Toll-Like Receptor 4 Participates in the Myelin Disruptions Associated with Chronic Alcohol Abuse SILVIA ALFONSO-LOECHES, 1 MARIA PASCUAL, 1 ULISES G  OMEZ-PINEDO, 2 MAYA PASCUAL-LUCAS, 1 JAIME RENAU-PIQUERAS, 3 AND CONSUELO GUERRI 1 * 1 Department of Cellular Pathology, Pr  ıncipe Felipe Research Centre, Valencia, Spain 2 Laboratory of Regenerative Medicine/Neurology and Neurosurgery, Neuroscience Institute, IdISSC, Hospital Cl  ınico San Carlos, Madrid, Spain 3 Section of Biology and Cellular Pathology, Centro Investigaci on Hospital La Fe, Valencia, Spain KEY WORDS ethanol intake; TLR4 receptors; myelin proteins; demyelin- ation; neuronal damage; live imaging ABSTRACT Alcohol abuse and alcoholism can cause brain damage, loss of white matter, myelin fiber disruption, and even neuronal injury. The underlying mechanisms of these alterations remain elusive. We have shown that chronic ethanol intake, by activating glial toll-like receptor 4 (TLR4) receptors, trig- gers the production of inflammatory mediators and can cause brain damage. Because neuroinflammation may be associ- ated with demyelination and neuronal damage, we evaluate whether the ethanol-induced TLR4-dependent proinflamma- tory environment in the brain could be involved in the mye- lin disruptions observed in alcoholics. Using brains from wild-type (WT) and TLR4 knockout (KO, TLR4 2/2 ) mice, we demonstrate that chronic ethanol treatment downregulated proteins involved in myelination [proteolipid protein (PLP), myelin basic protein (MBP), myelin-oligodendrocyte glyco- protein, 2,3-cyclic-nucleotide-3-phosphodiesterase, and mye- lin-associated glycoprotein], while increased chondroitin sul- fate proteoglycan NG2 (NG2)-proteoglycan in several brain regions of ethanol-treated WT mice. The immunohistochem- istry analysis also revealed that ethanol-treatment-altered myelin morphology reduced the number of MBP-positive fibers and caused oligodendrocyte death, as demonstrated by an increase in caspase-3-positive oligodendrocytes. The in vivo imaging system further confirmed that chronic ethanol intake markedly reduced the PLP in WT mice. Most myelin alterations were not observed in brains from ethanol-treated TLR4 2/2 mice. Electron microscopy studies revealed that although 41–47% of axons showed myelin sheath disarrange- ments in the cerebral cortex and corpus callosum of WT etha- nol-treated mice, respectively, small focal fiber disruptions were noticed in these brain areas of ethanol-treated TLR4 2/2 mice. In summary, the present results suggest that ethanol- induced neuroinflammation might be involved in myelin dis- ruptions and white matter loss observed in human alco- holics. V V C 2012 Wiley Periodicals, Inc. INTRODUCTION Alcohol is a neurotoxic compound and its abuse can cause structural and functional brain alterations and can even induce neurodegeneration (Harper and Matsu- moto, 2005). Neuroimaging and postmortem studies have demonstrated reductions in brain weight in alco- holics, effects that have been attributed to the loss of white matter (Kril et al., 1997; Wang et al., 2009). Like- wise, a downregulation in both glial fibrillary acidic pro- tein and myelin-associated genes (Lewohl et al., 2005; Liu et al., 2006) as well as transcallosal white matter fiber degradation associated with cognitive and motor impairments has been described in human alcoholics (Pfefferbaum et al., 2010; Schulte et al., 2010). The ethanol actions on the brain are complex, although the molecular mechanisms underlying ethanol- induced brain damage and demyelination remain unknown. Recent reports have revealed that alcohol- induced brain damage, and neurodegeneration through mechanisms involved in activating the innate immune system and glial cells, leads to neuroinflammatory proc- esses (Alfonso-Loeches et al., 2010; Blanco et al., 2005; Qin et al., 2008; Valles et al., 2004). Neuroinflammation participates in the pathophysiol- ogy of progressive neurodegenerative disorders, includ- ing those associated with white matter injury (Glass et al., 2010). The brain inflammatory response is usually associated with innate immunity activation and specifi- cally with toll-like receptors (TLRs), the major molecules in immune response regulation in infections and central nervous system (CNS) damage. Activation of TLRs trig- gers the downstream stimulation of nuclear factor-jB (NF-jB) and the induction of those genes encoding inflammation-associated molecules and cytokines (Akira and Takeda, 2004). Indeed, a number of recent studies have provided evidence for the role of TLRs in neuroin- flammation associated with neurodegeneration (Glass et al., 2010). We have recently reported that ethanol is ca- pable of acting as a TLR4 agonist to cause not only the activation of astrocytes (Blanco et al., 2005) and micro- Grant sponsor: Spanish Ministry of Science and Innovation; Grant number: SAF 2009-07503; Grant sponsor: PNSD; Grant number: 2010I037; Grant sponsor: Generalitat Valenciana-Conselleria de Educaci on; Grant number: PROMETEO/ 2009/072; Grant sponsor: The Spanish Ministry of Health, Carlos III Institute, FEDER Funds (RTA-Network), M. Lautenschal€ ager Award. *Correspondence to: Consuelo Guerri, Cellular Pathology Laboratory, Centro de Investigaci on Pr  ıncipe Felipe, Avda. Autopista del Saler, 16., 46012 Valencia, Spain. E-mail: guerri@cipf.es Received 10 November 2011; Accepted 24 February 2012 DOI 10.1002/glia.22327 Published online 19 March 2012 in Wiley Online Library (wileyonlinelibrary. com). GLIA 60:948–964 (2012) V V C 2012 Wiley Periodicals, Inc.