Percentage Of Neurite-bearing Cells Research Articles

Stimulation of neuroendocrine differentiation in prostate cancer cells by GHRH and its blockade by GHRH antagonists.

Prostate cancer is the second leading cause of cancer-related deaths among men in developed countries. Neuroendocrine prostate cancer, in particular, is associated with an aggressive phenotype and a poor prognosis. Neuroendocrine cells produce and secrete peptide hormones and growth factors in a paracrine/autocrine manner which promote the progression of the disease. Recent studies have demonstrated that extracellular vesicles or exosomes are released by prostate cancer cells, supporting the spread of prostate cancer. Hence, the aim of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) on neuroendocrine differentiation (NED) in the androgen-dependent prostate cancer cell line LNCaP and the molecular mechanisms underlying these effects. GHRH induced an increase in the percentage of neurite-bearing cells and in the protein levels of Neuron-Specific Enolase. Both effects were blocked by the GHRH receptor antagonist MIA-690. In addition, pretreatment of these cells with the calcium chelator BAPTA, the EGFR inhibitor AG-1478 or the HER2 inhibitor AG-825 reduced the effectof GHRH, suggesting that the GHRH-induced stimulation of NED involves calcium channel activation and EGFR/HER2 transactivation. Finally, PC3-derived exosomes led to an increase in NED, cell proliferation and cell adhesion. Altogether, these findings suggest that GHRH antagonists should be considered for in the management of neuroendocrine prostate cancer.

Preparation and characterization of conductive poly-dl-lactic-acid/tetra-aniline conduit for peripheral nerve regeneration

Defected peripheral nerve regeneration is still a challenge in clinical treatment. Conductive polymers show great potential in nerve tissue engineering because of their electrical property based on bioelectricity in vivo. In this study, conductive composite nerve conduit was synthesized with tetra-aniline and poly-dl-lactic acid. Their properties and the differentiation of rat pheochromocytoma 12 (PC12) cells in vitro stimulated with 200 mV for 1 h were investigated. Different amounts of tetra-aniline (0%, 5%, 10%, and 15%) were used to synthesize the conduits with different conductivities (0, 0.00625, 0.01, and 0.025 s/m, respectively), tensile strengths (2.45, 3.40, 4.45, and 5.50 MPa, respectively), and contact angles (80°, 78.5°, 62.5°, and 61.5°, respectively). The percentage of neurite-bearing cells and the median neurite length increased with an obvious raise of the content of tetra-aniline. In addition, the conduit with subcutaneous implantable experiments in vivo showed less inflammatory response. These promising results illustrated that this poly-dl-lactic acid/tetra-aniline conductive composite conduit had potential for nerve tissue engineering.

Calsenilin, a Presenilin Interactor, Regulates RhoA Signaling and Neurite Outgrowth.

Calsenilin modulates A-type potassium channels, regulates presenilin-mediated γ-secretase activity, and represses prodynorphin and c-fos genes expression. RhoA is involved in various cellular functions including proliferation, differentiation, migration, transcription, and regulation of the actin cytoskeleton. Although recent studies demonstrate that calsenilin can directly interact with RhoA and that RhoA inactivation is essential for neuritogenesis, it is uncertain whether there is a link between calsenilin and RhoA-regulated neuritogenesis. Here, we investigated the role of calsenilin in RhoA-regulated neuritogenesis using in vitro and in vivo systems. We found that calsenilin induced RhoA inactivation, which accompanied RhoA phosphorylation and the reduced phosphorylation levels of LIM kinase (LIMK) and cofilin. Interestingly, PC12 cells overexpressing either full-length (FL) or the caspase 3-derived C-terminal fragment (CTF) of calsenilin significantly inactivated RhoA through its interaction with RhoA and p190 Rho GTPase-activating protein (p190RhoGAP). In addition, cells expressing FL and the CTF of calsenilin had increased neurite outgrowth compared to cells expressing the N-terminal fragment (NTF) of calsenilin or vector alone. Moreover, Tat-C3 and Y27632 treatment significantly increased the percentage of neurite-bearing cells, neurite length, and the number of neurites in cells. Finally, calsenilin deficiency in the brains of calsenilin-knockout mice significantly interfered with RhoA inactivation. These findings suggest that calsenilin contributes to neuritogenesis through RhoA inactivation.

Regulation of NGF‐driven neurite outgrowth by Ins(1,4,5)P3 kinase is specifically associated with the two isoenzymes Itpka and Itpkb in a model of PC12 cells

Four inositol phosphate kinases catalyze phosphorylation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 ] to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4 ]: these enzymes comprise three isoenzymes of inositol 1,4,5-trisphosphate 3-kinase (Itpk), referred to as Itpka, Itpkb and Itpkc, and the inositol polyphosphate multikinase (IPMK). The four enzymes that act on Ins(1,4,5)P3 are all expressed in rat pheochromocytoma PC12 cells, a model that is used to study neurite outgrowth induced by nerve growth factor (NGF). We compared the effect of over-expression of the four GFP-tagged kinases on NGF-induced neurite outgrowth. Our data show that over-expression of the Itpka and Itpkb isoforms inhibits NGF-induced neurite outgrowth, but over-expression of Itpkc and IPMK does not. Surprisingly, over-expression of the N-terminal F-actin binding domain of Itpka, which lacks catalytic activity, was as effective at inhibiting neurite outgrowth as the full-length enzyme. Neurite length was also significantly decreased in cells over-expressing Itpka and Itpkb but not Itpkc or IPMK. This result did not depend on the over-expression level of any of the kinases. PC12 cells over-expressing GFP-tagged kinase-dead mutants Itpka/b have shorter neurites than GFP control cells. The decrease in neurite length was never as pronounced as observed with wild-type GFP-tagged Itpka/b. Finally, the percentage of neurite-bearing cells was increased in cells over-expressing the membranous type I Ins(1,4,5)P3 5-phosphatase. We conclude that Itpka and Itpkb inhibit neurite outgrowth through both F-actin binding and localized Ins(1,4,5)P3 3-kinase activity. Itpkc and IPMK do not influence neurite outgrowth or neurite length in this model.

Conductive PPY/PDLLA conduit for peripheral nerve regeneration

The significant drawbacks and lack of success associated with current methods to treat critically sized nerve defects have led to increased interest in neural tissue engineering. Conducting polymers show great promise due to their electrical properties, and in the case of polypyrrole (PPY), its cell compatibility as well. Thus, the goal of this study is to synthesize a conducting composite nerve conduit with PPY and poly(d, l-lactic acid) (PDLLA), assess its ability to support the differentiation of rat pheochromocytoma 12 (PC12) cells in vitro, and determine its ability to promote nerve regeneration in vivo. Different amounts of PPY (5%, 10%, and 15%) are used to synthesize the conduits resulting in different conductivities (5.65, 10.40, and 15.56 ms/cm, respectively). When PC12 cells are seeded on these conduits and stimulated with 100 mV for 2 h, there is a marked increase in both the percentage of neurite-bearing cells and the median neurite length as the content of PPY increased. More importantly, when the PPY/PDLLA nerve conduit was used to repair a rat sciatic nerve defect it performed similarly to the gold standard autologous graft. These promising results illustrate the potential that this PPY/PDLLA conducting composite conduit has for neural tissue engineering.

Arachidonic acid inhibits retinoic acid‐induced neuritogenesis of human neuroblastoma SH‐SY5Y cells

Polyisoprenylated proteins regulate intracellular processes such as cell growth, differentiation, oncogenic transformation and actin cytoskeletal organization. Recent discoveries show that polyunsaturated fatty acids (PUFAs) may be involved in neurodegenerative diseases. PUFAs such as arachidonic (AA), and eicosapentaenoic acids (EPA) induce apoptosis in cultured cells. This effect has recently been shown to correlate with the inhibition of polyisoprenylated methylated protein methyl esterase (PMPMEase). We propose that abnormally high levels of PUFAs may be deleterious to neuronal cells. Enzyme kinetics analysis with PMPMEase revealed a 2‐fold decrease in KM from 16.2 ± 1.34 to 8.60 ± 1.20 and 7.84 ± 1.02 μM in the presence of 10 μM AA or EPA, respectively. A relatively severe degenerative effect of AA on SH‐SY5Y cells was observed in the absence of 10% FBS (EC50 of 6.2 μM). AA (2 – 10 μM) caused concentration and time‐dependent inhibition of neuritogenesis in retinoic acid (RA)‐differentiated SH‐SY5Y cells. Significant decreases in the percentage of neurite‐bearing cells and neurite lengths were observed after 24 h with 10 μM AA. These results suggest that excessive PMPMEase inhibition may extend beyond controlling cell viability to the degeneration of vulnerable cells such as neurons.Supported by NIH/NIGMS/SCORE (GM 08111‐35) & NIH/NCRR (G12 RR0 3020).

Rapid differentiation of human embryonal carcinoma stem cells (NT2) into neurons for neurite outgrowth analysis.

Human neurons derived from stem cells can be employed as in vitro models to predict the potential of neurochemicals affecting neurodevelopmental cellular processes including proliferation, migration, and differentiation. Here, we developed a model of differentiating human neurons from well characterized human embryonal carcinoma stem cells (NT2). NT2 cells were induced to differentiate into neuronal phenotypes after 2 weeks of treatment with retinoic acid in aggregate culture. Nestin positive progenitor cells migrate out of NT2 aggregates and differentiate into βIII-tubulin expressing neuronal cells. Culturing the NT2 cells for an additional 7-14 days resulted in increased percentage of βIII-tubulin expressing cells, elaborating a long neurite that positively stained for axonal marker (Tau) and presynaptic protein (synapsin). We then asked whether neurite outgrowth from NT2 cells is modulated by bioactive chemicals. Since the cAMP/PKA pathway has been widely investigated as a regulator of neurite outgrowth/regeneration in several experimental systems, we used chemical activators and inhibitors of cAMP/PKA pathway in the culture. The adenylyl cyclase activator, forskolin, and cell-permeable analog of cAMP, 8-Br-cAMP increased the percentage of neurite bearing cells and neurite extension. Application of the protein kinase A inhibitors, H-89 and Rp-cAMP, blocked neurite formation. Taken together, NT2 aggregates undergo migration, differentiation, and neurite elaboration and can be used as a model of differentiating human neurons to screen neurochemicals and to understand cellular mechanisms of human nerve cell development.

Material properties and electrical stimulation regimens of polycaprolactone fumarate–polypyrrole scaffolds as potential conductive nerve conduits

The mechanical and electrical properties of polycaprolactone fumarate–polypyrrole (PCLF–PPy) scaffolds were studied under physiological conditions to evaluate their ability to maintain the material properties necessary for application as conductive nerve conduits. PC12 cells cultured on PCLF–PPy scaffolds were stimulated with regimens of 10μA of either a constant or a 20Hz frequency current passed through the scaffolds for 1h per day. PC12 cellular morphologies were analyzed by fluorescence microscopy after 48h. PCLF–PPy scaffolds exhibited excellent mechanical properties at 37°C which would allow suturing and flexibility. The surface resistivity of the scaffolds was 2kΩ and the scaffolds were electrically stable during the application of electrical stimulation (ES). In vitro studies showed significant increases in the percentage of neurite bearing cells, number of neurites per cell and neurite length in the presence of ES compared with no ES. Additionally, extending neurites were observed to align in the direction of the applied current. This study shows that electrically conductive PCLF–PPy scaffolds possess the material properties necessary for application as nerve conduits. Additionally, the capability to significantly enhance and direct neurite extension by passing an electrical current through PCLF–PPy scaffolds renders them even more promising as future therapeutic treatments for severe nerve injuries.

A Trifluoromethyl Analog of Verbenachalcone Promotes Neurite Outgrowth and Cell Proliferation of NeuroScreen-1 Cells

Past research has shown that natural products of plant and marine origins and their congeners enhance the actions of neuritogenic factors of the central nervous system (CNS) such as nerve growth factor (NGF). However, the role of fluorine substitutions in their structure-activity relationship (SAR) has not been explored. We have synthesized a trifluoromethyl analog of verbenachalcone (VC), a pharmacologically active natural compound previously shown to potentiate NGF activity. This analog, designated C278, enhances neurite outgrowth and proliferation of NeuroScreen-1™ (NS-1) cells, a subclone of PC12 pheochromocytoma cells. C278 increases the percentage of neurite bearing cells in the presence of suboptimal doses of NGF in comparison with controls treated with NGF alone. In addition, C278 stimulates cell growth in reduced serum and serum-free cell culture conditions based on our observation of increases in cell number and metabolic assessment with MTT reduction and resazurin assays. The addition of C278 partially restored inhibition of NGF-induced neurite outgrowth by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U0126. Short-term sequential exposure of cells to U0126, C278, and NGF enhanced phosphorylation of extracellular signal-regulated kinase (ERK) in comparison with cells treated with only the MEK inhibitor and NGF. C278 also attenuated cell growth arrest caused by exposure to PD98059, U0126 and the phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002 but did not alter phosphorylation of Akt, a classic downstream target of PI3K during cell survival. These data suggest that C278 promotes NGF-dependent neurite outgrowth in NS-1 cells through a MEK signaling pathway by a mechanism that alters short-term activation of ERK. In contrast, C278 promotes PI3K-mediated survival independently of Akt phosphorylation.

PPARγ stimulation promotes neurite outgrowth in SH-SY5Y human neuroblastoma cells

Several evidences indicate that PPARγ stimulation promotes neuronal differentiation. However, to date, no data describe the effects of PPARγ agonists on neurite outgrowth. Here we have evaluated the effects of pioglitazone, a synthetic PPARγ agonist, on differentiation and neurite outgrowth in SH-SY5Y human neuroblastoma cells. Our results show that pioglitazone promotes cell differentiation and the outgrowth of cell processes in a concentration-dependent manner with the maximal effect at 100 nM–1 μM. It significantly increases both the mean process length and the percentage of neurite-bearing cells. In addition, these effects are accompanied by significant activation of p42 and p44 mitogen-activated protein kinases. In conclusion, albeit preliminary, these findings suggest the possibility that PPARγ stimulation may contribute to the development and maintenance of a proper neuronal connectivity within neuronal networks.

LIM Kinase and Slingshot Are Critical for Neurite Extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.

Rin GTPase Couples Nerve Growth Factor Signaling to p38 and b-Raf/ERK Pathways to Promote Neuronal Differentiation

In neuronal precursor cells, the magnitude and longevity of mitogen-activated protein (MAP) kinase cascade activation contribute to the nature of the cellular response, differentiation, or proliferation. However, the mechanisms by which neurotrophins promote prolonged MAP kinase signaling are not well understood. Here we defined the Rin GTPase as a novel component of the regulatory machinery contributing to the selective integration of MAP kinase signaling and neuronal development. Rin is expressed exclusively in neurons and is activated by neurotrophin signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to nerve growth factor (NGF)-mediated neuronal differentiation. Most surprisingly, although Rin was unable to stimulate MAP kinase activity in NIH 3T3 cells, it potently activated isoform-specific p38alpha MAP kinase signaling and weakly stimulated ERK signaling in pheochromocytoma (PC6) cells. This cell-type specificity is explained in part by the finding that Rin binds and stimulates b-Raf but does not activate c-Raf. Accordingly, selective down-regulation of Rin in PC6 cells suppressed neurotrophin-elicited activation of b-Raf and p38, without obvious effects on NGF-induced ERK activation. Moreover, the ability of NGF to promote neurite outgrowth was inhibited by Rin knockdown. Together, these observations establish Rin as a neuronal specific regulator of neurotrophin signaling, required to couple NGF stimulation to sustain activation of p38 MAP kinase and b-Raf signaling cascades required for neuronal development.

Influence of pulsed electromagnetic field with different pulse duty cycles on neurite outgrowth in PC12 rat pheochromocytoma cells

The influence of low frequency (50 Hz repetition rate) pulsed electromagnetic field (EMF) on PC12 cell neurite outgrowth in vitro was investigated in this study. We studied the percentage of neurite bearing cells, average length of neurites, and directivity of neurite outgrowth in PC12 cells cultured for 96 h in the presence of nerve growth factor (NGF). PC12 cells were exposed in one incubator to pulsed EMF at 1.36 mT (peak value) generated by a pair of Helmholtz coils, and the control samples were placed in another identical incubator. We found that the pulse duty cycle had significant effect on neurite outgrowth. Low (10%) pulse on-time significantly inhibited the percentage of neurite bearing cells, but at the same time increased the average length of neurites, while 100% on-time (DC) had exactly the opposite effects. Furthermore, we found that neurites were prone to extend along the direction of pulsed EMF with 10% pulse on-time. Our studies show that neurite outgrowth in PC12 cells is sensitive to the pulse duty and this sensitivity was associated with NGF concentration.

Neurotrophin-dependent Tyrosine Phosphorylation of Ras Guanine-releasing Factor 1 and Associated Neurite Outgrowth Is Dependent on the HIKE Domain of TrkA

Ras guanine-releasing factor 1 (RasGrf1), a guanine nucleotide exchange factor for members of the Ras and Rho family of GTPases, is highly expressed in the brain. It is regulated by two separate mechanisms, calcium regulation through interaction with its calcium/calmodulin-binding IQ domain and serine and tyrosine phosphorylation. RasGrf1 is activated downstream of G-protein-coupled receptors and the non-receptor tyrosine kinases, Src and Ack1. Previously, we demonstrated a novel interaction between the intracellular domain of the nerve growth factor-regulated TrkA receptor tyrosine kinase and an N-terminal fragment of RasGrf1. We now show that RasGrf1 is phosphorylated and interacts with TrkA, -B, and -C in co-transfection studies. This interaction and phosphorylation of RasGrf1 is dependent on the HIKE domain of TrkA (a region shown to interact with pleckstrin homology domains) but not on any of the phosphotyrosine residues that act as docking sites for intracellular signaling molecules such as Shc and FRS-2. The PH1 domain alone of RasGrf1 is sufficient for phosphorylation by the TrkA receptor. A potential role for Trk activation of RasGrf1 is suggested through transfection studies in PC12 cells in which RasGrf1 significantly increases neurite outgrowth at low doses of neurotrophin stimulation. Notably, this neurite outgrowth is dependent on an intact HIKE domain, as nnr5-S10 cells expressing a TrkA HIKE domain mutant do not exhibit potentiated neurite outgrowth in the presence of RasGrf1. These studies identify RasGrf1 as a novel target of neurotrophin activation and suggest an additional pathway whereby neurotrophin-stimulated neurite outgrowth may be regulated.

Retinoic acid-induced neuritogenesis of human neuroblastoma SH-SY5Y cells is ERK independent and PKC dependent.

Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA-induced neuritogenesis, we used human neuroblastoma SH-SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal-regulated kinase (ERK) 1 and 2 in RA-induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA-induced neuritogenesis (percentage of neurite-bearing cells and neurite length) but significantly reduced cell survival. In addition, we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA-induced ERK phosphorylation does not follow the classic Raf kinase-dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI 3-K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3-K inhibitor wortmannin, we observed a significant dose-dependent reduction in ERK phosphorylation. RA-induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration-dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA-induced neuritogenesis.

Critical period-dependent reduction of the permissiveness of cat visual cortex tissue for neuronal adhesion and neurite growth.

During postnatal development, the visual cortex undergoes an experience-dependent refinement of its circuitry. This process includes synapse formation, as well as synapse elimination. Both mechanisms appear to be restricted to a limited 'critical period' which lasts for approximately 2 months in cats. We tested whether the termination of the critical period for cortical malleability is paralleled by changes in the growth permissiveness of the tissue. These changes may inhibit progressive reorganization of functional circuitries mediated by axon growth. Embryonic cortical neurons were cultured on unfixed cryostat sections of the visual cortex obtained from cats aged 2-50 weeks. After 2-3 days in vitro the distribution of viable cells and the percentage of neurite-bearing cells were determined and analysed with respect to the developmental age and subdivisions of the underlying tissue substrate. It was shown that cell adhesion and neurite formation are correlated with the developmental age of the substrate tissue and the time period of myelination. While embryonic neurons adhered and survived on grey and white matter tissue from 2- and 4-week-old kittens, there was a significant reduction in cell adhesion on the myelinated white matter regions of the tissue sections of older animals. Quantitative analyses showed that neurite formation by cultured neurons also became successively impaired on grey and white matter areas of tissue substrates, corresponding to the time course of the critical period for cortical malleability. On grey matter tissue this effect was most pronounced between the second and sixth postnatal weeks. The effects were not antagonized by coating the substrate sections with the growth-promoting molecule laminin. It is therefore proposed that neurite growth-inhibiting factors, most probably associated with central nervous system myelin, are gradually expressed postnatally and may contribute to the termination of the critical period in the visual cortex of cats.

Ethanol enhances neurite outgrowth in primary cultures of rat cerebellar macroneurons

Effects of ethanol on neurite outgrowth and morphometry were investigated in primary cultures of rat cerebella. Cell cultures were prepared from cerebella on embryonic day 17 (E17) for treatment with a series of ethanol concentrations (50, 75, 100, 150 and 200 mM). Ethanol did not reduce neuronal survival or attachment to the substrate at any of the concentrations that were used. Treatment with 75 mM ethanol significantly enhanced neurite outgrowth. Measurements from dissociated cultures exposed to 75 mM ethanol immediately after plating showed a significant increase in the percentage of neurite-bearing cells after 8 and 24 h in vitro. Measurements of the area and perimeter of neuronal cell bodies in dissociated cell cultures showed that the cell bodies of ethanol-treated neurons were also larger than those of control neurons. Ethanol was also associated with significant increases in the total neuritic length per cell and in the length of the longest neurite in each cell. The mean number of neurite branches was also greater in the ethanol-treated neurons. Measurements from suspension cell cultures, in which dissociated cells were suspended overnight in the presence of 75 mM ethanol prior to plating, corroborated these results. These findings suggest that ethanol may have distinct effects on neurite initiation and outgrowth and branching. The cellular mechanisms involved and the functional significance of these effects are currently not known. The present results also indicated that high concentrations of ethanol (150–200 mM) and long periods of exposure (4–7 days) were required to produce toxic effects on neurons and glial cells in this system.

Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats.

The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats.

Microinjection of a p21ras Antibody into PC12 Cells Inhibits Neurite Outgrowth Induced by Nerve Growth Factor and Basic Fibroblast Growth Factor

The role of p21ras in signal transduction in PC12 cells was studied using an antibody that blocks its function. Native cells were microinjected with either a control solution or a solution containing the monoclonal antibody Y13-259. Treatment of the cells with growth factors appeared to enhance the ability of the cells to survive the microinjection procedure. Of the cells microinjected with the control solution 66-69% of those treated with either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) were still present 24 h post-injection, compared with only 57% for those not treated with growth factor after microinjection. This effect of the growth factors was inhibited by introduction of the Y13-259 antibody, suggesting that it occurs through a pathway that involves p21ras. Similarly, introduction of the Y13-259 antibody into cells also resulted in a statistically significant decrease in the percentage of neurite-bearing cells; 25-36% of the cells microinjected with the control solution had neurites, whereas 12-14% of the cells microinjected with the antibody solution had neurites. This decrease suggests that the induction of neurite outgrowth and the maintenance of established neurites by these growth factors is dependent on a functional p21ras pathway. As well as complementing the finding that p21ras is apparently involved in the mechanism of action of NGF in PC12 cells, these results further establish (1) that p21ras is also involved in the mechanism of action of bFGF, and (2) that the effect of NGF and bFGF on the number of labeled cells still present 24 h postinjection requires a functional p21ras protein.

Serum vulnerability and time-dependent stabilization of neurites induced by nerve growth factor in PC12 pheochromocytoma cells.

Cultures of PC12 pheochromocytoma cells were established on a polyornithine substratum in medium supplemented with the chemically defined N1 mixture in the presence or absence of Nerve Growth Factor (NGF). Normal cell proliferation in the absence of NGF was equally competent when fetal calf serum (FCS) was replaced with N1-supplemented medium. The differentiation of PC12 cells, which occurs upon NGF treatment, ultimately results in cell death without the addition of 0.1% FCS to the N1-supplemented medium. The combination of N1, 0.1% FCS, and NGF permits the PC12 cells to develop a neuritic outgrowth much earlier than when higher (1-10%) FCS levels are used. Neurite retraction is caused in a dose-dependent manner by a delayed presentation of FCS. Within 2 days of serum presentation, however, neurites regrow to achieve that percentage of neurite-bearing cells which is seen without a serum challenge. Moreover, the retraction response becomes less pronounced with time over the 8-day culture period for any given serum concentration. Among the N1 ingredients, only insulin and transferrin are needed by PC12 cells for survival whether in the dividing state or not. Neurite growth was not dependent on any of the N1 components.