Electrophysiology of mammalian Schwann cells

Schwann cells are the satellite cell of the peripheral nervous system, and they surround axons and motor nerve terminals. The review summarises evidence for the ion channels expressed by mammalian Schwann cells, their molecular nature and known or speculated functions. In addition, the recent evidence for gap junctions and cytoplasmic diffusion pathways within the myelin and the functional consequences of a lower-resistance myelin sheath are discussed.

The main types of ion channel expressed by Schwann cells are K⁺ channels, Cl⁻ channels, Na⁺ channels and Ca²⁺ channels. Each is represented by a variety of sub-types. The molecular and biophysical characteristics of the cation channels expressed by Schwann cells are closely similar or identical to those of channels expressed in peripheral axons and elsewhere. In addition, Schwann cells express P₂X ligand-gated ion channels. Possible in vivo roles for each ion channel type are discussed. Ion channel expression in culture could have a special function in driving or controlling cell proliferation and recent evidence indicates that some Ca²⁺ channel and Kir channel expression in culture is dependent upon the presence of neurones and local electrical activity.     

Schwann Cell Surface Proteins and Glycoproteins

Abstract: To identify surface sialoglycoproteins of rat Schwann cells and to compare molecular weights of these sialoglycoproteins with those present in rat peripheral nervous system myelin, we prepared Schwann cells from sciatic nerves of 1–3-day-old rats and cultured them in monolayer. Surface sialoglycoproteins of the cultured cells were tritium-labeled by the periodateborohydride procedure and compared with sialoglycoproteins of adult rat peripheral nervous system myelin by fluorography following polyacrylamide slab gel electrophoresis in sodium dodecyl sulfate. Three radioactive bands with apparent molecular weights of 114,000–132,000, 105,000–115,000, and 44,000–56,000 were observed in both the Schwann cell and myelin preparations. Bands of similar apparent molecular weights were noted in Schwann cells metabolically radiolabeled with d -[1,6-³H]glucosamine. A band co-migrating with myelin P₀ glycoprotein was the most intensely radiolabeled of all peptides in periodate-B³H₄^?treated myelin, but was present in only trace amounts in periodate-B³H₄^? or d -[1,6-³H]glucosamine radiolabeled Schwann cells. Many presumably non-myelin glycoproteins were identified in the cultured Schwann cells by the periodate-borohydride procedure and by incubation of the cells with d -[1,6-³H]glucosamine. An immunoprecipitation technique was used to detect radiolabeled peptides in a nonionic detergent extract of freshly prepared, surface-radioiodinated Schwann cells that were bound by a rabbit anti-Schwann cell serum preabsorbed with rat fibroblasts. Many radioactive peptides were detected in the immunoprecipitate, but the two most intensely radiolabeled had apparent molecular weights of 105,000–115,000 and 95,000–106,000. This study has identified a number of glycoproteins synthesized by cultured rat Schwann cells which resemble in apparent molecular weight the glycoproteins expressed in rat peripheral nervous system myelin and has defined Schwann cell surface proteins recognized by a specific anti-rat Schwann cell antiserum.     

Developmental regulation of insulin receptor gene in sciatic nerves and role of insulin on glycoprotein P0 in the Schwann cells

In view of the observations that Schwann cells contain insulin receptors, in the present study, we have investigated the developmental regulation of insulin receptor gene in the sciatic nerves of different postnatal age group rats. We have also investigated the role of insulin in the expression of the major PNS myelin glycoprotein P zero (P0) in normal as well as high glucose conditions in primary rat Schwann cells. The expression of insulin receptor gene in sciatic nerves appeared to be differentially regulated. The steady-state levels of insulin receptor mRNA increased remarkably during development and after postnatal day 10, when the peak of myelin structural gene (P0) expression occur and slowly increased further until at least postnatal day 90 in parallel with the growth of the myelin sheath. By employing immunofluorescence and RT-PCR, we observed significant increase in the P0 protein and mRNA levels in Schwann cells in response to the insulin than in insulin deprived counterparts. The presence of insulin in the high glucose medium ameliorated the altered protein and mRNA of P0 in Schwann cells compared to the insulin deprived counterparts. These studies demonstrate the importance of insulin and its receptor as possible regulatory factors in the PNS and also emphasizes their novel therapeutic applications in demyelinating diseases, especially in diabetic poly-neuropathy.     

The Expression of Nerve Growth Factor Receptor on Schwann Cells and the Effect of These Cells on Regeneration of Axons in Traumatically Injured Human Spinal Cord

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Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule,N-cadherin

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2mM Ca⁺⁺ or 0.2 mM Ca⁺⁺, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca⁺⁺, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca⁺⁺ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca⁺⁺ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca⁺⁺ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca⁺⁺ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca⁺⁺, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca⁺⁺. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca⁺⁺ levels had similar effects as lowering the Ca⁺⁺ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Schwann Cell Proliferation Is Accompanied by Enhanced Inositol Phospholipid Metabolism

Inositol phospholipid metabolism during mitogen-induced Schwann cell proliferation has been examined. Addition of axolemma- and myelin-enriched membrane fractions (AXL and MYE, respectively) to cultured Schwann cells stimulated 32P incorporation into phosphatidylinositol 4-monophosphate [PtdIns(4)P] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. During the first 5 min of incubation with the mitogens, the amount of 32P incorporated into PtdIns(4)P and PtdIns(4,5)P2 was four- to fivefold above control values. The phosphorylation of the inositol phospholipids was dependent on the concentration of membrane mitogens and was maximal within 1 h. Schwann cells that were prelabeled with [3H]glycerol and then stimulated with AXL and MYE displayed a 30-70% increase in the amounts of [3H]PtdIns(4)P and [3H]PtdIns(4,5)P2 and a 60-80% increase in the amount of [3H]phosphatidic acid. A concomitant 20% decrease in the content of [3H]PtdIns was observed after stimulation. These results suggest that the increased metabolism of PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 may be one of the initial molecular events in the transduction of the mitogenic signal across the Schwann cell plasma membrane.     

Myelin Basic Protein and Myelin Basic Protein Peptides Induce the Proliferation of Schwann Cells Via Ganglioside GM1 and the FGF Receptor

Myelin basic protein (MBP) and two peptides derived from MBP (MBP_1–44 and MBP_152–167) stimulated Schwann cell (SC) proliferation in a cAMP-mediated process. The two mitogenic regions of MBP did not compete with one another for binding to SC suggesting a distinctive SC receptor for each mitogenic peptide. Neutralizing antibodies to the fibroblast growth factor receptor blocked the mitogenic effect of the myelin-related SC mitogen found in the supernatant of myelin-fed macrophages. The binding of ¹²⁵I-MBP to Schwann cells was specifically inhibited by basic fibroblast growth factor (bFGF) and conversely the binding of ¹²⁵I-bFGF was competitively inhibited by MBP. These data suggested that the mitogenic effect of one MBP peptide was mediated by a bFGF receptor. The binding of MBP to ganglioside GM1 and the ability of MBP peptides containing homology to the B subunit of cholera toxin (which binds ganglioside GM1) to compete for the binding of a mitogenic peptide (MBP_1–44) to SC, identified ganglioside GM1 as a second SC receptor. Based on these results, we conclude that MBP_1–44 and MBP_152–167 associate with ganglioside GM1 and the bFGF receptor respectively to stimulate SC mitosis.     

A screen for mutations in zebrafish that affect myelin gene expression in Schwann cells and oligodendrocytes

Myelin is the multi-layered glial sheath around axons in the vertebrate nervous system. Myelinating glia develop and function in intimate association with neurons and neuron-glial interactions control much of the life history of these cells. However, many of the factors that regulate key aspects of myelin development and maintenance remain unknown. To discover new molecules that are important for glial development and myelination, we undertook a screen of zebrafish mutants with previously characterized neural defects. We screened for myelin basic protein (mbp) mRNA by in situ hybridization and identified four mutants (neckless, motionless, iguana and doc) that lacked mbp expression in parts of the peripheral and central nervous systems (PNS or CNS), despite the presence of axons. In all four mutants electron microscopy revealed that myelin-forming glia were present and had formed loose wraps around axons but did not form compact myelin. We found that addition of exogenous retinoic acid (RA) rescued mbp expression in neckless mutant embryos, which lack endogenous RA synthesis. Timed application of the RA synthesis inhibitor DEAB to wild type embryos showed that RA signalling is required at least 48 h before the onset of myelin protein synthesis in both CNS and PNS.     

Rat brain Na channel mRNAs in non-excitable Schwann cells

The expression of rat brain voltage-sensitive Na⁺ channel mRNAs in Schwann cells was examined using in situ hybridization cytochemistry and RT-PCR. The mRNAs of rat brain Na⁺ channel subtype II and III, but not subtype I, were detected in cultured Schwann cells from sciatic nerve and in intact sciatic nerve, which contains Schwann cells but not neuronal cell bodies. These results indicate that rat brain Na⁺ channel mRNAs, which have been considered as mainly neuronal-type messages, are also expressed in glial cells in vitro and in vivo.     

Aging Schwann cells in vitro

Schwann cells (SCs) can support the regeneration of lesioned fiber tracts of the peripheral and central nervous system and have been transplanted alone or in combination with synthetic nerve guides. For neuronal tissue engineering purposes, the cells must be isolated from small biopsies and expanded in vitro. In this study we analyze the impact of cell expansion on 9 different cell parameters, comparing short- and long-term cultured rat SCs, which we refer to as 'young' and 'old' or 'aged' cells, respectively. In comparison to young SCs, old SCs doubled the axonal outgrowth from dorsal root ganglion explants and displayed only one-third as much adhesion to the gray and white matter of spinal cord cryosections. In a 3-dimensional extracellular matrix the two cell populations showed very different cellular responses with regard to cell morphology and cell-cell adhesion. Cell proliferation of old SCs was independent of serum components and was not hampered by contact inhibition. In addition, population doubling times were reduced by a factor of almost three compared to those of young SCs. Despite considerable karyotype changes, with an average of 68.7 chromosomes versus 42 in native rat cells, old SCs did not show any increase in telomerase activity and loss of anchorage dependence--characteristics that are typical of tumor cells. The data also provide biological insights into which cell characteristics (proliferation and adhesion, for example) are functionally clustered and either change or remain constant with aging in vitro. Though the data indicate a lack of tumorigenic transformation coupled with increased neurite outgrowth-promoting activity after extensive SC expansion in vitro, thus suggesting better regeneration qualities, we strongly recommend that in vitro aged rat SCs (>11 passages) should not be employed for tissue engineering.     

Evidence That Secondary Rat Schwann Cells in Culture Maintain Their Differentiated Phenotype

Schwann cells, on receiving the correct signal, will encircle an axon and wrap it with a myelin sheath. To begin examining some of the mechanisms underlying the process of myelination in vitro, we isolated Schwann cells from the sciatic nerves of neonatal rats and generated large cell populations with cholera toxin. The immunological and biochemical properties of these secondary Schwann cells were characterized after five to seven passages in the absence of axonal contact. These cells continued to express antigens found in both myelinating (P0 and 2',3'-cyclic nucleotide phosphohydrolase) and nonmyelinating cells in vivo (A5E3 and glial fibrillary acidic protein) in addition to the markers common to both types of cells (Ran-1, 217c, S-100, and laminin). Biochemical analyses showed that these cells synthesize the very-long-chain fatty acids (22-26 carbon atoms) found in myelin membranes. Moreover, the enzymes required for the synthesis of myelin glycolipids (including sphingosine acyltransferase, UDP-galactose:ceramide galactosyltransferase, and cerebroside sulfotransferase) were still active, and metabolic labeling studies showed that galactocerebroside and sulfatide were synthesized even though the galactocerebroside pool was insufficient to be detected by immunostaining. Secondary Schwann cells also synthesized four species of myelin basic protein and the major structural glycoprotein in myelin, P0. The pathway necessary for glycosylation of P0 protein remained active, and an analysis of the oligosaccharide chain revealed that approximately 70% was processed to a complex form. In summary, we found that secondary Schwann cells still express most of the immunological markers of differentiated cells and continue to synthesize low levels of myelin components. Therefore, Schwann cells do not dedifferentiate in culture, as previously believed.     

P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord

The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3–5 mM) or a P2X7R agonist, 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.     

低温保存许旺细胞对周围神经再生的作用

目的：比较原代培养许旺细胞（Schwann cells,SCs)和冷冻保存的SCs移植对损伤后坐骨神经再生的作用。方法：原代培养和液氮保存的SCs分别移植到桥接缺损坐骨神经的硅胶管内。在移植后不同时间（第6和8周末）,硅胶管远端神经干内注射HRP,逆行追踪背根神经节和脊髓前角的标记神经元数量;测量再生神经纤维的复合动作电位传导速度;电镜观察再生神经纤维的髓鞘形成。结果：原代培养和冷冻保存SCs在移植后不同时间其背根神经节和脊髓前角神经元HRP标记细胞数量、再生神经纤维的复合动作电位传导速度基本一致,再生神经纤维髓鞘的形成未见明显差别。结论：冷冻保存的SCs仍具有促进损伤后周围神经再生的能力。     

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.     

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.