Stimulate Schwann Cell Proliferation Research Articles

Inhibition of KLF7-Targeting MicroRNA 146b Promotes Sciatic Nerve Regeneration.

A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.

Chitosan Degradation Products Promote Nerve Regeneration by Stimulating Schwann Cell Proliferation via miR-27a/FOXO1 Axis.

Natural polysaccharides are biomaterials widely used for constructing scaffolds in tissue engineering. While natural polysaccharides have been shown to robustly promote tissue regeneration, the underlying molecular mechanism remains largely unknown. Here, we show that chitooligosaccharides (COS), the intermediate products of chitosan degradation, stimulate peripheral nerve regeneration in rats. Our experiment also shows that COS stimulate the proliferation of Schwann cells (SCs) during nerve regeneration. By analyzing the transcriptome and gene regulatory network, we identified the miR-27a/FOXO1 axis as the main signaling pathway for mediating the proliferative effects of COS on SCs. COS increase the expression level of miR-27a and cause a reduction of FOXO1, which subsequently accelerates the cell cycle and stimulates SC proliferation to stimulate nerve regeneration. These findings define a basic pathway for oligosaccharides-mediated cell proliferation and reveal a novel aspect of polysaccharide biomaterials in tissue engineering.

Axonal Neuregulin 1 Type III Activates NF-κB in Schwann Cells during Myelin Formation

The formation of myelin requires a series of complex signaling events initiated by the axon to surrounding glial cells, which ultimately respond by tightly wrapping the axon with layers of specialized plasma membrane thereby allowing for saltatory conduction. Activation of the transcription factor NF-kappaB in Schwann cells has been suggested to be critical for these cells to differentiate into a myelinating phenotype; however, the mechanisms by which it is activated have yet to be elucidated. Here, we demonstrate that axonal membranes are sufficient to promote NF-kappaB activation in cultured Schwann cells and identify neuregulin 1 (NRG1), specifically the membrane-bound type III isoform, as the signal responsible for activating this transcription factor. Surprisingly, neither membrane-bound type I nor the soluble NRG1 EGF domain could activate NF-kappaB, indicating that type III induces a qualitatively unique signal. The transcriptional activity of NF-kappaB was significantly enhanced by treatment with forskolin, indicating these two signals converge for maximal activation. Both ErbB2 and -3 receptors were required for transducing the NRG1 signal, because gene deletion of ErbB3 in Schwann cells or treatment with the ErbB2 selective inhibitor, PKI-166, prevented the stimulation of NF-kappaB by axonal membranes. Finally, PKI-166 blocked the activation of the transcription factor in myelinating neuron/Schwann cell co-cultures and in vivo, in developing sciatic nerves. Taken together, these data establish NRG1 type III as the activator of NF-kappaB during myelin formation.

Sustained Axon–Glial Signaling Induces Schwann Cell Hyperproliferation, Remak Bundle Myelination, and Tumorigenesis

Type III neuregulins exposed on axon surfaces control myelination of the peripheral nervous system. It has been shown, for example, that threshold levels of type III beta1a neuregulin dictate not only the myelination fate of axons but also myelin thickness. Here we show that another neuregulin isoform, type III-beta3, plays a distinct role in myelination. Neuronal overexpression of this isoform in mice stimulates Schwann cell proliferation and dramatically enlarges peripheral nerves and ganglia-which come to resemble plexiform neurofibromas-but have no effect on myelin thickness. The nerves display other neurofibroma-like properties, such as abundant collagen fibrils and abundant dissociated Schwann cells that in some cases produce big tumors. Moreover, the organization of Remak bundles is dramatically altered; the small-caliber axons of each bundle are no longer segregated from one another within the cytoplasm of a nonmyelinating Schwann cell but instead are close packed and the whole bundle wrapped as a single unit, frequently by a compact myelin sheath. Because Schwann cell hyperproliferation and Remak bundle degeneration are early hallmarks of type I neurofibromatosis, we suggest that sustained activation of the neuregulin pathway in Remak bundles can contribute to neurofibroma development.

In situ evidence of involvement of Schwann cells in ulcerative colitis: autocrine and paracrine signaling by A disintegrin and metalloprotease-17–mediated tumor necrosis factor α production

An increase in S100 protein-positive cells has been reported in inflammatory bowel diseases, mainly Crohn disease. These cells were interpreted as myeloid-derived dendritic cells, chiefly in follicular areas. We were prompted to assess the nature of these cells in interfollicular areas of inflamed colonic mucosa in ulcerative colitis and study their involvement in tumor necrosis factor alpha production, the main inflammatory cytokine in ulcerative colitis. The number and distribution of cells expressing S100 protein, nerve growth factor receptor, CD68, CD1a, CD83, and calretinin were studied in samples from 16 patients with active ulcerative colitis using simple and double immunohistochemistry. Then, the localization in S100 protein-positive cells of (1) tumor necrosis factor alpha, (2) its sheddase A disintegrin and metalloprotease-17, and (3) the receptors TNFR1 was assessed using double immunofluorescence followed by confocal microscopy. In active ulcerative colitis, there was an increased number in S100 protein-positive cells in interfollicular areas of colonic mucosa compared with quiescent ulcerative colitis, nonulcerative colitis, or normal mucosa. All S100 protein-positive cells ensheathed calretinin-positive axons, indicating their Schwann cell origin. No CD1a- or CD83-positive dendritic cells were detected. Double immunofluorescence studies showed that in normal colon, Schwann cells of the mucosa and submucosal plexuses weakly expressed A disintegrin and metalloprotease-17 but did not express tumor necrosis factor alpha. By contrast, in active ulcerative colitis, they expressed both A disintegrin and metalloprotease-17 and tumor necrosis factor alpha. Schwann cells as well as calretinin-positive axons strongly expressed TNFR1. This study shows (1) a Schwann cell proliferation in the inflamed colonic mucosa during active ulcerative colitis and (2) that Schwann cells produce tumor necrosis factor alpha. Tumor necrosis factor alpha is thus likely to stimulate Schwann cell proliferation through an autocrine/paracrine mechanism.

LPA and NRG‐1b synergistically promote proliferation and migration of wild‐type and neoplastic Schwann cells via an ERRFI1‐dependent mechanism

In some cell types, lysophosphatidic acid (LPA)‐induced proliferation and migration requires transactivation of the EGF receptor (erbB1). This led us to hypothesize that LPA stimulates the proliferation and migration of normal and neoplastic Schwann cells and does so via a mechanism involving the EGFR‐related NRG‐1 receptors erbB2, 3 and/or 4. We have found that LPA stimulates the proliferation and migration of wild‐type Schwann cells and malignant peripheral nerve sheath tumor (MPNST) cells in a concentration‐dependent manner. Co‐stimulation of Schwann cells with NRG‐1β and LPA increases mitogenesis to a greater extent than the combined effects of each factor alone, indicating that LPA and NRG‐1β synergistically enhance proliferation of these glia. Further, LPA‐induced Schwann cell migration is ablated by treatment with the erbB kinase inhibitor PD168393 or GM6001, a MMP inhibitor which blocks the release of soluble NRG‐1. Microarray analyses also show that LPA represses Schwann cell expression of the pan‐erbB inhibitor Erffi1, thereby potentially enhancing erbB action. These findings suggest that LPA stimulates Schwann cell proliferation and migration synergistically with NRG‐1β and does so via a previously unknown mechanism that involves modulation of Erffi1 expression. Funded by R01 NS048353 and CA122804.

Physiological role of basic FGF in peripheral nerve development and regeneration: potential for reconstruction approaches

According to expression studies and functional analyses in mutant mice and in rats, FGF-2 appears to be specifically involved during development of peripheral nerves and in de-/re-generating processes at the lesion site and in spinal ganglia. In the absence of FGF receptor (FGFR)3, axonal and myelin diameters of peripheral nerves are significantly reduced, suggesting that FGFR3 physiologically regulates axonal development. The normally occurring neuronal cell death in spinal ganglia after peripheral nerve axotomy does not take place in FGF-2 and FGFR3-deleted mice, respectively, suggesting that injury-induced apoptosis is mediated via FGF-2 binding to FGFR3. According to a bimodal function of FGF-2, lesion-induced neuron death in rat spinal ganglia can be prevented by application of FGF-2 to the proximal nerve stump, which could be mediated via FGFR1/2. At the lesion site, FGF-2 appears to be involved in stimulating Schwann cell proliferation, promoting neurite outgrowth, especially of sensory nerve fibers, and regulating remyelination.

Erythropoietin Promotes MCF-7 Breast Cancer Cell Migration by an ERK/Mitogen-activated Protein Kinase-dependent Pathway and Is Primarily Responsible for the Increase in Migration Observed in Hypoxia

Recent studies indicate that cancer cells express erythropoietin receptor (EpoR). In this study, we have shown that erythropoietin (Epo) activates the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and promotes migration in MCF-7 breast cancer cells. Epo-stimulated MCF-7 cell migration was blocked by the MEK inhibitor PD098059 and by dominant negative MEK-1, indicating an essential role for ERK. When MCF-7 cells were exposed to hypoxia (1.0% O(2)) for 3 h, the Epo mRNA level increased 2.4 +/- 0.5-fold, the basal level of ERK activation increased, and cell migration increased 2.0 +/- 0.1-fold. Soluble EpoR and Epo-neutralizing antibody significantly inhibited hypoxia-induced MCF-7 cell migration, suggesting a major role for autocrine EpoR cell signaling. MCF-7 cell migration under hypoxic conditions was also inhibited by PD098059. These experiments identify a novel pathway by which exogenously administered Epo, and Epo that is produced locally by cancer cells under hypoxic conditions, may stimulate cancer cell migration.

Effect of xymedone on posttraumatic survival of sensory neurons

The effect of pyrimidine derivative xymedone promoting survival of sensory neurons and stimulating Schwann cell proliferation on regeneration of myelinated nerve fibers was studied. In rats treated with xymedone, the number of neurons in ipsilateral L4–L5 spinal ganglia on days 60 and 90 after transection of the sciatic nerve increased by 22 and 26%, myelinated fibers by 21.3 and 14.7%, and Schwann cells by 35.7 and 44.1%, respectively.

Sensory and Motor Neuron-derived Factor Is a Transmembrane Heregulin That Is Expressed on the Plasma Membrane with the Active Domain Exposed to the Extracellular Environment

The beta-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the beta-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other beta-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor.

Synergistic regulation of Schwann cell proliferation by heregulin and forskolin.

A peptide corresponding to the epidermal growth factor homology domain of beta-heregulin stimulated autophosphorylation of the heregulin receptors erbB2 and erbB3 in Schwann cells and activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. Heregulin-dependent activation of PAK65, a component of the stress-activated signaling pathway, ribosomal S6 kinase, and a cyclic AMP (cAMP) response element binding protein (CREB) kinase, identified as p95(RSK2), was also observed. Receptor phosphorylation and activation of these kinases in response to heregulin occurred in the absence of forskolin stimulation and were not augmented in cells treated with forskolin, a direct activator of adenylyl cyclase. Schwann cell proliferation in response to heregulin was observed only when the cells were also exposed to an agent that elevates cAMP levels. In the absence of heregulin, elevation of cAMP levels failed to stimulate Schwann cell proliferation. Forskolin significantly enhanced heregulin-stimulated expression of cyclin D and phosphorylation of the retinoblastoma gene product. In cells treated with both heregulin and forskolin there was a sustained accumulation of phospho-CREB, which was not observed in cells treated with either agent alone. Heregulin and forskolin synergistically activated transcription of a cyclin D promoter construct. These results demonstrate that heregulin-stimulated activation of MAP kinase is not sufficient to induce maximal Schwann cell proliferation. Expression of critical cell cycle regulatory proteins and cell division require activation of both heregulin and cAMP-dependent processes.

Axon-induced mitogenesis of human Schwann cells involves heregulin and p185erbB2.

The ability of sensory axons to stimulate Schwann cell proliferation by contact was established in the 1970s. Although the mitogen responsible for this proliferation has been localized to the axon surface and biochemically characterized, it has yet to be identified. Recently a family of proteins known as heregulins (HRGs) has been isolated, characterized, and shown to interact with a number of class 1 receptor tyrosine kinases, including the erbB2, erbB3, and erbB4 gene products. These factors include glial growth factor, a Schwann cell mitogen. We have tested the effects of antibodies against components of this system (HRG beta 1 and p185erbB2) in cocultures of rat sensory neurons and human (or rat) Schwann cells to elucidate the role of these proteins in axon-induced Schwann cell proliferation. 2C4, a monoclonal antibody specific for the human p185erbB2 receptor tyrosine kinase, bound to the surface of human Schwann cells and reduced human Schwann cell incorporation of [3H]thymidine by > 90% compared with untreated controls in this coculture system. This antibody had no effect on rat Schwann cell incorporation of [3H]thymidine under similar conditions. A polyclonal antibody raised against HRG beta 1 reduced human and rat Schwann cell incorporation of [3H]thymidine by nearly 80% and up to 49%, respectively, relative to controls. These results imply that a HRG, or a HRG-like molecule, is a component of the axonal mitogen. This mitogen is presented to Schwann cells by axons and induces proliferation through an interaction that involves p185erbB2 on Schwann cells.

Serum activity induces Schwann cell proliferation in vitro.

The present series of experiments demonstrate that a polypeptide activity present in rat serum induces a proliferative response in cultured rat Schwann cells. Schwann cells in multi-well tissue culture plates were incubated in medium containing 10% heat-inactivated fetal bovine serum and serial dilutions of normal rat serum, and control preparations were incubated in the same culture medium without rat serum. Rates of cell proliferation were assayed by measuring DNA incorporation of tritiated thymidine using liquid scintillation counting. A prominent dose-dependent proliferative response was observed among Schwann cells incubated with rat serum and rat plasma dilutions as compared to controls; this activity is abolished by heat inactivation and by proteolytic digestion, and was not affected by dialysis against a cellulose ester membrane that excludes molecules larger than 10,000 daltons. In contrast, no increase in DNA uptake of tritiated thymidine was observed when astrocyte and oligodendrocyte cultures were incubated with serial dilutions of rat serum. No proliferative effect was observed when rat Schwann cells were incubated with a dilution of standard adult bovine serum. These results suggest there is an intravascular plasma polypeptide with a molecular weight greater than 10,000 daltons that specifically stimulates Schwann cell proliferation, and it is proposed that this factor may be the mitogen responsible for the Schwann cell proliferative response known to occur after nerve injury.

Schwann Cells Secrete a PDGF-like Factor: Evidence for an Autocrine Growth Mechanism involving PDGF.

We have investigated the influence of platelet-derived growth factor (PDGF) in peripheral nervous system gliogenesis using two types of Schwann cell cultures. Short-term Schwann cell cultures grow very slowly, but when maintained in culture for several months the division rate of some cells increases, and cell lines can be established. We show that Schwann cells in both short- and long-term culture possess PDGF receptors and synthesize DNA in response to PDGF. Competitive binding experiments show that Schwann cells express mainly PDGF beta-receptors and respond better to PDGF-BB than to PDGF-AA. Conditioned media from short- and long-term Schwann cell cultures contain PDGF-like mitogenic activity, and anti-PDGF immunoglobin partially inhibits DNA synthesis in long-term Schwann cell cultures. Antibody neutralization experiments and Northern blot analyses both indicate that the predominant PDGF isoform in these cultures is PDGF-BB. PDGF-like activity is also detected in extracts of rat sciatic nerve. Taken together, these results suggest that PDGF-BB may stimulate Schwann cell proliferation in an autocrine manner during normal development.

Mitogen accumulation in von Recklinghausen neurofibromatosis

Most, but not all, patients with von Recklinghausen neurofibromatosis develop tumors (neurofibromas) that contain large numbers of Schwann cells and fibroblasts. To begin to understand the molecular events that contribute to cell proliferation in these benign tumors, we have analyzed extracts of neurofibromas to determine whether they contain mitogens for Schwann cells or fibroblasts, or both. Schwann cell and fibroblast mitogens are present in neurofibroma extracts. All the neurofibromas analyzed contain a Schwann cell mitogen similar to a neuronal cell surface molecule known to stimulate Schwann cell proliferation during normal development; this mitogen also stimulates fibroblast proliferation. Basic fibroblast growth factor is present in 60% of tumors evaluated. Accumulation of mitogenic substances may contribute to the growth of neurofibromas.

Cerebellar granule cells contain a membrane mitogen for cultured Schwann cells.

Proliferation of Schwann cells is one of the first events that occurs after contact with a growing axon. To further define the distribution and properties of this axonal mitogen, we have (a) cocultured cerebellar granule cells, which lack glial ensheathment in vivo with Schwann cells; and (b) exposed Schwann cell cultures to isolated granule cell membranes. Schwann cells cocultured with granule cells had a 30-fold increase in the labeling index over Schwann cells cultured alone, suggesting that the mitogen is located on the granule cell surface. Inhibition of granule cell proteoglycan synthesis caused a decrease in the granule cells' ability to stimulate Schwann cell proliferation. Membranes isolated from cerebellar granule cells when added to Schwann cell cultures caused a 45-fold stimulation in [3H]thymidine incorporation. The granule cell mitogenic signal was heat and trypsin sensitive and did not require lysosomal processing by Schwann cells to elicit its proliferative effect. The ability of granule cells and their isolated membranes to stimulate Schwann cell proliferation suggests that the mitogenic signal for Schwann cells is a ubiquitous factor present on all axons regardless of their ultimate state of glial ensheathment.

PC12 cells as a source of neurite-derived cell surface mitogen, which stimulates Schwann cell division.

Primary cultures of rat dorsal root ganglion Schwann cells were used to assay the efficacy of PC12 cells in stimulating Schwann cell proliferation. Co-cultures of PC12 cells and Schwann cells assayed by [3H]thymidine labeling followed by autoradiography showed proliferation of Schwann cells only where contact occurred between PC12 neurites and Schwann cells. Membranes derived from PC12 cells were shown to have many characteristics similar to membranes derived from sensory neurons; both could mimic whole cells in stimulating Schwann cell division; both were inactivated by mild heat treatment and by trypsinization, and both elevated intracellular cyclic AMP concentrations in Schwann cells 16 h after addition of membranes. We conclude that PC12 cells will be a valuable source for the isolation of the neuronal cell surface component which controls proliferation of Schwann cells during development of the peripheral nervous system.

Fibronectin promotes rat Schwann cell growth and motility.

Techniques are now available for culturing well characterized and purified Schwann cells. Therefore, we investigated the role of fibronectin in the adhesion, growth, and migration of cultured rat Schwann cells. Double-immunolabeling shows that, in primary cultures of rat sciatic nerve, Schwann cells (90%) rarely express fibronectin, whereas fibroblasts (10%) exhibit a granular cytoplasmic and fibrillar surface-associated fibronectin. Secondary cultures of purified Schwann cells do not express fibronectin. Exogenous fibronectin has a small effect on promoting the adhesion of Schwann cells to the substrate and does not significantly affect cell morphology, but it produced a surface fibrillar network on fibronectin on the secondary Schwann cells. Tritiated thymidine autoradiography revealed that addition of fibronectin to the medium, even at low concentrations, markedly stimulates Schwann cell proliferation, in both primary and secondary cultures. In addition, when cell migration was measured in a Boyden chamber assay, fibronectin was found to moderately, but clearly, stimulate directed migration or chemotaxis.

Mitogenicity of Brain Axolemma Membranes and Soluble Factors for Dorsal Root Ganglion Schwann Cells

Previous studies in this laboratory have shown that membranes derived from dorsal root ganglia (DRG) neurites are mitogenic for cultured Schwann cells derived from the same source [Salzer et al (1980): J Cell Biol 84:767-778]. Improved procedures are described for preparing Schwann cells derived from dorsal root ganglia that are highly responsive to various mitogens. Under these conditions, the cells respond not only to the neurite mitogen but also to pituitary extracts, dibutyryl cyclic AMP, and cholera toxin that have been shown previously to be good mitogens for Schwann cells derived from sciatic nerve [Raff et al (1978): Cell 15:813-822], thus reconciling discrepancies in the response of these different Schwann cell preparations to mitogens. Searching for a source of membranes more suitable for biochemical characterization of the neurite mitogen, we found that bovine brain axolemma, prepared by the method of DeVries et al [(1977):Brain Res 147:339-352] is highly mitogenic for Schwann cells. The mitotic index of Schwann cells was increased by the addition of axolemma from 0.5%-2% to 30%-50% during 24-h incubation with [3H]thymidine. Half maximal effect was obtained at about 0.4 microgram axolemma protein per microwell containing 2-4 X 10(3) cells. The axolemma mitogen appears to be an integral membrane protein that remains bound to the membrane under various ionic conditions but can be extracted in a partially active form with deoxycholate. Like the DRG neurite mitogen, the mitogenic activity of axolemma was abolished by trypsin treatment. Unlike the neurite preparation, however, the mitogenic activity of axolemma was only partially inactivated by heat treatment (60%-70% inactivation). A significant difference between the mitogenic activity of axolemma membranes and neurite membranes is the fact that axolemma membranes fail to stimulate Schwann cell proliferation in a defined, serum-free medium (N-2), whereas neurites show significant mitogenic activity in this medium. These findings indicate a possible difference between DRG neurites and brain axolemma either in the mitogen itself or surface components responsible for recognition between the membranes and the cells.

Studies of Schwann cell proliferation. III. Evidence for the surface localization of the neurite mitogen.

In the preceding paper (Salzer et al., 1980, J. Cell Biol. 84:753--766), evidence was presented that a neurite membrane fraction could be used to stimulate Schwann cell proliferation in culture. In this study, we present evidence that the mitogenic signal by which intact neurites or neurite membranes stimulate Schwann cell proliferation is located at the neurite surface. This conclusion is based on the following observations: (a) stimulation of Schwann cell proliferation by neurons requires direct contact between neurites and Schwann cells, separation of the two cells by a permeable collagen diaphragm 6 microns thick prevents Schwann cell proliferation; (b) treatment of intact neurites with trypsin before preparation of neurite membranes abolishes the ability of these membranes to be mitogenic for Schwann cells; and (c) the mitogenic activity of neurite homogenates is exclusively localized in the particulate rather than the soluble fraction of the homogenate. The mitogenic component on the neurite surface is heat labile, and is inactivated by aldehyde fixation. Preliminary data suggest that the mitogenic effect of neurite on Schwann cells is not mediated by 3',5'-cyclic AMP.