Alcohol Promotes Exosome Biogenesis and Release via Modulating Rabs and miR-192 Expression in Human Hepatocytes

Exosomes are membrane vesicles released by various cell types into the extracellular space under different conditions including alcohol exposure. Exosomes are involved in intercellular communication and as mediators of various diseases. Alcohol use causes oxidative stress that promotes exosome secretion. Here, we elucidated the effects of alcohol on exosome biogenesis and secretion using human hepatocytes. We found that alcohol treatment induces the expression of genes involved in various steps of exosome formation. Expression of Rab proteins such as Rab1a, Rab5c, Rab6, Rab10, Rab11, Rab27a and Rab35 were increased at the mRNA level in primary human hepatocytes after alcohol treatment. Rab5, Rab6 and Rab11 showed significant induction in the livers of patients with alcohol-associated liver disease. Further, alcohol treatment also led to the induction of syntenin, vesicle-associated membrane proteins (VAMPs), and syntaxin that all play various roles in exosome biogenesis and secretion. VAMP3, VAMP5, VAPb, and syntaxin16 mRNA transcripts were increased in alcohol treated cells and in the livers of alcohol-associated liver disease (ALD) patients. Induction in these genes was associated with increases in exosome secretion in alcohol treated hepatocytes. We found that hepatocyte enriched miR-192 and miR-122 levels were significantly decreased in alcohol treated hepatocytes whereas their levels were increased in the cell-free supernatant. The primary transcripts of miR-192 and miR-122 were reduced in alcohol treated hepatocytes, suggesting alcohol partially affects these miRNAs at the transcriptional level. We found that miR-192 has putative binding sites for genes involved in exosome secretion. Inhibition of miR-192 in human hepatoma cells caused a significant increase in Rab27a, Rab35, syntaxin7 and syntaxin16 and a concurrent increase in exosome secretion, suggesting miR-192 regulates exosomes release in hepatocytes. Collectively, our results reveal that alcohol modulates Rabs, VAMPs and syntaxins directly and partly via miR-192 to induce exosome machinery and release.

Alcohol induced hepatic retinoid depletion is associated with the induction of multiple retinoid catabolizing cytochrome P450 enzymes

Chronic alcohol consumption leads to a spectrum of liver disease that is associated with significant global mortality and morbidity. Alcohol is known to deplete hepatic vitamin A content, which has been linked to the pathogenesis of alcoholic liver disease. It has been suggested that induction of Cytochrome P450 2E1 (CYP2E1) contributes to alcohol-induced hepatic vitamin A depletion, but the possible contributions of other retinoid-catabolizing CYPs have not been well studied. The main objective of this study was to better understand alcohol-induced hepatic vitamin A depletion and test the hypothesis that alcohol-induced depletion of hepatic vitamin A is due to CYP-mediated oxidative catabolism. This hypothesis was tested in a mouse model of chronic alcohol consumption, including wild type and Cyp2e1 -/- mice. Our results show that chronic alcohol consumption is associated with decreased levels of hepatic retinol, retinyl esters, and retinoic acid. Moreover, the depletion of hepatic retinoid is associated with the induction of multiple retinoid catabolizing CYPs, including CYP26A1, and CYP26B1 in alcohol fed wild type mice. In Cyp2e1 -/- mice, alcohol-induced retinol decline is blunted but retinyl esters undergo a change in their acyl composition and decline upon alcohol exposure like WT mice. In conclusion, the alcohol induced decline in hepatic vitamin A content is associated with increased expression of multiple retinoid-catabolizing CYPs, including the retinoic acid specific hydroxylases CYP26A1 and CYP26B1.

Chronic Alcohol Exposure Alters Gene Expression and Neurodegeneration Pathways in the Brain of Adult Mice

Background: Chronic alcohol consumption can alter the structure of the central nervous system and disrupt cognitive function. Alcoholics are more likely to develop neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). However, the role of alcohol in promoting neurotoxicity and neurodegeneration remains unclear. Objective: In this study, we aimed at estimating the effects of chronic binge alcohol exposure on brain transcriptome and behavior changes in a chronic “Drinking in the Dark” (DID) mouse model. Methods: The adult C57BL/6J male mice were exposed to alcohol for 4 weeks. RNA-seq was applied to assess the effects of chronic alcohol exposure on transcriptome in brain. The open field test and novel object recognition test were used to assess the changes of anxiety level, locomotive function, and short-term memory induced by alcohol. RNA-seq analysis revealed that chronic alcohol exposure caused significant change in the brain transcriptome, especially in prefrontal cortex. Results: The gene dysregulation caused by chronic alcohol exposure includes pathways related to mitochondrial energy metabolism (such as oxidative phosphorylation) and multiple neurodegenerative diseases (such as AD and PD). Furthermore, the pathway and network analyses suggest that the genes involved in mitochondrial energy metabolism, ubiquitin-proteasome system, Wnt signaling pathway, and microtubules may attribute to the neurotoxicity and neurodegeneration caused by chronic alcohol consumption. Additionally, locomotive function was also significantly impaired. Conclusion: This work provides gene transcriptional profile data for future research on alcohol-induced neurodegenerative diseases, especially AD and PD.

Association of Alcohol Use Diagnostic Codes in Pregnancy and Offspring Conotruncal and Endocardial Cushion Heart Defects

Background The pathogenesis of congenital heart disease (CHD) remains largely unknown, with only a small percentage explained solely by genetic causes. Modifiable environmental risk factors, such as alcohol, are suggested to play an important role in CHD pathogenesis. We sought to evaluate the association between prenatal alcohol exposure and CHD to gain insight into which components of cardiac development may be most vulnerable to the teratogenic effects of alcohol. Methods and Results This was a retrospective analysis of hospital discharge records from the California Office of Statewide Health Planning and Development and linked birth certificate records restricted to singleton, live‐born infants from 2005 to 2017. Of the 5 820 961 births included, 16 953 had an alcohol‐related International Classification of Diseases , Ninth and Tenth Revisions (ICD‐9; ICD‐10 ) code during pregnancy. Log linear regression was used to calculate risk ratios (RR) for CHD among individuals with an alcohol‐related ICD ‐9 and ICD10 code during pregnancy versus those without. Three models were created: (1) unadjusted, (2) adjusted for maternal demographic factors, and (3) adjusted for maternal demographic factors and comorbidities. Maternal alcohol‐related code was associated with an increased risk for CHD in all models (RR, 1.33 to 1.84); conotruncal (RR, 1.62 to 2.11) and endocardial cushion (RR, 2.71 to 3.59) defects were individually associated with elevated risk in all models. Conclusions Alcohol‐related diagnostic codes in pregnancy were associated with an increased risk of an offspring with a CHD, with a particular risk for endocardial cushion and conotruncal defects. The mechanistic basis for this phenotypic enrichment requires further investigation.

Impact of adolescent intermittent ethanol exposure on interneurons and their surrounding perineuronal nets in adulthood.

Background: Binge alcohol exposure during adolescence results in long-lasting alterations in brain and behavior. For example, adolescent intermittent ethanol (AIE) exposure in rodents results in long-term loss of functional connectivity among prefrontal cortex (PFC) and striatal regions as well as a variety of neurochemical, molecular, and epigenetic alterations. Interneurons in the PFC and striatum play critical roles in behavioral flexibility and functional connectivity. For example, parvalbumin (PV) interneurons are known to contribute to neural synchrony, and cholinergic interneurons contribute to strategy selection. Furthermore, extracellular perineuronal nets (PNNs) surround some interneurons, particularly PV+ interneurons, to further regulate cellular plasticity. The effect of AIE exposure on expression of these markers within the PFC is not well understood. Methods: The present study tested the hypothesis that AIE exposure reduces expression of PV+ and ChAT+ interneurons in the adult PFC and striatum and increases related expression of PNNs (marked by binding of Wisteria Floribunda agglutinin lectin; WFA) in adulthood. Male rats were exposed to AIE (5 g/kg/day, 2-days-on/2-days-off, i.g., P25-P54) or water (CON), and brain tissue was harvested in adulthood (> P80). Immunohistochemistry and co-immunofluorescence were used to assess expression of ChAT, PV, and WFA labeling within the adult PFC and striatum following AIE exposure. Results: ChAT and PV interneuron numbers in the striatum and PFC were unchanged after AIE exposure. However, WFA labeling in the PFC of AIE-exposed rats was increased compared to CON rats. Moreover, significantly more PV neurons were surrounded by WFA labeling in AIE-exposed subjects relative to controls in both PFC subregions assessed: the orbitofrontal cortex (CON = 34%; AIE = 40%) and the medial PFC (CON = 10%; AIE = 14%). Conclusions: These findings indicate that while PV interneuron expression in the adult PFC and striatum is unaltered following AIE exposure, PNNs surrounding these neurons (indicated by extracellular WFA binding) are increased. This increase in PNNs may restrict plasticity of the ensheathed neurons, thus contributing to impaired microcircuitry in frontostriatal connectivity and related behavioral impairments.

Prevalence of marijuana use in pregnant women with concurrent opioid use disorder or alcohol use in pregnancy

Background A quarter of pregnant women use alcohol, 6.5/1000 deliveries are affected by opioid use disorder (OUD), and the prevalence of cannabis use in pregnant women is increasing. However, marijuana co-exposure in polysubstance-using women is not well described. Methods The well-characterized ENRICH-1 cohort (n = 251), which focused on the effects of two primary exposures of interest—opioids and alcohol, was used to (1) estimate the prevalence/frequency of marijuana use in those with OUD and/or alcohol use, and (2) examined correlates of marijuana use. Participants were classified into an OUD group (n = 125), Alcohol group (n = 69), and concurrent OUD and Alcohol (OUD + Alcohol) group (n = 57). Self-report and biomarkers ascertained substance use. Multivariable logistic regression identified correlates of marijuana use. Results The prevalence of any marijuana use in pregnancy was 43.2%, 52.6%, and 46.4% in the OUD, OUD + Alcohol, and Alcohol groups, respectively. Correspondingly, weekly or daily use was reported by 19.4%, 21.0%, and 24.6% of participants. In the OUD and OUD + Alcohol groups, the proportion of women using marijuana was significantly higher in those taking buprenorphine (45.8% and 58.3%, respectively) compared to women using methadone (37.5% and 42.9%, respectively). Mean maternal age was lower in women who used marijuana in all three groups compared to non-marijuana users. Independent correlates of marijuana use (controlling for group, race/ethnicity, education, and smoking) were maternal age (adjusted Odds Ratio (aOR) per 5-year increment 0.61; (95% CI 0.47, 0.79)), and polysubstance use (aOR 2.02; 95% CI 1.11, 3.67). There was a significant interaction between partnership status and group: among women who were not in a partnership, those in the OUD and OUD + Alcohol groups had lower odds of marijuana use relative to the Alcohol group. For women in the Alcohol group, partnered women had lower odds of marijuana use than un-partnered women (aOR 0.12; 95% CI: 0.02, 0.68). Conclusions Results indicate a relatively high prevalence and frequency of marijuana use in pregnant women being treated for OUD and/or women consuming alcohol while pregnant. These results highlight the need for ongoing risk reduction strategies addressing marijuana use for pregnant women receiving OUD treatment and those with alcohol exposure.