The relationship between neurite extension and phospholipid metabolism in PC12 cells

NGF stimulates a variety of changes in PC12 cells. These include neurite extension and an increase in the metabolism of phosphatidylinositol and phosphatidic acid (the so-called 'PI effect'). Cyclic AMP has been postulated to mediate many of the effects of NGF on PC12 cells, and we recently found that a variety of agents which stimulate neurite extension also increase intracellular cyclic AMP. These agents included phospholipase A2 and phospholipase C. In an effort to clarify the relationship of the 'PI effect' to other NGF-induced events we investigated the effects of these agents and of cyclic AMP on phospholipid metabolism. All agents which elevate cyclic AMP stimulate phosphatidylinositol metabolism. In addition, elevated exogenous KCl, which does not stimulate cyclic AMP production, promotes neurite extension and also causes the 'PI effect'. These data suggest that an increase in the turnover of phosphatidylinositol may be a requisite event in neurite outgrowth.     

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.     

Induction of Neuronal Morphology in Adrenal Chromaffin Cells Cocultured with Denervated Schwann Cells

We have studied the interactions of adrenal chromaffin and Schwann cells in a coculture system to observe whether denervated Schwann cells induce and support chromaffin cell differentiation in a manner analogous to nerve growth factor (NGF). Schwann cells induce both the accumulation of intense clumps of cocultured chromaffin cells on their surfaces and intense neurite outgrowth. This interaction is not blocked by antibodies to NGF or laminin. Conditioned medium from Schwann cell cultures fosters neurite outgrowth in chromaffin cells in a fashion qualitatively similar to NGF. Our data indicate that denervated Schwann cells exert a profound aggregating and differentiating effect upon chromaffin cells, inducing the expression of a neuronal phenotype via a predominantly NGF-independent mechanism.     

Differential effects of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells

PC12 cells are a pheochromocytoma cell line that can be made to differentiate into sympatheticlike neurons by nerve growth factor (NGF). An essential component of the NGF-induced differentiation is the development of action potentials and sodium channels. Using whole-cell clamp we have confirmed that NGF produces a 5- to 6-fold increase in sodium channel density. The sodium channels induced by NGF are not different from those in cells not treated with NGF and are similar to those in other cell types. Basic fibroblast growth factor (FGF), another growth factor that causes PC12 cells to differentiate into sympathetic-like neurons, also produces a 5- to 6-fold increase in sodium current density with channels indistinguishable from those in PC12 cells treated and not treated with NGF. Basic FGF produces the same or somewhat larger increase in sodium channel density but much less neurite outgrowth. In contrast, epidermal growth factor does not produce neurite outgrowth but induces a small, reproducible increase in sodium channel density. Cyclic AMP produces spike-like processes but not neurites and results in a decrease in sodium current and sodium current density. Dexamethasone, a synthetic glucocorticoid, inhibits the increase in sodium current and sodium current density but does not antagonize the neurite outgrowth induced by NGF. Thus, although the increase in sodium channel expression induced by NGF and basic FGF parallels the changes in morphology that lead to neurite outgrowth, it clearly does not depend on them. The results show that different aspects of neuronal differentiation might be independently regulated by the microenvironment.     

Variant PC12 cell line that spontaneously differentiates and extends neuritic processes

The rat pheochromocytoma PC12 cells differentiate into neuronal-like cells in response to treatment with neurotrophins. The cells have been extensively used for investigating neuronal differentiation and axonal growth. Here we report the isolation of a variant PC12 cell line, named PC12-N1, which spontaneously differentiates and extends neuritic processes. The PC12-N1 cells expressed many neuronal specific proteins, including the synaptosomal associated protein of 25 kDa (SNAP-25), synaptotagmin, and synaptobrevin (also known as VAMP). The cells also expressed neurofilament protein of 68 kDa, a marker for differentiated neurons. In addition to the spontaneous neurite outgrowth, the PC12-N1 cells showed a marked increase in neurite outgrowth upon treatment with nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and cyclic AMP (cAMP). The activation of mitogen-activated protein (MAP) kinases was examined by immunoblot analysis using phospho-specific antibodies. No overactivation was observed with ERK1/2 or p38. However, the c-Jun N-terminal kinase JNK/SAPK was activated approximately 10-fold over the parental PC12 cells. These results suggest that activation of JNK/SAPK may be involved in the spontaneous neurite extension in the PC12-N1 cells. Moreover, the PC12-N1 cells may be used as a model for investigating molecular signaling mechanisms underlying neuronal differentiation and axonal outgrowth.     

Nerve growth factor and a fibroblast growth factor-like neurotrophic activity in cerebrospinal fluid of brain injured human patients

We report here the presence of nerve growth factor (NGF) in the cerebrospinal fluid (CSF) of some brain-injured human patients soon after injury. The NGF was quantified against a recombinant human NGF standard in a two-site enzyme-linked immunoabsorbant assay using antibodies against murine B NGF. None of the samples collected more than 2 days after injury contained detectable levels of NGF. When the CSF was assayed for the ability to promote neurite outgrowth from PC12 cells, neurite outgrowth was reduced, but not completely blocked, by antibodies to B NGF, suggesting that there were other biologically active factors present. Fibroblast growth factor (FGF) also promotes neurite outgrowth in PC12 cells. In an initial screening for the presence of FGF, we employed PC12 cells and NR119 cells, PC12 variants in which recombinant human B NGF, but not recombinant human basic FGF, promotes neurite outgrowth. CSF from brain injury patients promoted greater neurite outgrowth from PC12 cells than from NR119 cells, suggesting that some of the biological activity associated with the injury CSF may be due a FGF. This possibility is further supported by the observation that the biological activity of the injury CSF significantly reduced by batch absorption with heparin Sepharose, suggesting the presence of a heparin binding neurotrophic factor. Neurotrophic factors appear in CSF as a consequence of diverse types of brain injury, including head trauma, intracerebral hemorrhage and subarachnoid hemorrhage. The appearance of these factors may reflect important common elements in the complex series of cellular changes occuring in response to acute brain injury.     

Overexpression of neuronal intermediate filament protein alpha-internexin in PC12 cells

The neuronal intermediate filaments include not only the neurofilament triplet proteins but also peripherin and alpha-internexin. To determine whether neurite outgrowth is enhanced by alpha-internexin, the cDNA of rat alpha-internexin tagged with enhanced green fluorescent protein (EGFP) was transfected into a rat adrenal pheochromocytoma cell line PC12 that responds to nerve growth factor (NGF) by induction of the neuronal phenotype. Selected stable clones were induced by NGF and examined for expression patterns of neuronal intermediate filaments by Western blot and immunocytochemistry. Differentiating neurons were also collected after NGF induction for RT-PCR analysis. Overexpressed alpha-internexin-EGFPs were found mainly in cell bodies and the proximal part of neurites. It was also found that overexpression of alpha-internexin-EGFPs enhanced the neurite outgrowth of PC12 cells at the early stages of NGF induction. Meantime, NF-L and NF-M were upregulated by the overexpression of alpha-internexin-EGFPs. Interestingly, alpha-internexin-EGFP-transfected cells obviously detached from culture plates at the later stages of NGF induction. Massive IF accumulations, swelling mitochondria, and degenerating neurites with numerous electron-dense granules were observed ultrastructurally in the alpha-internexin-EGFP-transfected cells. In addition, neuronal death was also characterized positively by the TUNEL assay. These observations may imply that cell death was occurring in alpha-internexin-EGFP-transfected cells. From this study, it could be suggested that alpha-internexin plays an important role in neurite outgrowth and regulates the expression of other neurofilaments during neuronal development. Apoptosis-like cell death could also be induced by the overexpression of alpha-internexin-EGFP in PC12 cells after NGF induction.     

Dose-Dependent Effects of Recombinant Human NGF on Grafted Adult Adrenal Medullary Tissue

It has previously been shown that the chromaffin cells of the adrenal medulla respond to nerve growth factor (NGF) with neurite outgrowth and increased cell survival in tissue culture or after grafting. In the present study we evaluated the dose dependency in neurite outgrowth from chromaffin tissue to recombinant human NGF (rhNGF). Therefore, pieces of adrenal medullary tissue from adult rat were grafted into the anterior chamber of the eye of previously sympathectomized recepients. Survival time was 4 weeks. At grafting and at Days 7, 14, and 21 postgrafting, the eyes were injected with 5 μl of rhNGF at concentrations of 10, 30, 60, 100, 150, and 200 μg/ml, or with a control solution. All grafts, including the controls, survived well and became vascularized. At the low doses of rhNGF, 10 and 30 μg/ml, a small area of the irides was reinnervated and the density of the nerve fiber network was low. The maximal response was obtained at 100 μg rhNGF/ml. Using larger concentrations of 150 and 200 μg rhNGF/ml, the density of the nerve fiber network did not change, but the reinnervated area of the irides was significantly decreased compared to the outgrowth seen in irides treated with 100 μg/ml. In conclusion, adult rat chromaffin tissue responds to rhNGF in a dose-dependent manner. However, at the highest doses used, the outgrowth area was suboptimal, although nerve fiber density was maximal. These results indicate that to obtain maximal effects, the dose of NGF is critical.     

Nerve growth factor in combination with second messenger analogues causes neuronal differentiation of PC12 cells expressing a dominant inhibitory Ras protein without inducing activation of extracellular signal-regulated kinases

In the present work, nerve growth factor (NGF) was used in combination with the calcium ionophore, ionomycin or dibutyryl cyclic AMP (dbcAMP), to study the connection between neuronal differentiation and extracellular signal-regulated kinase (ERK) activation of PC12 rat pheochromocytoma cells expressing a dominant negative, Ha-Ras Asn17 protein. Due to the block of endogenous Ras activity, neurite outgrowth in response to NGF is completely inhibited in these cells. However, this blockade can be bypassed by combined treatment with NGF plus ionomycin or NGF plus dbcAMP. The mitogen-activated protein kinase (MAPK) /ERK kinase inhibitor, PD98059, proved to be insufficient in inhibiting the neurite outgrowth under these conditions. Moreover, although both long-term ERK activation and nuclear translocation of ERKs are believed to be key events in neuronal differentiation, neither detectable ERK phosphorylation, nor nuclear translocation of these enzymes, occurred upon combination treatments in our experimental system. However, the neuritogenesis induced by either the combination of NGF/ionomycin or NGF/dbcAMP was inhibited by the Trk inhibitor, K252a. Ras-independent pathways, originating from the NGF receptor, can thus synergize with second messenger analogues bypassing the ERK cascade but leading to the same biological result--neurite formation.     

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.     

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.     

Molecular and cellular mechanism of neuronal degeneration caused by nerve growth factor deprivation approached through PC12 cell culture.

1. Degenerative processes of PC12 cells after acute withdrawal of nerve growth factor (NGF) were characterized. PC12 cells treated with NGF share properties similar to sympathetic neurons, and only these cells responded to acute withdrawal of NGF by degeneration characterized by disappearance of growth cones, neurite disintegration, and eventually cell death. 2. The cell pool of ATP and release of lactate dehydrogenase (LDH) into medium were measured and taken as a quantitative measure of cell degeneration. 3. The release of LDH after NGF withdrawal was not completely blocked by cycloheximide or cordycepin, suggesting that active cell death occurred only partially in PC12 cells. Degeneration processes including neurite disintegration did not require protein synthesis. All the degenerative processes were completely prevented by chronic depolarization with high K+ and cyclic AMP. 4. Preliminary evidence was presented on a transient induction of the proto-oncogene c-fos upon acute withdrawal of NGF. Thus, one of the interesting aspects available from studies of PC12 cells would be to compare pathways initiated by NGF deprivation to those activated by NGF exposure to naive PC12 cells, since the latter pathways have been extensively studied. 5. These results suggest that PC12 cells serve as a model system for studying neuronal degeneration caused by acute withdrawal of NGF. We also suggest that NGF removal elicits divergent cellular signalling pathways leading to cell degeneration, some of which may be utilized in common with those activated by NGF exposure to naive PC12 cells.     

Expression and regulation of CNTF receptor-alpha in the in situ and in oculo grafted adult rat adrenal medulla

Cultured and transplanted adrenal medullary cells respond to ciliary neurotrophic factor (CNTF) with neurite formation and improved cell survival although the presence of the CNTF receptor-alpha (CNTFRalpha) has been unclear. This study show that CNTFRalpha mRNA was expressed in the postnatal day 1 as well as in the adult rat adrenal medulla. The highest CNTFRalpha mRNA signal was found in the ganglion cells of the adrenal medulla. After transplantation of adrenal medullary tissue the CNTFRalpha mRNA levels were down-regulated in the chromaffin cells. CNTF treatment of grafts did not normalize the receptor levels, but treatment with nerve growth factor (NGF) did. Thus, we demonstrate that CNTFRalpha mRNA is expressed in adrenal medulla, the levels becomes down-regulated after transplantation, but normalized after treatment with NGF.     

Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors

Neurite-promoting activity in feeding medium conditioned by rat astrocytes and Schwann cells in culture was examined. The conditioned medium (CM) from both types of glial cultures stimulated extensive neurite outgrowth from embryonic chick dorsal root ganglia (DRG) as well as pheochromocytoma (PC12) cells. Both the DRG and PC12 cells also produce neurite outgrowth in the presence of nerve growth factor (NGF). With the DRG, the neurite growth rates observed with the glial cell CM were identical to growth rates seen with NGF. Although anti-NGF antibody did not inhibit the neurite outgrowth produced by either of the glial CM, a nerve growth factor radioreceptor assay did detect an NGF-like molecule in both CM. Since the extensive neurite outgrowth stimulated by the glial CM was not mimicked by pure laminin alone, we conclude that the glial neurite promoting factors are distinct from laminin.     

Mutual induction of TGFbeta1 and NGF after treatment with NGF or TGFbeta1 in grafted chromaffin cells of the adrenal medulla

Chromaffin cells have been recognized for their ability to transform into sympathetic ganglion-like cells in response to nerve growth factor (NGF) or to stimulation of other neurotrophic factors. Transforming growth factor beta (TGFbeta) family members have been shown to potentiate the effect of different trophic factors. The aim of this study was to investigate if TGFbeta may influence NGF-induced neuronal transformation and regulation of NGF, TGFbeta1, and their receptors in the adult rat chromaffin tissue after grafting. Intraocular transplantation of adult chromaffin tissue was employed and grafts were treated with TGFbeta1 and/or NGF. Graft survival time was 18 days after which the grafts were processed for TGFbeta luciferase detection assay, NGF enzyme immunoassay, or in situ hybridization. In grafts stimulated with NGF, increased levels of TGFbeta1 and TGFbeta1 mRNA were detected. When grafts instead were treated with TGFbeta1, enhanced levels of NGF protein were found. Furthermore, a positive mRNA signal corresponding to the transforming growth factor II receptor (TbetaRII) was found in the chromaffin cells of the normal adrenal medulla as well as after grafting. No increase of TbetaRII mRNA levels was detected after transplantation or after TGFbeta1 treatment. Instead a reduction of TbetaRII mRNA expression was noted after NGF treatment. NGF stimulation of grafts increased the message for NGF receptors p75 and trkA in the chromaffin transplants. Grafts processed for evaluations of neurite outgrowth were allowed to survive for 28 days and were injected weekly with NGF and/or TGFbeta1. NGF treatment resulted in a robust innervation of the host irides. TGFbeta1 had no additive effect on nerve fiber formation when combined with NGF. Combined treatment of NGF and anti-TGFbeta1 resulted in a significantly larger area of reinnervation. In conclusion, it was found that NGF and TGFbeta1 may regulate the expression of each other's protein in adult chromaffin grafts. Furthermore, TbetaRII mRNA was present in the adult rat chromaffin cells and became downregulated as a result of NGF stimulation. Although no synergistic effects of TGFbeta1 were found on NGF-induced neurite outgrowth, it was found that TGFbeta1 and NGF signaling are closely linked in the chromaffin cells of the adrenal medulla.     

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.     

Inhibitory effects of brain chondroitin sulfate proteoglycans on neurite outgrowth from PC12D cells

Soluble chondroitin sulfate proteoglycans (CSPGs), prepared from 10-d-old rat brain, were added to the culture medium of PC12D cells containing NGF to examine the effects on NGF-induced neurite outgrowth from the cells. PC12D cells, a flat-shaped variant of PC12 pheochromocytoma cells, are characteristic of prompt neurite formation in response not only to NGF, but also to cAMP-enhancing reagents such as forskolin. Brain CSPGs inhibited the neurite elongation irreversibly in a dose-dependent manner; complete inhibition was observed at a concentration of 50 nmol uronic acid/ml. Closely similar dose-dependent inhibition was observed in the forskolin-induced neurite outgrowth from PC12D cells. NGF-induced neurite outgrowth from conventional PC12 cells was also inhibited completely by 50 nmol uronic acid/ml CSPGs. Some brain CSPGs seemed to be inhibitory, but the cartilage-unique CSPG did not show any inhibitory effect. Chondroitin sulfate, a polysaccharide moiety of CSPGs, did not show any inhibitory effect even at a concentration of 250 nmol uronic acid/ml, while core proteins prepared from brain CSPGs by digestion with chondroitinase ABC exhibited inhibitory activity similar to that of intact CSPGs. This indicates that the site of the inhibitory activity exists in the core protein moiety of brain CSPGs. From these observations, it is conceivable that brain CSPGs are involved in the regulation of neuronal differentiation.     

Phospholipases elevate cyclic AMP levels and promote neurite extension in a clonal nerve cell line

Phospholipases A2 and C increase choline acetyltransferase (CAT) activity and promote neurite extension in PC12 cells. Since these phospholipases also elevate intracellular cyclic AMP, they probably stimulate CAT and neurite outgrowth through their ability to increase cyclic AMP levels. Other possible modes of action of phospholipases such as the release of arachidonic acid or the production of lysolipids are less likely to play a role in this system.