Abstract
Excessive alcohol intake affects hippocampal function and neuronal communication through oxidative stress and mitochondrial impairment. Previous studies have suggested that the melanocortin system (MCS) plays an essential role in alcohol consumption and addiction. The MCS is a hypothalamic region involved in regulating inflammatory processes in the brain, and its pharmacological activation through the melanocortin-4 receptor (MC4R) reduces both alcohol consumption and the neuroinflammatory responses in the brain. However, the cellular mechanisms involved in the beneficial actions of MCS against ethanol toxicity are not entirely understood. The objective of this study was to investigate the protective role of the MC4R pharmacological activator RO27-3225 on oxidative damage and mitochondrial impairment present in hippocampal neuronal cultures acutely exposed to ethanol (50, 75 mM, 24 h). Pre-treatment with RO27-3225 (250 nM, 1 h) prevented reactive oxygen species (ROS) increase, dysregulation of cytosolic calcium homeostasis, and mitochondrial potential loss induced by ethanol. Improvement of mitochondrial failure produced by RO27-3225 was accompanied by a significant increase in ATP production in ethanol-treated neurons. More importantly, RO27-3225 promoted the activation of the antioxidant pathway Nrf-2, demonstrated by an increase in the expression and nuclear translocation of Nrf-2, and upregulation of mRNA levels of NAD(P)H quinone oxidoreductase 1 (NQO1), an antioxidant enzyme which expression is activated by this pathway. These results suggest that the stimulation of MC4R prevents oxidative damage and mitochondrial stress induced by ethanol through the activation of the Nrf-2 pathway in cultured hippocampal neurons. These results are novel and demonstrate the critical function of MC4R in promoting antioxidant defense and reducing mitochondrial damage produced by ethanol in the brain.
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References
Abrahao KP, Salinas AG, Lovinger DM (2017) Alcohol and the brain: neuronal molecular targets, synapses, and circuits. Neuron 96:1223–1238. https://doi.org/10.1016/j.neuron.2017.10.032
Ali T, Rehman SU, Shah FA, Kim MO (2018) Acute dose of melatonin via Nrf2 dependently prevents acute ethanol-induced neurotoxicity in the developing rodent brain. J Neuroinflammation 15:1–19. https://doi.org/10.1186/s12974-018-1157-x
Bitto A, Polito F, Irrera N, Calò M, Spaccapelo L, Marini HR, Giuliani D, Ottani A, Rinaldi M, Minutoli L, Guarini S, Squadrito F, Altavilla D (2012) Protective effects of melanocortins on short-term changes in a rat model of traumatic brain injury. Crit Care Med 40:945–951
Boyadjieva NI, Sarkar DK (2013) Microglia play a role in ethanol-induced oxidative stress and apoptosis in developing hypothalamic neurons. Alcohol Clin Exp Res 37:252–262. https://doi.org/10.1111/j.1530-0277.2012.01889.x
Caruso C, Durand D, Schioth HB, Rey R, Seilicovich A, Lasaga M (2007) Activation of melanocortin 4 receptors reduces the inflammatory response and prevents apoptosis induced by lipopoly- saccharide and interferon-gamma in astrocytes. Endocrinology. 148:4918–4926
Cone RD (2005) Anatomy and regulation of the central melanocortin system. Nat Neurosci 8:571–578. https://doi.org/10.1038/nn1455
Crews, F. T. (2000). Neurotoxicity of alcohol: excitotoxicity, oxidative stress, neurotrophic factors, apoptosis, and cell adhesion molecules. In Noronha M, Eckardt M, Warren K (eds). Review of NIAAA’s neuroscience and behavioral research portfolio. Monograph no. 34. Bethesda, MD: National Institutes of Health, 189–206
Dembele K (2006) Intrauterine ethanol exposure results in hypothalamic oxidative stress and neuroendocrine alterations in adult rat offspring. AJP: Regulatory, Integrative and Comparative Physiology 291(3):R796–R802. https://doi.org/10.1152/ajpregu.00633.2005
Dinkova-Kostova, A. T., & Abramov, A. Y. (2015). The emerging role of Nrf2 in mitochondrial function. Free Radical Biology & Medicine, 88(PB), 179–188. 10.1016/j.freeradbiomed.2015.04.036
Gan L, Johnson JA (2014) Oxidative damage and the Nrf2-ARE pathway in neurodegenerative diseases. BBA - Molecular Basis of Disease 1842:1208–1218. https://doi.org/10.1016/j.bbadis.2013.12.011
Gantz I, Fong TM (2003) The melanocortin system. Am J Physiol Endocrinol Metab 284:E468–E474. https://doi.org/10.1152/ajpendo.00434.2002
Haorah J, Ramirez SH, Floreani N, Gorantla S, Morsey B, Persidsky Y (2008) Mechanism of alcohol-induced oxidative stress and neuronal injury. Free Radic Biol Med 45(11):1542–1550. https://doi.org/10.1016/j.freeradbiomed.2008.08.030
Giuliani D, Mioni C, Altavilla D, Leone S, Bazzani C, Minutoli L, Bitto A, Cainazzo MM, Marini H, Zaffe D, Botticelli AR, Pizzala R, Savio M, Necchi D, Schiöth HB, Bertolini A, Squadrito F, Guarini S (2006) Both early and delayed treatment with melanocortin 4 receptor-stimulating melanocortins produces neuroprotection in cerebral ischemia. Endocrinology 147:1126–1135
Giuliani D, Minutoli L, Ottani A, Spaccapelo L, Bitto A, Galantucci M, Altavilla D, Squadrito F, Guarini S (2012) Melanocortins as potential therapeutic agents in severe hypoxic conditions. Front Neuroendocrinol 33(2):179–193. https://doi.org/10.1016/j.yfrne.2012.04.001
Harper C (1998) The neuropathology of alcohol-specific brain damage, or does alcohol damage the brain? Brain Behav. Immun 57:101–110. https://doi.org/10.1097/00005072-199802000-00001
Haskew-Layton RE, Payappilly JB, Smirnova NA, Ma TC, Chan KK, Murphy TH, Guo H, Langley B, Sultana R, Butterfield DA, Santagata S, Alldred MJ, Gazaryan IG, Bell GW, Ginsberg SD, Ratan RR (2010) Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway. Proc Natl Acad Sci U S A 107(40):17385–17390. https://doi.org/10.1073/pnas.1003996107
Heaton MB, Paiva M, Madorsky I, Shaw G (2003) Ethanol effects on neonatal rat cortex: comparative analyses of neurotrophic factors, apoptosis-related proteins, and oxidative processes during vulnerable and resistant periods. Brain Res Dev Brain Res 145:249–262. https://doi.org/10.1016/j.devbrainres.2003.08.005
Hill JW, Faulkner LD (2017) The role of the melanocortin system in metabolic disease: new developments and advances. Neuroendocrinology 104:330–346. https://doi.org/10.1159/000450649
Holmstrom KM, Baird L, Zhang Y, Hargreaves I, Chalasani A, Land JM, Stanyer L, Yamamoto M, Dinkova-Kostova AT, Abramov AY (2013) Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration. Biol Open 2:761–770
Huang Q, Tatro JB (2002) α-Melanocyte stimulating hormone suppresses intracerebral tumor necrosis factor-α, and interleukin-1b gene expression following transient cerebral ischemia in mice. Neurosci. Lett 334:186–190
Ikram M, Saeed K, Khan A, Muhammad T, Khan MS, Jo MG, Rehman SU, Kim MO (2019) Natural dietary supplementation of curcumin protects mice brains against ethanol-induced oxidative stress-mediated neurodegeneration and memory impairment via Nrf2/TLR4/RAGE signaling. Nutrients 11:1082–1018. https://doi.org/10.3390/nu11051082
Jara C, Aránguiz A, Cerpa W, Tapia-Rojas C, Quintanilla RA (2018) Genetic ablation of tau improves mitochondrial function and cognitive abilities in the hippocampus. Redox Biol 18:279–294. https://doi.org/10.1016/j.redox.2018.07.010
Joshi G, Gan KA, Johnson DA, Johnson JA (2014) Increased Alzheimer’s disease-like pathology in the APP/ PS1DeltaE9 mouse model lacking Nrf2 through modulation of autophagy. Neurobiol Aging 36:664–679. https://doi.org/10.1016/j.neurobiolaging.2014.09.004
Karadayian AG, Malanga G, Czerniczyniec A, Lombardi P, Bustamante J, Lores-Arnaiz S (2017) Free radical production and antioxidant status in brain cortex non-synaptic mitochondria and synaptosomes at alcohol hangover onset. Free Radic Biol Med 108:692–703. https://doi.org/10.1016/j.freeradbiomed.2017.04.344
Kapinya KJ, Harms U, Harms C, Blei K, Katchanov J, Dirnagl U, Hörtnagl H (2003) Role of NAD(P)H: quinone oxidoreductase in the progression of neuronal cell death in vitro and following cerebral ischemia in vivo. J Neurochem 84:1028–1039
Kishi T, Aschkenasi CJ, Lee CE, Mountjoy KG, Saper CB, Elmquist JK (2003) Expression of melanocortin 4 receptor mRNA in the central nervous system of the rat. J Comp Neurol 457:213–235. https://doi.org/10.1002/cne.10454
Kouzoukas DE, Li G, Takapoo M, Moninger T, Bhalla RC, Pantazis NJ (2013) Intracellular calcium plays a critical role in the alcohol-mediated death of cerebellar granule neurons. J Neurochem 124:323–335. https://doi.org/10.1111/jnc.12076
Kouzoukas DE, Bhalla RC, Pantazis NJ (2018) Activation of cyclic GMP-dependent protein kinase blocks alcohol-mediated cell death and calcium disruption in cerebellar granule neurons. Neurosci Lett 676:108–112. https://doi.org/10.1016/j.neulet.2018.04.026
Kovac S, Angelova PR, Holmström KM, Zhang Y, Dinkova-Kostova AT, Abramov AY (2014) Nrf2 regulates ROS production by mitochondria and NADPH oxidase. BBA - General Subjects 1850:1–9. https://doi.org/10.1016/j.bbagen.2014.11.021
Kumar A, Singh CK, Lavoie HA, Dipette DJ, Singh US (2011) Resveratrol restores Nrf2 level and prevents ethanol-induced toxic effects in the cerebellum of a rodent model of fetal alcohol spectrum disorders. Mol Pharmacol 80(3):446–457. https://doi.org/10.1124/mol.111.071126
Lamarche F, Carcenac C, Gonthier B, Cottet-Rousselle C, Chauvin C, Barret L, Leverve X, Savasta M, Fontaine E (2013) Mitochondrial permeability transition pore inhibitors prevent ethanol-induced neuronal death in mice. Chem Res Toxicol 26:78–88
Lee Y-J, Beak S-Y, Choi I, Sung J-S (2017) Quercetin and its metabolites protect hepatocytes against ethanol-induced oxidative stress by activation of Nrf2 and AP-1. Food Sci Biotechnol 27:653–661. https://doi.org/10.1007/s10068-017-0287-8
Lerma-Cabrera JM, Carvajal F, de la Torre L, de la Fuente L, Navarro M, Thiele TE, Cubero I (2012) Control of food intake by MC4-R signaling in the lateral hypothalamus, nucleus accumbens shell, and ventral tegmental area: interactions with ethanol. Behav Brain Res 234:51–60. https://doi.org/10.1016/j.bbr.2012.06.006
Lerma-Cabrera JM, Carvajal F, Chotro G, Gaztañaga M, Navarro M, Thiele TE, Cubero I (2013) MC4-R signaling within the nucleus accumbens shell, but not the lateral hypothalamus, modulates ethanol palatability in rats. Behav Brain Res 239:51–54. https://doi.org/10.1016/j.bbr.2012.10.055
Mountjoy KG, Mortrud MT, Low MJ, Simerly RB, Cone RD (1994) Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 8:1298–1308. https://doi.org/10.1210/mend.8.10.7854347
Narasimhan M, Mahimainathan L, Rathinam ML, Riar AK, Henderson GI (2011) Overexpression of Nrf2 protects cerebral cortical neurons from ethanol-induced apoptotic death. Mol Pharmacol 80:988–999
Navarro M, Cubero I, Knapp DJ, Thiele TE (2003) MTII-induced reduction of voluntary ethanol drinking is blocked by pretreatment with AgRP-(83-132). Neuropeptides 37:338–344. https://doi.org/10.1016/j.npep.2003.10.003
Navarro M, Lerma-Cabrera JM, Carvajal F, Lowery EG, Cubero I, Thiele TE (2011) Assessment of voluntary ethanol consumption and the effects of a melanocortin (MC) receptor agonist on ethanol intake in mutant C57BL/6J mice lacking the MC-4 receptor. Alcohol Clinic Exp Res 35:1058–1066. https://doi.org/10.1111/j.1530-0277.2011.01438.x
O’Malley PM, Johnston LD (2002) Epidemiology of alcohol and other drug use among American college students. J Stud Alcohol 14:23–39. https://doi.org/10.15288/jsas.2002.s14.23
Orellana JA, Cerpa W, Carvajal MF, Lerma-Cabrera JM, Karahanian E, Osorio-Fuentealba C, Quintanilla RA (2017) New implications for the melanocortin system in alcohol drinking behavior in adolescents: the glial dysfunction hypothesis. Front Cell Neurosci 11:4–23. https://doi.org/10.3389/fncel.2017.00090
Ottani A, Giuliani D, Mioni C, Galantucci M, Minutoli L, Bitto A, Altavilla D, Zaffe D, Botticelli AR, Squadrito F, Guarini S (2009) Vagus nerve mediates the protective effects of melanocortins against cerebral and systemic damage after ischemic stroke. J Cereb Blood Flow Metab 29:512–523
Pérez MJ, Quintanilla RA (2017) Development of disease_ duality of the mitochondrial permeability transition pore. Dev Biol 426:1–7
Pérez MJ, Jara C, Quintanilla RA (2018a) Contribution of tau pathology to mitochondrial impairment in neurodegeneration. Front Neurosci 12:441. https://doi.org/10.3389/fnins.2018.00441
Pérez MJ, Vergara-Pulgar K, Jara C, Cabezas-Opazo F, Quintanilla RA (2018b) Caspase-cleaved tau impairs mitochondrial dynamics in Alzheimer’s disease. Mol Neurobiol 55:1004–1018. https://doi.org/10.1007/s12035-017-0385-x
Pérez MJ, Ponce DP, Aránguiz A, Behrens MI, Quintanilla RA (2018c) Mitochondrial permeability transition pore contributes to mitochondrial dysfunction in fibroblasts of patients with sporadic Alzheimer’s disease. Redox Biol 19:290–300. https://doi.org/10.1016/j.redox.2018.09.001
Ploj K, Roman E, Kask A, Hyytiä P, Schiöth HB, Wikberg JES, Nylander I (2002) Effects of melanocortin receptor ligands on ethanol intake and opioid peptide levels in alcohol-preferring AA rats. Brain Res Bull 59:97–104. https://doi.org/10.1016/S0361-9230(02)00844-4
Qin L, Crews FT (2012) NADPH oxidase and reactive oxygen species contribute to alcohol-induced microglial activation and neurodegeneration. J Neuroinflammation 9:5. https://doi.org/10.1186/1742-2094-9-5
Quintanilla RA, Muñoz FJ, Metcalfe MJ, Hitschfeld M, Olivares G, Godoy JA, Inestrosa NC (2005) Trolox and 17beta-estradiol protect against amyloid beta-peptide neurotoxicity by a mechanism that involves modulation of the Wnt signaling pathway. J Biol Chem 280:11615–11625. https://doi.org/10.1074/jbc.M411936200
Quintanilla RA, Jin YN, Bernhardi v R, Johnson GVW (2013a) Mitochondrial permeability transition pore induces mitochondria injury in Huntington disease. Mol Neurodegener 8:45. https://doi.org/10.1186/1750-1326-8-45
Quintanilla RA, Godoy JA, Alfaro I, Cabezas D, Bernhardi v R, Bronfman M, Inestrosa NC (2013b) Thiazolidinediones promote axonal growth through the activation of the JNK pathway. PLoS One 8:e65140. https://doi.org/10.1371/journal.pone.0065140
Ramirez D, Saba J, Turati J, Carniglia L, Imsen M, Mohn C et al (2019) NDP-MSH reduces oxidative damage induced by palmitic acid in primary astrocytes. J Neuroendocrinol 31:e12673–e12614. https://doi.org/10.1111/jne.12673
Ramsey CP, Glass CA, Montgomery MB, Lindl KA, Ritson GP, Chia LA, Chu CT, Jordan-Sciutto KL (2007) Expression of Nrf2 in neurodegenerative diseases. J Neuropathol Exp Neurol 66:75–85. https://doi.org/10.1097/nen.0b013e31802d6da9
Rajput P, Jangra A, Kwatra M, Mishra A, Lahkar M (2017) Alcohol aggravates stress-induced cognitive deficits and hippocampal neurotoxicity: protective effect of melatonin. Biomedicine Et Pharmacotherapy 91:457–466. https://doi.org/10.1016/j.biopha.2017.04.077
Roberto M, Varodayan FP (2017) Synaptic targets: chronic alcohol actions. Neuropharmacology 122:85–99. https://doi.org/10.1016/j.neuropharm.2017.01.013
Saba LM, Flink SC, Vanderlinden LA, Israel Y, Tampier L, Colombo G, Kiianmaa K, Bell RL, Printz MP, Flodman P, Koob G, Richardson HN, Lombardo J, Hoffman PL, Tabakoff B (2015) The sequenced rat brain transcriptome - its use in identifying networks predisposing alcohol consumption. FEBS J 282:3556–3578. https://doi.org/10.1111/febs.13358
Schapira AH (2012) Mitochondrial diseases. Neuron 379(9828):1825–1834. https://doi.org/10.1016/S0140-6736(11)61305-6
Spaccapelo L, Bitto A, Galantucci M, Ottani A, Irrera N, Minutoli L, Altavilla D, Novellino E, Grieco P, Zaffe D, Squadrito F, Giuliani D, Guarini S (2011) Melanocortin MC4 receptor agonists counteract late inflammatory and apoptotic responses and improve neuronal functionality after cerebral ischemia. Eur J Pharmacol 670:479–486
Spear LP (2015) Adolescent alcohol exposure are there separable vulnerable periods within adolescence? Physiol Behav 148:122–130. https://doi.org/10.1016/j.physbeh.2015.01.027
Squeglia LM, Jacobus J, Tapert SF (2014) The effect of alcohol use on human adolescent brain structures and systems. Handb Clin Neurol 125:501–510. https://doi.org/10.1016/B978-0-444-62619-6.00028-8
Shield KD, Parry C, Rehm J (2013) Chronic diseases and conditions related to alcohol use. Alcohol Res 35:155–173
Tapia-Rojas, C., Mira, R. G., Torres, A. K., Jara, C., Pérez, M. J., Vergara, E. H., et al. (2017). Alcohol consumption during adolescence: a link between mitochondrial damage and ethanol brain intoxication. Birth Defec Res 109, 1623–1639. doi.org/10.1002/bdr2.1172
Tapia-Rojas C, Carvajal FJ, Mira RG, Arce C, Lerma-Cabrera JM, Orellana JA, Cerpa W, Quintanilla RA (2018) Adolescent binge alcohol exposure affects the brain function through mitochondrial impairment. Mol Neurobiol 55:4473–4491. https://doi.org/10.1007/s12035-017-0613-4
Tapia-Rojas C, Torres AK, Quintanilla RA (2019) Adolescence binge alcohol consumption induces hippocampal mitochondrial impairment that persists during the adulthood. Neuroscience 406:356–368. https://doi.org/10.1016/j.neuroscience.2019.03.018
Ullah I, Ullah N, Naseer MI, Lee HY, Kim MOK (2012) Neuroprotection with metformin and thymoquinone against ethanol-induced apoptotic neurodegeneration in prenatal rat cortical neurons. BMC Neurosci 13:11–11
Vargas MR, Johnson JA (2009) The Nrf2–ARE cytoprotective pathway in astrocytes. Expert Rev Mol Med 11:e17. https://doi.org/10.1017/S1462399409001094
Wang H, Wang X, Li Y, Yu H, Wang C, Feng C, Xu G, Chen J, You J, Wang P, Wu X, Zhao R, Zhang G (2018) Chronic ethanol exposure induces SK-N-SH cell apoptosis by increasing N-methyl-D-aspartic acid receptor expression and intracellular calcium. Experimental and Therapeutic Medicine:1–10. https://doi.org/10.3892/etm.2018.5902
Worhunsky PD, Dager AD, Meda SA, Khadka S, Stevens MS, Austad CS, Raskin SA, Tennen H, Wood RM, Fallahi CR, Potenza MN, Pearlson GD (2016) A preliminary prospective study of an escalation in ‘maximum daily drinks,’ frontoparietal circuitry, and impulsivity-related domains in young adult drinkers. Neuropsychopharmacology 41:1637–1647
World Health Organization. (2018). Global status report on alcohol and health 2018. https://www.who.int/substance_abuse/publications/global_alcohol_report/en/
Wozniak DF, Hartman RE, Boyle MP, Vogt SK, Brooks AR, Tenkova T, Young C, Olney JW, Muglia LJ (2004) Apoptotic neurodegeneration induced by ethanol in neonatal mice is associated with profound learning/memory deficits in juveniles followed by progressive functional recovery in adults. Neurobiol Dis 17:403–414
Yeligar SM, Machida K, Kalra VK (2010) Ethanol-induced HO-1 and NQO1 are differentially regulated by HIF-1α and Nrf2 to attenuate inflammatory cytokine expression. J Biol Chem 285:35359–35373
Zhang H-H, Liu J, Qin G-J, Li X-L, Du P-J, Hao X et al (2017) Melanocortin 4 receptor activation attenuates mitochondrial dysfunction in skeletal muscle of diabetic rats. J Cell Biochem 118:4072–4079. https://doi.org/10.1002/jcb.26062]
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This work was supported by Fondo de Ciencia y Tecnología (FONDECYT), Chile (Grant No. 1170441) and CONICYT-PIA Anillo ACT1411 (to RAQ), Chile
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RAQ and MJP conceived the study, performed the experiments, and analyzed the data; CTM, GM, and AA performed the experiments; RAQ wrote the manuscript. All authors read and approved the final version of the manuscript.
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Supplementary Figure 1
The melanocortin-4 receptor agonist RO27-3225 enhances the activation of the Nrf-2 antioxidant pathway in hippocampal neurons treated with ethanol. Complementary images of immunofluorescence studies for Nrf-2 showed in Fig.5. The bar represents 20 μm. (PNG 1736 kb)
Supplementary Figure 2.
Fluorescent background control for secondary antibody anti-Alexa 488 in fixed hippocampal neuronal cultures. (A) Hippocampal cultured neurons were submitted to the same immunofluorescence protocol (see material and methods) omitting anti-Nrf2 polyclonal antibody but loaded with DAPI. (B) Hippocampal cultured neurons were submitted to the same immunofluorescence protocol (see material and methods) omitting anti-Nrf2 polyclonal antibody. We did not detect significant fluorescence staining from Anti-Alexa 488 secondary antibody incubation. The bar represents 20 μm. (PNG 1366 kb) (PNG 1366 kb)
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Quintanilla, R.A., Pérez, M.J., Aranguiz, A. et al. Activation of the Melanocortin-4 Receptor Prevents Oxidative Damage and Mitochondrial Dysfunction in Cultured Hippocampal Neurons Exposed to Ethanol. Neurotox Res 38, 421–433 (2020). https://doi.org/10.1007/s12640-020-00204-1
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DOI: https://doi.org/10.1007/s12640-020-00204-1