECX neurodegeneration (Collins et al., 1996; Kelso et al., 2011; Obernier et al., 2002), which resembles corticolimbic patterns of brain damage in chronic alcoholics (Erdozain et al., 2014; Harper, 2009; Wilson et al., 2017). Our levels of FJB+ cells are comparable with those seen in other studies (Cippitelli et al., 2017; Kelso et al., 2011; Obernier et al., 2002). Although the current study used FJB, an often-used marker of degenerating neurons, it can also stain glial cells (Damjanac et al., 2007). Special care was taken to count cells that morphologically resembled neuronal soma. Previous studies (Collins et al., 1996; Obernier et al., 2002), using another marker of degenerating neurons, the amino cupric silver technique, in this same alcohol binge model, also incurred extensive staining in the HC and ECX structures indicating specific neurodegeneration.

Moreover, PARP inhibitor treatment (veliparib) prevented this damage in vivo. The current study extends earlier in vitro findings, which demonstrated 1) that alcohol increases enzymatic PARP activity in mouse cortical neuronal cultures (Gavin et al., 2016) and 2) that PARP inhibition protects against alcohol’s neurodamage in HC-ECX slice cultures (Tajuddin et al., 2018). The current study also complements our observations of elevated PARP-1 levels in highly vulnerable brain regions (HC and ECX) of moderately binged adult rats (~3 g/kg/d alcohol) (Tajuddin et al., 2013). Although the brain expresses multiple PARP isoforms, PARP-1 is responsible for approximately 90% of the total cellular PARylation activity after DNA damage (Horvath et al., 2011). Our findings are consistent with a growing literature indicating that DNA strand breaks after pro-oxidative brain insults (i.e., stroke or trauma) may overactivate PARP-1, with its poly(ADP-ribose) products eliciting neuroinflammation and neurodegeneration (Narne et al., 2017). They also agree with recent studies indicating alcohol binges elicit DNA damage (Moon et al., 2014; Suman et al., 2016).

Our prior studies demonstrate that binge alcohol treatment alter levels of PLA2 isoenzymes (Tajuddin et al., 2018; Tajuddin et al., 2014), which can lead to neuroinflammation (Ong et al., 2010). Cytosolic PLA2 acts on membrane phospholipids to mobilize arachidonic acid, which is subsequently converted by cyclooxygenases and lipoxygenases into potent pro-inflammatory mediators—e.g., prostaglandins and leukotrienes (Calder, 2006). These products and pathways can promote formation of reactive oxygen species that contribute to neurodegeneration (Ong et al., 2010).

In previous studies (Tajuddin et al., 2018; Tajuddin et al., 2014), alcohol elevated levels of cPLA2 and secretory PLA2 (sPLA2) in the HC and ECX of adult rats and brain slice cultures. Concurring with these PLA2 elevations, alcohol stimulated arachidonic acid release from brain slice cultures, while treatment with PLA2 inhibitors reduced binge alcohol-provoked neurodegeneration (Brown et al., 2009; Tajuddin et al., 2018). These data provide support for binge alcohol treatment promoting the neuroinflammatory cPLA2 pathway to facilitate neurodegeneration. It should be noted that ω−3 docosahexaenoic acid treatment also conferred neuroprotection while preventing alcohol-induced elevations in cPLA2 levels (Tajuddin et al., 2014). Together, these studies indicate that alcohol neurodegeneration relies on a cPLA2-dependent pathway.

Furthermore, alcohol-induced neuroinflammatory changes in PLA2 enzymes coincided with increased PARP levels in this laboratory’s past studies (Tajuddin et al., 2018; Tajuddin et al., 2014). Indeed, treatments with several PARP inhibitors normalized cPLA2 and sPLA2 levels while preventing alcohol-induced neurodegeneration in brain slice cultures (Tajuddin et al., 2018). This indicates alcohol’s effect on PARP is upstream of changes in PLA2. Veliparib co-treatment in the current in vivo study completely normalized alcohol-induced increases in cPLA2. Collectively, these factors indicate that alcohol potentiates PLA2-mediated neuroinflammation through a mechanism involving PARP, which then leads to neurodegeneration in vulnerable brain regions.

5. Conclusions

In conclusion, PARP inhibition by veliparib treatment prevented binge alcohol-induced neurodegeneration in the ventral HC and ECX, linking PARP activity to the mechanisms of binge alcohol-dependent brain neuroinjury. Alcohol binges elevated cPLA2 levels in the ventral HC and ECX, but not in the dorsal HC which lacked neurodamage. PARP inhibition also reduced related changes in levels of cPLA2 in the ventral HC and ECX. These in vivo results link PARP to neuroinflammatory cPLA2 activity in the mechanisms of alcohol-induced neurodegeneration. Given this connection, pharmacological inhibition of PARP might eventually be a valid neuroprotective strategy during the detoxification of chronic alcoholics, but much research remains. Future studies aimed at exploring this relationship may provide new insights into the mechanisms of alcohol-related brain damage and possible therapeutic interventions.

Supplementary Material

Supplement

Acknowledgements

Dr. Kimberly Nixon in the Division of Pharmacology & Toxicology, University of Texas at Austin, generously provided technical training for FJB staining and the alcohol binge model. Dr. Vidhya Rao and Mr. Jack Kwan are recognized for providing technical assistance. Dr. Gwendolyn Kartje of the Edward Hines Jr. VA Hospital and Loyola University Chicago gave an invaluable informal review.

The National Institutes of Health [U01 AA018279 to MAC, T32 AA013527 to Alcohol Research Program] supported this work. Additional support from the Dr. John P. and Therese E. Endowed Professorship in Ophthalmology (to SK) and the United States Department of Veteran Affairs [I21 RX003170 to DEK] are gratefully acknowledged. This work resulted from resources and facilities at Loyola University Chicago and the Edward Hines Jr. VA Hospital. The views expressed in this article are those of the authors and not necessarily that of the National Institutes of Health, the United States Department of Veterans Affairs, or the United States government.

Funding

Supported by the National Institutes of Health [U01-AA018279 to MAC, T32-AA013527 to Alcohol Research Program]. Additional support from the Dr. John P. and Therese E. Mulcahy Endowed Professorship in Ophthalmology (to SK) and United States Department of Veterans Affairs [I21-RX003170 to DEK] are gratefully acknowledged.

Footnotes

Conflicts of Interest

Stock/equity ownership: SK (Experimentica, Ltd.); SK (K&P Scientific, LLC); Consulting: SK (Experimentica, Ltd.). SK also conducts academic research in areas similar to the business interests of Experimentica, Ltd and K&P Scientific, LLC. This arrangement’s terms have been approved by Loyola University Chicago in accordance with its conflict of interest policy. This study has no other conflicts of interest.

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