Role of N-cadherin in Schwann cell precursors of growing nerves

In the present paper, we determine the localization and developmental regulation of N-cadherin in embryonic rat nerves and examine the role of N-cadherin in this system. We also identify a major transition in the architecture of embryonic nerves and relating it to N-cadherin expression. We find that in early embryonic nerves, N-cadherin is primarily expressed in Schwann cell precursors. Pronounced expression is seen at distal nerve fronts where these cells associate with growth cones, and the proximal nerve ends, in boundary cap cells. Unexpectedly, N-cadherin is downregulated as precursors generate Schwann cells, coinciding with the time at which most axons make target connections. Therefore, glial N-cadherin expression is essentially restricted to the period of axon outgrowth. We also provide evidence that N-cadherin supports the formation of contacts between Schwann cell precursors and show that these cells are a favorable substrate for axon growth, unlike N-cadherin-negative Schwann cells. Induction of N-cadherin expression in Schwann cells by neuregulin-1 restores their ability to form contacts and support axon growth. Finally, we show that the loss of glial N-cadherin during embryonic nerve development is accompanied by a transformation of nerve architecture, involving the appearance of endoneurial connective tissue space, fibroblasts, Schwann cell basal lamina, and blood vessels. Because N-cadherin is likely to promote the extensive glial contacts typical of the compact embryonic nerve, we suggest that N-cadherin loss at the time of Schwann cell generation allows endoneurial space to appear between the glial cells, a development that eventually permits the extensive interactions between connective tissue and individual axon-Schwann cell units necessary for myelination.     

Microglia enhance dorsal root ganglion outgrowth in Schwann cell cultures

Transplantation of cellular populations to facilitate regrowth of damaged axons is a common experimental therapy for spinal cord injury. Schwann cells (SC) or microglia grafted into injury sites can promote axonal regrowth of central projections of dorsal root ganglion (DRG) sensory neurons. We sought to determine whether the addition of microglia or microglia-derived secretory products alters DRG axon regrowth upon cultures of SC. Rat DRG explants were grown on monolayers consisting of either SC, microglia, SC exposed to microglia-conditioned medium (MCM), or co-cultures with different relative concentrations of microglia. Image analysis revealed that, compared to SC alone, the extent of neurite outgrowth was significantly greater on SC-microglia co-cultures. Immunocytochemistry for extracellular matrix molecules showed that microglial cells stained positively for growth-promoting thrombospondin, whereas laminin and the inhibitory chondroitin sulfate proteoglycans (CSPGs) were localized primarily to SC. Notably, immunoreactivity for CSPGs appeared reduced in areas associated with DRG outgrowth in co-cultures and SC exposed to MCM. These results show that microglia or their secreted products can augment SC-mediated DRG regrowth in vitro, indicating that co-grafting SC with microglia provides a novel approach to augment sensory fiber regeneration after spinal cord injury.     

Clinical implications of Schwann cell biology

The neuroglia of the peripheral nervous system (PNS) are derived from the neural crest and are a diverse family of cells. They consist of myelinating Schwann cells, non‐myelinating Schwann cells, satellite cells, and perisynaptic Schwann cells. Due to their prominent role in the formation of myelin, myelinating Schwann cells are the best recognised of these cells. However, Schwann cells and the other neuroglia of the PNS have many functions that are independent of myelination and contribute significantly to the functioning of the peripheral nerve in both health and disease. Here we discuss the contribution of PNS neuroglial cells to clinical deficit in neurodegenerative disease, peripheral neuropathy, and pain.     

Nerve expansion in nerve regeneration: Effect of time on induction of ornithine decarboxylase and Schwann cell proliferation

We studied the effect of initiation time of nerve expansion after nerve transection on the induction of ODC activity and Schwann cell proliferation in nerve tissue under Wallerian degeneration. The levels of ODC activity and Schwann cell proliferation decreased as the initiation time of nerve expansion was delayed after nerve transection, and peak levels of ODC activity following nerve expansion preceded peak levels of Schwann cell proliferation. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1314–1317, 1997     

Lithium promotes proliferation and suppresses migration of Schwann cells

Schwann cell proliferation,migration and remyelination of regenerating axons contribute to regeneration after peripheral nervous system injury.Lithium promotes remyelination by Schwann cells and improves peripheral nerve regeneration.However,whether lithium modulates other phenotypes of Schwann cells,especially their proliferation and migration remains elusive.In the current study,primary Schwann cells from rat sciatic nerve stumps were cultured and exposed to 0,5,10,15,or 30 mM lithium chloride(LiCl)for 24 hours.The effects of LiCl on Schwann cell proliferation and migration were examined using the Cell Counting Kit-8,5-ethynyl-2′-deoxyuridine,Transwell and wound healing assays.Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine assays showed that 5,10,15,and 30 mM LiCl significantly increased the viability and proliferation rate of Schwann cells.Transwell-based migration assays and wound healing assays showed that 10,15,and 30 mM LiCl suppressed the migratory ability of Schwann cells.Furthermore,the effects of LiCl on the proliferation and migration phenotypes of Schwann cells were mostly dose-dependent.These data indicate that lithium treatment significantly promotes the proliferation and inhibits the migratory ability of Schwann cells.This conclusion will inform strategies to promote the repair and regeneration of peripheral nerves.All of the animal experiments in this study were ethically approved by the Administration Committee of Experimental Animal Center of Nantong University,China(approval No.20170320-017)on March 2,2017.     

Two mitogenic regions of myelin basic protein interact with different receptors to induce Schwann cell proliferation in a cAMP dependent process

Previous studies have shown that myelin basic protein (MBP) is mitogenic for Schwann cells (SCs) in the presence of elevated intracellular cAMP. Two mitogenic regions of MBP have been identified: one mitogenic region within the first 44 residues of the aminoterminus (1–44) and the other mitogenic region within the terminal 15 residues of the carboxyl end of the molecule (152–167). Unlike the mitogenic effect of a myelin enriched fraction (MEF), the mitogenic effect of MBP was not reduced by the addition of the lysosomal inhibitor, ammonium chloride. These data indicate that MBP causes SC proliferation by direct interaction of MBP with a surface receptor. Using Scatchard analysis of the binding of MBP to SCs, we report that treatment with forskolin does not cause the upregulation of receptors for MBP. Moreover, MBP blocks the cross-linking of ¹²⁵I-bFGF with two fibroblast growth factor (FGF) receptors having apparent molecular weights of 140 kDa and 120 kDa, respectively. Since neither TGF-β nor PDGFBB displaced cell surface bound ¹²⁵I-MBP, we conclude that MBP binds to the FGF receptor rather than other growth factor receptors. Furthermore, only MBP_1–44 interacted with ganglioside GM1, whereas MBP_152–167 did not interact with this ganglioside. These results are consistent with the view that ganglioside GM1 mediates the mitogenic effects of MBP_1–44, while the FGF receptor mediates the mitogenic effect of MBP_152–67. Intracellular cAMP of SCs was transiently increased after the addition of macrophage conditioned medium, suggesting that macrophages may produce factors in vivo which can transiently elevate intracellular cAMP levels, allowing a wave of SC proliferation in response to MBP-related mitogens. ©1995 Wiley-Liss, Inc.     

Erythropoietin reduces Schwann cell TNF-alpha, Wallerian degeneration and pain-related behaviors after peripheral nerve injury

Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain-like behaviors. These effects are mediated in large part by pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF-alpha has been implicated in the development of pain-related behaviors, we measured TNF-alpha mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF-alpha mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF-alpha in Schwann cells, axoplasm and macrophages. In rhEpo-treated animals, TNF-alpha immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF-alpha in CCI by blocking expression of TNF-alpha in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF-alpha expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF-alphain vitro. These results indicated that rhEpo regulates TNF-alpha by multiple mechanisms; rhEpo regulates TNF-alpha mRNA expression by Schwann cells but also may directly counteract TNF-alpha signaling pathways that lead to injury, chronic pain and/or death.     

Neuron-glia signaling and the protection of axon function by Schwann cells

The interaction between neurons and glial cells is a feature of all higher nervous systems. In the vertebrate peripheral nervous system, Schwann cells ensheath and myelinate axons thereby allowing rapid saltatory conduction and ensuring axonal integrity. Recently, some of the key molecules in neuron–Schwann cell signaling have been identified. Neuregulin-1 (NRG1) type III presented on the axonal surface determines the myelination fate of axons and controls myelin sheath thickness. Recent observations suggest that NRG1 regulates myelination via the control of Schwann cell cholesterol biosynthesis. This concept is supported by the finding that high cholesterol levels in Schwann cells are a rate-limiting factor for myelin protein production and transport of the major myelin protein P0 from the endoplasmic reticulum into the growing myelin sheath. NRG1 type III activates ErbB receptors on the Schwann cell, which leads to an increase in intracellular PIP3 levels via the PI3-kinase pathway. Surprisingly, enforced elevation of PIP3 levels by inactivation of the phosphatase PTEN in developing and mature Schwann cells does not entirely mimic NRG1 type III stimulated myelin growth, but predominantly causes focal hypermyelination starting at Schmidt–Lanterman incisures and nodes of Ranvier. This indicates that the glial transduction of pro-myelinating signals has to be under tight and life-long control to preserve integrity of the myelinated axon. Understanding the cross talk between neurons and Schwann cells will help to further define the role of glia in preserving axonal integrity and to develop therapeutic strategies for peripheral neuropathies such as CMT1A.     

Inhibition of p38 mitogen-activated protein kinase interferes with cell shape changes and gene expression associated with Schwann cell myelination

In the present study we demonstrate that p38, a member of the mitogen-activated protein kinase (MAPK) family, is essential for ascorbate- and laminin-induced myelination in Schwann cell-dorsal root ganglion neuron cocultures. The inhibitory effect of the specific p38 blockers, PD 169316 and SB 203580, on ascorbate-induced myelination was exerted during the early stages (1-2 days) of ascorbate treatment. Inhibition of p38 was further shown to prevent the alignment of Schwann cells along axons in laminin-treated cocultures. The addition of laminin to Schwann cell-dorsal root ganglion neuron cocultures stimulated phosphorylation of p38, thereby demonstrating a link between laminin-induced myelination and p38 activation. Similarly, the small heat shock protein, Hsp27, which is phosphorylated by MAPKAPK2, a downstream substrate of p38, was phosphorylated in response to the addition of laminin to the cocultures. The p38 inhibitors did not affect the proliferation or survival of Schwann cells in the cocultures as assessed by BrdU incorporation and total cell counts. However, p38 inhibition interfered with an early stage in myelination, thereby preventing ascorbate-induced increases in the levels of mRNAs encoding MBP, MAG, and P(0) and reducing laminin deposition. These results indicate that activation of p38 by a signaling pathway(s) involving laminin and appropriate integrin receptor(s) is required for the alignment of Schwann cells with axons that precedes myelination.     

吡咯喹啉醌对许旺细胞增殖及Sox10表达的影响

背景：雪旺细胞是神经组织工程的种子细胞,但其体外增殖缓慢,难以满足科研与临床对其的需要,吡咯喹啉醌可以对多种细胞的增值产生促进作用,本课题研究吡咯喹啉醌对雪旺细胞增殖及Sox10表达的影响。 目的：研究吡咯喹啉醌对雪旺细胞的增殖作用并探讨其对雪旺细胞Sox10基因表达的影响。 设计、时间及地点：细胞增殖及基因表达检测,于2009-01/04在武汉大学人民医院中心实验室完成。 材料：出生3～4天SD大鼠20只,由武汉大学医学部实验动物中心提供,DMEM/F12培养基、胎牛血清由Gibco公司（USA）提供,Trizol、PCR引物由Invitrogen公司提供,RT-PCR试剂盒由Fermentas公司提供。 方法 雪旺细胞体外培养及纯化,S-100免疫荧光鉴定雪旺细胞;细胞经无血清培养12h后,加入10nM吡咯喹啉醌继续培养24h观察其形态学改变;加入不同浓度（0,1,10,100,1000,10000nM）吡咯喹啉醌于雪旺细胞培养24h,利用RT-PCR技术检测Sox10的mRNA表达。 主要观察指标：观察雪旺细胞形态学,并对雪旺细胞进行计数,应用RT-PCR方法检测Sox10的表达。 结果 吡咯喹啉醌促使雪旺细胞发生形态学改变,多数呈束状或并排生长,且细胞数目增多;1～1000nM吡咯喹啉醌可使雪旺细胞Sox10表达增高,100nM时表达最高;10000nM时对Sox10的表达表现为抑制作用（P<0.05）。 结论 吡咯喹啉醌可促进雪旺细胞增值并促使其发生形态学改变,且Sox10在PQQ促雪旺细胞增殖过程中表达上调。     

MMP‐9 controls Schwann cell proliferation and phenotypic remodeling via IGF‐1 and ErbB receptor‐mediated activation of MEK/ERK pathway

Phenotypic remodeling of Schwann cells is required to ensure successful regeneration of damaged peripheral axons. After nerve damage, Schwann cells produce an over 100‐fold increase in metalloproteinase‐9 (MMP‐9), and therapy with an MMP inhibitor increases the number of resident (but not infiltrating) cells in injured nerve. Here, we demonstrate that MMP‐9 regulates proliferation and trophic signaling of Schwann cells. Using in vivo BrdU incorporation studies of axotomized sciatic nerves of MMP‐9?/? mice, we found increased Schwann cell mitosis in regenerating (proximal) stump relative to wild‐type mice. Treatment of cultured primary Schwann cells with recombinant MMP‐9 suppressed their growth, mitogenic activity, and produced a dose‐dependent, biphasic, and selective activation of ERK1/2, but not JNK and p38 MAPK. MMP‐9 induced ERK1/2 signaling in both undifferentiated and differentiated (using dbcAMP) Schwann cells. Using inhibitors to MEK and trophic tyrosine kinase receptors, we established that MMP‐9 regulates Ras/Raf/MEK—ERK pathways through IGF‐1, ErbB, and PDGF receptors. We also report on the early changes of MMP‐9 mRNA expression (within 24 h) after axotomy. These studies establish that MMP‐9 controls critical trophic signal transduction pathways and phenotypic remodeling of Schwann cells. © 2009 Wiley‐Liss, Inc.     

Myelination of two axons by a single Schwann cell

Summary A Schwann cell can form only one internode of myelin around an axon. However, we observed the formation by a single Schwann cell of myelin around two axons of different diameters in the sural nerve of a 45-year-old man with mononeuritis multiplex. Schwann cell processes spiraled in the same direction around each axon, forming mesaxons. The findings in this case appear to be an undescribed type of aberrant myelination.     

Inhibition of cell proliferation of human gliomas by benzodiazepines in vitro

The effects of several benzodiazepines (diazepam, clonazepam, Ro 15-1788 and Ro 5-4864) on cell proliferation of 2 human gliomas were estimated in vitro by means of [3H]-thymidine uptake assay. It was found that all tested benzodiazepines suppressed [3H]-thymidine incorporation into the DNA of glioma cells, the effects being stronger in case of peripheral-type benzodiazepine receptor ligands. The results indicated that benzodiazepines might exert an antiproliferative action on glioma tumour cells growth.     

Effects of nerve segment supernatants on cultured Schwann cell proliferation and laminin production

Mouse sciatic nerves were transected and 3 hr to 16 days later proximal segments were removed and homogenized. Supernatants of these segments or of normal sciatic nerves were added to Schwann cells maintained in Dulbecco's modified Eagle's medium (DMEM) + 15% fetal calf serum (FCS). After 6 days, Schwann cells were solubilized and the protein content was measured using a Bio-Rad (Melville, NY) protein assay. Samples containing the same amounts of protein were then applied to microtiter plates and the laminin content was determined by enzymelinked immunosorbent assay (ELISA). Lysates of cultures treated with 24 hr proximal segment supernatants contained significantly higher levels of laminin than those prepared from other intervals, from distal segments, or from control nerves. Increased surface and cytoplasmic anti-laminin immunoreactivity also was found in Schwann cells treated with 24 hr supernatants. To identify the source(s) of this effect, proximal segments removed 24 hr after transection were bisected; supernatants were prepared from each half and tested. Significant increases in laminin production were produced by supernatants from both halves. When supernatants from proximal and distal halves were compared, the latter produced significantly higher laminin levels. Electron microscopic examination of both halves showed that distal halves contained sprouting neurites and growth cones ensheathed by Schwann cells which had a basal lamina and resembled those seen during development and regeneration. Proximal halves appeared normal. Schwann cell proliferation also was compared in supernatant-treated cultures by using a bromodeoxyuridine (BrdU) ELISA. The 24 hr and 2 day supernatants increased Schwann cell proliferation significantly; 12 hr, 4 day, and 8 day supernatants produced smaller increases. Our observations suggest that axons undergoing early regenerative changes are one of several possible sources of substance(s) in our proximal segment supernatants which increased Schwann cell proliferation and laminin production. © 1994 Wiley-Liss, Inc.     

Adhesion and proliferation are enhanced in vitro in Schwann cells from nerve undergoing Wallerian degeneration

Proliferation of Schwann cells during nerve degeneration or regeneration is well documented in vivo. We investigated whether the proliferative response of Schwann cells to injury is retained in vitro. Using 5-month-old male C57BL mice, Schwann cells were isolated from sciatic nerves under 3 experimental conditions: (1) uninjured, (2) after permanent nerve-transection, or (3) after nerve-crush, which permits axonal regeneration. Schwann cells rarely attached to polylysine-coated coverslips when isolated from uninjured or 1 day posttransection/crush nerves. The number of adherent cells increased when Schwann cells were isolated 3 days after nerve-transection or -crush. When cells were isolated from transected nerves, cell adhesion reached a peak 2 weeks after the injury and then declined. Maximal attachment of Schwann cells occurred when the cells were isolated 2-4 weeks after nerve-crush. The percentage of Schwann cells with spreading processes corresponded closely with the number of thymidine-labeled cells at 1 day in vitro. The in vitro capacity of cells to spread and incorporate thymidine reached maximal levels at 5 days posttransection/crush. Capacity of cells to spread and incorporate thymidine subsequently decreased with time following transection. However, a biphasic elevation in cell spreading and thymidine incorporation was observed in Schwann cells isolated from crushed nerves. Maximal growth of Schwann cells in vitro occurred at 1-2 weeks posttransection and at 1-4 weeks postcrush. Adhesion and spreading of Schwann cells were promoted by coating coverslips with laminin or fibronectin. Preincubation of Schwann cells with soluble laminin or fibronectin prevented the initial cell attachment induced by the corresponding protein. Our results suggest that Schwann cells from injured nerves possess binding sites for laminin and fibronectin, which are, in part, responsible for the enhanced adhesion of Schwann cells in vitro. This study provides a new method for preparation of Schwann cells from peripheral nerves of adult mice.     

Lipid composition and phospholipid asymmetry of membranes from a Schwann cell line

We report the total lipid composition and phospholipid asymmetry of a plasma membrane preparation isolated from a Schwann cell line (NF1T) derived from a human neurofibroma. The specific activities of three plasma membrane markers (5′-nucleotidase, Na-K-ATPase, and CNPase) were 8-fold, 12-fold, and 16-fold higher, respectively, in the plasma membrane fraction compared to the specific activities found in the total homogenate. The specific activities of the marker enzymes of intracellular membranes in the isolated plasma membrane fraction indicated little contamination with intracellular organelles. The enrichment of cholesterol (3-fold), sphingomyelin (3-fold), and glycolipids (cerebrosides 8-fold, sulfatides 5-fold) also indicated a high degree of purity of the plasma membrane fraction. The high content of phosphatidylinositol and phosphatidylcholine (10% and 44% of total phospholipid) and the low phosphatidylserine and phosphatidylethanolamine content (3% and 14% of the total phospholipid) were also characteristic of the plasma membrane fraction derived from this cell line. The transbilayer phospholipid distribution of the plasma membrane in intact cells and in the isolated plasma membrane fraction was investigated by using phospholipase A₂ (bee venom) and sphingomyelinase (S. aureus). The phospholipid asymmetry of NF1T plasma membrane followed the general features of phospholipid asymmetry in eukaryotic cells: sphingomyelin and phosphatidylcholine were preferentially located in the outer leaflet (90% and 89%, respectively) while the aminophospholipids phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol were in the inner half of the membrane (85%, 96%, and 69%, respectively). A high percentage of the total plasma membrane phosphatidylinositol (31%) was found in the outer side of the membrane indicating a decreased asymmetric distribution for this negatively charged phospholipid. The phospholipid asymmetry found in the plasma membrane vesicle fraction corroborated the phospholipid asymmetry of the intact cells, thus confirming that the plasma membrane vesicles maintained the original orientation and lipid asymmetry after homogenization and/or sonication. J. Neurosci. Res. 49:372–380, 1997. © 1997 Wiley-Liss, Inc.