Kazuhiro Takuma

Anti-apoptotic Effect of cGMP in Cultured Astrocytes INHIBITION BY cGMP-DEPENDENT PROTEIN KINASE OF MITOCHONDRIAL PERMEABLE TRANSITION PORE

Reperfusion of cultured astrocytes with normal medium after exposure to H2O2-containing medium causes apoptosis. We have recently shown that ibudilast, which has been used for bronchial asthma and cerebrovascular disorders, attenuated the H2O2-induced apoptosis of astrocytes via the cGMP signaling pathway. This study examines the mechanism underlying the protective effect of cGMP. The membrane-permeable cGMP analog dibutyryl-cGMP attenuated the H2O2-induced decrease in cell viability, DNA ladder formation, nuclear condensation, reduction of the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 activation in cultured astrocytes. These effects of dibutyryl-cGMP were almost completely inhibited by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. In isolated rat brain mitochondria, cGMP in the presence of cytosolic extract from astrocytes inhibited the mitochondrial permeability transition pore (PTP) as determined by monitoring Ca2+-induced mitochondrial swelling. This ability of the cytosolic extract was inactivated by heat treatment and was mimicked by exogenous PKG. The effect of cGMP on the mitochondrial swelling was blocked by KT5823. The PTP inhibitors cyclosporin A and bongkrekic acid prevented the H2O2-induced decrease in cell viability and caspase-3 activation. These findings demonstrate that cGMP inhibits the mitochondrial PTP via the activation of PKG, and the prevention of mitochondrial dysfunction contributes to its anti-apoptotic effect.

Apoptosis in Ca2 + reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-κB activation

We previously reported that incubation of cultured astrocytes in Ca2u200a+u200a‐containing medium after exposure to Ca2u200a+u200a‐free medium caused Ca2u200a+u200a influx followed by delayed cell death. Here, we studied the mechanisms underlying the Ca2u200a+u200a‐mediated injury of cultured astrocytes. Our results show that Ca2u200a+u200a reperfusion injury of astrocytes appears to be mediated by apoptosis, as demonstrated by DNA fragmentation and prevention of death by caspase‐3 inhibitors. Paradoxical Ca2u200a+u200a challenge stimulated rapidly reactive oxygen species (ROS) production. Ca2u200a+u200a reperfusion injury of astrocytes was influenced by several reagents which modified ROS production. When astrocytes were exposed to hydrogen peroxide (H2O2) for 30u2003min and then incubated without H2O2 for 1–5u2003days, cell toxicity including apoptosis was observed. Ca2u200a+u200a reperfusion injury induced by Ca2u200a+u200a depletion or H2O2 exposure was blocked by the iron chelator 1,10‐phenanthroline, the NF‐κB inhibitor pyrrolidinedithiocarbamate and the calcineurin inhibitor FK506. Incubation in normal medium after H2O2 exposure rapidly increased the level of nuclear NF‐κB p65 subunit, and the effect was blocked by 1,10‐phenanthroline, pyrrolidinedithiocarbamate and FK506. These findings indicate that Ca2u200a+u200a reperfusion‐induced apoptosis is mediated at least partly by ROS production and ROS cause NF‐κB activation in cultured astrocytes.

Involvement of Calcineurin in Ca2+ Paradox-Like Injury of Cultured Rat Astrocytes

Abstract: The Ca2+/calmodulin‐dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca2+‐containing medium after exposure to Ca2+‐free medium caused Ca2+ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca2+‐mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca2+ challenge‐induced injury in a dose‐dependent manner (10−10–10−8M). Cyclosporin A at 1 µM mimicked the effect of FK506. Rapamycin (1 µM) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 nM), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca2+ challenge‐induced increase in cytosolic Ca2+ level nor Na+‐Ca2+ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca2+ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.

Ibudilast attenuates astrocyte apoptosis via cyclic GMP signalling pathway in an in vitro reperfusion model

We examined the effect of 3‐isobutyryl‐2‐isopropylpyrazolo[1,5‐a]pyridine (ibudilast), which has been clinically used for bronchial asthma and cerebrovascular disorders, on cell viability induced in a model of reperfusion injury. Ibudilast at 10u2003–u2003100u2003μM significantly attenuated the H2O2‐induced decrease in cell viability. Ibudilast inhibited the H2O2‐induced cytochrome c release, caspase‐3 activation, DNA ladder formation and nuclear condensation, suggesting its anti‐apoptotic effect. Phosphodiesterase inhibitors such as theophylline, pentoxyfylline, vinpocetine, dipyridamole and zaprinast, which increased the guanosine‐3′,5′‐cyclic monophosphate (cyclic GMP) level, and dibutyryl cyclic GMP attenuated the H2O2‐induced injury in astrocytes. Ibudilast increased the cyclic GMP level in astrocytes. The cyclic GMP‐dependent protein kinase inhibitor KT5823 blocked the protective effects of ibudilast and dipyridamole on the H2O2‐induced decrease in cell viability, while the cyclic AMP‐dependent protein kinase inhibitor KT5720, the cyclic AMP antagonist Rp‐cyclic AMPS, the mitogen‐activated protein/extracellular signal‐regulated kinase inhibitor PD98059 and the leukotriene D4 antagonist LY 171883 did not. KT5823 also blocked the effect of ibudilast on the H2O2‐induced cytochrome c release and caspase‐3‐like protease activation. These findings suggest that ibudilast prevents the H2O2‐induced delayed apoptosis of astrocytes via a cyclic GMP, but not cyclic AMP, signalling pathway.

Roles of cathepsins in reperfusion-induced apoptosis in cultured astrocytes.

Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.

T-588 inhibits astrocyte apoptosis via mitogen-activated protein kinase signal pathway

The effect of (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan -1-ol hydrochloride (T-588), a cognition enhancer, on reperfusion injury was studied in cultured rat astrocytes. T-588 at 1-10 microM partially protected astrocytes against reperfusion injury after exposure to Ca(2+)-free medium or hydrogen peroxide. Nerve growth factor (NGF) had a similar protective effect. Addition of both T-588 and NGF resulted in complete protection against Ca(2+) reperfusion injury. T-588 did not stimulate NGF production in astrocytes. The effect of T-588 on Ca(2+) reperfusion injury including apoptosis was inhibited by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor 2-amino-3-methoxyflavone (PD98059), but not by the phosphoinositide 3-kinase inhibitor wortmannin. The effect of NGF was inhibited by PD98059 and wortmannin. T-588 stimulated rapidly the phosphorylation of ERK, but did not affect that of Akt in astrocytes. These findings suggest that the ERK MAP kinase pathway has a role in the protective effects of T-588 and NGF.

Heat shock inhibits hydrogen peroxide-induced apoptosis in cultured astrocytes.

Heat shock proteins (HSPs) have been shown to act as inhibitors of apoptosis, but this anti-apoptotic effect is not known in the central nervous system. Prior heat shock has been demonstrated to protect astrocytes from cell death in a model of reperfusion injury (Brain Res. 735 (1996) 265). The present study examines the mechanism underlying the protective effect of the heat shock. Preincubation of astrocytes at 40 degrees C for 10 min attenuated the hydrogen peroxide (H(2)O(2))-induced decrease in cell viability, DNA ladder formation and nuclear condensation, and these effects were blocked by the protein synthesis inhibitor cycloheximide. The thermal stress inhibited the H(2)O(2)-induced increase in caspase-3 like protease activity, but it did not affect the H(2)O(2)-induced loss of mitochondrial membrane potential. The cytosol prepared from preheated cells did not affect Ca(2+)-induced swelling of mitochondria, a marker of the permeable transition pore. The protective effect of the thermal stress on the H(2)O(2)-induced decrease in cell viability was not affected by the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor 2-amino-3-methoxyflavone, the phosphatidylinositol-3 kinase inhibitor wortmannin and the NF-kappaB inhibitor pyrrolidinedithiocarbamate. These findings suggest that HSPs inhibit apoptosis via an inhibition of caspase-3 activation without effect on mitochondrial dysfunction.

CV-2619 protects cultured astrocytes against reperfusion injury via nerve growth factor production

In this study, we examined the effect of the neuroprotective agent 2, 3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (CV-2619) on reperfusion injury in cultured rat astrocytes after exposure to hydrogen peroxide (H(2)O(2))-containing medium. CV-2619 (10 nM to 10 microM) significantly attenuated the reperfusion-induced decrease in cell viability. The compound showed an anti-apoptotic effect in this astrocyte injury model. Antioxidants such as ascorbic acid, alpha-tocopherol and reduced glutathione also inhibited H(2)O(2) exposure-induced cytotoxicity. CV-2619 did not affect the levels of reactive oxygen species, but it increased nerve growth factor (NGF) production. The effect of CV-2619 on H(2)O(2) exposure-induced cytotoxicity was blocked by cycloheximide and anti-NGF antibody. The protective effect of CV-2619 was antagonized by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor 2-amino-3-methoxyflavone and the phosphatidylinositol-3 kinase inhibitor wortmannin. These findings suggest that the effect of CV-2619 is mediated at least partly by NGF production in astrocytes and that ERK and phosphatidylinositol-3 kinases play a role in the downstream mechanism.

Possible involvement of astrocytes in neuroprotection by the cognitive enhancer T-588.

We have previously shown that the cognition enhancer (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (T-588) protects astrocytes against hydrogen peroxide (H2O2) injury via activation of extracellular signal-regulated kinase (ERK) pathway. The present study examines whether the effect of T-588 on astrocytes contributes to neuroprotection in neuronal injury models. Astrocyte-conditioned medium (ACM) protected against neuronal injury induced by amyloid-β protein (Aβ) in cultured cortical neurons. The effect of ACM on Aβ-induced injury was blocked by the ERK kinase inhibitor 2′-amino-3′-methoxyflavone. ACM stimulated ERK phosphorylation in cultured neurons. ACM derived from astrocytes exposed to H2O2 lost the activities to stimulate ERK phosphorylation and protect against neuronal injury. T-588 blocked the H2O2-induced loss of the activities of ACM. These results suggest that ACM protects against neuronal injury by an ERK-dependent mechanism, and the effect of T-588 on astrocytic injury results in neuroprotection.