Schwann Cell Motility Research Articles

GTPases and phosphatidylinositol 3-kinase are critical for insulin-like growth factor-I-mediated Schwann cell motility.

Previously, we reported insulin-like growth factor-I (IGF-I) promotes motility and focal adhesion kinase (FAK) activation in neuronal cells. In the current study, we examined the role of IGF-I in Schwann cell (SC) motility. IGF-I increases SC process extension and motility. In parallel, IGF-I activates IGF-I receptor, insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3 (PI-3)-kinase, and FAK. LY294002, a PI-3 kinase inhibitor, blocks IGF-I-induced motility and FAK phosphorylation. The Rho family of GTPases is important in the regulation of the cytoskeleton. Overexpression of constitutively active Leu-61 Cdc42 and Val-12 Rac1 enhances SC motility which is unaffected by LY294002. In parallel, stable transfection of SC with dominant negative Asn-17 Rac1 blocks IGF-I-mediated SC motility and FAK phosphorylation, implying Rac is an upstream regulator of FAK. Collectively our results suggest that IGF-I regulates SC motility by reorganization of the actin cytoskeleton via the downstream activation of a PI-3 kinase, small GTPase, and FAK pathway.

Src and phospho-FAK kinases are activated by allopregnanolone promoting Schwann cell motility, morphology and myelination.

Schwann cells' (SCs) development and maturation require coordinate and complementary activation of several signals and intracellular pathways. Among factors controlling these processes, the signalling intermediates Src tyrosine kinase and focal adhesion kinase (FAK) are relevant for SCs', participating in regulation of their adhesion, motility and migration. Recently, the progesterone metabolite allopregnanolone (ALLO) was proved to be synthesized by SCs, whereas it acts autocrinally on SCs motility and proliferation, which are crucial processes for nerve development, maturation and regeneration. Herein, we investigate the hypothesis that the molecular mechanisms behind the ALLO's action on SCs involve the signalling intermediates Src and FAK. We first demonstrated that ALLO 10-6 M regulates SCs morphology, motility and myelination, also increasing the internode distance in the invitro myelination model of neuron/SCs co-culture. ALLO's actions were mediated by the modulation of Src/FAK pathway, since they were counteracted by PP2 10-5 M, a selective inhibitor of Src kinase. Then, we proved that Src/FAK activation in SCs involves GABA-A dependent mechanisms and actin re-arrangements. In conclusion, our findings are the first to corroborate the importance of the neuroactive steroid ALLO in regulating SCs development and maturation via the Src and phospho-FAK signalling activation. Cover Image for this issue: doi: 10.1111/jnc.13795.

CAP1 was associated with actin and involved in Schwann cell differentiation and motility after sciatic nerve injury

Adenylate cyclase-associated protein 1 (CAP1), a member of cyclase-associated proteins that regulating actin dynamics, was shown to regulate actin filaments, localize to dynamic actin structures and mediate such processes as establishment of cell polarity, motility, morphogenesis, receptor-mediated endocytosis and mRNA location. But little is known about the role of CAP1 during peripheral nervous system injury. Here, we found the spatiotemporal protein expression of CAP1 after sciatic nerve crush. After crush, CAP1 had an increased protein expression level, reached a peak at about day 5 and then returned to the normal level at 4weeks, similar to Oct-6. Besides, in 5-day injured tissue, using double immunofluorescent staining we found CAP1 had a colocalization with S100 and Oct-6. In vitro, during the process of cAMP-induced Schwann cells differentiation, we observed enhanced expression of CAP1 and P0. Specially, CAP1-specific siRNA-tranfected SCs did not show significant actin structure which form cellure surface tension and protrusion shape after cAMP treatment. And we observed the interaction of CAP1 with actin and that CAP1-specific siRNA-transfected SCs had a decreased motility and migration. Together, all these data indicated that the change of CAP1 protein expression was associated with Schwann cells motility and differentiation after the crush of sciatic nerve.

Neuregulin‐1/glial growth factor stimulates Schwann cell migration by inducing α5 β1 integrin–ErbB2–focal adhesion kinase complex formation

After peripheral nerve injury, Schwann cells gain a migratory phenotype and remodel their extracellular matrix to provide a supportive environment for axonal regeneration. The soluble neuregulin-1 isoform, that is, glial growth factor (GGF), is expressed in regenerating axons of injured peripheral nerves and regulates Schwann cell motility by activating the ErbB family of tyrosine kinase receptors, but how GGF/ErbB signaling contributes to Schwann cell motility remains unclear. Here, we show that GGF stimulates Schwann cell migration by inducing the formation of a protein complex containing the fibronectin receptor α5β1 integrin, ErbB2, and focal adhesion kinase (FAK). ErbB2 co-localizes and co-immunoprecipitates with the focal complex members including α5β1 integrin and FAK after GGF treatment. These effects of GGF appear to involve FAK activation, which occurs downstream of ErbB2 stimulation. RNAi-mediated down-regulation of α5 integrin expression in primary cultured Schwann cells resulted in significantly decreased interaction between FAK and ErbB2, as well as decreased GGF-induced migration. An increase in the α5β1 integrin-ErbB2-FAK complex formation was observed in injured nerve Schwann cells, but not uninjured control. Taken together, these data suggest that GGF plays an important modulatory role in Schwann cell migration after nerve crush by inducing α5β1 integrin-ErbB2-FAK complex formation.

Measuring anisotropic cell motility on curved substrates

Schwann cell motility was observed on laminin-coated quartz cylinders with different curvatures over an 18 hour period. A new analysis based on difference images helped to determine the minimal radius of curvature, 46 μm, which restricted motility along the cylinder axis. The migration speed, measured by calculating differences between successive images in the time series, ranged between 0.3 to 0.8 μm per minute and is similar to previously reported rates for Schwann cells. Difference images provide a rapid and simple method for the analysis of cell motility on large populations of cells.

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus‐transduced mammalian cells enhance Schwann cell motility

For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies.

Bioengineered glial strands for nerve regeneration

Nerve guide implants approved for human application in the peripheral nervous system generally fail to bridge lesion gaps longer than 2cm and cannot match the clinical performance of autologous nerve transplants. Since current synthetic implants are simply hollow tubes, we aim to recreate the native microarchitecture of nerves inside the tubular implants. Most importantly, in the regenerating nerve, dedifferentiated Schwann cells align to form thousands of long glial strands, which act as guiding structures for the regrowing axons. In order to artificially induce the formation of Schwann cell strands, 28μm thick, endless poly-p-dioxanone filaments (PDO) were synthesized with longitudinal grooves. A polycationic coating on the PDO filaments rendered the polymer surface cell-permissive and induced the growth of highly oriented Schwann cells with polarized expression of N-cadherin at cell–cell contact sites. In vitro cell proliferation on three-dimensional PDO filaments was significantly increased in comparison to planar PDO substrates. Time lapse video recordings revealed high Schwann cell motility, which is advantageous for the repopulation of cell-free implants after implantation. In a pilot study we employed a novel microsurgical technique in vivo. All axon fascicles were selectively dissected from sciatic rat nerves, and the remaining epineural tube was filled with hundreds of PDO filaments. Histological analysis 6 weeks postoperatively showed no fibrosis or encapsulation but instead longitudinal cell alignment and axonal regrowth. The data suggest that the addition of microstructured PDO filaments to the lumen of synthetic tubular implants might significantly improve performance.

Schwann cell migration is integrin‐dependent and inhibited by astrocyte‐produced aggrecan

Schwann cells transplantation has considerable promise in spinal cord trauma to bridge the site of injury and for remyelination in demyelinating conditions. They support axonal regeneration and sprouting by secreting growth factors and providing a permissive surface and matrix molecules while shielding axons from the inhibitory environment of the central nervous system. However, following transplantation Schwann cells show limited migratory ability and they are unable to intermingle with the host astrocytes. This in turn leads to formation of a sharp boundary and an abrupt transition between the Schwann cell graft and the host tissue astrocytes, therefore preventing regenerating axons from exiting the graft. The objective of this study was to identify inhibitory elements on astrocytes involved in restricting Schwann cell migration. Using in vitro assays of cell migration, we show that aggrecan produced by astrocytes is involved in the inhibition of Schwann cell motility on astrocytic monolayers. Knockdown of this proteoglycan in astrocytes using RNAi or digestion of glycosaminglycan chains on aggrecan improves Schwann cell migration. We further show aggrecan mediates its effect by disruption of integrin function in Schwann cells, and that the inhibitory effects of aggrecan can overcome by activation of Schwann cell integrins.

Developmental loss of NT‐3 in vivo results in reduced levels of myelin‐specific proteins, a reduced extent of myelination and increased apoptosis of Schwann cells

This work investigates the role of NT-3 in peripheral myelination. Recent articles, based in vitro, propose that NT-3 acting through its high-affinity receptor TrkC may act to inhibit myelin formation by enhancing Schwann cell motility and/or migration. Here, we investigate this hypothesis in vivo by examining myelination formation in NT-3 mutant mice. On the day of birth, soon after the onset of myelination, axons showed normal ensheathment by Schwann cells, no change in the proportion of axons which had begun to myelinate, and no change in either myelin thickness or number of myelin lamellae. However in postnatal day 21 mice, when myelination is substantially complete, we observed an unexpected reduction in mRNA and protein levels for MAG and P(0), and in myelin thickness. This is the opposite result to that predicted from previous in vitro studies, where removal of an inhibitory NT-3 signal would have been expected to enhance myelination. These results suggest that, in vivo, the importance of NT-3 as a major support factor for Schwann cells (Meier et al., (1999) J Neurosci 19:3847-3859) over-rides its potential role as an myelin inhibitor, with the net effect that loss of NT-3 results in degradation of Schwann cell functions, including myelination. In support of this idea, Schwann cells of NT-3 null mutants showed increased expression of activated caspase-3. Finally, we observed significant reduction in width of the Schwann cell periaxonal collar in NT-3 mutant animals suggesting that loss of NT-3 and resulting reduction in MAG levels may alter signaling at the axon-glial interface.

Asymmetric ERM activation at the Schwann cell process tip is required in Axon‐Associated motility

Axon-associated Schwann cell (SC) motility and process dynamics are crucial in the development and regeneration of the peripheral nervous system (PNS). The bipolar morphology of SCs represents an unexplored conundrum in terms of directed motility. Using fluorescence time-lapse microscopy of transfected SCs within myelinating dorsal root ganglion (DRG) explants, we demonstrate cycling of SCs between bipolar and highly motile, unipolar morphologies as a result of asymmetric process retraction and extension. Unipolar SC motility appears nucleotaxic in nature, similar to the movement of neurons on radial glia during cortical development. We also show that asymmetric process retraction is associated with the activation of ERM (ezrin/radixin/moesin) proteins and subsequent recruitment of ezrin-binding phospho-protein 50 kDa (EBP50) at the retracting process tip. This activation occurs in response to localized synthesis of phosphatidylinositol (4,5)-bisphosphate (PIP2) at this site. Finally, we demonstrate that the activation of ERM proteins at the SC process tip is essential for motility and the maintenance of SC polarity, as ERM disruption yields a dysfunctional, multi-polar cell. These results demonstrate that specializations at the tips of SC processes regulate their dynamics, which in turn is associated with directed motility in these cells.

Upregulation of chondroitin 6-sulphotransferase-1 facilitates Schwann cell migration during axonal growth

Cell migration is central to development and post-traumatic regeneration. The differential increase in 6-sulphated chondroitins during axonal growth in both crushed sciatic nerves and brain development suggests that chondroitin 6-sulphotransferase-1 (C6ST-1) is a key enzyme that mediates cell migration in the process. We have cloned the cDNA of the C6ST-1 gene (C6st1) (GenBank accession number AF178689) from crushed sciatic nerves of adult rats and produced ribonucleotide probes accordingly to track signs of 6-sulphated chondroitins at the site of injury. We found C6st1 mRNA expression in Schwann cells emigrating from explants of both sciatic nerve segments and embryonic dorsal root ganglia. Immunocytochemistry indicated pericellular 6-sulphated chondroitin products around C6ST-1-expressing frontier cells. Motility analysis of frontier cells in cultures subjected to staged treatment with chondroitinase ABC indicated that freshly produced 6-sulphated chondroitin moieties facilitated Schwann cell motility, unlike restrictions resulting from proteoglycan interaction with matrix components. Sciatic nerve crush provided further evidence of in vivo upregulation of the C6ST-1 gene in mobile Schwann cells that guided axonal regrowth 1-14 days post crush; downregulation then accompanied declining mobility of Schwann cells as they engaged in the myelination of re-growing axons. These findings are the first to identify upregulated C6st1 gene expression correlating with the motility of Schwann cells that guide growing axons through both developmental and injured environments.

Effect of genetically modified Schwann cells with increased motility in end-to-side nerve grafting

Taking into account that Schwann-cell (SC) motility is a prerequisite for myelination during peripheral nerve regeneration, the present study was designed with the intention to increase SC motility in vitro and to evaluate the effect of transduced SC on nerve regeneration in vivo, through silicone tubes after end-to-side nerve repair. Our in vitro study demonstrated that SC transduction with the pREV-HW3 retrovirus, encoding for sialyl-transferase-X (STX), significantly increased their motility compared to the control. In the in vivo study, 45 Wistar rats were randomized into three groups of 15 each. In all animals, the left peroneal nerve was severed, and a 10-mm segment was removed. The distal stump of the peroneal nerve was connected end-to-side to a perineurial window in the ipsilateral tibial nerve with either a silicone tube lined with SC (group A) or a silicone tube lined with STX-transduced SC (groups B and C). Fluorescence and light microscopy in group C showed that SCs were viable the first critical 15 postoperative days. After 90 days, light microscopy in group B demonstrated that STX-transduced SCs with increased motility ensured nerve regeneration, through silicone tubes, in all cases. Furthermore, STX-transduced SCs increased significantly fiber diameter and myelin thickness, and most importantly enhanced significantly the functional outcome compared to non-transduced SCs.

Soluble forms of NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein tyrosine phosphatases zeta/beta as the putative F3 receptors on Schwann cells.

Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM-Fc and F3-Fc chimeras on Schwann cell (SC) migration. Binding sites were identified at the surface of cultured SCs by chimera coated fluorospheres. The functional effect of NCAM-Fc and F3-Fc binding was studied in two different SC migration models. In the first, migration is monitored at specific time intervals inside a 1-mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras significantly increased the extent of SC migration and this effect could be prevented by the corresponding anti-NCAM or anti-F3 blocking antibodies. Furthermore, antiproteoglycan-type protein tyrosine phosphatase zeta/beta (RPTPzeta/beta) antibodies identified the presence of RPTPzeta/beta on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3-Fc coated Sepharose beads precipitated RPTPzeta/beta from SC lysates. Altogether these data point to RPTPzeta/beta is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.

The neuron–glia signal β‐neuregulin promotes Schwann cell motility via the MAPK pathway

Neuregulins constitute a family of related growth factors that play important roles in Schwann cell development and maturation. We investigated the involvement of beta-neuregulin in Schwann cell migration, using a simple in vitro bioassay. Pure Schwann cells were prepared from the sciatic nerves of 5-day-old rats and were grown in defined medium, with or without serum, until a monolayer of confluent cells was formed. A cell-free area was then generated by inflicting a scratch resulting in a 1-mm-wide gap. Schwann cell migration within the gap was monitored microscopically at given time intervals and was quantified using an image analysis system. The extent of cell proliferation was estimated by BrdU incorporation, and cell migration was quantified both in the absence and presence of cytosine arabinoside. We found that, in the absence of serum, beta-neuregulin at a dose submaximal for proliferation increased the rate of Schwann cell migration by 84%. A more moderate effect was observed when beta-neuregulin was applied in the presence of serum which, however, is by itself responsible for increased Schwann cell motility. To assess the signal transduction pathways involved in this procedure we used one inhibitor of MAPK, PD098059, two inhibitors of PI-3-kinase, wortmannin, and LY0294002, and three different PKC inhibitors. Of these PD098059 inhibited the neuregulin-induced enhancement in Schwann cell migration by 40%, the two PI-3-kinase inhibitors yielded an approximately 20% inhibition while the PKC inhibitors were ineffective. Our data indicate that the action of beta-neuregulin on Schwann cell motility is primarily mediated via the MAPK pathway.

IGF-I promotes Schwann cell motility and survival via activation of Akt

We previously reported insulin-like growth factor-I (IGF-I) promotes Schwann cell (SC) motility and rescues SC from apoptosis induced by serum deprivation. This effect is mediated by phosphatidylinositol-3 (PI-3) kinase. In the current study, we examined the role of Akt, a downstream kinase of PI-3K, in SC motility and IGF-I mediated protection from apoptosis. IGF-I induces Akt phosphorylation at Ser473, an event which may be blocked by pretreatment with a PI-3K inhibitor, LY294002. In dominant negative K179M Akt (K179M) transfected SC, however, Akt is not activated in response to IGF-I. In addition, IGF-I is unable to promote SC motility and survival in K179M SC. These results suggest a critical role for Akt in IGF-I mediated motility and survival in SC.

Laminins of the neuromuscular system.

The mammalian neuromuscular system expresses seven laminin genes (alpha 1, alpha 2, alpha 4, alpha 5, beta 1, beta 2, and gamma 1), produces seven isoforms of the laminin trimer (laminins 1, 2, 4, 8, 9, 10, and 11), and distributes these trimers to at least seven distinct basal laminae (perineurial, endoneurial, terminal Schwann cell, myotendinous junction, synaptic cleft, synaptic fold, and extrajunctional muscle). The patterns of expression, assembly, and distribution are regulated during development, and primary and secondary changes in laminin expression occur in several neuromuscular genetic disorders. Functional studies using knockout and transgenic mice, and purified laminins and cell types, demonstrate that laminins are required components of basal laminae in the neuromuscular system. Collectively, laminins have both structural and signaling functions; individually, laminin isoforms have unique roles in regulating the behavior of nerve, muscle, and Schwann cell. Among them, laminin-2 (alpha 2 beta 1 gamma 1) plays an important structural role in supporting the muscle plasma membrane, laminin-4 regulates adhesion and differentiation of the myotendinous junction, and laminin-11 regulates nerve terminal differentiation and Schwann cell motility. Together, these observations reveal remarkable diversity in the formation and function of laminins and basal laminae, and suggest avenues for addressing some neuromuscular diseases.

CD9 plays a role in Schwann cell migration in vitro

To identify molecules that regulate Schwann cell migration, we have generated a panel of monoclonal antibodies against Schwann cell surface antigens that modulate Schwann cell migration in in vitro bioassays. One of these antibodies, SMRA1, recognizes a 26 kDa Schwann cell surface membrane protein identified here as CD9. SMRA1 enhances Schwann cell migration on two biologically relevant substrates: living axons of cultured dorsal root ganglion neurons, and cryostat sections of sciatic nerve. This CD9-induced regulation of Schwann cell motility is correlated with a rise in cytosolic calcium and enhanced tyrosine phosphorylation of several Schwann cell proteins. These results, together with the findings of Hadjiargyrou and Patterson (1994), implicate CD9 as an important regulator of Schwann cell behavior in peripheral nerve.