The Survival of Sympathetic Neurons Promoted by Potassium Depolarization, but Not by Cyclic AMP, Requires Phosphatidylinositol 3-Kinase and Akt

Phosphatidylinositol (PI) 3-kinase and Akt protein kinase mediate trophic factor-dependent survival in certain neurons. However, a role for these enzymes in neuronal survival promoted by other agents is unclear. We have tested PI 3-kinase and Akt for their role in survival promoted by membrane-depolarizing concentrations of extracellular potassium and the cell-permeable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (cpt-cAMP). Depolarization of sympathetic neurons resulted in an increase in the activities of both PI 3-kinase and Akt. In addition, the PI 3-kinase inhibitor LY294002 was a potent inducer of cell death in depolarized neurons. Stimulation with cpt-cAMP resulted in relatively small increases in PI 3-kinase and Akt activities, and neurons maintained with cpt-cAMP were more resistant to LY294002-induced death than were depolarized neurons. Expression of either dominant-negative PI 3-kinase or dominant-negative Akt blocked survival promoted by depolarization but not by cpt-cAMP. These results indicate that a PI 3-kinase/Akt pathway is required for survival of sympathetic neurons mediated by depolarization but not by cpt-cAMP. Thus, the survival of sympathetic neurons can be maintained through PI 3-kinase/Akt-dependent and -independent pathways.     

p75 neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12 cells and rat hippocampal neurones

We have previously shown that nerve growth factor (NGF)-induced activation of nuclear factor-kappaB increased neuronal expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. In the present study we determined the role of the p75 neurotrophin receptor in constitutive and NGF-induced survival signalling. Treatment of rat pheochromocytoma (PC12) cells with a blocking anti-rat p75 antibody or inhibition of p75 expression by antisense oligonucleotides reduced constitutive and NGF-induced bcl-xL expression. Treatment with the blocking anti-p75 antibody also inhibited NGF-induced activation of the survival kinase Akt. Inhibition of phosphatidylinositol-3-kinase (PI3 kinase) activity or overexpression of a dominant-negative mutant of Akt kinase inhibited NGF-induced nuclear factor-kappaB activation. Activation of Akt kinase by NGF was also observed in PC12nnr5 cells and cultured rat hippocampal neurones which both lack significant TrkA expression. Treatment of hippocampal neurones with the blocking anti-p75 antibody inhibited constitutive and NGF-induced Bcl-xL expression, activation of Akt, and blocked the protective effect of NGF against excitotoxic and apoptotic injury. Our data suggest that the p75 neurotrophin receptor mediates constitutive and NGF-induced survival signalling in PC12 cells and hippocampal neurones, and that these effects are mediated via the PI3-kinase pathway.     

Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal

Numerous studies reveal that phosphatidylinositol (PI) 3-kinase and Akt protein kinase are important mediators of cell survival. However, the survival-promoting mechanisms downstream of these enzymes remain uncharacterized. Glycogen synthase kinase-3 beta (GSK-3 beta), which is inhibited upon phosphorylation by Akt, was recently shown to function during cell death induced by PI 3-kinase inhibitors. In this study, we tested whether GSK-3 beta is critical for the death of sympathetic neurons caused by the withdrawal of their physiological survival factor, the nerve growth factor (NGF). Stimulation with NGF resulted in PI 3-kinase-dependent phosphorylation of GSK-3 beta and inhibition of its protein kinase activity, indicating that GSK-3 beta is targeted by PI 3-kinase/Akt in these neurons. Expression of the GSK-3 beta inhibitor Frat1, but not a mutant Frat1 protein that does not bind GSK-3 beta, rescued neurons from death caused by inhibiting PI 3-kinase. Similarly, expression of Frat1 or kinase-deficient GSK-3 beta reduced death caused by inhibiting Akt. In NGF-maintained neurons, overexpression of GSK-3 beta caused a small but significant decrease in survival. However, expression of neither Frat1, kinase-deficient GSK-3 beta, nor GSK-3-binding protein inhibited NGF withdrawal-induced death. Thus, although GSK-3 beta function is required for death caused by inactivation of PI 3-kinase and Akt, neuronal death caused by NGF withdrawal can proceed through GSK-3 beta-independent pathways.     

Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 T^ts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-x_L or inhibition of Jun kinase activity.     

Early decrease of survival signal-related proteins in spinal motor neurons of presymptomatic transgenic mice with a mutant SOD1 gene

The mechanisms of motor neuronal death in amyotrophic lateral sclerosis (ALS) remain to be unclear. Phosphatidy-linositol 3-kinase (PI3-K) and its main downstream effector, Akt/protein kinase B (PKB) have been shown to play a central role in neuronal survival against apoptosis supported by neurotrophic factors. In order to investigate a possible impairment of survival signaling, we examined expressions of PI3-K and Akt in the spinal cord of the transgenic mice overexpressing a mutant Cu/Zn superoxide dismutase (SOD1) gene, a valuable model for human ALS. Immunoblotting and immunohistochemical analyses showed that the majority of spinal motor neurons lost the immunoreactivities for both PI3-K and Akt in the early and presymptomatic stage that preceded significant loss of the neurons. The present results suggest that an early decrease of survival signal proteins in the spinal motor neurons may account for the subsequent motor neuronal loss in this animal model of ALS.     

Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.     

Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells

Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells.     

Activation of Akt is induced by heat shock and involved in suppression of heat-shock-induced apoptosis of NIH3T3 cells

Heat shock exposure to NIH3T3 cells for 15 min at 45 degrees C activated Akt, which is mediated by PI3-kinase, as evidenced by the significant inhibition of heat-shock-induced phosphorylation by specific inhibitors of PI3-kinase. The phosphorylated Akt was gradually decreased to the basal level within 9 h after heat shock. This resulted in growth arrest, but cell growth could be recovered within 24 h accompanied with a high rate of proliferation. However, heat shock for 60 min failed to activate Akt, resulting in apoptosis. The recovery of cell growth after heat-shock-inducing activation of Akt was completely blocked by wortmannin. Moreover, overexpression of a dominant-negative Akt mutant significantly inhibited the apoptosis-suppressive effect of heat shock, indicating the direct involvement of heat-shock-induced Akt activation in the apoptosis suppression. The results indicate that a signal transduction pathway, namely, PI3-kinase/Akt, may contribute to an apoptosis-suppressive function after heat shock in NIH3T3 cells.     

Activation of Akt Kinase Inhibits Apoptosis and Changes in Bcl-2 and Bax Expression Induced by Nitric Oxide in Primary Hippocampal Neurons

Emerging data indicate that growth factors such as insulin-like growth factor-1 (IGF-1) prevent neuronal death due to nitric oxide (NO) toxicity. On the other hand, growth factors can promote cell survival by acting on phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, serine-threonine kinase Akt, in various types of cells. Here, we examined the mechanism by which IGF-1 inhibits neuronal apoptosis induced by NO in primary hippocampal neurons. IGF-1 was capable of preventing apoptosis and caspase-3-like activation induced by a NO donor, sodium nitroprusside or 3-morpholin-osydnonimine. Incubation of neurons with a P13-kinase inhibitor, wortmannin or LY294002, blocked the effects of IGF-1 on NO-induced neurotoxicity and caspase-3-like activation. In addition, the P13-kinase inhibitors blocked the effect of IGF-1 on down-regulation in Bcl-2 and upregulation in Bax expression induced by NO. Adenovirus-mediated overexpression of the activated form of Akt significantly inhibited NO-induced cell death, caspase-3-like activation, and changes in Bcl-2 and Bax expression. Moreover, expression of the kinase-defective form of Akt almost completely blocked the effects of IGF-1. These findings suggest that activation of Akt is necessary and sufficient for the effect of IGF-1 and is capable of preventing NO-induced apoptosis by modulating the NO-induced changes in Bcl-2 and Bax expression.     

Role of Translation Initiation Factor 2B in Control of Cell Survival by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Signaling Pathway

The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway is an important mediator of growth factor-dependent survival of mammalian cells. A variety of targets of the Akt protein kinase have been implicated in cell survival, including the protein kinase glycogen synthase kinase 3beta (GSK-3beta). One of the targets of GSK-3beta is translation initiation factor 2B (eIF2B), linking global regulation of protein synthesis to PI 3-kinase/Akt signaling. Because of the central role of protein synthesis, we have investigated the involvement of eIF2B, which is inhibited as a result of GSK-3beta phosphorylation, in programmed cell death. We demonstrate that expression of eIF2B mutants lacking the GSK-3beta phosphorylation or priming sites is sufficient to protect both Rat-1 and PC12 cells from apoptosis induced by overexpression of GSK-3beta, inhibition of PI 3-kinase, or growth factor deprivation. Consistent with these effects on cell survival, expression of nonphosphorylatable eIF2B prevented inhibition of protein synthesis following treatment of cells with the PI 3-kinase inhibitor LY294002. Conversely, cycloheximide induced apoptosis of PC12 and Rat-1 cells, further indicating that protein synthesis was required for cell survival. Inhibition of translation resulting from treatment with cycloheximide led to the release of cytochrome c from mitochondria, similar to the effects of inhibition of PI 3-kinase. Expression of nonphosphorylatable eIF2B prevented cytochrome c release resulting from PI 3-kinase inhibition but did not affect cytochrome c release or apoptosis induced by cycloheximide. Regulation of translation resulting from phosphorylation of eIF2B by GSK-3beta thus appears to contribute to the control of cell survival by the PI 3-kinase/Akt signaling pathway, acting upstream of mitochondrial cytochrome c release.     

Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival.

In this report, we have examined the mechanisms whereby neurotrophins and neural activity coordinately regulate neuronal survival, focussing on sympathetic neurons, which require target-derived NGF and neural activity for survival during development. When sympathetic neurons were maintained in suboptimal concentrations of NGF, coincident depolarization with concentrations of KCl that on their own had no survival effect, synergistically enhanced survival. Biochemical analysis revealed that depolarization was sufficient to activate a Ras-phosphatidylinositol 3-kinase-Akt pathway (Ras-PI3-kinase-Akt), and function-blocking experiments using recombinant adenovirus indicated that this pathway was essential for approximately 50% of depolarization-mediated neuronal survival. At concentrations of NGF and KCl that promoted synergistic survival, these two stimuli converged to promote increased PI3-kinase-dependent Akt phosphorylation. This convergent PI3-kinase-Akt pathway was essential for synergistic survival. In contrast, inhibition of calcium/calmodulin-dependent protein kinase II revealed that, while this molecule was essential for depolarization-induced survival, it had no role in KCl- induced Akt phosphorylation, nor was it important for synergistic survival by NGF and KCl. Thus, NGF and depolarization together mediate survival of sympathetic neurons via intracellular convergence on a Ras-PI3-kinase-Akt pathway. This convergent regulation of Akt may provide a general mechanism for coordinating the effects of growth factors and neural activity on neuronal survival throughout the nervous system.     

Activated Phosphatidylinositol 3-Kinase and Akt Kinase Promote Survival of Superior Cervical Neurons

The signaling pathways that mediate the ability of NGF to support survival of dependent neurons are not yet completely clear. However previous work has shown that the c-Jun pathway is activated after NGF withdrawal, and blocking this pathway blocks neuronal cell death. In this paper we show that over-expression in sympathetic neurons of phosphatidylinositol (PI) 3-kinase or its downstream effector Akt kinase blocks cell death after NGF withdrawal, in spite of the fact that the c-Jun pathway is activated. Yet, neither the PI 3-kinase inhibitor LY294002 nor a dominant negative PI 3-kinase cause sympathetic neurons to die if they are maintained in NGF. Thus, although NGF may regulate multiple pathways involved in neuronal survival, stimulation of the PI 3-kinase pathway is sufficient to allow cells to survive in the absence of this factor.     

Early decrease of survival factors and DNA repair enzyme in spinal motor neurons of presymptomatic transgenic mice that express a mutant SOD1 gene

The primary pathogenetic mechanisms of amyotrophic lateral sclerosis (ALS) have been elusive. Some of the mechanisms would be implicated in an imbalance between death and survival factors, and impairment of DNA repair possibly caused by oxidative stress. Phosphatidylinositol 3-kinase (PI3-K) and its downstream effector, Akt/protein kinase B (PKB), have been shown to play a pivotal role in neuronal survival against apoptosis supported by neurotrophic factors. To elucidate the mechanisms of motor neuron death in ALS, we examined the expression of PI3-K, Akt, and the DNA repair enzyme redox factor-1 (Ref-1) protein in the spinal cord of transgenic mice with an ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) gene, a valuable model for human ALS. Immunoblotting and immunocytochemical analyses showed that most spinal motor neurons lost immunoreactivity for PI3-K, Akt, and Ref-1 in the presymptomatic stage that preceded a significant loss of neurons. These results suggest that an early decrease of survival signal proteins and a DNA repair enzyme in the spinal motor neurons may account for the mutant SOD1-mediated motor neuron death in this animal model of ALS.     

Monokine induced by interferon-gamma acts as a neurotrophic factor on PC12 cells and rat primary sympathetic neurons

We found that a monokine induced by interferon-gamma (Mig, CXCL9), which belongs to the CXC chemokine subfamily, acts as a neurotrophic factor on PC12 cells and rat primary sympathetic neurons. PC12 cells were shown to express a single class of high affinity binding sites for Mig (670 receptors/cell, Kd = 2.9 nm). Mig induced neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of extracellular signal-regulated kinase signaling pathways between Mig and nerve growth factor (NGF) revealed that these pathways are crucial for Mig action as well as NGF. K252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks) did not inhibit the action of Mig, suggesting that Mig action occurs via a different receptor from that of NGF. Furthermore, Mig as well as NGF promoted PC12 survival under serum-free conditions and activated Akt/protein kinase B downstream from phosphatidylinositol 3-kinase (PI3K). Because the PI3K inhibitor LY294002 prevented the Mig- and NGF-induced survival effect, this effect is probably mediated by the PI3K signaling pathway. Mig also promoted survival of rat primary sympathetic neurons that die when deprived of NGF. These results suggest that chemokines, including Mig (CXCL9) have neurotrophic effects on the nervous system.     

Midkine Inhibits Caspase-Dependent Apoptosis via the Activation of Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase in Cultured Neurons

Midkine (MK) is a new member of the heparin-binding neurotrophic factor family. MK plays important roles in development and carcinogenesis and has several important biological effects, including promotion of neurite extension and neuronal survival. However, the mechanism by which MK exerts its neurotrophic actions on neurons has not been elucidated to date. We have established an apoptosis induction system by serum deprivation in primary neuronal cultures isolated from mouse cerebral cortices. Neuronal apoptosis induced by serum deprivation was accompanied by the activation of caspase-3. MK, when added into the culture medium, inhibited the induction of apoptosis and activation of caspase-3 in a dose-dependent manner. Extracellular signal-regulated kinase (ERK) and Akt were not activated by serum deprivation, whereas ERK and Akt were rapidly activated by addition of MK. In addition, the trophic actions of MK of suppressing apoptosis and suppressing the activation of caspase-3 were abolished by concomitant treatment with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, and with wort-mannin or LY294002, specific inhibitors of phosphatidyl-inositol 3-kinase (PI 3-kinase). These PI 3-kinase inhibitors also inhibited the activation of ERK in response to MK, demonstrating a link between ERK and the caspase-3 pathway that is modulated by the PI 3-kinase activation. These results indicate that the ERK cascade plays a central role in MK-mediated neuronal survival via inhibition of caspase-3 activation.