Project description:Differential gene expression between mouse distal chromsome 1 congenic and background and reciprocal congenic and background Genetic factors significantly affect vulnerability to alcohol dependence (alcoholism). We previously identified a quantitative trait locus on chromosome 1 (Adw1) with a large effect on predisposition to alcohol physiological dependence and associated withdrawal following both chronic and acute alcohol exposure in mice. We fine-mapped these loci to a 1.1-1.7 Mb interval syntenic with human 1q23.2-23.3. Adw1 interval genes show remarkable genetic variation among mice derived from the C57BL/6J and DBA/2J strains, the two most widely studied genetic animal models for alcohol-related traits. Here, we report the creation of a novel recombinant Adw1 congenic model (R2) in which the Adw1 interval from a donor C57BL/6J strain is introgressed onto a uniform, inbred DBA/2J genetic background. As expected, R2 mice demonstrate significantly less severe alcohol withdrawal compared to wild-type littermates. Additionally, comparing R2 and background strain animals, as well as reciprocal congenic (R8) and appropriate background strain animals, we assessed Adw1 dependent brain gene expression using microarray and quantitative PCR analyses. To our knowledge this includes the first Weighted Gene Co-expression Network Analysis using reciprocal congenic models. Importantly, this allows detection of co-expression patterns limited to one or common to both genetic backgrounds with high and low predisposition to alcohol withdrawal severity. The gene expression patterns (modules) in common contain genes related to oxidative phosphorylation, building upon human and animal model studies that implicate involvement of oxidative phosphorylation in alcohol use disorders. Finally, we demonstrate that administration of N-acetylcysteine, an FDA-approved antioxidant, significantly reduces symptoms of alcohol withdrawal (convulsions) in mice, thus validating a phenotypic role for this network. Taken together, these studies support the importance of mitochondrial oxidative homeostasis in alcohol withdrawal and identify this network as a valuable therapeutic target in human alcohol use disorders.

Project description:Glial cells are central players in the pathogenesis of neurodevelopmental disorders like Fetal Alcohol Spectrum Disorder (FASD). Ethanol can modulate glial differentiation and astrocyte function imposing dramatic changes to the developing brain. Since ethanol can affect a vast number of intracellular targets and signaling pathways and many effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to a long-lasting change in astrocyte secretome in vitro. Animals were treated every other day with ethanol (5g/kg) or saline between post-natal day (P)10-30. At P31, astrocyte cultures were made and cells were submitted to stable isotope labeling by amino acids (SILAC). 24h-conditioned media of cells obtained from ethanol- or saline-treated animals (ET-CM or SAL-CM) were collected and analyzed by quantitative mass spectrometry in tandem with liquid chromatography. Here we show that 65 out of 280 quantifiable proteins displayed significant differences comparing ET-CM to SAL-CM. Among 59 proteins found reduced in ET-CM we found components of the extracellular matrix like Laminin subunits α2, α4, β1, β2 and γ1 and the proteoglycans Biglycan, Heparin Sulfate Proteoglycan 2 and Lumican. Proteins with trophic function like Insulin-Like Growth Factor Binding Protein 4, Pigment Epithelium-Derived Factor and Clusterin as well as proteins involved on modulation of proteolysis like TIMP-1 and PAI-1 were also found reduced. On the other hand, pro-synaptogeneic proteins like Thrombospondin-1, Hevin as well as the modulator of extracelular matrix expression, Angiotensinogen, were found increased in ET-CM. The analysis of interactome maps through Ingenuity Pathway Analysis demonstrated that the Amyloid beta A4 protein precursor (APP), which was found reduced in ET-CM, was previously shown to interact with ten other proteins that exhibited significant changes in the ET-CM, rendering it a suitable target for a more accurate analysis about the effects of ethanol on its biology. Taken together our results strongly suggest that early exposure to teratogens such alcohol may lead to an enduring change in astrocyte secretome, affecting mainly the expression of trophic and matricellular proteins, which in turn may lead to permanent alterations in brain function.

Project description:While women are more vulnerable than men to many of the medical consequences of alcohol abuse, the role of sex in the response to ethanol is controversial. Neuroadaptive responses that result in the hyperexcitability associated with withdrawal from chronic ethanol likely reflect gene expression changes. We have examined both genders for the effects of withdrawal on brain gene expression using mice with divergent withdrawal severity that have been selectively bred from a genetically heterogeneous population. A total of 295 genes were identified as ethanol regulated from each gender of each selected line by microarray analyses. Hierarchical cluster analysis of the arrays revealed that the transcriptional response correlated with sex rather than with the selected withdrawal phenotype. Consistent with this, gene ontology category over-representation analysis identified cell death and DNA/RNA binding as targeted classes of genes in females, while in males, protein degradation, and calcium ion binding pathways weremore altered by alcohol. Examination of ethanol regulated genes and these distinct signaling pathways suggested enhanced neurotoxicity in females. Histopathological analysis of brain damage following ethanol withdrawal confirmed elevated cell death in female but not male mice. The sexually dimorphic response was observed irrespective of withdrawal phenotype. Combined, these results indicate a fundamentally distinct neuroadaptive response in females compared to males during chronic ethanol withdrawal and are consistent with observations that female alcoholics may be more vulnerable than males to ethanol-induced brain damage associated with alcohol abuse. 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.

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Withdrawal from chronic alcohol exposure leads to tissue-specific recovery of the zebrafish transcriptome

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