Mechanisms of neuroimmune gene induction in alcoholism

RationaleAlcoholism is a primary, chronic relapsing disease of brain reward, motivation, memory, and related circuitry. It is characterized by an individual’s continued drinking despite negative consequences related to alcohol use, which is exemplified by alcohol use leading to clinically significant impairment or distress. Chronic alcohol consumption increases the expression of innate immune signaling molecules (ISMs) in the brain that alter cognitive processes and promote alcohol drinking.ObjectivesUnraveling the mechanisms of alcohol-induced neuroimmune gene induction is complicated by positive loops of multiple cytokines and other signaling molecules that converge on nuclear factor kappa-light-chain-enhancer of activated B cells and activator protein-1 leading to induction of additional neuroimmune signaling molecules that amplify and expand the expression of ISMs.ResultsStudies from our laboratory employing reverse transcription polymerase chain reaction (RT-PCR) to assess mRNA, immunohistochemistry and Western blot analysis to assess protein expression, and others suggest that ethanol increases brain neuroimmune gene and protein expression through two distinct mechanisms involving (1) systemic induction of innate immune molecules that are transported from blood to the brain and (2) the direct release of high-mobility group box 1 (HMGB1) from neurons in the brain. Released HMGB1 signals through multiple receptors, particularly Toll-like receptor (TLR) 4, that potentiate cytokine receptor responses leading to a hyperexcitable state that disrupts neuronal networks and increases excitotoxic neuronal death. Innate immune gene activation in brain is persistent, consistent with the chronic relapsing disease that is alcoholism. Expression of HMGB1, TLRs, and other ISMs is increased several-fold in the human orbital frontal cortex, and expression of these molecules is highly correlated with each other as well as lifetime alcohol consumption and age of drinking onset.ConclusionsThe persistent and cumulative nature of alcohol on HMGB1 and TLR gene induction support their involvement in alcohol-induced long-term changes in brain function and neurodegeneration

Toll-like receptor signaling and stages of addiction

Athina Markou and her colleagues discovered persistent changes in adult behavior following adolescent exposure to ethanol or nicotine consistent with increased risk for developing addiction. Building on Dr. Markou's important work and that of others in the field, researchers at the Bowles Center for Alcohol Studies have found that persistent changes in behavior following adolescent stress or alcohol exposure may be linked to induction of immune signaling in brain. This study aims to illuminate the critical interrelationship of the innate immune system (e.g., toll-like receptors [TLRs], high-mobility group box 1 [HMGB1]) in the neurobiology of addiction. This study reviews the relevant research regarding the relationship between the innate immune system and addiction. Emerging evidence indicates that TLRs in brain, particularly those on microglia, respond to endogenous innate immune agonists such as HMGB1 and microRNAs (miRNAs). Multiple TLRs, HMGB1, and miRNAs are induced in the brain by stress, alcohol, and other drugs of abuse and are increased in the postmortem human alcoholic brain. Enhanced TLR-innate immune signaling in brain leads to epigenetic modifications, alterations in synaptic plasticity, and loss of neuronal cell populations, which contribute to cognitive and emotive dysfunctions. Addiction involves progressive stages of drug binges and intoxication, withdrawal-negative affect, and ultimately compulsive drug use and abuse. Toll-like receptor signaling within cortical-limbic circuits is modified by alcohol and stress in a manner consistent with promoting progression through the stages of addiction

Binge ethanol exposure during adolescence leads to a persistent loss of neurogenesis in the dorsal and ventral hippocampus that is associated with impaired adult cognitive functioning

Adolescence is a developmental period that coincides with the maturation of adult cognitive faculties. Binge drinking is common during adolescence and can impact brain maturation. Using a rodent model of adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 20% EtOH w/v; 2 days on/2 days off from postnatal day [P]25 to P55), we discovered that AIE treatment reduced neurogenesis (i.e., doublecortin-immunoreactive [DCX + IR] cells) in both the dorsal and ventral hippocampal dentate gyrus of late adolescent (P56) male Wistar rats that persisted during abstinence into adulthood (P220). This reduction in neurogenesis was accompanied by a concomitant reduction in proliferating cells (Ki-67) and an increase in cell death (cleaved caspase-3). In the hippocampus, AIE treatment induced a long-term upregulation of neuroimmune genes, including Toll-like receptor 4 (TLR4) and its endogenous agonist high-mobility group box 1 as well as several proinflammatory signaling molecules. Administration of lipopolysaccharide, a gram-negative endotoxin agonist at TLR4, to young adult rats (P70) produced a similar reduction of DCX + IR cells that was observed in AIE-treated animals. Behaviorally, AIE treatment impaired object recognition on the novel object recognition task when assessed from P163 to P165. Interestingly, object recognition memory was positively correlated with DCX + IR in both the dorsal and ventral hippocampal dentate gyrus while latency to enter the center of the apparatus was negatively correlated with DCX + IR in the ventral dentate gyrus. Together, these data reveal that adolescent binge ethanol exposure persistently inhibits neurogenesis throughout the hippocampus, possibly through neuroimmune mechanisms, which might contribute to altered adult cognitive and emotive function

Cholinergic REST-G9a gene repression through HMGB1-TLR4 neuroimmune signaling regulates basal forebrain cholinergic neuron phenotype

Lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1) are Toll-like receptor (TLR4) agonists that activate proinflammatory neuroimmune signaling linked to loss of basal forebrain cholinergic neurons (BFCNs) and cognitive deficits. Loss of choline acetyltransferase immunoreactive (ChAT + IR) BFCNs is generally interpreted as cell death, but recent in vivo studies find anti-inflammatory interventions restore adolescent ethanol exposure-induced persistent loss of adult ChAT + IR neurons and cognitive deficits, suggesting proinflammatory signaling-induced reversible gene repression of ChAT in BFCNs. Using an ex vivo Wistar rat basal forebrain slice culture (FSC) model to investigate TLR4 involvement in repression of the BFCN phenotype, we report that direct TLR4 activation with LPS decreases expression of multiple BFCN markers in the absence of observable neuronal loss or cell death. Inhibition of HMGB1 blunts while inhibition of TLR4 blocks the LPS-induced loss of ChAT + IR neurons. TLR4 activation induces the transcriptional repressor RE1-silencing transcription factor (REST) and the methyltransferase G9a while increasing repressive histone 3 lysine 9 dimethylation and REST occupancy at cholinergic gene promoters. G9a inhibitors both prevent and reverse the LPS-induced loss of ChAT + IR whereas siRNA inhibition of REST blocks the LPS-induced loss of ChAT + IR BFCNs. These data suggest in vivo HMGB1-TLR4 signaling in BFCNs leads to a reversible loss of the cholinergic neuron phenotype through epigenetic gene repressive mechanisms

Adolescent intermittent ethanol reduces serotonin expression in the adult raphe nucleus and upregulates innate immune expression that is prevented by exercise

Serotonergic neurons of the raphe nucleus regulate sleep, mood, endocrine function, and other processes that mature during adolescence. Alcohol abuse and binge drinking are common during human adolescence. We tested the novel hypothesis that adolescent intermittent ethanol exposure would alter the serotonergic system that would persist into adulthood. Using a Wistar rat model of adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P]25 to P55), we found a loss of dorsal raphe nucleus (DRN) serotonin (5-HT)-immunoreactive (+IR) neurons that persisted from late adolescence (P56) into adulthood (P220). Hypothalamic and amygdalar DRN serotonergic projections were reduced following AIE. Tryptophan hydroxylase 2, the rate-limiting 5-HT synthesizing enzyme, and vesicular monoamine transporter 2, which packages 5-HT into synaptic vesicles, were also reduced in the young adult midbrain following AIE treatment. Adolescent intermittent ethanol treatment increased expression of phosphorylated (activated) NF-κB p65 as well as markers of microglial activation (i.e., Iba-1 and CD11b) in the adult DRN. Administration of lipopolysaccharide to mimic AIE-induced innate immune activation reduced 5-HT+IR and increased phosphorylated NF-κB p65+IR similar to AIE treatment. Voluntary exercise during adolescence through young adulthood blunted microglial marker and phosphorylated NF-κB p65+IR, and prevented the AIE-induced loss of 5-HT+IR neurons in the DRN. Together, these novel data reveal that AIE reduces 5-HT+IR neurons in the adult DRN, possibly through an innate immune mechanism, which might impact adult cognition, arousal, or reward sensitivity. Further, exercise prevents the deleterious effects of AIE on the serotonergic system

Adolescent Alcohol Exposure Persistently Impacts Adult Neurobiology and Behavior

Adolescence is a developmental period when physical and cognitive abilities are optimized, when social skills are consolidated, and when sexuality, adolescent behaviors, and frontal cortical functions mature to adult levels. Adolescents also have unique responses to alcohol compared with adults, being less sensitive to ethanol sedative–motor responses that most likely contribute to binge drinking and blackouts. Population studies find that an early age of drinking onset correlates with increased lifetime risks for the development of alcohol dependence, violence, and injuries. Brain synapses, myelination, and neural circuits mature in adolescence to adult levels in parallel with increased reflection on the consequence of actions and reduced impulsivity and thrill seeking. Alcohol binge drinking could alter human development, but variations in genetics, peer groups, family structure, early life experiences, and the emergence of psychopathology in humans confound studies. As adolescence is common to mammalian species, preclinical models of binge drinking provide insight into the direct impact of alcohol on adolescent development. This review relates human findings to basic science studies, particularly the preclinical studies of the Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium. These studies focus on persistent adult changes in neurobiology and behavior following adolescent intermittent ethanol (AIE), a model of underage drinking. NADIA studies and others find that AIE results in the following: increases in adult alcohol drinking, disinhibition, and social anxiety; altered adult synapses, cognition, and sleep; reduced adult neurogenesis, cholinergic, and serotonergic neurons; and increased neuroimmune gene expression and epigenetic modifiers of gene expression. Many of these effects are specific to adolescents and not found in parallel adult studies. AIE can cause a persistence of adolescent-like synaptic physiology, behavior, and sensitivity to alcohol into adulthood. Together, these findings support the hypothesis that adolescent binge drinking leads to long-lasting changes in the adult brain that increase risks of adult psychopathology, particularly for alcohol dependence

ATP-P2X7 receptor signaling controls basal and TNFα-stimulated glial cell proliferation

Activation and proliferation of glial cells and their progenitors is a key process of neuroinflammation associated with many neurodegenerative disorders. Under neuropathological conditions where glial cell activation and proliferation is evident, controlling the population of glia might be of therapeutic importance. The proliferative action of the cytokine tumor necrosis factor alpha (TNFα) on microglia has been reported, but the molecular mechanism of TNFα regulation of glial cell proliferation is largely unknown. Using a model of organotypic hippocampal-entorhinal cortex (HEC) slice culture, we investigated the role of ATP-P2X7 receptor signaling in glial proliferation by TNFα. Populations of proliferating cells in HEC culture were labeled with 5-bromo-2′-deoxyuridine (BrdU). Treatment with TNFα induced strong expression of P2X7 receptor mRNA and immunoreactivity in BrdU+ cells while markedly increasing proliferation of BrdU+ cells. In addition, TNFα increased aquaporin 4 (AQP4) expression, an ion channel involved in glial proliferation. The proliferative action of TNFα was attenuated by blocking the P2X7 receptors with the specific antagonists oxATP, BBG and KN62, or by lowering extracellular ATP with ATP hydrolysis apyrase. Basal proliferation of BrdU+ cells was also sensitive to blockade of ATP-P2X7 signaling. Furthermore, TNFα activation of P2X7 receptors appear to regulate AQP4 expression through protein kinase C cascade and down regulation of AQP4 expression can reduce TNFα-stimulated BrdU+ cell proliferation. Taken together, these novel findings demonstrate the importance of ATP-P2X7 signaling in controlling proliferation of glial progenitors under the pathological conditions associated with increased TNFα

Prognostic Value of Diagnostic Sonography in Patients With Plantar Fasciitis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135343/1/jum201534101729.pd

Increased receptor for advanced glycation end product expression in the human alcoholic prefrontal cortex is linked to adolescent drinking

Adolescence is characterized behaviorally by increased impulsivity and risk-taking that declines in parallel with maturation of the prefrontal cortex and executive function. In the brain, the receptor for advanced glycation end products (RAGE) is critically involved in neurodevelopment and neuropathology. In humans, the risk of alcoholism is greatly increased in those who begin drinking between 13 and 15 years of age, and adolescents binge drink more than any other age group. We have previously found that alcoholism is associated with increased expression of neuroimmune genes. This manuscript tested the hypothesis that adolescent binge drinking upregulates RAGE and Toll-like receptor (TLR) 4 as well as their endogenous agonist, high-mobility group box 1 (HMGB1). Immunohistochemistry, Western blot, and mRNA analyses found that RAGE expression was increased in the human post-mortem alcoholic orbitofrontal cortex (OFC). Further, an earlier age of drinking onset correlated with increased expression of RAGE, TLR4, and HMGB1. To determine if alcohol contributed to these changes, we used an adolescent binge ethanol model in rats (5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P] 25 to P55) and assessed neuroimmune gene expression. We found an age-associated decline of RAGE expression from late adolescence (P56) to young adulthood (P80). Adolescent intermittent ethanol exposure did not alter RAGE expression at P56, but increased RAGE in the young adult PFC (P80). Adolescent intermittent ethanol exposure also increased TLR4 and HMGB1 expression at P56 that persisted into young adulthood (P80). Assessment of young adult frontal cortex mRNA (RT-PCR) found increased expression of proinflammatory cytokines, oxidases, and neuroimmune agonists at P80, 25 days after ethanol treatment. Together, these human and animal data support the hypothesis that an early age of drinking onset upregulates RAGE/TLR4-HMGB1 and other neuroimmune genes that persist into young adulthood and could contribute to risk of alcoholism or other brain diseases associated with neuroinflammation

Alcohol and Stress Activation of Microglia and Neurons: Brain Regional Effects

BACKGROUND: Cycles of alcohol and stress are hypothesized to contribute to alcohol use disorders. How this occurs is poorly understood, although both alcohol and stress activate the neuroimmune system-the immune molecules and cells that interact with the nervous system. The effects of alcohol and stress on the neuroimmune system are mediated in part by peripheral signaling molecules. Alcohol and stress both enhance immunomodulatory molecules such as corticosterone and endotoxin to impact neuroimmune cells, such as microglia, and may subsequently impact neurons. In this study, we therefore examined the effects of acute and chronic ethanol (EtOH) on the corticosterone, endotoxin, and microglial and neuronal response to acute stress. METHODS: Male Wistar rats were treated intragastrically with acute EtOH and acutely stressed with restraint/water immersion. Another group of rats was treated intragastrically with chronic intermittent EtOH and acutely stressed following prolonged abstinence. Plasma corticosterone and endotoxin were measured, and immunohistochemical stains for the microglial marker CD11b and neuronal activation marker c-Fos were performed. RESULTS: Acute EtOH and acute stress interacted to increase plasma endotoxin and microglial CD11b, but not plasma corticosterone or neuronal c-Fos. Chronic EtOH caused a lasting sensitization of stress-induced plasma endotoxin, but not plasma corticosterone. Chronic EtOH also caused a lasting sensitization of stress-induced microglial CD11b, but not neuronal c-Fos. CONCLUSIONS: These results find acute EtOH combined with acute stress enhanced plasma endotoxin, as well as microglial CD11b in many brain regions. Chronic EtOH followed by acute stress also increased plasma endotoxin and microglial CD11b, suggesting a lasting sensitization to acute stress. Overall, these data suggest alcohol and stress interact to increase plasma endotoxin, resulting in enhanced microglial activation that could contribute to disease progression