Gene co-expression networks in human brain predict chromatin modifications in alcohol abusers
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ABSTRACT: A novel systems approach was applied to transcriptome profiling that included the traditional analysis of differentially expressed genes, gene co-expression networks, cell type - specific transcriptomes and a wide range of gene annotations. By integrating our data with previous findings, we generated the first systems hypothesis of human alcoholism that integrates epigenetic regulation of gene expression with structural and functional alterations in alcoholic brain. Total RNA was extracted from human post-mortem brain and gene expression profiles were compared between alcoholics and matched controls.
Project description:A novel systems approach was applied to transcriptome profiling that included the traditional analysis of differentially expressed genes, gene co-expression networks, cell type - specific transcriptomes and a wide range of gene annotations. By integrating our data with previous findings, we generated the first systems hypothesis of human alcoholism that integrates epigenetic regulation of gene expression with structural and functional alterations in alcoholic brain.
Project description:Alcohol affects gene expression in several brain regions. The amygdala is a key structure in the brainâs emotional system and in recent years the crucial importance of the amygdala in drug-seeking and relapse has been increasingly recognized. In this study gene expression screening was used to identify genes involved in alcoholism in the human basolateral amygdala. The results show that alcoholism affects a broad range of genes and many systems including genes involved in synaptic transmission, neurotransmitter transport, structural plasticity, metabolism, energy production, transcription and RNA processing and the circadian cycle. In particular, genes involved in the glutamate system were affected in the alcoholic patients. In the amygdala the glutamate system is involved in the acquisition, consolidation, expression and extinction of associative learning, which is a vital part of addiction, and in alcohol abusers it is associated with withdrawal anxiety and neurodegeneration. Downregulation of the excitatory amino acid transporters GLAST, GLT-1 and the AMPA glutamate receptor 2 (GluR2) revealed by the microarray were confirmed by Western blots. The decreased expression of GLAST, GLT-1 and GluR2 in the alcoholic patients may increase glutamate tone and activity in the basolateral amygdala and this may contribute to neurodegeneration as well as the expression of associative memories and anxiety which underlie continued drug-seeking and chronic relapse. Two-condition experiment, alcoholics vs controls. Biological replicates: 6 alcoholics 6 controls, one replicate per array.
Project description:Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated triidothyronine (T3) levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics. A total of 32 microarrays were run with 4 biological replicates per treatment, line, and sex. Selection replicates (i.e. WSP-1 and WSP-2) for each treatment, line, and sex were collapsed to improve statistical power (n=4) and to facilitate in the identification of phenotype related effects and exclude selection artifacts. For comparisons, EtOH regulation was determined by comparing 4 arrays from (for example) Male WSR EtOH treated versus 4 arrays from Male WSR Air treated arrays.
Project description:Compared gene expression in lymphoblasoid cell lines from alcholics and controls and 24 hr treatment with ethanol. To elucidate the effects of a controlled exposure to ethanol on gene expression, we studied lymphoblastoid cell lines (LCLs) from 21 alcoholics and 21 controls. We cultured each cell line for 24 h with and without 75 mM ethanol and measured gene expression using microarrays. Differences in expression between LCL from alcoholics and controls were modest, but included 13 genes previously identified as associated with alcoholism or related traits in one or more GWAS, including KCNA3, DICER1, ZNF415, Catalase and PPARGC1B. The paired design allowed us to detect very small changes due to ethanol treatment: ethanol altered the expression of 37% of the probe sets (51% of the unique named genes) detectably expressed in these LCLs, but most by very modest amounts. 99% of the named genes expressed in the LCLs were also expressed in at least 1 of 9 brain regions tested, suggesting that LCLs are a reasonable and accessible proxy for brain tissues. Key pathways affected by ethanol include cytokine, TNF and NF-kB signaling. Among the genes affected by ethanol were ANK3, EPHB1, SLC1A1, SLC9A9, NRD1, and SH3BP5, which were reported to be associated with alcoholism or related phenotypes in 2 genome wide association studies. Genes that either differed in expression between alcoholics and controls or were affected by ethanol exposure are candidates for further study. Lymphoblastoid cells from 21 alcoholics and 21 controls were treated with 75mM ethanol for 24 hours.
Project description:Comparison of gene expression in post-mortem hippocampus from 20 alcoholics and 19 controls. The chronic high-level alcohol consumption seen in alcoholism leads to dramatic effects on the hippocampus, including decreased white matter, loss of oligodendrocytes and other glial cells, and inhibition of neurogenesis. Examining gene expression in post mortem hippocampal tissue from 20 alcoholics and 19 controls allowed us to detect differentially expressed genes that may play a role in the risk for alcoholism or whose expression is modified by chronic consumption of alcohol. We identified 639 named genes whose expression significantly differed between alcoholics and controls at a False Discovery Rate (FDR) ? 0.20; 52% of these genes differed by at least 1.2-fold. Differentially expressed genes included the glucocorticoid receptor and the related gene FK506 binding protein 5 (FKBP5), UDP glycosyltransferase 8 (UGT8), urea transporter (SLC14A1), zinc transporter (SLC39A10), Interleukin 1 receptor type 1 (IL1R1), thioredoxin interacting protein (TXNIP), and many metallothioneins. Pathways related to inflammation, hypoxia, and stress showed activation, and pathways that play roles in neurogenesis and myelination showed decreases. The cortisol pathway dysregulation and increased inflammation identified here are seen in other stress-related conditions such as depression and post-traumatic stress disorder and most likely play a role in addiction. Many of the detrimental effects on the hippocampus appear to be mediated through NF-?B signaling. Twenty-four of the differentially regulated genes were previously identified by genome-wide association studies of alcohol use disorders; this raises the potential interest of genes not normally associated with alcoholism, such as suppression of tumorigenicity 18 (ST18), BCL2-associated athanogene 3 (BAG3), and von Willebrand factor (VWF). Comparison of gene expression from frozen human post-mortem hippocampus from 20 alcoholics and 19 controls using one array per sample. There were 6 females in each group. Sex and labeling batch were included in the analysis. The hippocampus samples came from the New South Wales Tissue Resource Centre at the University of Sydney, Australia, case ID provided for the sample. The total RNA samples were labeled in 2 balanced batches (indicated by labeling_batch). Brain sample QC provided by Tissue bank: brain pH and Post mortem interval in hours. Repoistory website: http://sydney.edu.au/medicine/pathology/trc/
Project description:Compared gene expression in lymphoblasoid cell lines from alcholics and controls and 24 hr treatment with ethanol. To elucidate the effects of a controlled exposure to ethanol on gene expression, we studied lymphoblastoid cell lines (LCLs) from 21 alcoholics and 21 controls. We cultured each cell line for 24 h with and without 75 mM ethanol and measured gene expression using microarrays. Differences in expression between LCL from alcoholics and controls were modest, but included 13 genes previously identified as associated with alcoholism or related traits in one or more GWAS, including KCNA3, DICER1, ZNF415, Catalase and PPARGC1B. The paired design allowed us to detect very small changes due to ethanol treatment: ethanol altered the expression of 37% of the probe sets (51% of the unique named genes) detectably expressed in these LCLs, but most by very modest amounts. 99% of the named genes expressed in the LCLs were also expressed in at least 1 of 9 brain regions tested, suggesting that LCLs are a reasonable and accessible proxy for brain tissues. Key pathways affected by ethanol include cytokine, TNF and NF-kB signaling. Among the genes affected by ethanol were ANK3, EPHB1, SLC1A1, SLC9A9, NRD1, and SH3BP5, which were reported to be associated with alcoholism or related phenotypes in 2 genome wide association studies. Genes that either differed in expression between alcoholics and controls or were affected by ethanol exposure are candidates for further study.
Project description:Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated triidothyronine (T3) levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics.
Project description:Chronic alcohol consumption can lead to alchohol-related brain damage (ARBD). Despite the well known acute effects of alcohol the mechanism responsible for chronic brain damage is largely unknown. Pathologically the major change is the loss of white matter while neuronal loss is mild and restricted to a few areas such as the prefrontal cortex. In order to improve our understanding of ARBD pathogenesis we used microarrays to explore the white matter transcriptome of alcoholics and controls. Our results suggest that hepatic encephalopathy, along with two confounders, gray matter contamination and low RNA quality, are major drivers of gene expression in ARBD. All three exceeded the effects of alcohol itself. In particular, low quality RNA samples were characterized by an upregulation of protein translation machinery while hepatic encephalopathy was associated with a downregulation of mitochondrial energy metabolism pathways. The findings in HE alcoholics are consistent with the metabolic acidosis seen in this condition. In contrast non-HE alcoholics had widespread but only subtle changes in gene expression in their white matter. The initial cohort was compromised of four alcoholics without hepatic encephalopathy (non-HE alcoholics), three alcoholics with HE (HE alcoholics) and three neurologically normal controls. For each indvidual frozen white matter was sampled in the superior frontal gyrus (prefrontal cortex) and the precentral gyrus (motor cortex). These two cortices experience either moderate (prefrontal cortex) or no neuronal loss (motor cortex) with alcohol-related brain damage. Each white matter sample was divided in two before RNA was extracted to give two 'biological' repeats and a total of 40 samples. Subsequently eight duplicates were removed due to their gray matter contamination or low RNA quality to leave a 32-sample cohort (23 alcoholic (including eight with HE ) and nine control samples.
Project description:Background: The incidence of alcohol and tobacco co-abuse is as high as 80%. The molecular mechanism underlying this comorbidity is virtually unknown but interactions between these drugs have important implications for the development of, and recovery from drug dependence. Methods: We investigated the effects of chronic tobacco and alcohol abuse and the interaction of the two behaviours on global gene expression in the human nucleus accumbens using cDNA microarrays and 20 alcoholic and control cases, with and without smoking comorbidity. Changes in gene expression were established by two-way ANOVA. Unsupervised hierarchical clustering was utilized to probe the strength of the data sets. Results: Subjecting the data sets derived from microarray gene expression screening to unsupervised hierarchical clustering tied the cases into distinct groups. When considering all alcohol-responsive genes, alcoholics were separated from non alcoholics with the exception of one control case. All smokers were distinguished from non smokers based on similarity in expression of smoking-sensitive genes. In the nucleus accumbens, alcohol-responsive genes were associated with transcription, lipid metabolism and signalling. Smoking-sensitive genes were predominantly assigned to functional groups concerned with RNA processing and the endoplasmic reticulum. Both drugs influenced the expression of genes involved in matrix remodelling, proliferation and cell morphogenesis. Additionally, a gene set encoding proteins involved in the canonical pathway ‘regulation of the actin cytoskeleton’ was induced in response to alcohol and tobacco co-abuse and included. Conclusions: The region-specific modulation of alcohol-sensitive gene expression by smoking may have important consequences for alcohol-induced aberrations within the mesolimbic dopaminergic system. The data set contained individual RNA samples extracted from the nucleus accumbens from 5 non smoking non drinking control case, 5 non smoking alcoholics, 5 non drinking smokers, and 5 smoking alcoholics. An aliquot if each sample was combined to generate a reference pool. Each sample was amplified individually and labelled with Cy3 in the process, the reference samples was amplified and labelled with Cy5. Each sample was hybridised competitively against the reference sample to 1 array.
Project description:Drugs of abuse including nicotine and alcohol elicit their effect by stimulating the mesocorticolimbic dopaminergic system. There is a high incidence of nicotine dependence in alcoholics. To date only limited data is available on the molecular mechanism underlying the action of alcohol and nicotine in the human brain. This study utilised gene expression screening to identify genes sensitive to chronic alcohol abuse within the ventral tegmental area of the human brain. Keywords: gene expression, brain, alcohol abuse, human, ventral tegmental area