Abeta(1-42)-induced JNK and ERK activation in rabbit hippocampus is differentially regulated by lithium but is not involved in the phosphorylation of tau

Administration of Abeta(1-42) into the rabbit brain induces apoptosis and phosphorylation of tau. These Abeta effects correlate with the activation of JNK and ERK, but not of p38. Treatment with 7 mM lithium inhibits apoptosis, modulates JNK and ERK and does not affect the phosphorylation of tau. Our results demonstrate that lithium, at this dose, effectively inhibits the Abeta-induced apoptosis but has no effect on tau phosphorylation, and that MAP kinases are not involved in the phosphorylation of tau.     

Genes associated with pro-apoptotic and protective mechanisms are affected differently on exposure of neuronal cell cultures to arsenite. No indication for endoplasmic reticulum stress despite activation of grp78 and gadd153 expression

The effect of arsenite exposure on cell viability, protein synthesis, energy metabolism and the expression of genes coding for cytoplasmic (hsp70) and endoplasmic reticulum (ER; gadd153, grp78, grp94) stress proteins was investigated in primary neuronal cell cultures. Furthermore, signs of ER stress were evaluated by investigating xbp1 mRNA processing. Arsenite levels of 30 and 100 microM induced severe cell injury. Protein synthesis was reduced to below 20% of control in cultures exposed to 30 and 100 microM arsenite for 1 h, and it remained markedly suppressed until 24 h of exposure. Arsenite induced a transient inhibition of energy metabolism after 1 h of exposure, but energy state recovered completely after 3 h. Arsenite exposure affected the expression and translation of genes coding for HSP70 and GRP78, GRP94, GADD153 to different extents. While hsp70 mRNA levels rose drastically, approximally 550-fold after 6 h exposure, HSP70 protein levels did not change over the first 6 h. On the other hand, gadd153 mRNA levels rose only approximately 14-fold after 6 h exposure, while GADD153 protein levels were markedly increased after 3 and 6 h exposure. HSP70 protein levels were markedly increased and GADD153 protein levels decreased to almost control levels in cultures left in arsenite solution for 24 h, i.e. when only a small fraction of cells had escaped arsenite toxicity. Arsenite exposure of neurons thus induced an imbalance between pro-apoptotic and survival-activating pathways. Despite the marked increase in gadd153 mRNA levels, we did not observe signs of xbp1 processing in arsenite exposed cultures, indicating that arsenite did not produce ER stress.     

Homocysteine-induced changes in mRNA levels of genes coding for cytoplasmic- and endoplasmic reticulum-resident stress proteins in neuronal cell cultures

Elevated homocysteine levels have been suggested to contribute to various pathological states of the brain. However, the basic mechanisms underlying homocysteine-induced neurotoxicity have not yet been fully elucidated. In the present series of experiments, we investigated the effect of homocysteine on mRNA levels of genes coding for cytoplasmic- or endoplasmic reticulum-resident stress proteins. Primary neuronal cell cultures were exposed to different homocysteine levels for 1-24 h. Cell injury was evaluated using the MTT assay, protein synthesis was studied by measuring the incorporation of L-[4,5-3H]leucine into proteins, mRNA levels of hsp70, gadd153, grp78, and grp94 were evaluated by quantitative PCR, and changes in protein levels of hsp70, grp78 and grp94 were analyzed by immunoblotting. Exposure of cells to 5 or 10 mM homocysteine for 24 h induced marked cell injury (decrease of viability to 58 or 45% of control respectively). After 6 h treatment, gadd153, grp78 and grp94 mRNA levels increased markedly, but only when cells were exposed to levels of homocysteine high enough to induce cell injury. In addition, hsp70 mRNA levels and protein synthesis were significantly reduced. At earlier (1 or 3 h) or later (12 or 24 h) time intervals, homocysteine exposure induced a marked increase in mRNA levels of all genes studied. GRP78 and GRP94 protein levels were increased in cells exposed to 5 mM homocysteine for 24 h but not in cells exposed to 10 mM homocysteine. HSP70 protein levels, in contrast, were decreased in cells exposed to homocysteine for different periods. The expression of genes coding for ER-resident stress proteins is specifically activated under conditions of ER stress. The close relationship between the extent of cell injury and increase in grp78 mRNA levels suggests that ER dysfunction may contribute to the pathological process. The results imply that the ER is an intracellular target of homocysteine toxicity.     

ER stress is involved in Abeta-induced GSK-3beta activation and tau phosphorylation

Intracellular neurofibrillary tangles, one of the characteristic hallmarks of Alzheimer's disease (AD), are mainly composed of hyperphosphorylated tau. The abnormal tau phosphorylation seems to be related to altered activity of kinases such as glycogen synthase kinase-3beta (GSK-3beta). Tau pathology is thought to be a later event during the progression of the disease, and it seems to occur as a consequence of amyloid-beta (Abeta) peptide accumulation. The aim of this work was to investigate whether soluble Abeta1-42, particularly oligomers that correspond to the neurotoxic species involved early in the development of AD, triggers tau phosphorylation by a mechanism involving activation of tau-kinase GSK-3beta. Several studies suggest that GSK-3beta plays a central role in signaling the downstream effects of endoplasmic reticulum (ER) stress. Therefore, the involvement of ER Ca(2+) release in GSK-3beta activation and tau phosphorylation induced by Abeta1-42 oligomers was evaluated using dantrolene, an inhibitor of Ca(2+) release through channels associated with ER ryanodine receptors. We observed that Abeta1-42 oligomers increase tau phosphorylation and compromises cell survival through a mechanism mediated by GSK-3beta activation. We also demonstrated that oligomeric Abeta1-42 induces ER stress and that ER Ca(2+) release is involved in oligomer-induced GSK-3beta activation and tau phosphorylation. This work suggests that GSK-3beta can be a promising target for therapeutic intervention in AD.     

Sodium tungstate decreases the phosphorylation of tau through GSK3 inactivation

Tungstate treatment increases the phosphorylation of glycogen synthase kinase-3beta (GSK3beta) at serine 9, which triggers its inactivation both in cultured neural cells and in vivo. GSK3 phosphorylation is dependent on the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) induced by tungstate. As a consequence of GSK3 inactivation, the phosphorylation of several GSK3-dependent sites of the microtubule-associated protein tau decreases. Tungstate reduces tau phosphorylation only in primed sequences, namely, those prephosphorylated by other kinases before GSK3beta modification, which are serines 198, 199, or 202 and threonine 231. The phosphorylation at these sites is involved in reduction of the interaction of tau with microtubules that occurs in Alzheimer's disease.     

Integrins mediate beta-amyloid-induced cell-cycle activation and neuronal death

Early intracellular events responsible for cell-cycle induction by beta-amyloid (A beta) in neurons have not been identified yet. Extracellular signal-regulated kinases 1/2 (ERK1/2) have been identified in this pathway, and inhibition of ERK activity prevents cell-cycle activation and reduces neuronal death induced by A beta. To identify upstream events responsible for ERK activation, attention has been focused on integrins. Treatment of SH-SY5Y cells, differentiated by long-term exposure to 10 microM retinoic acid with a neutralizing anti-alpha1-integrin antibody significantly reduced A beta-induced neuronal death. However, cell-cycle analysis showed that treatment with anti-alpha1-integrin per se produced changes in the distribution of cell populations, thus hampering any effect on A beta-induced cell-cycle activation. 4-Amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-D)pyramide, an inhibitor of src protein kinases that colocalizes with focal adhesion kinase (FAK) and is involved in integrin signaling, was effective in reducing activation of the cell cycle and preventing induction of neuronal death by A beta while inhibiting ERK1/2 phosphorylation. Similar results were obtained when FAK expression was down-regulated by siRNA silencing. The present study identifies a sequence of early events in the toxic effect of A beta in neuronal cultures that involves interaction with integrins, activation of FAK/src, enhanced phosphorylation of ERK1/2, and induction of the cell cycle, all leading to neuronal death.     

Modulation of ERK and JNK activity by transient forebrain ischemia in rats

The mitogen-activated protein (MAP) kinase families of ERK and JNK participate in numerous intracellular signaling pathways and are abundantly expressed in the CNS. Activation of ERK and JNK during reperfusion of ischemic tissue is implicated in promoting cell death, insofar as inhibition of either pathway reduces neuronal cell death. However, ERK or JNK activation provides protection in other neuronal injury models. In this study, we monitored the concurrent modulation of ERK and JNK activity in the hippocampus, neocortex, and striatum during ischemia and immediately upon reperfusion in a rat model of transient global ischemia. All three regions incur a similar reduction in blood flow during occlusion but show different extents and temporal patterns of injury following reperfusion. ERK and JNK were active in the normal rat forebrain, and phosphorylation was reduced by ischemia. Upon reperfusion, ERK was rapidly activated in the hippocampus, neocortex, and striatum, whereas JNK phosphorylation increased in the hippocampus and striatum but not in the neocortex. The response of JNK vs. ERK more closely reflects the susceptibility of these regions. JNK1 was the predominant phosphorylated isoform. A minor pool of phosphorylated JNK3 increased above the control level after reperfusion in hippocampal but not in neocortical particulate fractions. In addition, a novel 32-35-kDa c-Jun kinase activity was detected in the hippocampus, neocortex, and striatum. The results show that ERK and JNK activities are rapidly, but not identically, modulated by ischemia and reperfusion and indicate that the MAP kinase pathways contribute to regulating the response to acute CNS injury.     

Activation of NF-kB and ERK1/2 after permanent focal ischemia is abolished by simvastatin treatment

We investigated the effects of simvastatin treatment on the expression of IL-1beta and MCP-1, the activity of NF-kB, and the signaling pathways related to NF-kB activation in a rat model of permanent middle cerebral artery occlusion (pMCAO). IL-1beta and MCP-1 expression, determined using RT-PCR, was enhanced by pMCAO; this effect was inhibited by the administration of simvastatin before ischemia. Pre-treatment with simvastatin abolished the ischemia-induced activation of NF-kB observed in vehicle-treated animals. The evaluation of signal transduction pathways, including extracellular signal-regulated kinase (ERK1/2), SAPK/JNK 46/54 and p38, indicated that only ERK1/2 phosphorylation was enhanced by ischemia, and this activation was prevented by simvastatin. ERK1/2-inhibitor, U0126, reduced brain ischemia but not cytokine induction. These results provide evidence that the HMG-CoA reductase inhibitor induces its effect in the protection of ischemic brain damage with a more complex mechanism which also involve anti-inflammatory properties rather than simple inhibition of ERK1/2 signaling pathway.     

Mitogenic effect of glial cell line-derived neurotrophic factor is dependent on the activation of p70S6 kinase,but independent of the activation of ERK and up-regulation of Ret in SH-SY5Y cells

Glial cell line-derived neurotrophic factor (GDNF) activates c-Ret tyrosine kinase and several downstream intracellular pathways; the biological effects caused by the activation of each of these pathways, however, remain to be elucidated. Here we report the ability of GDNF to induce proliferation, rather than differentiation, of neuroblastoma cells (SH-SY5Y) by targeting the signaling pathway responsible for mediating this proliferative effect. GDNF induces the phosphorylation of Akt and p70S6 kinase (p70S6K) in SH-SY5Y cells in which Ret protein expression is relatively low. Interestingly, treating SH-SY5Y cells with retinoic acid greatly increases Ret protein levels and GDNF-induced Ret tyrosine phosphorylation, but does not affect the mitogenic action of GDNF and the activation of the Akt/p70S6K pathway. In contrast, the activation of the ERK pathway and the resulting induction of immediate-early genes parallel the increases in Ret protein levels. Rapamycin, a specific inhibitor of p70S6K activation by the mammalian target of rapamycin, completely prevents GDNF-induced proliferation and activation of p70S6K. These results suggest that GDNF promotes cell proliferation via the activation of p70S6K, independent of the ERK signaling pathway, and that GDNF activates the Akt/p70S6K pathway more efficiently than the ERK pathway in the cells in which Ret expression is low.     

MAPK信号通路在创伤性脑损伤中的作用

目的通过建立小鼠创伤性脑损伤(TBI)模型,研究丝裂原活化蛋白激酶(MAPKs)通路中的细胞外调节蛋白激酶1/2(ERK1/2)通路、JNK通路和p38通路的激活及在TBI中的作用及机制。方法建立小鼠TBI模型,通过Western blot检测ERK1/2、JNK和p38的相对磷酸化水平,确定TBI后MAPK通路的激活情况;分别加入ERK1/2通路抑制剂(PD98059,500μmol/L)、JNK通路抑制剂(SP600125,500μmol/L)和p38通路抑制剂(SB203580,500μmol/L),通过脑干湿重检测、神经功能学评分和TUNEL染色评估不同抑制剂对TBI的作用,并通过Western blot检测ERK1/2、JNK和p38的相对磷酸化水平,明确ERK1/2通路、JNK通路和p38通路之间的相互调节作用。结果 TBI可分别引起ERK1/2通路、JNK通路和p38通路的激活;抑制ERK通路和JNK通路可减轻TBI引起的脑水肿、神经功能损伤和细胞凋亡,而抑制p38通路则加重TBI引起的脑水肿、神经功能损伤和细胞凋亡;抑制JNK通路可减少ERK1/2的相对磷酸化水平,而抑制p38通路可增加ERK1/2的相对磷酸化水平。结论 TBI后,ERK1/2通路和JNK通路的激活发挥促进损伤形成的作用,而p38通路的激活则起到神经保护的作用;ERK1/2通路的激活受到JNK通路的促进和p38通路的抑制,表明MAPK通路之间存在相互调节。     

Glial-derived neurotrophic factor (GDNF) prevents ethanol-induced apoptosis and JUN kinase phosphorylation

Ethanol exposure during neural development leads to substantial neuronal loss in multiple brain regions. Our previous research indicated that exogenous glial-derived neurotrophic factor (GDNF) attenuated ethanol-induced cerebellar Purkinje cell loss. Additionally, ethanol decreased GDNF release suggesting that ethanol disrupts GDNF-signaling pathways. The present experiments utilized a homogeneous GDNF-responsive neuroblastoma cell line (SK-N-SH) to test the hypothesis that exogenous GDNF could attenuate ethanol-induced cell loss by suppressing cytotoxic signaling pathways and cell suicide. We measured two independently regulated markers of apoptosis, DNA fragmentation and the externalization of phosphatidylserine to the outer cell membrane leaflet. Ethanol induced a dose-related increase in both apoptosis and necrosis. Lower concentrations of ethanol (34 and 68 mM) specifically increased DNA fragmentation, while all concentrations (up to 137 mM) increased phosphatidylserine translocation, suggesting that ethanol induction of apoptosis is not a unitary process. Furthermore, only higher concentrations of ethanol (103 and 137 mM) induced necrosis. Additionally, ethanol specifically induced phosphorylation of c-jun N-terminal-kinase (JNK), a mitogen-activated protein (MAP) kinase selectively associated with apoptosis. In contrast, ethanol did not alter the phosphorylation of another MAP kinase, the extracellular signal-regulated kinases (ERK) that mediate cell survival. Thus, ethanol activated specific intracellular cell death-associated pathways and induced cell death. GDNF, in turn, prevented both ethanol-induced apoptosis and the activation of the death-associated JNK cascade. Therefore, GDNF may regulate multiple pathways to prevent ethanol-induced cell loss.     

Endoplasmic reticulum stress induces tau pathology and forms a vicious cycle: implication in Alzheimer's disease pathogenesis

Accumulation of unfolded proteins can disturb the functions of the endoplasmic reticulum (ER), leading to ER-stress or unfolded protein response (UPR). Recent data have shown that activation of UPR can be found in postmortem brains of Alzheimer's disease (AD) patients; and biological markers for activation of UPR are abundant in neurons with diffuse phosphorylated tau. Although these observations suggest a linkage between ER-stress and tau pathology, little is known of their relationship. In this study, we found that high levels of phosphorylated PKR-like ER-resident kinase (p-PERK) and phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2α) as markers for activation of UPR in the hippocampus of aged P301L mutant tau transgenic mice. The immunoreactivity of p-PERK was found to co-localize with that of phosphorylated tau. We then hypothesized that phosphorylation of tau could induce ER-stress and vice versa in promoting AD-like pathogenesis. By using the protein phosphatase 2A inhibitor okadaic acid (OA) as an inducer for phosphorylation of tau, we found that primary cultures of rat cortical neurons treated with OA triggered UPR as indicated by increased levels of p-PERK and p-eIF2α, splicing of mRNA for xbp-1 and elevated levels of mRNA for GADD153. On the other hand, thapsigargin as an ER-stress inducer stimulated phosphorylation of tau at Thr231, Ser262 and Ser396. Thapsigargin also induced activation of caspase-3 and cleavage of tau. These findings suggested that ER-stress and hyperphosphorylation of tau could be induced by each other to form a vicious cycle to propagate AD-like neurodegeneration.     

Hyperphosphorylation of tau is mediated by ERK activation during anticancer drug-induced apoptosis in neuroblastoma cells

Phosphorylated tau protein is the major component of paired helical filaments in Alzheimer disease (AD). We have previously shown that abnormal tau phosphorylation was induced in neuroblastoma SK-N-SH cells by the anticancer drug, paclitaxel, during apoptosis [Guise et al., 1999: Apoptosis 4:47-58]. In the present study, we first demonstrated a shift from fetal tau to hyperphosphorylated tau after incubation with paclitaxel, that showed some similarities with the hyperphosphorylated tau in AD, by using several tau antibodies, N-Term, Tau-1 and AT-8. Tau phosphorylation occurred independently of caspase-3 activation. We next showed that a sustained activation of ERK (extracellular signal-regulated kinase) induced both tau phosphorylation and apoptosis during paclitaxel treatment (1 microM). The inhibition of ERK activation by using the pharmacological MEK1/2 inhibitor, PD98059 (50 microM), or an antisense strategy, reduced tau phosphorylation and neuronal apoptosis (P < 0.001), indicating a link between ERK activation, tau phosphorylation and apoptosis. Doxorubicin (0.2 microM), an anticancer drug whose mechanism of action is independent of microtubules, also induced ERK activation, tau phosphorylation and apoptosis. Moreover, doxorubicin induced some morphological features of neurodegeneration such as loss of neurites and disorganization of the cytoskeleton in apoptotic neuroblastoma cells. Altogether, our results suggest that tau phosphorylation plays a significant role in apoptosis enhancing disruption of microtubules that in turn leads to formation of apoptotic bodies, suggesting that neurodegeneration and apoptosis are related.     

Ciliary neurotrophic factor and interleukin-6 differentially activate microglia

Studies have shown that cytokines released following CNS injury can affect the supportive or cytotoxic functions of microglia. Interleukin-6 (IL-6)-family cytokines are among the injury factors released. To understand how microglia respond to IL-6 family cytokines, we examined the effects of ciliary neurotrophic factor (CNTF) and IL-6 on primary cultures of rat microglia. To assess the functional state of the cells, we assayed the expression of tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), and cyclooxygenase 2 (COX-2) following stimulation. We show that CNTF reduces COX-2 levels, whereas IL-6 increases the expression of IL-1beta, TNFalpha, and Cox-2. We also examined trophic factor expression and found that CNTF enhances glial cell-line derived neurotrophic factor (GDNF) mRNA and protein secretion, whereas IL-6 has no effect. Correspondingly, conditioned media from CNTF-stimulated microglia promote motor neuron survival threefold beyond controls, whereas IL-6-stimulated microglia decrease neuronal survival twofold. To understand better the signaling mechanisms responsible for the opposite responses of these IL-6-family cytokines, we examined STAT-3 and ERK phosphorylation in CNTF- and IL-6-stimulated microglia. IL-6 markedly increases STAT-3 and ERK phosphorylation after 20 min of treatment, whereas these signal transducers are weakly stimulated by CNTF across a range of doses. We conclude that CNTF modifies microglial activation to support neuronal survival and that IL-6 enhances their capacity to do harm, as a result of different modes of intracellular signaling.     

Active c-jun N-terminal kinase induces caspase cleavage of tau and additional phosphorylation by GSK-3beta is required for tau aggregation

Neurofibrillary tangles (NFTs), comprising human intracellular microtubule-associated protein tau, are one of the hallmarks of tauopathies, including Alzheimer's disease. Recently, a report that caspase-cleaved tau is present in NFTs has led to the hypothesis that the mechanisms underlying NFT formation may involve the apoptosis cascade. Here, we show that adenoviral infection of tau into COS-7 cells induces activation of c-jun N-terminal kinase (JNK), followed by excessive phosphorylation of tau and its cleavage by caspase. However, JNK activation alone was insufficient to induce sodium dodecyl sulfate (SDS)-insoluble tau aggregation and additional phosphorylation by GSK-3β was required. In SH-SY5Y neuroblastoma cells, overexpression of active JNK and GSK-3β increased caspase-3 activation and cytotoxicity more than overexpression of tau alone. Taken together, these results indicate that, although JNK activation may be a primary inducing factor, further phosphorylation of tau is required for neuronal death and NFT formation in neurodegenerative diseases, including those characterized by tauopathy.     

The Inhibition of ERK Activation Mediates the Protection of Necrostatin-1 on Glutamate Toxicity in HT-22 Cells

Receptor-interacting protein 1 (RIP1), a molecular switch protein from apoptosis to necroptosis, is regarded to play an essential role in necroptotic cell death. Although the increased RIP1 activity induced by tumor necrosis factor α activates mitogen-activated protein kinases (MAPKs) including ERK and leads to apoptotic or necrotic cell death, it is unclear what is the role of ERK during the process of necroptosis. In this study, our data demonstrated that ERK inhibitors U0126 and PD98059 blocked glutamate-induced necroptosis in HT-22 cells, indicating the critical role of ERK activation in necroptosis. Further, we found glutamate treatment increased phosphorylated ERK1/2 level, but the specific necroptosis inhibitor Necrostatin-1 (Nec-1) significantly inhibited the phosphorylation of ERK1 (P44) at 5, 10, and 15 min after glutamate treatment; the phosphorylation of ERK2 (P42) level was also markedly reduced by Nec-1 at 10 min after glutamate treatment. The phosphorylation of JNK and P38, two other MAPK members, were slightly increased after glutamate treatment, but Nec-1 had no inhibitory effect on JNK and P38 activation. Our finding suggested that ERK activation may play an important role in necroptotic cell death and the inhibition of ERK activation mediated the protection of Nec-1 on glutamate-induced necroptosis. Since ERK is considered as a downstream of RIP1, the RIP1/ERK signal pathway may provide new therapeutic avenues for the treatment of ischemia–reperfusion damage and neurodegenerative diseases-containing necroptotic cell death.     

Glial-derived neurotrophic factor (GDNF) prevents ethanol (EtOH) induced B92 glial cell death by both PI3K/AKT and MEK/ERK signaling pathways

We investigated the neuroprotective effect of glial-derived neurotrophic factor (GDNF) upon alcohol-exposed B92 cultures, as well as the role of the cytoskeleton and mitogen-activated protein kinase (MAPK) pathways in this effect. Ethanol (EtOH) was added to cultures, either alone or in combination with 30 ng/ml GDNF. Exposure to EtOH (86 and 172 mM; 60 and 120 min) increased the frequency of apoptotic cells identified by nuclear DNA staining with 4,6-diamidino-2-phenylindole (DAPI). Cultures treated with GDNF showed a decrease in ethanol-induced apoptosis. A jun N-terminal kinase (JNK) pathway is activated by EtOH and their pharmacological inhibition (by SP600125) neutralized ethanol-induced apoptosis, suggesting a role for JNK in EtOH neurotoxicity. Immunocytochemically detected phospho-JNK (p-JNK) showed an unusual filamental expression, and localized together with actin stress fibers. Examination of the cytoskeleton showed that EtOH depolymerized actin filaments, inducing p-JNK dissociation and translocation to the nucleus, which suggests that released p-JNK may contribute to glial cell death after EtOH exposure. Treatment with GDNF, in turn, may neutralize the ethanol-induced cell death pathway. Either a phosphatidylinositol 3-kinase (PI3K)/AKT pathway inhibitor (LY294002) or an inhibitor of the extracellular signal-regulated kinase (ERK) 1, 2 pathways (UO126) failed to neutralize GDNF protective effects. However, the simultaneous use of both inhibitors blocked the protective effect of GDNF, suggesting a role for both signaling cascades in the GDNF protection. These findings provide further insight into the mechanism involved in ethanol-induced apoptosis and the neurotrophic protection of glial cells.