Molecular characterization of the Schwann cell myelin protein, SMP: structural similarities within the immunoglobulin superfamily.

The Schwann cell myelin protein (SMP), previously defined in quail and chick by a monoclonal antibody, is in vivo exclusively expressed by myelinating and nonmyelinating Schwann cells and oligodendrocytes. The isolation of the complete nucleotide sequence of SMP is reported here. The predicted polypeptide chain reveals that SMP is a transmembrane molecule of the immunoglobulin superfamily showing sequence similarities with several surface glycoproteins expressed in the nervous and immune systems. In spite of a 43.5% overall sequence identity between rat myelin-associated glycoprotein (MAG) and quail SMP, SMP does not seem to be the avian homolog of MAG, since their expression, regulation, and functions are significantly different. Unusual sequence arrangements shared by SMP, MAG, and two lymphoid antigens suggest the existence of a particular subgroup in the immunoglobulin superfamily.     

Cell lineage analysis of Schwann cells in the PNS determined by shiverer-normal mouse chimeras

The myelin of the peripheral nervous system from the shiverer mutant mice is characterized by the absence of myelin basic protein, while the other myelin protein components are present at normal levels. Myelin lamella formation is normal in the shiverer mutant. Therefore, by using antiserum against myelin basic protein, we can distinguish the shiverer from the wild-type control myelin immunohistochemically. To study the cell lineage of Schwann cells, chimeras produced by the aggregation of eight-cell embryos from wild-type mice and shiverer mice have been used. Using myelin basic protein as a marker, it was observed that Schwann cells in the sciatic nerve existed as patches of cells with like-genotype. The patches occurred in a linear array along the axons with some intermingling of Schwann cells. Complete randomization by intermingling of Schwann cells was not observed and clones of Schwann cells may persist as contiguous groups throughout peripheral nerve development.     

A Cyclic AMP Analogue Induces Synthesis of a Myelin-Specific Glycoprotein by Cultured Schwann Cells

Neonatal rat Schwann cells, cultured with agents which increase intracellular cyclic AMP, were prompted to resume synthesis of a 170,000 Mr glycoprotein which is specific to peripheral nervous system myelin and is herein referred to as P170K. We have shown previously that similar treatment induces the synthesis by Schwann cells of the myelin lipid, galactocerebroside. In contrast to P170K and galactocerebroside, syntheses of P0 and myelin basic protein were not induced. Intracellular cyclic AMP is thus likely to be a participant in the complex system regulating myelination.     

Peripheral Nervous System Myelin and Schwann Cell Glycoproteins: Identification by Lectin Binding and Partial Purification of a Peripheral Nervous System Myelin-Specific 170,000 Molecular Weight Glycoprotein

Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities. These included concanavalin A, Helix pomatia, Limulus polyphemus, Maclura pomifera, peanut, soybean, Ulex europaeus, and wheat germ agglutinins. Inclusion of the appropriate monosaccharide in the overlay solution (0.2 M) inhibited lectin binding to the nitrocellulose-fixed proteins. Fluorography permitted identification of 26 myelin glycoproteins and many more in Schwann cells. All lectins labeled a band present in myelin, but not Schwann cells, corresponding to the major PNS myelin protein, P0. Our attention focused on a high-molecular-weight myelin glycoprotein [apparent molecular weight (Mr) 170,000], which appeared abundant by Coomassie Blue staining and which was heavily labeled by all lectins except concanavalin A. A protein with approximately this Mr and lectin-binding pattern was present in human and bovine PNS myelin as well, but not detected in rat Schwann cells, CNS myelin, liver and fibroblast homogenates, or cultured bovine oligodendroglia. Hence this 170,000 Mr glycoprotein is apparently unique to PNS myelin.     

Autophagy aids membrane expansion by neuropathic Schwann cells

Demyelinating peripheral neuropathies associated with abnormal expression of peripheral myelin protein 22 (PMP22) involve the formation of cytosolic protein aggregates within Schwann cells. Towards developing a therapy for these progressive neurodegenerative diseases, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could prevent protein aggregation and enhance Schwann cell myelination. Indeed, we found that glial cells from neuropathic mice activate autophagy in response to RM and produce abundant myelin internodes. Lentivirus-mediated shRNA shutdown of Atg12 abrogates the improvements in myelin production, demonstrating that autophagy is critical for the observed benefits.     

Stimulation of a MR 80,000 protein phosphorylation by EGF in EGF receptor-hyperproducing human tumor cells

NA and Ca9-22 cells derived from squamous cell carcinomas of the tongue possess a large number of epidermal growth factor (EGF) receptors (2.0 X 10(6) and 1.3 X 10(6) receptors/cell, respectively). In these cell lines, EGF stimulated receptor autophosphorylation and phosphatidylinositol (PI) turnover. Furthermore, EGF enhanced the phosphorylation of an acidic protein of Mr 80,000. Phosphorylation of this protein was also stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a phorbol ester tumor promoter, and was mainly at serine residues. Phosphopeptide mapping using protease V8 or trypsin indicated that Mr 80,000 proteins isolated from the EGF- and TPA-treated cells were identical. The Mr 80,000 protein was present mainly in the cytosol, but it became closely associated with the membrane as a phosphorylated form upon EGF or TPA stimulation. These results suggest that the EGF-stimulated phosphorylation of the Mr 80,000 acidic phosphoprotein in EGF receptor-hyperproducing tumor cells is mediated through the activation of PI turnover and protein kinase C.     

N-WASP is required for membrane wrapping and myelination by Schwann cells

During peripheral nerve myelination, Schwann cells sort larger axons, ensheath them, and eventually wrap their membrane to form the myelin sheath. These processes involve extensive changes in cell shape, but the exact mechanisms involved are still unknown. Neural Wiskott-Aldrich syndrome protein (N-WASP) integrates various extracellular signals to control actin dynamics and cytoskeletal reorganization through activation of the Arp2/3 complex. By generating mice lacking N-WASP in myelinating Schwann cells, we show that N-WASP is crucial for myelination. In N-WASP-deficient nerves, Schwann cells sort and ensheath axons, but most of them fail to myelinate and arrest at the promyelinating stage. Yet, a limited number of Schwann cells form unusually short internodes, containing thin myelin sheaths, with the occasional appearance of myelin misfoldings. These data suggest that regulation of actin filament nucleation in Schwann cells by N-WASP is crucial for membrane wrapping, longitudinal extension, and myelination.     

Expression of Schwann cell markers by mammalian neural crest cells in vitro

During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.     

Gpr126 is essential for peripheral nerve development and myelination in mammals

In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.     

Schwann-like cell differentiation of rat adipose-derived stem cells by indirect co-culture with Schwann cells in vitro

Objectives: Schwann cell (SC) transplantation is a promising therapy for peripheral nerve transaction, however, clinical use of SCs is limited due to their very limited availability. Adipose‐derived stem cells (ADSCs) have been identified as an alternative source of adult stem cells in recent years. The aim of this study was to evaluate the feasibility of using ADSCs as a source of stem cells for differentiation into Schwann‐like cells by an indirect co‐culture approach, in vitro. Materials and methods: Multilineage differentiation potential of the obtained ADSCs was assayed by testing their ability to differentiate into osteoblasts and adipocytes. The ADSCs were co‐cultured with SCs to be induced into Schwann‐like cells through proximity, using a Millicell system. Expression of typical SC markers S‐100, GFAP and P75NTR of the treated ADSCs was determined by immunocytochemical staining, western blotting and RT‐PCR. Myelination capacity of the differentiated ADSCs (dADSCs) was evaluated in dADSC/dorsal root ganglia neuron (DRGN) co‐cultures. Results: The treated ADSCs adopted a spindle shaped‐like morphology after co‐cultured with SCs for 6 days. All results of immunocytochemical staining, western blotting and RT‐PCR showed that the treated cells expressed S‐100, GFAP and P75NTR, indications of differentiation. dADSCs could form Schwann‐like cell myelin in co‐culture with DRGNs. Undifferentiated ADSCs (uADSCs) did not form myelin compared to DRGNs cultured alone, but could produce neurite extension. Conclusions: These results demonstrate that this indirect co‐culture microenvironment could induce ADSCs to differentiate into Schwann‐like cells in vitro, which may be beneficial for treatment of peripheral nerve injuries in the near future.     

Human schwann cells in vitro. II. Myelination of sensory axons following extensive purification and heregulin-induced expansion

Co-culture conditions are well established in which Schwann cells (SCs) derived from immature or adult rats proliferate and form myelin in response to contact with sensory axons. In a companion article, we report that populations of adult-derived human Schwann cells (HASCs) fail to function under these co-culture conditions. Furthermore, we report progressive atrophy of neurons in co-cultures containing populations of either human fibroblasts. Two factors that might account for the insufficiency of the co-culture system to support HASC differentiation are the failure of many HASCs to proliferate and the influence of contaminating fibroblasts. To minimize fibroblast contamination of neuron-HASC co-cultures, we used fluorescence-activated cell sorting to highly purify HASC populations (to more than 99.8%). To stimulate expansion of the HASC population, a mitogenic mixture of heregulin (HRGβ1 amino acid residues 177-244; 10 nM), cholera toxin (100 ng/mL), and forskolin (1 μM) was used. When these purified and expanded HASCs were co-cultured with embryo-derived rat sensory neurons, neuronal shrinkage did not occur and after 4 to 6 weeks some myelin segments were seen in living co-cultures. This myelin was positively identified as human by immunostaining with a monoclonal antibody specific to the human peripheral myelin protein P₀ (antibody 592). Although this is the first reported observation of myelination by HASCs in tissue culture, it should be noted that myelination occurred more slowly and in much less abundance than in comparable cultures containing adult rat-derived SCs. We anticipate that further refinements of the HASC co-culture system that enhance myelin formation will provide insights into important aspects of human SC biology and provide new opportunities for studies of human peripheral neuropathies. © 1995 John Wiley & Sons, Inc.     

GAP-43 is expressed by nonmyelin-forming Schwann cells of the peripheral nervous system.

Recently it has been demonstrated that the growth-associated protein GAP-43 is not confined to neurons but is also expressed by certain central nervous system glial cells in tissue culture and in vivo. This study has extended these observations to the major class of glial cells in the peripheral nervous system, Schwann cells. Using immunohistochemical techniques, we show that GAP-43 immunoreactivity is present in Schwann cell precursors and in mature non-myelin-forming Schwann cells both in vitro and in vivo. This immunoreactivity is shown by Western blotting to be a membrane-associated protein that comigrates with purified central nervous system GAP-43. Furthermore, metabolic labeling experiments demonstrate definitively that Schwann cells in culture can synthesize GAP-43. Mature myelin-forming Schwann cells do not express GAP-43 but when Schwann cells are removed from axonal contact in vivo by nerve transection GAP-43 expression is upregulated in nearly all Schwann cells of the distal stump by 4 wk after denervation. In contrast, in cultured Schwann cells GAP-43 is not rapidly upregulated in cells that have been making myelin in vivo. Thus the regulation of GAP-43 appears to be complex and different from that of other proteins associated with nonmyelin-forming Schwann cells such as N-CAM, glial fibrillary acidic protein, A5E3, and nerve growth factor receptor, which are rapidly upregulated in myelin-forming cells after loss of axonal contact. These observations suggest that GAP-43 may play a more general role in the nervous system than previously supposed.     

Schwann cells infected with a recombinant retrovirus expressing myelin-associated glycoprotein antisense RNA do not form myelin.

To elucidate the role of myelin-associated glycoprotein (MAG) in the axon-Schwann cell interaction leading to myelination, neonatal rodent Schwann cells were infected in vitro with a recombinant retrovirus expressing MAG antisense RNA or MAG sense RNA. Stably infected Schwann cells and uninfected cells were then cocultured with purified sensory neurons under conditions permitting extensive myelination in vitro. A proportion of the Schwann cells infected with the MAG antisense virus did not myelinate axons and expressed lower levels of MAG than control myelinating Schwann cells, as measured by immunofluorescence. Electron microscopy revealed that the affected cells failed to segregate large axons and initiate a myelin spiral despite having formed a basal lamina, which normally triggers Schwann cell differentiation. Cells infected with the MAG sense virus formed normal compact myelin. These observations strongly suggest that MAG is the critical Schwann cell component induced by neuronal interaction that initiates peripheral myelination.     

Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture

We have used antibodies to identify Schwann cells and oligodendrocytes and to study the expression of myelin-specific glycolipids and proteins in these cells isolated from perinatal rats. Our findings suggest that only Schwann cells which have been induced to myelinate make detectable amounts of galactocerebroside (GC), sulfatide, myelin basic protein (BP), or the major peripheral myelin glycoprotein (P0). When rat Schwann cells were cultured, they stopped making detectable amounts of these myelin molecules, even when the cells were associated with neurites in short-term explant cultures of dorsal root ganglion. In contrast, oligodendrocytes in dissociated cell cultures of neonatal optic nerve, corpus callosum, or cerebellum continued to make GC, sulfatide and BP for many weeks, even in the absence of neurons. These findings suggest that while rat Schwann cells require a continuing signal from appropriate axons to make detectable amounts of myelin- specific glycolipids and proteins, oligodendrocytes do not. Schwann cells and oligodendrocytes also displayed very different morphologies in vitro which appeared to reflect their known differences in myelinating properties in vivo. Since these characteristic morphologies are maintained when Schwann cells and oligodendrocytes were grown together in mixed cultures and in the absence of neurons, we concluded that they are intrinsic properties of these two different myelin- forming cells.     

GLUT-3 expression in human skeletal muscle

Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.     

Diphtheria toxin receptor. Identification of specific diphtheria toxin-binding proteins on the surface of Vero and BS-C-1 cells

The biochemical characteristics of specific receptor molecules for diphtheria toxin on the surface of two toxin-sensitive cell lines (Vero and BS-C-1) were examined. Diphtheria toxin was found to bind to a number of different proteins in Nonidet P-40 solubilized extracts of 125I-labeled cells. In contrast, permitting diphtheria toxin to bind first to labeled intact cells, which were subsequently solubilized and subjected to immunoprecipitation with anti-diphtheria toxin, resulted in a far more restricted profile of diphtheria toxin-binding proteins that possessed Mrs in the range of 10,000-20,000. Direct chemical cross-linking of radioiodinated diphtheria toxin to cell surface proteins resulted in the appearance of several predominant bands possessing Mrs of approximately 80,000. The Mr approximately 80,000 complexes were shown to be composed of radiolabeled diphtheria toxin (Mr 60,000) and unlabeled Mr approximately 20,000 cellular proteins. These complexes were judged to be a result of specific binding in that their appearance could be preferentially inhibited by the addition of a 100-fold excess of unlabeled diphtheria toxin. The formation of the Mr approximately 80,000 complexes was sensitive to prior trypsin treatment of the cells and to known inhibitors of diphtheria toxin binding. Furthermore, prior incubation of the cells with diphtheria toxin at 37 degrees C ("down regulation") markedly and specifically reduced the subsequent formation of the Mr approximately 80,000 cross-linked complexes, and these down-regulated cells were less sensitive to diphtheria toxin in cytotoxicity assays. Further incubation of down-regulated cells at 37 degrees C restored their ability to form Mr approximately 80,000 complexes; this regeneration requires protein synthesis and restores the cells' sensitivity to diphtheria toxin-mediated cytotoxicity. These results strongly suggest that a Mr 10,000-20,000 cell surface protein is, or constitutes a portion of, the functional diphtheria toxin receptor.     

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.     

Rapid phosphorylation of MAP-2-related cytoplasmic and nuclear Mr 300,000 protein by serine kinases after growth stimulation in quiescent cells

Antibody against brain microtubule-associated protein 2 (MAP-2) immunoprecipitated Mr 300,000 and 80,000 proteins of cultured fibroblasts and kidney cells. These proteins were not appreciably phosphorylated in quiescent cells, but were rapidly phosphorylated after growth stimulation by insulin, epidermal and fibroblast growth factors, transferrin, phorbol ester and diacylglycerol in the presence of Ca2+, in a manner similar to that of MAP-1-related Mr 350,000 protein (J. Cell Biol. 100, 748-753). A Ca2+ ionophore, which is known to make the quiescent cell competent but not to enter into the growth cycle, did not induce the phosphorylation. In a chase experiment, decay half lives of labeled phosphoproteins were 5 h for Mr 350,000 and 300,000 proteins, and 1.5 h for Mr 80,000 protein. On subcellular fractionation, phosphorylated Mr 350,000 and 300,000 proteins were detected first mainly in the cytoplasm and then in the nucleus, while Mr 80,000 phosphoprotein was consistently detected in the cytoplasm. The phosphorylation of these proteins occurred on serine residues after stimulation with various factors. Thus, the phosphorylation of cytoskeleton-associated Mr 350,000 and 300,000 proteins by serine kinases seems to be a common second process after growth stimulation and to link cytoplasmic and intranuclear events.