Marion M. Friske

Alcohol consumption triggers neuroinflammation, potentially creating a feed-forward loop that increases drinking. Previous studies have shown that activation of the toll-like receptor 7 (TLR7) leads to escalated drinking. In this study, we aim to identify cell type-specific transcriptomic patterns underlying TLR7-induced neuroinflammation, potentially leading to escalated drinking. Therefore, male C57BL/6J mice were treated with the selective TLR7 agonist R848 every-other day for 20 days in total. After 10 treatment-free days, half of the cohort underwent two bottle-choice drinking; the other half was sacrificed and brains were collected for single-nucleus RNA-Sequencing (snRNA-Seq) in the medial prefrontal cortex (mPFC) and central amygdala (AMG). The AMG showed a greater number of differentially expressed genes (DEGs), primarily in inhibitory and excitatory neurons. Among glial cells, AMG astrocytes exhibited the greatest number of DEGs, which were involved in blood-brain barrier (BBB) regulation (e.g., Cldn5 , Mecom , Nrg1 ), a finding supported by secondary validation using Xenium in situ spatial transcriptomics on AMG-containing sections. BBB-regulatory genes, including those in the Wnt signaling pathway (e.g., Notch3 , Top2a , Aldoc ), were altered across multiple cell types in both regions. Together with the alterations observed in neurons, these findings suggest that repeated TLR7 activation induces persistent BBB and neuronal dysregulation, potentially leading to TLR7-induced escalated drinking.

Alcohol use disorder (AUD) is associated with widespread epigenetic alterations, including changes in DNA methylation (DNAm). This multi-cohort study validated and extended previous findings on DNAm of HECW2 and GDAP1 in AUD, assessed sex differences, and explored DNAm in blood and brain tissue in humans and rats. DNAm was measured via pyrosequencing in human blood (N_Ctrl = 341, N_AUD = 258), postmortem frontal cortex (Brodmann area 9; discovery cohort: N_Ctrl = 10, N_AUD = 13, replication cohort: N_Ctrl = 64, N_AUD = 55) and rat blood and medial prefrontal cortex (N_Ctrl = 16, N_AUD = 15). Gene expression was assessed in human postmortem brain by quantitative real-time PCR. AUD-associated DNAm differences in HECW2 and GDAP1 were replicated in human blood. While decreased GDAP1 DNAm was only observed in men, HECW2 hypomethylation was present in both sexes. In brain tissue, initial DNAm increases in AUD and HECW2 gene expression decreases were not validated in the replication cohort. In rats, HECW2 hypomethylation appeared in the prelimbic cortex but not in blood. Our findings support the involvement of HECW2 and GDAP1 DNAm in AUD, revealing sex-specific and tissue-dependent epigenetic patterns. The opposing DNAm directionality in blood and brain underscores the complexity of alcohol-related epigenetic modifications and suggests the need for multi-tissue, cross-species, and longitudinal studies to clarify causal mechanisms.

Currently available clinical treatments on alcohol use disorder (AUD) exhibit limited efficacy and new druggable targets are required. One promising approach to discover new molecular treatment targets involves the transcriptomic profiling of brain regions within the addiction neurocircuitry, utilizing animal models and postmortem brain tissue from deceased patients with AUD. Unfortunately, such studies suffer from large heterogeneity and small sample sizes. To address these limitations, we conducted a cross-species meta-analysis on transcriptome-wide data obtained from brain tissue of patients with AUD and animal models. We integrated 36 cross-species transcriptome-wide RNA-expression datasets with an alcohol-dependent phenotype vs. controls, following the PRISMA guidelines. In total, we meta-analyzed 964 samples – 502 samples from the prefrontal cortex (PFC), 282 nucleus accumbens (NAc) samples, and 180 from amygdala (AMY). The PFC had the highest number of differentially expressed genes (DEGs) across rodents, monkeys, and humans. Commonly dysregulated DEGs suggest conserved cross-species mechanisms for chronic alcohol consumption/AUD comprising MAPKs as well as STAT, IRF7, and TNF. Furthermore, we identified numerous unique gene sets that might contribute individually to these conserved mechanisms and also suggest novel molecular aspects of AUD. Validation of the transcriptomic alterations on the protein level revealed interesting targets for further investigation. Finally, we identified a combination of DEGs that are commonly regulated across different brain tissues as potential biomarkers for AUD. In summary, we provide a compendium of genes that are assessable via a shiny app, and describe signaling pathways, and physiological and cellular processes that are altered in AUD that require future studies for functional validation.

Background: Alcohol use disorder (AUD) is associated with increased mortality and morbidity risk. A reason for this could be accelerated biological aging, which is strongly influenced by disease processes such as inflammation. As recent studies of AUD show changes in DNA methylation and gene expression in neuroinflammation‐related pathways in the brain, biological aging represents a potentially important construct for understanding the adverse effects of substance use disorders. Epigenetic clocks have shown accelerated aging in blood samples from individuals with AUD. However, no systematic evaluation of biological age measures in AUD across different tissues and brain regions has been undertaken.Methods: As markers of biological aging (BioAge markers), we assessed Levine's and Horvath's epigenetic clocks, DNA methylation telomere length (DNAmTL), telomere length (TL), and mitochondrial DNA copy number (mtDNAcn) in postmortem brain samples from Brodmann Area 9 (BA9), caudate nucleus, and ventral striatum (N = 63–94), and in whole blood samples (N = 179) of individuals with and without AUD. To evaluate the association between AUD status and BioAge markers, we performed linear regression analyses while adjusting for covariates.Results: The majority of BioAge markers were significantly associated with chronological age in all samples. Levine's epigenetic clock and DNAmTL were indicative of accelerated biological aging in AUD in BA9 and whole blood samples, while Horvath's showed the opposite effect in BA9. No significant association of AUD with TL and mtDNAcn was detected. Measured TL and DNAmTL showed only small correlations in blood and none in brain.Conclusions: The present study is the first to simultaneously investigate epigenetic clocks, telomere length, and mtDNAcn in postmortem brain and whole blood samples in individuals with AUD. We found evidence for accelerated biological aging in AUD in blood and brain, as measured by Levine's epigenetic clock, and DNAmTL. Additional studies of different tissues from the same individuals are needed to draw valid conclusions about the congruence of biological aging in blood and brain.

During the COVID‐19 pandemic, many potential risk groups have been identified, such as those with obesity, diabetes, preexisting organ injuries, and several other conditions. Smoking is the most reported substance use disorder linked to increased COVID‐19 hospitalization rate and disease severity. In relation to smoking, we discuss the impairment of the innate and the adaptive immune systems as being among the main potential reasons for increased COVID‐19 infection risk and severity. Chronic alcohol consumption and alcohol use disorder (AUD) also have a negative impact on the immune system, but when it comes to COVID‐19 risk, they produce diverse outcomes. Some studies provide evidence that chronic alcohol consumption and AUD increase the risk of COVID‐19 infection and severe disease progression, while others report reduced hospitalization and death rates. In this review, we summarize the current state of epidemiological and molecular data concerning alcohol consumption and AUD as risk factors for COVID‐19 infection, hospitalization, and mortality.

Background: Chronic alcohol consumption and alcohol use disorder have a tremendous impact on the patient's psychological and physiological health. There is evidence that chronic alcohol consumption influences SARS‐CoV2 infection risk, but so far, the molecular mechanism underlying such an effect is unknown.Methods: We generated the expression data of SARS‐CoV2 infection‐relevant genes (Ace2, Tmprss2, and Mas) in different organs in rat models of chronic alcohol exposure and alcohol dependence. Ace2 and Tmprss2 represent the virus entry point, whereas Mas activates the anti‐inflammatory response once the cells are infected.Results: Across three different chronic alcohol test conditions, we found a consistent upregulation of Ace2 gene expression in the lung, which has been shown to be the most affected organ in COVID‐19 patients. Other organs such as liver, ileum, kidney, heart, and brain also showed upregulation of Ace2 and Mas gene expression but less consistently across the different animal models, while Tmprss2 expression was unaffected in all conditions.Conclusions: We conclude that alcohol‐induced upregulation of Ace2 gene expression can lead to an elevated stochastic probability of virus entry into cells and may thus confer a molecular risk for SARS‐CoV2 infection.

Alcohol use disorder (AUD) is a complex mental health condition. Currently available clinical treatments exhibit limited efficacy and new druggable targets are required. One promising approach to discover new molecular treatment targets involves the transcriptomic profiling of brain regions within the addiction neurocircuitry, utilizing animal models and post-mortem brain tissue from deceased AUD patients. Unfortunately, such studies suffer from large heterogeneity and small sample sizes. To address these limitations, we conducted a cross-species meta-analysis on transcriptome-wide data obtained from brain tissue of AUD patients and animal models. We integrated 36 cross-species transcriptome-wide RNA-expression datasets with an alcohol-dependent phenotype vs. controls, following the PRISMA guidelines. In total, we meta-analyzed 1,000 samples – 502 samples for the prefrontal cortex (PFC), 318 nucleus accumbens (NAc) samples, and 180 amygdala (AMY) samples. The PFC had the highest number of differentially expressed genes (DEGs) across rodents, monkeys, and humans. Commonly dysregulated DEGs pointed towards enrichment in inflammatory responses and alterations in BBB-regulatory mechanisms in astrocytes, microglia and endothelial cells. Gene set enrichment analysis further showed that MAPK/ERK-signaling plays a critical role in AUD and especially in monkeys Dusp4 as a major inhibitor of the MAPK pathway may be a main driver of these pathway alterations. Our data also suggest that the transcriptomic profile in the NAc is less vulnerable to the maintenance of AUD. Finally, we provide a combination of DEGs that are commonly regulated across different brain tissues as potential biomarker for AUD. In summary, we provide a compendium of genes, signaling pathways, and physiological and cellular processes that are altered in AUD and that require future studies for functional validation.

Cocaine use disorder (CUD) is characterized by a loss of control over drug intake and is associated with structural, functional, and molecular alterations in the brain. At the molecular level, epigenetic alterations are hypothesized to contribute to the higher-level functional and structural brain changes as observed in CUD. Most evidence of cocaine-associated epigenetic changes comes from animal studies while only a few studies have been performed using human tissue. We investigated epigenome-wide DNA methylation signatures of CUD in human postmortem brain tissue of Brodmann Area 9 (BA9). A total of N = 42 BA9 brain samples were obtained from N = 21 individuals with CUD and N = 21 individuals without a CUD diagnosis. We performed an epigenome-wide association study and analyzed CUD-associated differentially methylated regions (DMRs). To assess the functional role of CUD-associated differential methylation, we performed Gene Ontology enrichment analyses and characterized co-methylation networks using a weighted correlation network analysis. We further investigated epigenetic age in CUD using epigenetic clocks for the assessment of biological age. While no CpG site was associated with CUD at epigenome-wide significance in BA9, we detected a total of 20 CUD-associated DMRs. After annotation of DMRs to genes, we identified NPFFR2 and KALRN for which a previous role in the behavioral response to cocaine in rodents is known. Three of the four identified CUD-associated co-methylation modules were functionally related to neurotransmission and neuroplasticity. Protein-protein interaction networks derived from module hub genes revealed several addiction-related genes as highly connected nodes such as CACNA1C, NR3C1 , and JUN . In BA9, we observed a trend toward epigenetic age acceleration in individuals with CUD remaining stable even after adjustment for covariates. Results from our study highlight that CUD is associated with epigenome-wide differences in DNA methylation levels in BA9 particularly related to synaptic signaling and neuroplasticity. This supports findings from previous studies that report on the strong impact of cocaine on neurocircuits in the human prefrontal cortex. Further studies are needed to follow up on the role of epigenetic alterations in CUD focusing on the integration of epigenetic signatures with transcriptomic and proteomic data.

(1) Background: Epigenome-wide association studies (EWAS) in peripheral blood have repeatedly found associations between tobacco smoking and aberrant DNA methylation (DNAm), but little is known about DNAm signatures of smoking in the human brain, which may contribute to the pathophysiology of addictive behavior observed in chronic smokers. (2) Methods: We investigated the similarity of DNAm signatures in matched blood and postmortem brain samples (n = 10). In addition, we performed EWASs in five brain regions belonging to the neurocircuitry of addiction: anterior cingulate cortex (ACC), Brodmann Area 9, caudate nucleus, putamen, and ventral striatum (n = 38–72). (3) Results: cg15925993 within the LOC339975 gene was epigenome-wide significant in the ACC. Of 16 identified differentially methylated regions, two (PRSS50 and LINC00612/A2M-AS1) overlapped between multiple brain regions. Functional enrichment was detected for biological processes related to neuronal development, inflammatory signaling and immune cell migration. Additionally, our results indicate the association of the well-known AHRR CpG site cg05575921 with smoking in the brain. (4) Conclusion: The present study provides further evidence of the strong relationship between aberrant DNAm and smoking.

Chronic alcohol consumption and alcohol use disorder (AUD) have a tremendous impact on the patient’s psychological and physiological health. There is some evidence that chronic alcohol consumption influences SARS-CoV2 infection risk, but the molecular mechanism is unknown. Here, we generated expression data of SARS-CoV2 infection relevant genes (Ace2, Tmprss2 and Mas) in different organs in rat models of chronic alcohol exposure and alcohol dependence. ACE2 and TMPRSS2 represent the virus entry point whereas Mas is activating the anti-inflammatory response once the cells are infected. Across three different chronic alcohol test conditions, we found a consistent upregulation of Ace2 in the lung, which is the most affected organ in Covid-19 patients. Other organs such as liver, ileum, kidney, heart, and the brain showed also up-regulation of Ace2 and Mas but in a less consistent manner across the different animal models, while Tmprss2 was unaffected in all conditions. We suggest that alcohol-induced up-regulation of Ace2 can lead to an elevated stochastic probability of cellular virus entry and may thus confer a molecular risk factor for a SARS-CoV2 infection.