glutaminase and Necrosis

Topics: Blood Coagulation; Blood Coagulation Factors; Clinical Enzyme Tests; Enzymes; Glutaminase; Humans; Necrosis; Peptide Hydrolases; Thrombin

Microglia are macrophage-like cells that constantly sense the microenvironment within the central nervous system (CNS). In the event of neuronal stress or injury, microglial cells rapidly react and change their phenotype. This response may lead to a deleterious type of microglial activation, which is often associated with neuroinflammation and neurotoxicity in several neuropathological conditions. We investigated the molecular mechanisms underlying triggering of microglial activation by necrotic neuronal damage.. Primary cultures of microglia were used to study the effect of necrotic neurons on microglial inflammatory responses and toxicity towards cerebellar granule neurons (CGN). The mouse hippocampal cell line, HT22, was used in this study as the main source of necrotic neurons to stimulate microglia. To identify the signal transduction pathways activated in microglia, primary microglial cultures were obtained from mice deficient in Toll-like receptor (TLR) -2, -4, or in the TLR adapter protein MyD88.. Necrotic neurons, but not other necrotic cell types, induced microglial activation which was characterized by up-regulation of: i) MHC class II; ii) co-stimulatory molecules, i.e. CD40 and CD24; iii) beta2 integrin CD11b; iii) pro-inflammatory cytokines, i.e. interleukin 6 (IL-6), IL-12p40 and tumor-necrosis factor (TNF); iv) pro-inflammatory enzymes such as nitric oxide synthase (iNOS, type II NOS), indoleamine 2,3-dioxygenase (IDO) and cyclooxygenase-2 (COX-2) and increased microglial motility. Moreover, microglia-conditioned medium (MCM) obtained from cultures of activated microglia showed increased neurotoxicity mediated through the N-methyl-D-aspartate receptor (NMDAR). The activation of microglia by necrotic neurons was shown to be dependent on the TLR-associated adapter molecule myeloid differentiation primary response gene (MyD88). Furthermore, MyD88 mediated enhanced neurotoxicity by activated microglia through up-regulation of the expression and activity of glutaminase, an enzyme that produces glutamate, which is an NMDAR agonist.. These results show that necrotic neurons activate in microglia a MyD88-dependent pathway responsible for a pro-inflammatory response that also leads to increased neurotoxic activity through induction of glutaminase. This finding contributes to better understanding the mechanisms causing increased neuroinflammation and microglial neurotoxicity in a neurodegenerative environment.

Topics: Animals; Cells, Cultured; Culture Media, Conditioned; Cytokines; Encephalitis; Gliosis; Glutamic Acid; Glutaminase; Inflammation Mediators; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Myeloid Differentiation Factor 88; Necrosis; Nerve Degeneration; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Toll-Like Receptors; Up-Regulation

Synaptic increase of glutamate level, when not coupled to a heightened energy production, renders neurons susceptible to death. Astrocyte uptake and recycling of synaptic glutamate as glutamine is a major metabolic pathway dependent on energy metabolism, which inter-relationships are not fully understood and remain controversial. We examine how the glutamate-glutamine cycle and glucose metabolism are modified in two in vivo models of severe and mild brain injury. Graded reductions of glutaminase, the glutamate synthetic enzyme, were evidenced combined with increases in glutamine synthetase, the inactivating glutamate enzyme. Increased lactate dhydrogenase (LDH) activity was only present after a more severe injury. These results indicate an in vivo adaptation of the glutamate-glutamine cycle in order to increase the net glutamine output, reduce glutamate excitotoxicity, and avoid neuronal death. We conclude that the graded modification of the glutamate-glutamine correlation and neuronal lactate availability may be key factors in the apoptotic and necrotic neuronal demise, whose control may prove highly useful to potentiate neuronal survival.

Topics: Animals; Apoptosis; Brain; Brain Injuries; Cell Death; Cell Survival; Denervation; Disease Models, Animal; Energy Metabolism; Fornix, Brain; Glutamate-Ammonia Ligase; Glutamic Acid; Glutaminase; Glutamine; Hippocampus; L-Lactate Dehydrogenase; Lactic Acid; Male; Necrosis; Nerve Degeneration; Neurons; Neurotoxins; Rats; Rats, Sprague-Dawley

Nucleated cells are more resistant to complement-mediated cell death than anucleated cells such as erythrocytes. There are few reports concerning the metabolic response of nucleated cells subjected to sub-lethal complement attack. It is possible that the rate of utilization of specific metabolic fuels by the cell is increased to enhance cell defence. We have measured the maximum activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase and glutaminase in rat mesenteric lymphocytes exposed to sub-lethal concentrations of activated complement (present in zymosan-activated serum, ZAS). These enzymes were carefully selected as they indicate changes of flux in glycolysis, TCA cycle, pentose phosphate pathway and glutaminolysis, respectively. The only enzyme activity to change on exposure of lymphocytes to ZAS was glutaminase, which was enhanced approximately by two-fold. Although rates of both glutamine and glucose utilization were enhanced by exposure to ZAS, only the rate of oxidation of glutamine was increased. Complement kills anucleated cells by simple osmotic lysis. However, it is likely that some nucleated cells will display characteristics of an ordered death mechanism and we have demonstrated that the concentration of lymphocyte ATP is dramatically decreased by activated complement. Nevertheless, the extent of cell death could be significantly reduced by the addition of inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). We conclude that glutamine metabolism is not only important for lymphocyte proliferative responses but is also important for cell defence from sub-lethal concentrations of activated complement. The rapid rate of complement-induced lymphocyte death reported here is suggested to be a consequence of over-activation of the nuclear enzyme PARP and ATP depletion.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Cell Death; Cell Survival; Complement System Proteins; Dose-Response Relationship, Drug; Glucose; Glucosephosphate Dehydrogenase; Glutaminase; Glutamine; Hexokinase; Humans; Lymphocytes; Male; Necrosis; Oxygen; Pentose Phosphate Pathway; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar

Glutamate dehydrogenase (GDH) and glutaminase activity in the rat brain was altered by chronic ammonia intoxication, subacute liver injury induced by CCl4 or digalactosamine, and the combination of them. GDH activity was found to increase considerably in ammonia intoxication without liver damage, probably as a result of enzyme induction. GDH activity failed to increase, while glutaminase was appreciably elevated. when ammonia intoxication was associated with hepatic injury. Toxicological studies have indicated that these changes in brain enzyme levels may be involved in the mechanism of ammonia neurotoxicity.

Topics: Ammonia; Animals; Brain; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Galactosamine; Glutamate Dehydrogenase; Glutaminase; Necrosis; Neurologic Manifestations; Rats