Isolation and some characteristics of an adhesive factor of brain that enhances neurite outgrowth in central neurons

Fractionation of octyl glucoside-solubilized proteins from young rat brain was monitored using rat brain neurons, which were cultured in microwells coated with various protein fractions to be studied. An adhesive protein that promotes neurite outgrowth in rat brain neurons was isolated by chromatography on heparin-Sepharose followed by Affi-Gel blue. The apparent molecular mass of the protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions was about 30 kilodaltons (p30). Under nonreducing conditions a closely spaced doublet band was observed corresponding to 27-28-kilodalton size. Gel filtration in the presence of 4 M urea indicated the molecular size of 58 kilodaltons suggesting a dimeric structure. Western blotting experiments using affinity-purified rabbit antibodies detected p30 as an immunochemically distinct protein in brain and in N18 neuroblastoma cells. The p30 protein was also detected in the N18 cells by lactoperoxidase-catalyzed cell surface iodination. Western blotting of heparin-binding proteins solubilized from brains of rats of various age groups indicated that p30 is clearly more abundant in perinatal brain as compared to adult tissue. The neuron-binding and neurite outgrowth-promoting properties of p30 as well as the developmental regulation of its content in brain tissue suggest a role in neuronal growth.     

Expression of P30, a protein with adhesive properties, in Schwann cells and neurons of the developing and regenerating peripheral nerve

P30 is a heparin-binding protein with adhesive and neurite outgrowth-promoting properties present at high levels in the developing rat central nervous system (Rauvala, H., and R. Pihlaskari. 1987 J. Biol. Chem. 262:16625-16635). Partial sequencing of p30 has revealed homology or identity with HMG-1 (Rauvala, H., J. Merenmies, R. Pihlaskari, M. Korkolainen, M.-L. Huhtala, and P. Panula. 1988. J. Cell Biol. 107:2292-2305), a 28-kD protein that was originally purified from the thymus (Goodwin, G.H., C. Sanders, and E. W. Johns. 1973. Eur. J. Biochem. 38:14-19) which binds DNA in vitro. We have analyzed the distribution of p30 in the developing rat peripheral nervous system (PNS). P30 was detected by immunohistochemistry and Western blot analysis using antibodies raised against intact p30 and against a synthetic peptide corresponding to the amino terminus of the p30 molecule. P30 was localized to nonnuclear compartments of neurons and peripheral glial cells (Schwann cells). P30 immunoreactivity of PNS neurons persisted into adulthood. In contrast, Schwann cell staining decreased after the second postnatal week and was not detectable in adult animals. Neuron-Schwann cell contact was correlated with diminished p30 levels in Schwann cells. Schwann cells of the normal adult sciatic nerve did not express p30; however, when deprived of axonal contact by nerve transection, the Schwann cells of the distal nerve stained intensely for p30. In addition, when Schwann cells and dorsal root ganglion neurons were grown in coculture, Schwann cells that were associated with neurites were not as intensely stained by anti-p30 as Schwann cells that were not in contact with neurons. The pattern of p30 expression during development and regeneration, and its apparent regulation by cell-cell contact suggests that p30 plays a role in the interaction between neurons and Schwann cells during morphogenesis of peripheral nerves.     

30-kDa heparin-binding protein of brain (amphoterin) involved in neurite outgrowth. Amino acid sequence and localization in the filopodia of the advancing plasma membrane

A cDNA library constructed from mRNA of rat brain was used to clone the cDNA that encodes the 30-kDa heparin-binding protein (amphoterin) that is developmentally regulated in brain and enhances neurite outgrowth in cerebral neurons. cDNA and peptide sequencing identified a dipolar sequence that has been previously found in studies of high mobility group 1 protein: the 184-amino acid cationic region is followed by a cluster of 30 anionic residues. The mRNA encoding amphoterin is also developmentally regulated; it is strongly reduced in quantity after the rapid perinatal growth phase of the rat brain. Anti-synthetic peptide antibodies raised according to the sequence of amphoterin were shown to bind specifically to the protein isolated from brain, and were used to detect amphoterin in subcellular fractions and in immunostaining of cells. Amphoterin was found in the cytoplasm of the cell soma, in the cell processes, and the substrate-attached material. In cells that are at an active stage of spreading and extending their cytoplasmic processes amphoterin was especially associated with plasma membrane filopodia. The distinct localization to the filopodia of the advancing plasma membrane suggests that endogenous amphoterin has a role in the extension of neurite-type cytoplasmic processes in developing cells. This inference is further supported by the finding that both anti-amphoterin and the anti-synthetic peptide antibodies in the culture media strongly inhibit the outgrowth of cytoplasmic processes.     

Multiple and alternative adhesive responses on defined substrata of an immortalized dorsal root neuron hybrid cell line

Attachment and neurite extension processes have been evaluated for an immortalized derivative cell of a rat dorsal root neuron after fusion with a mouse neuroblastoma cell (the clonal F11 hybrid cell line) and these processes compared with previous studies of neuroblastoma cells, since both cell types may be derived from the neural crest of the developing embryo. Biochemically defined substrata were provided by human plasma fibronectin (pFN), the heparan sulfate-binding protein platelet factor-4 (PF4), and the ganglioside GM1-binding protein cholera toxin B subunit (CTB). While some attachment of unsupplemented cells was noted on CTB substrata, GM1 supplementation permitted F11 cells to attach as well on CTB as on pFN or PF4. On PF4, very few neurite processes were observed while on pFN two morphologically distinct types of neurites could be identified: short, linear processes in a low percentage of cells resembling those of neuroblastoma cells and long, irregular and narrow processes in a higher percentage of cells resembling those of dorsal root neurons. On CTB, neurites of the latter class were even more prominent; however, cell bodies on CTB failed to spread by cytoplasmic extension as commonly observed in F11 cells on pFN and, to some extent, on PF4. The formation of both neurite classes on either pFN or CTB was completely inhibited by low concentrations of an RGDS (Arg-Gly-Asp-Ser) peptide in the medium of cultures, indicating the significance of pFN's binding to cell surface integrin or ganglioside GM1's possible interaction with integrin for mediating the differentiative process. In contrast, neurite formation of neuroblastoma cells is refractile to the soluble peptide as reported previously. Neurite extensions of F11 cells on either pFN or CTB were comparably sensitive to low concentrations of cytochalasin D, revealing the mediation of microfilament reorganization in these processes. Treatment of F11 cells with cycloheximide failed to inhibit neurite extension on pFN but did partially inhibit extension on CTB; this contrasts with the very high sensitivity of neurite formation by neuroblastoma cells on CTB substrata reported previously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices

We have compared neurite outgrowth on extracellular matrix (ECM) constituents to outgrowth on glial and muscle cell surfaces. Embryonic chick ciliary ganglion (CG) neurons regenerate neurites rapidly on surfaces coated with laminin (LN), fibronectin (FN), conditioned media (CM) from several non-neuronal cell types that secrete LN, and on intact extracellular matrices. Neurite outgrowth on all of these substrates is blocked by two monoclonal antibodies, CSAT and JG22, that prevent the adhesion of many cells, including neurons, to the ECM constituents LN, FN, and collagen. Neurite outgrowth is inhibited even on mixed LN/poly-D-lysine substrates where neuronal attachment is independent of LN. Therefore, neuronal process outgrowth on extracellular matrices requires the function of neuronal cell surface molecules recognized by these antibodies. The surfaces of cultured astrocytes, Schwann cells, and skeletal myotubes also promote rapid process outgrowth from CG neurons. Neurite outgrowth on these surfaces, though, is not prevented by CSAT or JG22 antibodies. In addition, antibodies to a LN/proteoglycan complex that block neurite outgrowth on several LN-containing CM factors and on an ECM extract failed to inhibit cell surface-stimulated neurite outgrowth. After extraction with a nonionic detergent, Schwann cells and myotubes continue to support rapid neurite outgrowth. However, the activity associated with the detergent insoluble residue is blocked by CSAT and JG22 antibodies. Detergent extraction of astrocytes, in contrast, removes all neurite- promoting activity. These results provide evidence for at least two types of neuronal interactions with cells that promote neurite outgrowth. One involves adhesive proteins present in the ECM and ECM receptors on neurons. The second is mediated through detergent- extractable macromolecules present on non-neuronal cell surfaces and different, uncharacterized receptor(s) on neurons. Schwann cells and skeletal myotubes appear to promote neurite outgrowth by both mechanisms.     

AlphaII-spectrin participates in the surface expression of cell adhesion molecule L1 and neurite outgrowth

AlphaII-spectrin, a basic component of the spectrin-based scaffold which organizes and stabilizes membrane microdomains in most animal cells, has been recently implicated in cell adherence and actin dynamics. Here we investigated the contribution of αΙΙ-spectrin to neuritogenesis, a highly complex cellular process which requires continuous actin cytoskeleton remodeling and cross-talk between extracellular cues and their cell surface receptors, including cell adhesion molecules. Using RNA interference-mediated gene silencing to down-regulate αΙΙ-spectrin expression in human neuroblastoma SH-SY5Y cells, we observed major changes in neurite morphology and cell shape: (1) reduced mean length and a higher number of neurites per cell; occasional long neurites were thinner and displayed abnormal adhesiveness during cell migration resulting in frequent breaks; similar persisting adhesiveness and breaks were also observed in trailing edges of cell bodies; (2) irregular polygonal cell shape in parallel with loss of cortical F-actin from neuronal cell bodies; (3) reduction in protein levels of αΙ- and βΙ-spectrins, but not βΙΙ-spectrin (4) decreased global expression of adhesion molecule L1 and spectrin-binding adapter ankyrin-B, which links L1 to the plasma membrane. Remarkably, αΙΙ-spectrin depletion affected L1 – but not NCAM – cell surface expression, and L1 clustering at growth cones. This study demonstrates that αΙΙ-spectrin is implicated in normal morphology and adhesive properties of neuron cell bodies and neurites, and in cell surface expression and organization of adhesion molecule L1.     

Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1

BACKGROUND: On the basis of experiments suggesting that Notch and Delta have a role in axonal development in Drosophila neurons, we studied the ability of components of the Notch signaling pathway to modulate neurite formation in mammalian neuroblastoma cells in vitro. RESULTS: We observed that N2a neuroblastoma cells expressing an activated form of Notch, Notch1(IC), produced shorter neurites compared with controls, whereas N2a cell lines expressing a dominant-negative Notch1 or a dominant-negative Delta1 construct extended longer neurites with a greater number of primary neurites. We then compared the effects on neurites of contacting Delta1 on another cell and of overexpression of Delta1 in the neurite-extending cell itself. We found that N2a cells co-cultured with Delta1-expressing quail cells produced fewer and shorter neuritic processes. On the other hand, high levels of Delta1 expressed in the N2a cells themselves stimulated neurite extension, increased numbers of primary neurites and induced expression of Jagged1 and Notch1. CONCLUSIONS: These studies show that Notch signals can antagonize neurite outgrowth and that repressing endogenous Notch signals enhances neurite outgrowth in neuroblastoma cells. Notch signals therefore act as regulators of neuritic extension in neuroblastoma cells. The response of neuritic processes to Delta1 expressed in the neurite was opposite to that to Delta1 contacted on another cell, however. These results suggest a model in which developing neurons determine their extent of process outgrowth on the basis of the opposing influences on Notch signals of ligands contacted on another cell and ligands expressed in the same cell.     

Cooperativity of ganglioside-dependent with protein-dependent substratum adhesion and neurite extension of human neuroblastoma cells

The potential involvement of gangliosides in the adherence and neurite extension of human neuroblastoma cells (Platt and La-N1) was investigated on tissue culture substrata coated with the ganglioside GM1-binding protein, cholera toxin B (CTB) subunit, for comparison with similar processes on plasma fibronectin (pFN)-coated substrata. Cells attached with reduced efficiency on CTB substrata as compared with pFN substrata and required a much longer time to form neurite processes for a small percentage of cells on CTB. The specificity of these processes for GM1 binding was tested in a variety of ways. Supplementation of the cells with exogenous GM1, but not GD1a, identified a larger population of cells adherent on CTB (comparable to pFN-adherent cells) and dramatically increased the proportion of cells capable of forming neurites without reducing the time requirement. In ultrastructural studies using the scanning electron microscope (SEM) and immunofluorescence (IF) analyses to discriminate microtubule distributions, neurites of GM1-supplemented cells on CTB were virtually identical with pFN-adherent neurites, whereas unsupplemented cells on CTB generated processes with fine-structural differences. Treatment of cells during the GM1 supplementation period with cycloheximide completely abolished the ability of cells to generate neurites on CTB and decreased the adhesive capacity of cells as well; a similar treatment of cells had no adverse effect on adherence or neurite extension on pFN. The importance of one or more proteins in GM1-dependent processes was further confirmed by demonstrating the trypsin sensitivity of a cell surface component(s) required to achieve maximal attachment on CTB; in contrast, adherence and neurite extension on pFN were much more resistant to this treatment process. Therefore, these experiments demonstrate (a) that certain cell surface gangliosides are capable of mediating adherence and neurite outgrowth of human neuroblastoma cells on a suitable ganglioside-binding substratum; (b) this ganglioside dependence is cooperative with one or more cell surface proteins which can now be analysed. These results are discussed in light of the identification in ref. [16] (Exp cell res 169 (1987) 311) of a second ‘cell-binding’ domain on the pFN molecule competent for adherence and neurite extension of these neuroblastoma cells, as well as the potential role of pFN binding to a complex ganglioside on the surface of these neural tumor cells in these processes.     

Recombinant myelin-associated glycoprotein confers neural adhesion and neurite outgrowth function

Myelin-associated glycoprotein (MAG) cDNA clones for the small (p67) and large (p72) forms were expressed in heterologous cells. Purified recombinant MAG protein was incorporated into fluorescent liposomes, and both forms were shown to bind predominantly to neurites in DRG or spinal cord cultures. This adhesion was completely blocked by Fab fragments of monoclonal anti-MAG antibody. Liposomes prepared with the control protein glycophorin or no protein failed to bind neurites. Small cerebellar neurons, which are not myelinated in vivo, failed to bind MAG liposomes. In a second test of function, p67 MAG-transfected fibroblasts were markedly enhanced in their ability to promote DRG neurite extension over a 2 day culture period compared with control fibroblasts not expressing MAG. Neurite extension was blocked by anti-MAG antibodies. These results show that both forms of MAG can facilitate the interactions between glial cells and neurites that ultimately lead to myelin formation.     

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.     

Heterophilic interactions of DM-GRASP: GRASP-NgCAM interactions involved in neurite extension

DM-GRASP is an immunoglobulin superfamily cell adhesion molecule that is expressed in both the developing nervous and immune system. Specific populations of neurons respond to DM-GRASP substrates appears to require homophilic interactions between DM-GRASP molecules. We were interested in determining whether DM-GRASP interacts heterophilically with other ligands as well. We have found that eleven proteins from embryonic chick brain membranes consistently bind to and elute from a DM-GRASP-Sepharose affinity column. One of these proteins is DM-GRASP itself, consistent with its known homophilic binding. Another protein, at 130 kD, is immunoreactive with monoclonal antibodies to NgCAM. Other neural cell adhesion molecules were not detected in the eluate. The DM- GRASP-Sepharose eluate also contains a potent neurite stimulating activity, which cannot be accounted for by either DM-GRASP or NgCAM. To investigate the interaction of DM-GRASP and NgCAM, antibodies against DM-GRASP were added to neuronal cultures extending neurites on an NgCAM substrate. The presence of antibodies to DM-GRASP decreased neurite extension on laminin, suggesting that the antibody is not toxic or generally inhibiting motility. We present two possible models for the DM-GRASP-NgCAM association and a hypothesis for neural cell adhesion function that features the dimerization of cell adhesion molecules.     

Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.     

Effects of eosinophils on nerve cell morphology and development: the role of reactive oxygen species and p38 MAP kinase

The adhesion of eosinophils to nerve cells and the subsequent release of eosinophil products may contribute to the pathogenesis of conditions such as asthma and inflammatory bowel disease. In this study we have separately examined the consequences of eosinophil adhesion and degranulation for nerve cell morphology and development. Eosinophils induced neurite retraction of cultured guinea pig parasympathetic nerves and differentiated IMR32 cholinergic neuroblastoma cells. Inhibition of eosinophil adhesion to IMR32 cells attenuated this retraction. Eosinophil adhesion to IMR32 cells led to tyrosine phosphorylation of a number of nerve cell proteins, activation of p38 MAP kinase, and generation of neuronal reactive oxygen species (ROS). Inhibition of tyrosine kinases with genistein prevented both the generation of ROS in the nerve cells and neurite retraction. The p38 MAP kinase inhibitor SB-239063 prevented neurite retraction but had no effect on the induction of ROS. Thus eosinophils induced neurite retraction via two distinct pathways: by generation of tyrosine kinase-dependent ROS and by p38 MAP kinase. Eosinophils also prevented neurite outgrowth during differentiation of IMR32 cells. In contrast to their effect on neurite retraction, this effect was mimicked by medium containing products released from eosinophils and by eosinophil major basic protein. These results indicate that eosinophils modify the morphology of nerve cells by distinct mechanisms that involve adhesion and released proteins.     

Adhesion among neural cells of the chick embryo. IV. Role of the cell surface molecule CAM in the formation of neurite bundles in cultures of spinal ganglia

The cell adhesion molecule (CAM) is involved in adhesion among embryonic retinal and brain cells and has been detected in a variety of neural tissues. This paper describes the use of spinal ganglion cultures and specific anti-CAM antibodies to determine the distribution of CAM on plasma membranes of nerve processes, and to assess the results of perturbation of its function during the growth of neurites from ganglia. The results indicate that CAM is distributed over the entire surface of nerve processes, and that specific anti-CAM Fab' fragments alter the morphology of neurite outgrowth. In particular, it was observed that anti-CAM inhibits formation of nerve bundles, so that the ganglion becomes surrounded by a tangled net of fine processes. Growth cone functions, such as neurite elongation, motility, and attachment to the substratum, did not appear to be affected by the antibody. These studies suggest that one of the major functions of CAM is to mediate side-to-side adhesion between neurites to form fascicles, and raise the possibility that this molecule serves a key role in embryogenesis of nerve tissues.     

Purified N-cadherin is a potent substrate for the rapid induction of neurite outgrowth

N-cadherin is the predominant mediator of calcium-dependent adhesion in the nervous system (Takeichi, M. 1988. Development (Camb.). 102: 639-655). Investigations using antibodies to block N-cadherin function (Bixby, J.L., R.L. Pratt, J. Lilien, and L.F. Reichardt. 1987. Proc. Natl. Acad. Sci. USA. 84:2555-2569; Bixby, J.L., J. Lilien, and L.F. Reichardt. 1988. J. Cell Biol. 107:353-362; Tomaselli, K.J., K.N. Neugebauer, J.L. Bixby, J. Lilien, and L.F. Reichardt. 1988. Neuron. 1:33-43) or transfection of the N-cadherin gene into heterologous cell lines (Matsunaga, M., K. Hatta, A. Nagafuchi, and M. Takeichi. 1988. Nature (Lond.). 334:62-64) have provided evidence that N-cadherin, alone or in combination with other molecules, can participate in the induction of neurite extension. We have developed an affinity purification procedure for the isolation of whole N-cadherin from chick brain and have used the isolated protein as a substrate for neurite outgrowth. N-cadherin promotes the rapid extension of neurites from chick ciliary ganglion neurons, which extend few or no neurites on adhesive but noninducing substrates such as polylysine, tissue culture plastic, and collagens. N-cadherin is extremely potent, more so than the L1 adhesion molecule, and comparable to the extracellular matrix protein laminin. Compared to laminin, however. N-cadherin promotes outgrowth from ciliary ganglion neurons extremely rapidly and with a distinct morphology. These results provide a direct demonstration that N-cadherin is sufficient to induce neurite outgrowth when substrate bound and suggest that the mechanism(s) involved may differ from that induced by laminin.     

An 18-kd heparin-binding protein of developing brain that is distinct from fibroblast growth factors.

An 18-kd heparin-binding protein (p18) was isolated from perinatal rat brain. Although the protein closely resembles the fibroblast growth factors in its strong binding to heparin and in its apparent molecular mass, it has a distinct structure. This was concluded from the amino-terminal sequence analysis that identified a unique structure containing a cluster of lysine residues. Antipeptide antibodies were raised in rabbits according to the sequence analysis and affinity purified using a synthetic peptide. The antibodies were shown to bind specifically to p18, which was immunochemically distinct from the basic fibroblast growth factor. The antipeptide antibodies detected p18 in brain but not in liver, kidney, heart or skeletal muscle. The content of the protein was shown to undergo a remarkable developmental change corresponding to the time period of rapid sprouting of axons and dendrites in brain. The content of p18 was rapidly increased at the time of birth until the postnatal age of approximately 1 week, after which it was decreased to values less than 10% in young adults as compared to the content found in perinatal rats. p18 also enhanced neurite outgrowth in brain neurons in vitro. The protein was stained in neurons in cells dispersed from perinatal brain. The properties of p18 suggest that it has a role in the growth and maturation of brain.     

Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition

Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.     

JNK phosphorylation of paxillin, acting through the Rac1 and Cdc42 signaling cascade, mediates neurite extension in N1E-115 cells

Neurons extend neurites from the cell body before formation of the polarized processes of an axon and dendrites. Neurite outgrowth involves remodeling of the cytoskeletal components, which are initially regulated by small GTPases of the Rho family. Here we show that c-Jun N-terminal kinase (JNK), which is controlled by Rho GTPases Rac1 and Cdc42, is activated following neurite extension in mouse N1E-115 neuroblastoma cells as a model. The extension is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) and Clostridium difficile Toxin B, the inhibitor for Rho GTPases. Additionally, paxillin, the multifunctional focal adhesion protein, is phosphorylated at Ser 178 by upregulation of the Rac1/Cdc42/JNK cascade. Conversely, transfection of the paxillin construct harboring the Ser 178-to-Ala mutation into cells inhibits neurite extension. Taken together, these results suggest the novel role of the Rac1/Cdc42/JNK signaling cascade in neurite extension and indicate that the downstream target paxillin may be one of the convergent points of various signaling pathways underlying neurite extension.     

The role of cell adhesion molecules in neurite outgrowth on Müller cells

The roles of neural cell adhesion molecule (NCAM), L1, N-cadherin, and integrin in neurite outgrowth on various substrates were studied. Antibodies against these cell surface molecules were added to explants of chick retina and the neurites from retinal ganglion cells were examined for effects of the antibodies on neurite length and fasciculation. On laminin, an anti-integrin antibody completely inhibited neurite outgrowth. The same antibody did not inhibit neurite outgrowth on polylysine or Müller cells. Antibodies to NCAM, L1, and N-cadherin did not significantly inhibit neurite outgrowth on laminin but produced significant inhibition on Müller cells. The inhibition of neurite outgrowth on glia by anti-L1 antibodies supports the hypothesis that L1 is capable of acting in a heterophilic binding mechanism. On laminin, both anti-N-cadherin and anti-L1 caused defasciculation of neurites from retinal ganglion cells, while anti-NCAM did not. None of these antibodies produced defasciculation on Müller cells. The results indicate that these three cell adhesion molecules may be very important in interactions with glia as axons grow from the retina to the tectum and may be less important in axon-axon interactions along this pathway. No evidence was found supporting the role of integrins in axon growth on Müller cells.     

Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM-FGFR-dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM-dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM-responsive axons.