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Biological factors are known to influence disease trajectories and treatment effectiveness in alcohol addiction. One important biological factor influencing disease dynamics in alcohol use disorder is sex, as preclinical and clinical evidence suggests that gender influences disease trajectories in subjects with alcohol use disorder. Another critical factor is age at first intoxicating drink, which has been identified as a risk factor for later alcohol binging. Preclinical research allows prospective monitoring of rodents throughout the lifespan, providing very detailed information that cannot be acquired in humans. Lifetime monitoring in rodents can be conducted under highly controlled conditions, during which one can systematically introduce multiple biological and environmental factors that impact behaviors of interest.Here, we used the alcohol deprivation effect (ADE) rat model of alcohol addiction in a computerized drinkometer system to study disease dynamics and transition points of addictive behavior in cohorts of adolescent vs. adult as well as male vs. female rats in direct comparison. Early age of onset of drinking (postnatal day 40) in male rats had surprisingly little impact on the development of drinking behavior and compulsivity (quinine taste adulteration) when compared to rats that started drinking during early adulthood (postnatal day 72). However, female rats showed a stronger resistance to quinine taste adulteration after a history of ADEs, indicative of more pronounced compulsive behavior in females as compared to male rats. Specifically, female rats exhibit during quinine taste adulteration a higher frequency of drinking events and a larger access size, especially from the 20% alcohol solution, resulting in fast intoxication despite taste aversion in a relapse-like situation. Our results suggest that drinking patterns, and specifically solution preference as well as access size, provide a valuable indication into determining compulsive drinking. These findings provide a better understanding of sex and age factors involved in the development of drinking behavior that are important in preclinical development of models of addiction, drug development and exploration options for new treatments.

IntroductionFormal genetics studies show that smoking is influenced by genetic factors; exploring this on the molecular level can offer deeper insight into the etiology of smoking behaviours.MethodsSummary statistics from the GWAS and Sequencing Consortium of Alcohol and Nicotine (GSCAN) Consortium were used to calculate polygenic risk scores (PRS) in a sample of ∼2,200 smokers/never-smokers. The association of PRS for Smoking Initiation (i.e. Lifetime Smoking; SI-PRS) with smoking status, and PRS for Cigarettes per Day (CpD-PRS) and Smoking Cessation (SC-PRS) with Fagerström Test for Nicotine Dependence (FTND) score were examined, as were distinct/additive effects of parental smoking on smoking status.ResultsSI-PRS explained 6.65% of variance (Nagelkerke-R2) in smoking status (p=1.71×10−24). In smokers, CpD-PRS (R2=3.15%, p=1.82×10−8) and SC-PRS (R2=2.01%; p=7.18×10−6) were associated with FTND score. Parental smoking alone explained R2=3.06% (p=2.43×10−12) of smoking status, and 1.39% when added to the most informative SI-PRS model (total R²=8.04%).ConclusionThese results show the potential utility of molecular genetic data for research investigating smoking prevention. The fact that PRS explains more variance than family history highlights progress from formal to molecular genetics; the overlap and increased predictive value when using both suggests the importance of combining these approaches.ImplicationsThis study underlines the value of using PRS to predict smoking status/behaviour. It highlights the importance of molecular genetic methods in research investigating smoking prevention and points to the necessity of combining family history and molecular genetic data.

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 identifiedNPFFR2andKALRNfor 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 asCACNA1C, NR3C1, andJUN. 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.

The lack of understanding as to the cellular and molecular basis of clinical and genetic heterogeneity in progressive multiple sclerosis (MS) has hindered the search for new effective therapies and biomarkers. Here, to address this gap, we analysed 740,000 single nuclei RNAseq profiles of 165 samples of white matter (WM) lesions, normal appearing WM, grey matter (GM) lesions and normal appearing GM from 55 MS patients and 28 controls. We find that gene expression changes in response to MS are highly cell-type specific in WM and GM lesions but are largely shared within an individual cell-type across lesions, following a continuum rather than discrete lesion-specific molecular programs. The major biological determinants of variability in gene expression in MS samples relate to individual patient effects, rather than to lesion types or other metadata. Using multi-omics factor analysis (MOFA+), we identify three subgroups of MS patients with distinct oligodendrocyte composition and WM glial gene expression signatures, suggestive of engagement of different pathological/regenerative processes. The discovery of these three patterns significantly advances our mechanistic understanding of progressive MS, provides a framework to use molecular biomarkers to stratify patients for best therapeutic approaches for progressive MS, and highlights the need for precision-medicine approaches to address heterogeneity among MS patients.

The COVID-19 pandemic severely affected the lives of families, and the well-being of children and their parent. Prenatal stress, dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, and genetic factors might influence individuals’ well-being in the presence of a major stressor such as the COVID-19 pandemic.The present work is part of an ongoing birth cohort study and aims to investigate maternal perceived stress, early childhood HPA axis activity, and polygenic risk scores (PRSs) as predictors of emotional well-being during the COVID-19 pandemic. All participants are part of the ongoing birth cohort study POSEIDON. Emotional well-being of children (n=263) and mothers (n=241) was assessed during the COVID-19 pandemic using the CRISIS questionnaire in two waves between June 2020 and February 2021. Associations of well-being with previously assessed maternal perceived stress, children’s salivary and morning urine cortisol at 45 months, PRSs for depression, schizophrenia, loneliness were investigated.A positive association between the children’s and mothers’ emotional well-being was found. Lower emotional well-being was observed in both children and mothers during the pandemic compared to before. Children’s emotional well-being improved over the course of the pandemic. Prenatally assessed maternal perceived stress was associated with a decrease in children’s but not in the mothers’ well-being. Cortisol measures and PRSs were not significantly associated with emotional wellbeing.The present study confirms that emotional well-being of children and mothers are linked, and were negatively affected by the COVID-19 pandemic, with differences in development over time.

Alcohol Use Disorder (AUD) is a major contributor to global mortality and morbidity. Postmortem human brain tissue enables the investigation of molecular mechanisms of AUD in the neurocircuitry of addiction. We aimed to identify differentially expressed (DE) genes in the ventral and dorsal striatum between individuals with AUD and controls, and to integrate the results with findings from genome- and epigenome-wide association studies (GWAS/EWAS) to identify functionally relevant molecular mechanisms of AUD. DNA-methylation and gene expression (RNA-seq) data was generated from postmortem brain samples of 48 individuals with AUD and 51 controls from the ventral striatum (VS) and the dorsal striatal regions caudate nucleus (CN) and putamen (PUT). We identified DE genes using DESeq2, performed gene-set enrichment analysis (GSEA), and tested enrichment of DE genes in results of GWASs using MAGMA. Weighted correlation network analysis (WGCNA) was performed for DNA-methylation and gene expression data and gene overlap was tested. Differential gene expression was observed in the dorsal (FDRARHGEF15 gene was upregulated in all three brain regions. GSEA in CN and VS pointed towards cell-structure associated GO-terms and in PUT towards immune pathways. The WGCNA modules most strongly associated with AUD showed strong enrichment for immune response and inflammation pathways. Our integrated analysis of multi-omics data sets provides further evidence for the importance of immune-and inflammation-related processes in AUD.

BackgroundThe SARS-CoV-2 pandemic, with all its impacts on our way of life, is affecting our experiences and mental health. Notably, individuals with mental disorders have been reported to have a higher risk of contracting SARS-CoV-2. Personality traits could represent an important determinant of preventative health behavior and, therefore, the risk of contracting the virus.AimsWe examined overlapping genetic underpinnings between major psychiatric disorders, personality traits, and susceptibility to SARS-CoV-2 infection.MethodsLinkage disequilibrium score regression was used to explore the genetic correlations of COVID-19 susceptibility with psychiatric disorders and personality traits based on data from the largest available respective genome-wide association studies (GWAS). In two cohorts (the PsyCourse (n=1346) and the HeiDE (n=3266) study), polygenic risk scores were used to analyze if a genetic association between, psychiatric disorders, personality traits, and COVID-19 susceptibility exists in individual-level data.ResultsWe observed no significant genetic correlations of COVID-19 susceptibility with psychiatric disorders. For personality traits, there was a significant genetic correlation for COVID-19 susceptibility with extraversion (p=1.47×10-5; rg=0.284). Yet, this was not reflected in individual-level data from the PsyCourse and HeiDE studies.ConclusionsWe identified no significant correlation between genetic risk factors for severe psychiatric disorders and genetic risk for COVID-19 susceptibility. Among the personality traits, extraversion showed evidence for a positive genetic association with COVID-19 susceptibility, in one but not in another setting. Overall, these findings highlight a complex contribution of genetic and non-genetic components in the interaction between COVID-19 susceptibility and personality traits or mental disorders.

Prenatal, perinatal, and postnatal factors have been shown to shape neurobiological functioning and alter the risk for mental disorders later in life. The gut microbiome is established early in life, and interacts with the brain via the brain-immune-gut axis. However, little is known about how the microbiome relates to early-life cognitive functioning in children. The present study, where the fecal microbiome of 380 children was characterized using 16s rDNA and metagenomic sequencing aimed to investigate the association between the microbiota and cognitive functioning of children at the age of 45 months measured with the Wechsler Preschool and Primary Scale of Intelligence (WPPSI-III). Overall the microbiome profile showed a significant association with cognitive functioning. A strong correlation was found between cognitive functioning and the relative abundance of an unidentified genus of the family Enterobacteriaceae. Follow-up mediation analyses revealed significant mediation effects of the level of this genus on the association of maternal smoking during pregnancy and current cigarette smoking with cognitive function. Metagenomic sequencing of a subset of these samples indicated that the identified genus was most closely related to Enterobacter asburiae. Analysis of metabolic potential showed a nominally significant association of cognitive functioning with the microbial norspermidine biosynthesis pathway. Our results indicate that alteration of the gut microflora is associated with cognitive functioning in childhood. Furthermore, they suggest that the altered microflora might interact with other environmental factors such as maternal cigarette smoking by acting as a mediator between maternal smoking and child cognitive function. Interventions directed at altering the microbiome should be explored in terms of improving cognitive functioning in young children. Overall the microbiome profile showed a significant association with cognitive functioning. A strong correlation was found between cognitive functioning and the relative abundance of an unidentified genus of the family Enterobacteriaceae. Follow-up mediation analyses revealed significant mediation effects of the level of this genus on the associations of maternal smoking during pregnancy and current maternal smoking with cognitive functioning. Metagenomic sequencing of a subset of these samples indicated that the identified genus was most closely related to Enterobacter asburiae. Our results indicate that alteration of the gut microflora is associated with cognitive functioning in childhood. Furthermore, they suggest that the altered microflora might interact with other environmental factors such as maternal cigarette smoking by acting as a mediator between those factors and child cognitive function. Interventions directed at altering the microbiome should be explored in terms of improving cognitive functioning in young children.

BackgroundTherapeutic sleep deprivation (SD) rapidly induces robust, transient antidepressant effects in a large proportion of major mood disorder patients suffering from a depressive episode, but underlying biological factors remain poorly understood. Research suggests that these patients may have altered circadian molecular genetic ‘clocks’ and that SD functions through ‘resetting’ dysregulated genes; additional factors may be involved, warranting further investigation. Leveraging advances in microarray technology enabling the transcriptome-wide assessment of gene expression, this study aimed to examine gene expression changes accompanying SD and recovery sleep in patients suffering from an episode of depression.MethodsPatients (N=78) and controls (N=15) underwent SD, with blood taken at the same time of day before, after one night of SD and after recovery sleep. A transcriptome-wide gene-by-gene approach was used, with a targeted look also taken at circadian genes. Furthermore, gene set enrichment, and longitudinal gene set analyses including the time point after recovery sleep, were conducted.ResultsCircadian genes were significantly affected by SD, with patterns suggesting that molecular clocks of responders and non-responders, as well as patients and controls respond differently to chronobiologic stimuli. Notably, gene set analyses revealed a strong widespread effect of SD on pathways involved in immune function and inflammatory response, such as those involved in cytokine and especially in interleukin signalling. Longitudinal gene set analyses showed that in responders these pathways were upregulated after SD; in non-responders, little response was observed.ConclusionsOur findings emphasize the close relationship between circadian, immune and sleep systems and their link to etiology of depression at the transcriptomic level.

The purification of astrocytes by fluorescence-activated cell sorting (FACS) requires that an astrocyte-specific promoter drive the expression of the green fluorescent protein (GFP). Our laboratory uses FACS to acutely isolate astrocytes from young and old tissue as well as to isolate GFP-negative neurons at the end of the FACS sorting to conduct comparative unbiased, large-scale gene expression studies. Because of the relatively harsh nature of FACS sorting, few astrocytes or neurons survive long enough after the sort to be cultured.

We describe the use of immunopanning to purify rodent astrocytes. Immunopanning of astrocytes permits the prospective isolation of astrocytes from the rodent brain. Prospective isolation refers to the direct selection of cells without multiple steps that extend over a few days, thereby permitting the selection of a representative proportion of the astrocytes from the cortex. Because immunopanning is a very gentle process, the cells are viable at the end of the preparation and can be cultured in a serum-free medium containing heparin-binding EGF-like growth factor (HBEGF), the critical survival factor of astrocytes in vitro, for at least 2 wk. This protocol was initially established and verified with rats, but modifications for the purification of mouse cells are also described here.