The zinc finger transcription factor Zif268/Egr-1 is essential for Schwann cell expression of the p75 NGF receptor.

Nerve injury alters the function of Schwann cells from quiescent, myelin forming cells to proliferating cells that facilitate nerve repair. The transcription factor, Zif268, may be involved in transmitting injury-related signals since its expression is rapidly induced by nerve transection in vivo and without intervening protein synthesis by injury-related signals in vitro. Expression of the low-affinity p75 nerve growth factor receptor (NGFRp75) by Schwann cells after nerve injury closely correlated with the zif268 expression profile, and Zif268 transactivated the NGFRp75 promoter in transient transfection assays. Conversely, the NGFRp75 gene was not expressed when Zif268 protein was depleted by stable transfection of antisense cDNA. Moreover, nuclear proteins corresponding to Zif268 bound to the NGFRp75 promoter by Southwestern blotting, indicating that a direct interaction of Zif268 with the NGFR gene is required for its expression in Schwann cells.

Role of basal lamina in Schwann cell glycolipid biosynthesis.

Schwann cells that are deprived of axonal contact switch their glycolipid metabolic pathway from primarily galactocerebroside (GalCe) synthesis to the formation of glucocerebroside (GlcCe) and its homologs. The removal of axonal influence has a dual effect on Schwann cell phenotype; they lose the ability to assemble both myelin and basement membrane. To determine whether a loss of basement membrane directly affects glycolipid expression, we have examined lipid biosynthesis in Schwann cells which were allowed to interact with axons of dorsal root ganglion neurons but which were deprived of the ability to assemble basal lamina. These Schwann cells resemble those from myelinating nerve in that they synthesize a large amount of galactohydroxycerebroside. This suggests that axon contact, even in the absence of basement membrane, is sufficient to induce the GalCe metabolic pathway.

Subcellular Distribution of Sulfated Glucuronyl Glycolipids in Human Peripheral Motor and Sensory Nerves.

Sulfated glucuronyl glycolipids (SGGL) have been implicated as important target antigens in patients with demyelinating polyneuropathy and IgM paraproteinemia. Sulfated glucuronyl paragloboside (SGPG), a major species of SGGL, was identified in the subcellular fractions of human peripheral motor and sensory nerves using a simple and quantitative method. SGPG was found to be concentrated in the myelin-enriched fractions of both motor and sensory nerves (1.3 +/- 0.3 and 1.5 +/- 0.4 &mgr;g/mg protein, respectively), whereas its concentration was 0.9 +/- 0.2 and 1.8 +/- 0.6 &mgr;g/mg protein in the axolemma-enriched fractions of motor and sensory nerves, respectively. Our finding that SGPG is more abundant in the human sensory nerve axolemma-enriched fraction may account for the clinical and pathological observations that the lesions are more heavily concentrated in the sensory nerve than in other parts of the nerve tissues in this disorder. Copyright 1994 S. Karger AG, Basel

Association of glucocerebroside homolog biosynthesis with Schwann cell proliferation.

The biosynthesis of myelin-associated glycolipids was studied in quiescent secondary cultures of Schwann cells and in rapidly proliferating population of transfected Schwann cells (TSC) by in vitro incorporation of [3H]galactose. The TSC demonstrated a marked increase (> 10-fold) in [3H]galactose incorporation when compared to quiescent Schwann cells. The level (or amount) of [3H]galactose incorporation into lipids is dependent upon the number of TSC in culture. The majority of 3H-labeled lipids were oligohexosylceramides (GL-2, GL-3, and GL-4). Substrates that inhibit TSC proliferation, collagen type I and Matrigel, and artificial basement membrane, decrease the [3H]galactose incorporation by 25% and 80%, respectively. Our results indicate that the synthesis of glucocerebroside and its homologs is associated with Schwann cell proliferation.

Release of membrane-associated growth factors during neural injury.

The release of membrane-associated growth factors after neural injury may influence the outcome of the recovery. For example, for remyelination to occur after neural injury it is critical for the glial cell to proliferate prior to remyelination in both the PNS and CNS. In the CNS, the relative response of the oligodendrocytes and astroglia to growth factors mobilized during neural injury may play a role in the cellular dynamics of repair of neural injury or scarring and subsequent failure to repair neural injury. In support of this view, we have studied the mitotic potential and cell cycle kinetics of cultured adult oligodendrocytes and found that these adult cells respond only weakly to factors such as FGF which are known to be potent mitogens for neonatal cells. However, given the same dose of FGF, adult astrocytes are mitotically stimulated to a much greater degree than are the adult oligodendrocytes (Vick and De Vries, unpublished observations). Given the pathways which may be operative in the release of growth factors after injury, it has not escaped our attention that, provided the released factors are in equilibrium with easily accessible and peripheral body fluids, these released factors may serve as new markers for neural injury. Further experiments are in progress to explore this possibility.

Isolation and characterization of axolemma-enriched fractions from rabbit and bovine peripheral nerve.

Axolemma-enriched fractions were isolated from bovine spinal accessory nerves, bovine intradural dorsal roots, and rabbit sciatic nerve by differential centrifugation and separation on a linear 10-40% sucrose (w/w) gradient. The fractions were enriched 4 to 10 fold in acetylcholinesterase, a biochemical marker for axolemma. Axolemma-enriched fractions isolated from uniformly well-myelinated fibers (bovine spinal accessory nerve) contained lower CNPase activity and higher acetylcholinesterase activity than comparable fractions isolated from variably myelinated fibers (rabbit sciatic nerve and bovine intradural roots). Separation by polyacrylamide electrophoresis showed that the molecular weight distribution of all peripheral nerve axolemma-enriched fractions was similar and ranged from 20 to over 150 kilodaltons. All axolemma-enriched fractions appeared to contain a small but variable amount of myelin-specific proteins. Based on biochemical properties, peripheral nerves containing uniformly well-myelinated fibers yield an axolemma-enriched fraction which is least contaminated with myelin-related membranes.

Isolated growth cones stimulate proliferation of cultured Schwann cells.

A growth cone-enriched fraction was prepared from 3-4 day rat cerebra. Examination of the growth cone fraction by electron microscopy revealed numerous structures circular in appearance that contain a number of features common to neuronal growth cones in vivo. The isolated growth cones stimulated a dose-dependent incorporation of [3H]-thymidine into cultured Schwann cells in a manner similar to that observed with an axolemma-enriched fraction prepared from adult rat brainstem. The mitogenic activities of both the growth cone fraction and axolemma-enriched fraction were decreased 50% and 20%, respectively, by treatment with heparitinase I. The mitogen for Schwann cells present in the isolated growth cones appears to be similar to that found in axolemma-enriched fractions prepared from adult rats.

Axonal regulation of Schwann cell glycolipid biosynthesis.

Schwann cell biosynthesis of glycolipids was studied by in vitro incorporation of [3H]galactose into neonatal rat sciatic nerves before and after endoneurial explant culture and in culture of purified Schwann cells. In neonatal nerves prior to culture, [3H]galactose was actively incorporated into galactocerebrosides (GalCe), monogalactosyl diacylglycerol (MGDG), and the sulfatides (Su). In contrast, the incorporation of [3H]galactose into MGDG, GalCe, and Su was nearly undetected in endoneurial explants after 4 days in vitro (div). Instead, there was increased 3H-labeling of glucocerebrosides (GlcCe) and its homologues, with tetrahexosylceramides (GL-4) being a major product, which continued through 8 div. This shift in glycolipid biosynthesis was further demonstrated in the purified Schwann cell cultures. These observations, together with our early findings in the permanent transection paradigm support a direct role of axons in specifying Schwann cell biosynthesis of the GalCe, MGDG, and Su and that the absence of this Schwann cell-axon interaction results in the phenotypic expression of glucocerebroside homologues by the Schwann cell.

Proliferation and differentiation of a transfected Schwann cell line is altered by an artificial basement membrane.

Rapidly dividing transfected Schwann cells were grown on Matrigel, a reconstituted basement membrane gel. Matrigel decreased the proliferation of the cells by 75% when compared to sister cultures that were grown on an untreated plastic substrate. Some transfected cells plated onto a Matrigel substrate formed colonies similar to that observed when the cells were plated on a plastic substrate. Additionally, many cells on Matrigel assembled themselves into fascicles projecting away from the colonies. These fascicles were composed of transfected Schwann cells that had assumed a bipolar appearance reminiscent of quiescent secondary Schwann cells in culture. Transfected cells grown on Matrigel contained approximately 10-fold less glial fibrillary acidic protein when compared to sister cultures grown on an untreated plastic substrate. By indirect immunofluorescence laminin immunoreactivity appeared as globules within the cytoplasm of the cells which were cultured on a plastic substrate. However, cells that were grown on the Matrigel substrate appear to organize laminin in a linear array around themselves. These results demonstrate that the presence of an artificial basement membrane alters the morphology, rate of proliferation, and state of differentiation of a transfected Schwann cell line.

Morphological and proliferative responses of cultured Schwann cells following rapid phagocytosis of a myelin-enriched fraction.

Cultured Schwann cells were found to phagocytose exogenously applied myelin membranes within 1 h. However, the resulting proliferative response required an additional 9 h of incubation. Treatment with ammonium chloride, a lysosomal inhibitor, delayed the appearance of the proliferative response to the myelin membranes by 12 h. Processing of myelin within the Schwann cells was followed by the appearance of immunocytochemically detectable myelin basic protein which was first visible at 4 h. Similar to the proliferative response, the appearance of immunoreactive material was delayed by the addition of ammonium chloride. Schwann cells were observed initially to ingest myelin fragments at their distal-most tips after which time the myelin phagosomes collected in the perinuclear region and fused with lysosomes. Phagocytic Schwann cells had a notable increase in Golgi membranes and microfilaments and contained widely dilated, rough endoplasmic reticulum cisternae. In purified cell cultures, Schwann cells phagocytosed myelin slower than macrophages, but displayed phagocytic abilities much greater than fibroblasts. The ability of cultured Schwann cells to phagocytose myelin rapidly suggests that these cells may aid in the breakdown and removal of myelin during Wallerian degeneration. These data further confirm the mitogenic effect of myelin and its possible role during nerve regeneration.

Subcellular localization of sulfated glucuronic acid-containing glycolipids reacting with anti-myelin-associated glycoprotein antibody.

Peripheral nerve glycolipids, with which anti-myelin-associated glycoprotein (MAG) antibodies from patients with demyelinating neuropathy and plasma cell dyscrasia cross-react, proved to be novel glycosphingolipids containing a sulfated glucuronyl residue. Consequently, there has been much interest in the immunological role that these sulfated glucuronyl-glycosphingolipids (SGGLs) may play in the pathogenesis of this disorder. For the determination of the distribution of these glycolipids in various nervous tissues and, thereby, the elucidation of their pathogenicity, a quantitative immunostaining-TLC method for their detection has been devised. Using this method, we demonstrated that these glycolipids were distributed in greatly different amounts in the peripheral nerves from human, bovine, chicken, rat, and rabbit. Subcellular localization studies of bovine peripheral nerve also demonstrated that they were enriched in the axolemma-enriched fraction and present in glial-related membranes in lower concentrations. In addition, these glycolipids were present in bovine dura mater and transformed rat Schwann cells. These biochemical results suggest that not only myelin but also axons could be involved as targets of the anti-MAG antibody in macroglobulinemia neuropathy, and it may also be necessary to examine anti-SGGL activity in patients with axonal neuropathy associated with plasma cell dyscrasia.

Effect of lithium on Schwann cell proliferation stimulated by axolemma- and myelin-enriched fractions.

Cultured Schwann cells stimulated with an axolemma- or myelin-enriched fraction incorporated 2.5 to three times as much [3H]thymidine when 10 mM lithium was added to the extracellular medium. The ability of lithium to enhance the mitogenic activity of either fraction was dose dependent. This result was not due to an increase in osmolarity, because addition of 10 mM NaCl had no effect on the amount of labeled thymidine accumulated by Schwann cells treated with either membrane fraction. In an earlier study, the effect of either membrane fraction could be potentiated with active phorbol esters. Lithium significantly enhanced the incorporation of [3H]thymidine into Schwann cells treated with a myelin-enriched fraction and phorbol esters. In contrast, lithium slightly increased the amount of labeled thymidine incorporated into Schwann cells stimulated with an axolemma-enriched fraction and phorbol esters. The mitogenic activity of either membrane fraction was impaired when the calcium channel blockers Mn2+ and nifedipine were added. Addition of lithium stimulated an increase in the amount of [3H]thymidine accumulated by Schwann cells treated with either the axolemma- or myelin-enriched fraction in the presence of either Mn2+ or nifedipine.

Developmental changes in myelin-induced proliferation of cultured Schwann cells.

Schwann cell proliferation induced by a myelin-enriched fraction was examined in vitro. Although nearly all the Schwann cells contained material that was recognized by antisera to myelin basic protein after 24 h, only 1% of the cells were synthesizing DNA. 72 h after the addition of the mitogen a maximum of 10% of the cells incorporated [3H]thymidine. If the cultures were treated with the myelin-enriched fraction for 24 h and then washed, the number of proliferating Schwann cells decreased by 75% when compared with those cells that were incubated with the mitogen continuously. When Schwann cells were labeled with [14C]thymidine followed by a pulse of [3H]thymidine 24 h later, every Schwann cell labeled with [3H]thymidine was also labeled with [14C]thymidine. Although almost every Schwann cell can metabolize the myelin membranes within 24 h of exposure, a small population of cell initially utilizes the myelin as a mitogen, and this population continues to divide only if myelin is present in the extracellular media. The percentage of the Schwann cells that initially recognize the myelin-enriched fraction as a mitogen is dependent upon the age of the animal from which the cells were prepared.

Differential proliferative responses of cultured Schwann cells to axolemma and myelin-enriched fractions. II. Morphological studies.

Axolemma-enriched and myelin-enriched fractions were prepared from bovine CNS white matter and conjugated to fluorescein isothiocyanate (FITC). Both unlabelled and FITC-labelled axolemma and myelin were mitogenic for cultured rat Schwann cells. Treatment of Schwann cells with the FITC-labelled mitogens for up to 24 h resulted in two distinct morphological appearances. FITC-myelin-treated cells were filled with numerous round, fluorescent-labelled intracellular vesicles, while FITC-axolemma-treated cells appeared to be coated with a patchy, ill-defined fluorescence, primarily concentrated around the cell body but extending onto the cell processes. These observations were corroborated under phase microscopy. Electron microscopy revealed multiple, membrane-bound, membrane-containing phagosomes within myelin-treated cells and to a far lesser extent in axolemma-treated cells. The effect on the expression of the myelin-mediated and axolemma-mediated mitogenic signal when Schwann cells were treated with the lysosomal inhibitors, ammonium chloride and chloroquine, was evaluated. The mitogenicity of myelin was reduced 70-80% by these agents whereas the mitogenicity of axolemma was not significantly altered under these conditions. These results suggest that axolemma and myelin stimulate the proliferation of cultured Schwann cells by different mechanisms. Myelin requires endocytosis and lysosomal processing for expression of its mitogenic signal; in contrast, the mitogenicity of axolemma may be transduced at the Schwann cell surface.

Localization of 2',3'-cyclic nucleotide 3'-phosphodiesterase on cultured Schwann cells.

Primary cultures of Schwann cells were labeled by indirect immunofluorescence using an antibody directed against 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Schwann cells which had been maintained in culture for 8 weeks were labeled with this antibody. Immunoblot analysis of Schwann cell homogenates revealed a single band with a molecular weight of 54,000 daltons which corresponded to a single immunoreactive polypeptide present in myelin prepared from rat sciatic nerve. The subcellular localization of CNPase was examined by fractionation of cultured Schwann cell homogenates with linear sucrose gradients. The distribution of CNPase paralleled that of 5'-nucleotidase, a putative marker for plasma membranes. These results suggest that CNPase is localized on the plasma membranes of Schwann cells and is expressed by the cells in the absence of an axonal stimulus.

Differential proliferative responses of cultured Schwann cells to axolemma- and myelin-enriched fractions. I. Biochemical studies.

Cultured rat Schwann cells were treated for 72 h with axolemma- and myelin-enriched fractions prepared from rat brainstem. [3H]Thymidine was added to the cultures 48 h before the termination of the experiment. Although, both fractions produced a dose-dependent uptake of label into Schwann cells, the shape of the dose response curves and rates at which [3H]thymidine was incorporated were different. The axolemma-enriched fraction produced a sigmoid dose response curve with a Hill coefficient of 2.05. The dose response curve for myelin rose sharply and saturated at a level that was approximately 50% of the maximal response observed with axolemma. Schwann cells that had been treated with axolemma exhibited little change in the rate of [3H]thymidine incorporation from 36-72 h after the addition of the membranes. In contrast, Schwann cells accumulated label three times faster during the 48-72-h period following the addition of myelin to the cultures when compared with the rate during the preceding 12-h interval. Furthermore, the mitogenic activity of the myelin-enriched fraction was decreased by the addition of ammonium chloride, a lysosomal inhibitor, whereas the activity of the axolemmal fraction was not impaired.

Identification of an axolemma-enriched fraction from peripheral nerve.

A method has been devised for the fractionation of whole peripheral nerve. The procedure utilizes differential centrifugation and separation on a linear sucrose gradient (10-40%, wt/wt). A membrane fraction localized between 26% and 29% sucrose was not only enriched for the plasma membrane markers, 5'-nucleotidase and acetylcholinesterase (AChE), but also possessed the highest binding of [3H]saxitoxin, a specific marker for sodium channels. Neurons in the lumbar dorsal roots and ventral horns of rats were injected with [3H]fucose to label glycoproteins associated with the axolemma from sciatic nerve. Fractionation of the labeled nerves demonstrated a coincidence in the distribution of [3H]fucose-labeled material and AChE activity in the sucrose density gradient. The increase in the specific activity of marker enzymes for plasma membrane, sodium channels, and labeled membrane, previously demonstrated to be of axolemmal origin, identified the 26-29% region of the sucrose gradient as enriched for axolemma derived from peripheral nerve.

Leptinotarsin: a presynaptic neurotoxin that stimulates release of acetylcholine.

Leptinotarsin, a toxin found in the hemolymph of the beetle Leptinotarsa haldemani, can stimulate release of acetylcholine from synaptic termini. Leptinotarsin causes an increase in the frequency of miniature end plate potentials (mepps) of the rat phrenic nerve-diaphragm preparation. The increase in the frequency of mepps induced by leptinotarsin is biphasic: about 10% of the total mepps are released in an initial burst that lasts about 90 sec, after which the remaining mepps are released over a period of 10-20 min. Tetrodotoxin has no effect upon the release induced by leptinotarsin, but low-Ca(2+) conditions abolish the first phase. The two phases of release may represent two presynaptic pools of acetylcholine, both of which can be released in quantized form. In a second study, rat brain synaptosomes were incubated with [(3)H]choline and were immobilized on Millipore filters. Leptinotarsin induced release of [(3)H]acetylcholine from this preparation, confirming the release seen by using neurophysiological methods. The ability of leptinotarsin to induce release from either intact nerve terminals or synaptosomes was abolished when the toxin was heated. The releasing activity of leptinotarsin from synaptosomes was also partially dependent upon the presence of Ca(2+) in the perfusing solution. Release from synaptosomes followed first-order kinetics, and was not inhibited by commercial antibodies to black widow spider antigens. The data suggest that leptinotarsin acts as a presynaptic neurotoxin and may be of value as a mechanistic probe in understanding the storage and release of neurotransmitters.