Mitsuaki Moriyama

Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling

Brain edema and the consequent increase in intracranial pressure and brain herniation are major complications of acute liver failure (fulminant hepatic failure) and a major cause of death in this condition. Ammonia has been strongly implicated as an important factor, and astrocyte swelling appears to be primarily responsible for the edema. Ammonia is known to cause cell swelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter (NKCC1) has been shown to be involved in cell swelling in several neurological disorders. We therefore examined the effect of ammonia on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to ammonia (NH4Cl; 5 mm), and NKCC activity was measured. Ammonia increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished ammonia-induced astrocyte swelling. Ammonia also increased total as well as phosphorylated NKCC1. Treatment with cyclohexamide, a potent inhibitor of protein synthesis, diminished NKCC1 protein expression and NKCC activity. Since ammonia is known to induce oxidative/nitrosative stress, and antioxidants and nitric-oxide synthase inhibition diminish astrocyte swelling, we also examined whether ammonia caused oxidation and/or nitration of NKCC1. Cultures exposed to ammonia increased the state of oxidation and nitration of NKCC1, whereas the antioxidants N-nitro-l-arginine methyl ester and uric acid all significantly diminished NKCC activity. These agents also reduced phosphorylated NKCC1 expression. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by ammonia and that such activation appears to be mediated by NKCC1 abundance as well as by its oxidation/nitration and phosphorylation.

NFκB in the mechanism of ammonia-induced astrocyte swelling in culture

Astrocyte swelling and brain edema are major neuropathological findings in the acute form of hepatic encephalopathy (fulminant hepatic failure), and substantial evidence supports the view that elevated brain ammonia level is an important etiological factor in this condition. Although the mechanism by which ammonia brings about astrocyte swelling remains to be determined, oxidative/nitrosative stress and mitogen‐activated protein kinases (MAPKs) have been considered as important elements in this process. One factor known to be activated by both oxidative stress and MAPKs is nuclear factor κB (NFκB), a transcription factor that activates many genes, including inducible nitric oxide synthase (iNOS). As the product of iNOS, nitric oxide (NO), is known to cause astrocyte swelling, we examined the potential involvement of NFκB in ammonia‐induced astrocyte swelling. Western blot analysis of cultured astrocytes showed a significant increase in NFκB nuclear translocation (a measure of NFκB activation) from 12 h to 2 days after treatment with NH4Cl (5 mM). Cultures treated with anti‐oxidants, including superoxide dismutase, catalase, and vitamin E as well as the MAPKs inhibitors, SB239063 (an inhibitor of p38‐MAPK) and SP600125 (an inhibitor of c‐Jun N‐terminal kinase), significantly diminished NFκB activation by ammonia, supporting a role of oxidative stress and MAPKs in NFκB activation. The activation of NFκB was associated with increased iNOS protein expression and NO generation, and these changes were blocked by BAY 11–7082, an inhibitor of NFκB. Additionally, ammonia‐induced astrocyte swelling was inhibited by the NFκB inhibitors, BAY 11–7082 and SN‐50, thereby implicating NFκB in the mechanism of astrocyte swelling. Our studies indicate that cultured astrocytes exposed to ammonia display NFκB activation, which is likely to be a consequence of oxidative stress and activation of MAPKs. NFκB activation appears to contribute to the mechanism of ammonia‐induced astrocyte swelling, apparently through its up‐regulation of iNOS protein expression and the subsequent generation of NO.

Trauma-Induced Cell Swelling in Cultured Astrocytes

Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury that account for most early deaths after traumatic brain injury. An important component of brain edema after traumatic brain injury is astrocyte swelling (cytotoxic edema). To examine the pathophysiologic mechanisms of trauma-induced astrocyte swelling, we used an in vitro fluid percussion trauma model. Exposure of cultured rat astrocytes to 5 atm of pressure resulted in significant cell swelling at 1 to 24 hours posttrauma that was maximal at 3 hours. Because oxidative/nitrosative stress, mitochondrial permeability transition (mPT), and mitogen-activated protein kinases (MAPKs) have been implicated in astrocyte swelling in other neurologic conditions, we examined their potential roles in this model. We previously showed increased free radical generation after in vitro trauma and show here that trauma to astrocytes increased the production of nitric oxide. Trauma also induced mPT and increased phosphorylation (activation) of MAPKs (extracellular signal-regulated kinase 1/2, c-Jun-N-terminal kinase, and p38-MAPK); these changes were diminished by antioxidants and the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester. Antioxidants, N-nitro-l-arginine methyl ester, the mPT inhibitor cyclosporin A, and inhibitors of MAPKs all significantly diminished trauma-induced astrocyte swelling. These findings demonstrate that direct mechanical injury to cultured astrocytes brings about cell swelling, and that blockade of oxidative/nitrosative stress, mPT, and MAPKs significantly reduce such swelling.

Slc25a13-Knockout Mice Harbor Metabolic Deficits but Fail To Display Hallmarks of Adult-Onset Type II Citrullinemia

ABSTRACT Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn −/− mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn −/− mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.

Na-K-Cl cotransporter-1 in the mechanism of cell swelling in cultured astrocytes after fluid percussion injury.

J. Neurochem. (2011) 117, 437–448.

Citrin/Mitochondrial Glycerol-3-phosphate Dehydrogenase Double Knock-out Mice Recapitulate Features of Human Citrin Deficiency

Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol.Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD+ ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.

Neurotrophic Action of Interleukin 3 and Granulocyte-Macrophage Colony-Stimulating Factor on Murine Sympathetic Neurons

We investigated neurotrophic effects of interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on cultured sympathetic neurons obtained from mouse superior cervical ganglia. After 1 day of culture with physiological concentrations of mouse recombinant IL-3 and GM-CSF, the numbers of process-bearing neurons were increased. Maximum responses were elicited by 10 U/ml IL-3 and 1 U/ml GM-CSF, which were equivalent to the action of a submaximal dose (5 ng/ml) of nerve growth factor (NGF). The effects of IL-3 and GM-CSF were completely blocked by their corresponding antibodies, but not by anti-NGF, indicting their action is specific and completely independent of NGF. IL-3 and, to a lesser extent, GM-CSF were also able to protect NGF-differentiated neurons from apoptotic cell death caused by NGF withdrawal. The mitogen-activated protein (MAP) kinase signal transduction pathway is known to be involved in action of IL-3 and GM-CSF on hemopoietic cells, and thus we examined the participation of this pathway in the neurotrophic activities of IL-3 and GM-CSF. IL-3 and GM-CSF stimulation of the differentiated neurons was found to result in a rapid elevation of MAP kinase activity, and PD98059, an inhibitor of MAP kinase kinase activity, blocked both the neuritogenic and neuroprotective effects of IL-3 and GM-CSF. Immunocytochemical studies showed that IL-3 and GM-CSF receptors were present on the differentiated neurons. Thus, IL-3 and GM-CSF appear to be able to stimulate sympathetic nerve growth, via specific cytokine receptors on neurons, which lead to activation of the MAP kinase pathway that then mediates the observed neurotrophic effects.

Role of Mitogen-Activated Protein Kinases in the Mechanism of Oxidant-Induced Cell Swelling in Cultured Astrocytes

Cytotoxic brain edema, usually a consequence of astrocyte swelling, is an important complication of stroke, traumatic brain injury, hepatic encephalopathy, and other neurological disorders. Although mechanisms underlying astrocyte swelling are not fully understood, oxidative stress (OS) has generally been considered an important factor in its pathogenesis. To better understand the mechanism(s) by which OS causes cell swelling, we examined the potential involvement of mitogen‐activated protein kinases (MAPKs) in this process. Cultures exposed to theoxidant H2O2 (10, 25, 50 μM) for different time periods (1–24 hr) significantly increased cell swelling in a triphasic manner. Swelling was initially observed at 10 min (peaking at 30 min), which was followed by cell shrinkage at 1 hr. A subsequent increase in cell volume occurred at approximately 6 hr, and the rise lasted for at least 24 hr. Cultures exposed to H2O2 caused the activation of MAPKs (ERK1/2, JNK and p38‐MAPK), whereas inhibition of MAPKs diminished cell swelling induced by 10 and 25 μM H2O2. These findings suggest that activation of MAPKs is an important factor in the mediation of astrocyte swelling following oxidative stress.

The Myelin Vacuolation ( mv ) Rat with a Null Mutation in the Attractin Gene

We recently found a spontaneous tremor mutant in an outbred colony of Sprague-Dawley rats. The tremor behavior was exhibited from around 3 weeks of age and inherited as an autosomal recessive trait. The mutant rats had variously sized vacuoles in the neuropil and white matter throughout the central nervous system, especially in the brain stem, cerebellum, and spinal cord. Ultrastructurally these vacuoles mainly consisted of splitting of myelin lamella both in the periaxonal and intermyelinic spaces. Linkage analysis using intercross progeny between the myelin vacuolation (mv) rat, named after the pathologic characteristics, and normal control rat strains showed that the mv phenotypes were cosegregated with polymorphic markers adjacent to the Atrn (Attractin, formerly zi [zitter]) locus on rat chromosome 3. A test for allelism suggested that the mv mutation was a new allele in Atrn. In comparison with a marked decrease of Atrnzi/Arnzi, Northern blot analysis revealed no expression of Atrn mRNA in the brain of the mv rats. Finally, a genomic deletion including exon 1 of the mv rats was detected by genomic and sequence analyses. Discovery of the rat null mutation Atrnmv, different from Atrnzi, provides a new animal model for studying the functions of the attractin protein.

Transglutaminase 2 expression induced by lipopolysaccharide stimulation together with NO synthase induction in cultured astrocytes

Activation of glia has been observed in neurodegenerative diseases such as Parkinsons disease (PD), Alzheimers disease (AD), multiple sclerosis and brain ischemia. Excessive production of nitric oxide (NO), as a consequence of increased inducible NO synthase (iNOS) in glia, contributes to neurodegeneration. Transglutaminase 2 (TG2) is a cross-linking enzyme, which is activated in neurodegenerative diseases such as PD, AD and Huntingtons diseases. However, mechanisms contributing to the increased TG activity in neurodegenerative diseases remain to be clarified. In the present study, we examined the expression of TG2 in cultured rat hippocampal astrocytes activated with lipopolysaccharide (LPS), which is generally used for a stimulant of iNOS induction. The expressions of TG2 mRNA and protein were increased by stimulation with LPS in a dose-dependent manner. The LPS-induced TG2 expression was diminished by ammonium pyrrolidine-1-carbodithioate; an inhibitor for nuclear factor (NF)-κB activation, suggesting the factors involved. Both expressions of TG2 and iNOS induced by LPS stimulation were suppressed by an antioxidant, ethyl pyruvate, in a dose-dependent manner. Furthermore, they were also suppressed by cystamine, an inhibitor of TG activity. These results suggest that the level of TG2 expression is regulated by oxidative stress and the activity of TG itself, and that the induction of iNOS and NO production are closely associated with TG2 expression in LPS-stimulated activation of astrocytes.