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Research Component 5/8: Effects of Adolescent Alcohol on Adult Brain Neurocircuitry.

 

Principal Investigator: Fulton T. Crews, Ph.D., University of North Carolina at Chapel Hill

Co-Investigator: Ryan P. Vetreno, Ph.D., University of North Carolina at Chapel Hill

 

Abstract

Our studies find adolescent intermittent ethanol (AIE) exposure increases adult risky decisions, disinhibition, behavioral inflexibility, and reduces sensitivity to adult ethanol challenge as well as increasing alcohol drinking, all risk factors for human AUD. Acetylcholine is reduced by AIE and plays a critical role in cortical activation and integration as well as maturation of adolescent neurocircuits that could impact ethanol sensitivity. Although AIE does not change adult ethanol elimination, it does reduce adult ethanol motor-sedative, anxiolytic, PFC immediate early gene (IEG) c-fos, and cortical functional connectivity magnetic resonance imaging (fcMRI) responses. We discovered AIE increases adult HMGB1 and its receptors (i.e., Toll-like receptor 4 [TLR4] and receptor for advanced glycation end products [RAGE]) on neurons, and these signals are linked to epigenetic silencing of cholinergic phenotype genes (i.e., ChAT, VAChT, TrkA) and loss of hippocampal neurogenesis, which are examples of AIE-altered neurocircuitry. Low response to ethanol is associated with AUD, and these changes are found in post-mortem human adult AUD brain. Basal forebrain cholinergic neurons integrate neurocircuits, and reductions of these neurons may contribute to AIE pathology. We hypothesize AIE HMGB1TLR4/RAGE epigenetic silencing of cholinergic neurons alters basal forebrain-corticalhippocampal neurocircuitry, leading to reduced ethanol sensitivity and increased alcohol drinking in adulthood. Reductions of adult ChAT+ neurons caused by AIE were initially interpreted as neurodegeneration; however, the surprising discovery that AIE reductions of ChAT+ neurons are reversible by exercise and indomethacin supports HMGB1→TLR4/RAGE→NFκB epigenetic silencing of ChAT within neurons persisting into adulthood. Reversal of neuroimmune ChAT silencing suggests AIE pathology is persistent, but not permanent. However, it is not known if AIE-mediated adolescent-like low adult ethanol sensitivity can be prevented or reversed. This proposal therefore has the following Aims. AIM 1. Test the hypothesis that AIE decreases adult ethanol sensitivity to brain fcMRI changes, c-fos expression, ethanol response behaviors (ERBs), and increases adult alcohol drinking. AIM 2 will extend studies testing the hypothesis that HMGB1→TLR4/RAGE→NFκB signaling reduces basal forebrain ChAT+ cholinergic neurons by reversible epigenetic mechanisms. AIM 3 will test the hypothesis that adolescent ChAT silencing mimics adult AIE low ethanol response and other pathology. AIM 4 will test the hypothesis that anti-inflammatory treatment known to reverse AIE-altered ChAT, neurogenesis, and epigenetic changes, will also recover AIE-altered c-fos, fcMRI, ethanol low behavioral response sensitivity, and adult ethanol drinking. Identifying molecular mechanisms and key neurocircuits regulating low response to alcohol will contribute to our understanding of adolescent brain development and the mechanisms determining low response to ethanol challenge, a known risk factor for AUD, as well as possibly identifying treatment targets.

 

 

Specific Aims

Our studies find adolescent intermittent ethanol (AIE) exposure increases adult risky decisions, disinhibition, behavioral inflexibility, and reduces sensitivity to adult ethanol challenge as well as increasing alcohol drinking, all risk factors for human AUD. Acetylcholine is reduced by AIE and plays a critical role in cortical activation and integration as well as maturation of adolescent neurocircuits that could impact ethanol sensitivity. Although AIE does not change adult ethanol elimination, it does reduce adult ethanol sensitivity to ethanol motor-sedative, anxiolytic, PFC immediate early gene (IEG) c-fos, and cortical functional connectivity magnetic resonance imaging (fcMRI) responses. We discovered AIE increases adult HMGB1 and its receptors (i.e., Toll-like receptor 4 [TLR4] and receptor for advanced glycation end products [RAGE]), and these signals are linked to epigenetic silencing of cholinergic phenotype genes (i.e., ChAT, VAChT, TrkA) and loss of hippocampal neurogenesis, which are examples of AIE-altered neurocircuitry. Low response to ethanol is associated with AUD, and these changes are found in post-mortem human adult AUD brain. Basal forebrain cholinergic neurons integrate neurocircuits, and reductions of these neurons may contribute to AIE pathology. We hypothesize AIE HMGB1TLR4/RAGE epigenetic silencing of cholinergic neurons disrupts adult brain functional connectivity, thereby altering basal forebraincortical-hippocampal neurocircuitry, leading to reduced ethanol sensitivity and increased alcohol drinking in adulthood.

 Emerging studies find neuroimmune signals across glia and neurons regulate synaptic plasticity during brain maturation, and NADIA studies have established AIE reduces ethanol sensitivity, increases ethanol drinking, and disrupts cognitive function in adulthood. Our discovery that AIE reductions of cholinergic neurons are reversible by exercise, indomethacin, and galantamine suggests neuroimmune-epigenetic mechanisms mediate neuronal ChAT silencing. Indeed, AIE increases epigenetic silencing markers at ChAT and TrkA gene promoter regions with corresponding reductions of ChAT mRNA and ChAT+ neurons. AIE increases HMGB1→TLR4/ RAGE→NFκB signaling in ChAT+ neurons and hippocampal neuroprogenitors, reducing adult hippocampal neurogenesis. We find exercise, anti-inflammatory drugs, and epigenetic modifiers prevent and/or reverse AIE-induced proinflammatory gene induction, reductions of ChAT and neurogenesis, and impaired cognitive flexibility. It is unknown if AIE-reduced adult ethanol sensitivity can be prevented or reversed. We expect AIE will alter neurocircuits that regulate adult ethanol sensitivity. We propose the following AIMs:

 AIM 1. Test the hypothesis that AIE decreases adult ethanol sensitivity to brain fcMRI changes, cfos expression, ethanol response behaviors (ERBs), and increases adult alcohol drinking. AIE reduces adult acute ethanol challenge enhancement of functional connectivity (fcMRI) and PFC c-fos responses. AIE also reduces adult ethanol sensitivity (tilting plane/rotorod) and increases drinking. Adult sensitivity to acute ethanol challenge in males and females will be determined using a longitudinal design to link fcMRI connectivity to ERB (e.g., intoxication rating, hypothermia, tilting plane), alcohol drinking preference, and PFC c-fos expression. We expect that AIE brain regional fcMRI and c-fos responses to acute ethanol will identify altered adult neurocircuits linked to AIE-reduced ethanol sensitivity and increased drinking in adulthood.

 AIM 2. Test the hypothesis that HMGB1TLR4/RAGENFκB signaling reduces basal forebrain ChAT+ cholinergic neurons by epigenetic silencing. We find AIE increases HMGB1→TLR4/RAGE→NFκB in adult basal forebrain and cholinergic neurons. Exercise, indomethacin, and histone deacetylase inhibitors prevent and/or reverse adult AIE pathology. We propose to determine if neuron-specific CRISPR/dCas9-KRAB silencing of TLR4 and RAGE or specific pharmacological G9a inhibitors of H3K9 dimethylation prevent AIE pathology. We will also determine cholinergic phenotype (e.g., ChAT) and neuroimmune (e.g., TLR4, RAGE) gene chromatin accessibility using ATAC-Seq in the forebrain in collaboration with the Epigenetic/Molecular Scientific Core of the NADIA. We expect to identify AIE HMGB1→TLR4/RAGE→NFκB linked to ChAT silencing and/or enhancer responses of TLR4/RAGE that contribute to persistent adult pathology.

 AIM 3. Test the hypothesis that ChAT silencing mimics AIE pathology. Previous studies find ChAT loss is associated with reduced cognition and cortical function; however, it is unknown how this affects acute ethanol sensitivity and reduced neurogenesis. We will determine adult ethanol challenge responses (ERB, cfos,

fcMRI) and drinking following CRISPR/dCas9-KRAB ChAT H3K27me3 silencing. Adolescent reductions in ChAT+ neurons are expected to reduce hippocampal neurogenesis, mimic AIE-reduce sensitivity to ethanol challenge, and increase ethanol drinking in adulthood.

AIM 4. Test the hypothesis that anti-inflammatory treatment persistently reverses AIE-altered ChAT, neurogenesis, and epigenetic changes, and recovers AIE-altered c-fos, fcMRI ethanol challenge sensitivity, and adult ethanol drinking. Anti-inflammatory indomethacin treatment, known to acutely reverse AIE adult ChAT-neurogenesis pathology, will be assessed for persistent reversal of AIE-induced reductions of ChAT+ cholinergic neurons and hippocampal neurogenesis, increased neuroimmune signaling, and extended to reversal of adult ethanol challenge fcMRI, ERB, drinking, and c-fos. We expect that lasting reversal of AIE-induced neuroimmune-ChAT pathology will reverse AIE-induced adult ethanol low response sensitivity and increased drinking.

Identifying molecular mechanisms and key neurocircuits regulating low response to alcohol will contribute to our understanding of adolescent development and the low-response risk factor for AUD.