Neonatal Schwann Cells Research Articles

Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs.

Exosomes are small (30-150nm) vesicles of endosomal origin secreted by most cell types. Exosomes contain proteins, lipids, and RNA species including microRNA, mRNA, rRNA, and long noncoding RNAs. The mechanisms associated with exosome synthesis and cargo loading are still poorly understood. A role for exosomes in intercellular communication has been reported in physiological and pathological conditions both in vitro and in vivo. Previous studies have suggested that Schwann cell-derived exosomes regulate neuronal functions, but the mechanisms are still unclear. Here, we describe protocols to establish rat neonatal Schwann cell cultures and to isolate exosomes from the conditioned medium of these cultures by differential ultracentrifugation. To analyze the RNA content of Schwann cell-derived exosomes, we detail protocols for RNA extraction and next-generation sequencing using miRNA and mRNA libraries. The protocol also includes RNA sequencing of Schwann cells, which allows the comparison between RNA content from cells and the secreted exosomes. Identification of RNAs present in Schwann cell-derived exosomes is a valuable tool to understand novel roles of Schwann cells in neuronal function in health and disease.

C3-induced release of neurotrophic factors from Schwann cells – potential mechanism behind its regeneration promoting activity

Previous studies revealed a peripheral nerve regeneration (PNR)1 promoting activity of Clostridium botulinum C32 exoenzyme or a 26mer C-terminal peptide fragment covering amino acids 156–181 (C3156−181),3 when delivered as one-time injection at the lesion site. The current study was performed to 1) investigate if prolonged availability of C3 and C3156−181 at the lesion site can further enhance PNR in vivo and to 2) elucidate effects of C3 and C3156−181 on Schwann cells (SCs)4in vitro.For in vivo studies, 10 mm adult rat sciatic nerve gaps were reconstructed with the epineurial pouch technique or autologous nerve grafts. Epineurial pouches were filled with a hydrogel containing i) vehicle, ii) 40 μM C3 or iii) 40 μM C3156−181. Sensory and motor functional recovery was monitored over 12 weeks and the outcome of PNR further analyzed by nerve morphometry. In vitro, we compared gene expression profiles (microarray analysis) and neurotrophic factor expression (western blot analysis) of untreated rat neonatal SCs with those treated with C3 or C3156−181 for 72 h. Effects on neurotrophic factor expression levels were proven in adult human SCs.Unexpectedly, prolonged delivery of C3 and C3156−181 at the lesion site did not increase the outcome of PNR. Regarding the potential mechanism underlying their previously detected PNR promoting action, however, 6 genes were found to be commonly altered in SCs upon treatment with C3 or C3156−181. We demonstrate significant down-regulation of genes involved in glutamate uptake (Eaac1,5Grin2a6) and changes in neurotrophic factor expression (increase of FGF-27 and decrease of NGF8). Our microarray-based expression profiling revealed novel C3-regulated genes in SCs possibly involved in the axonotrophic (regeneration promoting) effects of C3 and C3156−181. Detection of altered neurotrophic factor expression by C3 or C3156−181 treated primary neonatal rat SCs and primary adult human SCs supports this hypothesis.

Schwann cells generated from neonatal skin-derived precursors or neonatal peripheral nerve improve functional recovery after acute transplantation into the partially injured cervical spinal cord of the rat.

The transplantation of Schwann cells (SCs) holds considerable promise as a therapy for spinal cord injury, but the optimal source of these cells and the best timing for intervention remains debatable. Previously, we demonstrated that delayed transplantation of SCs generated from neonatal mouse skin-derived precursors (SKP-SCs) promoted repair and functional recovery in rats with thoracic contusions. Here, we conducted two experiments using neonatal rat cells and an incomplete cervical injury model to examine the efficacy of acute SKP-SC transplantation versus media control (Experiment 1) and versus nerve-derived SC or dermal fibroblast (Fibro) transplantation (Experiment 2). Despite limited graft survival, by 10 weeks after injury, rats that received SCs from either source showed improved functional recovery compared with media- or fibroblast-treated animals. Compared with media treatment, SKP-SC-transplanted rats showed enhanced rubrospinal tract (RST) sparing/plasticity in the gray matter (GM) rostral to injury, particularly in the absence of immunosuppression. The functional benefits of SC transplantations over fibroblast treatment correlated with the enhanced preservation of host tissue, reduced RST atrophy, and/or increased RST sparing/plasticity in the GM. In summary, our results indicate that: (1) early transplantation of neonatal SCs generated from skin or nerve promotes repair and functional recovery after incomplete cervical crush injury; (2) either of these cell types is preferable to Fibros for these purposes; and (3) age-matched SCs from these two sources do not differ in terms of their reparative effects or functional efficacy after transplantation into the injured cervical spinal cord.

Platelet-Derived Growth Factors-BB and Fibroblast Growth Factors-Base Induced Proliferation of Schwann Cells in a 3D Environment

The proliferation of neonatal Schwann cells (SCs) in response to mitogenic agents has been well analyzed in vitro (mono-layer-culture method, 2D environment), but not in vivo (3D environment). To assess the mitogenic effect of platelet-derived growth factors-BB (PDGF-BB), Fibroblast Growth Factors-base (bFGF), and their combinations for SCs in collagen gel (three-dimensional, 3D environment), we have developed an integrated microfluidic device on which can reproducibly measure the proliferation from small number of cells (1-100). The rat SCs were cultured for 4 week at the different concentrations of growth factors generated by concentration gradient generator. In the collagen gel culture, almost all of the cells in colonies presented a round cell morphology and maintained their round morphology by the 4th week. The results showed that PDGF-BB and bFGF are all capable of moderately stimulating SCs growth and every group reached the peak in the growth curve at 3 weeks. Moreover, the proliferation test using the conventional method was performed simultaneously and revealed similar results. The biggest difference between 2D and 3D was that cells decrease more remarkable in 3D than that in 2D at 4 weeks. And at 2 and 3 weeks, the growth rate in the collagen gel with 7.14/2.86 and 8.57/1.43 ng/mL groups was higher than that in the mono-layer culture. Our results showed that PDGF-BB and bFGF are capable of moderately stimulating neonatal SCs growth, respectively and synergistically, and the microfluidic technique is highly controllable, contamination free, fully automatic, and inexpensive.

Synergistic effects of osteonectin and brain-derived neurotrophic factor on axotomized retinal ganglion cells neurite outgrowth via the mitogen-activated protein kinase-extracellular signal-regulated kinase1/2 pathways

Our previous study identified osteonectin (ON) in a screen of factors made by Schwann cells (SCs) which promoted peripheral and central neurons survival and neuritogenesis, however, the mechanisms of ON promoting effects are largely unknown. In the present study, we investigated the effects of ON-deficient SC-conditioned medium (SCCM) and molecular mechanisms of ON, in regulating retinal ganglion cells (RGCs) survival and neurite outgrowth. Neonatal rat RGCs and SCs were purified by immunopanning technique. RGC survival and neuritogenesis reduced significantly when treated with either ON-null mice SCCM or ON-immunodepleted (IP) SCCM ( P<0.05). In contrast to wild type SCCM, in the presence of a tyrosine kinase receptor (Trk) inhibitor (K252a), ON-null mice SCCM-induced neuritogenesis were further reduced by 24%. The Trk-mediated signaling pathways became more sensitive to K252a inhibition in the absence of ON. We also showed the synergistic effects of ON and brain-derived neurotrophic factor (BDNF) in promoting RGCs growth and the involvement of ON in two major neurotrophin-mediated signaling pathways, PI-3K-Akt and MAPK-Erk1/2. ON alone activated Akt phosphorylation and increased survival. Blockage of TrkB signalling pathway by TrkB-Fc chimera (BDNF scavenger) or K252a in ON-treated cultures reduced Akt-P level significantly. This suggests that ON induces BDNF synthesis and secretion from RGCs. The enhancement of neuritogenesis and Erk1/2 phosphorylation by ON in BDNF-treated cultures further demonstrate the signaling pathways responsible for the synergistic effect of ON on BDNF-induced neurite outgrowth. To the best of our knowledge, this is the first report showing the synergistic effects of ON on classical neurotrophins which participate in the same signalling pathways in regulating RGC neurite outgrowth.

Myelination in mouse dorsal root ganglion/Schwann cell cocultures

The established protocols for in vitro studies of peripheral nerve myelination with rat embryonic dorsal root ganglia (DRG) and postnatal Schwann cell cocultures do not work with mouse cells. Consequently, the full potential of this model, which would allow to perform cell type-specific, mixed genotype cocultures without cross-breeding the animals, cannot be exploited. We determined the conditions required to promote full myelination in cocultures of pre-purified mouse embryonic DRG and neonatal Schwann cells, and present a method which consistently yields 50–200 mature myelin sheaths/culture. Causes for the failure of the existing protocols to yield satisfactory results with mouse cells fell into three categories: the lack of adherent support provided by the substratum, growth factor and hormone deficiencies, and the high serum content of the media. For optimal results, mouse cocultures require a 3-dimensional substratum, a myelination-promoting culture medium containing pituitary extract, N2 supplement and forskolin, and a low serum concentration.

Calponin is expressed by subpopulations of connective tissue cells but not olfactory ensheathing cells in the neonatal olfactory mucosa

BackgroundDebate has been ongoing on the relative merits of olfactory ensheathing cells (OECs) and Schwann cells as candidates for transplant-mediate repair of CNS lesions. Both glial cells exhibit similar molecular and cellular properties and to date there has been no antigenic marker identified that can clearly distinguish the two cell types. This inability to distinguish between the two cells types prevents confirmation of a controversial statement that cultures of OECs are contaminated with Schwann cells. Recently, proteomic analysis of foetal OECs and adult Schwann cells identified an actin-binding protein, calponin, as a specific marker for OECs. However, at the same time a recent report suggested that adult OECs do not express calponin. It was not clear if this discrepancy was due to methodology, as cells had to be treated with proteinase K to maximize calponin staining or developmental differences with only foetal/neonatal OECs expressing calponin. For this reason we have examined calponin expression in the peripheral olfactory system of embryonic and neonatal rats in vivo and from cells in vitro to assess if calponin is expressed in a developmental manner.ResultsIn this study we show that: i) proteinase K pretreatment had no effect on calponin staining in both OECs and Schwann cells. ii) calponin immunoreactivity was not expressed by embryonic or neonatal OECs in vitro and in vivo although connective tissue from the olfactory mucosa was strongly positive in neonatal rats but not embryonic rats, iii) calponin expression in the olfactory mucosa was heterogeneous, defining subpopulations of connective tissue cells iv) using functional confrontation assays between OECs or Schwann cells with astrocytes, calponin was expressed heterogeneously by astrocytes.ConclusionIt is concluded that calponin is heterogeneously expressed by neonatal mucosal connective tissue but not expressed by neonatal OECs, embryonic OECs, and neonatal Schwann cells. Furthermore, we propose that calponin is not a specific marker for OECs generated from any developmental age.

Secretory products of central nervous system glial cells induce Schwann cell proliferation and protect from cytokine‐mediated death

There continues to be interest in Schwann cells (SC) as a possible source of myelinating cells for transplantation into the central nervous system (CNS) of patients with multiple sclerosis (MS) and spinal cord injury. It has been suggested that CNS glial cells interfere with SC migration, survival, maturation, and clinically significant remyelination in the CNS. To investigate the effects of CNS glial cells on SC, we examined the effects of serum-free supernatants obtained from rat mixed CNS glial cultures on rat neonatal SC cultures. Supernatants from 1-, 3-, and 5-day CNS glial cultures induced proliferation of SC assayed at 5 days in vitro but did not induce SC differentiation as measured by induction of surface expression of galactolipids (GalL). High concentrations of cAMP simulate many of the effects of axolemma on SC; CNS glial cell supernatants did not inhibit cAMP induction of SC differentiation. CNS glial cell supernatants had no apparent effect on SC viability at 48 hr as measured by trypan blue exclusion. We have previously demonstrated that incubation of SC with transforming growth factor-beta1 (TGF-beta1) + tumor necrosis factor-alpha (TNF-alpha) induces SC death via apoptosis. We now show that CNS glial supernatants inhibits TGF-beta1/TNF-alpha-induced SC death. Our data show that soluble products of CNS glial cells do not induce or inhibit SC differentiation or increase cell death but have the potential to increase proliferation of SC and their resistance to cytokine-mediated death, and thus may affect the outcome of SC transplantation into the CNS.

Multiple-channel scaffolds to promote spinal cord axon regeneration

As molecular, cellular, and tissue-level treatments for spinal cord injury are discovered, it is likely that combinations of such treatments will be necessary to elicit functional recovery in animal models or patients. We describe multiple-channel, biodegradable scaffolds that serve as the basis for a model to investigate simultaneously the effects on axon regeneration of scaffold architecture, transplanted cells, and locally delivered molecular agents. Poly(lactic- co-glycolic acid) (PLGA) with copolymer ratio 85:15 was used for these initial experiments. Injection molding with rapid solvent evaporation resulted in scaffolds with a plurality of distinct channels running parallel along the length of the scaffolds. The feasibility of creating scaffolds with various channel sizes and geometries was demonstrated. Walls separating open channels were found to possess void fractions as high as 89%, with accessible void fractions as high as 90% through connections 220 μm or larger. Scaffolds degraded in vitro over a period of 30 weeks, over which time-sustained delivery of a surrogate drug was observed for 12 weeks. Primary neonatal Schwann cells were distributed in the channels of the scaffold and remained viable in tissue culture for at least 48 h. Schwann-cell containing scaffolds implanted into transected adult rat spinal cords contained regenerating axons at one month post-operation. Axon regeneration was demonstrated by three-dimensional reconstruction of serial histological sections.

Glial cell line‐derived neurotrophic factor‐induced signaling in Schwann cells

Glial cell line-derived neurotrophic factor (GDNF), a known survival factor for neurons, has recently been shown to stimulate the migration of Schwann cells (SCs) and to enhance myelination. GDNF exerts its biological effects by activating the Ret tyrosine kinase in the presence of glycosylphosphatidylinositol-linked receptor, GDNF family receptor (GFR) alpha1. In Ret-negative cells, the alternative transmembrane coreceptor is the 140-kDa isoform of neural cell adhesion molecule (NCAM) associated with a non-receptor tyrosine kinase Fyn. We confirmed that GDNF, GFRalpha1 and NCAM are expressed in neonatal rat SCs. We found that GDNF induces an increase in the partitioning of NCAM and heparan sulfate proteoglycan agrin into lipid rafts and that heparinase inhibits GDNF-signaling in SCs. In addition to activation of extracellular signal-regulated kinases, and phosphorylation of cAMP response element binding protein, we found that cAMP-dependent protein kinase A and protein kinase C are involved in GDNF-mediated signaling in SCs. Although GDNF did not promote the differentiation of purified SCs into the myelinating phenotype, it enhanced myelination in neuron-SC cocultures. We conclude that GDNF utilizes NCAM signaling pathways to regulate SC function prior to myelination and at early stages of myelin formation.

Nerve Growth Factor Blocks the Glucose-induced Down-regulation of Caveolin-1 Expression in Schwann Cells via p75 Neurotrophin Receptor Signaling

Altered neurotrophism in diabetic peripheral neuropathy (DPN) is associated in part with substantial degenerative changes in Schwann cells (SCs) and an increased expression of the p75 neurotrophin receptor (p75NTR). Caveolin-1 (Cav-1) is highly expressed in adult SCs, and changes in its expression can regulate signaling through Erb B2, a co-receptor that mediates the effects of neuregulins in promoting SC growth and differentiation. We examined the hypothesis that hyperglycemia-induced changes in Cav-1 expression and p75NTR signaling may contribute to altered neurotrophism in DPN by modulating SC responses to neuregulins. In an animal model of type 1 diabetes, hyperglycemia induced a progressive decrease of Cav-1 in SCs of sciatic nerve that was reversed by insulin therapy. Treatment of primary neonatal SCs with 20-30 mm d-glucose, but not l-glucose, was sufficient to inhibit transcription from the Cav-1 promoter and decrease Cav-1 mRNA and protein expression. Hyperglycemia prolonged the kinetics of Erb B2 phosphorylation and significantly enhanced the mitogenic response of SCs to neuregulin1-beta1, and this effect was mimicked by the forced down-regulation of Cav-1. Intriguingly, nerve growth factor antagonized the enhanced mitogenic response of SCs to neuregulin1-beta1 and inhibited the glucose-induced down-regulation of Cav-1 transcription, mRNA, and protein expression through p75NTR-dependent activation of JNK. Our data suggest that Cav-1 down-regulation may contribute to altered neurotrophism in DPN by enhancing the response of SCs to neuregulins and that p75NTR-mediated JNK activation may provide a mechanism for the neurotrophic modulation of hyperglycemic stress.

A new approach to CNS repair using chimeric peripheral nerve grafts.

We have examined whether transplanted freeze-thawed peripheral nerve (PN) sheaths repopulated ex vivo with purified adult Schwann cells (SCs) support the regeneration of adult rat retinal ganglion cell (RGC) axons. Cultured adult SCs were derived from donor rats or from the host animals themselves. We also transplanted PN sheaths filled with neonatal SCs or donor adult olfactory ensheathing glia (OEG). 100,000 cells were injected into 1.5-cm lengths of freeze-thawed PN. After 2 days in culture, repopulated PN segments were grafted onto the transected optic nerve of adult Fischer rats. Three weeks later, 6% fluorogold (FG) was applied to distal PN. Retrogradely labeled RGCs were counted in retinal wholemounts and PN grafts were processed for immunohistochemistry. As expected, there was no RGC axon regeneration in cell-free grafts. Regrowth was also absent in neonatal SC- and adult OEG-filled grafts, which contained only small numbers of surviving donor cells. Many cells were, however, seen in adult SC repopulated PN grafts, intermingled with pan-neurofilament(+) and GAP-43(+) fibers. SCs were aligned along the grafts and were S-100(+), p75(+). Ultrastructurally, SCs were associated with myelinated and unmyelinated axons. Hundreds of FG-labeled RGCs were seen in retinas of rats with congeneic or allogeneic PN sheaths repopulated with either donor or autologous (host-derived) adult SCs. Intraocular CNTF injections significantly increased the number of regenerating RGCs in donor and autologous adult SC groups. The use of chimeric grafts to bridge CNS tissue defects could provide a clinical alternative to using multiple PN autografts, the harvesting of which would exacerbate peripheral dysfunction in already injured patients.

A novel biodegradable implant for neuronal rescue and regeneration after spinal cord injury

After spinal cord injury, the severed neuronal pathways fail to regenerate spontaneously. This study describes a biodegradable implant using poly-β-hydroxybutyrate (PHB) fibers as carrier scaffold for matrix components and cell lines supporting neuronal survival and regeneration after spinal cord injury.After cervical spinal cord injury in adult rats, a graft consisting of PHB fibers coated with alginate hydrogel+fibronectin was implanted in the lesion cavity. In control groups, PHB was omitted and only alginate hydrogel or fibronectin, or their combination, were used for grafting. In addition, comparisons were made with animals treated intrathecally after spinal cord injury with the neurotrophic factors BDNF or NT-3. The neurons of the rubrospinal tract served as experimental model. In untreated animals, 45% of the injured rubrospinal neurons were lost at 8 weeks postoperatively. Implantation of the PHB graft reduced this cell loss by 50%, a rescuing effect similar to that obtained after treatment with BDNF or NT-3. In the absence of PHB support, implants of only alginate hydrogel or fibronectin, or their combination, had no effect on neuronal survival. After addition of neonatal Schwann cells to the PHB graft, regenerating axons were seen to enter the graft from both ends and to extend along its entire length.These results show that implants using PHB as carrier scaffold and containing alginate hydrogel, fibronectin and Schwann cells can support neuronal survival and regeneration after spinal cord injury

A new type of biocompatible bridging structure supports axon regrowth after implantation into the lesioned rat optic tract.

We have developed a new type of polymer/cell/matrix implant and tested whether it can promote the regrowth of retinal ganglion cell (RGC) and other axons across surgically induced tissue defects in the CNS. The constructs, which consisted of 2-2.5-mm-long polycarbonate tubes filled with lens capsule-derived extracellular matrix coated with cultured neonatal Schwann cells, were implanted into lesion cavities made in the left optic tract (OT) of 18-21-day-old rats. In one group, to promote Schwann cell proliferation and perhaps also to stimulate axon regrowth, basic fibroblast growth factor (bFGF) was added to the lens capsule matrix prior to implantation. In another group, to determine whether application of growth factors to the somata of cells enhances the regrowth of distally injured axons, the neurotrophin NT-4/5 was injected into the eye contralateral to the OT lesion. NT-4/5 and bFGF treatments were combined in some rats. After medium-term (4-10 weeks) or long-term (15-20 weeks) survivals, axon growth into implants was assessed immunohistochemically using a neurofilament (RT97) antibody. RGC axons were visualized after injection of WGA/HRP into the right eye. Viable Schwann cells were present in implants at all times after transplantation. Large numbers of RT97+ axons were consistently found within the bridging implants, often associated with the peripheral glia. Axons were traced up to 1.7 mm from the nearest CNS neuropil and there was immunohistochemical evidence of myelination by Schwann cells and by host oligodendrocytes. There were fewer RGC axons in the implants, fibers growing up to 1.6 mm from the thalamus. Neither NT-4/5 nor bFGF, alone or in combination, significantly increased the extent of RGC axon growth within the implants. A group of OT-lesioned rats was implanted with polymer tubes filled with 2-2.5-mm-long pieces of predegenerate peripheral nerve. Surprisingly, polymer/cell/matrix constructs contained comparatively greater numbers of RGC and other axons and supported more extensive axon elongation. Thus, implants of this type may potentially be useful in bridging large tissue defects in the CNS.

Schwann cell survival mediated by the signaling phospholipid lysophosphatidic acid.

Lysophosphatidic acid (LPA) is a bioactive phospholipid with properties of an extracellular growth factor for many cell lines, including those derived from neuroblastomas. However, the relevance of LPA signaling to the normal, developing nervous system is unknown, in part reflecting the previous unavailability of cloned receptor genes. Recent studies of the first such gene, encoding the G protein-coupled receptor LPA1/VZG-1 (lysophospholipid receptor A1/ventricular zone gene-1), revealed a major locus of expression in oligodendrocytes and Schwann cells (SCs) during development, suggesting an influence of LPA on these myelinating cells. Here we report that LPA (>/=10 nM) is a potent survival factor for cultured neonatal SCs, with survival activity equaling the maximal effect of neuregulin, the major peptide SC survival factor. LPA activates a pharmacologically defined signaling pathway in SCs, involving Gi and phosphoinositide 3-kinase. Moreover, LPA's effect depends on Akt, a downstream kinase that can mediate phosphoinositide 3-kinase-dependent survival, as demonstrated by both Western blot and transfection analyses. Overexpression of functional epitope-tagged LPA1/VZG-1 protein decreases SC apoptosis in response to serum withdrawal. These data demonstrate a role for extracellular LPA and its receptor LPA1/VZG-1 in SC survival and, more broadly, implicate G protein-coupled receptor-mediated lysophospholipid signaling as a significant mechanism in neural development.

Control of Schwann Cell Survival and Proliferation: Autocrine Factors and Neuregulins

Postnatal rat Schwann cells secrete factors that prevent the programmed cell death (PCD) of low-density Schwann cells in serum-free culture. These autocrine survival signal(s) do not promote Schwann cell proliferation. Moreover, while NRG and bFGF, which promote proliferation, both rescue a subpopulation of neonatal Schwann cells from PCD, they do not rescue freshly isolated Schwann cells from older animals; other known protein factors tested also do not mimic the autocrine signal. These results suggest that Schwann cells switch their survival dependency around the time of birth from axonal signals such as NRG to autocrine signals. Such an arrangement would be advantageous for the regeneration of peripheral axons following injury. We also compared NRG-induced Schwann cell proliferation using autocrine signals or serum to promote survival. The autocrine signals increase the rate of NRG-stimulated proliferation of low-density Schwann cells in serum-free medium, whereas serum inhibits proliferation by inhibiting both the production of survival signals and the expression of erbB2 and erbB3 receptors; these inhibitions are all reversed by forskolin. In contrast, forskolin has no effect on proliferation when the cells are exposed to high levels of autocrine factors.

Age-induced decrease of glycoprotein Po and myelin basic protein gene expression in the rat sciatic nerve. Repair by steroid derivatives

The data here reported show that the gene expression of the glycoprotein Po and of the myelin basic protein, the major components of myelin in the peripheral nervous system, dramatically decreases with ageing in the sciatic nerve of normal male rats. A one-month treatment with dihydroprogesterone, the 5 α-reduced derivative of progesterone, is able to partially restore the fall in Po gene expression occuring in the sciatic nerve of aged male rats, without significantly modifying the gene expression of the myelin basic protein. In cultures of neonatal Schwann cells (the peripheral nervous system elements involved in the synthesis of myelin), the addition of progesterone and of dihydroprogesterone significantly increases Po gene expression; the 3 α-reduced metabolite of dihydroprogesterone, tetrahydroprogesterone proved to be even more effective. These data suggest that the effect of progesterone is linked to its conversion into dihydroprogesterone and especially into tetrahydroprogesterone, since Schwann cells possess the 5 α-reductase-3 α-hydroxysteroid dehydrogenase system. The data provide the first demonstration that ageing decreases the gene expression of two major components of the peripheral myelin in the sciatic nerve; they also show that this phenomenon may be partially reversed by progesterone derivatives, which might act by stimulating Po gene expression in the Schwann cells.

Response of Schwann cells to mitogens in vitro is determined by pre-exposure to serum, time in vitro, and developmental age

We compared the mitogenic response of Schwann cells freshly isolated from adult, neonatal, and embryonic nerves, and compared these cells with cells that had been cultured in serum for 5 days. DNA synthesis in response to growth factors was measured using bromodeoxyuridine and immunocytochemistry. Freshly isolated adult Schwann cells were unresponsive to growth factors with or without forskolin to elevate intracellular cAMP levels. After 5 days of culture in serum, or alternatively in defined medium containing fibroblast growth factor 2 plus forskolin, or neu-differentiation factor beta2, adult cells were responsive to mitogens, whereas cells cultured in defined medium alone remained unresponsive. Serum also increased expression of type 1 fibroblast growth factor receptor. Freshly isolated embryonic and neonatal Schwann cells in contrast responded to growth factors even in the absence of forskolin. This responsiveness changed with time in culture. Neonatal cells cultured for 5 days in defined medium in the presence or absence of serum no longer responded to FGF alone, but required forskolin for a mitogenic response. Thus, the response of freshly isolated cells to mitogens is developmentally regulated; extrinsic signals are required to render adult cells responsive to mitogens; and with time in culture, neonatal cells develop a requirement for cAMP elevation for mitogenic response.

Apoptosis regulates the number of Schwann cells at the premyelinating stage.

At the premyelinating stage, the Schwann cells of peripheral nerves are able to recognize the axon, to arrange themselves along it in a nonoverlapping manner, and finally to establish a one-to-one cell-axon relationship. The mechanism that regulates these processes is not known in detail. We found the existence of a significant Schwann cell apoptosis in vivo of rat postnatal sciatic nerve, peaking around postnatal day 3. More than 50% of the neonatal Schwann cells cultured in axon-free medium undergo a rapid apoptosis. The apoptosis can be suppressed by addition of survival factors such as Neu differentiation factors or by increasing the adhesion of Schwann cells to substratum. We suggest that in neonatal nerves in vivo, Schwann cells are highly susceptible to apoptosis, but they are saved from death by contact with axons. The dramatic increase in number of Schwann cells between postnatal day 0 and 3 overcomes the number of axons available for them. Consequently the Schwann cells that fail to contact an axon undergo apoptosis. In conclusion, the number of Schwann cells in the developing nerves is regulated by the apoptosis and clearly depends on the survival signals from axons.

TGF-betas upregulate NCAM and L1 expression in cultured Schwann cells, suppress cyclic AMP-induced expression of O4 and galactocerebroside, and are widely expressed in cells of the Schwann cell lineage in vivo.

We have examined both how the molecular phenotype of Schwann cells in vitro is regulated by transforming growth factor beta (TGF-beta), using immunohistochemistry and immunoblotting, and the distribution of TGF-beta 2 and 3 in embryonic and mature nerves and ganglia, using immunohistochemistry and in situ hybridisation. We find that TGF-beta 2 and -3 upregulate expression of the neural cell adhesion molecules NCAM and L1. In TGF-beta-treated cultures, in addition to the 140 and 120 kD isoforms known to be present in Schwann cells, small amounts of the 180 kD isoform can be detected. TGF-beta s also block cAMP-induced expression of the lipid antigens galactocerebroside (GalC) and O4, in addition to blocking expression of protein zero (P0), the major peripheral myelin glycoprotein, as previously shown. Using antibodies specific to TGF-beta 2 and -3, respectively, we confirm the presence of these proteins in myelin-forming Schwann cells and show also that TGF-beta 2 and -3 are clearly expressed by peripheral glia that are not involved in myelination. This includes Schwann cell precursors, embryonic Schwann cells, non-myelin-forming Schwann cells and satellite cells from adult nerves and ganglia, and neonatal Schwann cells in purified cultures without neurones. In situ hybridisation with a digoxygenin-labelled riboprobe reveals a strong TGF-beta 3 mRNA signal in Schwann cells, satellite cells, and some neurones. Schwann cells in culture also secrete TGF-beta in a latent form, whereas purified cultures of dorsal root ganglion neurones from 1-day-old rats secrete active TGF beta during the first 48 h in culture.