Mitogenic effect of glial cell line-derived neurotrophic factor is dependent on the activation of p70S6 kinase,but independent of the activation of ERK and up-regulation of Ret in SH-SY5Y cells

Glial cell line-derived neurotrophic factor (GDNF) activates c-Ret tyrosine kinase and several downstream intracellular pathways; the biological effects caused by the activation of each of these pathways, however, remain to be elucidated. Here we report the ability of GDNF to induce proliferation, rather than differentiation, of neuroblastoma cells (SH-SY5Y) by targeting the signaling pathway responsible for mediating this proliferative effect. GDNF induces the phosphorylation of Akt and p70S6 kinase (p70S6K) in SH-SY5Y cells in which Ret protein expression is relatively low. Interestingly, treating SH-SY5Y cells with retinoic acid greatly increases Ret protein levels and GDNF-induced Ret tyrosine phosphorylation, but does not affect the mitogenic action of GDNF and the activation of the Akt/p70S6K pathway. In contrast, the activation of the ERK pathway and the resulting induction of immediate-early genes parallel the increases in Ret protein levels. Rapamycin, a specific inhibitor of p70S6K activation by the mammalian target of rapamycin, completely prevents GDNF-induced proliferation and activation of p70S6K. These results suggest that GDNF promotes cell proliferation via the activation of p70S6K, independent of the ERK signaling pathway, and that GDNF activates the Akt/p70S6K pathway more efficiently than the ERK pathway in the cells in which Ret expression is low.     

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine enhance neurite outgrowth in PC12 cells via P13K/AKT, ERK, and pertussis toxin-sensitive pathways

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine, were found to enhance neurite outgrowth induced by nerve growth factor (NGF) in PC12 cells. These drugs increased the number of cells bearing neurites, the length of primary neurites, and the size of the cell body of NGF-differentiated PC12 cells. In addition, the drugs induced sprouting of neurite-like processes in PC12 cells in the absence of NGF. Olanzapine, quetiapine, and clozapine enhanced the phosphorylation of Akt and ERK in combination with NGF, and specific inhibitors of these pathways attenuated these effects. Pretreatment of cells overnight with pertussis toxin had no effect on NGF-induced differentiation but significantly decreased the effects of the antipsychotic drugs on neurite outgrowth, suggesting that Gi/Go-coupled receptors are involved in the response to drug. A better understanding of the mechanisms underlying the effects of the second-generation drugs might suggest new therapeutic targets for enhancement of neurite outgrowth.     

Zinc stimulates tau S214 phosphorylation by the activation of Raf/mitogen-activated protein kinase-kinase/extracellular signal-regulated kinase pathway

Hyperphosphorylated tau is a main component of neurofibrillary tangles, a pathological hallmark of Alzheimer's disease (AD). There is evidence that various protein kinases are involved in tau hyperphosphorylation. However, little is known about AD-related stimuli that activates tau kinases. We investigated the role of zinc, a metal involved in AD pathology, in tau phosphorylation. Zinc increased the phosphorylation of serine 214 (S214) in tau protein in human wild-type tau1-441-expressing SH-SY5Y cells. The phosphorylation was inhibited by suppressing the Ras-Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathway. Mutation of serine to alanine at residue 214 of tau reduced microtubule polymerization impairment by ERK phosphorylation. These data suggest that zinc induces S214 phosphorylation in tau through ERK activation and interferes with microtubule polymerization.     

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine enhance neurite outgrowth in PC12 cells via P13K/AKT, ERK, and pertussis toxin-sensitive pathways

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine, were found to enhance neurite outgrowth induced by nerve growth factor (NGF) in PC12 cells. These drugs increased the number of cells bearing neurites, the length of primary neurites, and the size of the cell body of NGF-differentiated PC12 cells. In addition, the drugs induced sprouting of neurite-like processes in PC12 cells in the absence of NGF. Olanzapine, quetiapine, and clozapine enhanced the phosphorylation of Akt and ERK in combination with NGF, and specific inhibitors of these pathways attenuated these effects. Pretreatment of cells overnight with pertussis toxin had no effect on NGF-induced differentiation but significantly decreased the effects of the antipsychotic drugs on neurite outgrowth, suggesting that G_i/G_o-coupled receptors are involved in the response to drug. A better understanding of the mechanisms underlying the effects of the second-generation drugs might suggest new therapeutic targets for enhancement of neurite outgrowth.     

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.     

Magnolol Protects Against Oxidative Stress-Mediated Neural Cell Damage by Modulating Mitochondrial Dysfunction and PI3K/Akt Signaling

Magnolol, an orally available compound from Magnolia officinalis used widely in traditional herbal medicine against a variety of neuronal diseases, possesses potent antioxidant properties and protects the brain against oxidative damage. The aim of the work is to examine the protective mechanisms of magnolol on human neuroblastoma SH-SY5Y cells against apoptosis induced by the neurotoxin acrolein, which can cause neurodegenerative disorders by inducing oxidative stress. By investigating the effect of magnolol on neural cell damage induced by the neurotoxin acrolein, we found that magnolol pretreatment significantly attenuated acrolein-induced oxidative stress through inhibiting reactive oxygen species accumulation caused by intracellular glutathione depletion and nicotinamide adenine dinucleotide phosphate oxidase activation. We next examined the signaling cascade(s) involved in magnolol-mediated antiapoptotic effects. The results showed that acrolein induced SH-SY5Y cell apoptosis by activating mitochondria/caspase and MEK/ERK signaling pathways. Our findings provide the first evidence that magnolol protects SH-SY5Y cells against acrolein-induced oxidative stress and prolongs SH-SY5Y cell survival through regulating JNK/mitochondria/caspase, PI3K/MEK/ERK, and PI3K/Akt/FoxO1 signaling pathways.     

Integrins mediate beta-amyloid-induced cell-cycle activation and neuronal death

Early intracellular events responsible for cell-cycle induction by beta-amyloid (A beta) in neurons have not been identified yet. Extracellular signal-regulated kinases 1/2 (ERK1/2) have been identified in this pathway, and inhibition of ERK activity prevents cell-cycle activation and reduces neuronal death induced by A beta. To identify upstream events responsible for ERK activation, attention has been focused on integrins. Treatment of SH-SY5Y cells, differentiated by long-term exposure to 10 microM retinoic acid with a neutralizing anti-alpha1-integrin antibody significantly reduced A beta-induced neuronal death. However, cell-cycle analysis showed that treatment with anti-alpha1-integrin per se produced changes in the distribution of cell populations, thus hampering any effect on A beta-induced cell-cycle activation. 4-Amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-D)pyramide, an inhibitor of src protein kinases that colocalizes with focal adhesion kinase (FAK) and is involved in integrin signaling, was effective in reducing activation of the cell cycle and preventing induction of neuronal death by A beta while inhibiting ERK1/2 phosphorylation. Similar results were obtained when FAK expression was down-regulated by siRNA silencing. The present study identifies a sequence of early events in the toxic effect of A beta in neuronal cultures that involves interaction with integrins, activation of FAK/src, enhanced phosphorylation of ERK1/2, and induction of the cell cycle, all leading to neuronal death.     

MEKK1 controls neurite regrowth after experimental injury by balancing ERK1/2 and JNK2 signaling

After injury, peripheral neuronal cells initiate complex signaling cascades to promote survival and regeneration. In the present study, we have identified the mitogen-activated protein kinase (MAPK) isoforms which are necessary for nerve growth factor (NGF)-induced neurite regrowth after injury of differentiated PC12 cells. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the usually pro-apoptotic c-Jun N-terminal kinase 2 (JNK2) are crucial for neurite regrowth, while p38 plays no role in this context. Surprisingly, the MEK1 inhibitors PD 98059 and U 0126 blocked both ERK1/2 and JNK phosphorylation, indicating a novel form of balancing MAPK cascade cross-talk. Results from RNAi experiments excluded direct ERK/JNK interactions. We identified the upstream kinase MEKK1 as an activator of both the ERK1/2 and JNK2 pathways, whereby the ERK1/2 kinase MEK1 and the JNK kinase MKK7 bind to MEKK1 in a competing fashion. Our findings suggest an important role of JNK2 and MAPK pathway cross-talk in neurite regeneration.     

Role of CDK5 in neuroprotection from serum deprivation by mu-opioid receptor agonist

Cyclin-dependent kinase 5 (CDK5), a unique member of the CDK family of cyclin-dependent kinases, is predominantly expressed in postmitotic neurons with proposed roles in both cell survival and programmed cell death. To understand how CDK5 participates in such disparate cellular outcomes, we investigated whether activation of CDK5 could mediate neuroprotection from serum deprivation by mu-opioid receptor agonist in differentiated SH-SY5Y cells and primary hippocampal neurons. We found that CDK5 kinase activity decreased following serum deprivation in differentiated SH-SY5Y cells coincident with increased cell loss and activation of caspases cascade activation, which was reversed by opioid antagonist. Overexpression of CDK5 in serum-free medium reversed activation of caspase cascade and augmented DAMGO neuroprotection. Blocking CDK5 activity by pharmacologic inhibitor, roscovitine or overexpression of dominant negative CDK5 augmented activation of cell death markers and diminished mu-opioid receptor agonist protection. Reduction in CDK5 activity corresponded to reduction in protein levels of CDK5 activator p35 during serum deprivation which was also reversed by mu-opioid receptor agonist. Phosphorylation of STAT3 at Serine 727 by CDK5 decreased during serum deprivation, and partly recovered by mu-opioid agonist. PI3K signaling pathway was not required for CDK5-mediated mu-opioid neuroprotection against serum deprivation. These findings indicate that neuroprotection by mu-opioid receptor agonist against serum deprivation is mediated by activation of CDK5 through up-regulation of p35 and phosphorylation of STAT3 by CDK5 may contribute to the neuroprotection.     

Methylmercury inhibits TrkA signaling through the ERK1/2 cascade after NGF stimulation of PC12 cells

Using PC12 cells as a model of neuronal differentiation, we have shown that acute exposure to methylmercury (CH3Hg) inhibits nerve growth factor (NGF)-induced activation of TrkA. In the present study, we examined the effects of CH3Hg on pathways activated by NGF. NGF-induced phosphorylation of ERK1/2 in PC12 cells was time-dependent. Concurrent exposure to CH3Hg and NGF for 2.5 min resulted in a concentration-dependent inhibition of ERK1/2 phosphorylation (EC50 = 0.018 microM). However, NGF-stimulated ERK1/2 phosphorylation was not altered after 5 min of exposure to CH3Hg. In vitro studies revealed that CH3Hg did not directly inhibit the ERK kinase MEK. As reported in other neuronal tissue, CH3Hg can inhibit PKC activity in vitro. Incubation of PC12 cell lysates with CH3Hg produced a concentration-dependent inhibition of PKC activity that was significant at 0.3-10 microM. Further studies using recombinant enzymes examined the effect of CH3Hg on PKC isoforms expressed in PC12 cells. CH3Hg inhibited PKCdelta, and zeta activity in a concentration-dependent manner at higher concentrations (3-10 microM), while a significant increase in PKCalpha activity was observed at lower concentrations (0.1 microM). However, CH3Hg had no affect on NGF-induced PKC activity in intact cells. These results show that CH3Hg inhibition of NGF-stimulated TrkA activation in PC12 cells decreases downstream signaling through the Raf/MEK/ERK cascade. In intact cells PKC does not appear to be a primary target for CH3Hg.     

MEK/ERKs signaling is essential for lithium-induced neurite outgrowth in N2a cells

Lithium, a drug used for the treatment of bipolar disorder, has been shown to affect different aspects of neuronal development such as neuritogenesis, neurogenesis and survival. The underlying mechanism responsible for lithium's influence on neuronal development, however, still remains to be elucidated. In the present study, we demonstrate that lithium increases the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt) and promotes neurite outgrowth in mouse N2a neuroblastoma cells (N2a). The inactivation of mitogen-activated protein kinase kinase (MEK)/ERKs signaling with a MEK inhibitor inhibits neurite outgrowth, but it enhances Akt activation in lithium-treated N2a cells. Furthermore, the inactivation of phosphoinositide-3-kinase (PI3K)/Akt signaling with a PI3K inhibitor increases both lithium-induced ERKs activation and lithium-induced neurite outgrowth. Taken together, our study suggests that lithium-induced neurite outgrowth in N2a cells is regulated by cross-talk between the MEK/ERKs and PI3K/Akt pathways and requires the activation of the MEK/ERKs signaling.     

Protective effects of olanzapine and haloperidol on serum withdrawal-induced apoptosis in SH-SY5Y cells

PURPOSE: Recent clinical studies have suggested that treatment with second generation antipsychotic drugs such as olanzapine may prevent progressive alterations of brain structure in patients with schizophrenia. However, the molecular mechanisms underlying these different effects remain to be determined. We investigated the mechanisms of action of olanzapine and haloperidol, on serum withdrawal apoptosis in human neuroblastoma SH-SY5Y cells. METHODS: SH-SY5Y cells were cultured with olanzapine and haloperidol in medium with or without serum. We determined the effects of the drugs on cell viability against serum withdrawal by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, to explore the drugs' actions, Western blot was performed to examine the expression of key genes involved in GSK-3beta-mediated signaling, notably GSK-3beta, beta-catenin, and Bcl-2. RESULTS: SH-SY5Y cells suffered about a 38% loss in cell number under serum-free conditions for 48 h. Olanzapine (10-200 muM) up to 100 muM significantly attenuated serum withdrawal-induced cell loss (p<0.01), and a dose of 100 muM also increased cell viability (p<0.05). In contrast, haloperidol (0.01-10 muM) did not affect cell viability but exacerbated cell death at 10 muM under serum-free conditions (p<0.01). Western blot analysis showed that olanzapine, but not haloperidol, prevented the serum withdrawal-induced decrease in levels of neuroprotective proteins such as p-GSK-3beta, beta-catenin, and Bcl-2 (p<0.01), whereas haloperidol robustly reduced the levels of these proteins at a 10 muM dose in serum-starved cells (p<0.05). Moreover, olanzapine alone significantly increased phosphorylation of GSK-3beta under normal conditions (p<0.05). CONCLUSIONS: This study showed that olanzapine may have neuroprotective effects, whereas haloperidol was apparently neurotoxic. The actions of signaling systems associated with GSK-3beta may be key targets for olanzapine and haloperidol, but their effects are distinct. These differences suggest different therapeutic effects of first and second generation antipsychotic drugs in patients with schizophrenia.     

ATP敏感性钾通道开放剂抑制PC12细胞凋亡的作用途径

Objective To investigate the effect of ATP-sensitive potassium channel (KATP) openers on ischemia-hypoxia-induced PC12 cell apoptosis and the mRNA and protein expression of Akt and Bcl-2, which should manifest the protective mechanism of KATP openers. Methods PC12 cells 3 days after passage were divided into group A (control group), B (ischemia-hypoxia group), C (KATP channel opener group) and D (KATP channel opener+blocker group). Apoptosis rate was detected using Annexin-v FITC/PI double staining flow cytometry; mRNA and protein levels of p-Akt and Bcl-2 were measured by immunofluorescent staining, Western-blotting and RT-PCR methods. Results After ischemia-hypoxia, number of apoptotic PC12 cells in group B, C and D gradually increased with time and peaked at 24h. Apoptotic cell numbers at each time point in group C were significantly different from that of group A, B and D (P<0.01). Apoptotic cell numbers in group B at different time points were not significantly different from that of group D (P>0.05). After ischemia-hypoxia, the mRNA and protein levels of p-Akt and Bcl-2 all increased and reached peak at 12h. The mRNA and protein levels of p-Akt and Bcl-2 in group C at different time points were significantly different from that of group A, B and D (P<0.05, or P<0.01). At contrast, no significant difference was seen in mRNA and protein levels of p-Akt and Bcl-2 between group B and D at different time points (P>0.05).Conclusion The protective mechanism of KATP openers on PC12 cells apoptosis after ischemia-hypoxia may be through activation of PI3K/Akt signaling pathway, which further activates the expression of downstream Bcl-2 gene.     

Tau phosphorylation during apoptosis of human SH-SY5Y neuroblastoma cells

In Alzheimer's Disease brain, the microtubule-associated protein tau is hyperphosphorylated at specific epitopes and abnormally aggregates into filamentous structures. In addition, there is significant neurodegeneration in Alzheimer's disease brain, and there is data to suggest that apoptotic-like processes may contribute to the neurodegeneration. It has been demonstrated that in PC12 cells undergoing apoptosis due trophic factor removal, tau is hyperphosphorylated prior to chromatin condensation. To establish that increased tau phosphorylation is a generalized outcome of the apoptotic process, and to examine the involvement of the protein kinase in these events, apoptosis was induced in retinoic-acid differentiated human SH-SY5Y neuroblastoma cells using the topoisomerase-1 inhibitor camptothecin. Treatment of the differentiated SH-SY5Y cells with camptothecin resulted in a time and concentration dependent activation of caspase-3 with a concomitant increase in the presence of apoptotic nuclei. Immunoblotting revealed that camptothecin treatment resulted in a significant increase in tau phosphorylation. Addition of a cyclin-dependent kinase inhibitor reduced camptothecin-induced cell death in the differentiated SH-SY5Y cells and decreased the effects of camptothecin on tau phosphorylation. In contrast, a general caspase inhibitor decreased camptothecin-induced cell death, but did not significantly decrease the increases in tau phosphorylation. These results suggest that increased tau phosphorylation is likely a generalized outcome of apoptotic processes in neuron-related cells, and that cyclin-dependent kinases probably play a role in this process.     

Acetaldehyde promotes rapamycin-dependent activation of p70(S6K) and glucose uptake despite inhibition of Akt and mTOR in dopaminergic SH-SY5Y human neuroblastoma cells

Alcohol intake is one of the important lifestyle factors for the risk of insulin resistance and type 2 diabetes. Acetaldehyde, the major ethanol metabolite which is far more reactive than ethanol, has been postulated to participate in alcohol-induced tissue injury although its direct impact on insulin signaling is unclear. This study was designed to examine the effect of acetaldehyde on glucose uptake and insulin signaling in human dopaminergic SH-SY5Y cells. Akt, mammalian target of rapamycin (mTOR), ribosomal-S6 kinase (p70(S6K)), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and insulin receptor substrate (IRS)-2 were evaluated by Western blot analysis. Glucose uptake and apoptosis were measured using [(3)H]-2-deoxyglucose uptake and caspase-3 assay, respectively. Short-term exposure (12 h) of acetaldehyde (150 muM) facilitated glucose uptake in a rapamycin-dependent manner without affecting apoptosis, IRS-2 expression and insulin-stimulated glucose uptake in SH-SY5Y cells. Acetaldehyde suppressed basal and insulin-stimulated Akt phosphorylation without affecting total Akt expression. Acetaldehyde inhibited mTOR phosphorylation without affecting total mTOR and insulin-elicited response on mTOR phosphorylation. Rapamycin, which inhibits mTOR leading to inactivation of p70(S6K), did not affect acetaldehyde-induced inhibition on phosphorylation of Akt and mTOR. Interestingly, acetaldehyde enhanced p70(S6K) activation and depressed 4E-BP1 phosphorylation, the effect of which was blunted and exaggerated, respectively, by rapamycin. Collectively, these data suggested that acetaldehyde did not adversely affect glucose uptake despite inhibition of insulin signaling cascade at the levels of Akt and mTOR, possibly due to presence of certain mechanism(s) responsible for enhanced p70(S6K) phosphorylation.     

The Predominant Protective Effect of Tianeptine Over Other Antidepressants in Models of Neuronal Apoptosis: The Effect Blocked by Inhibitors of MAPK/ERK1/2 and PI3-K/Akt Pathways

Tianeptine (Tian) possesses neuroprotective potential, however, little is known about the effect of this drug in models of neuronal apoptosis. In the present study, we aimed (1) to compare the neuroprotective capacities of some antidepressants (ADs) in the models of staurosporine (St)- and doxorubicin (Dox)-evoked cell death, activating the intracellular and the extracellular apoptotic pathway, respectively; (2) to identify the Tian-modulated steps underlying its neuroprotective action; (3) to test the effect of various ADs against Dox-evoked cell damage in glia cells. Primary neuronal and glia cell cultures and retinoic acid-differentiated human neuroblastoma SH-SY5Y (RA-SH-SY5Y) cells were co-treated with imipramine, fluoxetine, citalopram, reboxetine, mirtazapine or Tian and St or Dox. The data showed the predominant neuroprotective effect of Tian over other tested ADs against St- and Dox-induced cell damage in primary neurons and in RA-SH-SY5Y cells. This effect was shown to be caspase-3-independent but connected with attenuation of DNA fragmentation. Moreover, neuroprotection elicited by Tian was blocked by pharmacological inhibitors of MAPK/ERK1/2 and PI3-K/Akt signaling pathways as well by inhibitor of necroptosis, necrostatin-1. Interestingly, the protective effects of all tested ADs were demonstrated in primary glia cells against the Dox-evoked cell damage. The obtained data suggests the glial cells as a common target for protective action of various ADs whereas in relation to neuronal cells only Tian possesses such properties, at least against St- and Dox-induced cell damage. Moreover, this neuroprotective effect of Tian is caspase-3-independent and engages the regulation of survival pathways (MAPK/ERK1/2 and PI3-K/Akt).     

p21(ras) stimulates pathways in addition to ERK, p38, and Akt to induce elongation of neurites in PC12 cells

Initiation and elongation of neurites in PC12 cells has been shown to be stimulated by nerve growth factor (NGF). Initiation of NGF-stimulated neurites in a PC12 subclone (PC12-N09) is rapid, giving rise to short neurites that do not elongate after 1 day. To determine whether increasing activation of p21(ras) could restore neurite elongation in these cells and whether it would affect the phosphorylation of signaling proteins, the subclone PC12-N09 was transfected with constitutively active p21(ras61L) (PC12-N09ras61L) and neurite outgrowth with or without NGF was determined. Overexpression of wild-type p21(ras) (PC12-N09rasWT) did not lead to spontaneous neurite initiation but restored the ability of NGF to stimulate continuous neurite elongation. However, NGF-stimulated phosphorylation of ERK, p38, and Akt in PC12-N09rasWT cells is similar in duration to that in PC12-N09 cells, indicating that the p21(ras) signaling through ERK, p38, and Akt was not involved in the restoration of normal neurite elongation in PC12-N09 cells. These results show that p21(ras)-activated pathways other than ERK, p38, and Akt are necessary for appropriate NGF-stimulated neurite elongation in PC12 cells.     

Protein kinase B is involved in Nogo-66 inhibiting neurite outgrowth in PC12 cells

Nogo-A, a member of the reticulon family, is one of the most important myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the central nervous system. RhoA has been targeted pharmacologically to promote neurite outgrowth and functional recovery in the brain and spinal cord. However, the underlying mechanism of the inhibition of neurite outgrowth by Nogo-A has not yet been fully defined. Protein kinase B (PKB, also known as Akt) is a protein serine/threonine kinase that plays a key role in intracellular signaling and cellular homeostasis. This study reports the role of PKB signaling on Nogo-A-treated PC12 neuronal cells. An inhibitory fragment of Nogo-A (Nogo-66) activated RhoA and reduced the phosphorylation of PKB at Ser473 in a time-dependent manner. In contrast, pretreatment with Y27632, a specific inhibitor of Rho-A, resulted in an increase of the phosphorylation of PKB. Nogo-66 also inhibited the neurite outgrowth of PC12 cells, whereas pretreatment with LY294002, a specific inhibitor of PKB, ameliorated the neurite outgrowth. These data suggest that PKB is involved in the inhibition of neurite outgrowth by Nogo-A in PC12 cells.