Neurite outgrowth from progeny of epidermal growth factor-responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: effect of brain-derived neurotrophic factor

Epidermal growth factor (EGF)-responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self-renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF-responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF-generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain-derived neurotrophic factor (BDNF) (5 ng in 0.5 microL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF-responsive stem cell-derived neurons possess limited intrinsic capability for long-distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF-responsive stem cell-derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF.     

Neurite outgrowth from progeny of epidermal growth factor–responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: Effect of brain‐derived neurotrophic factor

Epidermal growth factor (EGF)–responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self‐renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF‐responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF‐generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain‐derived neurotrophic factor (BDNF) (5 ng in 0.5 μL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF‐responsive stem cell‐derived neurons possess limited intrinsic capability for long‐distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF‐responsive stem cell–derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 391–413, 1999     

Explant cultures of teleost spinal cord: source of neurite outgrowth

The source of neurite outgrowth in explant cultures of normal adult Apteronotus spinal cord was examined. Explants which contained the central region of spinal cord, including ependyma, showed neurite outgrowth in culture. Explants which did not contain ependyma showed no neurite outgrowth. It is concluded that the ependymal region is necessary for neurite outgrowth in these cultures of adult teleost spinal cord. In addition, our failure to observe axon outgrowth clearly attributable to fluorescently back-labeled electromotor neurons in these cultures suggests that the exuberant neurite outgrowth in vitro is most probably due to cells other than the electromotor neurons. This explant culture system provides a unique opportunity to study neuronal differentiation, regeneration, and neurogenesis in vitro.     

Cultured sympathetic neurons: Effects of cell-derived and synthetic substrata on survival and development

The long-term culture of dissociated rat sympathetic neurons requires strong adhesion of the neuronal processes to the culture substratum. A variety of artificial and cell-derived substrata was examined for their effects on the survival, neurite outgrowth, and neurotransmitter development of these neurons. Compared to dried collagen films, both three-dimensional hydrated collagen gels and surfaces coated with basic polymers provided a substratum highly adherent for developing neurons. Polylysine and polyornithine were most suitable for long-term culture when covalently linked with glutaraldehyde to an underlying layer of dried gelatin. Dissociated neurons also attached strongly to a substratum of killed nonneuronal cells fixed by paraformaldehyde, heat, ethanol, or trichloroacetic acid. In addition, an extracellular, substrate-associated material apparently produced by nonneuronal cells (rat cardiac myocytes and associated fibroblasts) promoted the long-term adhesion of growing neurites. The adhesive property of this microexudate was sensitive to trypsin, periodate, and alkali, but resistant to hyaluronidase, chondroitinase, 8 M urea, and 0.5 M acetic acid. Similar characteristics have been reported for fibronectin, an extracellular glycoprotein produced by many cells and cell lines. This protein, or one with similar features, may function in vivo in the extension and guidance of neuronal fibers. The choice and development of neurotransmitter function were unaffected by the various substrata tested, with one exception. Nonneuronal cells fixed with paraformaldehyde caused a significant induction of cholinergic properties similar to that seen with nonneuronal conditioned medium.     

Neurite outgrowth induced by the substrate associated material from nonneuronal cells

Cultured embryonic heart cells release a powerful inducer of neurite outgrowth into the surrounding medium. The present report demonstrates that these cells also deposit material which induces neurite outgrowth directly onto their culture substratum. Thus, embryonic heart cells condition both the culture medium and the culture substratum with respect to neurite outgrowth. Conditioned substrata were prepared by incubating heart cell monolayers in EDTA until the cells released from the substratum and were discarded. When dissociated neurons from ciliary or sympathetic chain ganglia were plated in fresh medium onto a conditioned substratum, neurite outgrowth was initiated in 80–95% of the neurons within 60 min. The neurite-inducing activity is trypsin sensitive, but is not inactivated by antibodies to the cell attachment protein fibronectin, by the membrane-solubilizing detergent Triton X-100, or by the enzymes collagenase, RNase, or DNase. The factor in conditioned medium which also induces neurite outgrowth depends for its activity on attachment to an artificial polyornithine substratum, under which condition it appears to promote adhesion of neuronal filopodia to the substratum. Thus, neurite outgrowth in these two culture systems occurs only if the substratum is conditioned by the appropriate extracellular materials: conditioned either directly by the deposition of heart cell products or indirectly by the binding of a conditioned medium factor to the polyornithine substratum. These substratum-conditioning factors may be related to those components of the extracellular matrix which support neurite outgrowth in vivo.     

Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis

The neurotransmitter serotonin has been shown to inhibit neurite outgrowth in specific identified neurons isolated from adult Helisoma. While in vivo experiments on Helisoma embryos have supported the hypothesis that endogenous serotonin regulates neurite outgrowth during embryonic development, direct effects of serotonin on embryonic neurons have not been measured. In the present study, cultures of dissociated embryonic neurons were used to test the direct actions of serotonin on developing embryonic neurons. Serotonin arrested neurite outgrowth in a significant percentage of elongating neurites in a dose-dependent manner. Furthermore, analysis of neurons with stable, nonelongating neurites revealed a novel response. Serotonin caused the reinitiation of neurite outgrowth in a significant percentage of nonelongating neurites. The arrestment of outgrowth and reinitiation of outgrowth occurred in similar percentages of elongating and nonelongating neurites, respectively. Parallel experiments on cultures of dissociated adult neurons were carried out to determine whether serotonin could also induce both inhibitory and stimulatory responses in adult cells. Serotonin arrested neurite outgrowth in a similar percentage of neurites to that observed in cultures of embryonic neurons. In contrast, serotonin did not reinitiate neurite outgrowth in a significant percentage of adult neurites. These data support the hypothesis that serotonin regulates neurite outgrowth in developing embryonic neurons. Furthermore, only some of these regulatory effects appear to be conserved from embryonic to adult neurons.     

Fibronectin-like immunoreactivity in Helisoma buccal ganglia: evidence that an endogenous fibronectin-like molecule promotes neurite outgrowth

We examined the distribution of fibronectin-like (FNL) immunoreactivity associated with intact buccal ganglia, cell-cultured buccal ganglia neurons and nonneuronal cells, and brain-conditioned medium from the snail Helisoma. In addition, the possible roles of fibronectin in the regulation of neurite outgrowth were studied. Immunofluorescent staining for FNL antigens revealed intense staining in patches and fibrous arrays over the connective tissue sheaths of buccal ganglia and nerve trunks. Within the ganglia, heavy staining was seen surrounding neurons and in track-like arrangements. In cell cultures, specific staining was associated with nonneuronal cell surfaces and to a lesser degree with the surface of identified neurons. In addition, a noncellular, substrate-bound component of brain-conditioned medium displayed FNL immunoreactivity. Since cultured Helisoma neurons require a substrate-associated, brain-derived conditioning factor (CF) in order to elaborate neurites with motile growth cones, we tested whether the FNL immunoreactive substance might act as a neuritotropic agent. Fibronectin antiserum suppressed, in a dose-dependent manner, the CF-induced sprouting of identified neurons in isolated cell culture. When added at increasing concentrations to neurons already growing in response to CF, fibronectin antiserum exerted a biphasic effect on neurite elongation; outgrowth was accelerated at low, but inhibited at high, antiserum concentrations. In contrast, growth cone structures associated with motility (filopodia and lamellipodia) were progressively reduced by increasing levels of antiserum. A short peptide derived from fibronectin's cell-binding domain (Arg-Gly-Asp-Ser) also greatly reduced neurite outgrowth. The combined results of this study indicate an abundance of FNL immunoreactive molecules within the CNS of Helisoma, their probable production by nonneuronal cells, and their function as a substrate-associated component of CF which promotes growth cone filopodial and lamellipodial activity.     

Characterization of a cell surface adhesion molecule expressed by a subset of developing chick neurons.

We have isolated a 105-kDa membrane glycoprotein expressed by subsets of developing chick neurons. This glycoprotein, identified by the JC7 monoclonal antibody, is present on the surface of axons and cell bodies of developing spinal motor neurons, dorsal root ganglion sensory neurons, sympathetic and parasympathetic neurons, and a small subset of brain neurons. Late in development the JC7 antigen is expressed at high levels on CNS nonneuronal glial-like cells. When attached to latex beads this glycoprotein can mediate homophilic adhesion and when used as a culture substrate stimulates a highly branched pattern of neurite outgrowth from dorsal root ganglion explants. The JC7 antigen appears to be identical to the SC1, BEN, and DM antigens. Its limited distribution, adhesive qualities, and ability to stimulate neurite outgrowth suggest it may play a role in the selective growth of neural processes during development.     

Interneurons versus efferent neurons: heterogeneity in their neurite outgrowth response to glia from several brain regions

We have studied in vitro the morphology of two populations of dopaminergic neurons from mouse embryos: the periglomerular interneurons from the olfactory bulb (DOBI) and the efferent neurons from the substantia nigra (DENN). The intrinsic potential of both neuronal types has been studied by comparing process outgrowth in a predominantly neuronal environment or in a glial environment that is endogenous or from other brain regions. Both populations exhibit in vitro different characteristics that reflect their phenotype in situ. In addition they greatly differ in their response to glial signals. DOBI maintain a constant stellate morphology with short processes under all culture conditions tested, whereas DENN exhibit a great plasticity and in particular respond to olfactory bulb glia with a striking increase in neurite length. The olfactory bulb glia differs from other brain region glia in two aspects: (a) in addition to type I astrocytes, common to all the glial monolayers that we have studied, it contains a population of fusiform astrocytes (GFAP+) that might represent the superficial glia (Raisman, 1985); and (b) both astrocytes and fusiform cells produce large amounts of laminin that is secreted in a thick extracellular matrix. DENN outgrowth on olfactory bulb glia, however, is not blocked by antilaminin antibodies that block outgrowth on a laminin substrate. Our results demonstrate that two neuronal populations sharing the same neurotransmitter present intrinsic differences in the control of cell shape. The fact that glia harvested from different brain regions supports varying extent of DENN neurite outgrowth suggests a heterogeneity of environmental signals throughout the developing brain.     

Mouse acetylcholinesterase enhances neurite outgrowth of rat R28 cells through interaction with laminin-1

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert 'non-classical', morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1.     

Cell cultures derived from early zebrafish embryos differentiate in vitro into neurons and astrocytes

The zebrafish is a polular nonmammalian model for studies of neural development. We have derived cell cultures, initiated from blastula-stage zebrafish embryos, that differentiate in vitro into neurons and astrocytes. Cultures were initiated in basal nutrient medium supplemented with bovine insulin, trout serum, trout embryo extract and fetal bovine serum. After two weeks in culture the cells exhibited extensive neurite outgrowth and possessed elevated levels of acetylcholinesterase enzyme activity. Ultrastructural analysis revealed that the neurites possessed microtubules, synaptic vessicles and areas exhibiting growth cone morphology. The cultures expressed proteins recognized by antibodies to the neuronal and astrocyte-specific markers, neurofilament and glial fibrillary acidic protein (GFAP). Poly-D-lysine substrate stimulated neurite outgrowth in the cultures and inhibited the growth of nonneuronal cells. Medium conditioned by the buffalo rat liver line, BRL, promoted the growth and survival of the cells in culture. Mitotically active cells were identified in cultures that had undergone extensive differentiation. The embryo cell cultures provide an in vitro system for investigations of biochemical parameters influencing zebrafish neuronal cell growth and differentiation.     

On the role of proteases, their inhibitors and the extracellular matrix in promoting neurite outgrowth

Using a novel method, a monoclonal antibody was produced which can directly block the activity of an extracellular matrix-associated neurite outgrowth promoting complex (Matthew and Patterson, 1983). Presumably binding at or near the active site, this antibody recognizes a determinant consisting of heparan sulfate and a larger molecule which is likely to be laminin (Matthew et al., in preparation). The antibody has been further used to localize this determinant in adult tissues in vivo. Extracellular binding is seen at sites known to promote axon regeneration in the peripheral nervous system and is not seen in the central nervous system (Matthew et al., in preparation). In investigating how neurons may modify their environment as they grow processes, we have recently found that sensory and sympathetic neurons spontaneously release a collagenase and a plasminogen activator from their distal processes and/or growth cones (Pittman, 1985). A 43 kD irreversible inhibitor of the plasminogen activator is secreted by cardiac myocytes and is found on the surfaces of cultured neurons (Pittman, 1984). This inhibitor is also released by nonneuronal cell cultures from peripheral, but not central, nerves (Pittman, unpublished). Of interest in relation to the proteoglycan neurite outgrowth promoting complex is the finding that the 43 kD inhibitor preparation binds heparin tightly and can displace laminin from its heparin binding site (Patterson and Pittman, unpublished). Thus it is possible that the protease/inhibitor system could affect outgrowth via interaction with the neurite outgrowth promoting complex in the extracellular matrix.     

Monoclonal M6 antibody interferes with neurite extension of cultured neurons.

Monoclonal M6 antibody binds to the surface of murine central nervous system neurons as well as to apical surfaces of epithelial cells in the choroid plexus and proximal tubules of the kidney. M6 antigen is expressed in the central nervous system as early as embryonic day 10, most strongly in the marginal zone of the neural tube, and remains detectable in adulthood. IgG or Fab fragments of M6 antibody interfere with the extension of neurites by cultured cerebellar neurons. Effects of the antibody on neurite extension are readily detectable after 24 h. No reduction of cell viability is detected during the first 3 days of antibody treatment. Cultures maintained in the presence of antibody for longer than 5 days exhibit reduced viability of neurons. This reduction in long-term viability in the presence of M6 antibody is largely avoided when 25 mM KCl is included in the culture medium. The antibody-mediated perturbation of neurite outgrowth is not blocked by the presence of elevated KCl. The unusually short and flattened appearance of neurites in these cultures suggests that the M6 antibody selectively affects neurite extension. Time-lapse cinematography of anti-M6-treated neurons reveals no apparent effect on movement of lamellipodia and filopodia of growth cones. Only the overall extension of the neurite appears to be inhibited. M6 antigen is a 35 kD glycoprotein that can be isolated from a deoxycholate- (DOC) solubilized membrane fraction from adult mouse brain.     

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5′ base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.     

Expression of Ndufb11 encoding the neuronal protein 15.6 during neurite outgrowth and development

Neurite outgrowth (e.g. axonal or dendrite outgrowth) of neurons is necessary for the development and functioning of the central nervous system. It is well accepted that the differentiation of neurons and neurite outgrowth involve alterations in gene expression. Furthermore, mitochondria play a role in different aspects of neurite outgrowth. Here we show that the expression of Ndufb11, a gene encoding the mitochondrial protein NP15.6 is decreased in the course of neuronal differentiation. NP15.6 is homologous to the bovine protein ESSS, a component of the mitochondrial complex 1. The homologous human NDUFB11 gene is localized to Xp11.3-Xp11.23, a region associated with neurogenetic disorders. The down-regulation of NP15.6 correlates with neurite outgrowth of PC12 cells induced by nerve growth factor. Furthermore, we analyzed the expression of Ndufb11 in the embryonic and adult mouse.     

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides

We have identified a synthetic peptide derived from the B2-chain of mouse laminin, Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (p20), which stimulates the neurite outgrowth-promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1-5 microM (1-5 micrograms/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (greater than 50 microM; greater than 50 micrograms/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth-promoting domain of laminin is located in the alpha-helical region of the B2-chain, and is active for both central and peripheral neurons.     

Characterization of a neuronal growth factor from mouse heart-cell-conditioned medium

A fraction of medium conditioned by embryonic mouse heart cells in culture promotes the growth of sympathetic and parasympathetic neurons in vitro. The factor stimulates neurite outgrowth, elevates specific activities of tyrosine hydroxylase and choline acetyltransferase in sympathetic ganglion explants, and enhances survival of dissociated sympathetic neurons in culture. The growth-promoting activity, which has a profound effect on survival of mouse sympathetic and parasympathetic neurons but little effect on mouse sensory neuron survival, is sensitive to trypsin and elevated temperature, suggesting association with a polypeptide or protein. Unlike nerve growth factor (NGF), the conditioned medium fraction is insensitive to anti-NGF antiserum, and fosters growth of mouse parasympathetic neurons. Consequently, the conditioned medium appears to contain a new nerve growth-promoting factor.     

Nerve growth factor-independent development of embryonic mouse sympathetic neurons in dissociated cell culture

Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth. To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development. Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF. By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture. During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent. Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival. The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.     

Monoclonal M6 antibody interferes with neurite extension of cultured neurons

Monoclonal M6 antibody binds to the surface of murine central nervous system neurons as well as to apical surfaces of epithelial cells in the choroid plexus and proximal tubules of the kidney. M6 antigen is expressed in the central nervous system as early as embryonic day 10, most strongly in the marginal zone of the neural tube, and remains detectable in adulthood. IgG or Fab fragments of M6 antibody interfere with the extension of neurites by cultured cerebellar neurons. Effects of the antibody on neurite extension are readily detectable after 24 h. No reduction of cell viability is detected during the first 3 days of antibody treatment. Cultures maintained in the presence of antibody for longer than 5 days exhibit reduced viability of neurons. This reduction in long-term viability in the presence of M6 antibody is largely avoided when 25 mM KCl is included in the culture medium. The antibody-mediated perturbation of neurite outgrowth is not blocked by the presence of elevated KCl. The unusually short and flattened appearance of neurites in these cultures suggests that the M6 antibody selectively affects neurite extension. Time-lapse cinematography of anti-M6-treated neurons reveals no apparent effect on movement of lamellipodia and filopdia of growth cones. Only the overall extension of the neurite appears to be inhibited. M6 antigen is a 35 kD glycoprotein that can be isolated from a deoxycholate- (DOC) solubilized membrane fraction from adult mouse brain.     

Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs

Different neuronal populations were used to compare the neurite outgrowth-promoting activities of N-CAM and N-cadherin expressed via gene transfer on the surface of nonneuronal cells. In contrast to a previously reported developmental loss of retinal ganglion cell responsiveness to N-CAM, these cells exhibited an increased and maintained responsiveness to N-cadherin over the same developmental period (E6-E11). N-CAM and N-cadherin responses could be specifically inhibited by their own antibodies, but not by antisera to the beta 1 integrin family or the L1/G4 glycoprotein. Cerebellar neurons showed qualitative differences in the nature of the dose-response curves for transfected N-CAM expression (highly cooperative) versus N-cadherin expression (linear). In addition "subthreshold" levels of N-CAM expression, which do not normally support neurite outgrowth, did so when coexpressed with functional levels of N-cadherin. These studies show fundamental differences in neuronal responsiveness to cell adhesion molecules and suggest a more dynamic regulation for N-CAM-dependent neurite outgrowth than for N-cadherin-dependent outgrowth.