β‐catenin mediates insulin‐like growth factor‐I actions to promote cyclin D1 mRNA expression,cell proliferation and survival in oligodendroglial cultures

By promoting cell proliferation, survival and maturation insulin‐like growth factor (IGF)‐I is essential to the normal growth and development of the central nervous system. It is clear that IGF‐I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)‐Akt kinases and MAP kinases signal many of IGF‐I‐IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether β‐catenin, a molecule that is a downstream target of glycogen synthase kinase‐3β (GSK3β) and plays a key role in the Wnt canonical signaling pathway, mediates IGF‐I actions. We found that IGF‐I increases β‐catenin protein abundance within an hour after IGF‐I‐induced phosphorylation of Akt and GSK3β. Inhibiting the PI3‐Akt pathway suppressed IGF‐I‐induced increases in β‐catenin and cyclin D1 mRNA, while suppression of GSK3β activity simulated IGF‐I actions. Knocking‐down β‐catenin mRNA by RNA interference suppressed IGF‐I‐stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that β‐catenin is an important downstream molecule in the PI3‐Akt‐GSK3β pathway, and as such it mediates IGF‐I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells. © 2010 Wiley‐Liss, Inc.     

Regulation of GSK‐3β by calpain in the 3‐nitropropionic acid model

Glycogen synthase kinase‐3β (GSK‐3β) is a crucial component in the cascade of events that culminate in a range of neurodegenerative diseases. It is controlled by several pathways, including calpain‐mediated cleavage. Calpain mediates in cell death induced by 3‐nitropropionic acid (3‐NP), but GSK‐3β regulation has not been demonstrated. Here we studied changes in total GSK‐3β protein levels and GSK‐3β phosphorylation at Ser‐9 in this model. The 3‐NP treatment induced GSK‐3β truncation. This regulation was dependent on calpain activation, since addition of calpeptin to the medium prevented this cleavage. While calpain inhibition prevented 3‐NP‐induced neuronal loss, inhibition of GSK‐3β by SB‐415286 did not. Furthermore, inhibition of cdk5, a known target of calpain involved in 3‐NP‐induced cell death, also failed to rescue neurons in our model. Our results point to a new target of calpain and indicate possible cross‐talk between calpain and GSK‐3β in the 3‐NP toxicity pathway. On the basis of our findings, we propose that calpain may modulate 3‐NP‐induced neuronal loss. © 2009 Wiley‐Liss, Inc.     

Differential regulation of AKT, MAPK and GSK3β during C2-ceramide-induced neuronal death

Evidence has implicated apoptosis as a mechanism underlying cell demise in diverse neurodegenerative diseases including Parkinson's disease (PD). Endogenous toxins and other stress signals activate the sphingomyelin pathway increasing the levels of ceramide, an important regulator of cell death.In the present paper we have analysed the contribution of PI3K/AKT-GSK3β and MAPK (ERK and JNK) pathways to cell death in a catecholaminergic cell line following exposure to C₂-ceramide. We also explored the potential neuroprotective action of insulin-like growth factor-1 (IGF-1) and neurotrophin-3 (NT3).We demonstrated that C₂-ceramide-induced cell death is associated to an early decrease in phosphorylation (inhibition) of PI3K/AKT and ERK, followed by phosphorylation (activation) of JNK and de-phosphorylation (activation) of glycogen synthase kinase-3 beta (GSK3β). NT3 and IGF-1 increased survival at early time points, but only IGF-1 is capable to attenuate C₂-ceramide-mediated neuronal death, and this neuroprotection is associated to strong and permanent activation of AKT and inhibition of GSK3β.In conclusion, C₂-ceramide initiates a series of events including an early inactivation of PI3K/AKT and ERK pathways followed by activation of JNK and activation of GSK3β and neuronal death, changes that are counteracted by IGF-1.     

Ischemia preconditioning protects astrocytes from ischemic injury through 14‐3‐3γ

Stroke is a leading cause of death and disability, and new strategies are required to reduce neuronal injury and improve prognosis. Ischemia preconditioning (IPC) is an intrinsic phenomenon that protects cells from subsequent ischemic injury and might provide promising mechanisms for clinical treatment. In this study, primary astrocytes exhibited significantly less cell death than control when exposed to different durations of IPC (15, 30, 60, or 120 min). A 15‐min duration was the most effective IPC to protect astrocytes from 8‐hr‐ischemia injury. The protective mechanisms of IPC involve the upregulation of protective proteins, including 14‐3‐3γ, and attenuation of malondialdehyde (MDA) content and ATP depletion. 14‐3‐3γ is an antiapoptotic intracellular protein that was significantly upregulated for up to 84 hr after IPC. In addition, IPC promoted activation of the c‐Jun N‐terminal kinase (JNK), extracellular signal‐related kinase (ERK)?1/2, p38, and protein kinase B (Akt) signaling pathways. When JNK was specifically inhibited with SP600125, the upregulation of 14‐3‐3γ induced by IPC was almost completely abolished; however, there was no effect on ATP or MDA levels. This suggests that, even though both energy preservation and 14‐3‐3γ up‐regulation were turned on by IPC, they were controlled by different pathways. The ERK1/2, p38, and Akt signaling pathways were not involved in the 14‐3‐3γ upregulation and energy preservation. These results indicate that IPC could protect astrocytes from ischemia injury by inducing 14‐3‐3γ and by alleviating energy depletion through different pathways, suggesting multiple protection of IPC and providing new insights into potential stroke therapies. © 2015 Wiley Periodicals, Inc.     

Positive modulation of AMPA receptors prevents downregulation of GluR2 expression and activates the Lyn‐ERK1/2‐CREB signaling in rat brain ischemia

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPARs) are responsible for excitotoxicity induced by ischemic injury in hippocampal CA1 neurons, whereas the molecular mechanisms responsible for their neurotrophic activities are much less studied. Here, we examined the neuroprotective effect of positive modeulation of AMPARs by coapplication of AMPA with PEPA, an allosteric potentiator of AMPARs. We showed that coapplication of AMPA with PEPA protected hippocampal CA1 neurons from brain ischemia‐induced death. Coapplication of AMPA with PEPA could prevent downregulated expression of GluR2 subunit caused by ischemia and increase BDNF expression via Lyn‐ERK1/2‐CREB signaling. Furthermore, TrkB receptor‐mediated PI3K/Akt signal pathway was activated after coapplication of AMPA with PEPA, which was related to MAPK pathway and protected CA1 neurons against ischemic insults through depression of JNK3 activity, release of cytochrome c to cytosol and depression of capase‐3 activity. Our results revealed that positive modulation of AMPARs could exert neuroprotective effects and the possible signaling pathways underlied. © 2009 Wiley‐Liss, Inc.     

Downregulation of the Wnt/β‐catenin signaling pathway is involved in manganese‐induced neurotoxicity in rat striatum and PC12 cells

Manganese (Mn) is an essential trace element. However, exposure to excessive Mn may cause neurodegenerative disorders called manganism. Accumulating evidence indicated that dysregulation of Wnt/β‐catenin signaling was tightly associated with the onset of neurodegenerative disorders. However, whether aberrant Wnt/β‐catenin signaling contributes to Mn‐induced neurotoxicity remains unknown. The present study investigates the involvement of Wnt/β‐catenin signaling in Mn‐induced neurotoxicity. Western blot and immunohistochemistry analyses showed a remarkable downregulation of p‐Ser9‐glycogen synthase kinase‐3β (GSK‐3β) and β‐catenin in rat striatum after Mn exposure. TUNEL assay revealed significant neuronal apoptosis following treatment with 25 mg/kg Mn. Immunofluorescent staining showed that β‐catenin was expressed predominantly in neurons, and colocalization of β‐catenin and active caspase‐3 was observed after Mn exposure. Furthermore, Mn exposure resulted in PC12 cells apoptosis, which was accompanied by reduced levels of cellular β‐catenin and p‐GSK‐3β. Accordingly, the mRNA level of the prosurvival factor survivin, a downstream target gene of β‐catenin, was synchronously decreased. More importantly, blockage of GSK‐3β activity with the GSK‐3β inhibitor lithium chloride could attenuate Mn‐induced downregulation of β‐catenin and survivin as well as neuronal apoptosis. Overall, the present study demonstrates that downregulation of Wnt/β‐catenin signaling pathway may be of vital importance in the neuropathological process of Mn‐induced neurotoxicity. © 2014 Wiley Periodicals, Inc.     

δ‐Catenin/NPRAP: A new member of the glycogen synthase kinase‐3β signaling complex that promotes β‐catenin turnover in neurons

Through a multiprotein complex, glycogen synthase kinase‐3β (GSK‐3β) phosphorylates and destabilizes β‐catenin, an important signaling event for neuronal growth and proper synaptic function. δ‐Catenin, or NPRAP (CTNND2), is a neural enriched member of the β‐catenin superfamily and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ‐catenin expression is also affected by GSK‐3β signaling and participates in the molecular complex regulating β‐catenin turnover in neurons. Immunofluorescent light microscopy revealed colocalization of δ‐catenin with members of the molecular destruction complex: GSK‐3β, β‐catenin, and adenomatous polyposis coli proteins in rat primary neurons. GSK‐3β formed a complex with δ‐catenin, and its inhibition resulted in increased δ‐catenin and β‐catenin expression levels. LY294002 and amyloid peptide, known activators of GSK‐3β signaling, reduced δ‐catenin expression levels. Furthermore, δ‐catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ‐catenin, like that of β‐catenin, is regulated by proteasome‐mediated degradation. Coimmunoprecipitation experiments showed that δ‐catenin overexpression promoted GSK‐3β and β‐catenin interactions. Primary cortical neurons and PC12 cells expressing δ‐catenin treated with proteasome inhibitors showed increased ubiquitinated β‐catenin forms. Consistent with the hypothesis that δ‐catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ‐catenin showed enhanced β‐catenin turnover. These studies identify δ‐catenin as a new member of the GSK‐3β signaling pathway and further suggest that δ‐catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β‐catenin in neuronal cells. © 2010 Wiley‐Liss, Inc.     

TrkB‐mediated activation of the phosphatidylinositol‐3‐kinase/Akt cascade reduces the damage inflicted by oxygen–glucose deprivation in area CA3 of the rat hippocampus

The selective vulnerability of hippocampal area CA1 to ischemia‐induced injury is a well‐known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen–glucose deprivation–reperfusion (OGD‐RP), an in vitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain‐derived neurotrophic factor (BDNF) or 7,8‐dihydroxyflavone (7,8‐DHF) exhibited reduced depression of the electrical activity triggered by OGD‐RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD‐RP. The protective effect of TrkB activation was limited to area CA3, as OGD‐RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3‐kinase/Akt (PI‐3‐K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD‐RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase‐3 activity in slices pretreated with BDNF and exposed to OGD‐RP. We propose that PI‐3‐K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.     

Tibolone modulates neuronal plasticity through regulating Tau,GSK3β/Akt/PI3K pathway and CDK5 p35/p25 complexes in the hippocampus of aged male mice

Aging is a key risk factor for cognitive decline and age-related neurodegenerative disorders. Also, an age-related decrease in sex steroid hormones may have a negative impact on the formation of neurofibrillary tangles(NFTs); these hormones can regulate Tau phosphorylation and the principal kinase GSK3β involved in this process. Hormone replacement therapy decreases NFTs, but it increases the risk of some types of cancer. However, other synthetic hormones such as tibolone(TIB) have been used for hormone replacement therapy. The aim of this work was to evaluate the long-term effects of TIB(0.01 mg/kg and 1 mg/kg, intragastrically for 12 weeks) on the content of total and hyperphosphorylated Tau(PHF-1) proteins and the regulation of GSK3β/Akt/PI3 K pathway and CDK5/p35/p25 complexes in the hippocampus of aged male mice. We observed that the content of PHF-1 decreased with TIB administration. In contrast, no changes were observed in the active form of GSK3β or PI3 K. TIB decreased the expression of the total and phosphorylated form of Akt while increased that of p110 and p85. The content of CDK5 was differentially modified with TIB: it was increased at low doses and decreased at high doses. When we analyzed the content of CDK5 activators, an increase was found on p35; however, the content of p25 decreased with administration of low dose of TIB. Our results suggest a possible mechanism of action of TIB in the hippocampus of aged male mice. Through the regulation of Tau and GSK3β/Akt/PI3 K pathway, and CDK5/p35/p25 complexes, TIB may modulate neuronal plasticity and regulate learning and memory processes.     

Remifentanil postconditioning improves global cerebral ischemia-induced spatial learning and memory deficit in rats via inhibition of neuronal apoptosis through the PI3K signaling pathway

Global cerebral ischemia followed by reperfusion, which leads to extensive neuronal damage, particularly the neurons in the hippocampal CA1 region. Apoptosis is one of the major mechanisms that lead to neuronal death after cerebral ischemia and reperfusion. The neuroprotective effects of remifentanil preconditioning against cerebral ischemia/reperfusion injury have been recently reported. Here we investigated whether remifentanil postconditioning exerts neuroprotective effects against global cerebral ischemia/reperfusion injury in rats and its potential mechanisms. Global cerebral ischemia was performed via 10 min of four-vessel occlusion. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells and expression of Bcl-2 and Bax in the hippocampal CA1 region were assessed after reperfusion. Morris water maze task was used to quantify spatial learning and memory deficits after reperfusion. We found remifentanil postconditioning markedly improved the spatial learning and memory as well as attenuated neuronal apoptosis in hippocampus caused by cerebral ischemia/reperfusion injury. In addition, remifentanil postconditioning enhanced the expression of anti-apoptotic gene Bcl-2 while suppressed the expression of pro-apoptotic gene Bax in hippocampal CA1 region. However, the neuroprotective effects of remifentanil postconditioning were abolished by pretreatment of the PI3K inhibitor LY294002. The results suggest that remifentanil postconditioning exhibits neuroprotective effects against global cerebral ischemia/reperfusion injury in rats, and its mechanisms might involve inhibition of neuronal apoptosis through the PI3K pathway.     

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.     

Implication of Akt‐dependent Prp19α/14‐3‐3β/Cdc5L complex formation in neuronal differentiation

PRP19α and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19α/14‐3‐3β/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)‐induced neuronal differentiation of PC12 cells. Analysis of PRP19α mutants revealed that the phosphorylation of PRP19α at Thr 193 by Akt was critical for its binding with 14‐3‐3β to translocate into the nuclei and for PRP19α/14‐3‐3β/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19α or sense 14‐3‐3β RNAs promoted NGF‐induced neuronal differentiation, whereas down‐regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19αT193A lost its binding ability with 14‐3‐3β and acted as a dominant‐negative mutant in neuronal differentiation. These results imply that Akt‐dependent phosphorylation of PRP19α at Thr193 triggers PRP19α/14‐3‐3β/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre‐mRNAs. © 2010 Wiley‐Liss, Inc.     

Insulin-like growth factor 1 prevents neuronal cell death induced by corticosterone through activation of the PI3k/Akt pathway

Corticosterone (CORT) is well known to induce neuronal damage in various brain regions including the hippocampus, but the precise mechanism(s) of action underlying these effects has yet to be fully established. Insulin-like growth factor-1 (IGF-1) is a trophic factor promoting cell survival by the activation of the phosphatidylinositide 3-kinase (PI3K)/Akt kinase pathway. We report that IGF-1 prevents neuronal cell death induced by CORT, likely via the stimulation of the PI3K/Akt pathway in primary hippocampal cultured neurons. CORT induced neuronal cell death at a minimal concentration of 50 nM. IGF-1 (10 nM) prevented cell death induced by CORT under serum-free conditions. The neuroprotective effect of IGF-1 was accompanied by reversal of the Akt pathway inhibition induced by CORT. The PI3 kinase inhibitor, LY29004, inhibited the neuroprotective effect of IGF-1 whereas the MEK (MAPK kinase) inhibitor PD98059, an upstream blocker of mitogen-activated protein (MAP) kinase, had no effect. These results suggest that IGF-1 can prevent neuronal cell death induced by CORT in hippocampal neurons by modulating the activity of the PI3K/Akt pathway.     

Protective effects of peroxisome proliferator‐activated receptors γ coactivator‐1α against neuronal cell death in the hippocampal CA1 subfield after transient global ischemia

Peroxisome proliferator‐activated receptors γ coactivator‐1α (PGC‐1α) may regulate the mitochondrial antioxidant defense system under many neuropathological settings. However, the exact role of PGC‐1α in ischemic brain damage is still under debate. Based on an experimental model of transient global ischemia (TGI), this study evaluated the hypothesis that the activation of PGC‐1α signaling pathway protects hippocampal CA1 neurons against delayed neuronal death after TGI. In Sprague‐Dawley rats, significantly increased content of oxidized proteins in the hippocampal CA1 tissue was observed as early as 30 min after TGI, followed by augmentation of PGC‐1α expression at 1 hr. Expression of uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2) in the hippocampal CA1 neurons was upregulated 4–48 hr after TGI. In addition, knock‐down of PGC‐1α expression by pretreatment with a specific antisense oligodeoxynucleotide in the hippocampal CA1 subfield downregulated the expression of UCP2 and SOD2 with resultant exacerbation of oxidative stress and augmentation of delayed neuronal cell death in the hippocampus after TGI. Overall, our results indicate that PGC‐1α is induced by cerebral ischemia leading to upregulation of UCP2 and SOD2, thereby providing a neuroprotective effect against ischemic brain injury in the hippocampus by ameliorating oxidative stress. © 2009 Wiley‐Liss, Inc.     

Amyloid‐β interrupts the PI3K‐Akt‐mTOR signaling pathway that could be involved in brain‐derived neurotrophic factor‐induced Arc expression in rat cortical neurons

The deposition of amyloid‐β (Aβ) contributes to the pathogenesis of Alzheimer's disease. Even at low levels, Aβ may interfere with various signaling cascades critical for the synaptic plasticity that underlies learning and memory. Brain‐derived neurotrophic factor (BDNF) is well known to be capable of inducing the synthesis of activity‐regulated cytoskeleton‐associated protein (Arc), which plays a fundamental role in modulating synaptic plasticity. Our recent study has demonstrated that treatment of fibrillar Aβ at a nonlethal level was sufficient to impair BDNF‐induced Arc expression in cultured rat cortical neurons. In this study, BDNF treatment alone induced the activation of the phosphatidylinositol 3‐kinase‐Akt‐mammlian target of rapamycin (PI3K‐Akt‐mTOR) signaling pathway, the phosphorylation of eukaryotic initiation factor 4E binding protein (4EBP1) and p70 ribosomal S6 kinase (p70S6K), the dephosphorylation of eukaryotic elongation factor 2 (eEF2), and the expression of Arc. Interrupting the PI3K‐Akt‐mTOR signaling pathway by inhibitors prevented the effects of BDNF, indicating the involvement of this pathway in BDNF‐induced 4EBP1 phosphorylation, p70S6K phosphorylation, eEF2 dephosphorylation, and Arc expression. Nonlethal Aβ pretreatment partially blocked these effects of BDNF. Double‐ immunofluorescent staining in rat cortical neurons further confirmed the coexistence of eEF2 dephosphorylation and Arc expression following BDNF treatment regardless of the presence of Aβ. These results reveal that, in cultured rat cortical neurons, Aβ interrupts the PI3K‐Akt‐mTOR signaling pathway that could be involved in BDNF‐induced Arc expression. Moreover, this study also provides the first evidence that there is a close correlation between BDNF‐induced eEF2 dephosphorylation and BDNF‐induced Arc expression. © 2009 Wiley‐Liss, Inc.     

Insulin activates the PI3K-Akt survival pathway in vulnerable neurons following global brain ischemia

Abstract

Insulin is neuroprotective following transient global brain ischemia; however, the mechanisms by which insulin exerts its salutary effects remain unclear.

Objective: We assessed insulin's effect on the PI3K-Akt survival system and consequent modulation of the pro-apoptotic proteins Bim, Bad and FoxO3a.

Methods: We utilized rats subjected to 10 minutes of global brain ischemia, with or without insulin administered at the onset of reperfusion.

Results: In sham-operated animals, minimal pAkt immunofluorescence was detected in the CA1. Moreover, at 30 minute reperfusion, there was no change in pAkt in CA1 neurons. Single bolus high-dose insulin treatment resulted in an early increase in pAkt after 30 minutes, preservation of CA1 neurons to 14 days of reperfusion and preservation of spatial learning ability. Insulin treatment increased cytoplasmic and nuclear staining for pAkt in both CA1 and cortex. Insulin-induced Akt phosphorylation was suppressed by the PI3K inhibitor wortmannin. Neither reperfusion nor insulin induced any change in the phosphorylation or subcellular localization of FoxO3a, Bim or Bad. A single bolus of high-dose insulin reduced CA1 neuronal cell death and thus represents a potential therapeutic intervention for global brain ischemia.

Discussion: These results reveal that proximal elements of a known cell-survival pathway are triggered by high-dose insulin during early reperfusion. Insulin induces robust PI3K-dependent phosphorylation of Akt by 30 minute reperfusion and results in improvement of hippocampal structure and function. However, the Akt substrates FoxO3a, Bim and Bad do not undergo corresponding changes in phosphorylation or subcellular localization in this model of global brain ischemia. The downstream components of insulin-induced Akt survival signaling after transient global brain ischemia remain to be identified.     

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.     

Early glycogen synthase kinase‐3β and protein phosphatase 2A independent tau dephosphorylation during global brain ischaemia and reperfusion following cardiac arrest and the role of the adenosine monophosphate kinase pathway

Abnormal tau phosphorylation (p‐tau) has been shown after hypoxic damage to the brain associated with traumatic brain injury and stroke. As the level of p‐tau is controlled by Glycogen Synthase Kinase (GSK)‐3β, Protein Phosphatase 2A (PP2A) and Adenosine Monophosphate Kinase (AMPK), different activity levels of these enzymes could be involved in tau phosphorylation following ischaemia. This study assessed the effects of global brain ischaemia/reperfusion on the immediate status of p‐tau in a rat model of cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We reported an early dephosphorylation of tau at its AMPK sensitive residues, Ser³⁹⁶ and Ser²⁶²after 2 min of ischaemia, which did not recover during the first two hours of reperfusion, while the tau phosphorylation at GSK‐3β sensitive but AMPK insensitive residues, Ser²⁰²/Thr²⁰⁵ (AT8), as well as the total amount of tau remained unchanged. Our data showed no alteration in the activities of GSK‐3β and PP2A during similar episodes of ischaemia of up to 8 min and reperfusion of up to 2 h, and 4 weeks recovery. Dephosphorylation of AMPK followed the same pattern as tau dephosphorylation during ischaemia/reperfusion. Catalase, another AMPK downstream substrate also showed a similar pattern of decline to p‐AMPK, in ischaemic/reperfusion groups. This suggests the involvement of AMPK in changing the p‐tau levels, indicating that tau dephosphorylation following ischaemia is not dependent on GSK‐3β or PP2A activity, but is associated with AMPK dephosphorylation. We propose that a reduction in AMPK activity is a possible early mechanism responsible for tau dephosphorylation.