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Minimal hepatic encephalopathy (MHE) is a neuropsychiatric syndrome produced by central nervous system dysfunction subsequent to liver disease. Hyperammonemia and inflammation act synergistically to alter neurotransmission, leading to the cognitive and motor alterations in MHE, which are reproduced in rat models of chronic hyperammonemia. Patients with MHE show altered functional connectivity in different neural networks and a reduced response in the cognitive potential mismatch negativity (MMN), which correlates with attention deficits. The mechanisms by which MMN is altered in MHE remain unknown. The objectives of this work are as follows: To assess if rats with chronic hyperammonemia reproduce the reduced response in the MMN found in patients with MHE. Analyze the functional connectivity between the areas (CA1 area of the dorsal hippocampus, prelimbic cortex, primary auditory cortex, and central inferior colliculus) involved in the generation of the MMN and its possible alterations in hyperammonemia. Granger causality analysis has been applied to detect the net flow of information between the population neuronal activities recorded from a local field potential approach. Analyze if altered MMN response in hyperammonemia is associated with alterations in glutamatergic and GABAergic neurotransmission. Extracellular levels of the neurotransmitters and/or membrane expression of their receptors have been analyzed after the tissue isolation of the four target sites. The results show that rats with chronic hyperammonemia show reduced MMN response in hippocampus, mimicking the reduced MMN response of patients with MHE. This is associated with altered functional connectivity between the areas involved in the generation of the MMN. Hyperammonemia also alters membrane expression of glutamate and GABA receptors in hippocampus and reduces the changes in extracellular GABA and glutamate induced by the MMN paradigm of auditory stimulus in hippocampus of control rats. The changes in glutamatergic and GABAergic neurotransmission and in functional connectivity between the brain areas analyzed would contribute to the impairment of the MMN response in rats with hyperammonemia and, likely, also in patients with MHE., (© 2019 International Society for Neurochemistry.)

Activation of NMDA receptors leads to activation of cAMP-dependent protein kinase (PKA). The main substrates phosphorylated by PKA following NMDA receptor activation remain unidentified. The aim of this work was to identify a major substrate phosphorylated by PKA following NMDA receptor activation in cerebellar neurones in culture, and to assess whether this phosphorylation may be involved in neuronal death induced by excessive NMDA receptor activation. The main PKA substrate following NMDA receptor activation was identified by MALDI-TOFF fingerprinting as the nuclear protein, matrin 3. PKA-mediated phosphorylation of matrin 3 is followed by its degradation. NMDA receptor activation in rat brain in vivo by ammonia injection also induced PKA-mediated matrin 3 phosphorylation and degradation in brain cell nuclei. Blocking NMDA receptors in brain in vivo with MK-801 reduced basal phosphorylation of matrin 3, suggesting that it is modulated by NMDA receptors. Inhibition of PKA with H-89 prevents NMDA-induced phosphorylation and degradation of matrin 3 as well as neuronal death. These results suggest that PKA-mediated phosphorylation of matrin 3 may serve as a rapid way of transferring information from synapses containing NMDA receptors to neuronal nuclei under physiological conditions, and may contribute to neuronal death under pathological conditions.

Hyperammonemia is responsible for most neurological alterations in patients with hepatic encephalopathy by mechanisms that remain unclear. Hyperammonemia alters phosphorylation of neuronal protein kinase C (PKC) substrates and impairs NMDA receptor-associated signal transduction. The aim of this work was to analyse the effects of hyperammonemia on the amount and intracellular distribution of PKC isoforms and on translocation of each isoform induced by NMDA receptor activation in cerebellar neurons. Chronic hyperammonemia alters differentially the intracellular distribution of PKC isoforms. The amount of all isoforms (except PKC zeta) was reduced (17-50%) in the particulate fraction. The contents of alpha, beta1, and epsilon isoforms decreased similarly in cytosol (65-78%) and membranes (66-83%), whereas gamma, delta, and theta; isoforms increased in cytosol but decreased in membranes, and zeta isoform increased in membranes and decreased in cytosol. Chronic hyperammonemia also affects differentially NMDA-induced translocation of PKC isoforms. NMDA-induced translocation of PKC alpha and beta is prevented by ammonia, whereas PKC gamma, delta, epsilon, or theta; translocation is not affected. Inhibition of phospholipase C did not affect PKC alpha translocation but reduced significantly PKC gamma translocation, indicating that NMDA-induced translocation of PKC alpha is mediated by Ca2+, whereas PKC gamma translocation is mediated by diacylglycerol. Chronic hyperammonemia reduces Ca+2-mediated but not diacylglycerol-mediated translocation of PKC isoforms induced by NMDA.

The signal transduction pathways involved in NMDA receptor modulation by other receptors remain unclear. cAMP could be involved in this modulation. The aim of this work was to analyse the contribution of cAMP to NMDA receptor modulation in cerebellar neurones in culture. Forskolin increases cAMP and results in increased intracellular calcium and cGMP that are prevented by blocking NMDA receptors. Similar effects were induced by two cAMP analogues, indicating that cAMP leads to NMDA receptor activation. It has been reported that phosphorylation of Ser897 of the NR1 subunit of NMDA receptors by cAMP-dependent protein kinase (PKA) activates the receptors. Forskolin increases Ser897 phosphorylation. Neither Ser897 phosphorylation nor cGMP increase induced by forskolin are prevented by four inhibitors of PKA, suggesting that NMDA receptor activation is dependent on cAMP but not on PKA. Inhibition of Akt prevents forskolin-induced phosphorylation of Ser897, suggesting a role for Akt in the mediation of the modulation of NMDA receptors by cAMP. Pituitary adenylate cyclase-activating polypeptide (PACAP) activates its receptors, increasing cAMP and also leading to phosphorylation of Ser897 of NR1 and activation of NMDA receptors. These results indicate that cAMP modulates NMDA receptor in cerebellar neurones and may play a role in NMDA receptor modulation by other receptors.

Acute ammonia toxicity is mediated by excessive activation of NMDA receptors. Activation of NMDA receptors leads to activation of poly(ADP-ribose) polymerase (PARP) which mediates NMDA excitotoxicity. PARP is activated following DNA damage and may lead to cell death via NAD+ and ATP depletion. The aim of the present work was to assess whether acute ammonia intoxication in vivo leads to increased PARP in brain cells nuclei and to altered NAD+ and superoxide metabolism and the contribution of NMDA receptors to these alterations. Acute ammonia intoxication increases PARP content twofold in brain cells nuclei.NAD+ content decreased by 55% in rats injected with ammonia. This was not due to decreased NAD+ synthetase nor increased NAD+ hydrolase activities and would be due to increased NAD+ consumption by PARP. Superoxide radical formation increased by 75% in nuclei of brains of rats injected with ammonia, that also induced protein nitrotyrosylation and DNA damage. Blocking NMDA receptors prevented ammonia-induced PARP, superoxide and nitrotyrosylation increase, DNA damage and NAD+ decrease. These results show that acute ammonia intoxication in vivo leads to activation of NMDA receptors, leading to increased superoxide formation and PARP content and depletion of NAD+ in brain cells nuclei that contribute to ammonia toxicity.

Metallothionein (MT)-III, a member of the MT family of metal-binding proteins, is mainly expressed in the CNS and is abundant in glutamatergic neurons. Results in genetically altered mice indicate that MT-III may play neuroprotective roles in the brain, but the mechanisms through which this protein functions have not been elucidated. The aim of this work was to assess whether MT-III is able to prevent glutamate neurotoxicity and to identify the step of the neurotoxic process interfered with by MT-III. Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive activation of glutamate receptors, increased intracellular calcium, and increased nitric oxide. It is shown that MT-III prevented glutamate- and nitric oxide-induced neurotoxicity in a dose-dependent manner, with nearly complete protection at 0.3-1 microgram/ml. MT-III did not prevent the glutamate-induced rise of intracellular calcium level but reduced significantly the nitric oxide-induced formation of cyclic GMP. Circular dichroism analysis revealed that nitric oxide triggers the release of the metals coordinated to the cysteine residues of MT-III, indicative of the S(Cys)-nitrosylation of the protein. Therefore, the present results indicate that MT-III can quench pathological levels of nitric oxide, thus preventing glutamate and nitric oxide neurotoxicity.

Exposure to aluminum (Al) produces neurotoxic effects in humans. However, the molecular mechanism of Al neurotoxicity remains unknown. Al interferes with glutamatergic neurotransmission and impairs the neuronal glutamate-nitric oxide-cyclic GMP (cGMP) pathway, especially in rats prenatally exposed to Al. The aim of this work was to assess whether Al interferes with processes associated with activation of NMDA receptors and to study the molecular basis for the Al-induced impairment of the glutamate-nitric oxide-cGMP pathway. We used primary cultures of cerebellar neurons prepared from control rats or from rats prenatally exposed to Al. Prenatal exposure to Al prevented glutamate-induced proteolysis of the microtubule-associated protein-2, disaggregation of microtubules, and neuronal death, indicating an impairment of NMDA receptor-associated signal transduction pathways. Prenatal exposure to Al reduced significantly the content of nitric oxide synthase and guanylate cyclase and increased the content of calmodulin both in cultured neurons and in the whole cerebellum. This effect was selective for proteins of the glutamate-nitric oxide-cGMP pathway as the content of mitogen-activated protein kinase and the synthesis of most proteins were not affected by prenatal exposure to Al. The alterations in the expression of proteins of the glutamate-nitric oxide-cGMP pathway could be responsible for some of the neurotoxic effects of Al.

Humans are exposed to aluminum from environmental sources and therapeutic treatments. However, aluminum is neurotoxic and is considered a possible etiologic factor in Alzheimer's disease and other neurological disorders. The molecular mechanism of aluminum neurotoxicity is not understood. We tested the effects of aluminum on the glutamate-nitric oxide-cyclic GMP pathway in cultured neurons. Neurons were exposed to 50 microM aluminum in culture medium for short-term (4 h) or long-term (8-14 days) periods, or rats were prenatally exposed, i.e., 3.7% aluminum sulfate in the drinking water, during gestation. Chronic (but not short-term) exposure of neurons to aluminum decreased glutamate-induced activation of nitric oxide synthase by 38% and the formation of cyclic GMP by 77%. The formation of cyclic GMP induced by the nitric oxide-generating agent S-nitroso-N-acetylpenicillamine was reduced by 33%. In neurons from rats prenatally exposed to aluminum but not exposed to it during culture, glutamate-induced formation of cyclic GMP was inhibited by 81%, and activation of nitric oxide synthase was decreased by 85%. The formation of cyclic GMP induced by S-nitroso-N-acetylpenicillamine was not affected. These results indicate that chronic exposure to aluminum impairs glutamate-induced activation of nitric oxide synthase and nitric oxide-induced activation of guanylate cyclase. Impairment of the glutamate-nitric oxide-cyclic GMP pathway in neurons may contribute to aluminum neurotoxicity.

The effect of chronic and in vitro ethanol exposure on brain oxygen radical formation and lipid peroxidation was analyzed. Ethanol induces a dose-dependent increase in lipid peroxidation in brain homogenates. The peroxidative effects of alcohol seem to be related to both cytochrome P450 and the ethanol-inducible form of cytochrome P450 (CYP2E1), because preincubation with metyrapone (an inhibitor of cytochrome P450) or with an antibody against CYP2E1 abolished the ethanol-increased lipid peroxidation. Using the formation of dichlorofluorescein, we also demonstrated that both in vitro and chronic alcohol exposure significantly enhanced the formation of oxygen radical species in synaptosomes. Chronic alcohol treatment also leads to an induction of cytochrome P450 (230%), NADPH cytochrome c reductase (180%), NADPH oxidation (184%), and CYP2E1 in brain microsomes. In addition, this treatment produced a decrease in the GSH/GSSG ratio in brain and significantly enhanced the levels of superoxide dismutase and catalase activities. This mechanism could be involved in the toxic effects of ethanol on brain and membrane alterations occurring after chronic ethanol intake.