Alcohol dependence can cause depression by affecting hippocampal functions. Animal models of depression display dendritic atrophy and decreased spine density in hippocampal CA3 pyramidal neurons. Astrocytes have been shown to play key roles in regulating structural and functional plasticity in neurons. Epigenetic regulation of gene expression can be modulated by histone acetylation and DNA methylation through the activity histone deacetylases (HDACs) and DNA methyltrasferases (DNMTs) respectively. We hypothesize that chronic ethanol and /or withdrawal induce epigenetic changes in astrocytes, affect the release of ECM proteins, inhibit neuronal plasticity in the hippocampus, and cause depressive-like behavior, effects that may be rescued by HDAC inhibition. We will carry out the following specific aims:
Aim 1) : Epigenetic regulation of astrocyte ECM proteins. We will study the effect of chronic ethanol, ethanol withdrawal, and the HDAC inhibitor SAHA on: 1.1) HDAC and DNMT isoforms' transcription and expression in astrocyte in vitro. 1.2) HDAC and DNMT isoforms' expression in vivo. 1.3) Laminin-1, fibronectin, PAI-1, and tPA protein and mRNA levels, promoter DNA methylation, promoter histone 3 lysine 9 (H3K9) acetylation in vivo.
Aim 2) : Mechanisms of astrocyte ECM-induced neuritogenesis and their inhibition by ethanol: integrins and small GTPases. ECM-mediated neuritogenesis depends on the binding of neuronal integrins to ECM proteins, which triggers the activation of Rho GTPases and the rearrangement of the cytoskeleton in neurons. We will explore: 2.1) the integrin subunits responsible for the neuritogenic effects of astrocytes; 2.2) the effect of ethanol/withdrawal in astrocytes on Rho GTPase activation in neurons; 2.3) the effect of the HDAC inhibitor SAHA and of astrocyte transfection with HDAC2 siRNA on fibronectin, laminin and PAI-1 levels in astrocytes and small GTPases activation and neuritogenesis in neurons.
Aim 3) : Effects of in vivo chronic ethanol, ethanol withdrawal, SAHA treatments, and HDAC2 siRNA infusions in the hippocampus CAS during ethanol withdrawal on depressive-like behavior and on hippocampal dendritic arborization and spine density.
Aim 3 will also test whether fibronectin siRNA infusion in the hippocampus CAS causes depression-like behavior and decreases dendritic arborization and spine density in pyramidal neurons.
Aim 4) : Genome-wide changes in mRNA levels by RNA-sequencing and histone acetylation (HSK9) patterns by ChlP-sequencing in the hippocampus of rats chronically exposed to ethanol and after ethanol withdrawal.
The proposed investigation of non-genetic (epigenetic) changes caused by alcohol to the DNA structure of non-neuronal brain cells, astrocytes, which may affect neuronal plasticity and may lead to the understanding of a novel mechanism of alcohol withdrawal-induced depressive behavior and to the identification of possible treatments to ameliorate ethanol-induced depression.
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