Involvement of SHP2 in focal adhesion, migration and differentiation of neural stem cells

Objectives

SHP2 (Src-homology-2 domain-containing protein tyrosine phosphatase) plays an important role in cell adhesion, migration and cell signaling. However, its role in focal adhesion, differentiation and migration of neural stem cells is still unclear.

Methods

In this study, rat neurospheres were cultured in suspension and differentiated neural stem cells were cultured on collagen-coated surfaces.

Results

The results showed that p-SHP2 co-localized with focal adhesion kinase (FAK) and paxillin in neurospheres and in differentiated neural precursor cells, astrocytes, neurons, and oligodendrocytes. Suppression of SHP2 activity by PTP4 or siRNA-mediated SHP2 silencing caused reduction in the cell migration and neurite outgrowth, and thinning of glial cell processes. Differentiation-induced activation of FAK, Src, paxillin, ERK1/2, and RhoA was decreased by SHP2 inactivation.

Conclusions

These results indicate that SHP2 is recruited in focal adhesions of neural stem cells and regulates focal adhesion formation. SHP2-mediated regulation of neural differentiation and migration may be related to formation of focal adhesions and RhoA and ERK1/2 activation.     

Differentiation of neural stem cells influences their chemotactic responses to vascular endothelial growth factor

Although much effort has been devoted to the delineation of factors involved in the migration of neural stem/progenitor cells (NSCs), the relationship between the chemotactic response and the differentiation status of these cells remains elusive. In the present study, we found that NSCs in varying differentiation states possess different chemotactic responses to vascular endothelial growth factor (VEGF): first, the number of chemotaxing NSCs and the optimal concentrations of VEGF that induced the peak migration vary greatly; second, time-lapse video analysis shows that NSCs at certain differentiation states migrate more efficiently toward VEGF, although the migration speed remains unchanged irrespective of cell states; third, the phosphorylation status of Akt, ERK1/2, SAPK/JNK, and p38MAPK is closely related to the differentiation levels of NSCs subjected to VEGF; and, finally, although inhibition of ERK1/2 signaling significantly attenuates VEGF-stimulated transfilter migration of both undifferentiated and differentiating NSCs, NSCs show normal chemotactic response after treatment with inhibitors of SAPK/JNK or p38MAPK. Meanwhile, interference with PI3K/Akt signaling prevents only NSCs of 12 hr differentiation, but not NSCs of 1 day or 3 days differentiation, from migrating in response to VEGF. Moreover, blocking of PI3K/Akt or MAPK signaling impairs the migration efficiency and/or speed, the extent of which depends on the cell differentiation status. Collectively, these results demonstrate that differentiation of NSCs influences their chemotactic responses to VEGF: NSCs in varying differentiation states have different migratory capacities, thereby shedding light on optimization of the therapeutic potential of NSCs to be employed for neural regeneration after injury.     

Mechanisms by which cleaved kininogen inhibits endothelial cell differentiation and signalling

We have shown that cleaved high-molecular-weight kininogen inhibits endothelial cell tube and vacuole formation in a concentration-dependent manner and this correlates with its recognised anti-angiogenic activity. The antibody against the urokinase plasminogen activator receptor (uPAR) mimicked the inhibitory effect of cleaved kininogen (HKa) on apoptosis (HKa: 30% and uPAR antibody: 26%) and tube formation. In tumour angiogenesis, cancer cells release angiogenic stimulators, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), thus stimulating the transformation of endogenous pro-uPA to uPA. The proteolytic enzyme urokinase plasminogen activator (uPA) then binds to its receptor in a complex with its inhibitor PAI-1, which results in the internalisation of this complex, and activates extracellular signal-regulated kinase (ERK). Recycling of the uPAR regulates the migration of endothelial cells (ECs). ERK activation stimulates migration and proliferation and suppresses apoptosis of ECs. HKa disrupted the uPA-uPAR complex, inhibited ERK activation, and blocked the internalization of uPAR, eventually resulting in cell death and cell motility arrest. Both are critical steps in angiogenesis.     

Brefeldin A-inhibited Guanine Exchange Factor 2 Regulates Filamin A Phosphorylation and Neuronal Migration

Periventricular heterotopia (PH) is a human malformation of cortical development associated with gene mutations in ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2 encodes for Big2 protein) and Filamin A (FLNA). PH is thought to derive from neuroependymal disruption, but the extent to which neuronal migration contributes to this phenotype is unknown. Here, we show that Arfgef2 null mice develop PH and exhibit impaired neural migration with increased protein expression for both FlnA and phosphoFlnA at Ser2152. Big2 physically interacts with FlnA and overexpression of phosphomimetic Ser2512 FLNA impairs neuronal migration. FlnA phosphorylation directs FlnA localization toward the cell cytoplasm, diminishes its binding affinity to actin skeleton, and alters the number and size of paxillin focal adhesions. Collectively, our results demonstrate a molecular mechanism whereby Big2 inhibition promotes phosphoFlnA (Ser2152) expression, and increased phosphoFlnA impairs its actin binding affinity and the distribution of focal adhesions, thereby disrupting cell intrinsic neuronal migration.     

Modulation of endothelial cell migration by extracellular nucleotides: involvement of focal adhesion kinase and phosphatidylinositol 3-kinase-mediated pathways

Extracellular nucleotides bind to type-2 purinergic/pyrimidinergic (P2) receptors that mediate various responses, such as cell activation, proliferation and apoptosis, implicated in inflammatory processes. The role of P2 receptors and their associated signal transduction pathways in endothelial cell responses has not been fully investigated. Here, it is shown that stimulation of human umbilical vein endothelial cells (HUVEC) with extracellular ATP or UTP increased intracellular free calcium ion concentrations ([Ca(2+)](i)), induced phosphorylation of focal adhesion kinase (FAK), p130(cas) and paxillin, and caused cytoskeletal rearrangements with consequent cell migration. Furthermore, UTP increased migration of HUVEC in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. BAPTA or thapsigargin inhibited the extracellular nucleotide-induced increase in [Ca(2+)](i), a response crucial for both FAK phosphorylation and cell migration. Furthermore, long-term exposure of HUVEC to ATP and UTP, agonists of the G protein-coupled P2Y2 and P2Y4 receptor subtypes, caused upregulation of alpha(v) integrin expression, a cell adhesion molecule known to directly interact with P2Y2 receptors. Our results suggest that extracellular nucleotides modulate signaling pathways in HUVEC influencing cell functions, such as cytoskeletal changes, cellular adhesion and motility, typically associated with integrin-activation and the action of growth factors. We propose that P2Y2 and possibly P2Y4 receptors mediate those responses that are important in vascular inflammation, atherosclerosis and angiogenesis.     

FAK-mediated inhibition of vascular smooth muscle cell migration by the tetraspanin CD9

Migration of vascular smooth muscle cells (SMC) towards the intima is a key event in vascular proliferative diseases. We investigated a potential role for the tetraspanin CD9 in this process in a wound migration assay. Aortic SMC from CD9 knock-out mice had higher migration rates and the presumably stimulatory anti-CD9 antibody ALMA-1 inhibited migration of human SMC. The signaling pathways responsible for this inhibitory effect were investigated. In migrating CD9-/- SMC, stress fiber formation was decreased and focal adhesions were smaller and more diffusely distributed, consistent with an inhibition of integrin clustering. In migrating mouse SMC expressing CD9, focal adhesion kinase (FAK) tyrosine phosphorylation was doubled. No differences in intracellular calcium signaling were observed between CD9+/+ and CD9-/- SMC during migration. We suggest that CD9 in hibits SMC migration by a stimulation of both stress fiber formation and integrin clustering, leading to a stimulation of FAK phosphorylation.     

Formation of a beta1 integrin signaling complex in Schwann cells is independent of rho

Schwann cell adhesion to basal lamina is essential for peripheral nerve development. beta(1) integrin receptors for extracellular matrix cooperate with other receptors to transmit signals that coordinate cell cycle progression and initiation of differentiation, including myelin-specific gene expression. In Schwann cell/sensory neuron cocultures, beta(1) integrins complex with focal adhesion kinase (FAK), fyn kinase, paxillin, and schwannomin in response to basal lamina adhesion. To study the assembly of this signaling complex in Schwann cells (SCs), we induced beta(1) integrin clustering on suspended cells using an immobilized antibody and recovered a complex containing beta(1) integrin, FAK, paxillin, and schwannomin. In adherent subconfluent cells, the proteins colocalized to filopodia, ruffling membranes and focal contacts. We assessed the role of rhoGTPase in the process of integrin complex assembly by introducing C3 transferase (C3T), a rho inhibitor, into the cells. Although C3T caused dose-dependent morphological abnormalities, FAK, paxillin, and schwannomin were able to coimmunoprecipitate with beta(1) integrin. Additionally, colocalization of FAK, paxillin, and schwannomin with beta(1) integrin in filopodia and small focal contacts remained unchanged. We conclude that SCs do not require active rho to recruit signaling and structural proteins to beta(1) integrins clustered at the plasma membrane. Rho is required to establish large focal adhesions and to spread and stabilize plasma membrane extensions.     

Synergistic induction of cyclin D1 in oligodendrocyte progenitor cells by IGF-I and FGF-2 requires differential stimulation of multiple signaling pathways

D-type cyclins are direct targets of extracellular signals and critical regulators of G(1) progression. Our previous data demonstrated that IGF-I and FGF-2 synergize to enhance cyclin D1 expression, cyclin E/cdk2 complex activation, and S-phase entry in OP cells. Here, we provide a mechanistic explanation for how two growth factor signaling pathways converge on a major cell cycle regulator. IGF-I and FGF-2 differentially activate signaling pathways to coordinately promote cyclin D1 expression. We show that the p44/p42 MAPK signaling pathway is essential for FGF-2 induction of cyclin D1 mRNA. In contrast, blocking the PI3-Kinase pathway results in loss of IGF-I/FGF-2 synergistic induction of cyclin D1 protein levels. Moreover, the presence of IGF-I significantly enhances nuclear localization of cyclin D1, which also requires PI3K signaling. GSK-3beta, a downstream target of the PI3K/Akt pathway, is phosphorylated in the presence of IGF-I in OPs. Consistent with a known role for GSK-3beta in cyclin D1 degradation, we show that proteasome inhibition in OPs exposed to FGF-2 increased cyclin D1 levels, equivalent to levels seen in IGF-I/FGF-2 treated cells. Thus, we provide a model for cyclin D1 coordinate regulation where FGF-2 stimulation of the MAPK pathway promotes cyclin D1 mRNA expression while IGF-I activation of the PI3K pathway inhibits proteasome degradation of cyclin D1 and enhances nuclear localization of cyclin D1.     

Tissue factor pathway inhibitor (TFPI) interferes with endothelial cell migration by inhibition of both the Erk pathway and focal adhesion proteins

Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that is mainly known for its inhibition of tissue factor-mediated coagulation. In addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. In this work we demonstrate that at concentrations within the physiological range, TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro. These effects were specific to endothelial cells since no inhibitory effect was observed on the migration of tumor (glioblastoma) cells. Inhibition of endothelial cell migration was correlated with a concomitant loss in cell adhesion, suggesting an alteration of focal adhesion complex integrity. Accordingly, we observed that TFPI inhibited the phosphorylation of focal adhesion kinase and paxillin, two key proteins involved in the scaffolding of these complexes, and that this effect was specific to endothelial cells. These results suggest that TFPI influences the angiogenic process via a non-haemostatic pathway, by downregulating the migratory mechanisms of endothelial cells.     

α-Lipoic Acid Interaction with Dopamine D2 Receptor-Dependent Activation of the Akt/GSK-3β Signaling Pathway Induced by Antipsychotics: Potential Relevance for the Treatment of Schizophrenia

Chronic administration of antipsychotics has been associated with dopamine D2 receptor (D2R) upregulation and tardive dyskinesia. We have previously shown that haloperidol, a first-generation antipsychotic (FGA), exerted an increase in D2R expression and oxidative stress and that (±)-α-lipoic acid reversed its effect. Previous studies have implicated the Akt/glycogen synthase kinase-3β (GSK-3β) signaling pathway in antipsychotic action. These findings led us to examine whether the Akt/GSK-3β pathway was involved in D2R upregulation and oxidative stress elicited by antipsychotics and, in (±)-α-lipoic acid-induced reversal of these phenomena, in SH-SY5Y cells. Antipsychotics increased phosphorylation of Akt and GSK-3β, and additive effects were observed with (±)-α-lipoic acid. GSK-3β inhibitors reversed haloperidol-induced overexpression of D2R mRNA levels but did not affect haloperidol-induced oxidative stress. Sustained antipsychotic treatment increased β-arrestin-2 and D2R receptor interaction. Regarding Akt/GSK-3β downstream targets, antipsychotics increased β-catenin levels, whereas (±)-α-lipoic acid induced an elevation of mTOR activation. These results suggest (1) that the effect of antipsychotics on the Akt/GSK-3β pathway in SH-SY5Y cells is reminiscent of their in vivo action, (2) that (±)-α-lipoic acid partially synergizes with antipsychotic drugs (APDs) on the same pathway, and (3) that the Akt/GSK-3β signaling cascade is not involved in the preventive effect of (±)-α-lipoic acid on antipsychotics-induced D2R upregulation.     

Nerve growth factor regulates human choroidal, but not retinal, endothelial cell migration and proliferation

We have previously shown that sympathetic denervation results in significant blood vessel growth of the choroid and retina. The mechanism of this growth remains unclear. Since sympathetic denervation can result in increased nerve growth factor (NGF) levels, it was the goal of this study to determine if choroidal and retinal endothelial cells in culture would respond to nerve growth factor and if nerve growth factor promote endothelial cell migration and proliferation, two components of angiogenesis. Western blotting with phospho-specific antibodies, cell migration, and cell proliferation assays were employed to determine NGF effects on both choroidal and retinal cell growth. NGF treatment produced phosphorylation of TrkA in choroidal and retinal endothelial cells. NGF stimulation resulted in activation of ERK1/2, Akt, and Src in choroidal endothelial cells, while little phosphorylation was noted following NGF treatment in retinal endothelial cells. NGF increased choroidal endothelial cell migration by 50% over control and this was inhibited by pretreatment with LY294002 (PI3K inhibitor), Akt inhibitor, and MMP2/9 inhibitor. KT5823, PD98059, and PP2 did not affect choroidal cell migration. NGF also produced a 47% increase in choroidal endothelial cell proliferation, which was blocked by PP2, LY294002, Akt inhibitor, KT5823, and PD98059. NGF stimulation did not alter retinal endothelial cell migration or proliferation. Thus, it appears that increased NGF levels that may be noted after sympathectomy are capable of producing some aspects of vascular remodeling via different signaling cascades in choroidal endothelial cells in culture.     

Caspase-3 is Involved in Aluminum-Induced Impairment of Long-Term Potentiation in Rats Through the Akt/GSK-3β Pathway

A number of studies have indicated that aluminum (Al) exposure can impair learning and memory function. The ability of Al to inhibit hippocampal long-term potentiation (LTP) suggests the possibility of Al impairing synaptic plasticity. LTP is dependent on the externalization of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPAR). The protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β) signaling pathway has been demonstrated to mediate AMPAR delivery. A mechanism by which caspase-3 cleaves Akt is involved in synaptic plasticity, but the underlying molecular mechanism involved has still not been elucidated. The purpose of this study was to investigate the mechanism of LTP impairment and the related signaling pathway disturbance induced by Al exposure. Our results reveal that Al treatment produces a dose-dependent suppression of LTP and decreases in the AMPAR subunits GluR1 and GluR2, in both membrane and total cell extracts. Al caused increased accumulation of active caspase-3 and a gradual decrease in Akt and pGSK-3β. Interestingly, Al depressed LTP and AMPAR protein concentration. N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (a caspase-3 inhibitor) reversed the Al-induced LTP inhibition, increased levels of active caspase-3, and decreased AMPAR levels in both total and membrane-enriched extracts. It also decreased Akt and pGSK-3β. The molecular mechanism of Al-induced LTP impairment might be related to the activation of caspase-3, cleavage of Akt, activation of GSK-3β, and inhibition of the externalization of AMPAR.     

Blockade of phosphodiesterase-III protects against oxygen-glucose deprivation in endothelial cells by upregulation of VE-cadherin

We recently reported that a phosphodiesterase-III inhibitor, cilostazol, prevented the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tissue plasminogen activator (tPA) and that it reversed tPA-induced cell damage by protecting the neurovascular unit, particularly endothelial cells. However, the mechanisms of cilostazol action are still not clearly defined. The adheren junction (AJ) protein, VE-cadherin, is a known mediator of endothelial barrier sealing and maintenance. Therefore, we tested whether cilostazol might promote expression of adhesion molecules in endothelial cells, thereby preventing deterioration of endothelial barrier functions. Human brain microvascular endothelial cells were exposed to 6-h oxygen-glucose deprivation (OGD). We compared cilostazol with aspirin treatments and examined 2 representative AJ proteins: VE-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1). A protein kinase A (PKA) inhibitor, LY294002 (a PI3-K inhibitor), db-cAMP, and RP-cAMPS were used to assess the roles of cAMP, PKA, and PI3-K signaling, respectively, in cilostazol-induced responses. Cilostazol and db-cAMP prevented OGD-stress injury in endothelial cells by promoting VE-cadherin expression, but not PECAM-1. Aspirin did not prevent cell damage. P13-K inhibition by LY294002 had no influence on the effects of cilostazol, but inhibition of cAMP/PKA with PKA inhibitor and Rp-cAMPS suppressed cilostazol-induced inhibition of cell damage and promotion of VE-cadherin expression. In contrast, OGD stress had no detectable effects on VEGF, VEGF receptor, or angiopoietin-1 levels. Cilostazol promotes VE-cadherin expression through cAMP/PKA-dependent pathways in brain endothelial cells; thus, cilostazol effects on adhesion molecule signaling may provide protection against OGD stress in endothelial cells.     

Astrocytes spatially restrict VEGF signaling by polarized secretion and incorporation of VEGF into the actively assembling extracellular matrix

The spatial organization of vascular endothelial growth factor (VEGF) signaling is a key determinant of vascular patterning during development and tissue repair. How VEGF signaling becomes spatially restricted and the role of VEGF secreting astrocytes in this process remains poorly understood. Using a VEGF‐GFP fusion protein and confocal time‐lapse microscopy, we observed the intracellular routing, secretion and immobilization of VEGF in scratch‐activated living astrocytes. We found VEGF to be directly transported to cell‐extracellular matrix attachments where it is incorporated into fibronectin fibrils. VEGF accumulated at β1 integrin containing fibrillar adhesions and was translocated along the cell surface prior to internalization and degradation. We also found that only the astrocyte‐derived, matrix‐bound, and not soluble VEGF decreases β1 integrin turnover in fibrillar adhesions. We suggest that polarized VEGF release and ECM remodeling by VEGF secreting cells is key to control the local concentration and signaling of VEGF. Our findings highlight the importance of astrocytes in directing VEGF functions and identify these mechanisms as promising target for angiogenic approaches. GLIA 2016;64:440–456     

Vascular endothelial growth factor enhances the expression of urokinase receptor in human endothelial cells via protein kinase C activation

Among other proteolytic enzymes, the urokinase-type plasminogen activator (u-PA)/plasmin cascade contributes to cell migration and the formation of capillary-like structures in a fibrinous exudate. The u-PA receptor (u-PAR) focuses proteolytical activity on the cell surface of the endothelial cell and hereby accelerates the pericellular matrix degradation. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 enhance u-PA receptor expression in human endothelial cells. In this paper we show that the protein kinase C (PKC) inhibitors Ro31-8220 and GF109203X inhibit VEGF165-induced u-PAR antigen expression in human endothelial cells, whereas PKC inhibition had no effect on FGF-2-induced u-PAR antigen enhancement. In addition, inhibition of PKC activity had no effect on VEGF165- or FGF-2-induced proliferation in human endothelial cells. We conclude that VEGF165 induces u-PAR via a PKC-dependent pathway, whereas proliferation is induced via a different pathway probably involving tyrosine phosphorylation of proteins downstream of the VEGF receptors.     

Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.     

Disruption of focal adhesions mediates detachment during neuronal apoptosis.

Apoptosis participates in the development of the nervous system and in neurodegeneration. The aim of this study was to investigate the mechanisms of detachment of neuronal cells from the extracellular matrix (ECM) during apoptosis. Detachment of Ntera2 neuronal cells was accompanied by decreased surface expression of the beta1 integrin and redistribution of proteins from focal adhesions (FA). FA proteins were cleaved in a discrete sequence: p130cas, then paxillin, then talin. Caspase inhibition prevented detachment and cleavage of paxillin and p130cas, whilst calpain inhibition blocked talin cleavage. Neuronal cells therefore detach as a result of redistribution and caspase-dependent cleavage of focal adhesion proteins. Cleavage occurs sequentially such that critical ECM-integrin survival signalling cascades are severed before disruption of focal adhesion-cytoskeletal links.     

Apoptosis Induced by Src-Family Tyrosine Kinase Inhibitors in Cultured Rat Cortical Cells

In the central nervous system, members of the Src family of tyrosine kinases (SFKs) are widely expressed and are abundant in neurons. The purpose of this study is to examine whether glycogen synthase-3 (GSK-3) is involved in SFK inhibitor-induced apoptosis. PP2 and SU6656, SFK inhibitors, increased apoptotic cell death with morphological changes that were characterized by cell shrinkage, chromatin condensation, or nuclear fragmentation. Moreover, both activation of caspase-9 and caspase-3 were accompanied by the cell death. GSK-3 inhibitors, such as alsterpaullone and SB216763, prevented the PP2-induced apoptosis. In addition, insulin-like growth factor-I prevented the PP2-induced cell death and PP2 inhibited phosphorylation of focal adhesion kinase (FAK). Phosphorylation of FAK on Tyr 576 by Src activates FAK. These results suggest that inhibition of SFK induces apoptosis possibly via blocking of FAK/phosphatidylinositol-3 kinase/Akt signaling pathway and activation of GSK-3 is involved in the cell death in rat cortical neurons.