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1.
J Neuroinflammation ; 21(1): 279, 2024 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-39478585

RESUMEN

BACKGROUND: Microglia, a type of resident immune cells within the central nervous system, have been implicated in 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 if microglial extracellular vesicles (exosomes) are involved in the ethanol-induced neuronal death of the ß-endorphin neuron via secreting elevated levels of the chemokine monocyte chemoattractant protein 1 (MCP1), a key regulator of neuroinflammation. METHODS: We employed an in vitro model, consisting of primary culture of hypothalamic microglia prepared from postnatal day 2 (PND2) rat hypothalami and treated with or without 50 mM ethanol for 24 h, and an in vivo animal model in which microglia were obtained from hypothalami of PND6 rats fed daily with 2.5 mg/kg ethanol or control milk formula for five days prior to use. Exosomes were extracted and characterized with nanosight tracking analysis (NTA), transmission electron microscopy and western blot. Chemokine multiplex immunoassay and ELISA were used for quantitative estimation of MCP1 level. Neurotoxic ability of exosome was tested using primary cultures of ß-endorphin neurons and employing nucleosome assay and immunocytochemistry. Elevated plus maze, open field and restraint tests were used to assess anxiety-related behaviors. RESULTS: Ethanol elevated MCP1 levels in microglial exosomes both in vitro and in vivo models. Ethanol-activated microglial exosomes when introduced into primary cultures of ß-endorphin neurons, increased cellular levels of MCP1 and the chemokine receptor CCR2 related signaling molecules including inflammatory cytokines and apoptotic genes as well as apoptotic death of ß-endorphin neurons. These effects of microglial exosomes on ß-endorphin neurons were suppressed by a CCR2 antagonist RS504393. Furthermore, RS504393 when injected in postnatal rats prior to feeding with ethanol it reduced alcohol-induced ß-endorphin neuronal death in the hypothalamus. RS504393 also suppressed corticosterone response to stress and anxiety-like behaviors in postnatally alcohol-fed rats during adult period. CONCLUSION: These data suggest that alcohol exposures during the developmental period elevates MCP1 levels in microglial exosomes that promote MCP1/CCR2 signaling to increase the apoptosis of ß-endorphin neurons and resulting in hormonal and behavioral stress responses.


Asunto(s)
Quimiocina CCL2 , Etanol , Exosomas , Hipotálamo , Microglía , Neuronas , Proopiomelanocortina , Transducción de Señal , Animales , Etanol/toxicidad , Etanol/farmacología , Ratas , Exosomas/metabolismo , Exosomas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Quimiocina CCL2/metabolismo , Hipotálamo/metabolismo , Hipotálamo/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ratas Sprague-Dawley , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Femenino , Depresores del Sistema Nervioso Central/farmacología , Depresores del Sistema Nervioso Central/toxicidad , betaendorfina/metabolismo
2.
Neuroendocrinology ; 113(8): 844-858, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36948162

RESUMEN

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.


Asunto(s)
Etanol , Proopiomelanocortina , Ratones , Masculino , Animales , Proopiomelanocortina/metabolismo , Etanol/metabolismo , Etanol/farmacología , Regiones no Traducidas 3' , Hipotálamo/metabolismo , Expresión Génica
3.
Breast Cancer Res ; 24(1): 33, 2022 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-35568869

RESUMEN

BACKGROUND: Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control the growth and proliferation of breast cancer cells. However, the underlying cell signaling mechanism remained unclear. METHODS: To determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, CRISPR or chemical inhibition or activation of beta-adrenergic receptors (B2AR) and mu-opioid receptors (MOR) gene, and PCR array technology and studied aggressive tumor phenotype and signaling cascades. RESULTS: We show here that in triple-negative breast cancer cells, the reduction in expression B2AR and MOR by genetic and pharmacological tools leads to a less aggressive phenotype of triple-negative breast cancer cells in vitro and in animal xenografts. Genomic analysis indicates the glycogen synthase kinase 3 (GSK3) pathway as a possible candidate messenger system involved in B2AR and MOR cross talk. GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation. CONCLUSIONS: These data confirm a role of B2AR and MOR interaction in the control of breast cancer cell growth and identify a possible role of the GSK3 signaling system in mediation of these two receptors' cross talk. Screening for ligands targeting B2AR and MOR interaction and/or the GSK3 system may help to identify novel drugs for the prevention of triple-negative breast cancer cell growth and metastasis.


Asunto(s)
Receptores Adrenérgicos beta 2 , Receptores Opioides mu , Neoplasias de la Mama Triple Negativas , Animales , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética
4.
J Neurosci ; 40(41): 7965-7979, 2020 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-32887744

RESUMEN

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.


Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Complemento C1q/farmacología , Etanol/farmacología , Exosomas/efectos de los fármacos , Trastornos del Espectro Alcohólico Fetal/patología , Microglía/efectos de los fármacos , Proopiomelanocortina/genética , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Femenino , Trastornos del Espectro Alcohólico Fetal/metabolismo , Hipotálamo/metabolismo , Hipotálamo/patología , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Embarazo , Proteómica , Ratas , Ratas Sprague-Dawley , betaendorfina/metabolismo
5.
Alcohol Clin Exp Res ; 43(2): 212-220, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30597578

RESUMEN

BACKGROUND: Epigenetic modifications of a gene have been shown to play a role in maintaining a long-lasting change in gene expression. We hypothesize that alcohol's modulating effect on DNA methylation on certain genes in blood is evident in binge and heavy alcohol drinkers and is associated with alcohol motivation. METHODS: Methylation-specific polymerase chain reaction (PCR) assays were used to measure changes in gene methylation of period 2 (PER2) and proopiomelanocortin (POMC) genes in peripheral blood samples collected from nonsmoking moderate, nonbinging, binge, and heavy social drinkers who participated in a 3-day behavioral alcohol motivation experiment of imagery exposure to either stress, neutral, or alcohol-related cues, 1 per day, presented on consecutive days in counterbalanced order. Following imagery exposure on each day, subjects were exposed to discrete alcoholic beer cues followed by an alcohol taste test (ATT) to assess behavioral motivation. Quantitative real-time PCR was used to measure gene expression of PER2 and POMC gene levels in blood samples across samples. RESULTS: In the sample of moderate, binge, and heavy drinkers, we found increased methylation of the PER2 and POMC DNA, reduced expression of these genes in the blood samples of the binge and heavy drinkers relative to the moderate, nonbinge drinkers. Increased PER2 and POMC DNA methylation was also significantly predictive of both increased levels of subjective alcohol craving immediately following imagery (p < 0.0001), and with presentation of the alcohol (2 beers) (p < 0.0001) prior to the ATT, as well as with alcohol amount consumed during the ATT (p < 0.003). CONCLUSIONS: These data establish significant association between binge or heavy levels of alcohol drinking and elevated levels of methylation and reduced levels of expression of POMC and PER2 genes. Furthermore, elevated methylation of POMC and PER2 genes is associated with greater subjective and behavioral motivation for alcohol.


Asunto(s)
Consumo de Bebidas Alcohólicas/metabolismo , Consumo de Bebidas Alcohólicas/psicología , Consumo Excesivo de Bebidas Alcohólicas/metabolismo , Metilación de ADN/efectos de los fármacos , Motivación , Proteínas Circadianas Period/metabolismo , Proopiomelanocortina/metabolismo , Adulto , Ansia/efectos de los fármacos , Señales (Psicología) , Epigénesis Genética , Etanol/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Proteínas Circadianas Period/sangre , Estimulación Luminosa , Proopiomelanocortina/sangre , Adulto Joven
6.
Alcohol Clin Exp Res ; 43(9): 1887-1897, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31329297

RESUMEN

BACKGROUND: We have recently shown that binge or heavy levels of alcohol drinking increase deoxyribonucleic acid (DNA) methylation and reduce gene expression of proopiomelanocortin (POMC) and period 2 (PER2) in adult human subjects (Gangisetty et al., Alcohol Clin Exp Res, 43, 2019, 212). One hypothesis would be that methylation of these 2 genes is consistently associated with alcohol exposure and could be used as biomarkers to predict risk of prenatal alcohol exposure (PAE). Results of the present study provided some support for this hypothesis. METHODS: We conducted a series of studies to determine DNA methylation changes in stress regulatory genes proopiomelanocortin (POMC) and period 2 (PER2) using biological samples from 3 separate cohorts of patients: (i) pregnant women who consumed moderate-to-high levels of alcohol or low/unexposed controls, (ii) children with PAE and non-alcohol-exposed controls, and (iii) children with PAE treated with or without choline. RESULTS: We found pregnant women who consumed moderate-to-high levels of alcohol and gave birth to PAE children had higher DNA methylation of POMC and PER2. PAE children also had increased methylation of POMC and PER2. The differences in the gene methylation of PER2 and POMC between PAE and controls did not differ by maternal smoking status. PAE children had increased levels of stress hormone cortisol and adrenocorticotropic hormone. Choline supplementation reduced DNA hypermethylation and increased expression of POMC and PER2 in children with PAE. CONCLUSIONS: These data suggest that PAE significantly elevates DNA methylation of POMC and PER2 and increases levels of stress hormones. Furthermore, these results suggest the possibility that measuring DNA methylation levels of PER2 and POMC in biological samples from pregnant women or from children may be useful for identification of a woman or a child with PAE.


Asunto(s)
Depresores del Sistema Nervioso Central/efectos adversos , Etanol/efectos adversos , Proteínas Circadianas Period/metabolismo , Efectos Tardíos de la Exposición Prenatal , Proopiomelanocortina/metabolismo , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Colina/farmacología , Colina/uso terapéutico , Metilación de ADN/efectos de los fármacos , Suplementos Dietéticos , Epigénesis Genética/efectos de los fármacos , Femenino , Trastornos del Espectro Alcohólico Fetal/metabolismo , Trastornos del Espectro Alcohólico Fetal/prevención & control , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lipotrópicos/farmacología , Lipotrópicos/uso terapéutico , Masculino , Embarazo
7.
J Neuroinflammation ; 14(1): 83, 2017 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-28407740

RESUMEN

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.


Asunto(s)
Etanol/toxicidad , Microglía/metabolismo , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Receptores Opioides delta/fisiología , Receptores Opioides mu/fisiología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células Cultivadas , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Femenino , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Hipotálamo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas
8.
Alcohol Clin Exp Res ; 40(1): 134-40, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26727531

RESUMEN

BACKGROUND: Fetal alcohol exposure (FAE) increases the susceptibility to carcinogen-induced mammary cancer progression in rodent models. FAE also decreases ß-endorphin (ß-EP) level and causes hyperstress response, which leads to inhibition of immune function against cancer. Previous studies have shown that injection of nanosphere-attached dibutyryl cyclic adenosine monophosphate (dbcAMP) into the third ventricle increases the number of ß-EP neurons in the hypothalamus. In this study, we assessed the therapeutic potential of stress regulation using methods to increase hypothalamic levels of ß-EP, a neuropeptide that inhibits stress axis activity, in treatment of carcinogen-induced mammary cancer in fetal alcohol exposed rats. METHODS: Fetal alcohol exposed and control Sprague Dawley rats were given a dose of N-Nitroso-N-methylurea (MNU) at postnatal day 50 to induce mammary cancer growth. Upon detection of mammary tumors, the animals were either transplanted with ß-EP neurons or injected with dbcAMP-delivering nanospheres into the hypothalamus to increase ß-EP peptide production. Spleen cytokines were detected using reverse transcription polymerase chain reaction assays. Metastasis study was done by injecting mammary cancer cells MADB106 into jugular vein of ß-EP-activated or control fetal alcohol exposed animals. RESULTS: Both transplantation of ß-EP neurons and injection of dbcAMP-delivering nanospheres inhibited MNU-induced mammary cancer growth in control rats, and reversed the effect of FAE on the susceptibility to mammary cancer. Similar to the previously reported immune-enhancing and stress-suppressive effects of ß-EP transplantation, injection of dbcAMP-delivering nanospheres increased the levels of interferon-γ and granzyme B and decreased the levels of epinephrine and norepinephrine in fetal alcohol exposed rats. Mammary cancer cell metastasis study also showed that FAE increased incidence of lung tumor retention, while ß-EP transplantation inhibited lung tumor growth in both normal and fetal alcohol exposed rats. CONCLUSIONS: Our results suggest that increase of ß-EP production in the hypothalamus may serve as a potential therapeutic strategy for treating the cancer growth in patients with chronic stress and compromised immune function, such as the patients with FAE.


Asunto(s)
Hipotálamo/metabolismo , Neoplasias Mamarias Experimentales/patología , Neuronas/metabolismo , Efectos Tardíos de la Exposición Prenatal , betaendorfina/metabolismo , Alquilantes/toxicidad , Animales , Bucladesina/farmacología , Depresores del Sistema Nervioso Central/farmacología , Citocinas/efectos de los fármacos , Citocinas/genética , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Epinefrina/metabolismo , Etanol/farmacología , Femenino , Granzimas/efectos de los fármacos , Granzimas/metabolismo , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Interferón gamma/efectos de los fármacos , Interferón gamma/metabolismo , Neoplasias Mamarias Experimentales/inducido químicamente , Metilnitrosourea/toxicidad , Neuronas/citología , Neuronas/trasplante , Norepinefrina/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
9.
Addict Biol ; 21(1): 23-34, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25581210

RESUMEN

Alcohol exposure during fetal and early postnatal development can lead to an increased incidence of later life adult-onset diseases. Examples include central nervous system dysfunction, depression, anxiety, hyperactivity, and an inability to deal with stressful situations, increased infection and cancer. Direct effects of alcohol leading to developmental abnormalities often involve epigenetic modifications of genes that regulate cellular functions. Epigenetic marks carried over from the parents are known to undergo molecular programming events that happen early in embryonic development by a wave of DNA demethylation, which leaves the embryo with a fresh genomic composition. The proopiomelanocortin (Pomc) gene controls neuroendocrine-immune functions and is imprinted by fetal alcohol exposure. Recently, this gene has been shown to be hypermethylated through three generations. Additionally, the alcohol epigenetic marks on the Pomc gene are maintained in the male but not in the female germline during this transgenerational transmission. These data suggest that the male-specific chromosome might be involved in transmitting alcohol epigenetic marks through multiple generations.


Asunto(s)
Epigénesis Genética/genética , Trastornos del Espectro Alcohólico Fetal/genética , Exposición Paterna , Efectos Tardíos de la Exposición Prenatal/genética , Proopiomelanocortina/genética , Espermatozoides/metabolismo , Animales , Metilación de ADN/genética , Femenino , Regulación de la Expresión Génica , Células Germinativas , Humanos , Masculino , Embarazo
10.
Alcohol Clin Exp Res ; 39(1): 146-57, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25623413

RESUMEN

BACKGROUND: Alcohol exposure has adverse effects on stress physiology and behavioral reactivity. This is suggested to be due, in part, to the effect of alcohol on ß-endorphin (ß-EP)-producing neurons in the hypothalamus. In response to stress, ß-EP normally provides negative feedback to the hypothalamic-pituitary-adrenal axis and interacts with other neurotransmitter systems in the amygdala to regulate behavior. We examined whether ß-EP neuronal function in the hypothalamus reduces the corticosterone response to acute stress, attenuates anxiety-like behaviors, and modulates alcohol drinking in rats. METHODS: To determine whether ß-EP neuronal transplants modulate the stress response, anxiety behavior, and alcohol drinking, we implanted differentiated ß-EP neurons into the paraventricular nucleus (PVN) of the hypothalamus of normal, prenatal alcohol-exposed, and alcohol-preferring (P) and alcohol-non-preferring (NP) rats. We then assessed corticosterone levels in response to acute restraint stress and other markers of stress response in the brain and anxiety-like behaviors in the elevated plus maze and open-field assays. RESULTS: We showed that ß-EP neuronal transplants into the PVN reduced the peripheral corticosterone response to acute stress and attenuated anxiety-like behaviors. Similar transplants completely reduced the hypercorticosterone response and elevated anxiety behaviors in prenatal alcohol-exposed adult rats. Moreover, we showed that ß-EP reduced anxiety behavior in P rats with minimal effects on alcohol drinking during and following restraint stress. CONCLUSIONS: These data further establish a role of ß-EP neurons in the hypothalamus for regulating physiological stress response and anxiety behavior and resemble a potential novel therapy for treating stress-related psychiatric disorders in prenatal alcohol-exposed children and those genetically predisposed to increased alcohol consumption.


Asunto(s)
Consumo de Bebidas Alcohólicas/terapia , Ansiedad/terapia , Neuronas/trasplante , Núcleo Hipotalámico Paraventricular/cirugía , Efectos Tardíos de la Exposición Prenatal/terapia , betaendorfina/uso terapéutico , Amígdala del Cerebelo/metabolismo , Animales , Corticosterona/sangre , Hormona Liberadora de Corticotropina/biosíntesis , Femenino , Masculino , Aprendizaje por Laberinto , Ratones Endogámicos , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Ratas , Receptores de Hormona Liberadora de Corticotropina/biosíntesis , Restricción Física , betaendorfina/metabolismo
11.
Adv Exp Med Biol ; 815: 389-402, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25427920

RESUMEN

The idea that exposure to adverse environmental conditions and lifestyle choices during pregnancy can result in fetal programming that underlies disease susceptibility in adulthood is now widely accepted. Fetal alcohol exposed offspring displays many behavioral and physiological abnormalities including neuroendocrine-immune functions, which often carry over into their adult life. Since the neuroendocrine-immune system plays an important role in controlling tumor surveillance, fetal alcohol exposed offspring can be vulnerable to develop cancer. Animal studies have recently showed increased cancer growth and progression in various tissues of fetal alcohol exposed offspring. I will detail in this chapter the recent evidence for increased prostate carcinogenesis in fetal alcohol exposed rats. I will also provide evidence for a role of excessive estrogenization during prostatic development in the increased incidence of prostatic carcinoma in these animals. Furthermore, I will discuss the additional possibility of the involvement of impaired stress regulation and resulting immune incompetence in the increased prostatic neoplasia in the fetal alcohol exposed offspring.


Asunto(s)
Etanol/toxicidad , Feto/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal , Neoplasias de la Próstata/inducido químicamente , Animales , Carcinogénesis , Susceptibilidad a Enfermedades , Estrógenos/biosíntesis , Femenino , Humanos , Masculino , Embarazo , Ratas
12.
Alcohol Clin Exp Res ; 38(9): 2323-30, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25069392

RESUMEN

Hypothalamic proopiomelanocortin (POMC) neurons, one of the major regulators of the hypothalamic-pituitary-adrenal (HPA) axis, immune functions, and energy homeostasis, are vulnerable to the adverse effects of fetal alcohol exposure (FAE). These effects are manifested in POMC neurons by a decrease in Pomc gene expression, a decrement in the levels of its derived peptide ß-endorphin and a dysregulation of the stress response in the adult offspring. The HPA axis is a major neuroendocrine system with pivotal physiological functions and mode of regulation. This system has been shown to be perturbed by prenatal alcohol exposure. It has been demonstrated that the perturbation of the HPA axis by FAE is long-lasting and is linked to molecular, neurophysiological, and behavioral changes in exposed individuals. Recently, we showed that the dysregulation of the POMC system function by FAE is induced by epigenetic mechanisms such as hypermethylation of Pomc gene promoter and an alteration in histone marks in POMC neurons. This developmental programming of the POMC system by FAE altered the transcriptome in POMC neurons and induced a hyperresponse to stress in adulthood. These long-lasting epigenetic changes influenced subsequent generations via the male germline. We also demonstrated that the epigenetic programming of the POMC system by FAE was reversed in adulthood with the application of the inhibitors of DNA methylation or histone modifications. Thus, prenatal environmental influences, such as alcohol exposure, could epigenetically modulate POMC neuronal circuits and function to shape adult behavioral patterns. Identifying specific epigenetic factors in hypothalamic POMC neurons that are modulated by fetal alcohol and target Pomc gene could be potentially useful for the development of new therapeutic approaches to treat stress-related diseases in patients with fetal alcohol spectrum disorders.


Asunto(s)
Epigénesis Genética/fisiología , Trastornos del Espectro Alcohólico Fetal/genética , Desarrollo Fetal/fisiología , Sistema Hipotálamo-Hipofisario/fisiología , Proopiomelanocortina/genética , Estrés Psicológico/genética , Adulto , Animales , Femenino , Trastornos del Espectro Alcohólico Fetal/epidemiología , Trastornos del Espectro Alcohólico Fetal/metabolismo , Humanos , Red Nerviosa/fisiología , Sistema Hipófiso-Suprarrenal/fisiología , Embarazo , Efectos Tardíos de la Exposición Prenatal/epidemiología , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/metabolismo , Proopiomelanocortina/metabolismo , Estrés Psicológico/epidemiología , Estrés Psicológico/metabolismo
13.
Alcohol Clin Exp Res ; 38(12): 2988-97, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25581653

RESUMEN

BACKGROUND: Recently, retrograde tracing has provided evidence for an influence of hypothalamic ß-endorphin (BEP) neurons on the liver, but functions of these neurons are not known. We evaluated the effect of BEP neuronal activation on alcohol-induced liver injury and hepatocellular cancer. METHODS: Male rats received either BEP neuron transplants or control transplants in the hypothalamus and were randomly assigned to feeding alcohol-containing liquid diet or control liquid diet for 8 weeks or to treatment of a carcinogen diethylnitrosamine (DEN). Liver tissues of these animals were analyzed histochemically and biochemically for tissue injuries or cancer. RESULTS: Alcohol feeding increased liver weight and induced several histopathological changes such as prominent microvesicular steatosis and hepatic fibrosis. Alcohol feeding also increased the levels of triglyceride, hepatic stellate cell (HSC) activation factors, and catecholamines in the liver and endotoxin levels in the plasma. However, these effects of alcohol on the liver were reduced in animals with BEP neuron transplants. BEP neuron transplants also suppressed carcinogen-induced liver histopathologies such as extensive fibrosis, large focus of inflammatory infiltration, hepatocellular carcinoma (HCC), collagen deposition, numbers of preneoplastic foci, levels of HSC activation factors and catecholamines, as well as inflammatory milieu and increased the levels of natural killer cell cytotoxic factors in the liver. CONCLUSIONS: These findings are the first evidence for a role of hypothalamic BEP neurons in influencing liver functions. Additionally, the data identify that BEP neuron transplantation prevents hepatocellular injury and HCC formation possibly via influencing the immune function.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Modelos Animales de Enfermedad , Etanol/toxicidad , Hipotálamo/trasplante , Neoplasias Hepáticas/prevención & control , Neuronas/trasplante , betaendorfina , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Etanol/administración & dosificación , Femenino , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/patología , Masculino , Embarazo , Distribución Aleatoria , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley
14.
J Immunol ; 188(6): 2583-91, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22308312

RESUMEN

Prolonged subjection to unstable work or lighting schedules, particularly in rotating shift-workers, is associated with an increased risk of immune-related diseases, including several cancers. Consequences of chronic circadian disruption may also extend to the innate immune system to promote cancer growth, as NK cell function is modulated by circadian mechanisms and plays a key role in lysis of tumor cells. To determine if NK cell function is disrupted by a model of human shift-work and jet-lag, Fischer (344) rats were exposed to either a standard 12:12 light-dark cycle or a chronic shift-lag paradigm consisting of 10 repeated 6-h photic advances occurring every 2 d, followed by 5-7 d of constant darkness. This model resulted in considerable circadian disruption, as assessed by circadian running-wheel activity. NK cells were enriched from control and shifted animals, and gene, protein, and cytolytic activity assays were performed. Chronic shift-lag altered the circadian expression of clock genes, Per2 and Bmal1, and cytolytic factors, perforin and granzyme B, as well as the cytokine, IFN-γ. These alterations were correlated with suppressed circadian expression of NK cytolytic activity. Further, chronic shift-lag attenuated NK cell cytolytic activity under stimulated in vivo conditions, and promoted lung tumor growth following i.v. injection of MADB106 tumor cells. Together, these findings suggest chronic circadian disruption promotes tumor growth by altering the circadian rhythms of NK cell function.


Asunto(s)
Trastornos Cronobiológicos/complicaciones , Relojes Circadianos/fisiología , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/etiología , Animales , Western Blotting , Proteínas CLOCK/inmunología , Proteínas CLOCK/metabolismo , Trastornos Cronobiológicos/inmunología , Neoplasias Pulmonares/inmunología , Masculino , Fotoperiodo , Ratas , Ratas Endogámicas F344 , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
15.
Artículo en Inglés | MEDLINE | ID: mdl-39343719

RESUMEN

BACKGROUND: Prenatal alcohol exposure poses significant risks to offspring mental health. However, the interplay between genetic predispositions to mental health disorders and prenatal alcohol exposure remains incompletely understood, limiting our ability to develop effective interventions for these conditions. METHODS: Data from the Adolescent Brain and Cognitive Development (ABCD) Study were analyzed to explore associations between polygenic risk scores (PRS) for mental disorders and maternal alcohol consumption during pregnancy. Logistic regression and structural equation modeling were utilized to assess these relationships. RESULTS: Maternal alcohol consumption after pregnancy awareness was significantly associated with an increased genetic risk for specific mental health disorders, particularly bipolar disorder in offspring. The relationship between maternal alcohol consumption and mental health outcomes was influenced by polygenic risk scores, with both externalizing and internalizing problems being affected. CONCLUSIONS: Our findings highlight the specific interaction between increased genetic risk for bipolar disorder and prenatal alcohol exposure in shaping offspring mental health outcomes. The significant associations we observed underscore the importance of considering both polygenic risk scores and prenatal alcohol exposure when assessing mental health risks in children. These insights emphasize the need for targeted interventions that address both genetic predispositions and environmental exposures to better understand and mitigate the impact on offspring mental health.

16.
Sci Rep ; 14(1): 19886, 2024 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-39191924

RESUMEN

Prenatal alcohol-exposed (AE) infants and children often demonstrate disrupted sleep patterns, including more frequent awakenings, reduced total sleep time, and more night-to-night sleep variability. Despite the strong connection between sleep patterns and circadian rhythmicity, relatively little is known about circadian rhythm disruptions in individuals with AE. Recently, several reports demonstrated that evaluating the expression patterns of human clock genes in biological fluids could reveal an individual's circadian phenotype. Human saliva offers an emerging and easily available physiological sample that can be collected non-invasively for core-clock gene transcript analyses. We compared the expression patterns of core-clock genes and their regulatory genes in salivary samples of children aged 6-10 years-old with and without AE during the light cycle between ZT0-ZT11. We isolated the RNA from the samples and measured the expression patterns of core clock genes and clock regulating genes using the human specific primers with quantitative real-time PCR. Analysis of core clock genes expression levels in saliva samples from AE children indicates significantly altered levels in expression of core-clock BMAL1, CLOCK, PER1-3 and CRY1,2, as compared to those in age-matched control children. We did not find any sex difference in levels of clock genes in AE and control groups. Cosinor analysis was used to evaluate the rhythmic pattern of these clock genes, which identified circadian patterns in the levels of core clock genes in the control group but absent in the AE group. The gene expression profile of a salivary circadian biomarker ARRB1 was rhythmic in saliva of control children but was arhythmic in AE children. Altered expression patterns were also observed in clock regulatory genes: NPAS2, NFL3, NR1D1, DEC1, DEC2, and DBP, as well as chromatin modifiers: MLL1, P300, SIRT1, EZH2, HDAC3, and ZR1D1, known to maintain rhythmic expression of core-clock genes. Overall, these findings provide the first evidence that AE disturbs the circadian patten expression of core clock genes and clock-regulatory chromatin modifiers in saliva.


Asunto(s)
Ritmo Circadiano , Epigénesis Genética , Trastornos del Espectro Alcohólico Fetal , Saliva , Humanos , Saliva/metabolismo , Niño , Femenino , Masculino , Trastornos del Espectro Alcohólico Fetal/genética , Trastornos del Espectro Alcohólico Fetal/metabolismo , Ritmo Circadiano/genética , Embarazo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Regulación de la Expresión Génica , Relojes Circadianos/genética
17.
Neuro Oncol ; 2024 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-39093695

RESUMEN

BACKGROUND: Pituitary neuroendocrine tumors, PitNETs, are often aggressive and precipitate in distant metastases that are refractory to current therapies. However, the molecular mechanism in PitNETs' aggressiveness is not well understood. Developmental pluripotency-associated 4 (DPPA4) is known as a stem cell regulatory gene and overexpressed in certain cancers, but its function in the context of PitNETs' aggressiveness is not known. METHODS: We employed both rat and human models of PitNETs. In the rat pituitary tumor model (RPT), we used prenatal-alcohol-exposed (PAE) female Fischer rats which developed aggressive PitNETs following estrogen treatment, while in the human pituitary tumor (HPT) model, we used aggressively proliferative cells from pituitary tumors of patients undergone surgery. Various molecular, cellular, and epigenetic techniques were used to determine the role of DPPA4 in PitNETs' aggressiveness. RESULTS: We show that DPPA4 is overexpressed in association with increased cell stemness factors in aggressive PitNETs of PAE rats and of human patients. Gene-editing experiments demonstrate that DPPA4 increases the expression of cell stemness and tumor aggressiveness genes and promotes proliferation, colonization, migration, and tumorigenic potential of PitNET cells. ChIP assays and receptor antagonism studies reveal that DPPA4 binds to canonical WINTs promoters and increases directly or indirectly the Wnt/ß-catenin control of cell stemness, tumor growth, and aggressiveness of PitNETs. Epigenetic studies show involvement of histone methyltransferase in alcohol activation of DPPA4. CONCLUSIONS: These findings support a role of DPPA4 in tumor stemness and aggressiveness and provide a preclinical rationale for modulating this stemness regulator for the treatment of PitNETs.

18.
J Biol Chem ; 287(20): 16734-47, 2012 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-22451667

RESUMEN

In the natural killer (NK) cells, δ-opiate receptor (DOR) and µ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.


Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Neoplasias Mamarias Animales/inmunología , Multimerización de Proteína/efectos de los fármacos , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides mu/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Femenino , Células Asesinas Naturales , Ligandos , Masculino , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/terapia , Multimerización de Proteína/inmunología , Ratas , Ratas Endogámicas F344 , Receptores Opioides delta/agonistas , Receptores Opioides delta/inmunología , Receptores Opioides delta/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/inmunología , Receptores Opioides mu/metabolismo
19.
Alcohol Clin Exp Res ; 37(8): 1370-9, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23550806

RESUMEN

BACKGROUND: We have previously shown that ethanol (EtOH) increases cellular apoptosis to developing neurons via the effects on oxidative stress of neurons directly and via increasing production of microglia-derived factors. To study further the mechanism of EtOH action on neuronal apoptosis, we determined the effects of 2 well-known PKA activators, dibutyryl cAMP (dbcAMP) and brain-derived neurotrophic factor (BDNF), on EtOH-activated oxidative stress and apoptotic processes in the hypothalamic neurons in the presence and absence of microglial cells' influence. METHODS: In enriched neuronal cells from fetal rat hypothalami treated with EtOH or with conditioned medium from EtOH-treated microglia, we measured cellular apoptosis by the free nucleosome assay and the levels of cAMP, BDNF, O²â», reactive oxygen species (ROS), nitrite, glutathione (GSH), and catalase following treatment with EtOH or EtOH-treated microglial culture conditioned medium. Additionally, we tested the effectiveness of dbcAMP and BDNF in preventing EtOH or EtOH-treated microglial conditioned medium on cellular apoptosis and oxidative stress in enriched hypothalamic neuronal cell in primary cultures. RESULTS: Neuronal cell cultures following treatment with EtOH or EtOH-activated microglial conditioned medium showed decreased production levels of cAMP and BDNF. EtOH also increased apoptotic death as well as oxidative status, as demonstrated by higher cellular levels of oxidants but lower levels of antioxidants, in neuronal cells. These effects of EtOH on oxidative stress and cell death were enhanced by the presence of microglia. Treatment with BDNF or dbcAMP decreased EtOH or EtOH-activated microglial conditioned medium-induced changes in the levels of intracellular free radicals, ROS and O²â», nitrite, GSH, and catalase. CONCLUSIONS: These data support the possibility that EtOH by acting directly and via increasing the production of microglial-derived factors reduces cellular levels of cAMP and BDNF to increase cellular oxidative status and apoptosis in hypothalamic neuronal cells in primary cultures.


Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , AMP Cíclico/fisiología , Etanol/metabolismo , Hipotálamo/metabolismo , Microglía/fisiología , Animales , Antioxidantes/metabolismo , Apoptosis/inmunología , Células Cultivadas , Depresores del Sistema Nervioso Central/efectos adversos , Etanol/efectos adversos , Femenino , Trastornos del Espectro Alcohólico Fetal/etiología , Hipotálamo/efectos de los fármacos , Microglía/efectos de los fármacos , Estrés Oxidativo/inmunología , Embarazo , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/fisiología
20.
Alcohol Clin Exp Res ; 37(2): 252-62, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22823548

RESUMEN

BACKGROUND: Animals exposed to alcohol during the developmental period develop many physiological and behavioral problems because of neuronal loss in various brain areas including the hypothalamus. Because alcohol exposure is known to induce oxidative stress in developing neurons, we tested whether hypothalamic cells from the fetal brain exposed to ethanol (EtOH) may alter the cell-cell communication between neurons and microglia, thereby leading to increased oxidative stress and the activation of apoptotic processes in the neuronal population in the hypothalamus. METHODS: Using enriched neuronal and microglial cells from fetal rat hypothalami, we measured cellular levels of various oxidants (O2 -, reactive oxygen species, nitrite), antioxidants (glutathione [GSH]), antioxidative enzymes (glutathione peroxidase [GSH-Px], catalase, superoxide dismutase) and apoptotic death in neurons in the presence and absence of EtOH or EtOH-treated microglial culture medium. Additionally, we tested the effectiveness of antioxidative agents in preventing EtOH or EtOH-treated microglial conditioned medium actions on oxidative stress and apoptosis in neuronal cell cultures. RESULTS: Neuronal cell cultures showed increased oxidative stress, as demonstrated by higher cellular levels of oxidants but lower levels of antioxidant and antioxidative enzymes, as well as, increased apoptotic death following treatment with EtOH. These effects of EtOH on oxidative stress and cell death were enhanced by the presence of microglia. Antioxidative agents protected developing hypothalamic neurons from oxidative stress and cellular apoptosis which is caused by EtOH or EtOH-treated microglial culture medium. CONCLUSIONS: These data suggest that exposure of developing hypothalamic neurons to EtOH increases cellular apoptosis via the effects on oxidative stress of neurons directly and via increasing production of microglial-derived factor(s).


Asunto(s)
Apoptosis/efectos de los fármacos , Etanol/efectos adversos , Feto/efectos de los fármacos , Hipotálamo/metabolismo , Microglía/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Cromanos/farmacología , Medios de Cultivo Condicionados/farmacología , Relación Dosis-Respuesta a Droga , Etanol/antagonistas & inhibidores , Femenino , Feto/metabolismo , Hipotálamo/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Compuestos Organometálicos/farmacología , Embarazo , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Salicilatos/farmacología
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