Synergistic effect of immobilized laminin and nerve growth factor on PC12 neurite outgrowth

Immobilized extracellular matrix proteins and neurotrophins have been extensively studied to enhance neuronal adhesion and proliferation on surfaces for applications in nerve tissue engineering and neuroprosthetic devices. This article describes how the coimmobilization of laminin, an extracellular matrix protein and nerve growth factor (NGF), a neurotrophin can enhance neurite outgrowth observed separately with each type of molecule. In the absence of immobilized NGF, PC12 neurite outgrowth is influenced strongly by the presence of NGF in solution and unaffected by significant increases in laminin surface density (18.7–93.5 ng/mm²). However, when both laminin and NGF are immobilized together, the surface density of laminin is an important factor in determining whether or not the neurite outgrowth‐promoting effect of NGF can be obtained. PC12 neurite outgrowth on surfaces with coimmobilized laminin and NGF with surface densities of 27.6 ng/mm² and 1.4 ng/mm², respectively, are similar to that observed on surfaces with immobilized laminin and dissolved NGF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The amyloid protein precursor of Alzheimer's disease is a mediator of the effects of nerve growth factor on neurite outgrowth.

The beta A4 protein, the major component of the amyloid deposition characterizing Alzheimer's disease, derives from the amyloid protein precursor (APP), an integral membrane protein with soluble derivatives. The function of APP is unknown. Both soluble and membrane-associated human brain APP (10(-10) M) significantly increased (P less than 0.025) neurite length and branching in pheochromocytoma PC12 cells, but did not affect the number of neurites per cell. At higher concentrations, APP was cytotoxic, with a half-maximal concentration of 5 x 10(-9) M. Nerve growth factor (NGF) is known to affect APP expression in vivo and in vitro. Antibodies to APP specifically diminished the effects of NGF on neurite length and branching. Thus APP may act to mediate neurite outgrowth promotion by NGF.     

Changes in the phosphorylation and distribution of vinculin during nerve growth factor induced neurite outgrowth

The mechanism of neurite initiation and elongation was studied using nerve growth factor (NGF) treatment of PC12 cells. The distribution of focal adhesion sites and of the cytoskeletal protein vinculin was determined in large, fused, multinucleated PC12 cells. In the absence of NGF, focal adhesion sites as seen by interference reflection microscopy were restricted to the cell periphery in a regular distribution. Vinculin assemblies (foci), observed by indirect immunofluorescence microscopy using affinity purified anti-vinculin antibodies, were restricted to the cell periphery at focal adhesion sites. Within 4 hr after NGF treatment of the cells, the distribution of both vinculin and focal adhesion sites began to change. Focal adhesion sites became restricted to discrete protruding portions of the cell periphery. Larger, brighter vinculin foci appeared at the tips of the cell margin extensions, concomitant with the loss of foci at locations between the protrusions. As neurites elongated focal adhesion sites and vinculin foci remained with the tips of the growth cone extensions. Both focal adhesion sites and vinculin foci were rarely seen in the perikarya of cells with elongating neurites, and these were always confined to extended portions of the cell body margin. Occasionally, vinculin foci could be seen at the proximal portion of the neurite, at bending elbows, and at discrete expansions along the length. By immunoprecipitation of vinculin from 32P-labeled cells, vinculin phosphorylation was found to be increased within 1 hr of NGF treatment. The role of vinculin phosphorylation and assembly in the formation and directional elongation of neuritic processes in response to NGF is discussed.     

Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

Neurite outgrowth and neuronal differentiation play a crucial role in the development of the nervous system. Understanding of neurotrophins induced neurite outgrowth was important to develop therapeutic strategy for axon regeneration in neurodegenerative diseases as well as after various nerve injuries. It has been reported that extension of neurite and differentiation of sympathetic neuron-like phenotype was modulated by nerve growth factor (NGF) in PC12 cells. In this study, NGF mediated neurite outgrowth was investigated in PC12 cells after liquiritin exposure. Liquiritin is a kind of flavonoids that is extracted from Glycyrrhizae radix, which is frequently used to treat injury or swelling for its life-enhancing properties as well as detoxification in traditional Oriental medicine. The result showed that liquiritin significantly promotes the neurite outgrowth stimulated by NGF in PC12 cells in dose dependant manners whereas the liquiritin alone did not induce neurite outgrowth. Oligo microarray and RT-PCR analysis further clarified that the neurotrophic effect of liquiritin was related to the overexpression of neural related genes such as neurogenin 3, neurofibromatosis 1, notch gene homolog 2, neuromedin U receptor 2 and neurotrophin 5. Thus, liquiritin may be a good candidate for treatment of various neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease.     

Effect of nerve growth factor and fibroblast growth factor on SCG10 and c-fos expression and neurite outgrowth in protein kinase C-depleted PC12 cells

The role of protein kinase C (PKC) in mediating nerve growth factor (NGF) or basic fibroblast growth factor (bFGF)-stimulated SCG10 and c-fos expression as well as neurite outgrowth was studied in PC12 cells. Activators of PKC such as phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl 2-acetyl glycerol mimicked the stimulatory effect of NGF and bFGF on SCG10 mRNA levels. Induction involved a protein synthesis-dependent mechanism and was maximal within 12-24 h of exposure. Chronic treatment of the cells with PMA for up to 8 days resulted in a substantial decrease (approximately 90%) in total PKC activity in the continued presence of PMA. PKC depletion did not affect NGF- or bFGF-stimulated SCG10 mRNA induction and bFGF-stimulated c-fos mRNA induction. However, NGF-stimulated c-fos mRNA induction was attenuated. In addition, induction of neurite outgrowth was not abolished in PKC-depleted cells. The results imply that PKC is not involved in NGF- and bFGF-stimulated SCG10 mRNA induction and neurite outgrowth. Furthermore, while the effect of bFGF on c-fos mRNA induction is PKC-independent, that of NGF is mediated by PKC-dependent and -independent pathways.     

Electrical stimulation promotes nerve growth factor-induced neurite outgrowth and signaling

Background

Neurotrophins are important regulators for neural development and regeneration. Nerve growth factor (NGF) therapy has been tested in various models of neural injury and degeneration. However, whether NGF can reach target tissues and maintain effective concentration for a certain period of time remains uncertain. To facilitate neural regeneration, we investigate the possibility of combining NGF and electrical stimulation (ES) in promoting neurite outgrowth, an essential process during neural regeneration.

Methods

PC12 cells were seeded on collagen and indium tin oxide (ITO)-coated area on the transparent conductive devices. Cells were then subjected to the combination of ES and NGF treatment. Neurite outgrowth was compared.

Results

Our findings suggest that ES of 100 mV/mm together with NGF provides optimal effect on neurite outgrowth of PC12 cells. ES increases NGF-induced neurite length but reduces neurite branching, indicative of its primary effect on neurite elongation instead of initiation. One mechanism that ES enhances neurite outgrowth is through increasing NGF-induced phosphorylation of ERK1/2 (pERK1/2) and expression of Egr1 gene. ES has previously been demonstrated to increase the activity of protein kinase C (PKC). Our result indicates that activating PKC further increases NGF-induced pERK1/2 and thus neurite outgrowth.

Conclusion

It is likely that ES promotes NGF-induced neurite outgrowth through modulating the activity of ERK1/2.

General significance

Findings from this study suggest that combining ES and NGF provides a promising strategy for promoting neurite outgrowth.     

Interactions between retinoic acid, nerve growth factor and sonic hedgehog signalling pathways in neurite outgrowth

Many studies have shown a role of retinoid signalling in neurite outgrowth in vitro, and that the retinoic acid receptor (RAR) beta2 is critical for this process. We show here that RARbeta2 is expressed predominantly in dorsal root ganglia (DRG) neuronal subtypes that express neurofilament (NF) 200 and calcitonin gene-related peptide (CGRP), and that these neurons extend neurites in response to RA. We demonstrate that retinoid signalling has a role in neurite outgrowth in vivo, by showing that in a peripheral nerve crush model there is less neurite outgrowth from RARbeta null DRG compared to wild-type. We identify sonic hedgehog (Shh) as a downstream target of the RARbeta2 signalling pathway as it is expressed in the injured DRG of wild-type but not RARbeta null mice. This regulation is direct as when RARbeta2 is overexpressed in adult motoneurons Shh is induced in them. Finally we show that Shh alone cannot induce neurite outgrowth but potentiates RARbeta2 signalling in this process.     

Reconstituted basement membrane enhances neurite outgrowth in PC12 cells induced by nerve growth factor

Rat pheochromocytoma cells (PC12) respond to nerve growth factor (NGF) by elaborating neurites. We have studied the effect of extracellular matrix proteins on responsiveness of PC12 cells to NGF. NGF elicited neurite outgrowth in cells plated on collagen, fibronectin, or laminin within 48-72 h. In contrast, cells entrapped in reconstituted basement membrane prepared from the Engelbreth-Holm-Swarm tumor (EHS-BM) responded within 24 h. Cells plated on collagen or fibronectin required a minimal dose of 50 ng/ml NGF to achieve the same effect as cells entrapped in EHS-BM at 1 ng/ml NGF. Neurites displayed by cells plated in EHS-BM were more extensively branched and remained intact up to 21 days following a single administration of NGF.     

Protein kinase C-epsilon plays a role in neurite outgrowth in response to epidermal growth factor and nerve growth factor in PC12 cells.

In this study, we examined the role of specific protein kinase C (PKC) isoforms in the differentiation of PC12 cells in response to nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells express PKC-alpha, -beta, -gamma, -delta, -epsilon, -mu, and -zeta. For PKC-delta, -epsilon, and -zeta, NGF and EGF exerted differential effects on translocation. Unlike overexpression of PKC-alpha and -delta, overexpression of PKC-epsilon caused enhanced neurite outgrowth in response to NGF. In the PKC-epsilon-overexpressing cells, EGF also dramatically induced neurite outgrowth, arrested cell proliferation, and induced a sustained phosphorylation of mitogen-activated protein kinase (MAPK), in contrast to its mitogenic effects on control cells or cells overexpressing PKC-alpha and -delta. The induction of neurite outgrowth by EGF was inhibited by the MAPK kinase inhibitor PD95098. In cells overexpressing a PKC-epsilon dominant negative mutant, NGF induced reduced neurite outgrowth and a more transient phosphorylation of MAPK than in controls. Our results suggest an important role for PKC-epsilon in neurite outgrowth in PC12 cells, probably via activation of the MAPK pathway.     

Modulation of protein synthesis by nerve growth factor.

The effects of nerve growth factor (NGF) and two analogs of cAMP have been studied in the PC12 line of rat pheochromocytoma cells. We have used two-dimensional gel electrophoresis to detect more than 800 proteins from control and NGF-treated cells. Of these proteins, none were qualitatively repressed in response to NGF, and no new proteins appeared after NGF treatment. Visual inspection of the gels showed that approximately 5% of the proteins were detectably increased or decreased in rate of synthesis by NGF, and each of these changes was mimicked by both cAMP analogs. The two-dimensional gel data were further analyzed by a computerized scanning system. This analysis has revealed many significant changes that are smaller than those detected by eye. Approximately 25 to 30% of the proteins analyzed were found to be altered in rate of synthesis by 30% or more. Statistical analysis has shown that the response to NGF and the response to dibutyryl cAMP are highly correlated, even down to changes as small as 30%. No proteins were found to be significantly altered by both dibutyryl cAMP and 8-bromo cAMP, but not by NGF. These results show that NGF causes only quantitative modulations of protein synthesis in PC12 cells, and these data strongly suggest that the response of PC12 cells to NGF is mediated by cAMP.     

Stage-dependent stimulation of neurite outgrowth exerted by nerve growth factor and chick heart in cultured embryonic ganglia

The ability of embryonic chick heart to elicit neuritic outgrowth in different ganglia was tested to examine (1) whether stimulative activity is possessed by the heart only at specific stages and (2) whether the ability of the ganglionic neurons to respond is limited to certain periods of development. As an assay, ganglia were explanted into thin collagen gels with ventricular tissue placed at a distance of about 1 mm. Neuritic outgrowth was measured after 2 days. Control ganglia and ganglia cultured with added nerve growth factor (NGF) were also scored. Four types of tested ganglia, including the ciliary ganglion, showed a peak in neuritic outgrowth when cultured with heart of embryonic Day 18, at about which age the heart becomes sympathetically innervated in ovo. No age-related size differences that could account for this temporal pattern were found among the heart explants when measuring their protein content. A peak in neuronal susceptibility to heart tissue was evident in the 6-day ciliary ganglion and in the 8-day paravertebral, Remak, and spinal ganglia, roughly coinciding with the onset of fibre outgrowth in ovo. Neurite extension is concluded to have been triggered by a factor spread from the heart explants and being distinct from the mouse type of NGF since anti-NGF did not at any stage block the events and since added NGF at all stages failed to evoke neurite formation in the ciliary ganglia. A testable hypothesis is that this factor regulates the growth of sympathetic and possibly parasympathetic and sensory fibres in the developing chick heart.     

Neuroserpin regulates neurite outgrowth in nerve growth factor-treated PC12 cells

Neuroserpin is a serine protease inhibitor widely expressed in the developing and adult nervous systems and implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration and axogenesis. We have analysed the effect of neuroserpin on growth factor-induced neurite outgrowth in PC12 cells. We show that small changes in neuroserpin expression result in changes to the number of cells extending neurites and total neurite length following NGF treatment. Increased expression of neuroserpin resulted in a decrease in the number of cells extending neurites and a reduction in total free neurite length whereas reduced levels of neuroserpin led to a small increase in the number of neurite extending cells and a significant increase in total free neurite length compared to the parent cell line. Neuroserpin also altered the response of PC12 cells to bFGF and EGF treatment. Neuroserpin was localised to dense cored secretory vesicles in PC12 cells but was unable to complex with its likely enzyme target, tissue plasminogen activator at the acidic pH found in these vesicles. These data suggest that modulation of neuroserpin levels at the extending neurite growth cone may play an important role in regulating axonal growth.     

Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation

We have developed an experimental paradigm to study the mechanism by which nerve growth factor (NGF) allows the survival of sympathetic neurons. Dissociated sympathetic neurons from embryonic day-21 rats were grown in vitro for 7 d in the presence of NGF. Neurons were then deprived of trophic support by adding anti-NGF antiserum, causing them to die between 24 and 48 h later. Ultrastructural changes included disruption of neurites, followed by cell body changes characterized by an accumulation of lipid droplets, changes in the nuclear membrane, and dilation of the rough endoplasmic reticulum. No primary alterations of mitochondria or lysosomes were observed. The death of NGF-deprived neurons was characterized biochemically by assessing [35S]methionine incorporation into TCA precipitable protein and by measuring the release of the cytosolic enzyme adenylate kinase into the culture medium. Methionine incorporation began to decrease approximately 18 h post-deprivation and was maximally depressed by 36 h. Adenylate kinase began to appear in the culture medium approximately 30 h after deprivation, reaching a maximum by 54 h. The death of NGF-deprived neurons was entirely prevented by inhibiting protein or RNA synthesis. Cycloheximide, puromycin, anisomycin, actinomycin-D, and dichlorobenzimidazole riboside all prevented neuronal death subsequent to NGF deprivation as assessed by the above morphologic and biochemical criteria. The fact that sympathetic neurons must synthesize protein and RNA to die when deprived of NGF indicates that NGF, and presumably other neurotrophic factors, maintains neuronal survival by suppressing an endogenous, active death program.     

Schwann cells promote neurite outgrowth of dorsal root ganglion neurons through secretion of nerve growth factor

The transplantation of Schwann cells (SCs) could successfully promote axonal regeneration. This is likely to attribute to the adhesion molecules expression and growth factors secretion of SCs. But which factor(s) play a key role has not been precisely studied. In this study, an outgrowth assay using dorsal root ganglia (DRG) neuron-SC co-culture system in vitro was performed. Co-culture of SCs or application of SC-conditioned medium (CM) substantially and significantly increased DRG neurite outgrowth. Further, nerve growth factor and NGF receptor (TrkA) mRNA were highly expressed in Schwann cells and DRG neuron, respectively. The high concentration of NGF protein was detected in SC-CM. When K-252a, a specific inhibitor of NGF receptor was added, DRG neurite outgrowth was significantly decreased in a concentration-dependent manner. These data strongly suggest that SCs play important roles in neurite outgrowth of DRG neurons by secreted NGF.     

Stochastic dynamics of the nerve growth cone and its microtubules during neurite outgrowth

The controlled extension of neurites is essential not only for nervous system development, but also for effective nerve regeneration after injury. This process is critically dependent on microtubule assembly since axons fail to elongate in the presence of drugs which disrupt normal assembly dynamics. For this reason, neurite outgrowth is potentially controllable by manipulation of the assembly state of the intracellular array of microtubules. Therefore, understanding how microtubule assembly dynamics and neurite outgrowth are coupled, in the absence of drugs, can lend valuable insight into the control and guidance of the outgrowth process. In the present study we characterized the stochastic dynamics of neurite outgrowth and its corresponding microtubule array, which advances concomitantly with the advance of the nerve growth cone, the highly motile structure at the terminus of the growing neurite, using reported fluorescent microscopic image sequences (Tanaka and Kirschner, 1991, J. Cell Biol. 115:345-363). Although previously modeled as an uncorrelated random walk, the stochastic advance of the growth cone was found to be anticorrelated over a time scale of approximately 4 min, meaning that growth cone advances tended to be followed by growth cone retractions approximately 4 min later. The observed anticorrelation most likely reflects the periodic stops and starts of neurite outgrowth that have been reported anecdotally. A strikingly similar pattern of anticorrelation was also identified in the advance of the growth cone's microtubule array. Cross-correlation analysis showed that growth cone dynamics tended to precede microtubule dynamics on a time scale of approximately 0-2 min, while microtubules tended to precede growth cone dynamics on a approximately 0-20-s time scale, indicating a close temporal coupling between microtubule and growth cone dynamics. Finally, the scaling of the mean-squared displacements with time for both the growth cone and microtubules suggested a fractional Brownian motion model which accounts for the observed anticorrelation of growth cone and microtubule advance. (c) 1996 John Wiley & Sons, Inc.     

Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells

The outgrowth of neurites from rat PC12 cells stimulated by combined treatment of nerve growth factor (NGF) with cAMP is significantly more rapid and extensive than the outgrowth induced by either factor alone. We have compared the responses of PC12 cells under three different growth conditions, NGF alone, cAMP alone, and combined treatment, with respect to surface morphology, rapidity of neurite outgrowth, and stability of neurite microtubules, to understand the synergistic action of NGF and cAMP on PC12. Surface events at early times in these growth conditions varied, suggesting divergent pathways of action of NGF and cAMP. This suggestion is strongly supported by the finding that cells exposed to saturating levels of dibutyryl cAMP without substantial neurite outgrowth initiated neurites within 5 min of NGF. This response has been adopted as a convenient assay for NGF. Neurites that regenerated in the three growth conditions showed marked differences in stability to treatments that depolymerize microtubules. The results indicate that microtubules in cells treated with both NGF and cAMP are significantly more stable than in either growth factor alone. We suggest that a shift of the assembly equilibrium favoring tubulin assembly is a necessary prerequisite for the initiation of neurites by PC12.     

Structure-based discovery of low molecular weight compounds that stimulate neurite outgrowth and substitute for nerve growth factor

Olanzapine, an atypical antipsychotic drug, was previously shown to protect neuronal cells against nutrient deprivation and to enhance neurite outgrowth. In an effort to identify small molecules with greater potency, the structure of olanzapine was used as a template to search commercially available chemical inventories for compounds with similar features. These compounds were evaluated for their ability to protect cells against glutamine deprivation and low-serum conditions. Positive compounds, 'hits' from initial screening, were then tested for stimulation of neurite outgrowth, alone and in combination with suboptimum concentrations of nerve growth factor (NGF). Numerous neuroprotective compounds (mw < 550 Da) were identified that significantly stimulated neurite outgrowth in PC12 cells. These included 4', 6'-diamidino-2-phenylindole, a nuclear stain; staurosporine, an antibiotic and kinase inhibitor; and 2-phenylamino-adenosine, an adenosine analog. The small molecules were comparable with NGF, and in fact, replaced NGF in outgrowth assays. Pharmacophore analysis of the hits led to the design and synthesis of an active compound, LSU-D84, which represented an initial lead for drug discovery efforts.     

Regulation of microtubule composition and stability during nerve growth factor-promoted neurite outgrowth

We have used the nerve growth factor (NGF)-responsive line of PC12 pheochromocytoma cells as a model system to study microtubule specializations associated with neurite outgrowth. PC12 cells treated with NGF cease proliferating and extend neurites. Long-term NGF treatment results in a two- to threefold increase in the proportion of total cellular tubulin that is polymerized in PC12 cells. The increase in this parameter first becomes apparent at 2-4 d with NGF and increases steadily thereafter. Several changes in microtubule-associated proteins (MAPs) of PC12 cells also occur after exposure to NGF. In immunoprecipitation assays, we observed the levels of MAP-2 to increase by at least several-fold after treatment with NGF. We also found that the compositions of three MAP classes with apparent Mr of 64K, 67K, and 80K are altered by NGF treatment. These MAPs, recently designated "chartins," are biochemically and immunologically distinct from the similarly-sized tau MAPs (Peng et al., 1985 Brain Res. 361: 200; Magendantz and Solomon, 1985 Proc. Natl. Acad. Sci. 82: 6581). In two-dimensional isoelectric focusing x SDS polyacrylamide gels, each chartin MAP class resolves into a set of proteins of similar apparent Mr but distinct pI. Peptide mapping analyses confirm that the isoelectric variants comprising each chartin MAP class are closely related in primary structure. Several striking differences in the composition of the chartin MAPs of PC12 cells grown with or without NGF were consistently observed. In particular, following longterm NGF treatment, the abundances of the more acidic variants of each chartin MAP class were markedly enhanced relative to the more basic members. This occurs without substantial changes in the abundance of each MAP class as a whole relative to total cell protein. The combined results of in vivo phosphorylation and peptide mapping experiments indicate that the NGF-inducible chartin MAP species are not primary translation products, but are generated posttranslationally, apparently by differential phosphorylation of other chartin MAPs. These observations suggest that NGF treatment of PC12 cells leads to changes in the posttranslational processing of the chartin MAPs. The time course of these changes closely resembles that for the increase in the proportion of cellular tubulin that is polymerized and for neurite outgrowth. One of the important events in the growth and stabilization of neurites appears to be the formation of microtubule bundles that extend from the cell body to the tips of the neurites.(ABSTRACT TRUNCATED AT 400 WORDS)