Nuzhath F. Tajuddin

IN UTERO ETHANOL EXPOSURE DECREASED THE DENSITY OF SEROTONIN NEURONS. MATERNAL IPSAPIRONE TREATMENT EXERTED A PROTECTIVE EFFECT

Prior studies from this laboratory showed that in utero ethanol exposure severely retards the development of the serotonin (5-HT) system; we demonstrated a reduced concentration of 5-HT and 5-HT reuptake sites and alterations in the concentration of 5-HT(1A) receptors in ethanol-exposed offspring. These investigations also found that maternal treatment with a 5-HT(1A) agonist, buspirone, prevented most of the ethanol-associated damage to the developing 5-HT system. In the present investigation, we investigated whether the ethanol-associated changes in the 5-HT system are due to a reduction of 5-HT neurons and whether any changes in the density of 5-HT neurons can be prevented by maternal treatment with another 5-HT(1A) agonist, ipsapirone. Using immunocytochemistry, we found that in utero ethanol exposure reduced the density of 5-HT immunopositive neurons in the dorsal raphe, median raphe and B9 neurons of postnatal day 5 (PN5) rats. In all three brain areas, the offspring of ethanol-fed, saline-treated dams exhibited a 28%-40% reduction in 5-HT neurons. Ipsapirone prevented the ethanol-induced reduction in 5-HT immunopositive neurons in the dorsal raphe, median raphe and B9 neurons. In the dorsal and median raphe of control offspring, ipsapirone did not alter the concentration of 5-HT neurons. However, this drug did reduce 5-HT neurons in the B9 region of the offspring of control-fed rats.

Chronic maternal ethanol consumption results in decreased serotonergic 5-HT1 sites in cerebral cortical regions from offspring

This laboratory previously demonstrated that chronic maternal ethanol consumption results in a marked deficiency of cortical serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) and of 5-HT uptake sites in the 19- and 35-day-old offspring. In order to determine whether in utero exposure to ethanol similarly affects other components of the serotonergic system we examined the influence of chronic maternal ethanol consumption on cortical, serotonergic 5-HT1 binding sites in developing offspring. Female Sprague-Dawley rats were pair-fed, using control or 6.6% (v/v) ethanol liquid diets on a chronic basis prior to parturition. Serotonergic 5-HT1 sites were measured in synaptosomal membranes from whole cortex and cortical regions from developing offspring. Serotonergic 5-HT1 sites were assessed by measuring the binding of [3H]-5-HT to synaptosomal membranes in the presence and absence of nonradioactive 5-HT. Serotonergic 5-HT2 sites were blocked by including 100 nM spiperone in the assay buffer. The results demonstrated that the 19- and 37-day-old offspring of ethanol-fed rats had a significant (approximately 10-40%) reduction in the Bmax for serotonergic 5-HT1 binding sites on synaptosomal membranes from whole cortex (p less than 0.025), motor cortex (p less than 0.01), and somatosensory cortex (p less than 0.025). However, the binding affinity (Kd) for serotonin was not significantly altered (p greater than 0.05). These results emphasize the sensitivity of the developing cortical serotonergic system to prenatal ethanol exposure.

A persistent deficit of serotonin neurons in the offspring of ethanol-fed dams: protective effects of maternal ipsapirone treatment

An earlier study from this laboratory found a significant reduction in the density of serotonin (5-HT) neurons in the dorsal and median raphe and in the B9 complex of postnatal day 5 (PN5) offspring of female rats that consumed ethanol on a chronic basis prior to parturition. In addition, we demonstrated that maternal treatment with the 5-HT(1A) agonist ipsapirone (3 mg/kg) prevented the ethanol-associated reduction in 5-HT neurons. The present investigation examined whether there was a persistent deficit of 5-HT-immunopositive neurons in the dorsal and median raphe of the offspring of ethanol-fed dams. We also evaluated whether a lower ipsapirone dose (1 mg/kg) was protective to developing 5-HT neurons in the offspring of ethanol-fed dams. The offspring of ethanol-fed dams exhibited an apparent lasting reduction in the density of 5-HT neurons in the dorsal and median raphe. The density of 5-HT neurons in control offspring was comparable at PN5 and PN19, but at both ages the offspring of ethanol-fed dams had a significant deficit of 5-HT neurons in the dorsal and median raphe. The lack of recovery in the density of 5-HT-immunopositive neurons in the offspring of ethanol-fed dams between PN5 and PN19 suggests and that the reduction was long lasting. The protective effects of ipsapirone appeared to be dose dependent. The density of 5-HT neurons in the dorsal and median raphe of PN5 (prior study) and PN19 offspring of ethanol-fed dams that were treated with 3 mg/kg of ipsapirone between gestational day 13 (G13) and G20 was comparable to that of control offspring. However, the effects of maternal treatment of ethanol-fed dams with the 1 mg/kg dose were variable, and some abnormalities were detected in the offspring of ipsapirone-treated control dams.

Neuroinflammation and Neurodegeneration in Adult Rat Brain from Binge Ethanol Exposure: Abrogation by Docosahexaenoic Acid

Evidence that brain edema and aquaporin-4 (AQP4) water channels have roles in experimental binge ethanol-induced neurodegeneration has stimulated interest in swelling/edema-linked neuroinflammatory pathways leading to oxidative stress. We report here that neurotoxic binge ethanol exposure produces comparable significant effects in vivo and in vitro on adult rat brain levels of AQP4 as well as neuroinflammation-linked enzymes: key phospholipase A2 (PLA2) family members and poly (ADP-ribose) polymerase-1 (PARP-1). In adult male rats, repetitive ethanol intoxication (3 gavages/d for 4 d, ∼9 g/kg/d, achieving blood ethanol levels ∼375 mg/dl; “Majchrowicz” model) significantly increased AQP4, Ca+2-dependent PLA2 GIVA (cPLA2), phospho-cPLA2 GIVA (p-cPLA2), secretory PLA2 GIIA (sPLA2) and PARP-1 in regions incurring extensive neurodegeneration in this model—hippocampus, entorhinal cortex, and olfactory bulb—but not in two regions typically lacking neurodamage, frontal cortex and cerebellum. Also, ethanol reduced hippocampal Ca+2-independent PLA2 GVIA (iPLA2) levels and increased brain “oxidative stress footprints” (4-hydroxynonenal-adducted proteins). For in vitro studies, organotypic cultures of rat hippocampal-entorhinocortical slices of adult age (∼60 d) were ethanol-binged (100 mM or ∼450 mg/dl) for 4 d, which augments AQP4 and causes neurodegeneration (Collins et al. 2013). Reproducing the in vivo results, cPLA2, p-cPLA2, sPLA2 and PARP-1 were significantly elevated while iPLA2 was decreased. Furthermore, supplementation with docosahexaenoic acid (DHA; 22:6n-3), known to quell AQP4 and neurodegeneration in ethanol-treated slices, blocked PARP-1 and PLA2 changes while counteracting endogenous DHA reduction and increases in oxidative stress footprints (3-nitrotyrosinated proteins). Notably, the PARP-1 inhibitor PJ-34 suppressed binge ethanol-dependent neurodegeneration, indicating PARP upstream involvement. The results with corresponding models support involvement of AQP4- and PLA2-associated neuroinflammatory pro-oxidative pathways in the neurodamage, with potential regulation by PARP-1 as well. Furthermore, DHA emerges as an effective inhibitor of these binge ethanol-dependent neuroinflammatory pathways as well as associated neurodegeneration in adult-age brain.

Effects of ethanol and ipsapirone on the expression of genes encoding anti-apoptotic proteins and an antioxidant enzyme in ethanol-treated neurons

Previously, this laboratory found that apoptosis was augmented significantly in fetal rhombencephalic neurons when they were treated with 50 mM ethanol for 24 h. These changes were associated temporally with a reduction in the phosphatidylinositol 3-kinase (PI3K) pro-survival pathway and in the downstream expression of several NF-kappaB dependent anti-apoptotic genes. The serotonin-1A agonist ipsapirone prevented ethanol-associated apoptosis; it also activated the PI3K-->pAkt pro-survival pathway and the expression of specific NF-kappaB dependent anti-apoptotic genes in ethanol-treated neurons. The present study investigated the temporal effects of both ethanol and ipsapirone on the expression of three NF-kappaB dependent genes, XIAP, Bcl-XL and catalase; these genes encode proteins that could potentially attenuate ethanol-induced apoptosis. Catalase activity was also measured. All three genes demonstrated an early activation by ethanol. After a brief treatment with 50 mM ethanol, i.e., 2 to 8 h depending on the gene, the expression of XIAP, Bcl-XL, and catalase was significantly increased, possibly as an initial attempt to survive. An ethanol-associated increase in catalase was followed by a rise in catalase activity. However, when ethanol treatment was continued for a longer time, there was a significant reduction in both XIAP and Bcl-XL. In addition, both catalase expression and activity returned to levels found in unstressed controls. Importantly, treatment with ipsapirone augmented the activity of catalase and the expression of Bcl-XL, XIAP, and catalase in ethanol-treated neurons at later time points. The latter effects are likely to contribute to the pro-survival effects of ipsapirone.

Effects of ethanol and ipsapirone on the development of midline raphe glial cells and astrocytes

Previously, results of studies from our laboratory have shown that the offspring of ethanol-fed female rats have a significant decrease in serotonin (5-HT) neurons and glia that contain S100B, an essential trophic factor for the development of 5-HT neurons. The deficiency of S100B-immunopositive glia was detected during the vulnerable period in 5-HT neuron development and in brain areas proximal to these neurons. The reductions of both 5-HT neurons and S100B-positive glia were prevented by maternal treatment with a 5-HT(1A) agonist (i.e., ipsapirone or buspirone). In the current study, we investigated whether the offspring of ethanol-fed rats had a general decrease in the density of glial cells in the brain areas that contain 5-HT neurons, and we determined whether these changes were prevented by maternal treatment with ipsapirone between gestational days (GDs) 13 and 20. We estimated the density of vimentin-positive glia of the midline raphe glial structure (MRGS) at GD 20 and postnatal day (PND) 5 and of glial fibrillary acidic protein (GFAP)-positive astrocytes proximal to the dorsal and median raphe at PNDs 5 and 19. The results of this study provide evidence that in utero ethanol exposure is associated with a reduced density of GFAP-immunopositive astrocytes proximal to the dorsal and median raphe. Maternal ipsapirone treatment significantly increased astroglial density in the dorsal raphe at PNDs 5 and 19 and in the median raphe at PND 5, such that it either prevented (dorsal raphe, PNDs 5 and 19) or blunted (median raphe, PND 5) the effects of ethanol.

Alcohol, Phospholipase A2-associated Neuroinflammation, and ω3 Docosahexaenoic Acid Protection

Chronic alcohol (ethanol) abuse causes neuroinflammation and brain damage that can give rise to alcoholic dementia. Insightfully, Dr. Albert Sun was an early proponent of oxidative stress as a key factor in alcoholism-related brain deterioration. In fact, oxidative stress has proven to be critical to the hippocampal and temporal cortical neurodamage resulting from repetitive “binge” alcohol exposure in adult rat models. Although the underlying mechanisms are uncertain, our immunoelectrophoretic and related assays in binge alcohol experiments in vivo (adult male rats) and in vitro (rat organotypic hippocampal-entorhinal cortical slice cultures) have implicated phospholipase A2 (PLA2)-activated neuroinflammatory pathways, release of pro-oxidative arachidonic acid (20:4 ω6), and elevated oxidative stress adducts (i.e., 4-hydroxynonenal-protein adducts). Also, significantly increased by the binge alcohol treatments was aquaporin-4 (AQP4), a water channel enriched in astrocytes that, when augmented, may trigger brain (esp. cellular) edema and neuroinflammation; of relevance, glial swelling is known to provoke increased PLA2 activities or levels. Concomitant with PLA2 activation, the results have further implicated binge alcohol-elevated poly (ADP-ribose) polymerase-1 (PARP-1), an oxidative stress-responsive DNA repair enzyme linked to parthanatos, a necrotic-like neuronal death process. Importantly, supplementation of the brain slice cultures with docosahexaenoic acid (22:6 ω3) exerted potent suppression of the induced changes in PLA2 isoforms, AQP4, PARP-1 and oxidative stress footprints, and prevention of the binge alcohol neurotoxicity, by as yet unknown mechanisms. These neuroinflammatory findings from our binge alcohol studies and supportive rat binge studies in the literature are reviewed.

PARP inhibition prevents ethanol-induced neuroinflammatory signaling and neurodegeneration in adult-age rat brain slice cultures

Using rat adult-age hippocampal-entorhinal cortical (HEC) slice cultures, we examined the role of poly [ADP-ribose] polymerase (PARP) in binge ethanol’s brain inflammatory and neurodegenerative mechanisms. Activated by DNA strand breaks, PARP (principally PARP1 in the brain) promotes DNA repair via poly [ADP-ribose] (PAR) products, but PARP overactivation triggers regulated neuronal necrosis (e.g., parthanatos). Previously, we found that brain PARP1 levels were upregulated by neurotoxic ethanol binges in adult rats and HEC slices, and PARP inhibitor PJ34 abrogated slice neurodegeneration. Binged HEC slices also exhibited increased Ca+2-dependent phospholipase A2 (PLA2) isoenzymes (cPLA2 IVA and sPLA2 IIA) that mobilize proinflammatory ω6 arachidonic acid (ARA). We now find in 4-day–binged HEC slice cultures (100 mM ethanol) that PARP1 elevations after two overnight binges precede PAR, cPLA2, and sPLA2 enhancements by 1 day and high-mobility group box-1 (HMGB1), an ethanol-responsive alarmin that augments proinflammatory cytokines via toll-like receptor-4 (TLR4), by 2 days. After verifying that PJ34 effectively blocks PARP activity (↑PAR), we demonstrated that, like PJ34, three other PARP inhibitors—olaparib, veliparib, and 4-aminobenzamide—provided neuroprotection from ethanol. Importantly, PJ34 and olaparib also prevented ethanol’s amplification of the PLA2 isoenzymes, and two PLA2 inhibitors were neuroprotective—thus coupling PARP to PLA2, with PLA2 activity promoting neurodegeneration. Also, PJ34 and olaparib blocked ethanol-induced HMGB1 elevations, linking brain PARP induction to TLR4 activation. The results provide evidence in adult brains that induction of PARP1 may mediate dual neuroinflammatory pathways (PLA2→phospholipid→ARA and HMGB1→TLR4→proinflammatory cytokines) that are complicit in binge ethanol-induced neurodegeneration.