Friday, April 19, 2024
4/19/2024
Chronic alcohol consumption induces transcriptional changes in genes encoding structural components and regulators of the extracellular matrix (ECM). The molecular mechanisms underlying these alterations and the functional implications of altered ECM gene expression are not well understood in the context of excessive alcohol drinking. We hypothesize that activation of the immune response-related transcription factor STAT3 in astrocytes results in transcriptional changes in genes encoding ECM components, resulting in alterations in ECM structure and function, synaptic changes, and increased ethanol intake. The proposed experiments will address this hypothesis with three specific aims. In Specific Aim 1, we will use an unbiased approach to determine changes in the cortical ECM proteome, or “matrisome” in mice that have chronically consumed ethanol in a binge-like drinking protocol (drinking in the dark) for 6 weeks. Cutting-edge proteomics and glycomics will be performed to quantitatively measure changes in cortical ECM. These results will be compared to changes in ECM-related genes in existing and new INIA transcriptome datasets and the genes analyzed for STAT3 binding motifs in their promoters. In Specific Aim 2, we will determine the role of astrocyte-expressed STAT3 in alcohol consumption by conditionally knocking out Stat3 in astrocytes using floxed Stat3 mice and the astrocyte-specific Cre line, Aldh1l1-CreERT2. Mice will be tested for binge-like ethanol consumption and dependence-induced escalation of ethanol intake. Association of activated STAT3 with gene promoters in the prefrontal cortex will also be determined after chronic binge-like drinking using chromatin immunoprecipitation with an antibody to phosphorylated STAT3, followed by whole-genome DNA sequencing (ChIP-Seq). Finally, we will determine the role of a specific ECM gene and putative STAT3 target, brevican (Bcan) in alcohol consumption, by conditionally knocking out Bcan in astrocytes as described above. We will also reduce the expression of Bcan in the prefrontal cortex of mice using viral-delivered shRNA and measure binge-like ethanol intake. Brevican is localized perisynaptically and regulates synaptic plasticity. To determine if brevican is more highly associated with synapses after chronic ethanol drinking and if synapse structure in the PFC is altered by chronic ethanol, we will perform super-resolution microscopy with brevican antibody and antibodies to excitatory and inhibitory synaptic markers in mice after chronic binge-like drinking. Within each Specific Aim, we will integrate and collaborate with INIA-Neuroimmune and INIA-Stress investigators. The completion of these Specific Aims will contribute to fundamental knowledge of the molecular and cellular mechanisms by which chronic alcohol drinking alters the ECM and potentially provide new cellular targets to reduce excessive alcohol consumption.
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