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

AUTHORS

Ryan P. Vetreno, Liya Qin, Leon G. Coleman Jr, Fulton T. Crews

ABSTRACT

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 Tolllike 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 HMGB1as 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

Loss of Basal Forebrain Cholinergic Neurons Following Adolescent Binge Ethanol Exposure: Recovery With the Cholinesterase Inhibitor Galantamine

AUTHORS

Fulton T. Crews, Rachael Fisher, Chloe Deason and Ryan P. Vetreno

ABSTRACT

Binge drinking and alcohol abuse are common during adolescence and cause both cognitive deficits and lasting cholinergic pathology in the adult basal forebrain. Acetylcholine is anti-inflammatory and studies using the preclinical adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2 day on/2 day off from postnatal day [P]25 to P54) model of human adolescent binge drinking report decreased basal forebrain cholinergic neurons (BFCNs) and induction of proinflammatory genes that persist long into adulthood. Recent studies link AIE-induced neuroimmune activation to cholinergic pathology, but the underlying mechanisms contributing to the persistent loss of BFCNs are unknown. We report that treatment with the cholinesterase inhibitor galantamine (4.0 mg/kg, i.p.) administered during AIE (i.e., P25–P54) or following the conclusion of AIE (i.e., P57–P72) recovered the persistent loss of cholinergic neuron phenotype markers (i.e., ChAT, TrkA, and p75NTR) and somal shrinkage of residual ChAT + neurons known to persist in AIE-exposed adults. Galantamine treatment also recovered the AIE-increased expression of the proinflammatory receptors TLR4 and RAGE, the endogenous TLR4/RAGE agonist HMGB1, and the transcription activation marker pNF-κB p65. Interestingly, we find BFCNs express TLR4 and RAGE, and that AIE treatment increased pNF-κB p65 expression in adult ChAT + IR neurons, consistent with intracellular HMGB1-TLR4/RAGE signaling within BFCNs. AIE increased epigenetic transcription silencing markers (i.e., H3K9me2 and H3K9me3) in the adult basal forebrain and H3K9me2 occupancy at cholinergic phenotype gene promoters (i.e., ChAT and TrkA). The finding of no AIE-induced changes in total basal forebrain NeuN + neurons with galantamine reversal of AIE-induced ChAT + neuron loss, TLR4/RAGE-pNF-κB p65 signals, and epigenetic transcription silencing markers suggests that AIE does not cause cell death, but rather the loss of the cholinergic phenotype. Together, these data suggest that AIE induces HMGB1-TLR4/RAGE-pNF-κB p65 signals, causing the loss of cholinergic phenotype (i.e., ChAT, TrkA, and p75NTR) through epigenetic histone transcription silencing that result in the loss of the BFCN phenotype that can be prevented and restored by galantamine.

Chronic ethanol modulates delta and mu-opioid receptor expression in rat CNS: immunohistochemical analysis with quantitiative confocal microscopy

AUTHORS

L.C. Saland, C.M. Hastings, A. Abeyta, J.B. Chavez

ABSTRACT

Ethanol consumption affects levels of endogenous opioids as well as opioid receptors in both animals and humans. We studied the expression of delta (δ) and mu (μ) opioid receptors (ORs) in brain sections of adult male Sprague–Dawley rats after 2 weeks of consuming ethanol in a liquid diet, with comparisons to sections from pair-fed control animals. Immunohistochemical staining for the ORs, using selective antibodies, and quantitation of confocal images, revealed increased expression of δ-ORs in hippocampal CA1 of the chronic ethanol-treated rats. In contrast, μ-ORs decreased in their expression after ethanol treatment in multiple brain areas, including cortex, hippocampus, midbrain colliculi, striatum and nucleus accumbens. The alterations in immunoreactive OR expression may be related to reduced functional coupling of the ORs to G-proteins, as found in prior studies in several brain regions, using the same chronic ethanol diet protocol. Changes in OR expression and functional coupling in the CNS may be factors in ongoing ethanol consumption and tolerance.