Growth inhibition and induction of differentiation by panaxydol in rat C6 glioma cells

OBJECTIVES: Panaxydol is a naturally occurring non-peptidyl small molecule isolated from the lipophilic fractions of Panax notoginseng, a well-known Chinese traditional medicine. In this study, we aimed to investigate the effects of panaxydol on growth inhibition and its mechanisms in C6 rat glioma cells. METHODS: The effects of panaxydol on cell proliferation, morphologic changes, glial fibrillary acidic protein (GFAP) expression and cell cycle regulation in rat C6 cells were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, hematoxylin and eosin (HE) staining, immunocytochemistry, flow cytometric analysis and Western blot respectively. RESULTS: Panaxydol markedly inhibited the proliferation of C6 cells in a dose-dependent manner with IC50 of 39.5 +/- 2.3 microM. In addition, the cell morphologic changes and increased expression of GFAP in C6 cells in the presence of panaxydol implied a cellular differentiation. Flow cytometric analysis revealed that panaxydol-treated cells accumulated in G0/G1 phase with a marked decrease in the number of C6 cells at S phase. Western blot analysis demonstrated that panaxydol resulted in an increase in the protein expression of p27 in C6 cells as early as 3 hours after treatment consistent with the differentiation response, but protein expression of p53, p21, p16 and pRb remained unchanged. CONCLUSION: These findings suggest that panaxydol inhibits the proliferation of C6 cells via G0/G1 cell cycle arrest in association with induction of p27 expression and differentiation.     

IGF-I has a direct proliferative effect in adult hippocampal progenitor cells

The aim of the present study was to investigate the potential direct effects of insulin-like growth factor-I (IGF-I) on adult rat hippocampal stem/progenitor cells (AHPs). IGF-I-treated cultures showed a dose-dependent increase in thymidine incorporation, total number of cells, and number of cells entering the mitosis phase. Pretreatment with fibroblast growth factor-2 (FGF-2) increased the IGF-I receptor (IGF-IR) expression, and both FGF-2 and IGF-I were required for maximal proliferation. Time-lapse recordings showed that IGF-I at 100 ng/ml decreased differentiation and increased proliferation of single AHPs. Specific inhibition of mitogen-activated protein kinase kinase (MAPKK), phosphatidylinositol 3-kinase (PI3-K), or the downstream effector of the PI3-K pathway, serine/threonine p70 S6 kinase (p70(S6K)), showed that both the MAPK and the PI3-K pathways participate in IGF-I-induced proliferation but that the MAPK activation is obligatory. These results were confirmed with dominant-negative constructs for these pathways. Stimulation of differentiation was found at a low dose (1 ng/ml) of IGF-I, clonal analysis indicating an instructive component of IGF-I signaling.     

Differential involvement of phosphatidylinositol 3-kinase and p42/p44 mitogen activated protein kinase pathways in brain-derived neurotrophic factor-induced trophic effects on cultured striatal neurons

Brain-derived neurotrophic factor (BDNF) is a potent trophic factor for striatal cells that promotes survival and/or differentiation of GABAergic neurons in vitro. In the present study, we show that the stimulation of cultured striatal cells with BDNF increased the phosphorylation of Akt and p42/p44. This effect was specifically blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-K) pathways (LY294002 and wortmannin) or p42/p44 mitogen-activated protein (MAP) kinase (PD98059 and U0126). BDNF treatment induced an increase in the number of calbindin-positive neurons but not in the number of GABAergic or total cells. Furthermore, BDNF increased the degree of dendritic arborization, soma area and axon length of striatal neurons. However, PD98059 was more effective blocking BDNF effects on calbindin- than on GABA-positive neurons, whereas LY294002 inhibited morphological differentiation in both neuronal populations. Moreover, BDNF induced neuronal survival only through the activation of the PI3-K pathway.     

Correlation between delayed neuronal cell death and selective decrease in phosphatidylinositol 4-kinase expression in the CA1 subfield of the hippocampus after transient forebrain ischemia.

Transient forebrain ischemia induces a delayed neuronal death in the CA1 area of the hippocampus. However, the mechanism leading to this phenomenon has yet to be established. The authors used an mRNA differential-display method to isolate genes for which mRNA levels change only in the hippocampus during ischemia/reperfusion. They succeeded in identifying the product of one down-regulated gene as phosphatidylinositol 4-kinase (PI 4-K). Compared with control levels, PI 4-K mRNA expression in the hippocampus, but not the cerebral cortex, was significantly decreased by 30% and about 80% 1 and 7 days after ischemia/reperfusion, respectively. Interestingly, PI 4-K and PI bisphosphate levels were selectively decreased in the CA1 region, but not other regions, whereas TUNEL-positive cells could be detected 3 days after ischemia. Consistent with these results, PI 4-K expression was suppressed by hypoxia in SK-N-MC neuroblastoma cells before loss of cell viability. Overexpression of wild-type PI 4-K, but not the kinase-negative mutant of PI 4-K (K1789A), recovered the loss of viability induced by hypoxia. These findings strongly suggest that a prior decrease in PI 4-K and PI bisphosphate levels caused by brain ischemia/hypoxia is partly involved in delayed neuronal cell death.     

Signalling pathways involved in the chemotactic activity of CXCL12 in cultured rat cerebellar neurons and CHP100 neuroepithelioma cells

We compared the signal transduction pathways activated by stromal cell-derived factor-1 (CXCL12) chemokine in two different cell systems: primary cultures of rat cerebellar granule neurons (CGN) and human neuroepithelioma CHP100 cells. Both cell types express functional CXC chemokine receptor 4 (CXCR4), which is coupled both to extracellular signal-regulated kinase (ERK) and Akt phosphorylation pathways. The activation of ERK shows different dependency on the phosphatidylinositol 3-kinase (PI3-K) pathway and different sensitivity to pertussis toxin (PTX) treatment, indicative of coupling to different G proteins in the two cell systems considered. We demonstrate that the inhibition of either the ERK kinase or the PI3-K pathways blocks the CXCL12 induced-chemotaxis in CHP100 cells; while only PI3-K activity is stringently necessary for CGN migration.     

壮骨镇痛胶囊含药血浆培养乳腺癌细胞rac-1，PI3-K转录和细胞伪足的变化

背景：前期实验已证实壮骨镇痛胶囊能显著抑制体外培养乳腺癌细胞的迁移和增殖。 目的：进一步观察壮骨镇痛胶囊对高转移人乳腺癌MDA-MB-231细胞rac-1,PI3-K转录和细胞伪足生长的影响。 方法：SD大鼠以3倍剂量灌服壮骨镇痛胶囊连续7 d,以大鼠含药血浆培养MDA-MB-231细胞24 h后,采用扫描电镜法观察不同浓度壮骨镇痛胶囊含药血浆对MDA-MB-231细胞伪足生长的影响,采用RT-PCR法检测含药血浆对MDA-MB-231细胞中rac-1,PI3-K转录的影响。 结果与结论：5.0%和1.25%壮骨镇痛胶囊含药血浆能显著抑制MDA-MB-231细胞伪足的形成和生长以及rac-1和PI3-K基因的转录(P < 0.05);0.63%壮骨镇痛胶囊含药血浆对伪足生长和rac-1基因转录无显著影响,但可显著抑制了PI3-K基因的转录(P < 0.05)。由此推测壮骨镇痛胶囊含药血浆抑制MDA-MB-231细胞迁移可能与其抑制rac-1和PI3-K基因转录进而影响细胞伪足形成和生长密切有关,并且该作用可能与浓度相关。     

Insulin-like growth factor-1-dependent maintenance of neuronal metabolism through the phosphatidylinositol 3-kinase-Akt pathway is inhibited by C2-ceramide in CAD cells

Ceramide is a lipid second-messenger generated in response to stimuli associated with neurodegeneration that induces apoptosis, a mechanism underlying neuronal death in Parkinson's disease. We tested the hypothesis that insulin-like growth factor-1 (IGF-1) could mediate a metabolic response in CAD cells, a dopaminergic cell line of mesencephalic origin that differentiate into a neuronal-like phenotype upon serum removal, extend processes resembling neurites, synthesize abundant dopamine and noradrenaline and express the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase, and that this process was phosphatidylinositol 3-kinase (PI 3-K)-Akt-dependent and could be inhibited by C(2)-ceramide. The metabolic response was evaluated as real-time changes in extracellular acidification rate (ECAR) using microphysiometry. The IGF-1-induced ECAR response was associated with increased glycolysis, determined by increased NAD(P)H reduction, elevated hexokinase activity and Akt phosphorylation. C(2)-ceramide inhibited all these changes in a dose-dependent manner, and was specific, as it was not induced by the inactive C(2)-ceramide analogue C(2)-dihydroceramide. Inhibition of the upstream kinase, PI 3-K, also inhibited Akt phosphorylation and the metabolic response to IGF-1, similar to C(2)-ceramide. Decreased mitochondrial membrane potential occurred after loss of Akt phosphorylation. These results show that IGF-1 can rapidly modulate neuronal metabolism through PI 3-K-Akt and that early metabolic inhibition induced by C(2)-ceramide involves blockade of the PI 3-K-Akt pathway, and may compromise the first step of glycolysis. This may represent a new early event in the C(2)-ceramide-induced cell death pathway that could coordinate subsequent changes in mitochondria and commitment of neurons to apoptosis.     

Brain-derived neurotrophic factor effects on oligodendrocyte progenitors of the basal forebrain are mediated through trkB and the MAP kinase pathway

Previous work has indicated that BDNF increases the differentiation of basal forebrain (BF) oligodendrocytes (OLGs) in culture through the mediation of trkB and the MAPK pathway (Du et al. [ 2006a, b] Mol. Cell. Neurosci. 31:366-375; J. Neurosci. Res. 84:1692-1702). In the present work, effects of BDNF on BF OLG progenitor cells (OPCs) were examined. BDNF increased DNA synthesis of OPCs, as assessed by thymidine and bromodeoxyuridine incorporation. Effects of BDNF on DNA synthesis were mediated through the trkB receptor and not the p75 receptor, as shown by inhibitors that block neurotrophin binding to the receptors and by the phosphorylation of trkB. TrkB can activate the mitogen- activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3-K), and phospholipase C-gamma (PLC-gamma) pathways. BDNF elicited the phosphorylation of MAPK and Akt, a kinase downstream of PI3K, but not PLC-gamma in OPCs. Through the use of specific inhibitors to the MAPK and PI3-K pathways, it was found that the MAPK pathway was responsible for the effect of BDNF on DNA synthesis. These data indicate that BDNF affects OPC proliferation and development through the mediation of trkB and the MAPK pathway.     

磷脂酰肌醇3-激酶/蛋白激酶B磷酸化是胰岛素受体后信号转导通路控制PC12细胞凋亡的机制

目的:研究胰岛素抵抗1-甲基-4苯基砒啶(MPP )诱导的PC12细胞凋亡的信号转导途径中,磷脂酰肌醇3-激酶/蛋白激酶B(PI3-K/PKB)的活力变化。方法:应用Wortmannin(PI3-K抑制剂),比较用药前后细胞生存率的变化;应用Western印迹分析检测此间PKB及其磷酸化水平的变化。结果:Wortmannin预处理组细胞生存率较之胰岛素干预组明显下降;PKB的特异性磷酸化程度(Ser473磷酸化程度/激酶蛋白量)与细胞生存率的变化有关。结论:胰岛素主要通过调节PI3-K活性后再促进PKB的磷酸化,从而促进PKB的激活,并导致生物学效应的变化,但是尚不能排除其他机制的参与。     

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.     

Retinoic acid-induced neuritogenesis of human neuroblastoma SH-SY5Y cells is ERK independent and PKC dependent

Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA-induced neuritogenesis, we used human neuroblastoma SH-SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal-regulated kinase (ERK) 1 and 2 in RA-induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA-induced neuritogenesis (percentage of neurite-bearing cells and neurite length) but significantly reduced cell survival. In addition, we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA-induced ERK phosphorylation does not follow the classic Raf kinase-dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI 3-K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3-K inhibitor wortmannin, we observed a significant dose-dependent reduction in ERK phosphorylation. RA-induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration-dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA-induced neuritogenesis.     

VEGF-induced activation of the PI3-K/Akt pathway reduces mutant SOD1-mediated motor neuron cell death

The increased oxidative stress induced by mutant SOD1 is associated with motor neuron degeneration in both human ALS and transgenic mice expressing mutant SOD1. Vascular endothelial growth factor (VEGF) is neurotrophic and also protects from hypoxia-induced neuronal injury. The potential role of VEGF in preventing mutant SOD1-mediated motor neuron cell death was examined using a mouse NSC34 motor neuron-like cell culture system. Infection with adenovirus containing mutant G93A-SOD1, but not vector control or wild-type SOD1, increased cellular oxidative stress and motor neuron-like cell death. However, NSC34 cells pretreated with VEGF displayed a dose-dependent resistance to oxidative damage from hydrogen peroxide, TNF-alpha, and mutant G93A-SOD1. VEGF activated both PI3-K and MAPK activities in mouse NSC34 motor neuron-like cells. Pharmacological inhibitors and constitutively active as well as dominant negative mutants of MAPK and PI3-K revealed that the protective effects of VEGF were mediated via the PI3-K activity, and that MAPK activation was not associated with NSC34 cell survival. Furthermore, VEGF-induced downstream Akt activation promoted motor neuron-like NSC34 cell survival in the presence of mutant G93A-SOD1. Thus, VEGF protected mouse NSC34 motor neuron-like cell death from mutant G93A-SOD1 effects via PI3-K/Akt activation.     

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.     

Lysophosphatidic acid regulates the proliferation and migration of olfactory ensheathing cells in vitro

Olfactory ensheathing cells (OECs) are a unique type of macroglia with axonal growth-promoting properties. However, our understanding of the factors that regulate OECs is at early stages. Lysophosphatidic acid (LPA) is a lipid that influences diverse functions in the nervous system. Recent studies suggest that glial cells, including astrocytes and Schwann cells, are important targets of LPA. However, the influences of LPA on OECs are not known. To address if LPA can influence OECs, we examined effects of LPA on the proliferation and migration of OECs and intracellular effector events. Initially, we observed that OECs expressed the genes for LPA1, LPA2, and LPA3 receptors. When OECs were treated with LPA, we observed stimulated proliferation and migration of OECs. Treatment of OECs with LPA also induced actin cytoskeleton reorganization and focal adhesion assembly. These effects of LPA were blocked by treatment with C3 exoenzyme or Y-27632, which inhibit Rho-GTPase and Rho-associated kinase, respectively. Effects of LPA on OEC proliferation were blocked by the MEK inhibitors PD098059 and U0126 and by the phosphotidylinositol 3-kinase (PI 3-K) inhibitors LY0294002 and wortmannin. These results show that LPA acts via Rho-GTPase, MAPK, and PI 3-K signaling cascades to influence OEC proliferation, migration, and cytoskeleton assembly.     

Signaling pathways activated by chemokine receptor CXCR2 and AMPA-type glutamate receptors and involvement in granule cells survival

We show that treatment of cerebellar granules with interleukin-8 (IL-8), growth-related gene product beta (GRObeta) or AMPA induced activation of PI3-K/Akt and of ERK pathways, the latter being independent of PI3-K and dependent on PTX-sensitive G proteins. We also show that AMPA-mediated neuron survival was abolished both by ERK kinase inhibitor PD98059 and AMPA-Rs blocker CNQX, and that chemokine-mediated survival was blocked by the PI3-K inhibitors LY294002 and wortmannin. We conclude that the neurotrophic effects of AMPA need the contemporary activation of ERKs and stimulation of AMPA-Rs, and that PI3-K/Akt activation is a determinant pathway for the IL-8/GRObeta anti-apoptotic activity.     

Role of phosphatidylinositol 3-kinase in neuronal survival and axonal outgrowth of adult mouse dorsal root ganglia explants

Adult ganglionic peripheral neurons have lost dependence on target-derived neurotrophin signaling for survival and regeneration after injury. To understand the mechanisms required to sustain such processes at maturity, we are studying neuronal survival and axonal outgrowth of adult mouse dorsal root ganglia (DRG) explants. We have here examined the role of phosphatidylinositol 3-kinase (PI3-K) activity. Both neuronal survival and axonal outgrowth of spontaneously growing preparations were decreased significantly by the PI3-K inhibitor LY294002 as was the increased outgrowth caused by nerve growth factor or glial cell line-derived factor. Inhibition of PI3-K activity promoted neuronal cell death to the same extent in the presence as in the absence of a growth factor, whereas inhibition of mitogen-activated protein kinase, MAPK, lacked effect. Using a compartmentalized system, it could be shown that only axonal outgrowth was decreased when the outgrowth region only was exposed to LY294002. Already-formed growth cones showed morphological changes within 5-10 min after exposure to LY294002. Akt (PKB) is one downstream effector of PI3-K. Immunofluorescence revealed the presence of activated Akt in DRG cell bodies and in axonal growth cones. Immunoreactivity was decreased by PI3-K inhibition. The results suggest that Akt is constitutively active in adult DRG neurons, and that PI3-K mediated processes are involved in neuronal survival of one or more DRG neuronal subpopulations and also in axonal elongation. The possible significance of Akt signaling for these effects is discussed.     

Bad as a converging signaling molecule between survival PI3-K/Akt and death JNK in neurons after transient focal cerebral ischemia in rats.

Bad, a proapoptotic Bcl-2 family protein, plays a critical role in determining cell death/survival. The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the c-Jun N-terminal kinase (JNK) pathway are thought to be involved in regulation of Bad. Therefore, the present study was performed to clarify the role of Bad as a common target of the PI3-K/Akt and JNK pathways after transient focal cerebral ischemia (tFCI) in rats. We found that Akt activity increased at 3 h and then decreased, whereas JNK activity increased 7 to 24 h in the peripheral area after tFCI. Administration of LY294002, a PI3-K-specific inhibitor, exacerbated DNA fragmentation, whereas administration of SP600125, a JNK-specific inhibitor, attenuated it. Inhibited by LY294002, phospho-Bad (Ser136) expression increased in the peripheral area 3 h after tFCI, with suppression of Akt activity. Furthermore, phospho-Bad (Ser136) and phospho-Akt (Ser473) were colocalized. Decreases in phospho-Bad (Ser136) and Bad/14-3-3 dimerization and increases in Bcl-X(L)/Bad or Bcl-2/Bad dimerization observed 7 to 24 h after tFCI, were prevented by SP600125 administration, with inhibition of JNK activity. The present study indicates that signal predominance varies from PI3-K/Akt-mediated survival signaling to JNK-mediated death signaling with the development of neuronal damage in the peripheral area after tFCI. This study also suggests that PI3-K/Akt has a role in Bad inactivation, whereas the JNK pathway is involved in Bad activation. We conclude that Bad may be an integrated checkpoint of PI3-K/Akt-mediated survival signaling and JNK-mediated death signaling and that it contributes to cell fate in the peripheral area after cerebral ischemia.     

Comparison of inhibitory effects of brain-derived neurotrophic factor and insulin-like growth factor on low potassium-induced apoptosis and activation of p38 MAPK and c-Jun in cultured cerebellar granule neurons

On cell maturation following culture in medium containing 26 mM potassium (high K+; HK), a change to medium containing 5 mM potassium (low K+; LK) rapidly induces apoptosis in rat cerebellar granule neurons. Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) have survival-promoting effects on the neurons via PI3-K. However, it remains unclear how they prevent the apoptosis in the pathway downstream of phosphatidylinositol-3 kinase (PI3-K). Recently, we have reported that PI3-K-ASK1 pathway is involved in signal-transduction to p38 MAPK (p38)-c-Jun pathway. Here we found that IGF-1 had a greater survival-promoting effect than BDNF, and activated PI3-K to a higher level and maintained the level for a longer time. BDNF and IGF-1 suppressed the activation of p38 and c-Jun, but not of c-Jun N-terminal kinase (JNK), caused by lowering the potassium concentration. The inhibitory effects of IGF-1 were much greater than those of BDNF. In addition, LY294002, a specific inhibitor of PI3-K, cancelled the inhibitory effects of BDNF and IGF-1. These results suggest that the greater inhibitory effects of IGF-1 than BDNF, on activation of p38 and c-Jun and apoptosis, are caused by the higher level of PI3-K activation during LK-induced apoptosis of cultured cerebellar granule neurons.     

Induction of Akt by endogenous neurosteroids and calcium sequestration in P19 derived neurons

Neuronal cell death caused by pathophysiological over-activation of glutamate receptors and the subsequent CaII overloading, has been implicated in neurodegeneration after stroke, cerebral trauma and epileptic seizures. Recent findings suggest that certain progesterone metabolites (neurosteroids) such as allopregnanolone and dehydroepiandrosterone can protect neuronal cells from such insults. In the present study, murine P19 cells were induced to differentiate into postmitotic neurons expressing specific neuronal markers, including GABA(A) and NMDA receptors. Activation of NMDA receptors in P19-N neurons resulted in excitotoxic cell death, which involved suppression of the phosphorylation of the survival kinase PKB/Akt. Allopregnanolone and DHEA induced a rapid and prolonged phosphorylation of the Akt kinase and they were able to reverse the NMDA-induced suppression of the PI3-K/Akt pathway. The specificity of the neuroprotective effects of these neurosteroids was confirmed by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, as well as by the GABA(A) receptor antagonist, bicuculline. The neurotoxic effect of NMDA on P19-N neurons was directly correlated with increased CaII entry, since the addition of EGTA or BAPTA-AM, significantly suppressed the NMDA-induced decrease of phospho-Akt and subsequent neuronal death. These results suggest that neurosteroids are able to act as survival factors on P19-N neurons, promoting the activation of the PI3-K/Akt pathway through a calcium-entry dependent mechanism.