miR-140-3p suppresses the proliferation and migration of macrophages

Macrophages benefit myelin debris removal, blood vessel formation, and Schwann cell activation following peripheral nerve injury. Identifying factors that modulate macrophage phenotype may advantage the repair and regeneration of injured peripheral nerves. microRNAs (miRNAs) are important regulators of many physiological and pathological processes, including peripheral nerve regeneration. Herein, we investigated the regulatory roles of miR-140-3p, a miRNA that was differentially expressed in injured rat sciatic nerves, in macrophage RAW264.7 cells. Observations from EdU proliferation assay demonstrated that elevated miR-140-3p decreased the proliferation rates of RAW264.7 cells while suppressed miR-140-3p increased the proliferation rates of RAW264.7 cells. Transwell-based migration assay showed that up-regulated and down-regulated miR-140-3p led to elevated and reduced migration abilities, respectively. However, the abundances of numerous phenotypic markers of M1 and M2 macrophages were not significantly altered by miR-140-3p mimic or inhibitor transfection. Bioinformatic analysis and miR-140-3p-induced gene suppression examination suggested that Smad3 might be the target gene of miR-140-3p. These findings illuminate the inhibitory effects of miR-140-3p on the proliferation and migration of macrophages and contribute to the cognition of the essential roles of miRNAs during peripheral nerve regeneration.     

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.     

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.     

Tropic 1808基因在大鼠损伤神经组织中的表达

目的观察Tropic 1808基因在大鼠正常和损伤坐骨神经组织中的表达,探讨Tropic 1808基因在周围神经损伤与再生过程中的作用.方法采用地高辛标记的Tropic 1808 cDNA探针、抗大鼠S-100蛋白抗体,以原位杂交和免疫组织化学双重染色法,观察Tropic 1808基因在正常和损伤大鼠坐骨神经组织中的表达.结果免疫组化结果显示,大鼠正常坐骨神经可表达S-100蛋白,但表达量较低;神经损伤后,其远侧端S-100蛋白的表达量明显增加.原位杂交结果显示,大鼠正常坐骨神经组织未见Tropic 1808 mRNA杂交信号;损伤神经的远侧端呈现较强的阳性信号,而且在部分S-100强阳性反应区可见Tropic 1808 mRNA杂交信号.结论 Tropic 1808基因在正常坐骨神经组织中未见表达;坐骨神经损伤后,其远侧端增殖的雪旺氏细胞可表达Tropic 1808 mRNA.提示,Tropic 1808是一种周围神经损伤后特异表达的基因.     

Inhibition of Ras extracellular-signal-regulated kinase (ERK) mediated signaling promotes ciliary neurotrophic factor (CNTF) expression in Schwann cells

Ciliary neurotrophic factor (CNTF) can prevent injury-induced motor neuron death. However, it is also evident that expression of CNTF in Schwann cells is suppressed during nerve regeneration. In this report, we have addressed the mechanism underlying the down-regulation of CNTF expression in injured nerves using a mouse Schwann cell line IMS32 and mouse sciatic nerve. In IMS32 cells, activation of the Ras extracellular-signal-regulated kinase (ERK) pathway by adenoviral vector-mediated expression of dominant active MEK1 did not alter a basal level of CNTF expression, whereas inhibition of the Ras-ERK pathway by using adenoviral vectors resulted in a marked increase in CNTF expression. This inverse relation between before and after axotomy was also observed in mouse sciatic nerve. In the axotomized sciatic nerve, the phosphorylated ERK was markedly increased; in contrast, the expression of CNTF was markedly decreased. These findings suggest that an inactive state of ERK is crucial for the CNTF expression in Schwann cells, and that activation of ERK following nerve injury critically influences the expression of CNTF. This might well explain why CNTF is highly expressed in quiescent Schwann cells in the peripheral nervous system, and also why CNTF is not abundant in axotomized nerves or cultured Schwann cells in which the proliferation signal is obviously active.     

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。     

Nidogen is a prosurvival and promigratory factor for adult Schwann cells

Schwann cells provide a favorable microenvironment for successful regeneration of the injured peripheral nerve. Even though the roles of extracellular matrix proteins in the Schwann cell physiology have long been studied, the precise function of nidogen, a ubiquitous component of the basal lamina, in Schwann cells is unknown. In this study, we show that the protein and mRNA messages for nidogens are up-regulated in the sciatic nerve after sciatic nerve transection. We demonstrate that recombinant nidogen-1 increased the process formation of Schwann cells cultured from adult rat sciatic nerves and that nidogen-1 prevented Schwann cells from serum-deprivation-induced death. In addition, nidogen-1 promoted spontaneous migration of Schwann cells in two-independent migration assays. The Schwann cell responses to the recombinant nidogen-1 were specific because the nidogen-binding ectodomain of tumor endothelial marker 7 inhibited the nidogen responses without affecting Schwann cell response to laminin. Finally, we found that beta1 subunit-containing integrins play a key role in the nidogen-induced process formation, survival, and migration of Schwann cells. Altogether, these results indicate that nidogen has a prosurvival and promigratory activity on Schwann cells in the peripheral nerve.     

Leukemia Inhibitory Factor Is an Autocrine Survival Factor for Schwann Cells

Abstract: Schwann cells play a major role in promoting nerve survival and regeneration after injury. Their activities include providing neurotrophic factors and increasing the production of extracellular matrix components and cell surface adhesion molecules to promote axon regeneration. Following nerve transection, leukemia inhibitory factor (LIF) is up-regulated by Schwann cells at the injury site. LIF receptors are also up-regulated at the nerve injury site, but their cellular localization and function have not been fully characterized. We demonstrate that Schwann cells express mRNAs for LIF and the LIF receptor components LIF receptor subunit β and glycoprotein 130 in vitro. We also show that although LIF is not required for the genesis of Schwann cells, it can potentiate the survival of differentiated Schwann cells in the context of neuregulin support. Not only does exogenous LIF promote survival under these conditions, but addition of the soluble LIF receptor (LIF binding protein) and anti-LIF antibodies significantly reduced cell survival, suggesting that LIF exerts autocrine effects. These results suggest that Schwann cell survival following nerve injury is potentially modulated by LIF.     

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.     

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

Desert hedgehog-patched 2 expression in peripheral nerves during Wallerian degeneration and regeneration

Hedgehog proteins are important in the development of the nervous system. As Desert hedgehog (Dhh) is involved in the development of peripheral nerves and is expressed in adult nerves, it may play a role in the maintenance of adult nerves and degeneration and regeneration after injury. We firstly investigated the Dhh-receptors, which are expressed in mouse adult nerves. The Dhh receptor patched(ptc)2 was detected in adult sciatic nerves using RT-PCR, however, ptc1 was undetectable under the same experimental condition. Using RT-PCR in purified cultures of mouse Schwann cells and fibroblasts, we found ptc2 mRNA in Schwann cells, and at much lower levels, in fibroblasts. By immunohistochemistry, Ptc2 protein was seen on unmyelinated nerve fibers. Then we induced crush injury to the sciatic nerves of wild-type (WT) and dhh-null mice and the distal stumps of injured nerves were analyzed morphologically at different time points and expression of dhh and related receptors was also measured by RT-PCR in WT mice. In dhh-null mice, degeneration of myelinated fibers was more severe than in WT mice. Furthermore, in regenerated nerves of dhh-null mice, minifascicular formation was even more extensive than in dhh-null intact nerves. Both dhh and ptc2 mRNA levels were down-regulated during the degenerative phase postinjury in WT mice, while levels rose again during the phase of nerve regeneration. These results suggest that the Dhh-Ptc2 signaling pathway may be involved in the maintenance of adult nerves and may be one of the factors that directly or indirectly determines the response of peripheral nerves to injury.     

Signaling pathways regulating dose-dependent dual effects of TNF-α on primary cultured Schwann cells

After peripheral nerve injury, Schwann cells are rapidly activated to participate in the regenerative process and modulate local immune reactions. Tumor necrosis factor-α (TNF-α), one of the major initiators of the inflammatory cascade, has been known to exert pleiotropic functions during peripheral nerve injury and regeneration. In this study, we aimed to investigate the in vitro effects of TNF-α on peripheral neural cells. First, gene-microarray analysis was applied to the RNA samples extracted from injured peripheral nerves, providing the information of gene interactions post nerve injury. Then, after primary cultured Schwann cells were treated with increasing dosages (0–40 ng/ml) of TNF-α, cell proliferation and migration were examined by EdU incorporation and a transwell-based assay, and cell apoptosis was observed and quantified by electron microscopy and Annexin V-FITC assay, respectively. The results showed that lower dosages of TNF-α increased cell proliferation and migration, whereas higher dosages of TNF-α decreased cell proliferation and migration and enhanced cell apoptosis. The tests using a chemical inhibitor of TNF-α further confirmed the above effects of TNF-α. To understand how TNF-α produced the dose-dependent dual effects on primary cultured Schwann cells, we performed co-immunoprecipitation, Western blot analysis, and immunocytochemistry to decipher the complex network of biochemical pathways involving many signaling molecules, i.e., TNF receptor-associated death domain, Fas-associated death domain, receptor interacting protein, JNK, NF-κB p65, and caspases, thus assuming the mechanisms by which TNF-α activated the death and survival pathways and achieved a balance between the two opposite actions in primary cultured Schwann cells.     

Pleiotrophin cellular localization in nerve regeneration after peripheral nerve injury.

Pleiotrophin (PTN) is a member of the family of heparin-binding growth factors that displays mitogenic activities and promotes neurite outgrowth in vitro. In vivo, PTN is widely expressed along pathways of developing axons during the late embryonic and early postnatal period. Although the level of PTN gene expression is very low during adulthood, activation of the gene may occur during recovery from injury and seems to play an important role in tissue regeneration processes. In this study, we investigated whether PTN was involved in the regenerative process of injured peripheral nerves. To refer localization of the fluorescent markers to myelinated axons, we developed a specific computer tool for colocalization of fluorescence images with phase contrast images. Immunohistochemical analysis showed PTN in different types of nonneural cells in distal nerve segments, including Schwann cells, macrophages, and endothelial cells, but not in axons. Schwann cells exhibited PTN immunoreactivity as early as 2 days after injury, whereas PTN-positive macrophages were found 1 week later. Strong PTN immunoreactivity was noted in endothelial cells at all time points. These findings support the idea that PTN participates in the adaptive response to peripheral nerve injury. A better understanding of its contribution may suggest new strategies for enhancing peripheral nerve regeneration.     

It is all about the support — The role of the extracellular matrix in regenerating axon guidance

Although it is known for long time that the peripheral nervous system has the capacity for self-regeneration, the molecular mechanisms by which Schwann cells and extracellular matrix (ECM) guide the injured axons to regrow along their original path, remains a poorly understood process. Due to the importance of ECM molecules during development, constitutive mutant organisms display increased lethality, therefore, conditional or inducible strategies have been used to increase the survival of the organisms and allow the study of the role of ECM proteins. In a recent report published in Neuron, Isaacman-Beck and colleagues (2015) used these pioneering genetic studies on zebrafish combined with in vivo fluorescent imaging, to investigate the micro-environmental conditions required for targeted regeneration of the dorsal motor nerve of zebrafish larvae after laser-transection. A candidate gene approach targeting lh3 basal laminar collagen substrates revealed that the lh3 substrate col4α5 regulates dorsal nerve regeneration by destabilizing misdirected axons. Col4α5 was upregulated in a small population of lh3 expressing Schwann cells located ventrally and ventro-laterally to the injury site and found to co-localize with the molecule slit guidance ligand 1 (slit1a). Capitalizing on the crucial observations of mistargeted regeneration of dorsal nerves in mutant larvae, they put forward a model in which Schwann cells shape an environment that allows and directs axonal regeneration to their original synaptic target. In the light of Isaacman-Beck and colleagues (2015) findings, we will review how their study contributes to the research field, and comment on its potential implications for promoting nerve regeneration after injury.     

Regulation of Hemopexin Synthesis in Degenerating and Regenerating Rat Sciatic Nerve

Abstract : In injured peripheral nerves, hemopexin mRNA is expressed by fibroblasts, Schwann cells, and invading blood macrophages, and the protein accumulates in the extracellular matrix. This and its absence of regulation in injured central optic nerve suggest that hemopexin could play a positive role in peripheral nerve repair. Here, we studied the regulation of hemopexin expression in degenerating and regenerating nerves. After a sciatic nerve injury, both the synthesis of hemopexin and the level of its mRNA increase sharply during the first 2 days, leading to an accumulation of hemopexin in the nerve. Afterward, hemopexin expression decreases progressively in regenerating nerves. In permanently degenerated nerves, it is again transiently increased and then strongly decreased, whereas hemopexin from blood origin is accumulating. As part of the elucidation of the complex regulation of hemopexin expression in injured nerves, we demonstrate that interleukin-6 increases hemopexin synthesis in intact nerves, whereas adult rat serum, but not purified hemopexin, inhibits it in degenerated nerves. Hemopexin, known as acute-phase protein, is therefore one of the molecules rapidly and specifically up-regulated in injured peripheral nerves. More generally, our findings suggest that the acute phase could be not only a systemic liverspecific response but also a reaction of injured tissues themselves.     

Schwann-spheres derived from injured peripheral nerves in adult mice--their in vitro characterization and therapeutic potential

Multipotent somatic stem cells have been identified in various adult tissues. However, the stem/progenitor cells of the peripheral nerves have been isolated only from fetal tissues. Here, we isolated Schwann-cell precursors/immature Schwann cells from the injured peripheral nerves of adult mice using a floating culture technique that we call "Schwann-spheres." The Schwann-spheres were derived from de-differentiated mature Schwann cells harvested 24 hours to 6 weeks after peripheral nerve injury. They had extensive self-renewal and differentiation capabilities. They strongly expressed the immature-Schwann-cell marker p75, and differentiated only into the Schwann-cell lineage. The spheres showed enhanced myelin formation and neurite growth compared to mature Schwann cells in vitro. Mature Schwann cells have been considered a promising candidate for cell-transplantation therapies to repair the damaged nervous system, whereas these "Schwann-spheres" would provide a more potential autologous cell source for such transplantation.     

Extrinsic cellular and molecular mediators of peripheral axonal regeneration

The ability of injured peripheral nerves to regenerate and reinnervate their original targets is a characteristic feature of the peripheral nervous system (PNS). On the other hand, neurons of the central nervous system (CNS), including retinal ganglion cell (RGC) axons, are incapable of spontaneous regeneration. In the adult PNS, axonal regeneration after injury depends on well-orchestrated cellular and molecular processes that comprise a highly reproducible series of degenerative reactions distal to the site of injury. During this fine-tuned process, named Wallerian degeneration, a remodeling of the distal nerve fragment prepares a permissive microenvironment that permits successful axonal regrowth originating from the proximal nerve fragment. Therefore, a multitude of adjusted intrinsic and extrinsic factors are important for surviving neurons, Schwann cells, macrophages and fibroblasts as well as endothelial cells in order to achieve successful regeneration. The aim of this review is to summarize relevant extrinsic cellular and molecular determinants of successful axonal regeneration in rodents that contribute to the regenerative microenvironment of the PNS.     

Deletion of p75NTR impairs regeneration of peripheral nerves in mice

AimsAfter peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves.Main methodsIn p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry.Key findingsThe results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice.SignificanceOur data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.     

<Emphasis Type="Italic">miR-3075</Emphasis> Inhibited the Migration of Schwann Cells by Targeting Cntn2

Peripheral nerve injury is a complex biological process that involves the expression changes of various coding and non-coding RNAs. Previously, a number of novel miRNAs that were dysregulated in rat sciatic nerve stumps after peripheral nerve injury were identified and functionally annotated by Solexa sequencing. In the current study, we studied one of these identified novel miRNAs, miR-3075, in depth. Results of transwell-based cell migration assay showed that increased expression of miR-3075 suppressed the migration rate of Schwann cells while decreased expression of miR-3075 elevated the migration rate of Schwann cells, demonstrating that miR-3075 inhibited Schwann cell migration. Results of BrdU cell proliferation assay showed that neither miR-3075 mimic nor miR-3075 inhibitor would affect Schwann cell proliferation. We further studied candidate target genes of miR-3075 by using bioinformatic tools and analyzing gene expression patterns and found that miR-3075 might target contactin 2 (Cntn2). Previous study showed that Cntn2 regulated cell migration and myelination. Our current observation suggested that the biological effects of miR-3075 on Schwann cell phenotype might by through the negative regulation of Cntn2. Overall, our study revealed the function of a novel miRNA, miR-3075, and expanded our current understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.