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Microglia depletion and alcohol: Transcriptome and behavioral

ABSTRACT: Purpose: Alcohol abuse induces changes in microglia morphology and immune function, but whether microglia initiate or simply amplify the harmful effects of alcohol exposure is still a matter of debate. Here we determined microglia function in acute and voluntary drinking behaviors using a colony stimulating factor 1 receptor inhibitor (PLX5622) and 3’UTR biased-sequencing. Therefore, The purpose of this study was to provide insight regarding microglia depletion and voluntary alcohol consumption. Methods: We performed 3’UTR biased transcriptome sequencing (3’Tag-seq) on total homogenate isolated from the prefrontal cortex (PFC) of C57BL6/J mice following microglia depletion and chronic every-other-day alcohol consumption. Results: Differential expression analysis and WGCNA network analysis revealed that although many immune genes have been implicated in alcohol abuse, downregulation of microglia genes does not necessitate changes in alcohol intake. Finally, we show that microglia depletion and chronic alcohol result in compensatory upregulation of ethanol-responsive, reactive astrocyte genes, indicating astrocytes may play a critical role in regulation of these alcohol behaviors. Conclusion:Taken together our behavioral and transcriptional data indicate that microglia are not the primary effector cell responsible for regulation of acute and voluntary alcohol behaviors. In addition, our data represents a novel resource for groups interested in transcriptional effects of microglia depletion after alcohol consumption.

ORGANISM(S): Mus musculus

PROVIDER: GSE132516 | GEO | 2020/06/01

REPOSITORIES: GEO

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Project description:Action of alcohol on synaptic mRNA in the amygdala of mice Chronic alcohol consumption induces changes in gene expression, causing persistent long-term neuro-adaptations and the remodeling of synaptic structures. These alcohol-induced synaptic changes may rely specifically on the local translation of mRNAs in the synaptic compartments of the cell. We profiled the transcriptome from synaptoneurosomes (SN) and paired total homogenates (TH) of amygdala to analyze the synaptic adaptations induced by chronic voluntary alcohol consumption in mice. In the SN both the number of alcohol-responsive mRNAs and the magnitude of fold-change were greater than in the TH. Accordingly, the SN detected many genes with coordinated patterns of expression producing a highly connected mRNA network in gene co-expression analysis. The greater sensitivity of the SN preparation allowed for improved cell-type specificity analysis, revealing an up-regulation of alcohol-responsive astrocytic and microglial modules that correlated with alcohol consumption. Alcohol was found to induce changes in the SN functionally important biological pathways, including long-term potentiation, long-term potentiation depression, glutamate pathway, neuro-immune, RNA-processing and translational machineries and provided overlap with changes seen in human alcoholic brain. Transposable elements were responsive to alcohol and found in the down-regulated neuronal mRNA module, which may underlie some of the coordinated gene expression changes associated with alcohol. We provide evidence that enrichment of synaptic components reveals a more intricate network of coordinated gene expression. Increased resolution captures the molecular effects of synaptic manipulations and provides an improved technique for identifying therapeutic targets for alcohol abuse. Synaptoneurosomes (SN) and Paired total homogenates (TH) were prepared from the same homogenate. 42 microarrays were used in total for the alcohol-control analysis (8 alcohol treated mice and 13 controls), 21 SN and 21 paired TH. 2 TH (control) samples were found outliers and were removed from the analysis. For the SN vs. TH analysis, the 2 TH samples found outliers were removed with the 2 paired SN samples.

Project description:Introduction: Though heavy alcohol drinking has been well characterized as causing a variety of injuries, recent epidemiological evidence in humans suggests moderate consumption may provide beneficial effects. For example, there exists a J- or U-shaped relationship between the level of alcohol intake and cardiovascular disease risk. We investigated the underlying mechanisms of these positive consequences by identifying which genes are responsive to moderate alcohol intake in the liver, the primary site for alcohol metabolism. Methods: Twelve female, inbred, alcohol-preferring (iP10a) rats were split equally between chronic water exposure and voluntary chronic ethanol exposure. Hepatic cholesterol and triglyceride levels were analyzed both histologically and biochemically. Hepatic transcriptomes were paired-end sequenced on the Illumina HiScanSQ system. Reads were analyzed and mapped using CLCbio Genomics Workbench 4.9. We confirmed altered expression of a subset of genes using TaqMan-based qRT-PCR. To quantify DNA methylation, we first digested DNA with methylation sensitive restriction enzymes and then performed qPCR using TaqMan assays surrounding the digest sites. Calculating M-NM-^TCt between a mock digest and digest determines the percent methylation in that locus. Results: Voluntary alcohol consumption in iP10a rats modeled moderate consumption in humans. These levels did not result in intrahepatic fat accumulation. Sequencing produced ~1.2 Gb of sequence per sample, and 65% of reads mapped uniquely. Using a FDR corrected p value of 0.05 we found 250 altered transcripts. Ontology analysis of genes with a fold change M-bM-^IM-%1.3 identified many cholesterol synthesis genes and cytoskeleton subunit genes, all of which were down-regulated. Of the 28 genes reanalyzed by qRT-PCR, altered expression was confirmed in 24 genes including the majority of the cholesterol synthesis and cytoskeleton subunit genes. Lastly, we profiled methylation throughout the promoter and gene body of four genes elicited in the RNA-Seq experiment. We found that alcohol caused demethylation at all loci; however this loss happened in a site-specific, tightly regulated manner. Conclusion: Voluntary consumption in the iP10a animals models moderate consumption in humans, does not produce intrahepatic fat accumulation, and causes down-regulation of a majority of cholesterol synthesis genes. Moderate alcohol also results in a tightly-regulated demethylation effect. Our results explain, at least in part, the J- or U-shaped relationship between level of alcohol intake and cardiovascular disease risk. We sequenced 12 female iP10a rat hepatic transcriptomes providing 6 biological replicates for water control and 6 for ethanol treatment.

Project description:Introduction: Though heavy alcohol drinking has been well characterized as causing a variety of injuries, recent epidemiological evidence in humans suggests moderate consumption may provide beneficial effects. For example, there exists a J- or U-shaped relationship between the level of alcohol intake and cardiovascular disease risk. We investigated the underlying mechanisms of these positive consequences by identifying which genes are responsive to moderate alcohol intake in the liver, the primary site for alcohol metabolism. Methods: Twelve female, inbred, alcohol-preferring (iP10a) rats were split equally between chronic water exposure and voluntary chronic ethanol exposure. Hepatic cholesterol and triglyceride levels were analyzed both histologically and biochemically. Hepatic transcriptomes were paired-end sequenced on the Illumina HiScanSQ system. Reads were analyzed and mapped using CLCbio Genomics Workbench 4.9. We confirmed altered expression of a subset of genes using TaqMan-based qRT-PCR. To quantify DNA methylation, we first digested DNA with methylation sensitive restriction enzymes and then performed qPCR using TaqMan assays surrounding the digest sites. Calculating ΔCt between a mock digest and digest determines the percent methylation in that locus. Results: Voluntary alcohol consumption in iP10a rats modeled moderate consumption in humans. These levels did not result in intrahepatic fat accumulation. Sequencing produced ~1.2 Gb of sequence per sample, and 65% of reads mapped uniquely. Using a FDR corrected p value of 0.05 we found 250 altered transcripts. Ontology analysis of genes with a fold change ≥1.3 identified many cholesterol synthesis genes and cytoskeleton subunit genes, all of which were down-regulated. Of the 28 genes reanalyzed by qRT-PCR, altered expression was confirmed in 24 genes including the majority of the cholesterol synthesis and cytoskeleton subunit genes. Lastly, we profiled methylation throughout the promoter and gene body of four genes elicited in the RNA-Seq experiment. We found that alcohol caused demethylation at all loci; however this loss happened in a site-specific, tightly regulated manner. Conclusion: Voluntary consumption in the iP10a animals models moderate consumption in humans, does not produce intrahepatic fat accumulation, and causes down-regulation of a majority of cholesterol synthesis genes. Moderate alcohol also results in a tightly-regulated demethylation effect. Our results explain, at least in part, the J- or U-shaped relationship between level of alcohol intake and cardiovascular disease risk.

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Microglia depletion and alcohol: Transcriptome and behavioral

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