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.     

GTP and guanosine synergistically enhance NGF-induced neurite outgrowth from PC12 cells

Six per cent of rat pheochromocytoma (PC12) cells extended neurites (processes greater than one cell diameter in length) in the presence of 300 μM extracellular GTP or 300 μM guanosine for 48 hr, compared to only 2.5% of cells in control cultures. In the presence of 40 ng/ml of 2.5S NGF, about 20–35% of PC12 cells had neurites after 48 hr, and the addition of 300 μM guanosine or GTP together with NGF synergistically increased the proportion of cells with neurites to 40–65%. GTP and guanosine also increased the average number of branches per neurite, from 0.6 in NGF-treated cultures to 1.2 (guanosine) or 1.5 (GTP). Neurites formed after exposure to NGF alone had axonal characteristics as determined by immunocytochemistry with antibody, SMI-31, against axonal-specific polyphosphorylated neurofilament epitopes. Neurites generated with the addition of both guanosine or GTP had the same characteristics.GTP probably did not exert its effects via the P_2X or P_2Y purinoceptors because the adenine nucleotides ATP, ATPγS, ADPβS, and ADP, which are all agonists of these receptors, inhibited rather than enhanced, NGF-induced neurite outgrowth. UTP also enhanced the proportion of cells with neurites, although not to the same degree as did GTP. This may indicate activity through a P_2U-like nucleotide receptor. However, the response profile obtained, GTP > UTP ? ATP, does not fit the profile of any known P_2Y, P_2X or P_2U receptor. The poorly hydrolyzable GTP analogues, GTPγS and GDPβs were also unable to enhance the proportion of cells with neurites. This implied that GTP may produce its effects through a GTP-specific ectoenzyme or kinase. This idea was supported by results showing that another poorly hydrolyzable analogue, GMP-PCP, competitively inhibited the effects of GTP on neurite outgrowth. GTP did not exert its effects after hydrolysis to guanosine since the metabolic intermediates GDP and GMP were also ineffective in enhancing the proportion of cells with neurites. Moreover, the effects of GTP and guanosine were mutually additive, implying that these two purines utilized different signal transduction mechanisms.The effects of guanosine were not affected by the nucleoside uptake inhibitors nitrobenzylthioinosine (NBTI) and dipyridamole, indicating that a transport mechanism was not involved. Guanosine also did not activate the purinergic P₁ receptors, because the A2 receptor antagonists, 1, 3-dipropyl-7-methylxanthine (DPMX) or CGS15943, and the At receptor antagonist, 1, 3-dipropyl-8-(2-amino-4-chloro)xanthine (PACPX) did not inhibit its reaction. Therefore guanosine enhanced neurite outgrowth by a signal transduction mechanism that does not include the activation of the Pt purinoceptors.The enhancement of the neuritogenic effects of NGF by GTP and guanosine may have physiological implications in sprouting and functional recovery after neuronal injury in the CNS, due to the high levels of nucleosides and nucleotides released from dead or injured cells.     

Lead enhances NGF-induced neurite outgrowth in PC12 cells by potentiating ERK/MAPK activation

Although the neurotoxicity of lead exposure is well documented, the cellular and molecular mechanisms underlying lead neurotoxicity have not been well defined. We have investigated the effect of lead on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells and the role in this process of extracellular signal regulated protein kinase (ERK), a key component of NGF-induced differentiation. We found that exposure of cells to lead acetate (0.1-100 microM) resulted in enhanced NGF-induced neurite outgrowth. Lead exposure also promoted formation of multiple neurites per cell in NGF-treated cells. However, lead alone did not cause neurite outgrowth. Lead also enhanced NGF-induced ERK phosphorylation and activation, but lead alone did not stimulate ERK. The MAP kinase kinase (MEK) inhibitor, PD98059, significantly decreased the effect of lead on NGF-induced neurite outgrowth and ERK activation. These findings indicate that exposure of cells to low, toxic levels of lead amplifies growth factor-induced neurite outgrowth by means of an ERK-dependent signaling pathway.     

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.     

Inhibition of glucose transport in PC12 cells by the atypical antipsychotic drugs risperidone and clozapine, and structural analogs of clozapine

Treatment of schizophrenics with some antipsychotic drugs has been associated with an increased incidence of hyperglycemia and new-onset type 2 diabetes. Some of these drugs also inhibit glucose transport in rat pheochromocytoma (PC12) cells. The current study was designed to examine the effects of the atypical antipsychotic drugs--risperidone, clozapine and analogs of clozapine on glucose uptake in PC12 cells. Glucose transport was measured in cells incubated with vehicle or drug over a range of concentrations (0.2-100 microM). Uptake of 3H-2-deoxyglucose was measured over 5 min and the data were normalized on the basis of total cell protein. Risperidone and clozapine inhibited glucose transport in a dose-dependent fashion with IC(50)'s estimated to be 35 and 20 microM, respectively. The clozapine metabolite, desmethylclozapine, was considerably more potent than the parent drug, whereas clozapine N-oxide was essentially inactive. The structural analogs of clozapine, loxapine and amoxapine, both inhibited glucose transport with amoxapine being the least potent. The ability of the drugs to inhibit glucose transport was significantly decreased by including 2-deoxyglucose (5 mM) in the uptake medium. Schild analysis of the glucose sensitivity of clozapine, loxapine and risperidone indicated that 2-deoxyglucose non-competitively antagonized the inhibitory effects of these drugs. Moreover, clozapine and fluphenazine inhibited glucose transport in the rat muscle cell line, L6. These studies suggest that the drugs may block glucose accumulation directly at the level of the glucose transporter (GLUT) protein in cells derived from both peripheral and brain tissue. Furthermore, this work may provide clues about how the antipsychotic drugs produce hyperglycemia in vivo.     

Methylmercury decreases NGF-induced TrkA autophosphorylation and neurite outgrowth in PC12 cells

Neurotrophin signaling through Trk receptors is important for differentiation and survival in the developing nervous system. The present study examined the effects of CH(3)Hg on (125)I-nerve growth factor (NGF) binding to the TrkA receptor, NGF-induced activation of the TrkA receptor, and neurite outgrowth in an in vitro model of differentiation using PC12 cells. Whole-cell binding assays using (125)I-NGF revealed a single binding site with a K(d) of approximately 1 nM. Methylmercury (CH(3)Hg) at 30 nM (EC(50) for neurite outgrowth inhibition) did not affect NGF binding to TrkA. TrkA autophosphorylation was measured by immunoblotting with a phospho-specific antibody. TrkA autophosphorylation peaked between 2.5 and 5 min of exposure and then decreased but was still detectable at 60 min. Concurrent exposure to CH(3)Hg and NGF for 2.5 min resulted in a concentration-dependent decrease in TrkA autophosphorylation, which was significant at 100 nM CH(3)Hg. To determine whether the observed inhibition of TrkA was sufficient to alter cell differentiation, NGF-stimulated neurite outgrowth was examined in PC12 cells after exposure to 30 nM CH(3)Hg, a concentration that inhibited TrkA autophosphorylation by approximately 50%. For comparison, a separate group of PC12 cells were exposed to a concentration of the selective Trk inhibitor K252a (30 nM), which had been shown to produce significant inhibition of TrkA autophosphorylation. Twenty-four hour exposure to either CH(3)Hg or K252a reduced neurite outgrowth to a similar degree. Our results suggest that CH(3)Hg may inhibit differentiation of PC12 cells by interfering with NGF-stimulated TrkA autophosphorylation.     

Activation of ERK1 and ERK2 is required for manganese-induced neurite outgrowth in rat pheochromocytoma (PC12) cells.

Mn(2+) treatment has been shown to promote neurite outgrowth in rat pheochromocytoma (PC12) cells in a time- and dose-dependent manner. This process is mediated through the interactions of extracellular matrix (ECM) proteins and integrin receptors. Studies were performed to determine whether the phosphorylation of the MAP kinases, ERK1 and 2, is required for Mn(2+)-induced neurite outgrowth. A time- and dose-dependent increase in phosphorylation of both ERK1 and 2 was observed upon treatment of PC12 cells with Mn(2+). Phosphorylation of the ERKs occurred as early as 2 hr after initiating treatment, with a maximum increase occurring at approximately 24 hr. Inhibition of MEK with the specific inhibitor, PD98059, blocked the phosphorylation of ERK1 and 2 and increased Mn(2+) toxicity. When cells were grown in serum-free defined medium, Mn(2+)-induced phosphorylation of ERK1 and ERK2 occurred in cells grown on surfaces treated with growth serum or fibronectin but not on surfaces treated with poly-L-lysine. In addition, the pentapeptide GRGDS, which blocks RGD-mediated interactions, inhibited Mn(2+)-induced phosphorylation of ERK1 and 2. The Mn(2+)-induced increase in phosphorylated ERK1 and 2 was not seen in a PC12 cell line that does not respond to Mn(2+). These data support the hypothesis that integrin-mediated activation of the MAPK signal transduction pathway leading to the activation of ERK1 and 2 is required for Mn(2+)-induced PC12 differentiation and neurite outgrowth.     

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.     

Effects of tunicamycin on NGF binding and neurite outgrowth in PC12 cells

The effects of inhibition of glycosylation on nerve growth factor (NGF) binding and neurite outgrowth response of PC12 cells have been examined. Exposure to tunicamycin (1-10 micrograms/ml) for 24-36 hr eliminates the rapidly dissociating component of NGF binding and decreases the proportion of PC12 cells capable of elaborating neurites in a dose-dependent manner. These decreased cellular responses are probably due to an underglycosylation of the NGF receptor, since the effects of tunicamycin are correlated with a decrease in 3H-fucose incorporation rather than a general decline in cellular metabolism as measured by viability and protein synthesis. These results suggest that carbohydrate side chains are important for the function and/or orientation of the NGF receptor in PC12 cells and that the rapidly dissociating component of NGF binding may be associated with a minimum concentration of functional receptors per cell required for the full biologic response.     

Manganese induces neurite outgrowth in PC12 cells via upregulation of αv 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 β₁ integrins block the neurite-promoting activity of Mn by 90–95%. Bioassays and biochemical studies with antisera specific for the α_v, α₅, or α₈ integrin subunit suggest that the α_vβ₁ heterodimer is one of the principal β₁ 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 α_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 α_v indicate that Mn increases the surface expression of α_v integrins. This increase appears to be due in part to synthesis of α_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 α_v integrins, suggesting that Mn potentially represents an additional mechanism for regulating the rate and direction of neurite outgrowth during development and regeneration.     

Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth

This study demonstrates that the Rho-kinase (ROCK) inhibitor, Y-27632, potentiates not only the effect of cobalt chloride (CoCl(2)) but also that of deferoxamine, another HIF-1 inducer, on mesenchymal stem cell (MSC) neuronal differentiation. HIF-1 is essential for CoCl(2)+/-Y-27632-induced MSC neuronal differentiation, since agents inhibiting HIF-1 abolish the changes of morphology and cell cycle arrest-related gene or protein expressions (p21, cyclin D1) and the increase of neuronal marker expressions (Tuj1, NSE). Y-27632 potentiates the CoCl(2)-induced decrease of cyclin D1 and nestin expressions, the increase of HIF-1 activation and EPO expression, and decreases pVHL expression. Interestingly, CoCl(2) decreases RhoA expression, an effect potentiated by Y-27632, revealing crosstalk between HIF-1 and RhoA/ROCK pathways. Moreover, we demonstrate a synergistic effect of CoCl(2) and Y-27632 on neurosphere differentiation into neurons and PC12 neurite outgrowth underlining that a co-treatment targeting both HIF-1 and ROCK pathways might be relevant to differentiate stem cells into neurons.     

NGF induces neurite outgrowth via a decrease in phosphorylation of myosin light chain in PC12 cells.

The relationship between phosphorylation of myosin light chain (MLC) and neurite outgrowth induced by nerve growth factor (NGF) was studied in PC12 cells. Inhibitors of Rho kinase, HA-1077 or Y-27632 also induced neurite outgrowth. As already reported botulinum exoenzyme C3 which inactivates Rho protein also induced neurite outgrowth. Calyeulin A, an inhibitor of phosphatase counteracted both NGF- and C3- induced neurite outgrowth. Treatments of both NGF and C3 resulted in significant and transient decrease in phosphorylated MLC. These results suggest that NGF induces neurite outgrowth of PC12 by a transient decrease in phosphorylated MLC which is brought about by activation of MLC phosphatase via inhibition of Rho-Rho kinase pathway.     

Enhancement of NGF- and cholera toxin-induced neurite outgrowth by butyrate in PC12 cells

It has been shown that sodium butyrate (NaBu) does not elicit neurite outgrowth of PC12, one of the most widely used cell lines as a model of neuronal differentiation. In this study, the effects of NaBu on nerve growth factor (NGF)- and cholera toxin-induced neurite outgrowth in PC12 cells were examined. NaBu dose-dependently enhanced neurite formation induced by both agents. The maximum responses obtained at 0.5 mM NaBu were nearly twice those of the inducers alone. Propionate and valerate were also effective, but acetate and caproate were ineffective. Among the butyrate analogs with a moiety of three to five carbon atoms tested, isobutyrate, isovalerate, vinylacetate and 3-chloropropionate enhanced neurite outgrowth promoted by both inducers. However, neither alpha-, beta-, and gamma-aminobutyrates nor alpha-, beta-, and gamma-hydroxybutyrates were effective. All of the effective short-chain fatty acids and their analogs increased the level of histone acetylation, while ineffective ones did not. Furthermore, Helminthosporium carbonum toxin (HC toxin), a structurally dissimilar inhibitor of histone deacetylase, mimicked the effect of butyrate. These results suggest that NaBu enhances neurite outgrowth induced by NGF and cholera toxin in PC12 cells through a mechanism involving an increase in the level of histone acetylation.     

Quantitation of the growth-associated protein B-50/GAP-43 and neurite outgrowth in PC12 cells

A combined assay to measure neurite outgrowth and B-50/GAP-43 levels in PC12 cells is reported. During NGF-induced neuritogenesis, B-50/GAP-43 expression was monitored by enzyme-linked immunosorbent assay (ELISA). Neurite outgrowth was quantified at the same time by the use of video image analysis. Sensitivity and reliability of the methods are shown with a dose-response and time curve of beta-NGF-induced neuritogenesis. A linear increase in total length of neurites was induced by concentrations of beta-NGF greater than or equal to 5 ng/ml and was accompanied by a linear increase in the amount of B-50/GAP-43. The combined methods presented here can conveniently and reliably establish subtle changes in neurite outgrowth and intracellular protein contents.     

Heat shock induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway

We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.     

Repeated,intermittent treatment with amphetamine induces neurite outgrowth in rat pheochromocytoma cells (PC12 cells)

Repeated, intermittent treatment with amphetamine (AMPH) leads to long-term neurobiological adaptations in rat brain including an increased number and branching of dendritic spines. This effect depends upon several different cell types in the intact brain. Here we demonstrate that repeated, intermittent AMPH treatment induces neurite outgrowth in cultured PC12 cells without the requirement for integrated synaptic pathways. PC12 cells were treated with 1 micro M AMPH for 5 min a day, for 5 days. After 10 days of withdrawal, there was an increase in the percentage of cells with neurites ( approximately 30%) and the length of neurites as well as an increase in the level of GAP-43 and neurofilament-M. Neurite outgrowth was enhanced as withdrawal time was increased. Neurite outgrowth was much greater following repeated, intermittent treatment with AMPH compared to continuous or single treatment with AMPH. Pretreatment with cocaine, a monoamine transporter blocker, inhibited the AMPH-mediated increase in neurite outgrowth. Neither NGF antibody nor DA receptor antagonists blocked AMPH-induced neurite outgrowth, demonstrating that AMPH-induced neurite outgrowth is not dependent on endogenous NGF release or DA receptors. Thus we have demonstrated that repeated, intermittent treatment with AMPH has a neurotrophic effect in PC12 cells. The effect requires the action of AMPH on the norepinephrine transporter, and shares characteristics in its development with other forms of sensitization but does not require an intact neuroanatomy.     

Insulin-like growth factor-I receptor and estrogen receptor crosstalk mediates hormone-induced neurite outgrowth in PC12 cells

Estradiol (E(2)) and insulin-like growth factor-I (IGF-I) can act independently or in concert to promote neurite outgrowth in vivo and in cultured neurons. This study examined the role of crosstalk between estrogen receptor (ER)alpha and the IGF-I receptor as a critical mediator of hormone- and growth factor-dependent neurite outgrowth in a homogenous cell system. We used control PC12 cells and PC12 cells stably transfected with ER alpha, both of which express IGF-I receptor. Cells were treated for 1 week with vehicle, 1 nM E(2) or 100 ng/ml IGF-I alone or with E(2) or IGF-I in the presence of either the IGF-I receptor antagonist JB1 or the ER antagonist ICI 182,780. IGF-I significantly increased neurite outgrowth, as measured by the percentage of process-bearing cells, and absolute neurite length per cell in both control and ER alpha-transfected PC12 cells. In contrast, E(2) increased process formation and extension only in PC12 cells that were stably transfected with ER alpha. ICI 182,780 and JB1 blocked the IGF-I-induced increases in neurite length in both cell types. The efficacy of ICI 182,780 in control PC12 cells may have been due to the upregulation of ER alpha in these cells by the 7-day treatment with IGF-I. The ER and IGF-I receptor antagonists similarly blocked the E(2)-induced increase in neurite lengths in ER alpha-transfected cells. Immunofluorescent analysis of the cellular distribution of an axonal marker, phospho-neurofilament, verified that the processes extended by PC12 cells were neurites. These data suggest that receptor crosstalk between IGF-I receptors and ER alpha has an important role in neurite formation and extension even in a single-cell system.