Abstract
The NADIA consortium is dedicated to identifying the mechanisms underlying the long-term effects of adolescent intermittent ethanol exposure (AIE) on brain and behavior and exploring approaches to prevent or reverse them. Component 1 has focused on the enduring effects of AIE on hippocampal structure and function and their behavioral sequelae. Based on our findings, our overarching hypothesis AIE causes an enduring but reversible compromise of hippocampal synaptic function driven by interacting epigenetic, neuroinflammatory, and glial mechanisms. During the last NADIA funding cycle we found that AIE produces a pro-inflammatory milieu in the hippocampal formation, alterations of dendritic spine density and morphology, altered histone acetylation and methylation, diminished astrocyte-synaptic proximity, decreases in neurogenesis, and increases in the mobilization cell death molecular machinery. Through NADIA collaborations, we further identified epigenetic changes that suggest specific molecular mechanisms underlying those effects.
Importantly, we have also shown that many of those effects are reversible in adulthood with pharmacological agents in current clinical use. Our recent findings indicate that AIE results in a propensity toward anxiety-like behavior after environmental challenge, thus suggesting the need to expand our assessments to the ventral hippocampus. This has obvious translational implications because anxiety and stress reactivity are strong predictors of AUD development and relapse, and suggests that some of the AIE-induced changes we have observed in the dorsal hippocampus may be similar in the ventral region. Against this backdrop we propose three Specific Aims that will more fully characterize the AIE-induced phenotypes we have identified, identify mechanisms underlying them, and explore treatments to reverse them.
Aim 1 will address the role of histone H3K27 acetylation, and other possible molecular mechanisms, in AIE-induced dendritic spine changes, and further characterize our previously-reported reversal of AIE-induced spine changes by donepezil. Aim 2 will follow-up our recent findings to identify mechanisms underlying AIE-induced induction of hippocampal inflammatory markers and corresponding reductions of neurogenesis and increased cell death cascade markers. Aim 3 is founded on preliminary data indicating that AIE causes a decrease in astrocyte-synaptic proximity that could be of functional synaptic significance. We will confirm and expand that finding, and assess its pharmacological reversibility in adulthood. Our goal is to explore possible interlacing mechanisms underlying AIE effects on synaptic, inflammatory, and glial function. These proposed studies are all based on distinct sets of preliminary data, will address specific epigenetic and molecular mechanisms underlying AIE effects on hippocampal structure and function, and will expand our understanding of translationally-relevant AIE-induced phenotypes.
Specific Aims
This Component is focused on the enduring effects of adolescent intermittent ethanol (AIE) exposure on hippocampal structure and function and the behavioral sequelae of those effects. Our goals are 1) to characterize those AIE-induced phenotypes, 2) to identify the mechanisms underlying them, and 3) to contribute to the development of treatments to reverse or prevent them. We have recently found that AIE alters hippocampal dendritic spine density and morphology in parallel with altered histone acetylation and methylation; produces a pro-inflammatory milieu in the hippocampal formation in parallel with decreased neurogenesis and mobilization of cell death machinery; and increases astrocyte reactivity while reducing astrocyte-synaptic proximity. We have also shown that many of those effects are normalized in adulthood with medications in current clinical use, donepezil (Aricept) or gabapentin (Neurontin). Additional preliminary data indicate a propensity toward anxiety-like behavior in response to environmental challenge after AIE, suggesting changes in the ventral hippocampus (vHPC) that may parallel those we have observed in the dorsal hippocampus (dHPC), and providing a new direction with strong translational relevance to alcohol use disorder. Thus, our central hypothesis is that AIE causes an enduring but reversible compromise of hippocampal synaptic function driven by interacting epigenetic, neuroinflammatory, and astroglial mechanisms – resulting in disruption of excitatory/inhibitory balance in hippocampal circuits that drive behavioral change.
Specific Aim 1: To assess the molecular mechanisms underlying AIE-induced alterations of dendritic spine density and morphology in the hippocampal formation. We have reported AIE-induced acetylation of histone H3K27 at the CREB binding site on the Fmr1 gene in the dentate gyrus, correlated with increased Fmr1 mRNA expression and reduced dendritic spine density. We hypothesize that suppressing H3K27ac on the Fmr1 gene after AIE will decrease aberrant Fmr1 expression and reverse AIE effects on dendritic spine morphology and density. Because H3K27ac is likely not the only synaptically-related epigenetic target of AIE, we will collaborate with the Epigenetic Core to use ATAC-seq to identify epigenetically dysregulated genes in the hippocampus, and CRISPR/dcas9-KRAB to generate lentivirus for hippocampal microinjection after AIE to suppress H3K27ac, and any ATAC-seq-identified epigenetically regulated additional gene targets to assess their effects on AIE-induced dendritic spine changes. We will also assess the duration, sub-region specificity, and sex specificity of AIE-induced spine changes and their reversal.
Specific Aim 2: To identify mechanisms underlying AIE-induced induction of hippocampal inflammatory markers and corresponding alterations of neurogenesis and cell death cascades. We have reported that AIE promotes a proinflammatory milieu and cell death cascades in the dentate gyrus, while inhibiting neurogenesis. We will address three possible mechanisms underlying these effects: Complement protein activation, ephrin dysregulation, and epigenetic changes. We will use Western blot and qRT-PCR to assess AIE effects on complement protein expression and ephrin A3/A4, which regulate dendritic spine development and plasticity. We have shown that AIE both reduces EphA4 expression (Fig. 7) and increases RAGE expression (Fig. 2). Thus, in collaboration with the Epigenetics Core we will also test the hypothesis that knockdown of RAGE expression with CRISPR/dCas9-KRAB will reverse the pro-inflammatory and antineurogenic effects of AIE. Additional experiments will further characterize the duration, hippocampal subregion, and sex-specificity of our previously reported AIE effects on proinflammatory signaling, neurogenic impairment, or neurodegeneration.
Specific Aim 3: To test the hypothesis that AIE alters astrocyte morphology and proximity to neuronal synaptic markers in the hippocampal formation and assess the reversibility of that effect in adulthood. Astrocytes regulate synaptogenesis, synaptic function, and neuroimmune responses. Thus, ourprevious finding of their enduring reactivity in the hippocampus after AIE suggests involvement in both the synaptic (Aim 1) and neuroinflammatory (Aim 2) effects of AIE in adulthood. Preliminary data indicate that AIE diminishes astrocyte proximity to neuronal synaptic sites in the dHPC. We will extend those findings to the vHPC, assess their sex-specificity, and determine if their reversal by gabapentin endures beyond the brief timeframe we have previously assessed. With Component 6, we will also measure AIE-induced alteration of direct astrocyte contact with dendritic spines, and possible reversal by gabapentin, using a newly developed and validated approach involving viral labelling of neurons of astrocytes, immuno-amplification, superresolution confocal microscopy, and three-dimensional reconstruction.
Together, these Aims explore three distinct, but intersecting mechanisms based on our previous and preliminary studies. The hypothesized epigenetic mediation of AIE effects on dendritic spines (Aim 1) and neuroinflammation (Aim 2), and the involvement of astrocyte function (Aim 3) in those processes suggest the convergence of AIE effects across cell types resulting in a broad compromise of hippocampal function. Using a reversal-based strategy for testing specific hypotheses, the proposed experiments will yield information that is of both mechanistic and translational significance, while other experiments will provide a fine-grained characterization of AIE phenotypes that will set the stage for future studies.