HMGB1 Neuroimmune Signaling and REST-G9a Gene Repression Contribute to Ethanol-induced Reversible Suppression of the Cholinergic Neuron Phenotype

Adolescent binge drinking increases Toll-like receptor 4 (TLR4), receptor for advanced glycation end products (RAGE), the endogenous TLR4/RAGE agonist high-mobility group box 1 (HMGB1), and proinflammatory neuroimmune signaling in the adult basal forebrain in association with persistent reductions of basal forebrain cholinergic neurons (BFCNs). In vivo preclinical adolescent intermittent ethanol (AIE) studies find anti-inflammatory interventions post-AIE reverse HMGB1-TLR4/RAGE neuroimmune signaling and loss of BFCNs in adulthood, suggesting proinflammatory signaling causes epigenetic repression of the cholinergic neuron phenotype. Reversible loss of BFCN phenotype in vivo is linked to increased repressive histone 3 lysine 9 dimethylation (H3K9me2) occupancy at cholinergic gene promoters, and HMGB1-TLR4/RAGE proinflammatory signaling is linked to epigenetic repression of the cholinergic phenotype. Using an ex vivo basal forebrain slice culture (FSC) model, we report EtOH recapitulates the in vivo AIE-induced loss of ChAT+IR BFCNs, somal shrinkage of the remaining ChAT+ neurons, and reduction of BFCN phenotype genes. Targeted inhibition of EtOH-induced proinflammatory HMGB1 blocked ChAT+IR loss while disulfide HMBG1-TLR4 and fully reduced HMGB1-RAGE signaling decreased ChAT+IR BFCNs. EtOH increased expression of the transcriptional repressor RE1-silencing transcription factor (REST) and the H3K9 methyltransferase G9a that was accompanied by increased repressive H3K9me2 and REST occupancy at promoter regions of the BFCN phenotype genes Chat and Trka as well as the lineage transcription factor Lhx8. REST expression was similarly increased in the post-mortem human basal forebrain of individuals with alcohol use disorder, which is negatively correlated with ChAT expression. Administration of REST siRNA and the G9a inhibitor UNC0642 blocked and reversed the EtOH-induced loss of ChAT+IR BFCNs, directly linking REST-G9a transcriptional repression to suppression of the cholinergic neuron phenotype. These data suggest that EtOH induces a novel neuroplastic process involving neuroimmune signaling and transcriptional epigenetic gene repression resulting in the reversible suppression of the cholinergic neuron phenotype.