The inhibition of glycogen synthase kinase 3beta by a metabotropic glutamate receptor 5 mediated pathway confers neuroprotection to Abeta peptides

Activation of glycogen synthase kinase 3beta (Gsk3beta) has been shown to be a key component in signaling pathways that underlie neurodegeneration and neurodegenerative disease. Conversely, inactivation of Gsk3beta by phosphoinositide 3-kinase (PI3K)/Akt is an important neuroprotective mechanism. Previous studies have shown that agonist activation of group I metabotropic glutamate receptors (mGluRs) can increase neuronal survival and prevent apoptosis. However, little is known about the signaling pathways that couple mGluR5 to neuroprotection. In this report, we investigated whether activation of the PI3K/Akt/Gsk3beta pathway, which has been shown to have an important neuroprotective mechanism, is required for mGluR5 activation mediated neuroprotection against beta-amyloid. We found that brief incubations of mouse hippocampal slices with (R,S)-3,5-dihydroxyphenylglycine (DHPG) resulted in increased phosphorylation of Akt and Gsk3beta. The PI3K inhibitors, LY294002 and wortmannin, blocked the DHPG-induced increased phosphorylation of Akt and Gsk3beta. Similar results were observed in rat primary hippocampal cultures. Finally, we found that the PI3K inhibitor LY294002 can block (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) mediated neuroprotection against beta-amyloid. Thus, these findings suggest that mGluR5 can modulate the PI3K/Akt/Gsk3beta pathway in the hippocampus, and that modulation of this signaling pathway can reverse beta-amyloid-induced neuronal toxicity.     

Pyrroloquinoline quinone rescues hippocampal neurons from glutamate-induced cell death through activation of Nrf2 and up-regulation of antioxidant genes

Pyrroloquinoline quinone (PQQ) has been shown to protect primary cultured hippocampal neurons from glutamate-induced cell apoptosis by scavenging reactive oxygen species (ROS) and activating phosphatidylinositol-3-kinase (PI3K)/Akt signaling. We investigated the downstream pathways of PI3K/Akt involved in PQQ protection of glutamate-injured hippocampal neurons. Western blot analysis indicated that PQQ treatment following glutamate stimulation triggers phosphorylation of glycogen synthase kinase 3β, accompanied by maintenance of Akt activation. Immunostaining and quantitative RT-PCR revealed that PQQ treatment promotes nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), and up-regulates mRNA expression of Nrf2 and the antioxidant enzyme genes, heme oxygenase-1 and glutamate cysteine ligase catalytic in glutamate-injured hippocampal neurons; this is a process dependent on the PI3K/Akt pathway, as evidenced by blocking experiments with PI3K inhibitors. In addition, increased ROS production and decreased glutathione levels in glutamate-injured hippocampal neurons were found to be reduced by PQQ treatment. Collectively, our findings suggest that PQQ exerts neuroprotective activity, possibly through PI3K/Akt-dependent activation of Nrf2 and up-regulation of antioxidant genes. However, the ability of PQQ to scavenge ROS was not totally regulated by PI3K/Akt signaling; possibly it is governed by other mechanisms.     

Protective effect of erythropoietin against ketamine-induced apoptosis in cultured rat cortical neurons: Involvement of PI3K/Akt and GSK-3 beta pathway

Erythropoietin (EPO) prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals in models of neurodegenerative diseases. Here we investigated the neuroprotective effect of EPO in ketamine-induced neurotoxicity in primary cortical neurons. EPO in combination with ketamine greatly increased the cell viability and reduced the number of TUNEL-positive cells. To elucidate a possible mechanism by which EPO exerts its neuroprotective effect, we investigated the phosphoinositide3-kinase pathway using LY294002. The neuroprotection of EPO was prevented by LY294002. Immunoblotting revealed that EPO induced the phosphorylation/activation of Akt and phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK-3β). Moreover, the caspase-3-like activity was increased by addition of ketamine, and decreased by administration of ketamine with EPO. Decreased caspase-3-like activity by administration of ketamine with EPO was restored by LY294002. Our results suggest that PI3K/Akt and GSK-3β pathway are involved in the neuroprotective effect of EPO. You Shang and Yan Wu have contributed equally to this work.     

Differential neuroprotective activity of two different grape seed extracts

Glutamate excitotoxicity is one of the major events that takes place during various neurotoxic injuries such as brain ischemia. We prepared grape seed extracts, from two different varieties, containing high amounts of polyphenols but little resveratrol. Their neuroprotective effects were investigated using primary culture of neonatal mouse hippocampal neurons treated with an excitotoxic concentration of glutamate. Koshu, a white, local variety of V. vinifera, alleviated the acute inactivation of Erk1/2 and dendrite retraction in cultured hippocampal neurons exposed to a toxic concentration of glutamate (1.0 ng/ml). By contrast, Muscat Bailey A, a red, hybrid variety (Muscat Humburg × Bailey), failed to show any neuroprotective effect. Unlike brain-derived neurotrophic factor and other neuroprotective cytokines, Koshu extract did not induce Akt phosphorylation. Koshu extract also augmented neuron survival rate 24 hours after glutamate toxicity. The comparison of polyphenols between the two samples by liquid chromatography/time-of-flight mass spectrometry demonstrated that Koshu had higher amounts of low molecular weight polyphenols along with several Koshu-specific procyanidin oligomers. These data suggest the presence of high affinity molecular targets for polyphenols in hippocampal neurons, which induce neuroprotective effects in a manner different from BDNF, and the importance of low molecular weight polyphenols and/or procyanidin oligomers for neuroprotection.     

Inhibition of PTEN by peroxynitrite activates the phosphoinositide-3-kinase/Akt neuroprotective signaling pathway

Peroxynitrite is usually considered as a neurotoxic nitric oxide-derivative. However, an increasing body of evidence suggests that, at low concentrations, peroxynitrite affords transient cytoprotection, both in vitro and in vivo. Here, we addressed the signaling mechanism responsible for this effect, and found that rat cortical neurons in primary culture acutely exposed to peroxynitrite (0.1 mmol/L) rapidly elicited Akt-Ser(473) phosphorylation. Inhibition of phosphoinositide-3-kinase (PI3K)/Akt pathway with wortmannin or Akt small hairpin RNA (shRNA) abolished the ability of peroxynitrite to prevent etoposide-induced apoptotic death. Endogenous peroxynitrite formation by short-term incubation of neurons with glutamate stimulated Akt-Ser(473) phosphorylation, whereas Akt shRNA enhanced the vulnerability of neurons against glutamate. We further show that Akt-Ser(473) phosphorylation was consequence of the oxidizing, but not the nitrating properties of peroxynitrite. Peroxynitrite failed to nitrate or phosphorylate neurotrophin tyrosine kinase receptors (Trks), and it did not modify the ability of brain-derived neurotrophic factor (BDNF), to phosphorylate its cognate receptor, TrkB; however, peroxynitrite enhanced BDNF-mediated Akt-Ser(473) phosphorylation. Finally, we found that peroxynitrite-stimulated Akt-Ser(473) phosphorylation was associated with an increased proportion of oxidized phosphoinositide phosphatase, PTEN, in neurons. Moreover, peroxynitrite prevented the increase of apoptotic neuronal death caused by over-expression of PTEN. Thus, peroxynitrite exerts neuroprotection by inhibiting PTEN, hence activating the anti-apoptotic PI3K/Akt pathway in primary neurons.     

Growth hormone-stimulated insulin-like growth factor-1 expression in rainbow trout (Oncorhynchus mykiss) hepatocytes is mediated by ERK, PI3K-AKT, and JAK-STAT

Growth hormone (GH) initiates many of its growth-promoting actions by binding to GH receptors (GHR) and stimulating the synthesis and secretion of insulin-like growth factor-1 (IGF-1) from the liver and other sites. In this study, we used hepatocytes isolated from rainbow trout as a model system in which to determine the molecular signaling events of GH in fish. GH directly stimulated the phosphorylation of ERK, protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), JAK2, and STAT5 in hepatocytes incubated in vitro. Activation of ERK, Akt, JAK2, and STAT5 was rapid, occurring within 5-10 min, and was concentration dependent. GH-induced ERK activation was completely blocked by the ERK pathway inhibitor, U0126, and the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex), and was partially blocked by the PI3K inhibitor LY294002. GH-stimulated Akt activation was completely blocked by LY294002 and Hex, but was not affected by U0126; whereas, STAT5 activation by GH was blocked only by Hex, and was not affected by either U0126 or LY294002. GH stimulated hepatic expression of IGF-1 mRNA as well as the secretion of IGF-1, effects that were partially or completely blocked by U0126, LY294002, and Hex. These results indicate that GHR linkage to the ERK, PI3K/Akt, or STAT pathways in trout liver cells requires activation of JAK2, and that GH-stimulated IGF-1 synthesis and secretion is mediated through the ERK, PI3K/Akt, and JAK-STAT pathways.     

Insulin/PI3K signaling protects dentate neurons from oxygen–glucose deprivation in organotypic slice cultures

It is known that ischemia/reperfusion induces neurodegeneration in the hippocampus in a subregion‐dependent manner. This study investigated the mechanism of selective resistance/vulnerability to oxygen–glucose deprivation (OGD) using mouse organotypic hippocampal cultures. Analysis of propidium iodide uptake showed that OGD‐induced duration‐ and subregion‐dependent neuronal injury. When compared with the CA1–3 subregions, dentate neuronal survival was more sensitive to inhibition of phosphatidylinositol 3‐kinase (PI3K)/Akt signaling under basal conditions. Dentate neuronal sensitivity to PI3K/Akt signaling activation was inversely related to its vulnerability to OGD‐induced injury; insulin/insulin‐like growth factor 1 pre‐treatment conferred neuroprotection to dentate neurons via activation of PI3K/Akt signaling. In contrast, CA1 and CA3 neurons were less sensitive to disruptions of endogenous PI3K/Akt signaling and protective effects of insulin/insulin‐like growth factor 1, but more vulnerable to OGD. OGD‐induced injury in CA1 was reduced by inhibition of NMDA receptor or mitogen‐activated protein kinase signaling, and was prevented by blocking NMDA receptor in the presence of insulin. The CA2 subregion was distinctive in its response to glutamate, OGD, and insulin, compared with other CA subregions. CA2 neurons were sensitive to the protective effects of insulin against OGD‐induced injury, but more resistant to glutamate. Distinctive distribution of insulin receptor β and basal phospho‐Akt was detected in our slice cultures. Our results suggest a role for insulin signaling in subregional resistance/vulnerability to cerebral ischemia.     

Mechanism of guanosine-induced neuroprotection in rat hippocampal slices submitted to oxygen-glucose deprivation

Guanine derivates have been implicated in many relevant extracellular roles, such as modulation of glutamate transmission, protecting neurons against excitotoxic damage. Guanine derivatives are spontaneously released to the extracellular space from cultured astrocytes during oxygen-glucose deprivation (OGD) and may act as trophic factors, glutamate receptors blockers or glutamate transport modulators, thus promoting neuroprotection. The aim of this study was to evaluate the mechanisms involved in the neuroprotective role of the nucleoside guanosine in rat hippocampal slices submitted to OGD, identifying a putative extracellular binding site and the intracellular signaling pathways related to guanosine-induced neuroprotection. Cell damage to hippocampal slices submitted to 15 min of OGD followed by 2 h of reperfusion was decreased by the addition of guanosine (100 microM) or guanosine-5'-monophosphate (GMP, 100 microM). The neuroprotective effect of guanosine was not altered by the addition of adenosine receptor antagonists, nucleosides transport inhibitor, glutamate receptor antagonists, glutamate transport inhibitors, and a non-selective Na(+) and Ca(2+) channel blocker. However, in a Ca(2+)-free medium (by adding EGTA), guanosine was ineffective. Nifedipine (a Ca(2+) channel blocker) increased the neuroprotective effect of guanosine and 4-aminopyridine, a K(+) channel blocker, reversed the neuroprotective effect of guanosine. Evaluation of the intracellular signaling pathways associated with guanosine-induced neuroprotection showed the involvement of PKA, PKC, MEK and PI-3 K pathways, but not CaMKII. Therefore, this study shows guanosine is acting via K(+) channels activation, depending on extracellular Ca(2+) levels and via modulation of the PKA, PKC, MEK and/or PI-3 K pathways.     

Neuroprotective effects of astaxanthin against oxygen and glucose deprivation damage via the PI3K/Akt/GSK3β/Nrf2 signalling pathway in vitro

Astaxanthin (ATX), which is the most abundant flavonoid in propolis, has previously shown neuroprotective properties against cerebral ischaemia‐induced apoptosis. However, the mechanisms by which ATX mediates its therapeutic effects are unclear. At present, we explored the underlying mechanisms involved in the protective effects of ATX via the phosphoinositide 3‐kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor erythroid 2‐related factor 2 (Nrf2) signalling pathway in SH‐SY5Y cells. The PI3K/Akt inhibitor LY294002 and GSK3β inhibitor LiCl were employed in this study. Pre‐treatment with ATX for 24 hours significantly decreased the oxygen and glucose deprivation (OGD)‐induced viability loss, reduced the proportion of apoptosis and regulated OGD‐mediated reactive oxygen species (ROS) production. Furthermore, ATX suppressed OGD‐caused mitochondrial membrane potential and decomposition of caspase‐3 to cleaved caspase‐3, and heightened the B‐cell lymphoma 2 (Bcl‐2)/Bax ratio. PI3K/Akt/GSK3β/Nrf2 signalling pathway activation in SH‐SY5Y cells was verified by Western blot. ATX and LiCl treatment raised the protein levels of p‐Akt, p‐GSK3β, nucleus Nrf2 and haeme oxygenase 1 (HO‐1). However, these protein expression levels decreased by treatment of LY294002. The above in vitro data indicate that ATX can confer neuroprotection against OGD‐induced apoptosis via the PI3K/Akt/GSK3β/Nrf2 signalling pathway.     

Inhibition of MLK3-MKK4/7-JNK1/2 pathway by Akt1 in exogenous estrogen-induced neuroprotection against transient global cerebral ischemia by a non-genomic mechanism in male rats

Numerous studies have demonstrated the neuroprotective effects of estrogen in experimental cerebral ischemia. To investigate molecular mechanisms of estrogen neuroprotection in global ischemia, immunoblotting, immunohistochemistry and Nissel-staining analysis were used. Our results showed that chronic pretreatment with beta-estradiol 3-benzoate (E2) enhanced Akt1 activation and reduced the activation of mixed-lineage kinase 3 (MLK3), mitogen-activated protein kinase kinase 4/7 (MKK4/7), and c-Jun N-terminal kinase 1/2 (JNK1/2) in the hippocampal CA1 subfield during reperfusion after 15 min of global ischemia. In addition, E2 reduced downstream JNK nuclear and non-nuclear components, c-Jun and Bcl-2 phosphorylation and Fas ligand protein expression induced by ischemia/reperfusion. Administration of phosphoinositide 3-kinase (PI3K) inhibitor LY 294,002 prevented both activation of Akt1 and inhibition of MLK3, MKK4/7 and JNK1/2. The interaction between ERalpha and the p85 subunit of PI3K was also examined. E2 and antiestrogen ICI 182,780 promoted and prevented this interaction, respectively. Furthermore, ICI 182,780 blocked both the activation of Akt1 and the inhibition of MLK3, MKK4/7 and JNK1/2. Photomicrographs of cresyl violet-stained brain sections showed that E2 reduced CA1 neuron loss after 5 days of reperfusion, which was abolished by ICI 182,780 and LY 294,002. Our data indicate that in response to estrogen, ERalpha interacts with PI3K to activate Akt1, which may inhibit the MLK3-MKK4/7-JNK1/2 pathway to protect hippocampal CA1 neurons against global cerebral ischemia in male rats.     

Essential role for integrin linked kinase in Akt-mediated integrin survival signaling in hippocampal neurons

Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin-linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the alpha chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a beta1 integrin-dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin-activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide-3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor-induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin-mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate-associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.     

Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization

Leptin plays a pivotal role in the regulation of energy homeostasis and metabolism, primarily by acting on neurons in the hypothalamus that control food intake. However, leptin receptors are more widely expressed in the brain suggesting additional, as yet unknown, functions of leptin. Here we show that both embryonic and adult hippocampal neurons express leptin receptors coupled to activation of STAT3 and phosphatidylinositol 3-kinase-Akt signaling pathways. Leptin protects hippocampal neurons against cell death induced by neurotrophic factor withdrawal and excitotoxic and oxidative insults. The neuroprotective effect of leptin is antagonized by the JAK2-STAT3 inhibitor AG-490, STAT3 decoy DNA, and phosphatidylinositol 3-kinase/Akt inhibitors but not by an inhibitor of MAPK. Leptin induces the production of manganese superoxide dismutase and the anti-apoptotic protein Bcl-xL, and stabilizes mitochondrial membrane potential and lessens mitochondrial oxidative stress. Leptin receptor-deficient mice (db/db mice) are more vulnerable to seizure-induced hippocampal damage, and intraventricular administration of leptin protects neurons against seizures. By enhancing mitochondrial resistance to apoptosis and excitotoxicity, our findings suggest that leptin signaling serves a neurotrophic function in the developing and adult hippocampus.     

The excitoprotective effect of N-methyl-D-aspartate receptors is mediated by a brain-derived neurotrophic factor autocrine loop in cultured hippocampal neurons

The neuroprotective effect and molecular mechanisms underlying preconditioning with N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons have not been described. Pre-incubation with subtoxic concentrations of the endogenous neurotransmitter glutamate protects vulnerable neurons against NMDA receptor-mediated excitotoxicity. As a result of physiological preconditioning, NMDA significantly antagonizes the neurotoxicity resulting from subsequent exposure to an excitotoxic concentration of glutamate. The protective effect of glutamate or NMDA is time- and concentration-dependent, suggesting that sufficient agonist and time are required to establish an intracellular neuroprotective state. In these cells, the TrkB ligand, brain-derived neurotrophic factor (BDNF) attenuates glutamate toxicity. Therefore, we tested the hypothesis that NMDA protects neurons via a BDNF-dependent mechanism. Exposure of hippocampal cultures to a neuroprotective concentration of NMDA (50 microM) evoked the release of BDNF within 2 min without attendant changes in BDNF protein or gene expression. The accumulated increase of BDNF in the medium is followed by an increase in the phosphorylation (activation) of TrkB receptors and a later increase in exon 4-specific BDNF mRNA. The neuroprotective effect of NMDA was attenuated by pre-incubation with a BDNF-blocking antibody and TrkB-IgG, a fusion protein known to inhibit the activity of extracellular BDNF, suggesting that BDNF plays a major role in NMDA-mediated survival. These results demonstrate that low level stimulation of NMDA receptors protect neurons against glutamate excitotoxicity via a BDNF autocrine loop in hippocampal neurons and suggest that activation of neurotrophin signaling pathways plays a key role in the neuroprotection of NMDA.     

The upregulation of GLAST-1 is an indirect antiapoptotic mechanism of GDNF and neurturin in the adult CNS

Glial cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) protect retinal ganglion cells (RGCs) from axotomy-induced apoptosis. It is likely that neuroprotection by GDNF or NTN in the adult central nervous system (CNS) involves indirect mechanisms and independent signal transduction events. Extracellular glutamate is a trigger of apoptosis in injured RGCs, and glutamate transporter levels can be upregulated by GDNF. Therefore, GDNF may indirectly protect RGCs by enhancing glutamate uptake in the retina. We studied the upregulation of the glutamate transporters GLAST-1 and GLT-1 by GDNF and NTN, and the intracellular pathways required for GDNF/NTN neuroprotection. GDNF required phosphoinositide-3 kinase (PI3K) and Src activity to upregulate GLAST-1 and GLT-1. NTN required PI3K activity to upregulate GLAST-1 and did not affect GLT-1 levels. PI3K activity was also important for GDNF and NTN neuroprotection following optic nerve transection. However, GDNF also required Src and mitogen-activated protein kinase activity to prevent RGC apoptosis. RNA interference demonstrated that the upregulation of GLAST-1 by GDNF and NTN is required to rescue RGCs. Thus, additional independent signal transduction events, together with the upregulation of GLT-1 by GDNF, differentiate the biological activity of GDNF from NTN. Furthermore, the upregulation of the glial glutamate transporter GLAST-1 by both factors is an indirect neuroprotective mechanism in the CNS.     

Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways

Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate-induced apoptosis through regulations of p38 and Akt pathways. Pre-treatment of cerebellar granule neurons (CGNs) with minocycline (10-100 microm) elicited a dose-dependent reduction of glutamate excitotoxicity and blocked glutamate-induced nuclear condensation and DNA fragmentations. Using patch-clamping and fluorescence Ca2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamate-caused rises in intracellular Ca2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38-specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3beta (GSK3beta) attenuated glutamate-induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3beta caused by glutamate, as were abolished by PI3-K inhibitors. These results demonstrate that minocycline prevents glutamate-induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3-K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.