miRNA-383 and miRNA-384 Suppress Proopiomelanocortin Gene Expression in the Hypothalamus: Effects of Early Life Ethanol Exposure.

INTRODUCTION: Early life ethanol exposure is known to program hypothalamic proopiomelanocortin (POMC) neurons to express a reduced level of POMC and its control of stress axis functions throughout the life span. In this study, we tested whether miRNAs contribute to the ethanol-induced suppression of Pomc gene expression during the developmental period. METHODS: In in vivo studies, POMC-EGFP male mice were fed with 2.5 g/kg ethanol using milk formula (AF), pair-fed isocaloric milk formula, or left in the litter during postnatal days (PNDs) 2-6. In in vitro studies, mHypoA-POMC/GFP cells were treated with ethanol (50 mM) for a 24-h period. Hypothalamic tissues or cell extracts were used for measurement of miRNAs and POMC mRNA. RESULTS: Determination of genome-wide microRNA expression profile identified 40 miRNAs significantly altered in hypothalamic tissues of AF mice. In silico analysis further identified miRNA-383, -384, and -488 have putative binding sites at the POMC 3'UTR. However, only miR-383 and miR-384 are identified to be responsive to ethanol. Administration of miR-383 or -384 inhibitor oligos suppressed ethanol-stimulated miR-383 or -384 expression and restored Pomc mRNA and protein expression in AF mice. mHypoA-POMC/GFP cells when treated with ethanol showed elevated levels of miR-383 and miR-384 and reduced level of Pomc mRNA. Treatment with miR-383 or -384 mimic oligos reduced the level of Pomc mRNA, while treatment with miR-383 or -384 inhibitor oligos increased the level of Pomc mRNA. Reporter assay further confirms the binding specificity of miR-383 and miR-384 to Pomc 3'UTR. CONCLUSION: These data suggest that miR-383 and miR-384 suppress Pomc gene expression and may contribute to the ethanol-induced alteration of the stress axis functions.

Alcohol Increases Exosome Release from Microglia to Promote Complement C1q-Induced Cellular Death of Proopiomelanocortin Neurons in the Hypothalamus in a Rat Model of Fetal Alcohol Spectrum Disorders.

Microglia, a type of CNS immune cell, have been shown to contribute to ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing ß-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined whether the microglial extracellular vesicle exosome is involved in the ethanol-induced neuronal death of the ß-endorphin neuron. Extracellular vesicles were prepared from hypothalamic tissues collected from postnatal rats (both males and females) fed daily with 2.5 mg/kg ethanol or control milk formula for 5 d or from hypothalamic microglia cells obtained from postnatal rats, grown in cultures for several days, and then challenged with ethanol or vehicle for 24 h. Nanoparticle tracking analysis and transmission electron microscopy indicated that these vesicles had the size range and shape of exosomes. Ethanol treatments increased the number and the ß-endorphin neuronal killing activity of microglial exosomes both in vivo and in vitro Proteomics analyses of exosomes of cultured microglial cells identified a large number of proteins, including various complements, which were elevated following ethanol treatment. Proteomics data involving complements were reconfirmed using quantitative protein assays. Ethanol treatments also increased deposition of the complement protein C1q in ß-endorphin neuronal cells in both in vitro and in vivo systems. Recombinant C1q protein increased while C1q blockers reduced ethanol-induced C3a/b, C4, and membrane attack complex/C5b9 formations; ROS production; and ultimately cellular death of ß-endorphin neurons. These data suggest that the complement system involving C1q-C3-C4-membrane attack complex and ROS regulates exosome-mediated, ethanol-induced ß-endorphin neuronal death.SIGNIFICANCE STATEMENT Neurotoxic action of alcohol during the developmental period is recognized for its involvement in fetal alcohol spectrum disorders, but the lack of clear understanding of the mechanism of alcohol action has delayed the progress in therapeutic intervention of this disease. Proopiomelanocortin neurons known to regulate stress, energy homeostasis, and immune functions are reported to be killed by developmental alcohol exposure because of activation of microglial immune cells in the brain. While microglia are known to use extracellular vesicles to communicate with neurons for maintaining homeostasis, we show here that ethanol exposure during the developmental period hijacks this system to spread apoptotic factors, including complement protein C1q, to induce the membrane attack complex and reactive super-oxygen species for proopiomelanocortin neuronal killing.

Early life alcohol exposure primes hypothalamic microglia to later-life hypersensitivity to immune stress: possible epigenetic mechanism.

Growing evidence has shown that developmental alcohol exposure induces central nervous system inflammation and microglia activation, which may contribute to long-term health conditions, such as fetal alcohol spectrum disorders. These studies sought to investigate whether neonatal alcohol exposure during postnatal days (PND) 2-6 in rats (third trimester human equivalent) leads to long-term disruption of the neuroimmune response by microglia. Exposure to neonatal alcohol resulted in acute increases in activation and inflammatory gene expression in hypothalamic microglia including tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Adults with neonatal alcohol pre-exposure (alcohol fed; AF) animals showed an exaggerated peripheral stress hormonal response to an immune challenge (lipopolysaccharides; LPS). In addition, there were significantly more microglia present in the hypothalamus of adult AF animals, and their hypothalamic microglia showed more cluster of differentiation molecule 11b (Cd11b) activation, TNF-α expression, and IL-6 expression in response to LPS. Interestingly, blocking microglia activation with minocycline treatment during PND 2-6 alcohol exposure ameliorated the hormonal and microglial hypersensitivity to LPS in AF adult animals. Investigation of possible epigenetic programming mechanisms by alcohol revealed neonatal alcohol decreased several repressive regulators of transcription in hypothalamic microglia, while concomitantly increasing histone H3 acetyl lysine 9 (H3K9ac) enrichment at TNF-α and IL-6 promoter regions. Importantly, adult hypothalamic microglia from AF animals showed enduring increases in H3K9ac enrichment of TNF-α and IL-6 promoters both at baseline and after LPS exposure, suggesting a possible epigenetic mechanism for the long-term immune disruption due to hypothalamic microglial priming.

Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol.

BACKGROUND: Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus. METHODS: Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2-6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons. RESULTS: In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol's ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons. CONCLUSIONS: These results suggest that ethanol's neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.