7/8 NADIA U01 Recovery of Adolescent Alcohol Disruption of Basal Forebrain-Cortical Projection Circuits

Research Component 7/8: Recovery of Adolescent Alcohol Disruption of Basal Forebrain-Cortical Projection Circuits

Principal Investigator: Lisa Savage, Ph.D., Binghamton Duke University

Abstract

Heavy alcohol consumption during adolescence is associated with persistent changes in brain structure, connectivity, and adult cortical-mediated cognitive function. Critical pathological changes consistently observed in rodent models of adolescent binge ethanol exposure (Adolescent Intermittent Ethanol; AIE) are a reduction of cortical nerve growth factor (NGF), suppression of the cholinergic phenotypes, and a long-term decrease in the number of functional cholinergic basal forebrain neurons (CBFNs). The cholinergic projection neurons within the nucleus basalis magnocellularis complex (NbM) provide acetylcholine (ACh) to the frontal cortex, including the medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC). Behaviorally-stimulated efflux of ACh in both the mPFC and OFC are blunted following AIE, and there is a loss of cognitive and behavioral flexibility. Our recent data demonstrate that AIE-induced loss of CBFNs is due to epigenetic silencing of cholinergic phenotypic markers, but exercise, potentially through a NGF mechanism, can reverse these epigenetic changes and rescue cholinergic neurons. This demonstrates that AIE is initially marked by a reduction of the expression of cholinergic neuronal phenotypes, rather than neuronal cell death. Thus, mechanisms should be explored to recover CFBN pathology following AIE. Our overarching hypothesis is that AIE-induced reductions of NGF in cortical projection sites leads to epigenetic silencing of cholinergic phenotypes with the NbM, retraction and/or dysfunctional re-innervation of the cortical-NbM cholinergic connectome, causing blunting of cortical cholinergic tone and cognitive dysfunction. However, appropriately timed NGF-based therapies (exercise, NGF gene therapy, CRISPR/dCas9-P300 editing) following AIE will reinvigorate cholinergic forebrain circuitry through the revitalization of cholinergic genes, which will rescue cortical ACh and recover AIE-induced impairments in cortical-dependent behaviors. Our goals are to map and rescue cholinergic forebrain and cortical circuit pathology seen following AIE (Aim 1), restore cognitive functioning by exercise, NGF-gene therapy, or selective chemogenetic stimulation of cholinergic neurons (Aim 2), and identify the central role of NGF deficits, through CRISPR/dCas9 editing, in AIE-induced epigenetic silencing of cholinergic phenotype genes (Aim 3). Mounting evidence supports the neuroprotective effects of NGF as a course to rescue vulnerable CBFNs undergoing neurodegeneration, and we have strong evidence that AIE-induced cholinergic pathology can be reversed. Understanding the mechanisms of cholinergic neuronal recovery will aid in the development of more effective therapies to treat cognitive dysfunction associated with alcohol-related brain damage and other neurodegenerative disorders.

 Specific Aims

A key feature of alcohol-related brain damage associated with adolescent intermittent ethanol (AIE) exposure is a decrease of functional cholinergic neurons in the basal forebrain as well as a reduction of their key trophic mediator, nerve growth factor (NGF), in projection sites. In early adolescence, cortical NGF and cortical cholinergic innervation from the basalis magnocellularis complex (NbM) peak. The NbM is the primary source of acetylcholine (ACh) to the prefrontal cortex (PFC) (i.e., medial prefrontal cortex [mPFC] and orbitofrontal cortex [OFC]), and coordinates multiple cognition functions. AIE, which models human adolescent binge drinking, causes persistent reductions of both cortical NGF and NbM cholinergic phenotype neuronal markers (e.g., ChAT, TrkA, Nestin, p75NTR). AIE reductions of cortical NGF likely contribute to decreased expression of NbM cholinergic phenotype markers as NGF is require for maintenance of cholinergic neurons. Adult AIE-treated rats display reduced behaviorally-evoked ACh efflux in the mPFC and OFC, which is paralleled by impaired frontocortical-dependent behaviors (cognitive and behavioral flexibility). Thus, we propose to test the overarching hypothesis that AIE-induced reductions of NGF launches epigenetic silencing of the cholinergic phenotype leading to dysfunctional innervation of the PFC that produces persistent frontocortical behavioral dysfunction. Recent data from our research team demonstrate that AIE leads to epigenetic silencing of several cholinergic phenotypic markers, but post-AIE exercise therapy can correct the epigenetic changes and restore cholinergic neurons. Our goals are to map and rescue cholinergic PFC circuit pathology seen following AIE (Aim 1), restore NbM cholinergic neurons and cognitive functioning by exercise, NGF-gene therapy, or selective chemogenetic stimulation (Aim 2), and identify the role of cortical NGF deficits in AIE-induced epigenetic silencing of cholinergic phenotype genes (Aim 3).

 Aim 1. Map and quantify cholinergic projections, including cholinergic cell subtypes, within the NbM-mPFC and NbM-OFC circuits to identify AIE-induced cortical circuit dysfunction and use NGFgene therapy to restore NbM→FC innervation. Neurocircuit dissection will be done in the ChAT::Cre rat model with rAVV2-retro-DIO-tdTomato into the PFC and OFC. Quantification of changes in connectivity will be determined by counting NbM neurons expressing tdTomato and/or GFP as well as analysis of cholinergic fiber density patterns within the mPFC and OFC. We will counterstain with ChAT, TrkA, p75 NTR, and Nestin to determine phenotypes of the altered cholinergic neurons, and reveal the profile of cholinergic cell diversity underlying the organization in these specific networks. We hypothesize that AIE will disrupt NbM→mPFC/OFC cholinergic projections, but that restoration of NGF will correct forebrain cholinergic innervation of the PFC.

 Aim 2. Test whether AIE-induced deficits in ACh efflux, and cognitive and behavioral flexibility will be reversed by exercise, NGF-gene therapy, and selective excitation of the NbM-PFC and the NbMOFC cholinergic projections. Exercise can recover the cholinergic phenotype, and there is evidence that NGF is critical for this process. NGF-gene therapy rescues cholinergic neurons in aged rats and monkeys, and is in clinical trial for Alzheimer’s disease. We hypothesize that both exercise and NGF-gene therapy will rescue NbM cholinergic neuronal phenotype silencing after AIE, and restore cognitive functions dependent on the mPFC (working memory, attentional set-shifting) and OFC (reversal learning and delayed discounting). We also predict that stimulation of spared NbM cholinergic neurons will recover AIE-induced deficits in ACh efflux and behavior. Using the ChAT::Cre rat model coupled with LS1L-retro-DREADDs, we will selectively activate cholinergic forebrain projection circuits that modulate cognitive (mPFC) and behavioral flexibility (OFC) to determine their unique role of cholinergic functioning on AIE-induced cognitive dysfunction.

 Aim 3. Determine if reductions of NGF in the cortical NbM projection sites during adolescence promotes the loss of ChAT+ neurons and drives epigenetic silencing of cholinergic phenotype genes. NbM ChAT+ neurons are dependent on cortical NGF for maintenance of cholinergic phenotypes, and AIE reduces NGF in the PFC. We discovered the AIE reductions of forebrain ChAT+ neurons is due to reversible epigenetic silencing of cholinergic phenotype genes. We hypothesize that NGF reductions in the adolescent PFC leads to suppression of NbM ChAT+ neurons through epigenetic silencing of cholinergic phenotype genes. ATACseq will determine NGF and NGF signaling molecule gene chromatin accessibility in the OFC of CON- and AIE-treated subjects. We expect NGF CRISPR-dCas9 KRAB knockdown of NGF in the adolescent OFC to mimic AIE reductions of NbM ChAT+ neurons and H3K9me2 silencing of cholinergic phenotype genes (TrkA, ChAT, Nestin). Exercise and NGF CRISPR-dCas9-P300 will be used post-AIE to enhance PFC NGF expression to restore AIE-induced reductions of NbM ChAT+ neurons and reverse silencing of cholinergic phenotype genes revealing the critical role of NGF in recovering the cholinergic phenotype. We will utilize innovative viral tracing techniques, chemogenetics, translational NGF-gene therapy, and CRISPR editing tools to identify NGF involvement in AIE-induced frontocortical structural and functional deficits as well as epigenetic gene silencing of the cholinergic phenotype. Our studies complement the Crews component focused on neuroimmune-epigenetics-neuroimaging, the Chandler component focused on PFC circuitry, and the Pandey component that focuses on epigenetic regulation of anxiety and drinking.