Glutamate and Brain Glutaminases in Drug Addiction

Glutamate is the principal excitatory neurotransmitter in the central nervous system and its actions are related to the behavioral effects of psychostimulant drugs. In the last two decades, basic neuroscience research and preclinical studies with animal models are suggesting a critical role for glutamate transmission in drug reward, reinforcement, and relapse. Although most of the interest has been centered in post-synaptic glutamate receptors, the presynaptic synthesis of glutamate through brain glutaminases may also contribute to imbalances in glutamate homeostasis, a key feature of the glutamatergic hypothesis of addiction. Glutaminases are the main glutamate-producing enzymes in brain and dysregulation of their function have been associated with neurodegenerative diseases and neurological disorders; however, the possible implication of these enzymes in drug addiction remains largely unknown. This mini-review focuses on brain glutaminase isozymes and their alterations by in vivo exposure to drugs of abuse, which are discussed in the context of the glutamate homeostasis theory of addiction. Recent findings from mouse models have shown that drugs induce changes in the expression profiles of key glutamatergic transmission genes, although the molecular mechanisms that regulate drug-induced neuronal sensitization and behavioral plasticity are not clear.This work was financially supported by Grants RD12/0028/0013 (JM) and RD12/0028/0001 (FRF) of the RTA RETICS network from the Spanish Health Institute Carlos III, Grant SAF2015-64501-R from the Spanish Ministry of Economy and Competitivity (to JM and JMM) and Excellence Grant CVI-6656 (Regional Andalusian government) (to JM)

Biochemical parameters of silver catfish (Rhamdia quelen) after transport with eugenol or essential oil of Lippia alba added to the water

The transport of live fish is a routine practice in aquaculture and constitutes a considerable source of stress to the animals. The addition of anesthetic to the water used for fish transport can prevent or mitigate the deleterious effects of transport stress. This study investigated the effects of the addition of eugenol (EUG) (1.5 or 3.0 mu L L-1) and essential oil of Lippia alba (EOL) (10 or 20 mu L L-1) on metabolic parameters (glycogen, lactate and total protein levels) in liver and muscle, acetylcholinesterase activity (AChE) in muscle and brain, and the levels of protein carbonyl (PC), thiobarbituric acid reactive substances (TBARS) and nonprotein thiol groups (NPSH) and activity of glutathione-S-transferase in the liver of silver catfish (Rhamdia quelen; Quoy and Gaimard, 1824) transported for four hours in plastic bags (loading density of 169.2 g L-1). The addition of various concentrations of EUG (1.5 or 3.0 mu L L-1) and EOL (10 or 20 mu L L-1) to the transport water is advisable for the transportation of silver catfish, since both concentrations of these substances increased the levels of NPSH antioxidant and decreased the TBARS levels in the liver. In addition, the lower liver levels of glycogen and lactate in these groups and lower AChE activity in the brain (EOL 10 or 20 mu L L-1) compared to the control group indicate that the energetic metabolism and neurotransmission were lower after administration of anesthetics, contributing to the maintenance of homeostasis and sedation status.Fundacao de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS/PRONEX) [10/0016-8]; Conselho Nacional de Pesquisa e Desenvolvimento Cientifico (CNPq) [470964/2009-0]; Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES); CNPqinfo:eu-repo/semantics/publishedVersio

Analysis of Tumor Metabolism Reveals Mitochondrial Glucose Oxidation in Genetically Diverse Human Glioblastomas in the Mouse Brain In Vivo

SummaryDysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused 13C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo

Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism

Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene.We demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed.This is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was species- and tissue-specific providing evidence of a differential regulation of GLS2 transcripts in mammals

Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase

Cerium Oxide Nanoparticles Protect Cardiac Progenitor Cells from Oxidative Stress

Cardiac progenitor cells (CPCs) are a promising autologous source of cells for cardiac regenerative medicine. However, CPC culture in vitro requires the presence of microenvironmental conditions (a complex array of bioactive substance concentration, mechanostructural factors, and physicochemical factors) closely mimicking the natural cell surrounding in vivo, including the capability to uphold reactive oxygen species (ROS) within physiological levels in vitro. Cerium oxide nanoparticles (nanoceria) are redox-active and could represent a potent tool to control the oxidative stress in isolated CPCs. Here, we report that 24 h exposure to 5, 10, and 50 !g/mL of nanoceria did not a!ect cell growth and function in cardiac progenitor cells, while being able to protect CPCs from H2O2-induced cytotoxicity for at least 7 days, indicating that nanoceria in an e!ective antioxidant. Therefore, these "ndings con"rm the great potential of nanoceria for controlling ROS-induced cell damage

Basal ganglia volume predicts speed processing performance in obese adolescents

La obesidad se ha relacionado con cambios estructurales en los ganglios basales y alteraciones en dominios transversales como la velocidad de procesamiento. El objetivo del presente estudio es investigar la relación entre velocidad de procesamiento y volumen de los ganglios basales en adolescentes con y sin obesidad. Treinta y tres participantes obesos y treinta y tres con peso normal fueron resonados (Siemens 3T Trio) y evaluados neuropsicológicamente. Se compararon los resultados de un índice de velocidad y la ratio de los ganglios basales controlando por edad, sexo y tamaño de la cabeza. Se realizaron regresiones lineales con las estructuras relacionadas con este índice. Se encontraron diferencias en el índice de velocidad, pero no en la ratio de los ganglios basales. El grupo con obesidad mostró una correlación positiva entre velocidad y ratio del globo pálido derecho (r= .41; p= .021). No hallamos relaciones significativas en el grupo normopeso. La ratio del globo pálido derecho predijo parte del rendimiento en velocidad en adolescentes obesos. Las diferencias en velocidad de procesamiento entre adolescentes con y sin obesidad podrían estar moderadas por el tamaño de estructuras subcorticales como el globo pálido.Obesity is related to structural changes in basal ganglia and alterations among transversal domains such as speed processing. The aim of the study is to address the relationship between speed processing and basal ganglia volumes in adolescents with and without obesity. Thirty-three obese and 33 normal-weight participants underwent MRI acquisition (Siemens 3T Trio) and neuropsychological assessment to obtain an index of speed processing. Speed processing index and basal ganglia ratios (VolBrain software) were compared controlling for age, sex and head size. Linear regressions were conducted in the structures related to such index. Groups differed for speed processing index, but did not differ for basal ganglia ratios. The obese group showed positive correlations between speed processing index and the right globus pallidum ratio (r= .41; p= .021). None relationship was found among normal-weight adolescents. Concretely, the right globus pallidum ratio predicted part of the performance in speed in obese participants. The differences in speed processing between adolescents with and without obesity might be mediated by the size of subcortical structures such as the globus pallidum

Pyruvate: immunonutritional effects on neutrophil intracellular amino or alpha-keto acid profiles and reactive oxygen species production

For the first time the immunonutritional role of pyruvate on neutrophils (PMN), free α-keto and amino acid profiles, important reactive oxygen species (ROS) produced [superoxide anion (O2−), hydrogen peroxide (H2O2)] as well as released myeloperoxidase (MPO) acitivity has been investigated. Exogenous pyruvate significantly increased PMN pyruvate, α-ketoglutarate, asparagine, glutamine, aspartate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Moreover, increases in O2− formation, H2O2-generation and MPO acitivity in parallel with intracellular pyruvate changes have also been detected. Regarding the interesting findings presented here we believe, that pyruvate fulfils considerably the criteria for a potent immunonutritional molecule in the regulation of the PMN dynamic α-keto and amino acid pools. Moreover it also plays an important role in parallel modulation of the granulocyte-dependent innate immune regulation. Although further research is necessary to clarify pyruvate’s sole therapeutical role in critically ill patients’ immunonutrition, the first scientific successes seem to be very promising

A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity

The coordinated regulation of mitochondrial and nuclear activities is essential for cellular respiration and its disruption leads to mitochondrial dysfunction, a hallmark of ageing. Mitochondria communicate with nuclei through retrograde signalling pathways that modulate nuclear gene expression to maintain mitochondrial homeostasis. The monooxygenase CLK-1 (human homologue COQ7) was previously reported to be mitochondrial, with a role in respiration and longevity. We have uncovered a distinct nuclear form of CLK-1 that independently regulates lifespan. Nuclear CLK-1 mediates a retrograde signalling pathway that is conserved from Caenorhabditis elegans to humans and is responsive to mitochondrial reactive oxygen species, thus acting as a barometer of oxidative metabolism. We show that, through modulation of gene expression, the pathway regulates both mitochondrial reactive oxygen species metabolism and the mitochondrial unfolded protein response. Our results demonstrate that a respiratory enzyme acts in the nucleus to control mitochondrial stress responses and longevity

Vitamin C Enhances Vitamin E Status and Reduces Oxidative Stress Indicators in Sea Bass Larvae Fed High DHA Microdiets

Docosahexaenoic acid (DHA) is an essential fatty acid necessary for many biochemical, cellular and physiological functions in fish. However, high dietary levels of DHA increase free radical injury in sea bass (Dicentrarchus labrax) larvae muscle, even when vitamin E (α-tocopherol, α-TOH) is increased. Therefore, the inclusion of other nutrients with complementary antioxidant functions, such as vitamin C (ascorbic acid, vitC), could further contribute to prevent these lesions. The objective of the present study was to determine the effect of vitC inclusion (3,600 mg/kg) in high DHA (5 % DW) and α-TOH (3,000 mg/kg) microdiets (diets 5/3,000 and 5/3,000 + vitC) in comparison to a control diet (1 % DHA DW and 1,500 mg/kg of α-TOH; diet 1/1,500) on sea bass larvae growth, survival, whole body biochemical composition and thiobarbituric acid reactive substances (TBARS) content, muscle morphology, skeletal deformities and antioxidant enzymes, insulin-like growth factors (IGFs) and myosin expression (MyHC). Larvae fed diet 1/1,500 showed the best performance in terms of total length, incidence of muscular lesions and ossification degree. IGFs gene expression was elevated in 5/3,000 diet larvae, suggesting an increased muscle mitogenesis that was confirmed by the increase in the mRNA copies of MyHC. vitC effectively controlled oxidative damages in muscle, increased α-TOH larval contents and reduced TBARS content and the occurrence of skull deformities. The results of the present study showed the antioxidant synergism between vitamins E and C when high contents of DHA are included in sea bass larvae diets