Acquired brain damage and varying degrees of dementia are often consequences of chronic ethanol (EtOH) dependence and alcoholism (Fein et al., 2002; Gupta and Warner, 2008). Animal and human studies now indicate that neuroinflammatory processes underlie aspects of brain damage (neurodegeneration) from chronic high EtOH levels, particularly in binge exposures (Alfonso-Loeches et al., 2010; Alikunju et al., 2011; Qin et al., 2008; Sullivan and Zahr, 2008; Tajuddin et al., 2013). A binge drinking pattern in EtOH-dependent individuals increases the risk of brain damage (Hunt, 1993); brain trauma (from falls, etc.), concomitant malnutrition, and perhaps severe stress might be important contributing factors in some cases.
Our experiments using repetitive binge EtOH treatments have focused on phospholipid-dependent neuroinflammatory pathways potentially triggered by brain edema and neurodamaging oxidative stress. Brain edema's involvement is indicated by the facts that significant brain water elevations occur in chronic binge EtOH-intoxicated adult rats and that a diuretic (furosemide), in preventing the edema, reduces neurodegeneration in hippocampal and entorhinal cortical regions (Collins et al., 1998). Antagonism of glutamate receptors affords negligible neuroprotection (Collins et al., 1998; Hamelink et al., 2005), indicating that excitotoxicity is not a key mechanism. Oxidative stress, a potential outcome of brain edema (Jayakumar et al., 2008), is implicated in the above rat binge intoxication model based on evidence that selected antioxidants provide neuroprotection (Crews et al., 2006; Hamelink et al., 2005). Similarly, in rat organotypic slice cultures comprising the above 2 vulnerable brain regions, chronic binge EtOH exposure causes significant edema and neuronal damage that are reduced by furosemide or unrelated diuretics such as acetazolamide (Collins et al., 1998; Sripathirathan et al., 2009).
When mobilized excessively from brain membrane phospholipids by stressors or insults, the essential omega-6 polyunsaturated fatty acid, arachidonic acid (AA), can promote oxidative stress and neurodegeneration through enzymatic and nonenzymatic routes (Sun et al., 2012). Physiological levels of free brain AA, typically <10 μM, increase approximately 50-fold in response to pathophysiological insult, for example severe ischemia (Rehncrona et al., 1982). A key AA-mobilizing enzyme activity, phospholipase A2 (PLA2), can be stimulated by cellular deformation, edema, and/or swelling (Basavappa et al., 1998; Lambert et al., 2006). In our experiments with organotypic hippocampal–entorhinal cortical (HEC) slice cultures in which chronic binge EtOH exposure causes edema, PLA2 blockade with mepacrine, a broad spectrum inhibitor, significantly antagonizes EtOH-induced neurodegeneration (Brown et al., 2009).
PLA2 gene products are composed of at least 3 families—notably, Ca2+-dependent cytosolic PLA2 (cPLA2), Ca2+-independent cytosolic PLA2 (iPLA2), and secretory (also Ca2+-dependent) PLA2 (sPLA2)—that are expressed in brain (Sun et al., 2012). Multiple PLA2 isoforms or groups within these 3 families are implicated to varying extents in causal brain damage mechanisms distinct from EtOH, with the cPLA2 family frequently linked to neurodegeneration from insults such as ischemia or excitotoxicity. Also, the major brain endocannabinoid, monoarachidonoylglycerol, is a recently appreciated potential source of neuroinflammation-liberated AA via monoacylglycerol lipase (MAGL; Nomura et al., 2011). We considered it tenable that MAGL activity might also contribute to binge EtOH-induced neurodegeneration.
As with earlier studies, these experiments utilized organotypic HEC slice cultures which retain the cytoarchitecture of intact (albeit developing, approximately 3 to 4 weeks of age) brain and thus have unique advantages over mixed primary brain cultures. Moreover, unlike much more frequently employed slices of solely hippocampus, HEC slice cultures encompass 2 regions that are very susceptible to binge EtOH neurotoxicity (Collins et al., 1996), and retain functional perforant pathways (Del Turco and Deller, 2007) that might be important in hippocampal–cortical neuroinflammation. With these slice cultures, we sought to confirm with inhibitors whether PLA2 is critical for oxidative stress due to binge EtOH exposure and to determine the enzyme sources of AA involved in neuronal damage in the HEC complex.
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- Materials and Methods
As reported (Brown et al., 2009), cotreatment of developing HEC slice cultures with a broad spectrum PLA2 inhibitor, mepacrine, prevented chronic binge EtOH-induced AA release relatively early and suppressed later degeneration of hippocampal dentate granule and pyramidal cortical neurons. To verify that tissue oxidative stress is substantial and is linked to PLA2 activity during the binge EtOH exposure timecourse, neuroinflammatory oxidative stress indicators of 3-nitrotyrosinated (3-NT)- and 4-hydroxy-2-nonenal (4-HNE)-adducted proteins were determined in HEC slices with and without mepacrine. In Fig. 1A are representative immunoblots of 3-NT proteins between 3 and 6 days of binge EtOH exposure, a treatment causing neurodamage (Brown et al., 2009). Control slices showed 3NT proteins, reflecting basal oxidative stress. Quantitation of 3-NT protein blots (Fig. 1B) confirmed EtOH-dependent increases above controls at the 5- and 6-day timepoints in proteins from 30 to 160 kDa, but particularly those in the range of 55 kDa, which likely constitutes a number of tyrosine-nitrosylated proteins based on 2-dimensional gel studies (Moon et al., 2006). Mepacrine cotreatment did not alter control 3-NT protein levels, suggesting that basal oxidative stress in vitro was not PLA2 activity-derived, but it abolished binge EtOH-induced 6-day increases (Fig. 1C; EtOH + MEPA). In Fig. 2A are representative immunoblots for 4-HNE-adducted proteins, and controls contained basal levels of adducts (Fig. 2B). The 4-HNE adducts in EtOH-treated cultures, ranging between 35 and 95 kDa and concentrated at approximately 50 kDa, were significantly elevated by 5 to 6 days of binge exposure, as with 3-NT proteins. Mepacrine cotreatment blocked EtOH's potentiation of 4-HNE-adducted proteins while not affecting control levels (Fig. 2C; EtOH + MEPA), again indicating that basal oxidative stress did not arise from PLA2 activity.
Figure 1. Binge ethanol (EtOH) exposure significantly increases 3-nitrotyrosinated (3-NT)-protein levels in organotypic hippocampal–entorhinal cortical (HEC) slice cultures, and PLA2 inhibition by mepacrine prevents the increase. (A) Representative immunoblots of 3-NT proteins and GAPDH in HEC slice cultures during the daytime withdrawal periods following overnight binge treatment with EtOH (100 mM) for 3, 4, 5, or 6 successive days. (B) 3-NT proteins as represented in immunoblots in (A) are significantly increased after binge EtOH exposure (100 mM) for 5 or 6 days (n = 3; 6 to 9 slices/group). *p < 0.05 versus control. (C) Mepacrine (MEPA, 1 μM) cotreatment abolishes the increase in 3-NT proteins after 6 days of binge treatment with 100 mM EtOH (n = 3; 6 to 9 slices/group). *p < 0.05 versus control. #p < 0.05 versus EtOH.
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Figure 2. Binge ethanol (EtOH) exposure significantly increases 4-hydroxy-2-nonenal (4-HNE)-protein adduct levels in organotypic hippocampal–entorhinal cortical (HEC) slice cultures, and PLA2 inhibitor mepacrine abolishes the increase. (A) Representative immunoblots of 4-HNE-proteins and GAPDH in HEC slice cultures during the daytime withdrawal periods following overnight binge EtOH exposure (100 mM) for 3, 4, 5, and 6 successive days. (B) 4-HNE-proteins as represented in immunoblots in (A) were significantly increased after binge EtOH exposure (100 mM) for 5 and 6 days (n = 3; 6 to 9 slices/group). *p < 0.05 versus control. (C) Mepacrine (MEPA, 1 μM) cotreatment abolished the increase in 4-HNE-adducted proteins after 6 days of binge treatment with 100 mM EtOH (n = 3; 6 to 9 slices/group). *p < 0.05 versus control. #p < 0.05 versus EtOH.
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To address what phospholipase enzyme activities have roles in binge EtOH-induced neurodegeneration in these developing HEC cultures, relatively specific inhibitors were used. Whether sPLA2 activity is involved was examined with manoalide, which irreversibly inhibits most sPLA2 isoforms (Lombardo and Dennis, 1985). Representative photomicrograph images of PI staining of HEC slices (Fig. 3A) indicated extensive neurodegeneration due to binge exposure at 100 mM EtOH in entorhinal cortical and hippocampal regions relative to control slice regions. Cotreatment with 5 μM manoalide (EtOH + manoalide) largely suppressed the EtOH-induced PI fluorescence. Quantitation of slice fluorescence in Fig. 3B showed that robust neurodegeneration due to binge EtOH was almost completely abolished by manoalide (suppressed relative to mano alone), indicating that sPLA2 activity has an important role. However, as shown in Fig. 3C, ATA (10 μM), an inhibitor of groups I + IIA-B sPLA2 isoforms (Moreno, 1993), was ineffective, suggesting that sPLA2 activities in groups other than I and IIA-B are responsible in these developing brain slice cultures.
Figure 3. Effect of cotreatment with manoalide or aristolochic acid (ATA) on binge ethanol (EtOH)-induced neurodegeneration in rat developing hippocampal–entorhinal cortical (HEC) slice cultures. (A) Representative photomicrographs of propidium iodide (PI) staining in representative HEC slices indicating neuroprotection by manoalide: control, binge EtOH (100 mM), manoalide (5 μM)-treated, and EtOH + manoalide. (B) Quantitation of PI staining shows that chronic binge EtOH exposure caused increased neurodegeneration that was completely prevented by manoalide (EtOH + manoalide, 5 μM). n = 6, *p < 0.05 versus control. #p < 0.05 versus EtOH. (C) Chronic binge EtOH exposure caused increased PI staining (EtOH) that was not reduced by the copresence of 10 μM ATA (EtOH + ATA). n = 6, *p < 0.05 versus control or ATA.
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In Fig. 4A, the percent PI-labeled neurons was determined in binge EtOH-treated HEC slice cultures cotreated with MAFP, an irreversible inhibitor of iPLA2 and cPLA2 families (Lio et al., 1996). Quantitation of PI staining confirmed increased neurodegeneration due to binge EtOH, which was reduced approximately 60% by MAFP (20 μM); a lower MAFP concentration (1 μM) was ineffective (not shown). To examine more specifically the involvement of IVA/B (85 kDa) cPLA2 enzymes, HEC slice cultures with and without EtOH exposure were treated with the selective cPLA2 inhibitor, arachidonyl trifluoromethylketone (AACOCF3; Street et al., 1993). Figure 4B shows that AACOCF3 (10 μM) failed to inhibit or suppress neurodegeneration; also, a lower AACOCF3 concentration (1 μM) was not protective (not shown). Group VI iPLA2 was then examined with (±) BEL (5 μM), an irreversible suicide inhibitor of this enzyme class (Ackermann et al., 1995). As shown (Fig. 4C), (±) BEL reduced neurodegeneration by approximately 50%, indicating that the iPLA2 family is likely to be important, in contrast to cPLA2, in binge EtOH's neuronal damaging mechanism in these adolescent-age HEC slices.
Figure 4. Effect of cotreatment with methyl arachidonyl fluorophosphonate (MAFP), arachidonyl trifluoromethylketone (AACOCF3) or bromoenol lactone (±BEL) on binge ethanol (EtOH)-induced neurodegeneration in rat developing hippocampal–entorhinal cortical (HEC) slice cultures. (A) Chronic binge EtOH exposure (100 mM) for 6 days caused increased propidium iodide (PI) staining that was significantly reduced by 20 μM MAFP (EtOH + MAFP). n = 6, *p < 0.05 versus control or MAFP. #p < 0.05 versus EtOH. (B) Chronic binge EtOH exposure (100 mM) caused increased PI staining that was not reduced by 10 μM AACOCF3 (AACOCF3 + EtOH). n = 6, *p < 0.05 versus control or AACOCF3. (C) Chronic binge EtOH exposure (100 mM) caused increased PI staining that was significantly reduced by 5 μM BEL (EtOH + BEL). n = 6, *p < 0.05 versus control or BEL. #p < 0.05 versus EtOH.
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To better understand why inhibitors of PLA2 generally and sPLA2 specifically are neuroprotective while cPLA2 inhibition is not, the effects of mepacrine, manoalide, or AACOCF3 on levels of AA in withdrawal media during binge EtOH exposure, which indicates AA release, were examined (Fig. 5). As shown, the above inhibitors alone did not affect AA levels in withdrawal media. Concurring with our previous experiments using 3H-AA release (Brown et al., 2009), binge EtOH + withdrawal significantly increased media AA levels above control values. Mepacrine and manoalide cotreatments modestly but significantly reduced EtOH-dependent media AA levels, reflecting inhibition of release—which corresponds with their respective neuroprotective effects. However, AACOCF3 did not significantly change EtOH-induced AA release, consistent with the inhibitor's lack of neuroprotection and further implying that cPLA2 is not critical to AA mobilization and neurodegeneration in the EtOH-exposed developing HEC slices.
Figure 5. Effects of selected PLA2 inhibitors on media levels (release) of arachidonic acid (AA) in binge ethanol (EtOH)-exposed adolescent-age hippocampal–entorhinal cortical slice cultures. Binge EtOH exposure (100 mM) for 6 days significantly increased levels of AA in media pooled from the 4 withdrawal episodes (EtOH, *p < 0.05 vs. Control; n = 3). Mepacrine (MEPA; 1 μM) significantly reduced EtOH withdrawal media AA levels (EtOH + MEPA vs. EtOH, #p < 0.05). Manoalide (5 μM) significantly lowered EtOH withdrawal media AA levels (EtOH + manoalide vs. EtOH, #p < 0.05). AACOCF3 (10 μM) had no effect on withdrawal media AA levels (EtOH + AACOCF3 vs. EtOH, n.s.).
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The possibility that an alternate (non-PLA2) AA-mobilizing pathway, the augmented activity of MAGL, is involved in binge EtOH-induced neurodegeneration was examined with the specific MAGL inhibitor, JZL184. The experiments (Fig. 6) showed that the extent of PI labeling after binge EtOH treatment was not significantly reduced by cotreatment with 50 nM (not shown) or 1 μM JZL184 (concentration range that prevents endotoxin-induced AA-derived eicosanoid production; Nomura et al., 2011). The results thus argue against MAGL activity as a neuroinflammatory source of AA in this binge EtOH-treated organotypic brain slice model.
Figure 6. Effect of monoacylglycerol lipase inhibitor JZL184 on binge ethanol (EtOH)-induced neurodegeneration in adolescent-age hippocampal–entorhinal cortical slice cultures. Chronic binge EtOH exposure (100 mM) for 6 days caused increased propidium iodide staining that was not significantly reduced by 1 μM JZL184 (EtOH + JZL184). n = 6, *p < 0.05 versus control or JZL184.
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- Materials and Methods
These experiments were predicated on the idea that neuroinflammatory AA mobilization plays a critical role in oxidative stress and neurodegeneration caused by repetitive binge EtOH exposure. Previously, we reported that global inhibition of PLA2 activities with mepacrine during binge EtOH treatment of adolescent-age HEC slice cultures prevented neurodegeneration (Brown et al., 2009). From experiments with added antioxidants, oxidative stress has been linked in vivo to binge EtOH-induced neurodegeneration (Collins and Neafsey, 2012); however, it is not clear whether PLA2 contributes significantly to increased oxidative stress in the organotypic slice model. In dispersed brain cultures, 3-NT and 4-HNE protein adducts are indicative of EtOH-dependent, oxidative/peroxidative stress-induced damage (“inflammatory footprints”) to proteins and ω-6 polyunsaturated membrane lipids (e.g., AA; Alikunju et al., 2011). Interestingly, assays of these fingerprint proteins in organotypic slice cultures appear to be lacking, with the exception of 3-NT protein assays in a parkinsonian neurotoxin model (Larsen et al., 2008).
Binge EtOH treatment significantly increased the oxidative protein footprints concomitant with HEC slice neurodegeneration (5 to 6 days). Results with mepacrine verified that PLA2 activity was responsible, at least in part, for binge EtOH-induced generation of oxidative stress and resulting protein–lipid oxidation. However, it should be emphasized that other reactive oxygen species–generating pathways stimulated by EtOH, for example NADPH oxidase, are likely to have roles, as others have found (Alikunju et al., 2011; Qin and Crews, 2012b).
The inhibitory results by PLA2 inhibitors indicated that an atypical combination of sPLA2 and iPLA2 is upstream of neurodegeneration in developing HEC slices repetitively binge exposed to EtOH. These activities can give rise to AA and its eicosanoid products—notably, prostaglandins and leukotrienes that might be deleterious second (or third) messengers (Sun et al., 2012). Cyclooxygenase (COX) and its prostaglandin products receive much attention in the neuroinflammatory literature, and inducible COX-2 is increased in chronic EtOH-binged mice (Qin et al., 2008). However, the intermediacy of leukotrienes via multiple lipoxygenase (LOX) activities is unexplored with regard to EtOH's neuropathological effects. Indeed, LOX activity is the most prominent source of oxidative stress in neuronal cultures under apoptotic conditions such as high potassium chloride (Bobba et al., 2008). In addition, when directly added to neuronal cultures, AA exerts procytotoxic, apoptotic, necrotic, and/or excitatory effects (Chen and Chang, 2009). Mechanistically, AA may induce neuronal apoptosis via cytosolic cationic overload (Fang et al., 2008). In hepatoma or liver cells, AA causes apoptosis via cytochrome P450-mediated metabolism (Caro and Cederbaum, 2006), and loss of mitochondrial transmembrane potential (MTP; Scorrano et al., 2001); in fibroblasts, the omega-6 fatty acid was the most damaging among unsaturated fatty acids tested with regard to MTP (Maia et al., 2006).
The most effective neuroprotection was observed with manoalide. While this supports the involvement of sPLA2, neuroprotection could also involve suppression of NADPH oxidase activity already mentioned, because such an effect was reported in stressed nonneuronal cultures to be downstream of manoalide/PLA2 inhibition (de Carvalho et al., 2008). Nevertheless, the fact that manoalide significantly counters EtOH-induced AA media elevations is consistent with a key role for sPLA2. The observed partial protection with MAFP, a dual iPLA2/cPLA2 inhibitor, we consider to be mainly iPLA2-related, as BEL, a specific suicide inhibitor of iPLA2, was similarly effective, while the selective cPLA2 inhibitor, AACOCF3, provided no protection. Reports suggest that the iPLA2 family has a homeostatic or housekeeping role in membrane phospholipid turnover (Winstead et al., 2000), but iPLA2 activation in brain mitochondria has been linked to proapoptotic signaling (Brustovetsky et al., 2005) including that initiated by Fas receptors (Atsumi et al., 1998). In contrast, whereas cPLA2 family isoforms play important roles in AA mobilization and downstream neuroinflammation in neurodegenerative conditions other than EtOH (Sun et al., 2012), in these EtOH-treated developing rat brain slices the cPLA2 family does not appear important for neurodegeneration. This conclusion is further supported by the failure of the cPLA2 inhibitor AACOCF3 to suppress binge EtOH's augmentation of AA media levels (Fig. 5) in the HEC slices.
Concerning sPLA2 family members, evaluations of ischemic or excitotoxic neurodegeneration mechanisms have frequently implicated these small phospholipases in brain necrosis and/or apoptosis. In certain circumstances, sPLA2 groups I and II are particularly involved (Kolko et al., 2003), but in our binge EtOH model, an inhibitor of group I/IIA-B, ATA, was ineffective. However, at least 6 sPLA2 groups are expressed in the brain, and sPLA2 group III (DeCoster, 2003) and perhaps others such as sPLA2 groups IIF and V, which are induced in rodent brain by inflammatory endotoxin (Hamaguchi et al., 2003), could be responding to chronic binge EtOH exposure with up-regulation/secretion. In view of evidence that EtOH can alter brain levels of endotoxin-linked Toll-like receptors and their endogenous brain protein agonists (Alfonso-Loeches et al., 2010; Qin and Crews, 2012a), downstream activation of these sPLA2's via neuroimmune pathways is an attractive possibility.
A recent finding on AA mobilization is through a non-PLA2 route during exposure to neuroinflammatory stressors such as endotoxin—that is, endogenous cannabinoid hydrolysis involving MAGL activity on brain 2-monoarachidonoylglycerol (Nomura et al., 2011). We used MAGL inhibitor JZL184 to test whether binge EtOH-induced neurodegeneration involved this AA route, in addition to PLA2 pathways. Although relatively specific for MAGL, this inhibitor displays some cross-reactivity with fatty acid amide hydrolase (Chang et al., 2012), the enzyme releasing AA from the second key brain endocannabinoid, anandamide. Interestingly, if significant suppression of neurodegeneration with JZL184 had occurred, it could also have been explained by neuroprotective elevations in the 2 above-mentioned endocannabinoids, and not just by reduced AA mobilization. At any rate, results with JZL inhibitor provided no evidence that MAGL activity was involved in binge EtOH-induced neurodegeneration in this developing brain slice model.
Reiterating, these inhibition studies indicate that excessive liberation of AA by sPLA2 and iPLA2 is important in neuropathological signaling induced by chronic binge EtOH treatment of developing brain in vitro, whereas cPLA2 has little or no role. The findings are unusual, because as previously mentioned, in other degenerative or inflammatory insults, cPLA2 often is a critical enzymatic activity mobilizing AA, either alone or in combination with sPLA2 family members. Also, the finding is unexpected because brain edema, which is triggered by repeated binges of EtOH and is potentially upstream of PLA2 activation, is viewed as largely astrocytic—based on histochemical indications, on the increased expression of the astroglial-enriched water channel, aquaporin-4 (Sripathirathan et al., 2009), and on evidence that furosemide largely blocks astrocytic swelling (Hochman et al., 1995). Indeed, group IV cPLA2 isoforms are known to be activated as a consequence of brain cell swelling (Basavappa et al., 1998), and chronic EtOH treatment in mice is reported to increase brain cPLA2 activity (Basavarajappa et al., 1998); in addition, cPLA2 is reported to be increased in chronic EtOH-treated astrocytes (Floreani et al., 2010).
Furthermore, the PLA2 inhibitor findings in these developing HEC slices—possible neurotoxic role for iPLA2 but none for cPLA2 resulting from binge EtOH exposure—are inconsistent with our quantitative immunoblot results from binge EtOH-exposed adult rats (Tajuddin et al., 2013). In that study, binge pattern intoxicated adult rats showed depleted iPLA2 levels in concert with significant elevations in cPLA2 and (activated) phospho-cPLA2 in hippocampal and entorhinal cortical tissues. It is perhaps relevant that reduction in iPLA2 levels agrees with studies that have found brain iPLA2 depletion/inactivation, as opposed to elevations, during neurotoxic insults in adult rodents (Wilkins and Barbour, 2008).
At this juncture, it appears that binge EtOH exposure in developing adolescent-age rat brain (i.e., organotypic slices in culture) stimulates neuroinflammatory PLA2 isoform activation pathways that may differ from those of PLA2 families in brain of similarly exposed adult rats. Developmentally, rat brain levels of cPLA2 are unchanged between neonatal and adult ages (Yoshihara et al., 1992), so further research on EtOH-induced signaling differences among PLA2 families is needed to understand the respective mechanisms. Integrating in vitro results with adult in vivo studies should permit elucidation of the PLA2 families and groups that are important in neuroinflammatory oxidative signaling mechanisms triggered by chronic binge EtOH abuse.