TLR4 elimination prevents synaptic and myelin alterations and long-term cognitive dysfunctions in adolescent mice with intermittent ethanol treatment
Introduction
Alcohol binge drinking is prevalent in adolescence, and a high percentage of European and USA teenagers have reported heavy episodic drinking, defined as consuming five or more drinks on one occasion in the past 2 weeks (Danielsson et al., 2012, Johnston et al., 2013). These high rates of heavy alcohol use are concerning since many studies have indicated that heavy alcohol consumption can impair neurocognitive development which affects several neuropsychological domains, including memory, executive functioning, visuospatial skills, and sustained attention (Brown et al., 2000, Giancola et al., 2001). Longitudinal studies have also shown adverse effects of adolescent drinking on the development of visuospatial processing, attention and working memory (Hanson et al., 2011, Squeglia et al., 2009, Tapert et al., 2002).
Adolescence is an important brain maturation period during which some brain regions undergo remodeling and functional changes, which increase neuronal connectivity and change synaptic plasticity (Alfonso-Loeches and Guerri, 2011, Toga et al., 2006). The prefrontal cortex (PFC), which coordinates higher-order cognitive processes and executive functions, is the last brain region to mature (Mills et al., 2014). Gray matter reduction begins primarily in early adolescence (≈12–14 years) in posterior brain regions and progresses to more anterior regions (Gogtay et al., 2004), such as the PFC, to continue into early adulthood (mid-20s) (Sowell et al., 2001). Reduction in gray matter, which is particularly evident in the PFC, is associated with the pruning of excess neurons (Paus, 2005), and with synapse stabilization. Concomitantly, the white matter volume and myelination of fiber tracts increases in the PFC during adolescence, event associated with enhanced neuronal conduction and communication (Barnea-Goraly et al., 2005, Giedd et al., 1999, Pfefferbaum, 2004). All these processes are an integral component of neurocognitive development and generate efficient information processing and improved cognition (Squeglia et al., 2013). These extensive developing changes in brain maturation might explain the adolescent brain’s vulnerability to the deleterious effects of ethanol (Alfonso-Loeches and Guerri, 2011, Jacobus and Tapert, 2013). PFC-dependent behaviors have been described to be altered in human alcoholics (Fortier et al., 2009, Kamarajan et al., 2010) and also in PFC-damaged rodents (Bissonette et al., 2008), or after an acute ethanol challenge (Brown et al., 2007). Neuroimaging studies have also reported changes in microstructural and functional myelin integrity in different brain areas in adolescents with alcohol abuse (Bava et al., 2009a, Schweinsburg et al., 2010). These myelin alterations may be related with attention and spatial working memory deficits in human adolescents who participate in heavy alcohol abuse (Tapert et al., 1999, Tapert et al., 2004).
PFC is one of the brain regions more affected by ethanol drinking during adolescence and can cause PFC-mediated control deficits in adulthood (Gass et al., 2014). The underlying mechanisms of neurotoxic and behavioral effects of ethanol in adolescence are presently unknown. Our previous studies indicated that by activating the innate immune system, particularly the toll-like receptor 4 (TLR4) signaling response, ethanol triggers the activation of transcription factors NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) and AP-1 (activating protein-1), which induce the production of cytokines and inflammatory mediators, that cause brain damage. Indeed, we have shown that by activating TLR4 responses in glial cells, chronic alcohol consumption induces neuroinflammation, gliosis, demyelination and brain damage (Alfonso-Loeches et al., 2010, Alfonso-Loeches et al., 2012, Blanco et al., 2005, Fernandez-Lizarbe et al., 2009), and these effects have been associated with behavioral impairments (Pascual et al., 2011).
Our previous studies have also provided evidence that binge-like ethanol treatment in adolescent rats induces some inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and ciclooxygenase-2 (COX-2), in the cerebral cortex and the hippocampus, causing neural death and cognitive alterations (Pascual et al., 2007). Neural death can, in turn, enhance neuroinflammation by the release of danger-associated molecular pattern (DAMP) molecules, such as high-mobility group box-1 (HMGB1), a nuclear protein which is passively released during stress, cell injury and necrosis, or is actively secreted as a cytokine (Andersson and Tracey, 2011). HMGB1 can bind and activate several receptors, including TLRs, receptors for advanced glycation end products (RAGE) and C-X-C chemokine receptor 4 (CXCR4) (Yang et al., 2013). Recent studies have demonstrated that ethanol treatment induces the release of HMGB1 in the brain and liver, which contributes to ethanol-induced pathology in both organs (Ge et al., 2014, Vetreno et al., 2013, Whitman et al., 2013).
Despite the involvement of TLR4 in adult neuroinflammation, whether TLR4 activation participates in the myelin and synaptic dysfunction, and long-term neuroinflammatory and cognitive behavioral impairments associated with binge drinking in adolescence is presently unknown. This study reveals, for the first time, the critical role of TLR4 receptors in neuroinflammation and brain damage induced by intermittent ethanol treatment in the PFC of adolescent wild-type (WT) mice since changes in neither the production of cytokines and inflammatory mediators nor in myelin and synaptic structures were observed in TLR4-deficient (TLR4-KO) mice treated with alcohol. Elimination of the TLR4 function also prevents ethanol-induced long-lasting cognitive alterations, which suggests the role of TLR4 signaling and neuroinflammation in ethanol-induced brain alterations in adolescence.
Section snippets
Animals and treatments
Female C57BL/6 WT (Harlan Ibérica, Barcelona, Spain) and TLR4-KO knockout (KO) mice (C57BL/6 background, kindly provided by Dr. S. Akira, Osaka University, Suita, Japan) aged 30 days were used. All the animals were kept under controlled light and dark (12/12 h), temperature (23 °C), and humidity (60%) conditions. All the experimental procedures were carried out in accordance with the guidelines approved by the European Communities Council Directive (86/609/ECC) and by Spanish Royal Decree
Intermittent ethanol treatment activates TLR4 signaling pathways and promotes inflammatory mediators in the PFC of adolescent mice
To demonstrate the involvement of TLR4 signaling in the inflammatory response induced by intermittent ethanol treatment in adolescent animals, we evaluated the activation of different kinases, which are associated with the activation of the TLR4 signaling pathway (Akira and Takeda, 2004), including MAPK and NFκB. Fig. 1A shows that intermittent ethanol treatment promotes both the phosphorylation of ERK, p38 and JNK and the activation of transcription factor NFκB in the PFC of WT mice. Since the
Discussion
Increasing evidence supports the role of the neuroimmune response in the pathophysiology of many neurological and psychiatric disorders, such as neurodegeneration, depression and epilepsy (Capuron and Miller, 2011, Jones and Thomsen, 2013), and in alcohol abuse disorders (Mayfield et al., 2013). Studies done in the last decade have also indicated the adverse consequences of heavy drinking in adolescence, which can induce myelin alterations and other structural and functional brain dysfunctions
Acknowledgments
We would like to thank C. Guarch for her contribution in some experiments. We also thank the Electron Microscopy and Cytomics Services at the Centro de Investigación Príncipe Felipe. This work has been supported by grants from the European Foundation for Alcohol Research (EA-13-08), Plan Nacional sobre Drogas (PNSD 2010-I010), Institute Carlos III and FEDER funds (RTA-Network,RD12/0028/0007;RD12/0028/0005), GV-Conselleria d’Educació: PROMETEO II/2014-063. J. Montesinos and A. Pla were supported
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