Leukemia inhibitory factor regulates Schwann cell proliferation and migration and affects peripheral nerve regeneration

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that stimulates neuronal development and survival. Our previous study has demonstrated that LIF mRNA is dysregulated in the peripheral nerve segments after nerve injury. Here, we show that LIF protein is abundantly expressed in Schwann cells after rat sciatic nerve injury. Functionally, suppressed or elevated LIF increases or decreases the proliferation rate and migration ability of Schwann cells, respectively. Morphological observations demonstrate that in vivo application of siRNA against LIF after peripheral nerve injury promotes Schwann cell migration and proliferation, axon elongation, and myelin formation. Electrophysiological and behavior assessments disclose that knockdown of LIF benefits the function recovery of injured peripheral nerves. Differentially expressed LIF affects the metabolism of Schwann cells and negatively regulates ERFE (Erythroferrone). Collectively, our observations reveal the essential roles for LIF in regulating the proliferation and migration of Schwann cells and the regeneration of injured peripheral nerves, discover ERFE as a downstream effector of LIF, and extend our understanding of the molecular mechanisms underlying peripheral nerve regeneration.Subject terms: Cell migration, Regeneration and repair in the nervous system     

Role of laminin terminal globular domains in basement membrane assembly

Laminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted alpha1-, beta1-, or gamma1-LN domains or with duplicated beta1- or alpha1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of alpha-, beta-, and gamma-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin.     

Brain lipid binding protein in axon-Schwann cell interactions and peripheral nerve tumorigenesis

Loss of axonal contact characterizes Schwann cells in benign and malignant peripheral nerve sheath tumors (MPNST) from neurofibromatosis type 1 (NF1) patients. Tumor Schwann cells demonstrate NF1 mutations, elevated Ras activity, and aberrant epidermal growth factor receptor (EGFR) expression. Using cDNA microarrays, we found that brain lipid binding protein (BLBP) is elevated in an EGFR-positive subpopulation of Nf1 mutant mouse Schwann cells (Nf1(-/-) TXF) that grows away from axons; BLBP expression was not affected by farnesyltransferase inhibitor, an inhibitor of H-Ras. BLBP was also detected in EGFR-positive cell lines derived from Nf1:p53 double mutant mice and human MPNST. BLBP expression was induced in normal Schwann cells following transfection with EGFR but not H-Ras12V. Furthermore, EGFR-mediated BLBP expression was not inhibited by dominant-negative H-Ras, indicating that BLBP expression is downstream of Ras-independent EGFR signaling. BLBP-blocking antibodies enabled process outgrowth from Nf1(-/-) TXF cells and restored interaction with axons, without affecting cell proliferation or migration. Following injury, BLBP expression was induced in normal sciatic nerves when nonmyelinating Schwann cells remodeled their processes. These data suggest that BLBP, stimulated by Ras-independent pathways, regulates Schwann cell-axon interactions in normal peripheral nerve and peripheral nerve tumors.     

Polyglycolic acid filaments guide Schwann cell migration in vitro and in vivo

Nerve conduits filled with longitudinal aligned filaments have demonstrated a better regenerative outcome for bridging large peripheral nerve gaps than hollow nerve conduits. In the present study, we investigated the in vitro and in vitro cellular behavior of Schwann cells on polyglycolic acid (PGA) filaments by immunocyto/histochemistry and light/electron microscopy. After 1-3-week culture of rat dorsal root ganglia (DRGs) onto PGA filaments, Schwann cells from rat DRGs adhered to and migrated along PGA filaments. Twenty-four rats received implantation of chitosan conduits inserted with PGA filaments to bridge 10-mm-long sciatic nerve gaps. At 1, 2, 3 and 4 weeks post-implantation (n = 6, each time point), Schwann cells were found to migrate along PGA filaments and form cell columns resembling bands of Büngner. These results suggest that PGA filaments may play a contact guidance role in Schwann cell migration and thus serve as a promising conduit-filling material to facilitate peripheral nerve repair.     

Dynamic Change of Prohibitin2 Expression in Rat Sciatic Nerve After Crush

As a novel cell cycle inhibitor, PHB2 controls the G1/S transition in cycling cells in a complex manner. Its aberrant expression is closely related to cell carcinogenesis. While its expression and role in peripheral nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve crush (SNC) model in adult rats to examine the dynamic changes of PHB2. Temporally, PHB2 expression was sharply decreased after sciatic nerve crush and reached a valley at day 5. Spatially, PHB2 was widely expressed in the normal sciatic nerve including axons and Schwann cells. While after injury, PHB2 expression decreased predominantly in Schwann cells. The alteration was due to the decreased expression of PHB2 in Schwann cells after SNC. PHB2 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, PHB2 largely localized with GAP43 in axons in the crushed segment. Collectively, we suggested that PHB2 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Temporal-Spatial Expressions of Spy1 in Rat Sciatic Nerve After Crush

As a novel cell cycle protein, Spy1 enhances cell proliferation, promotes the G1/S transition as well as inhibits apoptosis in response to UV irradiation. Spy1 levels are tightly regulated during mammary development, and overexpression of Spy1 accelerates tumorigenesis in vivo. But little is known about the role of Spy1 in the pathological process of damage and regeneration of the peripheral nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of Spy1. Spy1 expression was elevated gradually after sciatic nerve crush and peaked at day 3. The alteration was due to the increased expression of Spy1 in axons and Schwann cells after SNC. Spy1 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, Spy1 largely localized in axons in the crushed segment, but rarely co-localized with GAP43. These findings suggested that Spy1 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

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.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.     

Transcriptional profile of the human peripheral nervous system by serial analysis of gene expression

The peripheral nerve contains both nonmyelinating and myelinating Schwann cells. The interactions between axons, surrounding myelin, and Schwann cells are thought to be important for the correct functioning of the nervous system. To get insight into the genes involved in human myelination and maintenance of the myelin sheath and nerve, we performed a serial analysis of gene expression of human sciatic nerve and cultured Schwann cells. In the sciatic nerve library, we found high expression of genes encoding proteins related to lipid metabolism, the complement system, and the cell cycle, while cultured Schwann cells showed mainly high expression of genes encoding extracellular matrix proteins. The results of our study will assist in the identification of genes involved in maintenance of myelin and peripheral nerve and of genes involved in inherited peripheral neuropathies.     

人脐带干细胞来源的外泌体促进体外培养雪旺细胞迁移的研究

目的:外泌体是活细胞分泌的来源于多囊泡体的膜性囊泡,其主要作用包括携带与运输。雪旺细胞是周围神经再生中非常优秀的种子细胞,但其迁移能力较差,影响修复效果。本文旨在探讨外泌体和雪旺细胞共培养是否可以促进雪旺细胞迁移。方法:本实验通过分离纯化人脐带干细胞外泌体和大鼠坐骨神经雪旺细胞并鉴定,随后将其共培养于Transwell小室观察雪旺细胞迁移率。结果:通过人脐带干细胞超高速离心法得到的外泌体高表达干细胞标志物CD44(92.2±3.6%)、CD73(99.1±0.6%),并且低表达单核细胞表面抗原CD14(0.5±0.06%)以及造血干细胞表面抗原CD34(0.4±0.07%),外泌体鉴定高表达CD81和CD9;雪旺细胞培养鉴定纯度达(92.3±2.7)%;均符合实验要求。通过Transwell小室实验发现外泌体可以明显促进雪旺细胞的迁移,并且具有一定剂量关系。结论:外泌体可以提高雪旺细胞的迁移能力,从而使雪旺细胞在组织工程领域中的应用产生巨大突破。     

Up-Regulation of HDAC4 is Associated with Schwann Cell Proliferation After Sciatic Nerve Crush

Histone deacetylase 4 (HDAC4), a member of the class IIa HDACs subfamily, has emerged as a critical regulator of cell growth, differentiation, and migration in various cell types. It was reported that HDAC4 stimulated colon cell proliferation via repression of p21. Also, HDAC4 contributes to platelet-derived growth factor-BB-induced proliferation and migration of vascular smooth muscle cells. Furthermore, HDAC4 may play an important role in the regulation of neuronal differentiation and survival. However, the role of HDAC4 in the process of peripheral nervous system regeneration after injury remains virtually unknown. Herein, we investigated the spatiotemporal expression of HDAC4 in a rat sciatic nerve crush model. We found that sciatic nerve crush induced up-regulated expression of HDAC4 in Schwann cells. Moreover, the expression of the proliferation marker Ki-67 exhibited a similar tendency with that of HDAC4. In cell cultures, we observed increased expression of HDAC4 during the process of TNF-α-induced Schwann cell proliferation, whereas the protein level of p21 was down-regulated. Interference of HDAC4 led to enhanced expression of p21 and impaired proliferation of Schwan cells. Taken together, our findings implicated that HDAC4 was up-regulated in the sciatic nerve after crush, which was associated with proliferation of Schwann cells.     

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.     

Netrin-1 induces proliferation of Schwann cells through Unc5b receptor

Netrin and its receptors, DCC (Deleted in Colorectal Cancer) and Unc5, are proposed to be involved in the axon guidance and neuroglial migration during development. However, accumulating evidence implies that they may also participate in the cell survival and apoptosis. Here, we show that netrin-1 induces proliferation of Schwann cells. Unc5b is the sole receptor expressed in RT4 schwannoma cells and adult primary Schwann cells, and netrin-1 and Unc5b are found to be expressed in the injured sciatic nerve. It was also found that the netrin-1-induced Schwann cell proliferation was blocked by the specific inhibition of Unc5b expression with RNAi. These data suggest that netrin-1 could be an endogenous trophic factor for Schwann cells in the injured peripheral nerves.     

周围神经损伤后神经组织中Par-3蛋白表达和分布的变化

目的观察极性蛋白Par-3在损伤后神经组织中的表达和分布,探讨Par-3蛋白在周围神经损伤后髓鞘再生中的作用。方法 32只Sprague Dawley大鼠随机分为正常对照组、损伤组(坐骨神经损伤后第1、2、4、8周)。制备坐骨神经挤压伤模型,分别于损伤后各时间点,采用免疫组织化学法检测坐骨神经损伤远端Par-3蛋白的表达和分布。结果正常大鼠坐骨神经组织中即存在Par-3蛋白,但表达量少,且仅分布于Schwann细胞核内。坐骨神经损伤后,Par-3蛋白的表达和分布发生变化。损伤后1周,Par-3蛋白表达开始升高,Par-3散在分布于Schwann细胞核和细胞浆内。损伤后2周,神经组织中的Par-3蛋白达峰值,在Schwann细胞浆内呈不对称性分布似包绕轴突,呈新月形或C形。损伤后4周和8周,Par-3蛋白表达显著降低,神经组织中Par-3蛋白主要分布于Schwann细胞核内,胞浆内很少。结果 极性蛋白Par-3可能参与周围神经损伤后Schwann细胞的髓鞘再生。     

Down‐regulation of long non‐coding RNA MEG3 promotes Schwann cell proliferation and migration and repairs sciatic nerve injury in rats

Peripheral nerve injury and regeneration are complex processes and involve multiple molecular and signalling components. However, the involvement of long non‐coding RNA (lncRNA) in this process is not fully clarified. In this study, we evaluated the expression of the lncRNA maternally expressed gene 3 (MEG3) in rats after sciatic nerve transection and explored its potential mechanisms. The expression of lncRNA MEG3 was up‐regulated following sciatic nerve injury and observed in Schwann cells (SCs). The down‐regulation of lncRNA MEG3 in SCs enhanced the proliferation and migration of SCs via the PTEN/PI3K/AKT pathway. The silencing of lncRNA MEG3 promoted the migration of SCs and axon outgrowth in rats after sciatic nerve transection and facilitated rat nerve regeneration and functional recovery. Our findings indicated that lncRNA MEG3 may be involved in nerve injury and injured nerve regeneration in rats with sciatic nerve defects by regulating the proliferation and migration of SCs. This gene may provide a potential therapeutic target for improving peripheral nerve injury.     

ProBDNF inhibits collective migration and chemotaxis of rat Schwann cells

Schwann cell migration, including collective migration and chemotaxis, is essential for the formation of coordinate interactions between Schwann cells and axons during peripheral nerve development and regeneration. Moreover, limited migration of Schwann cells imposed a serious obstacle on Schwann cell-astrocytes intermingling and spinal cord repair after Schwann cell transplantation into injured spinal cords. Recent studies have shown that mature brain-derived neurotrophic factor, a member of the neurotrophin family, inhibits Schwann cell migration. The precursor form of brain-derived neurotrophic factor, proBDNF, was expressed in the developing or degenerating peripheral nerves and the injured spinal cords. Since “the yin and yang of neurotrophin action” has been established as a common sense, proBDNF would be expected to promote Schwann cell migration. However, we found, in the present study, that exogenous proBDNF also inhibited in vitro collective migration and chemotaxis of RSC 96 cells, a spontaneously immortalized rat Schwann cell line. Moreover, proBDNF suppressed adhesion and spreading of those cells. At molecular level, proBDNF inhibits F-actin polymerization and focal adhesion dynamics in cultured RSC 96 cells. Therefore, our results suggested a special case against the classical opinion of “the yin and yang of neurotrophin action” and implied that proBDNF might modulate peripheral nerve development or regeneration and spinal cord repair through perturbing native or transplanted Schwann cell migration.