Targeting Persistent Changes in Neuroimmune and Epigenetic Signaling in Adolescent Drinking to Treat Alcohol Use Disorder in Adulthood.

Studies universally find early age of drinking onset is linked to lifelong risks of alcohol problems and alcohol use disorder (AUD). Assessment of the lasting effect of drinking during adolescent development in humans is confounded by the diversity of environmental and genetic factors that affect adolescent development, including emerging personality disorders and progressive increases in drinking trajectories into adulthood. Preclinical studies using an adolescent intermittent ethanol (AIE) exposure rat model of underage binge drinking avoid the human confounds and support lifelong changes that increase risks. AIE increases adult alcohol drinking, risky decision-making, reward-seeking, and anxiety as well as reductions in executive function that all increase risks for the development of an AUD. AIE causes persistent increases in brain neuroimmune signaling high-mobility group box 1 (HMGB1), Toll-like receptor, receptor for advanced glycation end products, and innate immune genes that are also found to be increased in human AUD brain. HMGB1 is released from cells by ethanol, both free and within extracellular vesicles, that act on neurons and glia, shifting transcription and cellular phenotype. AIE-induced decreases in adult hippocampal neurogenesis and loss of basal forebrain cholinergic neurons are reviewed as examples of persistent AIE-induced pathology. Both are prevented and reversed by anti-inflammatory and epigenetic drugs. Findings suggest AIE-increased HMGB1 signaling induces the RE-1 silencing transcript blunting cholinergic gene expression, shifting neuronal phenotype. Inhibition of HMGB1 neuroimmune signaling, histone methylation enzymes, and galantamine, the cholinesterase inhibitor, both prevent and reverse AIE pathology. These findings provide new targets that may reverse AUD neuropathology as well as other brain diseases linked to neuroimmune signaling. SIGNIFICANCE STATEMENT: Adolescent underage binge drinking studies find that earlier adolescent drinking is associated with lifelong alcohol problems including high levels of lifetime alcohol use disorder (AUD). Preclinical studies find the underage binge drinking adolescent intermittent ethanol (AIE) model causes lasting changes in adults that increase risks of developing adult alcohol problems. Loss of hippocampal neurogenesis and loss of basal forebrain cholinergic neurons provide examples of how AIE-induced epigenetic and neuroimmune signaling provide novel therapeutic targets for adult AUD.

Extracellular microvesicles promote microglia-mediated pro-inflammatory responses to ethanol.

Alcohol use disorder (AUD) pathology features pro-inflammatory gene induction and microglial activation. The underlying cellular processes that promote this activation remain unclear. Previously considered cellular debris, extracellular vesicles (EVs) have emerged as mediators of inflammatory signaling in several disease states. We investigated the role of microvesicles (MVs, 50 nm-100 µm diameter EVs) in pro-inflammatory and microglial functional gene expression using primary organotypic brain slice culture (OBSC). Ethanol caused a unique immune gene signature that featured: temporal induction of pro-inflammatory TNF-α and IL-1ß, reduction of homeostatic microglia state gene Tmem119, progressive increases in purinergic receptor P2RY12 and the microglial inhibitory fractalkine receptor CX3CR1, an increase in the microglial presynaptic gene C1q, and a reduction in the phagocytic gene TREM2. MV signaling was implicated in this response as reduction of MV secretion by imipramine blocked pro-inflammatory TNF-α and IL-1ß induction by ethanol, and ethanol-conditioned MVs (EtOH-MVs) reproduced the ethanol-associated immune gene signature in naïve OBSC slices. Depletion of microglia prior to ethanol treatment prevented pro-inflammatory activity of EtOH-MVs, as did incubation of EtOH-MVs with the HMGB1 inhibitor glycyrrhizin. Ethanol caused HMGB1 secretion from cultured BV2 microglia in MVs through activation of PI3 kinase. In summary, these studies find MVs modulate pro-inflammatory gene induction and microglial activation changes associated with ethanol. Thus, MVs may represent a novel therapeutic target to reduce neuroinflammation in the setting of alcohol abuse or other diseases that feature a neuroimmune component. [Correction added on 5 April 2021, after first online publication: The copyright line was changed.].

Characterization of extracellular vesicle miRNA identified in peripheral blood of chronic pancreatitis patients.

Plasma-derived extracellular vesicles (EV) can serve as markers of cell damage/disease but can also have therapeutic utility depending on the nature of their cargo, such as miRNA. Currently, there are challenges and lack of innovations regarding early diagnosis and therapeutic options within different aspects of management of patients suffering from chronic pancreatitis (CP). Use of EV as biomarkers for pancreatic health and/or as adjuvant therapy would make a difference in management of these patients. The aim of this study was to characterize the miRNA cargo of EV purified from the plasma of CP patients and compared to those of healthy participants. EVs were isolated from plasma of 15 CP patients and 10 healthy controls. Nanoparticle tracking analysis was used to determine frequency and size, while NanoString technology was used to characterize the miRNA cargo. Relevant clinical parameters were correlated with EV miRNA cargo. ~ 30 miRNA species were identified to have significantly (p < 0.05) different expression in EV from individuals with CP compared to healthy individuals; ~ 40 miRNA were differentially expressed in EV from pre-diabetic versus non-diabetic CP patients. miR-579-3p, while exhibiting significantly lower (~ 16-fold) expression in CP compared to healthy and lower (~ 24-fold) in CP narcotic users compared to the non-users, is actually enriched (~ 32-fold) within EV in pre-diabetic CP patients compared to non-diabetic CP patients. A unique pattern was identified in female CP patients. These data support the prospect of using a plasma-derived EV cargo to assess pancreatic health and its therapeutic potential in CP patients.

Increased Toll-like Receptor-MyD88-NFκB-Proinflammatory neuroimmune signaling in the orbitofrontal cortex of humans with alcohol use disorder.

BACKGROUND: Many brain disorders, including alcohol use disorder (AUD), are associated with induction of multiple proinflammatory genes. One aspect of proinflammatory signaling is progressive increases in expression across cells and induction of other innate immune genes. High-mobility group box 1 (HMGB1) heteromers contribute to amplification by potentiating multiple proinflammatory responses, including Toll-like receptors (TLRs). TLR signaling recruits coupling proteins linked to nuclear transcription factors that induce proinflammatory cytokines and chemokines and their respective receptors. We tested the hypothesis that AUD induction of TLR expression increases levels of proinflammatory genes and cellular signaling cascades in association with neurodegeneration in the orbitofrontal cortex (OFC). METHODS: Postmortem human OFC tissue samples (n = 10) from males diagnosed with AUD were compared to age-matched moderate drinking controls (CON). Neuroimmune signaling molecules were assessed using immunohistochemistry for protein and reverse transcription polymerase chain reaction for messenger RNA (mRNA). RESULTS: In the AUD OFC, we report induction of the endogenous TLR agonist HMGB1 as well as all TLRs assessed (i.e., TLR2-TLR9) except TLR1. This was accompanied by increased expression of the TLR adaptor protein myeloid differentiation primary response 88 (MyD88), activation of the proinflammatory nuclear transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and downstream induction of proinflammatory cytokines, chemokines, and their corresponding receptors. Several of these proinflammatory signaling markers are expressed in glia and neurons. The induction of HMGB1-TLR-MyD88-NFκB proinflammatory signaling pathways correlates with neurodegeneration (i.e., Fluoro-Jade B), lifetime alcohol consumption, and age of drinking onset. CONCLUSION: These data implicate the induction of HMGB1-TLR-MyD88-NFκB cascades through coordinated glial and neuronal signaling as contributors to the neurodegeneration seen in the postmortem human OFC of individuals with AUD.

TRAIL Mediates Neuronal Death in AUD: A Link between Neuroinflammation and Neurodegeneration.

Although the cause of progressive neurodegeneration is often unclear, neuronal death can occur through several mechanisms. In conditions such as Alzheimer's or alcohol use disorder (AUD), Toll-like receptor (TLR) induction is observed with neurodegeneration. However, links between TLR activation and neurodegeneration are lacking. We report a role of apoptotic neuronal death in AUD through TLR7-mediated induction of death receptor signaling. In postmortem human cortex, a two-fold increase in apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in neurons was found in AUD versus controls. This occurred with the increased expression of TLR7 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors. Binge ethanol treatment in C57BL/6 mice increased TLR7 and induced neuronal apoptosis in cortical regions that was blocked by TLR7 antagonism. Mechanistic studies in primary organotypic brain slice culture (OBSC) found that the inhibition of TLR7 and its endogenous ligand let-7b blocked ethanol-induced neuronal cell death. Both IMQ and ethanol induced the expression of TRAIL and its death receptor. In addition, TRAIL-neutralizing monoclonal antibodies blocked both imiquimod (IMQ) and ethanol induced neuronal death. These findings implicate TRAIL as a mediator of neuronal apoptosis downstream of TLR7 activation. TLR7 and neuronal apoptosis are implicated in other neurodegenerative diseases, including Alzheimer's disease. Therefore, TRAIL may represent a therapeutic target to slow neurodegeneration in multiple diseases.

Burn Injury Induces Proinflammatory Plasma Extracellular Vesicles That Associate with Length of Hospital Stay in Women: CRP and SAA1 as Potential Prognostic Indicators.

Severe burn injury is a devastating form of trauma that results in persistent immune dysfunction with associated morbidity and mortality. The underlying drivers of this immune dysfunction remain elusive, and there are no prognostic markers to identify at-risk patients. Extracellular vesicles (EVs) are emerging as drivers of immune dysfunction as well as biomarkers. We investigated if EVs after burn injury promote macrophage activation and assessed if EV contents can predict length of hospital stay. EVs isolated early from mice that received a 20% total body surface area (TBSA) burn promoted proinflammatory responses in cultured splenic macrophages. Unbiased LC-MS/MS proteomic analysis of early EVs (<72 h post-injury) from mice and humans showed some similarities including enrichment of acute phase response proteins such as CRP and SAA1. Semi-unbiased assessment of early human burn patient EVs found alterations consistent with increased proinflammatory signaling and loss of inhibition of CRP expression. In a sample of 50 patients with large burn injury, EV SAA1 and CRP were correlated with TBSA injury in both sexes and were correlated with length of hospital stay in women. These findings suggest that EVs are drivers of immune responses after burn injury and their content may predict hospital course.

Microglial depletion and repopulation in brain slice culture normalizes sensitized proinflammatory signaling.

BACKGROUND: Microglia are critical mediators of neuroimmune pathology across multiple neurologic disorders. Microglia can be persistently activated or "primed" by Toll-like receptor (TLR) activation, ethanol, stress, and other insults. Thus, strategies to prevent or reverse microglial priming may be beneficial for conditions that involve progressively increasing microglial activation. Microglial depletion with repopulation is emerging as a potential therapy to normalize chronic immune activation. Primary organotypic hippocampal slice culture (OHSC) allows for the study of neuroimmune activation as well as microglial depletion and repopulation without involvement of peripheral immune activation. OHSC undergoes functional maturation and retains cytoarchitecture similar to in vivo. METHODS: OHSC underwent microglial depletion with the CSF1R antagonist PLX3397 with or without repopulation after removal of PLX3397. Immune, trophic, and synaptic gene changes in response to agonists of TLRs 2, 3, 4, 7, and 9 as well as ethanol were assessed in the settings of microglial depletion and repopulation. Gi-DREADD inhibition of microglia was used to confirm select findings seen with depletion. The ability of microglial repopulation to prevent progressive proinflammatory gene induction by chronic ethanol was also investigated. RESULTS: Microglia were depleted (> 90%) by PLX3397 in OHSC. Microglial depletion blunted proinflammatory responses to several TLR agonists as well as ethanol, which was mimicked by Gi-DREADD inhibition of OHSC microglia. Removal of PLX3397 was followed by complete repopulation of microglia. OHSCs with repopulated microglia showed increased baseline expression of anti-inflammatory cytokines (e.g., IL-10), microglial inhibitory signals (e.g., CX3CL1), and growth factors (e.g., BDNF). This was associated with blunted induction (~ 50%) of TNFα and IL-1ß in response to agonists to TLR4 and TLR7. Further, chronic cycled ethanol from 4 days in vitro (DIV) to 16DIV caused immediate 2-fold inductions of TNFα and IL-1ß that grew to ~4-fold of age-matched control slices by 40DIV. This persistent inflammatory gene expression was completely reversed by microglial depletion and repopulation after chronic ethanol. CONCLUSIONS: Microglia in OHSCs mediate proinflammatory responses to TLR agonists and ethanol. Microglial repopulation promoted an anti-inflammatory, trophic neuroenvironment and normalized proinflammatory gene expression. This supports the possibility of microglial depletion with repopulation as a strategy to reverse chronic neuroimmune activation.

HMGB1/IL-1ß complexes regulate neuroimmune responses in alcoholism.

Neuroimmune activation is a key feature of the pathologies of numerous psychiatric disorders including alcoholism, depression, and anxiety. Both HMGB1 and IL-1ß have been implicated in brain disorders. Previous studies find HMGB1 andIL-1ß form heterocomplexes in vitro with enhanced immune responses, lead to our hypothesis that HMGB1 and IL-1ß heterocomplexes formed in vivo to contribute to the pathology of alcoholism. HMGB1/IL-1ß heterocomplexes were prepared in vitro and found to potentiate IL-1ß receptor proinflammatory gene induction compared to IL-1ß alone in hippocampal brain slice culture. These HMGB1/IL-1ß complexes were found to be increased in post-mortem human alcoholic hippocampus by co-immunoprecipiation. In mice, acute binge ethanol induced both HMGB1 and IL-1ß in the brain and plasma. HMGB1 and IL-1ß complexes were found only in mouse brain, with confocal microscopy revealing an ethanol-induced HMGB1 and IL-1ß cytoplasmic co-localization. Surprisingly, IL-1ß was found primarily in neurons. Studies in hippocampal brain slice culture found ethanol increased HMGB1/IL-1ß complexes in the media. These studies suggest a novel neuroimmune mechanism in the pathology of alcoholism. Immunogenic HMGB1/IL-1ß complexes represent a novel target for immune modulatory therapy in alcohol use disorders, and should be investigated in other psychiatric diseases that involve a neuroimmune component.

Innate Immune Signaling and Alcohol Use Disorders.

Innate immune signaling is an important feature in the pathology of alcohol use disorders. Alcohol abuse causes persistent innate immune activation in the brain. This is seen in postmortem human alcoholic brain specimens, as well as in primate and rodent models of alcohol consumption. Further, in vitro models of alcohol exposure in neurons and glia also demonstrate innate immune activation. The activation of the innate immune system seems to be important in the development of alcohol use pathology, as anti-immune therapies reduce pathology and ethanol self-administration in rodent models. Further, innate immune activation has been identified in each of the stages of addiction: binge/intoxication, withdrawal/negative affect, and preoccupation/craving. This suggests that innate immune activation may play a role both in the development and maintenance of alcoholic pathology. In this chapter, we discuss the known contributions of innate immune signaling in the pathology of alcohol use disorders, and present potential therapeutic interventions that may be beneficial for alcohol use disorders.

Correction to: Innate Immune Signaling and Alcohol Use Disorders.

In the second paragraph of section 1.3 on line 27, the text appears incorrect as glutamatergic and neurons. It should read as glutamatergic and GABAergic neurons. The original chapter was corrected.

Microglial-derived miRNA let-7 and HMGB1 contribute to ethanol-induced neurotoxicity via TLR7.

BACKGROUND: Toll-like receptor (TLR) signaling is emerging as an important component of neurodegeneration. TLR7 senses viral RNA and certain endogenous miRNAs to initiate innate immune responses leading to neurodegeneration. Alcoholism is associated with hippocampal degeneration, with preclinical studies linking ethanol-induced neurodegeneration with central innate immune induction and TLR activation. The endogenous miRNA let-7b binds TLR7 to cause neurodegeneration. METHODS: TLR7 and other immune markers were assessed in postmortem human hippocampal tissue that was obtained from the New South Wales Tissue Bank. Rat hippocampal-entorhinal cortex (HEC) slice culture was used to assess specific effects of ethanol on TLR7, let-7b, and microvesicles. RESULTS: We report here that hippocampal tissue from postmortem human alcoholic brains shows increased expression of TLR7 and increased microglial activation. Using HEC slice culture, we found that ethanol induces TLR7 and let-7b expression. Ethanol caused TLR7-associated neuroimmune gene induction and initiated the release let-7b in microvesicles (MVs), enhancing TLR7-mediated neurotoxicity. Further, ethanol increased let-7b binding to the danger signaling molecule high mobility group box-1 (HMGB1) in MVs, while reducing let-7 binding to classical chaperone protein argonaute (Ago2). Flow cytometric analysis of MVs from HEC media and analysis of MVs from brain cell culture lines found that microglia were the primary source of let-7b and HMGB1-containing MVs. CONCLUSIONS: Our results identify that ethanol induces neuroimmune pathology involving the release of let-7b/HMGB1 complexes in microglia-derived microvesicles. This contributes to hippocampal neurodegeneration and may play a role in the pathology of alcoholism.

Loss of neuronal lysosomal acid lipase drives amyloid pathology in Alzheimer's disease.

Underlying drivers of late-onset Alzheimer's disease (LOAD) pathology remain unknown. However, multiple biologically diverse risk factors share a common pathological progression. To identify convergent molecular abnormalities that drive LOAD pathogenesis we compared two common midlife risk factors for LOAD, heavy alcohol use and obesity. This revealed that disrupted lipophagy is an underlying cause of LOAD pathogenesis. Both exposures reduced lysosomal flux, with a loss of neuronal lysosomal acid lipase (LAL). This resulted in neuronal lysosomal lipid (NLL) accumulation, which opposed Aß localization to lysosomes. Neuronal LAL loss both preceded (with aging) and promoted (targeted knockdown) Aß pathology and cognitive deficits in AD mice. The addition of recombinant LAL ex vivo and neuronal LAL overexpression in vivo prevented amyloid increases and improved cognition. In WT mice, neuronal LAL declined with aging and correlated negatively with entorhinal Aß. In healthy human brain, LAL also declined with age, suggesting this contributes to the age-related vulnerability for AD. In human LOAD LAL was further reduced, correlated negatively with Aß1-42, and occurred with polymerase pausing at the LAL gene. Together, this finds that the loss of neuronal LAL promotes NLL accumulation to impede degradation of Aß in neuronal lysosomes to drive AD amyloid pathology.

EXTRACELLULAR VESICLES AS REGULATORS OF IMMUNE FUNCTION IN TRAUMATIC INJURIES AND SEPSIS.

ABSTRACT: Despite advancements in critical care and resuscitation, traumatic injuries are one of the leading causes of death around the world and can bring about long-term disabilities in survivors. One of the primary causes of death for trauma patients are secondary phase complications that can develop weeks or months after the initial insult. These secondary complications typically occur because of systemic immune dysfunction that develops in response to injury, which can lead to immunosuppression, coagulopathy, multiple organ failure, unregulated inflammation, and potentially sepsis in patients. Recently, extracellular vesicles (EVs) have been identified as mediators of these processes because their levels are increased in circulation after traumatic injury and they encapsulate cargo that can aggravate these secondary complications. In this review, we will discuss the role of EVs in the posttrauma pathologies that arise after burn injuries, trauma to the central nervous system, and infection. In addition, we will examine the use of EVs as biomarkers for predicting late-stage trauma outcomes and as therapeutics for reversing the pathological processes that develop after trauma. Overall, EVs have emerged as critical mediators of trauma-associated pathology and their use as a therapeutic agent represents an exciting new field of biomedicine.

Involvement of extracellular vesicles in the progression, diagnosis, treatment, and prevention of whole-body ionizing radiation-induced immune dysfunction.

Acute radiation syndrome (ARS) develops after exposure to high doses of ionizing radiation and features immune suppression and organ failure. Currently, there are no diagnostics to identify the occurrence or severity of exposure and there are limited treatments and preventative strategies to mitigate ARS. Extracellular vesicles (EVs) are mediators of intercellular communication that contribute to immune dysfunction across many diseases. We investigated if EV cargo can identify whole body irradiation (WBIR) exposure and if EVs promote ARS immune dysfunction. We hypothesized that beneficial EVs derived from mesenchymal stem cells (MSC-EVs) would blunt ARS immune dysfunction and might serve as prophylactic radioprotectants. Mice received WBIR (2 or 9 Gy) with assessment of EVs at 3 and 7 days after exposure. LC-MS/MS proteomic analysis of WBIR-EVs found dose-related changes as well as candidate proteins that were increased with both doses and timepoints (34 total) such as Thromboxane-A Synthase and lymphocyte cytosolic protein 2. Suprabasin and Sarcalumenin were increased only after 9 Gy suggesting these proteins may indicate high dose/lethal exposure. Analysis of EV miRNAs identified miR-376 and miR-136, which were increased up to 200- and 60-fold respectively by both doses of WBIR and select miRNAs such as miR-1839 and miR-664 were increased only with 9 Gy. WBIR-EVs (9 Gy) were biologically active and blunted immune responses to LPS in RAW264.7 macrophages, inhibiting canonical signaling pathways associated with wound healing and phagosome formation. When given 3 days after exposure, MSC-EVs slightly modified immune gene expression changes in the spleens of mice in response to WBIR and in a combined radiation plus burn injury exposure (RCI). MSC-EVs normalized the expression of certain key immune genes such as NFκBia and Cxcr4 (WBIR), Map4k1, Ccr9 and Cxcl12 (RCI) and lowered plasma TNFα cytokine levels after RCI. When given prophylactically (24 and 3 hours before exposure), MSC-EVs prolonged survival to the 9 Gy lethal exposure. Thus, EVs are important participants in ARS. EV cargo might be used to diagnose WBIR exposure, and MSC-EVs might serve as radioprotectants to blunt the impact of toxic radiation exposure.

Activation of hypothalamic-enhanced adult-born neurons restores cognitive and affective function in Alzheimer's disease.

Patients with Alzheimer's disease (AD) exhibit progressive memory loss, depression, and anxiety, accompanied by impaired adult hippocampal neurogenesis (AHN). Whether AHN can be enhanced in impaired AD brain to restore cognitive and affective function remains elusive. Here, we report that patterned optogenetic stimulation of the hypothalamic supramammillary nucleus (SuM) enhances AHN in two distinct AD mouse models, 5×FAD and 3×Tg-AD. Strikingly, the chemogenetic activation of SuM-enhanced adult-born neurons (ABNs) rescues memory and emotion deficits in these AD mice. By contrast, SuM stimulation alone or activation of ABNs without SuM modification fails to restore behavioral deficits. Furthermore, quantitative phosphoproteomics analyses reveal activation of the canonical pathways related to synaptic plasticity and microglia phagocytosis of plaques following acute chemogenetic activation of SuM-enhanced (vs. control) ABNs. Our study establishes the activity-dependent contribution of SuM-enhanced ABNs in modulating AD-related deficits and informs signaling mechanisms mediated by the activation of SuM-enhanced ABNs.

A critical review of recent knowledge of alcohol's effects on the immunological response in different tissues.

Alcohol misuse contributes to the dysregulation of immune responses and multiorgan dysfunction across various tissues, which are associated with higher risk of morbidity and mortality in people with alcohol use disorders. Organ-specific immune cells, including microglia in the brain, alveolar macrophages in the lungs, and Kupffer cells in the liver, play vital functions in host immune defense through tissue repair and maintenance of homeostasis. However, binge drinking and chronic alcohol misuse impair these immune cells' abilities to regulate inflammatory signaling and metabolism, thus contributing to multiorgan dysfunction. Further complicating these delicate systems, immune cell dysfunction associated with alcohol misuse is exacerbated by aging and gut barrier leakage. This critical review describes recent advances in elucidating the potential mechanisms by which alcohol misuse leads to derangements in host immunity and highlights current gaps in knowledge that may be the focus of future investigations.

Alcohol and Immunology: Mechanisms of multi-organ damage. Summary of the 2022 alcohol and Immunology research interest group (AIRIG) meeting.

On October 26th, 2022 the annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held as a satellite symposium at the annual meeting of the Society for Leukocyte Biology in Hawaii. The 2022 meeting focused broadly on the immunological consequences of acute, chronic, and prenatal alcohol exposure and how these contribute to damage in multiple organs and tissues. These included alcohol-induced neuroinflammation, impaired lung immunity, intestinal dysfunction, and decreased anti-microbial and anti-viral responses. In addition, research presented covered multiple pathways behind alcohol-induced cellular dysfunction, including mitochondrial metabolism, cellular bioenergetics, gene regulation, and epigenetics. Finally, the work presented highlighted potential biomarkers and novel avenues of treatment for alcohol-induced organ damage.

Chronic Ethanol Causes Persistent Increases in Alzheimer's Tau Pathology in Female 3xTg-AD Mice: A Potential Role for Lysosomal Impairment.

Epidemiological studies have found that heavy alcohol use is associated with increased risk for Alzheimer's disease (AD), with frequent drinking earlier in adulthood increasing risk. The increases in neuroinflammation featured in both heavy alcohol use and AD may be partially responsible for this link. However, it is unknown if abstinence mitigates this risk. We hypothesized that binge ethanol during mid adult life would persistently increase AD pathology even after prolonged abstinence. Male and female 3xTg-AD mice (APPSwe, tauP301, Psen1tm1Mpm) which feature progressive amyloid (Aß) and tau pathology, received chronic binge ethanol (5g/kg/day, 5-days-on/2-days-off, i.g.) or water during adulthood (from 5.5 to 9 months of age), followed by abstinence and assessment at 14 months of age. The effects of ethanol on protective AD genes (e.g., APOE and TREM2) as well as proinflammatory genes were measured by PCR. Levels of pathologic tau and Aß were measured by immunohistochemistry and western blot. Ethanol caused persistent reductions in protective AD genes: APOE (25% reduction, *p < 0.05), TREM2 (28%, *p < 0.05), LPL (40%, ** p < 0.01), and CTSD (24%, *p < 0.05) and promoted a proinflammatory gene signature in female, but not male cortex. Concurrently, ethanol increased total and hyperphosphorylated tau (AT8) in piriform cortex and hippocampus of females, but not males. Levels of AT8 were negatively correlated with APOE (R = -0.67, *p < 0.05) and TREM2 (R = -0.78, **p < 0.005) suggesting protective roles in pathogenesis. No differences were found in levels of main regulators of tau phosphorylation state (GSK3ß, PKA, PP2A), suggesting ethanol disrupted clearance of tau. Therefore, we measured the effect of ethanol on lysosomes, which degrade tau, and lysosomal localization of tau using co-immunofluorescence. In females, ethanol caused a persistent reduction in mature LAMP1 lysosomes in CA1 of hippocampus (35%, *p < 0.05), along with a 60% increase in total tau (*p < 0.05). Thus, chronic binge ethanol during mid adult life causes a persistent enhancement of tau pathology in cortical and hippocampal brain regions of females. Persistent AD pathology was associated with an increased proinflammatory signature and a reduction of mature lysosomes. This implicates binge ethanol exposure with increased risk of AD pathologic progression in females.

Ethanol Induces Secretion of Proinflammatory Extracellular Vesicles That Inhibit Adult Hippocampal Neurogenesis Through G9a/GLP-Epigenetic Signaling.

Adult hippocampal neurogenesis (AHN) is involved in learning and memory as well as regulation of mood. Binge ethanol reduces AHN, though the mechanism is unknown. Microglia in the neurogenic niche are important regulators of AHN, and ethanol promotes proinflammatory microglia activation. We recently reported that extracellular vesicles (EVs) mediate ethanol-induced inflammatory signaling in microglia. Therefore, we investigated the role of EVs in ethanol-induced loss of adult hippocampal neurogenesis. At rest, microglia promoted neurogenesis through the secretion of pro-neurogenic extracellular vesicles (pn-EVs). Depletion of microglia using colony-stimulating factor 1 receptor (CSFR1) inhibition in vivo or using ex vivo organotypic brain slice cultures (OBSCs) caused a 30% and 56% loss of neurogenesis in the dentate, respectively, as measured by immunohistochemistry for doublecortin (DCX). Likewise, chemogenetic inhibition of microglia using a CD68.hM4di construct caused a 77% loss in OBSC, indicating a pro-neurogenic resting microglial phenotype. EVs from control OBSC were pro-neurogenic (pn-EVs), enhancing neurogenesis when transferred to other naive OBSC and restoring neurogenesis in microglia-depleted cultures. Ethanol inhibited neurogenesis and caused secretion of proinflammatory EVs (EtOH-EVs). EtOH-EVs reduced hippocampal neurogenesis in naïve OBSC by levels similar to ethanol. Neurogenesis involves complex regulation of chromatin structure that could involve EV signaling. Accordingly, EtOH-EVs were found to be enriched with mRNA for the euchromatin histone lysine methyltransferase (Ehm2t/G9a), an enzyme that reduces chromatin accessibility through histone-3 lysine-9 di-methylation (H3K9me2). EtOH-EVs induced G9a and H3K9me2 by 2-fold relative to pn-EVs in naïve OBSCs. Pharmacological inhibition of G9a with either BIX-01294 or UNC0642 prevented loss of neurogenesis caused by both EtOH and EtOH-EVs. Thus, this work finds that proinflammatory EtOH-EVs promote the loss of adult hippocampal neurogenesis through G9a-mediated epigenetic modification of chromatin structure.

Adolescent Binge Alcohol Enhances Early Alzheimer's Disease Pathology in Adulthood Through Proinflammatory Neuroimmune Activation.

Epidemiological studies suggest that heavy alcohol use early in life is associated with increased risk for Alzheimer's disease (AD). However, mechanisms connecting AD with alcohol use have not been identified. Both heavy alcohol use and AD feature increased proinflammatory signaling. Therefore, we hypothesized that adolescent binge ethanol would increase AD molecular and behavioral pathology in adulthood through proinflammatory signaling. The 3xTg-AD mouse model (APPSwe, tauP301, Psen1tm1Mpm) which features amyloid (Aß) and tau pathology beginning at 6-12 months underwent adolescent intermittent ethanol (AIE, 5 g/kg/d, i.g., P25-55) with assessment of AD pathologic mediators at P200. A second group of mice received AIE +/- minocycline (30 mg/kg/d, IP) followed by behavioral testing in adulthood. Behavioral testing and age of testing included: locomotor activity and exploration (27-28 weeks), novel object recognition (NORT, 28-30 weeks), 3-chamber sociability and social memory (29-31 weeks), prepulse inhibition (PPI, 30-32 weeks), Morris Water Maze with reversal (MWM, 31-35 weeks), and Piezo sleep monitoring (35-37 weeks). We found that AIE increased levels of neurotoxic Aß1-42 in adult female hippocampus as well as intraneuronal Aß1-42 in amygdala and entorhinal cortex. Phosphorylated tau at residue Thr181 (p-tau-181) was also increased in female hippocampus by AIE. Several proinflammatory genes were persistently increased by AIE in the female hippocampus, including IL-1ß, MCP-1, IL-6, and IFNα. Expression of these genes was strongly correlated with the levels of Aß1-42 and p-tau-181 in hippocampus. AIE caused persistent decreases in locomotor activity (open-field and NORT habituation) and increased anxiety-like behavior (thigmotaxis) while reducing memory retention. Treatment with the anti-inflammatory compound minocycline during AIE blocked persistent increases in Aß1-42 in amygdala and p-tau-181 in hippocampus, and prevented AIE-induced thigmotaxis and memory loss. Together, these data find that adolescent binge ethanol enhances AD molecular and behavioral pathology in adulthood through proinflammatory signaling. Blockade of proinflammatory signaling during ethanol exposure prevents ethanol-induced effects on pathologic accumulation of AD-associated proteins and persistent behavior changes relevant to human AD.