Caspase-3 activation induced by inhibition of mitochondrial complex I is facilitated by glycogen synthase kinase-3β and attenuated by lithium

The compound 1-methyl-4-phenylpyridinium (MPP) is a selective inhibitor of mitochondrial complex I, and is widely used in model systems to elicit neurochemical alterations that may be associated with Parkinson’s disease. In the present study treatment of human neuroblastoma SH-SY5Y cells with MPP resulted in a time- and concentration-dependent activation of the apoptosis-associated cysteine protease caspase-3, and caused morphological changes characteristic of apoptosis. To test if the activation state of the cell survival-promoting phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway affects MPP-induced caspase-3 activation, PI3K was inhibited with LY294002, or activated with insulin-like growth factor-1. MPP-induced caspase-3 activation was increased by inhibition of PI3K, and decreased by stimulation of PI3K, indicative of anti-apoptotic signaling by the PI3K/Akt pathway. To test if glycogen synthase kinase-3β (GSK3β), a pro-apoptotic kinase that is inhibited by Akt, is involved in regulating MPP-induced apoptosis, overexpression of GSK3β and lithium, a selective inhibitor of GSK3β, were used to directly alter GSK3β activity. MPP-induced caspase-3 activity was increased by overexpression of GSK3β. Conversely, the GSK3β inhibitor lithium attenuated MPP-induced caspase-3 activation. To test if these regulatory interactions applied to other mitochondrial complex I inhibitors, cells were treated with rotenone. Rotenone-induced activation of caspase-3 was enhanced by inhibition of PI3K or increased GSK3β activity, and was attenuated by inhibiting GSK3β with lithium. Overall, these results indicate that inhibition of GSK3β provides protection against the toxic effects of agents, such as MPP and rotenone, that impair mitochondrial function.     

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate attenuates glutamate-induced caspase-3 cleavage via regulation of glycogen synthase kinase 3beta

Preconditioning of sublethal ischemia exhibits neuroprotection against subsequent ischemia-induced neuronal death. It has been indicated that glutamate, an excitatory amino acid, is involved in the pathogenesis of ischemia-induced neuronal death or neurodegeneration. To elucidate whether prestimulation of glutamate receptor could counter ischemia-induced neuronal death or neurodegeneration, we examined the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), an ionotropic subtype of glutamate receptor, on excess glutamate-induced excitotoxicity using primary cortical neuronal cultures. We found that AMPA exerted a neuroprotective effect in a time- and concentration-dependent manner. A blocker of phosphatidylinositol-3 kinase (PI3K), LY294002 (10 microM), significantly attenuated AMPA-induced protection. In addition, Ser473 of Akt/PKB, a downstream target of PI3K, was phosphorylated by AMPA administration (10 microM). Glycogen synthase kinase 3beta (GSK3beta), which has been reported to be inactivated by Akt, was phosphorylated at Ser9 by AMPA. Ser9-phosphorylated GSK3beta or inactivated form would be a key molecule for neuroprotection, insofar as lithium chloride (100 microM) and SB216763 (10 microM), inhibitors of GSK3beta, also induced phosphorylation of GSK3beta at Ser9 and exerted neuroprotection, respectively. Glutamate (100 microM) increased cleaved caspase-3, an apoptosis-related cysteine protease, and caspase-3 inhibitor (Ac-DEVD-CHO; 1 microM) blocked glutamate-induced excitotoxicity in our culture. AMPA (10 microM, 24 hr) and SB216763 (10 microM) prominently decreased glutamate-induced caspase-3 cleavage. These findings suggest that AMPA activates PI3K-Akt and subsequently inhibits GSK3beta and that inactivated GSK3beta attenuates glutamate-induced caspase-3 cleavage and neurotoxicity.     

Lamotrigine inhibition of rotenone- or 1-methyl-4-phenylpyridinium-induced mitochondrial damage and cell death

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the effect of antiepileptic lamotrigine against the cytotoxicity of mitochondrial respiratory complex I inhibitors rotenone and 1-methyl-4-phenylpyridinium (MPP+) in relation to the mitochondria-mediated cell death process and oxidative stress. Both rotenone and MPP+ induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Lamotrigine significantly attenuated the rotenone- or MPP+-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. The preventive effect of lamotrigine against the toxicity of rotenone was greater than its effect on that of MPP+. The results show that lamotrigine seems to reduce the cytotoxicity of rotenone and MPP+ by suppressing the mitochondrial permeability transition formation, leading to cytochrome c release and subsequent activation of caspase-3. The preventive effect may be ascribed to its inhibitory action on the formation of reactive oxygen species and depletion of GSH. Lamotrigine seems to exert a protective effect against the neuronal cell injury due to the mitochondrial respiratory complex I inhibition.     

PTEN, Akt, and GSK3beta signalling in rat primary cortical neuronal cultures following tumor necrosis factor-alpha and trans-4-hydroxy-2-nonenal treatments

PTEN is a dual phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway important for cell survival. We determined effects of the inflammation and oxidative stresses of tumor necrosis factor-alpha (TNFalpha) and trans-4-hydroxy-2-nonenal (HNE), respectively, on PTEN, Akt, and GSK3beta signalling in rat primary cortical neurons. The inhibitors bisperoxovanadium [bpV(Pic)] and LY294002 were also used to determine PTEN and PI3K involvement in TNFalpha and HNE modulation of neuronal cell death. PTEN inhibition with bpV(Pic) alone did not affect Ser(473)Akt or Ser(9)GSK3beta phosphorylation. Instead, effects of this inhibitor were manifest when it was used together with TNFalpha and to a lesser extent with HNE. TNFalpha together with PTEN inhibition increased phosphorylation of Ser(473)Akt and Ser(9)GSK3beta. TNFalpha and HNE both gave decreased numbers of viable and increased numbers of early apoptotic neurons. PTEN inhibition partially reversed the toxic effect of TNFalpha as shown by an increased number of viable and a decreased number of early apoptotic neurons. All effects were reversed by PI3K inhibition. HNE together with inhibition of PTEN gave increased Ser(473)Akt but not Ser(9)GSK3beta phosphorylation and no effects on the number of viable or early apoptotic cells. In conclusion, PTEN inhibition gives a mild reversal of TNFalpha- but not HNE-induced cell death via the PI3K pathway.     

过表达Mfn2基因通过PI3K/AKT通路抑制鱼藤酮诱导的帕金森病细胞模型的细胞凋亡

目的 探讨过表达线粒体融合蛋白2(Mfn2)基因通过PI3K/AKT通路抑制鱼藤酮诱导的帕金森病细胞模型的细胞凋亡。方法 培养SH-SY5Y细胞并分组,对照组用不含药物及质粒的DMEM干预; 鱼藤酮组用含有20 μmol/L鱼藤酮的DMEM干预; 鱼藤酮+空白质粒组用含有20 μmol/L鱼藤酮的DMEM干预并转染空白的pcDNA 3.1质粒; 鱼藤酮+Mfn2质粒组用含有20 μmol/L鱼藤酮的DMEM干预并转染表达Mfn2的pcDNA3.1质粒; 鱼藤酮+Mfn2质粒+LY组用含有20 μmol/L鱼藤酮、10 μmol/L LY294002的DMEM干预并转染表达Mfn2的pcDNA3.1质粒; 检测细胞活力、凋亡率及线粒体凋亡基因、p-PI3K、p-AKT的表达水平。结果 鱼藤酮组的细胞活力及细胞中p-PI3K、p-AKT的表达水平均低于对照组,凋亡率及细胞中caspase-9、cleaved caspase-3的表达水平均高于对照组; 鱼藤酮+Mfn2质粒组的细胞活力及细胞中p-PI3K、p-AKT的表达水平均高于鱼藤酮组,凋亡率及细胞中caspase-9、cleaved caspase-3的表达水平均低于鱼藤酮组; 鱼藤酮+Mfn2质粒+LY组的细胞活力及细胞中p-PI3K、p-AKT的表达水平均低于鱼藤酮+Mfn2质粒组,凋亡率及细胞中caspase-9、cleaved caspase-3的表达水平均高于鱼藤酮+Mfn2质粒组。结论 过表达Mfn2基因对鱼藤酮诱导的帕金森病细胞模型的细胞凋亡具有抑制作用,且该作用可能与抑制PI3K/AKT通路有关     

ErbB4 activation inhibits MPP+-induced cell death in PC12-ErbB4 cells

The neuroprotective effects of neuregulin (NRG), a polypeptide growth factor, on 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell death and oxidative stress in PC12-ErbB4 cells were investigated. Treatment of PC12-ErbB4 cells with MPP+induced cell death that was markedly attenuated by NRG. The PI3K/PKB/Akt and Ras/MapK signaling pathways probably mediate the survival effect of NRG. NRG induces prolonged activation of PKB/Akt and Erk. Moreover, inhibition of the PI3K and MEK activities prevented the NRG-induced survival effect. Over-expression of constitutively active PI3K or H-Ras (12V) inhibited MPP+-mediated cell death. In addition, MPP+-mediated reactive oxygen species (ROS) elevation was also inhibited by NRG. The effect of NRG on ROS levels was blocked by PI3K and MEK inhibitors, indicating that both signaling pathways can regulate the toxic ROS levels induced by MPP+. Taken together, these results indicate that in PC12-ErbB4 cells, the NRG-induced neuroprotective effect from MPP+treatment, requires PI3K/PKB/Akt and Ras/MapK signaling networks. These authors contributed equally to this work.     

IGF-1 protects oligodendrocyte progenitors against TNFalpha-induced damage by activation of PI3K/Akt and interruption of the mitochondrial apoptotic pathway

Proinflammatory cytokine-mediated injury to oligodendrocyte progenitor cells (OPCs) has been proposed as a cause of periventricular leukomalacia (PVL), the most common brain injury found in preterm infants. Preventing death of OPCs is a potential strategy to prevent or treat PVL. In the current study, we utilized an in vitro cell culture system to investigate the effect of insulin-like growth factor-1 (IGF-1) on tumor necrosis factor-alpha (TNFalpha)-induced OPC injury and the possible mechanisms involved. OPCs were isolated from neonatal rat optic nerves and cultured in chemically defined medium (CDM) supplemented with platelet-derived growth factor and basic fibroblast growth factor. Exposure to TNFalpha resulted in death of OPCs. IGF-1 protected OPCs from TNFalpha cytotoxicity in a dose-dependent manner as measured by the XTT and TUNEL assays. IGF-1 activates both the PI3K/Akt and the extracellular signal-regulated kinase (ERK) pathway. However, IGF-1-enhanced cell survival signals were mediated by the PI3K/Akt, but not by the ERK pathway, as evidenced by the observation that IGF-1-enhanced cell survival was partially abrogated by Akti, the Akt inhibitor, or wortmannin, the PI3K inhibitor, but not by PD98,059, the MAPK kinase/ERK kinase inhibitor. The downstream events of IGF-1-triggered survival signals included phosphorylation of BAD, blockade of TNFalpha-induced translocation of Bax from the cytosol to the mitochondrial membrane, and suppression of caspase-9 and caspase-3 activation. These observations indicate that the protection of OPCs by IGF-1 is mediated, at least partially, by interruption of the mitochondrial apoptotic pathway via activation of PI3K/Akt.     

IGF‐I neuroprotection in the immature brain after hypoxia‐ischemia,involvement of Akt and GSK3β?

Insulin-like growth factor I (IGF-I) is a neurotrophic factor that promotes neuronal growth, differentiation and survival. Neuroprotective effects of IGF-I have previously been shown in adult and juvenile rat models of brain injury. We wanted to investigate the neuroprotective effect of IGF-I after hypoxia-ischemia (HI) in 7-day-old neonatal rats and the mechanisms of IGF-I actions in vivo. We also wanted to study effects of HI and/or IGF-I on the serine/threonine kinases Akt and glycogen synthase kinase 3beta (GSK3beta) in the phophatidylinositol-3 kinase (PI3K) pathway. Immediately after HI, phosphorylated Akt (pAkt) and phosphorylated GSK3beta (pGSK3beta) immunoreactivity was lost in the ipsilateral and reduced in the contralateral hemisphere. After 45 min, pAkt levels were restored to control values, whereas pGSK3beta remained low 4 h after HI. Administration of IGF-I (50 microg i.c.v.) after HI resulted in a 40% reduction in brain damage (loss of microtubule-associated protein) compared with vehicle-treated animals. IGF-I treatment without HI was shown to increase pAkt whereas pGSK3beta decreased in the cytosol, but increased in the nuclear fraction. IGF-I treatment after HI increased pAkt in the cytosol and pGSK3beta in both the cytosol and the nuclear fraction in the ipsilateral hemisphere compared with vehicle-treated rats, concomitant with a reduced caspase-3- and caspase-9-like activity. In conclusion, IGF-I induces activation of Akt during recovery after HI which, in combination with inactivation of GSK3beta, may explain the attenuated activation of caspases and reduction of injury in the immature brain.     

Econazole attenuates cytotoxicity of 1-methyl-4-phenylpyridinium by suppressing mitochondrial membrane permeability transition

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The effect of econazole against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells was assessed in relation to the mitochondrial membrane permeability changes. Treatment of PC12 cells with MPP(+) resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. Econazole (0.25-2.5 microM) inhibited the cytotoxicity of MPP(+) or rotenone. The addition of econazole (0.5 microM) significantly attenuated the MPP(+)-induced mitochondrial damage, elevation of intracellular Ca(2+) level and cell death. However, because of the cytotoxicity, econazole at 5 microM did not attenuate the toxicity of MPP(+). The results show that econazole at the low concentrations may reduce the MPP(+)-induced viability loss in PC12 cells by suppressing the mitochondrial permeability transition, leading to activation of caspase-3 and the elevation of intracellular Ca(2+) levels, which are associated with the increased formation of ROS and depletion of GSH.     

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK‐3β and CREB through PI3‐K/Akt pathways

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK‐3β) and cAMP response element‐binding protein (CREB) through phosphatidylinositol 3‐kinase (PI3‐K)‐dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four‐vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK‐3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3‐K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK‐3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3‐K, compromised the neuroprotective effect of resveratrol and decreased the level of p‐Akt, p‐GSK‐3β and p‐CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro‐survival states of Akt, GSK‐3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3‐K/Akt signaling pathway, subsequently downregulating expression of GSK‐3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.     

Cesium chloride protects cerebellar granule neurons from apoptosis induced by low potassium

Neuronal apoptosis plays a critical role in the pathogenesis of neurodegenerative disorders, and neuroprotective agents targeting apoptotic signaling could have therapeutic use. Here we report that cesium chloride, an alternative medicine in treating radiological poison and cancer, has neuroprotective actions. Serum and potassium deprivation induced cerebellar granule neurons to undergo apoptosis, which correlated with the activation of caspase-3. Cesium prevented both the activation of caspase-3 and neuronal apoptosis in a dose-dependent manner. Cesium at 8 mM increased the survival of neurons from 45 +/- 3% to 91 +/- 5% of control. Cesium's neuroprotection was not mediated by PI3/Akt or MAPK signaling pathways, since it was unable to activate either Akt or MAPK by phosphorylation. In addition, specific inhibitors of PI3 kinase and MAP kinase did not block cesium's neuroprotective effects. On the other hand, cesium inactivated GSK3beta by phosphorylation of serine-9 and GSK3beta-specific inhibitor SB415286 prevented neuronal apoptosis. These data indicate that cesium's neuroprotection is likely via inactivating GSK3beta. Furthermore, cesium also prevented H(2)O(2)-induced neuronal death (increased the survival of neurons from 72 +/- 4% to 89 +/- 3% of control). Given its relative safety and good penetration of the brain blood barrier, our findings support the potential therapeutic use of cesium in neurodegenerative diseases.     

Inhibition of GSK3beta is a common event in neuroprotection by different survival factors

Depolarizing concentrations of potassium (HK, 25 mM), cyclic AMP elevating agents and analogs (cAMP), insulin-like growth factor-1 (IGF-1), or lithium can maintain the survival of cultured rat cerebellar granule neurons (CGNs). We investigated the possibility that the signal transduction pathways utilized by these four survival factors converge in regulating a common molecular target. We targeted the regulation of the kinase GSK3beta as the critical event in the survival directed by the four survival factors. We found that treatment of CGNs with HK, the cAMP-elevating agent forskolin, IGF-1, and lithium resulted in phosphorylation of GSK3beta at serine-9 and thus its inactivation. Furthermore, pharmacological inhibition of core components in the survival signaling cascades initiated by HK, forskolin, IGF-1, and lithium causes apoptosis and activation of GSK3beta accompanies this death. Finally, we examined the pharmacological inhibitors of GSK3beta, GSK3 inhibitor I, TDZD-8, and SB-415286, for their ability to prevent low potassium (LK)-induced apoptosis. Although previous reports demonstrate inhibition of GSK3beta in in vitro kinase assays with GSK3 inhibitor I and TDZD-8, we were unable to detect inhibition of GSK3beta in neuronal cultures treated with these compounds and thus no protection from LK-induced apoptosis. SB-415286 on the other hand, was able to rescue CGNs from cell death. Taken together, we conclude that regulation of GSK3beta is a critical convergence event in the promotion of CGN survival by different factors.     

Epigallocatechin gallate protects nerve growth factor differentiated PC12 cells from oxidative-radical-stress-induced apoptosis through its effect on phosphoinositide 3-kinase/Akt and glycogen synthase kinase-3

The effects of epigallocatechin gallate (EGCG) on the phosphoinositide 3-kinase (PI3K)/Akt and glycogen synthase kinase-3 (GSK-3) pathway during oxidative-stress-induced injury were studied using H2O2-treated PC12 cells, which were differentiated by nerve growth factor (NGF). Following 100 microM H2O2 exposure, the viability of differentiated PC12 cells (EGCG or z-VAD-fmk pretreated vs. not pretreated) was evaluated the number of viable cell with Trypan blue and 3,4,5-dimethylthiazol-2-yl (MTT). Additionally, expression of cytochrome c, caspase-3, poly(ADP-ribose) polymerase (PARP), PI3K/Akt and GSK-3 was examined using Western blot analyses. EGCG or z-VAD-fmk-pretreated PC12 cells showed an increase of viability compared to untreated PC12 cells, and pretreatment of PC12 cells with either agent induced a dose-dependent inhibition of caspase-3 activation and PARP cleavage. However, inhibition of cytochrome c release was only detected in EGCG-pretreated cells. Upon examination of the PI3K/Akt and GSK-3 upstream pathway, Western blots of EGCG pretreated cells showed decreased immunoreactivity (IR) of Akt and GSK-3 and increased IR of p85a PI3K, phosphorylated Akt and phosphorylated GSK-3. In contrast, no changes were seen in z-VAD-fmk-pretreated cells. These results show that EGCG affects the PI3K/Akt, GSK-3 pathway as well as downstream signaling, including the cytochrome c and caspase-3 pathways. Therefore, it is suggested that EGCG-mediated activation of PI3K/Akt and inhibition of GSK-3 could be a new potential therapeutic strategy for neurodegenerative diseases associated with oxidative injury.     

Activation of the Akt/GSK3beta signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rats.

Recent studies have revealed that the phosphatidylinositol 3-kinase (PI3-K) pathway is involved in apoptotic cell death after experimental cerebral ischemia. The serine-threonine kinase, Akt, functions in the PI3-K pathway and prevents apoptosis by phosphorylation at Ser473 after a variety of cell death stimuli. After phosphorylation, activated Akt inactivates other apoptogenic factors, including glycogen synthase kinase-3beta (GSK3beta), thereby inhibiting cell death. However, the role of Akt/GSK3beta signaling in the delayed death of hippocampal neurons in the CA1 subregion after transient global cerebral ischemia (tGCI) has not been clarified. Transient global cerebral ischemia for 5 mins was induced by bilateral common carotid artery occlusion combined with hypotension. Western blot analysis showed a significant increase in phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) in the hippocampal CA1 subregion after tGCI. Immunohistochemistry showed that expression of phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) was markedly increased in the vulnerable CA1 subregion, but not in the ischemic-tolerant CA3 subregion. Double staining with phospho-GSK3beta (Ser9) and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed different cellular distributions in the CA1 subregion 3 days after tGCI. Phosphorylation of Akt and GSK3beta was prevented by LY294002, a PI3-K inhibitor, which facilitated subsequent DNA fragmentation 3 days after tGCI. Moreover, transgenic rats that overexpress copper/zinc-superoxide dismutase, which is known to be neuroprotective against delayed hippocampal CA1 injury after tGCI, had enhanced and persistent phosphorylation of both Akt and GSK3beta after tGCI. These findings suggest that activation of the Akt/GSK3beta signaling pathway may mediate survival of vulnerable hippocampal CA1 neurons after tGCI.     

Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.     

Modulation of 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells by K<Subscript>ATP</Subscript> channel block

The present study investigated the effect of 5-hydroxydecanoate, a selective mitochondrial K(ATP) channel blocker, on the cytotoxicity of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells. 5-Hydroxydecanoate and glibenclamide (a cell surface and mitochondrial K(ATP) channel inhibitor) reduced the MPP(+)-induced cell death and GSH depletion and showed a maximal inhibitory effect at 5 and 10 microM, respectively. Addition of 5-hydroxydecanoate attenuated the MPP(+)-induced nuclear damage, changes in the mitochondrial membrane permeability and increase in the reactive oxygen species formation in PC12 cells. The results show that 5-hydroxydecanote may prevent the MPP(+)-induced viability loss in PC12 cells by suppressing formation of the mitochondrial permeability transition, leading to the cytochrome c release and caspase-3 activation. This effect appears to be accomplished by the inhibitory action on the formation of reactive oxygen species and the depletion of GSH. The blockade of mitochondrial K(ATP) channels seems to prevent the MPP(+)-induced neuronal cell damage.     

PIAS3过表达抑制人脑胶质瘤U251细胞生长

目的 探讨PIAS3 过表达对人脑胶质瘤U251 细胞生长的作用及其可能的机制.方法 通过实时荧光定量PCR 检测PIAS3 在正常脑细胞和脑胶质瘤U251 细胞中的表达情况.转染PIAS3 过表达载体,提高U251 细胞中PIAS3 表达水平,通过噻唑蓝(MTT)试验检测细胞增殖情况.Western Blot 验证PIAS3 表达与增殖相关基因PI3K 及Akt 间关系.结果 PIAS3 在正常脑组织中相对高表达,而在人脑胶质瘤U251 细胞中表达明显下降(P ＜0.01).体外转染PIAS3 过表达载体能明显抑制U251 细胞生长,并且有效抑制PI3K 活性及p-Akt 表达,但对总的Akt 无明显影响.结论 胶质瘤U251 细胞中异常低表达的PIAS3 发挥着重要的增殖促进作用,过表达PIAS3 可通过抑制PI3K/Akt 通路有效抑制U251 细胞增殖.     

Morphine promotes Jurkat cell apoptosis through pro-apoptotic FADD/P53 and anti-apoptotic PI3K/Akt/NF-kappaB pathways

Opiates have been shown to inhibit cell growth and trigger apoptosis, but the underlying molecular mechanisms remain unclear. We have previously shown that morphine induces Fas expression and promotes Fas-mediated apoptosis. Here, we investigated the mechanisms by which morphine modulates apoptosis in human Jurkat cells. Morphine-induced apoptosis was inhibited by transfection with a dominant negative Fas-associated death domain (FADD) plasmid, revealing that morphine-induced apoptosis is dependent on FADD. Furthermore, suppression of endogenous p53 expression by RNA interference technology considerably attenuated the morphine-induced apoptosis. In addition, morphine-induced apoptosis seems to be dependent on the activation of phosphatidylinositol 3-kinase (PI3K), as PI3K inhibition by the PI3K inhibitor LY294002 significantly enhanced morphine-induced apoptosis. Moreover, inhibition of Akt or nuclear factor-kappaB (NF-kappaB) expression by RNA interference technology also dramatically increased morphine-induced apoptosis. Our study thus demonstrates that morphine induces Jurkat cell apoptosis through FADD/p53, anti-apoptotic PI3K/Akt and NF-kappaB pathways.     

Neuroprotective effect of guanosine against glutamate-induced cell death in rat hippocampal slices is mediated by the phosphatidylinositol-3 kinase/Akt/ glycogen synthase kinase 3β pathway activation and inducible nitric oxide synthase inhibition

Excitotoxicity and cell death induced by glutamate are involved in many neurodegenerative disorders. We have previously demonstrated that excitotoxicity induced by millimolar concentrations of glutamate in hippocampal slices involves apoptotic features and glutamate-induced glutamate release. Guanosine, an endogenous guanine nucleoside, prevents excitotoxicity by its ability to modulate glutamate transport. In this study, we have evaluated the neuroprotective effect of guanosine against glutamate-induced toxicity in hippocampal slices and the mechanism involved in such an effect. We have found that guanosine (100 μM) was neuroprotective against 1 mM glutamate-induced cell death through the inhibition of glutamate release induced by glutamate. Guanosine also induced the phosphorylation and, thus, activation of protein kinase B (PKB/Akt), a downstream target of phosphatidylinositol-3 kinase (PI3K), as well as phosphorylation of glycogen synthase kinase 3β, which has been reported to be inactivated by Akt after phosphorylation at Ser9. Glutamate treated hippocampal slices showed increased inducible nitric oxide synthase (iNOS) expression that was prevented by guanosine. Slices preincubated with SNAP (an NO donor), inhibited the protective effect of guanosine. LY294002 (30 μM), a PI3K inhibitor, attenuated guanosine-induced neuroprotection, guanosine prevention of glutamate release, and guanosine-induced GSK3β(Ser9) phosphorylation but not guanosine reduction of glutamate-induced iNOS expression. Taken together, the results of this study show that guanosine protects hippocampal slices by a mechanism that involves the PI3K/Akt/GSK3β(Ser9) pathway and prevention of glutamate-induced glutamate release. Furthermore, guanosine also reduces glutamate-induced iNOS by a PI3K/Akt-independent mechanism.     

Serotonin stimulates mitochondrial transport in hippocampal neurons

Axonal transport of mitochondria is critical for proper neuronal function. However, little is known about the extracellular signals that regulate this process. In the present study, we show that the neuromodulator serotonin (5-HT) greatly enhances mitochondrial movement in the axons of rat hippocampal neurons in vitro. Administration of a 5-HT1A receptor antagonist inhibited mitochondrial movement, whereas addition of fluoxetine, a selective serotonin reuptake inhibitor, promoted mitochondrial movement. 5-HT receptors are known to activate the Akt/Protein kinase B pathway. Consistent with this, directional mitochondrial movement was almost completely blocked by a specific Akt inhibitor. Moreover, an inhibitor of glycogen synthase kinase-3beta (GSK3beta), a kinase whose activity is blocked by Akt-mediated phosphorylation, promoted mitochondrial movement. These findings show that 5-HT1A receptor activation stimulates mitochondrial movement in hippocampal neurons by inhibiting GSK3beta activity via Akt. Our findings suggest that 5-HT may mediate the redistribution of energy sources within responsive neurons, a possibility that has significant implications for understanding the global biological effects of this important neuromodulator.