Notch signaling inhibits PC12 cell neurite outgrowth via RBP-J-dependent and -independent mechanisms

The Notch signaling pathway has been implicated in the control of neurite extension, although the mechanisms are unknown. In this report, we studied the role of RBP-J/CBF-1 activation, the primary mediator of Notch signaling, in Notch-mediated regulation of neurite outgrowth in PC12 cells. Expression of constitutively active Notch proteins decreased neurite length and number after NGF treatment. In contrast, an inactive Notch protein had no effect on neurite extension. A dominant negative RBP-J construct prevented the reduction of neurite outgrowth by Notch. Conversely, an activated form of RBP-J decreased neurite length but failed to reduce neurite number. In summary, Notch activation inhibited PC12 cell neurite outgrowth by both RBP-J-dependent and -independent pathways.     

Neurotrophic factor-α1 modulates NGF-induced neurite outgrowth through interaction with Wnt-3a and Wnt-5a in PC12 cells and cortical neurons

Wnt-3a and Wnt-5a signaling activities inhibit and promote neurite outgrowth, respectively, to regulate dendritic and axonal genesis during neurodevelopment. NF-α1, a neurotrophic factor, has been shown to modulate dendritic remodeling and negatively regulate the canonical Wnt-3a pathway. Here, we investigated whether NF-α1 could modify nerve growth factor (NGF)-induced neurite outgrowth through interaction with Wnt-3a and Wnt-5a in PC12 cells and mouse primary cortical neurons. We showed that NGF-induced neurite outgrowth was inhibited by Wnt-3a, and this inhibition was prevented by NF-α1. Western blot analysis revealed that NF-α1 reduced the expression of both β-catenin in the canonical Wnt-3a pathway and Rho, a downstream effector of Wnt-3a's non-canonical signaling pathway. Treatment of PC12 cells with a ROCK inhibitor prevented the inhibition of NGF-induced neurite outgrowth by Wnt-3a, suggesting that NF-α1 promotes neurite outgrowth in the presence of Wnt-3a by down-regulating its canonical and non-canonical activities. Interestingly, treatment of PC12 cells with Wnt-5a, which formed a complex with NF-α1, induced neurite outgrowth that was enhanced by treatment with the combination of Wnt-5a, NGF, and NF-α1. These effects of NF-α1 on Wnt 3a's and Wnt 5a's regulation of neurite outgrowth in PC12 cells were also demonstrated in primary cultures of mouse cortical neurons. In addition, we showed in PC12 cells that NF-α1 acts by upregulating adenomatous polyposis coli (APC) accumulation at neurite tips, thereby providing positive and negative Wnt-3a/Wnt-5a mediated cues to modulate neurite outgrowth, a process important during neurodevelopment.     

Neuritin (cpg15) enhances the differentiating effect of NGF on neuronal PC12 cells

Neuritin is a small, highly conserved GPI-anchored protein involved in neurite outgrowth. We have analyzed the involvement of neuritin in NGF-induced differentiation of PC12 cells by investigating the time-course of neuritin expression, the effects of its overexpression or silencing, and the possible mechanisms of its regulation and action. Real-time PCR analysis has shown that neuritin gene is upregulated by NGF in PC12 cells hours before neurite outgrowth becomes appreciable. PC12 cells transfected with a plasmid expressing neuritin display a significant increase in the response to NGF: 1) in the levels of SMI312 positive phosphorylated neurofilament proteins (markers for axonal processes) and tyrosine hydroxylase; 2) in the percentage of cells bearing neurites; as well as 3) in the average length of neurites when compared to control cells. On the contrary, neuritin silencing significantly reduces neurite outgrowth. These data suggest that neuritin is a modulator of NGF-induced neurite extension in PC12 cells. We also showed that neuritin potentiated the NGF-induced differentiation of PC12 cells without affecting TrkA or EGF receptor mRNAs expression. Moreover, the S-methylisothiourea (MIU), a potent inhibitor of inducible nitric oxide synthases, partially counteracts the NGF-mediated neuritin induction. These data suggest that NGF regulates neuritin expression in PC12 cells via the signaling pathway triggered by NO. This study reports the first evidence that neuritin plays a role in modulating neurite outgrowth during the progression of NGF-induced differentiation of PC12 cells. PC12 cells could be considered a valuable model to unravel the mechanism of action of neuritin on neurite outgrowth. (c) 2007 Wiley-Liss, Inc.     

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.     

Cocaine inhibition of neuronal differentiation in NGF-induced PC12 cells is independent of ras signaling

In utero exposure to cocaine may result in altered neuronal development. Our previous studies demonstrated cocaine inhibits neurite outgrowth in NGF-induced PC12 cells through dopamine, by activation of D₁ receptors. This study examined where cocaine interferes in the NGF signaling cascade. GSras1 cells that inducibly express activated forms of Ras upon treatment with dexamethasone were used. Morphological differentiation was quantified by counting cells bearing neurite-like processes after 72 h exposure to either dexamethasone or NGF alone, or with cocaine, dopamine or SKF-38393. Cocaine, dopamine, and the D₁ agonist inhibited neurite-like process outgrowth in both dexamethasone and NGF-induced GSras1 cells. GAP-43 expression, used as a measure for biochemical differentiation was severely diminished in NGF and dexamethasone-induced GSras1 cells treated with cocaine. These results suggest that cocaine, dopamine and activation of D₁ receptors affect the NGF signaling downstream, independent of ras expression, leading to altered neuronal differentiation.     

Manganese induces neurite outgrowth in PC12 cells via upregulation of alpha(v) integrins

Previous studies have demonstrated that the divalent cation manganese (Mn) causes PC12 cells to form neurites in the absence of NGF. Since divalent cations modulate the binding affinity and specificity of integrins, and integrin function affects neurite outgrowth, we tested the hypothesis that Mn induces neurite outgrowth through an integrin-dependent signaling pathway. Our studies support this hypothesis. Function-blocking antisera specific for beta(1) integrins block the neurite-promoting activity of Mn by 90-95%. Bioassays and biochemical studies with antisera specific for the alpha(v), alpha(5), or alpha(8) integrin subunit suggest that the alpha(v)beta(1) heterodimer is one of the principal beta(1) integrins mediating the response of PC12 cells to Mn. This is corroborated by studies in which Mn failed to induce neurite outgrowth in a clone of PC12 cells that does not express alpha(v) at levels detectable by immunoprecipitation or immunocytochemistry. SDS-PAGE analysis of biotinylated surface proteins immunoprecipitated from Mn-responsive PC12 cells, as well as confocal laser microscopy of PC12 immunostained for surface alpha(v) indicate that Mn increases the surface expression of alpha(v) integrins. This increase appears to be due in part to synthesis of alpha(v) since specific inhibitors of RNA and protein synthesis block the neurite-promoting activity of Mn. These data indicate that Mn induces neurite outgrowth in PC12 cells by upregulating alpha(v) integrins, suggesting that Mn potentially represents an additional mechanism for regulating the rate and direction of neurite outgrowth during development and regeneration.     

Sargaquinoic acid promotes neurite outgrowth via protein kinase A and MAP kinases-mediated signaling pathways in PC12D cells

We previously isolated a nerve growth factor (NGF)-dependent neurite outgrowth promoting substance MC14 (sargaquinoic acid) from a marine brown alga, Sargassum macrocarpum. In the present study, the NGF-potentiating activity of MC14 to neural differentiation of PC12D cells was investigated in detail. The treatment of cells with 3 microg/ml MC14 in the presence of 1.25-100 ng/ml NGF markedly enhanced the proportion of neurite-bearing cells compared with the NGF-only controls. In addition, MC14 significantly elevated the NGF-induced specific acetylcholinesterase (AchE) activity in PC12D cells, suggesting that MC14 could morphologically and biochemically promote the differentiation of PC12D cells. The mechanism of action of MC14 was further investigated by pharmacological inhibition of several intracellular signaling molecules. Results indicated that the neurite outgrowth promoting activity of MC14 was almost completely blocked by 10 microM PD98059, suggesting that a TrkA-dependent MAP kinases-mediated signaling pathway may play a crucial role in modulating the effect of MC14. Besides, the MC14-enhanced neurite outgrowth was substantially suppressed by the pretreatment with 10 ng/ml protein kinase A (PKA) inhibitor, demonstrating that the adenylate cyclase-PKA signaling cascade was also involved in the action of MC14. In contrast, a PKC inhibitor chelerythrine chloride did not inhibit the neurite outgrowth promoting activity of MC14. Altogether, these results demonstrate that MC14 enhances the neurite outgrowth by cooperating at least two separated signaling pathways, a TrkA-MAP kinases pathway and an adenylate cyclase-PKA pathway, in PC12D cells.     

Cocaine inhibits NGF-induced PC12 cells differentiation through D(1)-type dopamine receptors

In utero cocaine exposure can adversely affect CNS development. Previous studies showed that cocaine inhibits neuronal differentiation in a dose-dependent fashion in nerve growth factor (NGF)-stimulated PC12 cells. Cocaine binds with high affinity to several neurotransmitter transporters, resulting in elevated neurotransmitter levels in nerve endings. To determine if cocaine inhibits neurite outgrowth through the effects of these neurotransmitters, we applied dopamine, norepinephrine, serotonin, and acetylcholine to NGF-induced PC12 cells. Dopamine was the only neurotransmitter to inhibit neurite outgrowth significantly in a dose-dependent pattern without affecting cell viability. Norepinephrine and acetylcholine did not affect neurite outgrowth, while serotonin enhanced it. Furthermore, GBR 12909, a potent dopamine transporter (DAT) inhibitor, yielded similar effects. We then showed PC12 cells express D(1) and D(2) receptors and DAT proteins. Dopamine uptake measured over time was significantly blocked by cocaine and GBR 12909 which may result in elevated extracellular dopamine. The role of dopamine receptors in PC12 differentiation was further examined by using D(1) and D(2) specific receptor agonists. Only the D(1) agonist, SKF-38393, had a significant dose-dependent inhibitory effect. In addition, a D(1) antagonist produced significant recovery of neurite outgrowth in cocaine-treated cells. These findings suggest that cocaine inhibitory effects on neuronal differentiation are mediated through its binding to the dopamine transporter, resulting in increased dopamine level in the synapses. Subsequently, up regulation of D(1) receptors alters NGF signaling pathways.     

Nardosinone, the first enhancer of neurite outgrowth-promoting activity of staurosporine and dibutyryl cyclic AMP in PC12D cells

Nardosinone was isolated as an enhancer of nerve growth factor (NGF) from Nardostachys chinensis [Neurosci. Lett. 273 (1999) 53]. Nardosinone (0.1-100 microM) enhanced dibutyryl cyclic AMP (dbcAMP, 0.3 mM)- and staurosporine (10 nM)-induced neurite outgrowth from PC12D cells in a concentration-dependent manner. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, partially blocked enhancements of dbcAMP (0.3 mM)- or staurosporine (10 nM)-induced neurite outgrowth by nardosinone. Nardosinone alone had no effect on the phosphorylation of MAP kinase. The dbcAMP-induced increase in phosphorylation of MAP kinase was not affected by nardosinone. Staurosporine almost unaffected the phosphorylation of MAP kinase, and nardosinone potentiated the staurosporine-induced neurite outgrowth without stimulation of the phosphorylation of MAP kinase. Since it is known that MAP kinase signaling is required for neurite outgrowth in PC12D cells, these results suggest that nardosinone enhances staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying both the MAP kinase-dependent and -independent signaling pathways of dbcAMP and staurosporine. It is also suggested that nardosinone enhances a downstream step of MAP kinase in the MAP kinase-dependent signaling pathway. Nardosinone is the first enhancer of the neuritogenic action of dbcAMP and staurosporine and may become a useful pharmacological tool for studying the mechanism of action of not only NGF but also both the neuritogenic substances.     

Role of protein tyrosine phosphorylation in the NGF response

The role of protein tyrosine phosphorylation in the response of PC12 cells to NGF was investigated by using a variety of agents which affect NGF-induced neurite outgrowth. K-252a, a kinase inhibitor, was previously found to selectively inhibit many of the actions of NGF on PC12 cells. In the present study, it was shown to inhibit NGF-induced protein tyrosine phosphorylation. However, sphingosine, an inhibitor of protein kinase C and NGF-induced differentiation of PC 12 cells, did not alter the phosphorylation of proteins on tyrosine stimulated by NGF. Disruption of either actin microfilaments or microtubules also had no effect on NGF-induced protein tyrosine phosphorylation in PC12 cells. The effect of vanadate, an inhibitor of phosphotyrosyl phosphatases, on the differentiation of PC12 cells was also examined. Vanadate did not promote neurite outgrowth but did stimulate protein tyrosine phosphorylation. Taken together, these results suggest that protein tyrosine phosphorylation is one of the first events in the NGF pathway in PC12 cells but alone is not sufficient to induce morphological differentiation. Finally, the distribution of phosphotyrosine-containing proteins in untreated and NGF-treated cells was examined by immunofluorescence microscopy. The distribution of these proteins was altered by treatment of the cells with NGF and appeared to correlate with the distribution of actin filaments, particularly in growth cones.     

Depletion of a fatty acid-binding protein impairs neurite outgrowth in PC12 cells

Epithelial fatty acid-binding protein (E-FABP) is up-regulated in rat dorsal root ganglia after sciatic nerve crush and in differentiating neurons during development. The present study investigates the role of E-FABP during nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells. Undifferentiated PC12 cells express low levels of E-FABP, while NGF triggers a 6- and 8-fold induction of E-FABP mRNA and protein, respectively. Up-regulation of E-FABP mRNA occurs as early as 24 h after NGF treatment and remains highly expressed over the course of several days, corresponding to NGF-mediated neurite outgrowth. Withdrawal of NGF leads to down-regulation of E-FABP mRNA and retraction of neurites. Immunofluorescence microscopy reveals E-FABP immunoreactivity in the perinuclear cytoplasm, neurites and growth cones of NGF-differentiated cells. To examine the role of E-FABP during neurite outgrowth, PC12 cells were transfected with a constitutive antisense E-FABP vector to create the E-FABP-deficient line PC12-AS. By morphometric analysis, PC12-AS cells treated for 2, 4, and 7 days with NGF exhibited significantly decreased neurite expression relative to control (mock-transfected) cells. Taken together, these data indicate that E-FABP is important in normal NGF-mediated neurite outgrowth in PC12 cells, a finding that is consistent with a potential role in axonal development and regeneration.     

PKC activators (phorbol ester or bryostatin) stimulate outgrowth of NGF-dependent neurites in a subline of PC12 cells

Nerve growth factor (NGF) stimulation of PC12 cells activates signaling pathways leading to new protein expression and growth of neurites. In wild type PC12 cells, incubation with phorbol ester (PMA) will activate protein kinase C (PKC) leading to the expression of many proteins necessary for neurite outgrowth, but this activation of PKC alone will not stimulate growth of long neurites. Here, we show in the subline of PC12-N09, which lacks NGF-stimulated growth of long neurites, that a brief incubation with PKC activators, PMA or bryostatin 1 (bryostatin), before NGF incubation, stimulates the growth of long neurites. However, incubation in the reverse order is ineffective. A short incubation with PMA or bryostatin followed by NGF induced tyrosine phosphorylation of MAP kinase (MAPK), which is of the same duration as that induced by NGF alone. Thus, PMA preincubation did not increase the length NGF activation of MAPK. Twenty-four hr after incubation with PMA or bryostatin, PKC isoforms were downregulated but PKC isoforms δ-, and ϵ- were still present. In these cells chronically treated with either PMA or bryostatin to downregulate PKC, NGF incubation preceded by PMA preincubation still led to long neurite outgrowth. These results suggest that a PMA or bryostatin incubation followed by NGF activates PKC isoforms δ-, and ϵ-leading to outgrowth of long neurites, and that the PMA signaling is independent of the MAPK pathway. J. Neurosci. Res. 53:214–222, 1998. © 1998 Wiley-Liss, Inc.     

Nerve growth factor stimulates GAP-43 expression in PC12 cell clones independently of neurite outgrowth

Expression of the growth associated protein GAP-43 (B-50, F1, neuromodulin) increases with the onset of neuronal development as seen by the growth of axons. To investigate the relationship of the signaling events leading to GAP-43 expression and neurite outgrowth, we examined PC12 clones with different phenotypes. Three clones, PC12-NO9, PC12-N15, and PC12-N21, responded to NGF with increased expression of GAP-43, but only two clones, PC12-N15 and PC12-N21, responded with growth of neurites. Similar increases in expression of GAP-43 were obtained when these clones were exposed to the phorbol ester PMA. Thus, NGF and PMA induced GAP-43 expression in PC12-NO9 cells in the absence of neurite outgrowth. In contrast, all three clones, were able to respond to forskolin (FOR) by initiation of long neurites which had synaptophysin in the growth cones, but showed only low levels of GAP-43. Combined stimualtion of PC12-NO9 cells with FOR and PMA both initiated neurites and increased expression of GAP-43 as seen in normal PC12 clones were also able to respond to FOR with increased neurite outgrowth in the presence of low levels of GAP-43. The dissociation of GAP-43 expression and growth of neurites observed in PC12-NO9 cells suggests that signaling mechanisms can independently regulate GAP-43 expression and neurite outgrowth during neuronal differentiation. © 1993 Wiley-Liss, Inc.     

Cocaine inhibits NGF-induced PC12 cells differentiation through D1-type dopamine receptors

In utero cocaine exposure can adversely affect CNS development. Previous studies showed that cocaine inhibits neuronal differentiation in a dose-dependent fashion in nerve growth factor (NGF)-stimulated PC12 cells. Cocaine binds with high affinity to several neurotransmitter transporters, resulting in elevated neurotransmitter levels in nerve endings. To determine if cocaine inhibits neurite outgrowth through the effects of these neurotransmitters, we applied dopamine, norepinephrine, serotonin, and acetylcholine to NGF-induced PC12 cells. Dopamine was the only neurotransmitter to inhibit neurite outgrowth significantly in a dose-dependent pattern without affecting cell viability. Norepinephrine and acetylcholine did not affect neurite outgrowth, while serotonin enhanced it. Furthermore, GBR 12909, a potent dopamine transporter (DAT) inhibitor, yielded similar effects. We then showed PC12 cells express D₁ and D₂ receptors and DAT proteins. Dopamine uptake measured over time was significantly blocked by cocaine and GBR 12909 which may result in elevated extracellular dopamine. The role of dopamine receptors in PC12 differentiation was further examined by using D₁ and D₂ specific receptor agonists. Only the D₁ agonist, SKF-38393, had a significant dose-dependent inhibitory effect. In addition, a D₁ antagonist produced significant recovery of neurite outgrowth in cocaine-treated cells. These findings suggest that cocaine inhibitory effects on neuronal differentiation are mediated through its binding to the dopamine transporter, resulting in increased dopamine level in the synapses. Subsequently, up regulation of D₁ receptors alters NGF signaling pathways.     

Purine analogs inhibit nerve growth factor-promoted neurite outgrowth by sympathetic and sensory neurons

Past studies have shown that purine analogs block certain, but not all, responses of cultured rat PC12 pheochromocytoma cells to nerve growth factor (NGF). In the present work, newborn rat sympathetic and sensory neurons were exposed to NGF in the presence or absence of the purine analogs 6-thioguanine and 2-aminopurine. These compounds reversibly suppressed NGF-dependent neurite outgrowth by the neurons and did so at concentrations comparable to those effective on PC12 cells. In contrast to their effects on neurites, neither compound significantly blocked NGF-promoted neuronal survival. Similar effects were seen with cultures of chick embryo sympathetic ganglia. These findings show that purine analog effects on NGF responses can be extended to mammalian and avian neurons. Moreover, the differential effects of the analogs on neurite outgrowth and survival indicate that these 2 actions of NGF can be dissected from one another and may represent different mechanistic pathways.     

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.