Adolescent binge alcohol exposure accelerates Alzheimer’s disease-associated basal forebrain neuropathology through proinflammatory HMGB1 signaling

AUTHORS

Rachael P. Fisher, Lindsay Matheny, Sarrah Ankeny, Liya Qin, Leon G. Coleman Jr., Ryan P. Vetreno

ABSTRACT

Human studies suggest that heavy alcohol use may be an etiological factor contributing to the development of Alzheimer’s disease (AD) neuropathology. Both alcohol use disorder (AUD) and AD share common underlying neuropathology, including proinflammatory high-mobility group box 1 (HMGB1)-mediated neuroimmune signaling and basal forebrain cholinergic neuron degeneration. Adolescent onset of binge drinking represents a significant risk factor for later development of an AUD, and accumulating evidence suggests that adolescent initiation of heavy alcohol use induces HMGB1 signaling and causes degeneration of the basal forebrain cholinergic system that persists into adulthood. However, it is unknown whether adolescent binge drinking confers increased risk for later development of AD-associated neuropathology through persistent induction of proinflammatory HMGB1 neuroimmune signaling. To investigate this question, we first (Experiment 1) assessed AD-associated neuropathology in the post-mortem human basal forebrain of individuals with AUD and an adolescent age of drinking onset relative to age-matched moderate drinking controls (CONs). In Experiment 2, we treated non-transgenic and 5xFAD male and female mice, which overexpress both mutant human APP and PS1, with adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g. 2-days on/2-days off; postnatal day [P]30 – P55), and assessed AD-associated neuropathology in the adult (P100) basal forebrain. In Experiment 3, 5xFAD female mice received AIE treatment followed by glycyrrhizic acid (150 mg/L), an HMGB1 inhibitor, in drinking water from P56 to P100, and basal forebrain tissue was collected on P100 for assessment of AD-associated neuropathology. In the post-mortem human AUD basal forebrain (Experiment 1), we report upregulation of Hmgb1 and the HMGB1 receptors Rage and Tlr4 as well as microglial activation and increased intraneuronal Aβ1–42 accumulation in association with reduced cholinergic neuron marker expression (ChAT). In the 5xFAD mouse model (Experiment 2), AIE accelerated AD-associated induction of Hmgb1 proinflammatory neuroimmune genes, microglial activation, and reductions of ChAT+ basal forebrain cholinergic neurons in the adult female, but not male, basal forebrain. In Experiment 3, post-AIE treatment with glycyrrhizic acid rescued the AIE-induced acceleration of AD-associated increases in proinflammatory HMGB1 neuroimmune signaling, microglial activation, and persistent reductions of basal forebrain cholinergic neurons in adult 5xFAD female mice. Together, these findings suggest that adolescent binge ethanol exposure may represent an underappreciated etiological factor contributing to onset of AD-associated neuropathology in adulthood through HMGB1- mediated neuroimmune signaling.

Sympathetic Overdrive and Unrestrained Adipose Lipolysis Drive Alcohol-Induced Hepatic Steatosis in Rodents

AUTHORS

Chunxue Zhou 1, Henry H. Ruiz, Li Ling, Giulia Maurizi, Kenichi Sakamoto, Claudia Liberini, Ling Wang, Adrien Stanley, Hale E. Egritag, Sofia M. Sanz, Claudia Lindtner, Mary A. Butera, Christoph Buettner

ABSTRACT

Objective

Hepatic steatosis is a key initiating event in the pathogenesis of alcohol-associated liver disease (ALD), the most detrimental organ damage resulting from alcohol use disorder. However, the mechanisms by which alcohol induces steatosis remain incompletely understood. We have previously found that alcohol binging impairs brain insulin action, resulting in increased adipose tissue lipolysis by unrestraining sympathetic nervous system (SNS) outflow. Here, we examined whether an impaired brain-SNS-adipose tissue axis drives hepatic steatosis through unrestrained adipose tissue lipolysis and increased lipid flux to the liver.

Methods

We examined the role of lipolysis, and the brain-SNS-adipose tissue axis and stress in alcohol induced hepatic triglyceride accumulation in a series of rodent models: pharmacological inhibition of the negative regulator of insulin signaling protein-tyrosine phosphatase 1β (PTP1b) in the rat brain, tyrosine hydroxylase (TH) knockout mice as a pharmacogenetic model of sympathectomy, adipocyte specific adipose triglyceride lipase (ATGL) knockout mice, wildtype (WT) mice treated with β3 adrenergic agonist or undergoing restraint stress.

Results

Intracerebral administration of a PTP1b inhibitor, inhibition of adipose tissue lipolysis and reduction of sympathetic outflow ameliorated alcohol induced steatosis. Conversely, induction of adipose tissue lipolysis through β3 adrenergic agonism or by restraint stress worsened alcohol induced steatosis.

Conclusions

Brain insulin resistance through upregulation of PTP1b, increased sympathetic activity, and unrestrained adipose tissue lipolysis are key drivers of alcoholic steatosis. Targeting these drivers of steatosis may provide effective therapeutic strategies to ameliorate ALD.

HMGB1 neuroimmune signaling and REST-G9a gene repression contribute to ethanol-induced reversible suppression of the cholinergic neuron phenotype

AUTHORS

Fulton T. Crews, Rachael P. Fisher, Liya Qin & Ryan P. Vetreno

ABSTRACT

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.

NADPH oxidase and endoplasmic reticulum stress is associated with neuronal degeneration in orbitofrontal cortex of individuals with alcohol use disorder

AUTHORS

Liya Qin, Ryan P. Vetreno, Fulton T. Crews

ABSTRACT

Many disorders of the central nervous system (CNS), including alcohol use disorder (AUD), are associated with induction of proinflammatory neuroimmune signalling and neurodegeneration. In previous studies, we found increased expression of Toll-like receptors (TLRs), activated NF-κB p65 (RELA), and other proinflammatory signalling molecules. Proinflammatory NADPH oxidases generate reactive oxygen species, which are linked to neurodegeneration. We tested the hypothesis that AUD increased RELA activation increases NADPH oxidase-oxidative stress and endoplasmic reticulum (ER) stress cell death cascades in association with neuronal cell death in the human orbitofrontal cortex (OFC). In the AUD OFC, we report mRNA induction of several NADPH oxidases, the dual oxidase DUOX2, and the oxidative stress lipid peroxidation marker 4-HNE and the DNA oxidation marker 8-OHdG that correlate with RELA, a marker of proinflammatory NF-κB activation. This was accompanied by increased expression of the ER stress-associated regulator protein glucose-regulated protein 78 (GRP78), transmembrane sensors activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and inositol-requiring kinase/endonuclease 1 (pIRE1), and the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Expression of NADPH oxidase-oxidative stress markers correlate with ER stress-associated molecules. Induction of oxidative stress and ER stress signalling pathways correlate with expression of cell death-associated caspases and neuronal cell loss. These data support the hypothesis that proinflammatory RELA-mediated induction of NADPH oxidase-oxidative stress and ER stress-associated signalling cascades is associated with neuronal cell death in the post-mortem human OFC of individuals with AUD.

Dose-Related Reduction in Hippocampal Neuronal Populations in Fetal Alcohol Exposed Vervet Monkeys

AUTHORS

Mark W. Burke, Hocine Slimani, Maurice Ptito, Frank R. Ervin, Roberta M. Palmour

ABSTRACT

Fetal alcohol spectrum disorder (FASD) is a chronic debilitating condition resulting in behavioral and intellectual impairments and is considered the most prevalent form of preventable mental retardation in the industrialized world. We previously reported that 2-year-old offspring of vervet monkey (Chlorocebus sabeus) dams drinking, on average, 2.3 ± 0.49 g ethanol per Kg maternal body weight 4 days per week during the last third of pregnancy had significantly lower numbers of CA1 (−51.6%), CA2 (−51.2%) and CA3 (−42.8%) hippocampal neurons, as compared to age-matched sucrose controls. Fetal alcohol-exposed (FAE) offspring also showed significantly lower volumes for these structures at 2 years of age. In the present study, we examined these same parameters in 12 FAE offspring with a similar average but a larger range of ethanol exposures (1.01–2.98 g/Kg/day; total ethanol exposure 24–158 g/Kg). Design-based stereology was performed on cresyl violet-stained and doublecortin (DCX)-immunostained sections of the hippocampus. We report here significant neuronal deficits in the hippocampus with a significant negative correlation between daily dose and neuronal population in CA1 (r2 = 0.486), CA2 (r2 = 0.492), and CA3 (r2 = 0.469). There were also significant correlations between DCX population in the dentate gyrus and daily dose (r2 = 0.560). Both correlations were consistent with linear dose-response models. This study illustrates that neuroanatomical sequelae of fetal ethanol exposure are dose-responsive and suggests that there may be a threshold for this effect.

Impact of adolescent intermittent ethanol exposure on interneurons and their surrounding perineuronal nets in adulthood

AUTHORS

Carol A. Dannenhoffer, Alexander Gómez-A, Victoria A. Macht, Rayyanoor Jawad, E. Blake Sutherland, Ryan P. Vetreno, Fulton T. Crews, Charlotte A. Boettiger, Donita L. Robinson

ABSTRACT

Background Binge alcohol exposure during adolescence results in long-lasting alterations in brain and behavior. For example, adolescent intermittent ethanol (AIE) exposure in rodents results in long-term loss of functional connectivity among prefrontal cortex (PFC) and striatal regions as well as a variety of neurochemical, molecular, and epigenetic alterations. Interneurons in the PFC and striatum play critical roles in behavioral flexibility and functional connectivity. For example, parvalbumin (PV) interneurons are known to contribute to neural synchrony, and cholinergic interneurons contribute to strategy selection. Furthermore, extracellular perineuronal nets (PNNs) surround some interneurons, particularly PV+ interneurons, to further regulate cellular plasticity. The effect of AIE exposure on expression of these markers within the PFC is not well understood.

Methods The present study tested the hypothesis that AIE exposure reduces expression of PV+ and ChAT+ interneurons in the adult PFC and striatum and increases related expression of PNNs (marked by binding of Wisteria Floribunda agglutinin lectin; WFA) in adulthood. Male rats were exposed to AIE (5 g/kg/day, 2-days-on/2-days-off, i.g., P25-P54) or water (CON), and brain tissue was harvested in adulthood (> P80). Immunohistochemistry and co-immunofluorescence were used to assess expression of ChAT, PV, and WFA labeling within the adult PFC and striatum following AIE exposure.

Results ChAT and PV interneuron numbers in the striatum and PFC were unchanged after AIE exposure. However, WFA labeling in the PFC of AIE-exposed rats was increased compared to CON rats. Moreover, significantly more PV neurons were surrounded by WFA labeling in AIE-exposed subjects relative to controls in both PFC subregions assessed: the orbitofrontal cortex (CON = 34%; AIE = 40%) and the medial PFC (CON = 10%; AIE = 14%).

Conclusions These findings indicate that while PV interneuron expression in the adult PFC and striatum is unaltered following AIE exposure, PNNs surrounding these neurons (indicated by extracellular WFA binding) are increased. This increase in PNNs may restrict plasticity of the ensheathed neurons, thus contributing to impaired microcircuitry in frontostriatal connectivity and related behavioral impairments.