María Belén Pascual

Involvement of TLR4 in the long-term epigenetic changes, rewarding and anxiety effects induced by intermittent ethanol treatment in adolescence

Studies in humans and experimental animals have demonstrated the vulnerability of the adolescent brain to actions of ethanol and the long-term consequences of binge drinking, including the behavioral and cognitive deficits that result from alcohol neurotoxicity, and increased risk to alcohol abuse and dependence. Although the mechanisms that participate in these effects are largely unknown, we have shown that ethanol by activating innate immune receptors, toll-like receptor 4 (TLR4), induces neuroinflammation, impairs myelin proteins and causes cognitive dysfunctions in adolescent mice. Since neuroimmune signaling is also involved in alcohol abuse, the aim of this study was to assess whether ethanol treatment in adolescence promotes the long-term synaptic and molecular events associated with alcohol abuse and addiction. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3g/kg) for 2 weeks, we showed that binge-like ethanol treatment in adolescent mice promotes short- and long-term alterations in synaptic plasticity and epigenetic changes in the promoter region of bdnf and fosb, which increased their expression in the mPFC of young adult animals. These molecular events were associated with long-term rewarding and anxiogenic-related behavioral effects, along with increased alcohol preference. Our results further showed the participation of neuroimmune system activation and the TLR4 signaling response since deficient mice in TLR4 (TLR4-KO) are protected against molecular and behavioral alterations of ethanol in the adolescent brain. Our results highlight a new role of the neuroimmune function and open up new avenues to develop pharmacological treatments that can normalize the immune signaling responsible for long-term effects in adolescence, including alcohol abuse and related disorders.

Autophagy Constitutes a Protective Mechanism against Ethanol Toxicity in Mouse Astrocytes and Neurons

Ethanol induces brain damage and neurodegeneration by triggering inflammatory processes in glial cells through activation of Toll-like receptor 4 (TLR4) signaling. Recent evidence indicates the role of protein degradation pathways in neurodegeneration and alcoholic liver disease, but how these processes affect the brain remains elusive. We have demonstrated that chronic ethanol consumption impairs proteolytic pathways in mouse brain, and the immune response mediated by TLR4 receptors participates in these dysfunctions. We evaluate the in vitro effects of an acute ethanol dose on the autophagy-lysosome pathway (ALP) on WT and TLR4-/- mouse astrocytes and neurons in primary culture, and how these changes affect cell survival. Our results show that ethanol induces overexpression of several autophagy markers (ATG12, LC3-II, CTSB), and increases the number of lysosomes in WT astrocytes, effects accompanied by a basification of lysosomal pH and by lowered phosphorylation levels of autophagy inhibitor mTOR, along with activation of complexes beclin-1 and ULK1. Notably, we found only minor changes between control and ethanol-treated TLR4-/- mouse astroglial cells. Ethanol also triggers the expression of the inflammatory mediators iNOS and COX-2, but induces astroglial death only slightly. Blocking autophagy by using specific inhibitors increases both inflammation and cell death. Conversely, in neurons, ethanol down-regulates the autophagy pathway and triggers cell death, which is partially recovered by using autophagy enhancers. These results support the protective role of the ALP against ethanol-induced astroglial cell damage in a TLR4-dependent manner, and provide new insight into the mechanisms that underlie ethanol-induced brain damage and are neuronal sensitive to the ethanol effects.

Gender differences in the inflammatory cytokine and chemokine profiles induced by binge ethanol drinking in adolescence

Heavy binge drinking in adolescence can cause long‐term cognitive and behavioral dysfunctions. Recent experimental evidence indicates the participation of immune system activation in the effects of ethanol in the adolescent brain and suggests gender differences. The present study aims to assess plasma cytokine and chemokine levels in male and female adolescents and young adults during acute alcohol intoxication and to correlate these results with the toll‐like receptor 4 (TLR4) response. The potential role of the TLR4 signaling response was also assessed in plasma and prefrontal cortex (PFC) of adolescent wild‐type and TLR4‐knockout male and female mice with binge ethanol treatment. The results showed that alcohol intoxication increased the plasma levels of several cytokine and chemokine [interferon‐γ, interleukin (IL)‐10, IL‐17A, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, fractalkine, monocyte chemoattractant protein 1 (MCP‐1) and macrophage inflammatory protein 1α (MIP‐1α)] and the upregulation of TLR4 mRNA levels occurred in intoxicated females, while elevation of colony‐stimulating factor was only observed in the plasma of males. In wild‐type female adolescent mice, intermittent ethanol treatment increased the levels of several cytokines (IL‐17A and IL‐1β) and chemokines (MCP‐1, MIP‐1α and fractalkine) in PFC and in serum (IL‐17A, MCP‐1 and MIP‐1α), but significant differences in the fractalkine levels in PFC were observed only in male mice. No changes in serum or prefrontal cortex cytokine and chemokine levels were noted in ethanol‐treated male or female TLR4‐knockout mice. Our findings revealed that females are more vulnerable than males to inflammatory effects of binge ethanol drinking and suggested that TLR4 is an important target of ethanol‐induced inflammation and neuroinflammation in adolescence.

Characterization and developmental expression of a glutamate decarboxylase from maritime pine

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a key enzyme in the synthesis of γ-aminobutyric acid (GABA) in higher plants. A complete cDNA encoding glutamate decarboxylase (GAD, EC 4.1.1.15) was characterized from Pinus pinaster Ait, and its expression pattern was studied to gain insight into the role of GAD in the differentiation of the vascular system. Pine GAD contained a C-terminal region with conserved residues and a predicted secondary structure similar to the calmodulin (CaM)-binding domains of angiosperm GADs. The enzyme was able to bind to a bovine CaM-agarose column and GAD activity was higher at acidic pH, suggesting that the pine GAD can be regulated in vivo by Ca2+/CaM and pH. A polyclonal antiserum was prepared against the pine protein. GAD expression was studied at activity, protein, and mRNA level and was compared with the expression of other genes during the differentiation of the hypocotyl and induction of reaction wood. In seedling organs, GABA levels closely matched GAD expression, with high levels in the root and during lignification of the hypocotyl. GAD expression was also induced in response to the production of compression wood and its expression matched the pattern of other genes involved in ethylene and 2-oxoglutarate synthesis. The results suggest of a role of GAD in hypocotyl and stem development in pine.

Biosynthesis and Metabolic Fate of Phenylalanine in Conifers.

The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.

A putative role for γ-aminobutyric acid (GABA) in vascular development in pine seedlings

Main conclusionA model for GABA synthesis in stems of pine seedlings is proposed. The localization of GABA in differentiating tracheids suggests a link between GABA production and vascular development.Abstractγ-aminobutyric acid (GABA) is a non-proteinogenic amino acid present in both prokaryotic and eukaryotic organisms. GABA plays a fundamental role as a signal molecule in the central nervous system in animals. In plants, GABA has been correlated with cellular elongation, plant development, gene expression regulation, synthesis of ethylene and other hormones, and signaling. Considering the physiological importance of GABA in plants, the lack of works about GABA localization in this kingdom seems surprising. In this work, the immunolocalization of GABA in root and hypocotyl during seedling development and in bent stem showing compression xylem has been studied. In the seedling root, the GABA signal was very high and restricted to the stele supporting previous evidences indicating a potential role for this amino acid in root growth and nutrient transport. In hypocotyl, GABA was localized in vascular tissues, including differentiating xylem, ray parenchyma and epithelial resin duct cells, drawing also a role for GABA in vascular development, communication and defense. During the production of compression wood, a special lignified wood produced when the stem loss its vertical position, a clear GABA signal was found in the new differentiating xylem cells showing a gradient-like pattern with higher signal in less differentiated elements. The results are in accordance with a previous work indicating that glutamate decarboxylase and GABA production are associated to vascular differentiation in pine Molina-Rueda et al. (Planta 232: 1471–1483, 2010). A model for GABA synthesis in vascular differentiation, communication, and defense is proposed in the stem of pine seedlings.

Fetal alcohol effects : Potential treatments from basic science

This article represents the proceedings of a symposium presented at the 2004 annual meeting of the International Society for Biomedical Research on Alcoholism, held in Mannheim, Germany. The presentations were as follows: 1) “Antioxidants Prevent Ethanol-Induced Cell Death in Developing Brain and in Cultured Neural Cells” by M. Pascual, M. C. Garcia-Minguillan, and Consuelo Guerri; 2) “Rational Development of Ethanol Antagonists” by Michael E. Charness and Michael F. Wilkemeyer; 3) “Choline Supplementation as a Treatment for Fetal Alcohol Effects” by Jennifer D. Thomas and Hector D. Dominguez; 4) “Cerebellar and Cortical Plasticity After Neonatal Alcohol Exposure: Model of Intervention” by Anna Y. Klintsova, Charles R. Goodlett, and William T. Greenough; and 5) “Circadian Rhythms in Prenatally Ethanol-Exposed Rats” by Hiromi Sakata-Haga.

Role of the innate immune system in the neuropathological consequences induced by adolescent binge drinking

Adolescence is a critical stage of brain maturation in which important plastic and dynamic processes take place in different brain regions, leading to development of the adult brain. Ethanol drinking in adolescence disrupts brain plasticity and causes structural and functional changes in immature brain areas (prefrontal cortex, limbic system) that result in cognitive and behavioral deficits. These changes, along with secretion of sexual and stress‐related hormones in adolescence, may impact self‐control, decision making, and risk‐taking behaviors that contribute to anxiety and initiation of alcohol consumption. New data support the participation of the neuroimmune system in the effects of ethanol on the developing and adult brain. This article reviews the potential pathological bases that underlie the effects of alcohol on the adolescent brain, such as the contribution of genetic background, the perturbation of epigenetic programming, and the influence of the neuroimmune response. Special emphasis is given to the actions of ethanol in the innate immune receptor toll‐like receptor 4 (TLR4), since recent studies have demonstrated that by activating the inflammatory TLR4/NFκB signaling response in glial cells, binge drinking of ethanol triggers the release of cytokines/chemokines and free radicals, which exacerbate the immune response that causes neuroinflammation/neural damage as well as short‐ and long‐term neurophysiological, cognitive, and behavioral dysfunction. Finally, potential treatments that target the neuroimmune response to treat the neuropathological and behavioral consequences of adolescent alcohol abuse are discussed.

Overexpression of a pine Dof transcription factor in hybrid poplars: A comparative study in trees growing under controlled and natural conditions

In this work, the role of the pine transcriptional regulator Dof 5 in carbon and nitrogen metabolism has been examined in poplar trees. The overexpression of the gene and potential effects on growth and biomass production were compared between trees growing in a growth chamber under controlled conditions and trees growing in a field trial during two growth seasons. Ten-week-old transgenic poplars exhibited higher growth than untransformed controls and exhibited enhanced capacity for inorganic nitrogen uptake in the form of nitrate. Furthermore, the transgenic trees accumulated significantly more carbohydrates such as glucose, fructose, sucrose and starch. Lignin content increased in the basal part of the stem likely due to the thicker stem of the transformed plants. The enhanced levels of lignin were correlated with higher expression of the PAL1 and GS1.3 genes, which encode key enzymes involved in the phenylalanine deamination required for lignin biosynthesis. However, the results in the field trial experiment diverged from those observed in the chamber system. The lines overexpressing PpDof5 showed attenuated growth during the two growing seasons and no modification of carbon or nitrogen metabolism. These results were not associated with a decrease in the expression of the transgene, but they can be ascribed to the nitrogen available in the field soil compared to that available for growth under controlled conditions. This work highlights the paramount importance of testing transgenic lines in field trials.

PpNAC1, a main regulator of phenylalanine biosynthesis and utilization in maritime pine

Summary The transcriptional regulation of phenylalanine metabolism is particularly important in conifers, long‐lived species that use large amounts of carbon in wood. Here, we show that the Pinus pinaster transcription factor, PpNAC1, is a main regulator of phenylalanine biosynthesis and utilization. A phylogenetic analysis classified PpNAC1 in the NST proteins group and was selected for functional characterization. PpNAC1 is predominantly expressed in the secondary xylem and compression wood of adult trees. Silencing of PpNAC1 in P. pinaster results in the alteration of stem vascular radial patterning and the down‐regulation of several genes associated with cell wall biogenesis and secondary metabolism. Furthermore, transactivation and EMSA analyses showed that PpNAC1 is able to activate its own expression and PpMyb4 promoter, while PpMyb4 is able to activate PpMyb8, a transcriptional regulator of phenylalanine and lignin biosynthesis in maritime pine. Together, these results suggest that PpNAC1 is a functional ortholog of the Arabidopsis SND1 and NST1 genes and support the idea that key regulators governing secondary cell wall formation could be conserved between gymnosperms and angiosperms. Understanding the molecular switches controlling wood formation is of paramount importance for fundamental tree biology and paves the way for applications in conifer biotechnology.