EtOH-induced Alterations Research Articles

Voluntary Exercise Ameliorates Chronic Ethanol Withdrawal-Induced Adaptations of Opioid Receptor Expression in the Nucleus Accumbens, Dopamine Release, and Ethanol Consumption.

Exercise has increasingly been recognized as an adjunctive therapy for alcohol-use disorder (AUD), yet our understanding of its underlying neurological mechanisms remains limited. This knowledge gap impedes the development of evidence-based exercise guidelines for AUD treatment. Chronic ethanol (EtOH) exposure has been shown to upregulate and sensitize kappa opioid receptors (KORs) in the nucleus accumbens (NAc), which is innervated by dopamine (DA) neurons in the midbrain ventral tegmental area (VTA), which may contribute to AUD-related behaviors. In this study, we investigated the impact of voluntary exercise in EtOH-dependent mice on EtOH consumption, KOR and delta opioid receptor (DOR) expression in the NAc and VTA, and functional effects on EtOH-induced alterations in DA release in the NAc. Our findings reveal that voluntary exercise reduces EtOH consumption, reduces KOR and enhances DOR expression in the NAc, and modifies EtOH-induced adaptations in DA release, suggesting a competitive interaction between exercise-induced and EtOH-induced alterations in KOR expression. We also found changes to DOR expression in the NAc and VTA with voluntary exercise but no significant changes to DA release. These findings elucidate the complex interplay of AUD-related neurobiological processes, highlighting the potential for exercise as a therapeutic intervention for AUD.

Ethanol disrupts hepatocellular lipophagy by altering Rab5-centric LD-lysosome trafficking.

Previous reports suggest that lipid droplets (LDs) in the hepatocyte can be catabolized by a direct engulfment from nearby endolysosomes (microlipophagy). Further, it is likely that this process is compromised by chronic ethanol (EtOH) exposure leading to hepatic steatosis. This study investigates the hepatocellular machinery supporting microlipophagy and EtOH-induced alterations in this process with a focus on the small, endosome-associated, GTPase Rab5. Here we report that this small Ras-related GTPase is a resident component of LDs, and its activity is important for hepatocellular LD-lysosome proximity and physical interactions. We find that Rab5 siRNA knockdown causes an accumulation of LDs in hepatocytes by inhibiting lysosome dependent LD catabolism. Importantly, Rab5 appears to support this process by mediating the recruitment of early endosomal and or multivesicular body compartments to the LD surface before lysosome fusion. Interestingly, while wild-type or a constituently active GTPase form (Q79L) of Rab5 supports LD-lysosome transport, this process is markedly reduced in cells expressing a GTPase dead (S34N) Rab5 protein or in hepatocytes exposed to chronic EtOH. These findings support the novel premise of an early endosomal/multivesicular body intermediate compartment on the LD surface that provides a "docking" site for lysosomal trafficking, not unlike the process that occurs during the hepatocellular degradation of endocytosed ligands that is also known to be compromised by EtOH exposure.

Impact of alcohol exposure on neural development and network formation in human cortical organoids

Prenatal alcohol exposure is the foremost preventable etiology of intellectual disability and leads to a collection of diagnoses known as Fetal Alcohol Spectrum Disorders (FASD). Alcohol (EtOH) impacts diverse neural cell types and activity, but the precise functional pathophysiological effects on the human fetal cerebral cortex are unclear. Here, we used human cortical organoids to study the effects of EtOH on neurogenesis and validated our findings in primary human fetal neurons. EtOH exposure produced temporally dependent cellular effects on proliferation, cell cycle, and apoptosis. In addition, we identified EtOH-induced alterations in post-translational histone modifications and chromatin accessibility, leading to impairment of cAMP and calcium signaling, glutamatergic synaptic development, and astrocytic function. Proteomic spatial profiling of cortical organoids showed region-specific, EtOH-induced alterations linked to changes in cytoskeleton, gliogenesis, and impaired synaptogenesis. Finally, multi-electrode array electrophysiology recordings confirmed the deleterious impact of EtOH on neural network formation and activity in cortical organoids, which was validated in primary human fetal tissues. Our findings demonstrate progress in defining the human molecular and cellular phenotypic signatures of prenatal alcohol exposure on functional neurodevelopment, increasing our knowledge for potential therapeutic interventions targeting FASD symptoms.

Heightened Exploratory Behavior Following Chronic Excessive Ethanol Drinking: Mediation by Neurotensin Receptor Type 2 in the Anterior Paraventricular Thalamus.

Chronic, excessive alcohol drinkers, even without dependence, can exhibit changes in behavior and neurochemical systems. Identifying these changes and their relationship with one another could provide novel avenues for the prevention and treatment of alcohol use disorder. We recently demonstrated, in rats, that neurotensin (NTS) in the paraventricular thalamus (PVT) regulates excessive ethanol (EtOH) drinking. Here, we investigate the effects of chronic EtOH drinking on the PVT-NTS system and its contribution to EtOH-induced behavioral changes. We gave adult male Long-Evans rats 20% EtOH under the intermittent access 2-bottle-choice paradigm or maintained them on chow and water for up to 11weeks. Prior to EtOH exposure and following several weeks of access, during acute abstinence, we tested these groups for multiple behaviors. In the 12th week, during acute abstinence, we examined gene expression and peptide levels of NTS and its receptors in the anterior and posterior subregions of the PVT. Finally, in chronic EtOH drinkers, during acute abstinence, we microinjected the NTS receptor type 2 (NTS2R) agonist, JMV-431, in the anterior PVT (aPVT) and examined subsequent EtOH intake and behavior. Following chronic intermittent EtOH access, rats were classified by cluster analysis as high or low EtOH drinkers. High EtOH drinkers spent more time in the light chamber of a light-dark box and open arms of an elevated plus maze and entered fewer familiar holes in a hole-board apparatus. These differences were absent prior to EtOH exposure but were detectable as early as 4weeks into drinking. Time in the light chamber following chronic drinking also predicted level of subsequent drinking. High EtOH drinkers also showed elevated protein levels of NTS2R in the aPVT, and pharmacological stimulation of aPVT NTS2R in low drinkers mimicked the increased time spent in the light chamber that was observed in high drinkers. Our findings suggest that chronic, excessive, but not lower level, EtOH drinking induces heightened or flexible exploratory behavior, which predicts future EtOH drinking and is partly mediated by elevated NTS2R signaling in the aPVT. These EtOH-induced alterations represent adaptations that could perpetuate excessive drinking and lead to the development of EtOH dependence.

Single Ethanol Withdrawal Regulates Extrasynaptic δ-GABAA Receptors Via PKCδ Activation.

Alcohol (ethanol, EtOH) is one of the most widely abused drugs with profound effects on brain function and behavior. GABAA receptors (GABAARs) are one of the major targets for EtOH in the brain. Temporary plastic changes in GABAARs after withdrawal from a single EtOH exposure occur both in vivo and in vitro, which may be the basis for chronic EtOH addiction, tolerance and withdrawal symptoms. Extrasynaptic δ-GABAAR endocytosis is implicated in EtOH-induced GABAAR plasticity, but the mechanisms by which the relative abundance and localization of specific GABAARs are altered by EtOH exposure and withdrawal remain unclear. In this study, we investigated the mechanisms underlying rapid regulation of extrasynaptic δ-GABAAR by a single EtOH withdrawal in cultured rat hippocampal neurons. Thirty-minutes EtOH (60 mM) exposure increased extrasynaptic tonic current (Itonic) amplitude without affecting synaptic GABAAR function in neurons. In contrast, at 30 min after withdrawal, Itonic amplitude and responsiveness to acute EtOH were both reduced. Similar results occurred in neurons with okadaic acid (OA) or phorbol 12,13-dibutyrate (PDBu) exposure. Protein kinase C (PKC) inhibition prevented the reduction of Itonic amplitude and the tolerance to acute EtOH, as well as the reduction of GABAAR-δ subunit abundance induced by a single EtOH withdrawal. Moreover, EtOH withdrawal selectively increased PKCδ level, whereas PKCδ inhibition specifically rescued the EtOH-induced alterations in GABAAR-δ subunit level and δ-GABAAR function. Together, we provided strong evidence for the important roles of PKCδ in the rapid regulation of extrasynaptic δ-GABAAR induced by a single EtOH withdrawal.

Adamts1 mediates ethanol-induced alterations in collagen and elastin via a FoxO1-sestrin3-AMPK signaling cascade in myocytes.

A variety of stressors including alcohol (EtOH) are known to induce collagen production and fibrotic diseases. Matrix metalloproteinases (MMP) play an important role in regulating fibrosis, but little is known regarding the relationship between EtOH and MMPs. In addition, the signaling cascades involved in this process have not been elucidated. We have identified the MMP Adamts1 as a target of EtOH regulation. To characterize the function of Adamts1, we examined EtOH-induced alterations in collagen I and elastin protein levels in C2C12 myocytes. Incubation of myocytes with 100 mM EtOH decreased elastin and increased collagen content, respectively, and these changes were associated with increased O-GLcNAc modification of Adamts1. Conversely, silencing of Adamts1 by siRNA blocked the adverse effects of EtOH on collagen and elastin levels. Similar results were obtained after treatment with a pharmacological inhibitor of MMP. Changes in collagen were due, at least in part, to a decreased interaction of Adamts1 with its endogenous inhibitor TIMP3. The AMPK inhibitor compound C blocked the EtOH-induced stimulation of collagen and O-GLcNAc Adamts1 protein. Changes in AMPK appear linked to FoxO1, since inhibition of FoxO1 blocked the effects of EtOH on AMPK phosphorylation and O-GLcNAc levels. These FoxO-dependent modifications were associated with an upregulation of the FoxO1 transcription target sestrin 3, as well as increased binding of sestrin 3 with AMPK. Collectively, these data indicate that EtOH regulates the collagen I and elastin content in an Adamts1-dependent manner in myocytes. Furthermore, Adamts1 appears to be controlled by the FoxO1-sestrin 3-AMPK signaling cascade.

Gene expression signatures affected by alcohol-induced DNA methylomic deregulation in human embryonic stem cells

Stem cells, especially human embryonic stem cells (hESCs), are useful models to study molecular mechanisms of human disorders that originate during gestation. Alcohol (ethanol, EtOH) consumption during pregnancy causes a variety of prenatal and postnatal disorders collectively referred to as fetal alcohol spectrum disorders (FASDs). To better understand the molecular events leading to FASDs, we performed a genome-wide analysis of EtOH's effects on the maintenance and differentiation of hESCs in culture. Gene Co-expression Network Analysis showed significant alterations in gene profiles of EtOH-treated differentiated or undifferentiated hESCs, particularly those associated with molecular pathways for metabolic processes, oxidative stress, and neuronal properties of stem cells. A genome-wide DNA methylome analysis revealed widespread EtOH-induced alterations with significant hypermethylation of many regions of chromosomes. Undifferentiated hESCs were more vulnerable to EtOH's effect than their differentiated counterparts, with methylation on the promoter regions of chromosomes 2, 16 and 18 in undifferentiated hESCs most affected by EtOH exposure. Combined transcriptomic and DNA methylomic analysis produced a list of differentiation-related genes dysregulated by EtOH-induced DNA methylation changes, which likely play a role in EtOH-induced decreases in hESC pluripotency. DNA sequence motif analysis of genes epigenetically altered by EtOH identified major motifs representing potential binding sites for transcription factors. These findings should help in deciphering the precise mechanisms of alcohol-induced teratogenesis.

Calpain Inhibition Prevents Ethanol-Induced Alterations in Spinal Motoneurons

Long-term exposure of ethanol (EtOH) alters the structure and function in brain and spinal cord. The present study addresses the mechanisms of EtOH-induced damaging effects on spinal motoneurons in vitro. Altered morphology and biochemical changes of such damage were demonstrated by in situ Wright staining and DNA ladder assay. EtOH at low to moderate (25-50mM) concentrations induced damaging effects in the motoneuronal scaffold which involved activation of proteases like μ-calpain and caspase-3. Caspase-8 was seen only at higher (100mM) EtOH concentration. Further, pretreatment with calpeptin, a potent calpain inhibitor, confirmed the involvement of active proteases in EtOH-induced damage to motoneurons. The lysosomal enzyme cathepsin D was also elevated in the motoneurons by EtOH, and this effect was significantly attenuated by inhibitor treatment. Overall, EtOH exposure rendered spinal motoneurons vulnerable to damage, and calpeptin provided protection, suggesting a critical role of calpain activation in EtOH-induced alterations in spinal motoneurons.

Alcohol-Induced Epigenetic Alterations to Developmentally Crucial Genes Regulating Neural Stemness and Differentiation

From studies using a diverse range of model organisms, we now acknowledge that epigenetic changes to chromatin structure provide a plausible link between environmental teratogens and alterations in gene expression leading to disease. Observations from a number of independent laboratories indicate that ethanol (EtOH) has the capacity to act as a powerful epigenetic disruptor and potentially derail the coordinated processes of cellular differentiation. In this study, we sought to examine whether primary neurospheres cultured under conditions maintaining stemness were susceptible to alcohol-induced alterations in the histone code. We focused our studies on trimethylated histone 3 lysine 4 and trimethylated histone 3 lysine 27, as these are 2 of the most prominent posttranslational histone modifications regulating stem cell maintenance and neural differentiation. Primary neurosphere cultures were maintained under conditions promoting the stem cell state and treated with EtOH for 5 days. Control and EtOH-treated cellular extracts were examined using a combination of quantitative RT-PCR and chromatin immunoprecipitation techniques. We find that the regulatory regions of genes controlling both neural precursor cell identity and processes of differentiation exhibited significant declines in the enrichment of the chromatin marks examined. Despite these widespread changes in chromatin structure, only a small subset of genes including Dlx2, Fabp7, Nestin, Olig2, and Pax6 displayed EtOH-induced alterations in transcription. Unexpectedly, the majority of chromatin-modifying enzymes examined including members of the Polycomb Repressive Complex displayed minimal changes in expression and localization. Only transcripts encoding Dnmt1, Uhrf1, Ehmt1, Ash2 l, Wdr5, and Kdm1b exhibited significant differences. Our results indicate that primary neurospheres maintained as stem cells in vitro are susceptible to alcohol-induced perturbation of the histone code and errors in the epigenetic program. These observations indicate that alterations to chromatin structure may represent a crucial component of alcohol teratogenesis and progress toward a better understanding of the developmental origins of fetal alcohol spectrum disorders.

Effects of Chronic Ethanol Feeding on Murine Dendritic Cell Numbers, Turnover Rate, and Dendropoiesis

Chronic alcoholics have increased susceptibility to and severity of infection, which are likely to be a result of impaired immune defense mechanisms. The contribution of dendritic cells (DC) to these immune defense changes is not well understood. Alterations in DC numbers, dendropoiesis, and lifespan have not been specifically studied in vivo in chronic ethanol (EtOH) exposure models. As DC play an essential role in initiating immune responses, alterations in these DC characteristics would help explain changes observed in adaptive immune responses. Mice received 20% EtOH (w/v) in the drinking water for up to 28 weeks, with mouse chow ad libitum. In EtOH-fed and water control mice, DC were enumerated by flow cytometry. The effect of EtOH on DC precursor numbers was determined by differentiation in vitro in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, and the effect of an EtOH environment on untreated DC differentiation was measured following bone marrow transfer to irradiated hosts. DC turnover rate was also examined by bromodeoxyuridine incorporation and loss. The percentage and absolute numbers of DC were decreased in spleen and increased in thymus beginning as early as 4 weeks of EtOH feeding. In addition, the overall cellularity of spleen and thymus were altered by this regimen. However, chronic EtOH consumption did not adversely affect DC precursor numbers, differentiation abilities, or turnover rates. Decreased splenic DC numbers observed following chronic murine EtOH consumption are not because of altered DC precursor numbers or differentiation, nor increased DC turnover rate. Similarly, increased thymic DC numbers are not the result of alterations in DC precursor differentiation or turnover rate. Compartment size plays a role in determining splenic and thymic DC numbers following chronic EtOH feeding. EtOH-induced alterations in total DC numbers provide several mechanisms to partially explain why chronic alcoholics have increased susceptibility to infections.

Chronic Ethanol Exposure Stimulates Endothelial Cell Nitric Oxide Production through PI-3 Kinase-and hsp90-dependent Mechanisms

Chronic ethanol (EtOH) ingestion increases the incidence of the Acute Respiratory Distress Syndrome (ARDS), a severe form of acute lung injury characterized by endothelial and epithelial barrier dysfunction. The regulated production of nitric oxide (NO) by the endothelium plays a central role in normal vascular function, and alterations in NO production have been implicated in barrier dysfunction. Although previous reports examined the impact of acute EtOH stimulation on endothelial NO production, this study extends those observations to clarify mechanisms of chronic EtOH-mediated alterations in endothelial nitric oxide synthase (eNOS) expression and NO production. Porcine pulmonary artery endothelial cells (PAEC) were treated with EtOH (0.04-0.16%, w/v) for 72 hr in sealed chambers to prevent evaporation. NO release and eNOS expression were determined to examine the effect of chronic EtOH stimulation on endothelial NO metabolism. While there was no change in the extent of phosphorylated eNOS at ser, chronic EtOH stimulation caused dose-dependent increases in NO production and increased eNOS expression, effects that were attenuated by the transcriptional inhibitor, alpha-amanitin (AA), and wortmannin, a specific phosphatidylinositol 3 kinase (PI3 K) inhibitor. EtOH stimulation also increased eNOS interaction with heat shock protein (hsp90), a molecular chaperone known to enhance eNOS activity. Geldanamycin, an hsp90 inhibitor, attenuated chronic EtOH-mediated increases in NO production. These results indicate that chronic EtOH exposure increases endothelial NO production by increasing eNOS protein levels through PI3 K-dependent up regulation of eNOS gene transcription and by increasing interactions between eNOS and hsp90. These findings clarify mechanisms by which chronic EtOH stimulation modulates vascular endothelial function and suggest new targets for investigation and intervention in EtOH-induced alterations in susceptibility to lung injury.

Effects of ethanol on intraovarian nitric oxide production in the prepubertal rat.

Nitric oxide (NO) has been shown to contribute to ovarian development and function. In non-ovarian tissues NO can be altered by ethanol (ETOH), a drug considered to be a gonadal toxin in men as well as male and female rats. The present study was undertaken to determine if some of the detrimental effects of chronic ETOH exposure on prepubertal ovarian function could be due to ETOH-induced alterations in the intraovarian NO system. Rats were implanted with intragastric cannulae on day 24 and began receiving control or ETOH diets on day 29. All rats were killed on day 34, determined to be in the late juvenile stage of development, and their ovaries and blood were collected. We analyzed the expression of the two constitutive forms of nitric oxide synthase (NOS), i.e. neuronal (n) NOS and endothelial (e) NOS, as well as the inducible (i) form of NOS protein in the ovaries of control and ETOH-treated rats by Western immunoblotting. Results demonstrate that eNOS protein increased markedly (P<0.02; 140 kDa) in ETOH-treated rats compared with controls. ETOH treatment did not alter the protein expression of nNOS (155 kDa) and only slightly increased that of iNOS (130 kDa). We also assessed NOS activity as determined by nitrite accumulation and by the conversion of L-[14C]arginine to L-[14C]citrulline. In this regard, the ETOH-treated animals showed an increase in ovarian nitrite generation (P<0.05), as well as an increase in ovarian citrulline formation (P<0.0001), when compared with control animals. Along with the above described ETOH-induced increases in ovarian eNOS and NO activity, the serum levels of estradiol were concomitantly suppressed (P<0.001) in the ETOH-treated rats. These results demonstrate for the first time the ETOH-induced changes in the prepubertal ovarian NO/NOS system, and suggest that these alterations contribute to the detrimental actions of the drug on prepubertal ovarian development and function.

Ethanol‐Induced Alterations in the Posttranslational Processing, But Not Secretion of Luteinizing Hormone‐Releasing Hormone In Vitro

The effects of ethanol (EtOH) on the male hypothalamic pituitary reproductive axis are multiple and varied. Although direct gonadal toxicity has been reported, hypothalamic-pituitary perturbations have also been noted. The difficulty of sampling the hypothalamus has made direct investigation of EtOH-induced alterations on luteinizing hormone-releasing hormone (LHRH) fraught with interpretation problems. To circumvent this, we have conducted a series of experiments exploring the effect of 200 mg% EtOH in vitro on GT1-7 cells, a newly developed LHRH secreting neural cell line. Cell lines were treated with EtOH-containing or EtOH-free media for 2, 6, 24, or 48 hr. EtOH caused no significant change in LHRH secretion at any time point, although there was a trend to increased secretion after 2 hr EtOH exposure when compared with control. Significantly increased total (i.e., cellular plus secreted) pro-LHRH coupled with significantly reduced cellular LHRH after 6 hr only of EtOH exposure suggested that EtOH caused a transient decrease in processing from bioinactive pro-LHRH to bioactive LHRH. However, even at this time point, LHRH secretion from these EtOH-exposed cells was no different than from control cells. Steady-state LHRH mRNA levels were not changed by EtOH at any time point. These findings are concordant with previous in vitro data using hypothalamic tissue that has similarly demonstrated no effect of EtOH on LHRH secretion. Taken together with the in vivo demonstration that EtOH reduces hypothalamic-pituitary portal blood levels of LHRH, these data indicate that EtOH exerts its effect either at an extrahypothalamic locus and/or on non-LHRH-producing cells within the hypothalamus.

VITAMIN A AT PHARMACOLOGIC DOSES AMELIORATES THE MEMBRANE LIPID PEROXIDATION INJURY AND TESTICULAR ATROPHY THAT OCCURS WITH CHRONIC ALCOHOL FEEDING IN RATS

The interaction of ethanol (ETOH) with testicular subcellular membranes contributes, at least in part, to alcohol-induced gonadal dysfunction. Vitamin A reaches the testes via the circulation as the retinyl ester and is converted to the free alcohol (retinol) and then to the aldehyde (retinal); retinal is the form of the vitamin which is essential for normal spermato-genesis. Because retinol can function as a free radical scavenger, testicular mitochondria were evaluated for evidence of a protective role provided by supplemental dietary vitamin A on ETOH-induced alterations in testicular structure and function in rats. Lipid peroxidation was evaluated by measurement of malonaldehyde formation and glutathione content of the testes. Compared to isocalorically matched dextrimaltose-fed controls (ISO) receiving a modified vitamin A containing diet, rats fed the corresponding ETOH diet for 50 days had a reduced testes/body ratio (ETOH: 0.0114 +/- 0.0004 vs ISO: 0.0128 +/- 0.0004). Mitochondrial enriched extracts obtained from the testes of these ETOH-fed rats showed significant increases in malonaldehyde formation; moreover, glutathione levels were reduced in the testes of the alcohol-fed animals when compared to their isocaloric controls. In contrast, no evidence for testicular atrophy was present in ETOH-fed rats receiving a standard vitamin A enriched diet; moreover, such ETOH-fed rats had a reduced rate of malonaldehyde formation as compared to their respective controls. Similarly, glutathione levels were not depleted in the testes of the ETOH-fed rats receiving the vitamin A enriched diet. Taken together, these data suggest that lipid peroxidation is a consequence of ethanol metabolism which can be attenuated, at least in part, by vitamin A.

The relationship between ethanol-induced locomotor activation and narcosis in long-sleep and short-sleep mice.

Mice selectively bred for marked response to hypnotic doses of ethanol (long-sleep, LS) respond to subhypnotic doses of ethanol (ETOH) with less stimulation of locomotor activity than their short-sleep (SS) counterparts. This assessment was made by comparing ETOH-induced alterations in locomotor activity to an untreated baseline within individual subjects, and to a saline-treated control group. A correlational study, using the same method in F2 generation hybrids of the LS and SS lines, produced a negative correlation (-.36) between locomotor stimulant effects of a subhypnotic dose of ETOH and length of loss of the righting reflex following a hypnotic dose. This relationship also appeared in a factor analysis of baseline locomotor activity, ETOH-stimulated activity, and depressant response variables. The genetic selection for LS and SS mice appears to have differentiated loci that influence more than one type of behavioral response to ETOH, an example of pleiotropism.