An 82-Kilodalton Membrane Protein that Inhibits the Activity of Neurite Outgrowth Factor

Neurite outgrowth factor (NOF), an extracellular matrix glycoprotein of 700 kilodaltons (kDa), promoted neurite outgrowth from cultured ciliary ganglion (CG) neurons of chicken embryo. A fraction solubilized with Nonidet P-40 of chicken gizzard muscle membranes inhibited the neurite-promoting activity of NOF in a dose-dependent manner, but not that of laminin. Binding of CG neurons to the substratum and their survival were not affected by the extract. The inhibitory activity of the extract was abolished by treatment with trypsin or heat. The molecular size was determined to be about 82 kDa by ligand blotting. The active component was partially purified by column chromatography. It is suggested that this molecule interacts with the domain of NOF responsible for its neurite-promoting activity and may modulate NOF activity during development in vivo.     

Laminin promotes neuritic regeneration from cultured peripheral and central neurons

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co- purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.     

A neurite-promoting factor from muscle supports the survival of cultured chicken spinal motor neurons.

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.     

Laminin fragment E8 mediates PC12 cell neurite outgrowth by binding to cell surface beta 1,4 galactosyltransferase

A number of cell surface receptors bind to distinct laminin domains, thereby mediating laminin's diverse biological activities. Cell surface beta 1,4-galactosyltransferase (GalTase) functions as one of these laminin receptors, facilitating mesenchymal cell migration and PC12 cell neurite outgrowth on laminin. In this study, the GalTase binding site within laminin was identified as the E8 fragment by assaying purified fragments and by immunoprecipitating and immunoblotting galactosylated laminin using E8-reactive antibodies. Compared with intact laminin and other laminin fragments, E8 possessed the highest GalTase binding activity, using both membrane-bound and solubilized GalTase. More significantly, the neurite-promoting activity of fragment E8 was shown to be dependent upon its interaction with GalTase. Pregalactosylating purified E8 eliminated subsequent GalTase binding and consequently inhibited neurite initiation; parallel studies on laminin fragments E1-4 or E1 failed to affect neurite outgrowth. Furthermore, anti-GalTase IgG inhibited neurite initiation on purified E8 substrates; control IgG had no effect. These results localize the predominant GalTase binding domain in laminin to fragment E8 and demonstrate that the neurite-promoting activity of E8 is dependent upon its interaction with GalTase.     

A neurite-promting factor from muscle supports the survival of cultured chicken spinal motor neurons

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%–6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutininhorseradish peroxidase or the fluorescent dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.     

Core Protein of Chondroitin Sulfate Proteoglycan Promotes Neurite Outgrowth from Cultured Neocortical Neurons

Chondroitin sulfate proteoglycan (CS-PG) was purified from rat brain and examined for its effect on neurite outgrowth in primary cultures of embryonic rat neocortical neurons. Neurite outgrowth was increased in culture wells coated with CS-PG. The core protein and glycosaminoglycan (GAG) prepared from the CS-PG were also examined for neurite-promoting activity. The activity was observed in culture wells coated with the core protein but not with GAG. These results suggest that CS-PG stimulates neurite outgrowth from the cultured neurons via its core protein.     

Effects of astroglia on the development of cultured neurons from embryonic rat cerebral cortex.

1. Previous studies from our laboratory demonstrated that subcultured astroglia enhance neurite outgrowth and survival of cultured neurons from embryonic rat cerebral cortex, but suppress proliferation of neuroblasts. 2. In the present study, the mechanisms of these three effects were further investigated. 3. Dissociated neurons were seeded on poly-L-lysine-coated coverslips which were plated on subcultured astroglia, and the survival, proliferation and neurite outgrowth of the neurons were investigated. Under these conditions, survival and antimitotic effects were also observed, while neurite extension was not stimulated. 4. The results clearly indicate that neuronal survival and proliferation are regulated by soluble factors produced by astroglia. 5. We also postulated that the neurite-promoting effect of astroglia is mediated by cell-cell contact. 6. This idea was confirmed by the finding that neurite extension was enhanced when the neurons were cultured directly on heat-treated astroglia. 7. The neurite-promoting effect was found to be specific to astroglia. 8. We preliminarily characterized the astroglial surface neurite-promoting factors (ASNPFs). 9. The relationship of laminin to ASNPFs was examined by using antibody to laminin. Laminin antibody did not inhibit the ASNPF activity. 10. The effect of digestion of heat-treated astroglia with enzymes (sialidase and endo-beta-galactosidase) on the ASNPF activity was also examined. 11. These enzyme treatments did not inhibit the ASNPF activity. 12. These results suggest that enhancement of the neurite-promoting activity is not associated with the sugar moiety of ASNPFs.     

Identification of molecules in leech extracellular matrix that promote neurite outgrowth

The molecular composition of the substrate is of critical importance for neurite extension by isolated identified leech nerve cells in culture. One substrate upon which rapid growth occurs in defined medium is a cell-free extract of extracellular matrix (ECM) that surrounds the leech central nervous system (CNS). Here we report the co-purification of neurite-promoting activity with a laminin-like molecule. High molecular mass proteins from leech ECM purified by gel filtration exhibited increased specific activity for promoting neurite outgrowth. The most active fractions contained three major polypeptide bands of ca. 340, 250 and 220 kDa. Electron microscopy of rotary-shadowed samples showed three macromolecules, one of which had a cross-shaped structure similar to vertebrate laminin. A second six-armed molecule resembled vertebrate tenascin and a third rod-like molecule resembled vertebrate collagen type IV. The most active fractions contained a protein of ca. 1 MDa on non-reducing gels with disulphide-linked subunits of ca. 220 and 340 kDa, with cross-shaped laminin-like molecules. We conclude that a laminin-like molecule represents a major neurite promoting component present in leech ECM. The experiments represent a first step in determining the location of leech laminin within the CNS and assessing its role in neurite outgrowth during development and regeneration.     

Purification and characterization of a neurite outgrowth factor from chicken gizzard smooth muscle

Chicken gizzard extract contains a macromolecular glycoprotein that promotes neurite outgrowth of dissociated neurons from the ciliary ganglia of chick embryos. Using conventional purification procedures, the factor responsible for the neurite outgrowth (neurite outgrowth factor (NOF)) was purified about 2000-fold to an apparent single protein band (as judged by agarose-polyacrylamide gel electrophoresis). Twenty fmol/cm2 of the purified NOF bound to the culture well was sufficient to exert maximal neuritic response of cultured ciliary ganglia neurons from 8-day-old chick embryos. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that NOF migrated as a single polypeptide of 700 and 210 kDa under nonreducing and reducing conditions, respectively. NOF stained with periodic acid-Schiff reagent and had a sedimentation coefficient of 12 s, a Stokes radius of 114 A, and an isoelectric point of about 5.1. Gizzard NOF was trypsin-sensitive, but resistant to treatment with heparinase, beta-galactosidase, and neuraminidase. Antibody prepared against the purified NOF blocked NOF activity in a dose-dependent manner. The antibody did not inhibit the biological activity of mouse laminin, although it cross-reacted weakly with laminin. Immunohistochemical analysis showed that the antibody against NOF strongly stained the extracellular matrix of cells in thin sections of gizzard, skeletal muscle, heart, liver, and ciliary ganglion, and also the membrane and the cytoplasm of cultured gizzard muscle cells. The present data suggest that gizzard NOF is a novel extracellular matrix glycoprotein which has a role in neurite outgrowth promotion from peripheral neurons in vivo. Although unlikely, the possibility that the NOF is a chick laminin could not be excluded.     

Merosin promotes cell attachment and neurite outgrowth and is a component of the neurite-promoting factor of RN22 schwannoma cells.

The laminin-like protein merosin was purified from human placenta in intact form and as pepsin fragments and compared to laminin in heparin affinity chromatography and cell binding assays. Intact merosin and a small fragment of merosin comprising the last two repeats of the heavy chain g domain bind to heparin. Intact merosin and large pepsin fragments of merosin, but not the small C-terminal fragment, mediate the attachment and spreading of several types of cells and promote neurite outgrowth from neuronal cells similar to laminin and its corresponding fragments. Cells with various integrin-type receptors for laminin attached equally well to merosin and laminin, suggesting that several of the known laminin binding receptors also bind to merosin. Antibodies to the beta 1 subunit of integrins inhibited neurite outgrowth on merosin as well as on laminin, confirming the involvement of integrin-mediated interaction of cells with both merosin and laminin. Schwannoma cells, which have previously been shown to produce a laminin-like, neurite-promoting factor, synthesize merosin in vivo and in vitro as shown by protein and mRNA analysis. The results suggest that merosin, which is the more abundant basement membrane protein in the laminin family, has properties very similar to laminin despite differences in the structure of the heavy chain. Furthermore, merosin may be identical to or a component of the neurite-promoting factors previously reported from heart, muscle, and Schwann cells.     

Laminin-like immunoreactivity in the snail Helisoma: involvement of approximately 300 kD extracellular matrix protein in promoting outgrowth from identified neurons

Polyclonal antibodies directed against laminin (LM), and against the A and B chains of reduced LM were used to identify antigenically related proteins in the extracellular matrix (ECM) of the snail Helisoma trivolvis. Immunofluorescence of snail central ganglionic rings using either the anti-LM or anti-B chain antibodies labeled the ECM within ganglionic sheaths as well as basal laminae surrounding the ganglia. Both the anti-LM and anti-B chain antibodies recognized a prominent, approximately 300-kD protein on immunoblots of a snail central ganglion preparation enriched in ECM components. The anti-A chain antibody failed to label any structures in sections of snail ganglia or to recognize any proteins on immunoblots of ganglionic ECM. A polyclonal antibody was raised against the approximately 300-kD snail protein. Immunofluorescence of snail ganglia with the anti- approximately 300-kD antibody gave a distribution of labeled structures comparable to that obtained with the anti-LM antibody. Immunofluorescent labeling of sections of snail muscle and salivary gland with the anti- approximately 300-kD antibody revealed a distribution of reactive protein characteristic of an ECM component. Probing immunoblots of ganglionic ECM with the anti- approximately 300-kD antibody revealed the recognition of the same approximately 300-kD protein as identified by the anti-LM antibodies. Media conditioned by Helisoma central ganglionic rings (CM) contains an unidentified neurite outgrowth promoting factor (NOPF). Immunoblots of CM probed with the anti-B chain and anti- approximately 300-kD antibodies reveal the recognition of a soluble approximately 300-kD protein similar to the approximately 300-kD protein identified in snail ECM. The ganglionic ECM preparation containing the approximately 300-kD protein supported outgrowth from cultured snail buccal neurons B5, and addition of anti- approximately 300-kD Fab fragments to CM abolished its outgrowth promoting activity. These results suggest that the approximately 300-kD ECM protein may be the NOPF in CM and/or functions in promoting neurite outgrowth.     

Laminin-like immunoreactivity in the snail Helisoma: Involvement of a ∼300 kD extracellular matrix protein in promoting outgrowth from identified neurons

Polyclonal antibodies directed against laminin (LM), and against the A and B chains of reduced LM were used to identify antigenically related proteins in the extracellular matrix (ECM) of the snail Helisoma trivolvis Immunofluorescence of snail central ganglionic rings using either the anti-LM or anti-B chain antibodies labeled the ECM within ganglionic sheaths as well as basal laminae surrounding the ganglia. Both the anti-LM and anti-B chain antibodies recognized a prominent, ～300-kD protein on immunoblots of a snail central ganglion preparation enriched in ECM components. The anti-A chain antibody failed to label any structures in sections of snail ganglia or to recognize any proteins on immunoblots of ganglionic ECM. A polyclonal antibody was raised against the ～300-kD snail protein. Immunofluorescence of snail ganglia with the anti-～300-kD antibody gave a distribution of labeled structures comparable to that obtained with the anti-LM antibody. Immunofluorescent labeling of sections of snail muscle and salivary gland with the anti-～300-kD antibody revealed a distribution of reactive protein characteristic of an ECM component. Probing immunoblots of ganglionic ECM with the anti- ～300-kD antibody revealed the recognition of the same ～ 300-kD protein as identified by the anti-LM antibodies. Media conditioned by Helisoma central ganglionic rings (CM) contains an unidentified neurite outgrowth promoting factor (NOPF). Immunoblots of CM probed with the anti-B chain and anti- ～300-kD antibodies reveal the recognition of a soluble ～300-kD protein similar to the ～300-kD protein identified in snail ECM. The ganglionic ECM preparation containing the ～300-kD protein supported outgrowth from cultured snail buccal neurons B5, and addition of anti- ～300-kD Fab fragments to CM abolished its outgrowth promoting activity. These results suggest that the ～300-kD ECM protein may be the NOPF in CM and /or functions in promoting neurite outgrowth.     

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.     

Thrombospondin promotes process outgrowth in neurons from the peripheral and central nervous systems.

Thrombospondin (TSP) is a prominent constituent of the extracellular matrix of the developing nervous system. We have examined the effects of TSP on the morphological differentiation of neurons. In short-term cultures (less than or equal to 24 hr) of embryonic rat sympathetic neurons, TSP stimulated neurite outgrowth, causing significant increase in the number of processes and their length. Similar effects were observed in cultures of rat dorsal root ganglion, hippocampal, and cerebral cortical neurons. Moreover, in cultures of central neurons, TSP was more effective than laminin in enhancing process extension. Analysis of long-term (5-7 days) cultures of sympathetic neurons indicated that processes formed in the presence of TSP had the cytochemical characteristics of axons. Thus, TSP can influence neuronal development by selectively enhancing axonal growth. The neurite-promoting region of the molecule was identified using a panel of monoclonal antibodies targeted to different regions of the protein. Process outgrowth could be totally inhibited with antibody A4.1, which recognizes the stalk region of TSP. These data suggest that the neurite-promoting activity is localized to a single region of the TSP molecule.     

Rat sciatic nerve schwann cell microcultures: Responses to mitogens and production of trophic and neurite-promoting factors

During embryonic development and in response to injury, the growing axons of peripheral neurons may influence the migration and proliferation of Schwann cells which, in return, may present neurons with a critical supply of factors required for neuronal survival, growth and differentiation. The identification and characterization of agents influencing the proliferation of Schwann cells as well as Schwann cell production of factors affecting neurons is greatly facilitated by the use of in vitro techniques. We describe here a simplified method of obtaining large numbers of purified neonatal rat sciatic nerve Schwann cells for use in generating large numbers of replicate microcultures. We then illustrate the use of these microcultures to examine Schwann cell: i) morphology and survival; ii) proliferation; and iii) production of neuronotrophic and neurite-promoting activities. We report that rat Schwann cells in microculture proliferate in response to serum, laminin and fibronectin, cholera toxin, and chick embryo parasympathetic ciliary neurons. Also, extracts of Schwann cell microcultures contain independently regulated activities which support the survival and neurite outgrowth of peripheral ganglionic neurons.Special issue dedicated to Dr. Paola S. Timiras     

Antiserum Against Neurite Outgrowth Factor in Chick Gizzard Extract and Its Inhibitory Effect on Neuritic Response in Cultured Ciliary Neurons

Abstract: Antiserum against a neurite outgrowth factor (NOF) of gizzard extract that promotes neurite outgrowth from dissociated ciliary ganglionic neurons (CG neurons) of 8-day-old chick embryo was prepared to determine whether or not the antiserum inhibits neurite outgrowth from cultured neurons or explants of chick and murine tissues. When CG neurons were cultured on a polyornithine-coated well exposed to NOF (NOF-bound POR well), marked neurite outgrowth was observed. When NOF-bound POR wells were exposed to antiserum, neurite outgrowth from CG neurons was gradually inhibited with increasing amounts of antiserum, while exposure to preimmune serum did not prevent neurite outgrowth. Antiserum had no effect on neuronal survival during a 48-h incubation. The diluted antiserum, which produced nearly 100% inhibition of the NOF activity, was almost equally active in suppressing the activity of NOFs in conditioned media (CM) of various chick embryo tissues, but showed much less inhibitory effects on NOFs in CM of murine tissues. The appearance of neurites from explants of spinal cord, dorsal root ganglion, or retina of chick embryo was also inhibited by the antiserum. These results indicate that antiserum against NOF from gizzard extract suppressed the activity of NOFs from various sources, and that there are species differences in NOFs, at least between chick and murine.     

Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG).

Sulfated proteoglycans (PGs) may play a significant role in the regulation of neurite outgrowth. They are present in axon-free regions of the developing nervous system and repel elongating neurites in a concentration-dependent manner in vitro. The addition of growth-promoting molecules, such as laminin, can modify the inhibitory effect of PGs on neurite outgrowth (Snow, Steindler, and Silver, 1990b). Substrata containing a high-PG/low-laminin ratio completely inhibit neurite outgrowth, while normal, unimpeded outgrowth is observed on low-PG/high-laminin substrata. Therefore, different patterns of neurite outgrowth may result from regulation of the ratio of growth-promoting molecules to growth-inhibiting molecules. Using video microscopy, embryonic chicken dorsal root ganglia neurons (DRG), chicken retinal ganglia neurons (RGC), and rat forebrain neurons (FB) were analyzed as they extended processes from a substratum consisting of laminin alone onto a step gradient of increasing concentrations of chondroitin sulfate proteoglycan (CS-PG) bound to laminin. In contrast to neurite outgrowth inhibition that occurs at the border of a single stripe of high concentration of CS-PG (Snow et al., 1990b and this study), growth cones grew onto and up CS-PG presented in a step-wise graded distribution. Although the behavior of the different cell types was unique, a common behavior of each cell type was a decrease in the rate of neurite outgrowth with increasing CS-PG concentration. These data suggest that appropriate concentrations of growth-promoting molecules combined with growth-inhibiting molecules may regulate the direction and possibly the timing of neurite outgrowth in vivo. The different responses of different neuronal types suggest that the presence of sulfated PG may have varying effects on different aspects of neuronal development.     

Protein kinase C is involved in laminin stimulation of neurite outgrowth

We are investigating the intracellular events involved in the induction of neurite outgrowth. The phorbol ester TPA, an activator of protein kinase C, potentiates neurite outgrowth from ciliary ganglion neurons cultured on suboptimal laminin concentrations, but not on optimal laminin concentrations. TPA also stimulates growth on fibronectin and collagen similar to that observed on laminin under control conditions. Manipulations that elevate intracellular cAMP levels (expected to activate A kinase) reduce neurite outgrowth on laminin. The protein kinase C inhibitors H7 and sphingosine inhibit neurite outgrowth on laminin in a reversible and dose-dependent manner. H7 does not inhibit the process outgrowth induced by concanavalin A in the same neurons. The results suggest that activation of protein kinase C is an important step in the neurite outgrowth caused by laminin binding to its receptor(s).     

Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules

When culture medium, conditioned by any of several cell types, is applied to a polycationic substratum, a substance is adsorbed that causes neurons cultured on that substratum to extend processes (neurites) rapidly and profusely. We have purified the factor responsible for this effect from medium conditioned by bovine corneal endothelial cells, and have shown that it is composed of the glycoprotein laminin and two associated laminin-binding molecules: a sulfated protein known as entactin, and a large heparan sulfate proteoglycan. Of these molecules, only laminin was found to be present throughout the purification in all fractions possessing neurite outgrowth-promoting activity and absent from all fractions lacking activity. Laminin, purified from other sources, has been shown previously to promote extensive outgrowth by cultured neurons. These and other data presented here support the conclusion that laminin is responsible for the neurite outgrowth-promoting activity of the conditioned medium factor. Evidence is also presented that the association of a proteoglycan with laminin promotes efficient attachment of laminin to polycationic substrata, particularly in the presence of competing molecules.     

A neuronal cell line (PC12) expresses two beta 1-class integrins-alpha 1 beta 1 and alpha 3 beta 1-that recognize different neurite outgrowth-promoting domains in laminin

Integrins mediate neuronal process outgrowth on components of the ECM. Integrin alpha subunit-specific antibodies have been used to examine the roles of individual beta 1 integrins in attachment and neurite outgrowth by the neuronal cell line, PC12, in response to laminin and collagen. alpha 1 beta 1 and alpha 3 beta 1 were identified as the major beta 1 integrins expressed by PC12 cells. In functional assays, both alpha 1 beta 1 and alpha 3 beta 1 mediated PC12 cell interactions with laminin, whereas alpha 1 beta 1 alone mediated responses to collagen types I and IV. alpha 1 beta 1 and alpha 3 beta 1 were shown to recognize two different neurite-promoting sites in laminin: alpha 1 beta 1 interacted with the cross-region of laminin present in proteolytic fragments E1-4 and E1; alpha 3 beta 1 recognized a site in the long arm contained in laminin fragment E8. Thus, PC12 cells express two beta 1 integrins, which together function in attachment and neurite outgrowth on laminin and collagen. These integrins are candidates for mediating neurite outgrowth of sympathetic and other neurons in response to these ECM components.