Jae n Ryu

Induction of matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide-stimulated primary astrocytes is mediated by extracellular signal-regulated protein kinase 1/2 (Erk1/2).

In the present study, we investigated whether the activation of protein kinase C (PKC) and extracellular signal‐regulated kinase 1/2 (Erk1/2) are involved in the induction of MMP‐9 in lipopolysaccharide (LPS)–stimulated primary astrocytes. The expression of MMP‐9 but not MMP‐2 was increased by LPS. LPS treatment induced activation of Erk1/2 within 30 min, which was dose‐dependently inhibited by PD98059, a specific inhibitor of the Erk kinase (MEK). In this condition, PD98059 blocked the increase in MMP‐9 protein and mRNA level as well as gelatin‐digesting activity. Inhibition of PKC activity blocked the LPS‐induced activation of Erk1/2 as well as MMP‐9 expression. In addition, activation of PKC by phorbol myristoyl acetate (PMA) activated Erk1/2 with concomitant increase in MMP‐9 production. Moreover, treatment of PD98059 dose‐dependently decreased the PMA‐induced MMP‐9 expression. The results from the present study suggest that induction of MMP‐9 by LPS in rat primary astrocytes is mediated, at least in part, by the sequential activation of PKC and Erk1/2. The Erk1/2‐mediated MMP‐9 induction may provide insights into the regulation of MMP‐9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation. GLIA 41:15–24, 2003.

Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death.

Abstract: Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO‐‐producing reagent 3‐morpholinosydnonimine (SIN‐1). Here we show that glucose deprivation and ONOO‐ synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A‐sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN‐1 (200 μM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose‐deprived/SIN‐1‐treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO‐ scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO‐ was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO‐) was markedly enhanced in glucose‐deprived/SIN‐1‐treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose‐deprived/SIN‐1‐treated astrocytes. The glutathione synthase inhibitor L‐buthionine‐(S,R)‐sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN‐1‐treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose‐deprived/SIN‐1‐treated astrocytes.

Depletion of intracellular glutathione mediates zinc-induced cell death in rat primary astrocytes

In the present study, we investigated the possible mechanisms of cellular injury induced by zinc in rat primary astrocytes and C6 glioma cells. Reactive oxygen species (ROS) production, cellular glutathione (GSH) level and mitochondrial transmembrane potential were examined. Exposure to 200–300 μM Zn2+ for 24 h resulted in significant lactate dehydrogenase (LDH) release in rat primary astrocytes and C6 glioma cells. An exposure of 200 μM Zn2+ resulted in profound morphological changes, for example, shrunken and fragmented nuclei. Pretreatment of a protein synthesis inhibitor, cycloheximide, did not attenuate cellular toxicity induced by Zn2+. Zn2+ exposure increased intracellular ROS levels by about 250%, and depleted cellular GSH within 2 h, which preceded observable LDH release from the cell. Addition of GSH, N-acetylcysteine (NAC) and ascorbic acid substantially attenuated cellular death induced by Zn2+ in a concentration dependent manner. ROS production and morphological changes induced by zinc were also inhibited by co-treatment of GSH or NAC with Zn2+. Zn2+ significantly depolarized mitochondrial transmembrane potential, which was reversed by co-treatment of GSH or NAC with zinc. In summary, ROS generation, GSH depletion and mitochondrial dysfunction may be key factors in Zn2+-induced glial toxicity.

Oxidative damage causes formation of lipofuscin-like substances in the hippocampus of the senescence-accelerated mouse after kainate treatment.

We have demonstrated that seizures induced by kainic acid (KA) are, at least in part, mediated via oxidative stress in rats [Life. Sci. 61 (1997) PL373; Brain Res. 853 (2000) 215; Brain Res. 874 (2000) 15; Neurosci. Lett. 281 (2000) 65]. In order to extend our findings, we employed the rodent aging model in this study. After KA treatments (once a day for 5 days; 20,20,20,20 and 40 mg/kg, i.p.), several parameters reflecting neurotoxic behaviors, oxidative stress [malondialdehyde (MDA) and protein carbonyl] and aging (lipofuscin-like substances) were compared between senile-prone (P8) and resistant (R1) strains of 9-month-old male senescence-accelerated mice (SAM). KA-induced neurotoxic signs as shown by mortality and seizure activity were more accentuated in the SAM-P8 than in the SAM-R1. Levels of MDA and carbonyl are consistently higher in the hippocampus of SAM-P8 than that of SAM-R1. Significant increases in the values of MDA and carbonyl were observed 4 h or 2 days after the final KA administration. This finding was more pronounced in the SAM-P8 than in the SAM-R1. Although a significant loss of hippocampal neurons was observed 7 days post-KA, at this time the MDA and carbonyl content had returned to near control levels. In contrast, fluorescent lipofuscin-like substances and lipofuscin granules were significantly increased 7 days after KA treatments. Therefore, our data suggests that mice in the senescence model are more susceptible to KA-induced seizures/oxidative damage, and that oxidative damage could be one of the casual factors in the accumulation of lipofuscin.

Immunostimulation of rat primary astrocytes decreases intracellular ATP level

In this study we investigated the effect of immunostimulation on intracellular ATP level in rat glial cells. Rat primary astrocytes or C6 glioma cells were treated for 48 h with IFN-gamma, LPS or IFN-gamma plus LPS. These treatments increased NO production from the cells and a synergistic increase in NO production was observed with IFN-gamma plus LPS. Intracellular ATP level was decreased to about half the control level at the highest concentration of IFN-gamma (100 U/ml) plus LPS (1 microg/ml) without affecting cell viability. The level of intracellular ATP was inversely correlated with the extent of NO production from the glial cells. The increase in NO production is at least 6 h ahead of the initiation of ATP depletion, and NOS inhibitor N(G)-nitro-L-arginine (NNA) or Nomega-nitro-L-arginine methyl ester (L-NAME) inhibited NO production and ATP depletion. Exogenous addition of peroxynitrite generator 3-morpholinosydnonimine (SIN-1) and to a lesser extent NO generator S-nitroso-N-acetylpenicillamine (SNAP) depleted intracellular ATP level in a dose-dependent manner. The results from the present study imply that immunostimulation of rat glial cells decreases the intracellular ATP level without affecting cell viability. Considering the role of astrocytes as an essential regulator of the extracellular environment in the brain, the immunostimulation-induced decrease in intracellular ATP level may participate in the pathogenesis of various neurological diseases.

Activation of p38 MAPK induced peroxynitrite generation in LPS plus IFN-γ-stimulated rat primary astrocytes via activation of iNOS and NADPH oxidase

We have shown that immunostimulated astrocytes produce excess nitric oxide (NO) and eventually peroxynitrite (ONOO(-)) that was closely associated with the glucose deprivation-potentiated death of astrocytes. The present study shows that activated p38 MAPK regulates ONOO(-) generation from lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-stimulated astrocytes. LPS+IFN-gamma-induced p38 MAPK activation and ONOO(-) generation were attenuated by SB203580 or SKF-86002, specific inhibitors of p38 MAPK. ONOO(-) generation was blocked by NADPH oxidase inhibitor, diphenyleneiodonium chloride, and nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester, suggesting both enzymes are involved in ONOO(-) generation. Inhibition of p38 MAPK suppressed LPS+IFN-gamma-induced NO production through down-regulating inducible form of NOS expression. It also suppressed LPS+IFN-gamma-induced NADPH oxidase activation and eventually, the inducible form of superoxide production. Transfection with dominant negative vector of p38 alpha reduced LPS+IFN-gamma-induced ONOO(-) generation through blocking both iNOS-derived NO production and NADPH oxidase-derived O2(-) production. Our results suggest that activated p38 MAPK may serve as a potential signaling molecule in ONOO(-) generation through dual regulatory mechanisms, involving iNOS induction and NADPH oxidase activation.

Microglial activation and tyrosine hydroxylase immunoreactivity in the substantia nigral region following transient focal ischemia in rats.

The temporal profiles of the changes of dopaminergic cells and microglial activation induced by transient cerebral ischemia were investigated in the substantia nigra pars compacta (SNc) located outside ischemic areas of rat brain. Transient cerebral ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 2 h and reperfusion was continued for 1, 2, 3, 4, 7, 10, 14, 28, 60, and 120 days. Dopaminergic cells immunostained with tyrosine hydroxylase (TH)-antibody in the ipsilateral SNc were significantly decreased at 7 days post-ischemia compared with those in the contralateral side (P<0.05). However, at 60 and 120 days, there were no significant differences between ipsilateral and contralateral side of the SNc. Unlike the TH immunoreactivity, activated microglial cells immunostained with OX-42 antibody were significantly increased at 2 and 3 days and then decreased gradually until 10 days post-ischemia. Activated microglial cells were increased at 2 weeks post-ischemia, and this pattern remained until 60 days. These results suggest that the transient changes of TH-immunoreactive cells in the SNc caused by transient focal ischemia are correlated with a biphasic microglial cell activation response.

Activation of microglial cells via protease-activated receptor 2 mediates neuronal cell death in cultured rat primary neuron

The role of protease-activated receptor (PARs) in the regulation of microglial activation process is increasingly evident. In the present study, we have investigated the role of PAR-2, which can be activated by trypsin-like proteases, in microglial activation and neuronal cell death. In cultured rat primary microglia, activation of PAR-2 induced nitrite production by PKC- and MAPKs-dependent mechanism. Among the three members of MAPK pathway, ERK and JNK but not p38 mediated PAR-2-induced microglial activation. The down-stream regulator of PAR-2-PKC-MAPK pathway-induced microglial activation was NF-kappaB pathway. Besides nitrite, PAR-2 activation increased production of a variety of inflammatory mediators such as ROS and pro-inflammatory cytokines including TNF-alpha and IL-1beta. The addition of culture spent media from PAR-2 activated microglia induced neuronal cell death in primary rat cortical neuron cultures with apoptotic features such as increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive neurons, dissipation of mitochondrial membrane potential, increased expression of pro-apoptotic Bax, decreased expression of anti-apoptotic Bcl-2, Bcl-X(L), and activation of caspase-3 in neurons. Interestingly, the increased production of cytoactive molecules as well as the neuronal cell death was normalized by PAR-2 or trypsin inhibitor or an NO synthase inhibitor, N(G)-nitro-l-arginine-methyl ester. Taken together, these results suggest that overt PAR-2 activation may induce microglial activation, which contributes to neuronal cell death.

Adenosine and Purine Nucleosides Protect Rat Primary Astrocytes from Peroxynitrite-Potentiated, Glucose Deprivation-Induced Death: Preservation of Intracellular ATP Level

Previously we have reported that immunostimulated astrocytes became highly vulnerable to glucose deprivation. In the present study we examined the effect of various kinds of nucleosides on the augmented death of glucose-deprived immunostimulated astrocytes. Preincubation with interferon-gamma (100 U/ml) and lipopolysaccharide (1 microg/ml) for 48 h and continuous exposure to glucose deprivation (4 h) significantly induced the lactate dehydrogenase (LDH) release, as a marker of cell injury or death, from astrocytes. The glucose deprivation-induced augmented cell death in immunostimulated astrocytes was mimicked by exogenous peroxynitrite generator 3-morpholinosydnonimine (SIN-1). The increased death in immunostimulated or SIN-1-treated astrocytes deprived of glucose was blocked by adenosine and ATP. Other purine nucleos(t)ides, not pyrimidine nucleotides, also showed similar protective effects. Adenosine receptor agonist R(-)-N-(2-phenylisopropyl)-adenosine or N-cyclohexyladenosine did not alter the augmented cell death. Adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener or 3,7-dimethyl-1-propargylxanthine also did not reverse the protective effect of adenosine. Intracellular ATP levels rapidly decreased prior to the LDH release in glucose-deprived immunostimulated astrocytes. The loss of intracellular ATP was prevented by adenosine and other purine nucleotides. The present results suggest that adenosine and their metabolites may protect astrocytes from peroxynitrite-potentiated, glucose deprivation-induced death by serving as substrates for intracellular ATP generation.

Evidence that protease-activated receptor-2 mediates trypsin-induced reversal of stellation in cultured rat astrocytes

Serine proteases such as thrombin and trypsin play a key role in the development and repair processes in the central nervous system. Molecular actions of serine proteases include multiple cellular events like activation of protease-activated receptors (PARs). PARs belong to a family of G protein-coupled receptors that can be stimulated through their proteolytic cleavage by ligands. PAR-2 has been implicated in neurodegenerative diseases including astrogliosis. Although recent studies have shown that low concentration of trypsin activates PAR-2, its role in morphological changes in primary astrocytes has not been studied. In the present study, we investigated the effects of PAR-2 in astrocyte stellation in rat primary astrocyte culture. Both trypsin (0.1-1 U/ml) and a PAR-2-activating peptide SLIGRL-NH2 (1-50 microM) significantly reversed the stellation induced by serum deprivation in rat astrocytes. Treatment of astrocytes with trypsin or SLIGRL-NH2 resulted in a transient rise of the intracellular Ca2+ level and trypsin-induced morphological changes were blocked by BAPTA, a Ca2+ chelator. In addition, a protein kinase C (PKC) inhibitor, bisindolylmaleimide significantly inhibited the trypsin-induced morphological changes, whereas activation of PKC by phorbol-12-myristate-13-acetate acted as trypsin. Taken together, these results suggest that activation of PAR-2 by trypsin caused reversal of stellation in cultured astrocytes, in part, via the mobilization of intracellular Ca2+ and activation of PKC.