Myelination determines the caliber of dorsal root ganglion neurons in culture

In order to understand the relationship of supporting cells to the differentiation of neurons in culture, we have used morphometry to study myelination of dorsal root ganglion (DRG) neurons by central or peripheral supporting cells. Dissociated DRG cultures from 15-day rat embryos, free of Schwann cells and fibroblasts, were prepared, and supporting cells were added from spinal cord or DRG; myelination commenced after 2 weeks. Control cultures received no supporting cells. At 7, 14, and 24 days, a total of 22 cultures were processed for electron microscopy. Three fascicles from defined points were sampled from each culture. In cultures containing glial cells, smaller fibers (p less than 0.001) were myelinated (mean of median diameter, 1.13 +/- 0.13 (SD) micron) than in cultures containing Schwann cells (1.67 +/- 0.17 micron), although there was no difference (p greater than 0.1) in the degree of myelination expressed as number of myelin lamellae/fiber. A new finding concerned the relationship of axonal diameter to the presence or absence of myelinating cells. In control cultures without supporting cells or in areas where supporting cells were absent, the range of neurite diameter (0.05 to 1.25 micron) and the median diameter (mean of median, 0.24 +/- 0.03 micron) were similar at different times (7, 14, and 24 days), demonstrating a stable population of neurite diameters throughout the period. In myelinated fascicles, a different distribution of neurite diameters was present. Myelinated neurites had a greater median diameter (measured to inner border of myelin) and a different range of fiber diameters compared to bare neurites. For Schwann cells, this range was 0.7 to 3.4 micron, and the mean of median diameters was 1.67 +/- 0.17 micron; for glial cells, the range was 0.6 to 2.4 micron, and the mean of median diameters 1.13 +/- 0.13 micron. Differences between myelinated and bare fibers were all highly significant (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cadm3 (Necl‐1) interferes with the activation of the PI3 kinase/Akt signaling cascade and inhibits Schwann cell myelination in vitro

Axo‐glial interactions are critical for myelination and the domain organization of myelinated fibers. Cell adhesion molecules belonging to the Cadm family, and in particular Cadm3 (axonal) and its heterophilic binding partner Cadm4 (Schwann cell), mediate these interactions along the internode. Using targeted shRNA‐mediated knockdown, we show that the removal of axonal Cadm3 promotes Schwann cell myelination in the in vitro DRG neuron/Schwann cell myelinating system. Conversely, over‐expressing Cadm3 on the surface of DRG neuron axons results in an almost complete inability by Schwann cells to form myelin segments. Axons of superior cervical ganglion (SCG) neurons, which do not normally support the formation of myelin segments by Schwann cells, express higher levels of Cadm3 compared to DRG neurons. Knocking down Cadm3 in SCG neurons promotes myelination. Finally, the extracellular domain of Cadm3 interferes in a dose‐dependent manner with the activation of ErbB3 and of the pro‐myelinating PI3K/Akt pathway, but does not interfere with the activation of the Mek/Erk1/2 pathway. While not in direct contradiction, these in vitro results shed lights on the apparent lack of phenotype that was reported from in vivo studies of Cadm3^−/− mice. Our results suggest that Cadm3 may act as a negative regulator of PNS myelination, potentially through the selective regulation of the signaling cascades activated in Schwann cells by axonal contact, and in particular by type III Nrg‐1. Further analyses of peripheral nerves in the Cadm^−/− mice will be needed to determine the exact role of axonal Cadm3 in PNS myelination. GLIA 2016;64:2247–2262     

Short‐term low‐frequency electrical stimulation enhanced remyelination of injured peripheral nerves by inducing the promyelination effect of brain‐derived neurotrophic factor on Schwann cell polarization

Electrical stimulation (ES) has been found to aid repair of nerve injuries and have been shown to increase and direct neurite outgrowth during stimulation. However, the effect of ES on peripheral remyelination after nerve damage has been investigated less well, and the mechanism underlying its action remains unclear. In the present study, the crush‐injured sciatic nerves in rats were subjected to 1 hr of continuous ES (20 Hz, 100 μsec, 3 V). Electron microscopy and nerve morphometry were performed to investigate the extent of regenerated nerve myelination. The expression profiles of P0, Par‐3, and brain‐derived neurotrophic factor (BDNF) in the injuried sciatic nerves and in the dorsal root ganglion neuron/Schwann cell cocultures were examined by Western blotting. Par‐3 localization in the sciatic nerves was determined by immunohistochemistry to demonstrate Schwann cell polarization during myelination. We reported that 20‐Hz ES increased the number of myelinated fibers and the thickness myelin sheath at 4 and 8 weeks postinjury. P0 level in the ES‐treated groups, both in vitro and in vivo, was enhanced compared with the controls. The earlier peak of Par‐3 in the ES‐treated groups indicated an earlier initiation of Schwann cell myelination. Additionally, ES significantly elevated BDNF expression in nerve tissues and in cocultures. ES on the site of nerve injury potentiates axonal regrowth and myelin maturation during peripheral nerve regeneration. Furthermore, the therapeutic actions of ES on myelination are mediated via enhanced BDNF signals, which drive the promyelination effect on Schwann cells at the onset of myelination. © 2010 Wiley‐Liss, Inc.     

Isolation, purification and expansion of myelination-competent, neonatal mouse Schwann cells

Most studies of peripheral nerve myelination using culture models are performed with dorsal root ganglion neurons and Schwann cells pre-purified from the rat. The potential of this model is severely compromised by the lack of rat myelin mutants and the published protocols work poorly with mouse cells, for which numerous myelin mutants are available. This is partly due to difficulties in obtaining sufficient quantities of myelination-competent mouse Schwann cells. Here, we describe the isolation, purification and expansion of wild-type, myelination-competent Schwann cells from the sciatic nerves of 4-day-old mouse pups. The method consistently yields 1.9-3.3 x 10(6) of approximately 95% pure Schwann cells from the sciatic nerves of 12-15 4-day-old mouse pups, within 14-20 days. The Schwann cell proliferation rate ranges from 2.7- to 4.30-fold growth/week. Proliferation ceases within 4 weeks, when the cells become quiescent. Growth is reinduced by the presence of neurons; neuregulin is not sufficient for this effect. The Schwann cells isolated by this protocol are able to form compact myelin in culture, as judged by the segregated expression patterns of early (myelin-associated glycoprotein) and late (myelin basic protein) myelination markers in a three-dimensional neuron/Schwann cell coculture model. The Schwann cell batch yields are sufficient to perform 100-150 individual myelinating coculture assays. Employing mixed phenotype/genotype mouse neuron/Schwann cell cocultures, it will be possible to analyse the cell specificity of a mutation, and the cumulative effects of different mutations, without having to cross-breed the animals.     

Axonal regulation of Schwann cell proliferation and survival and the initial events of myelination requires PI 3-kinase activity.

In this report, we have investigated the signaling pathways that are activated by, and mediate the effects of, the neuregulins and axonal contact in Schwann cells. Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase kinase (MAPK kinase) are strongly activated in Schwann cells by glial growth factor (GGF), a soluble neuregulin, and by contact with neurite membranes; both kinase activities are also detected in Schwann cell-DRG neuron cocultures. Inhibition of the PI 3-kinase, but not the MAP kinase, pathway reversibly inhibited Schwann cell proliferation induced by GGF and neurites. Cultured Schwann cells undergo apoptosis after serum deprivation and can be rescued by GGF or contact with neurites; these survival effects were also blocked by inhibition of PI 3-kinase. Finally, we have examined the role of these signaling pathways in Schwann cell differentiation in cocultures. At early stages of coculture, inhibition of PI 3-kinase, but not MAPK kinase, blocked Schwann cell elongation and subsequent myelination but did not affect laminin deposition. Later, after Schwann cells established a one-to-one relationship with axons, inhibition of PI 3-kinase did not block myelin formation, but the myelin sheaths that formed were shorter, and the rate of myelin protein accumulation was markedly decreased. PI 3-kinase inhibition had no observable effect on the maintenance of myelin sheaths in mature myelinated cocultures. These results indicate that activation of PI 3-kinase by axonal factors, including the neuregulins, promotes Schwann cell proliferation and survival and implicate PI 3-kinase in the early events of myelination.     

E‐Cadherin enhances neuregulin signaling and promotes Schwann cell myelination

In myelinating Schwann cells, E‐cadherin is a component of the adherens junctions that stabilize the architecture of the noncompact myelin region. In other cell types, E‐cadherin has been considered as a signaling receptor that modulates intracellular signal transduction and cellular responses. To determine whether E‐cadherin plays a regulatory role during Schwann cell myelination, we investigated the effects of E‐cadherin deletion and over‐expression in Schwann cells. In vivo, Schwann cell‐specific E‐cadherin ablation results in an early myelination delay. In Schwann cell‐dorsal root ganglia neuron co‐cultures, E‐cadherin deletion attenuates myelin formation and shortens the myelin segment length. When over‐expressed in Schwann cells, E‐cadherin improves myelination on Nrg1 type III^+/? neurons and induces myelination on normally non‐myelinated axons of sympathetic neurons. The pro‐myelinating effect of E‐cadherin is associated with an enhanced Nrg1‐erbB receptor signaling, including activation of the downstream Akt and Rac. Accordingly, in the absence of E‐cadherin, Nrg1‐signaling is diminished in Schwann cells. Our data also show that E‐cadherin expression in Schwann cell is induced by axonal Nrg1 type III, indicating a reciprocal interaction between E‐cadherin and the Nrg1 signaling. Altogether, our data suggest a regulatory function of E‐cadherin that modulates Nrg1 signaling and promotes Schwann cell myelin formation. GLIA 2015;63:1522–1536     

Recombinant peripheral myelin protein P0 confers both adhesion and neurite outgrowth-promoting properties.

To probe into the functional properties of the major peripheral myelin cell surface glycoprotein P0, its ability to confer adhesion and neurite outgrowth-promoting properties was studied in cell culture. To this aim, P0 was expressed as integral membrane glycoprotein at the surface of CV-1 cells with the help of a recombinant vaccinia virus expression system. Furthermore, the immunoglobulin-like extracellular domain of P0 (P0-ED) was expressed as soluble protein in a bacterial expression system and used as substrate coated to plastic dishes or as competitor in cell adhesion and neurite outgrowth-promoting assays. The adhesion of P0-expressing CV-1 cells to P0-ED substrate was specifically inhibitable by polyclonal P0 antibodies (54% +/- 6%). In addition, the specific interaction between P0 molecules could be reduced (49% +/- 8%) by adding soluble P0-ED to the culture medium, demonstrating that the homophilic interaction between recombinant P0 molecules can be mediated, at least on one partner of interacting molecules, by the unglycosylated Ig-like domain. Substrate-coated P0-ED also conferred adhesion and neurite outgrowth ability to dorsal root ganglion neurons with neurites of a mean length of about 150 microns. This neurite outgrowth was specifically inhibitable by soluble P0 (74% +/- 14%) and P0 antibodies (65% +/- 9%). These observations indicate that P0 is capable of displaying two different types of functional roles in the myelination process of peripheral nerves: The heterophilic interaction with neurons may be responsible for the recognition between axon and myelinating Schwann cell at the onset of myelination, whereas the homophilic interaction may indicate its role in the self-recognition of the apposing loops of Schwann cell surface membranes during the myelination process and in the mature compact myelin sheath.     

The dorsal root ganglion in Friedreich’s ataxia

Atrophy of dorsal root ganglia (DRG) and thinning of dorsal roots (DR) are hallmarks of Friedreich’s ataxia (FRDA). Many previous authors also emphasized the selective vulnerability of larger neurons in DRG and thicker myelinated DR axons. This report is based on a systematic reexamination of DRG, DR and ventral roots (VR) in 19 genetically confirmed cases of FRDA by immunocytochemistry and single- and double-label immunofluorescence with antibodies to specific proteins of myelin, neurons and axons; S-100α as a marker of satellite and Schwann cells; laminin; and the iron-responsive proteins ferritin, mitochondrial ferritin, and ferroportin. Confocal images of axons and myelin allowed the quantitative analysis of fiber density and size, and the extent of DR and VR myelination. A novel technology, high-definition X-ray fluorescence (HDXRF) of polyethylene glycol-embedded fixed tissue, was used to “map” iron in DRG. Unfixed frozen tissue of DRG in three cases was available for the chemical assay of total iron. Proliferation of S-100α-positive satellite cells accompanied neuronal destruction in DRG of all FRDA cases. Double-label visualization of peripheral nerve myelin protein 22 and phosphorylated neurofilament protein confirmed the known loss of large myelinated DR fibers, but quantitative fiber counts per unit area did not change. The ratio of myelinated to neurofilament-positive fibers in DR rose significantly from 0.55 to 0.66. In VR of FRDA patients, fiber counts and degree of myelination did not differ from normal. Pooled histograms of axonal perimeters disclosed a shift to thinner fibers in DR, but also a modest excess of smaller axons in VR. Schwann cell cytoplasm in DR of FRDA was depleted while laminin reaction product remained prominent. Numerous small axons clustered around fewer Schwann cells. Ferritin in normal DRG localized to satellite cells, and proliferation of these cells in FRDA caused wide rims of reaction product about degenerating nerve cells. Mitochondrial ferritin was not detectable. Ferroportin was present in the cytoplasm of normal satellite cells and neurons, and in large axons of DR and VR. In FRDA, some DRG neurons lost their cytoplasmic ferroportin immunoreactivity, whereas the cytoplasm of satellite cells remained ferroportin positive. Ferroportin in DR axons disappeared in parallel with atrophy of large fibers. HDXRF of DRG detected regional and diffuse increases in iron fluorescence that matched ferritin expression in satellite cells. The observations support the conclusions that satellite cells and DRG neurons are affected by iron dysmetabolism; and that regeneration and inappropriate myelination of small axons in DR are characteristic of the disease.     

Motor and sensory Schwann cell phenotype commitment is diminished by extracorporeal shockwave treatment in vitro

The gold standard for peripheral nerve regeneration uses a sensory autograft to bridge a motor/sensory defect site. For motor nerves to regenerate, Schwann cells (SC) myelinate the newly grown axon. Sensory SCs have a reduced ability to produce myelin, partially explaining low success rates of autografts. This issue is masked in pre‐clinical research by the excessive use of the rat sciatic nerve defect model, utilizing a mixed nerve with motor and sensory SCs. Aim of this study was to utilize extracorporeal shockwave treatment as a novel tool to influence SC phenotype. SCs were isolated from motor, sensory and mixed rat nerves and in vitro differences between them were assessed concerning initial cell number, proliferation rate, neurite outgrowth as well as ability to express myelin. We verified the inferior capacity of sensory SCs to promote neurite outgrowth and express myelin‐associated proteins. Motor Schwann cells demonstrated low proliferation rates, but strongly reacted to pro‐myelination stimuli. It is noteworthy for pre‐clinical research that sciatic SCs are a strongly mixed culture, not representing one or the other. Extracorporeal shockwave treatment (ESWT), induced in motor SCs an increased proliferation profile, while sensory SCs gained the ability to promote neurite outgrowth and express myelin‐associated markers. We demonstrate a strong phenotype commitment of sciatic, motor, and sensory SCs in vitro, proposing the experimental use of SCs from pure cultures to better mimic clinical situations. Furthermore we provide arguments for using ESWT on autografts to improve the regenerative capacity of sensory SCs.     

Cthrc1 is a negative regulator of myelination in Schwann cells

The analysis of the molecular mechanisms involved in the initial interaction between neurons and Schwann cells is a key issue in understanding the myelination process. We recently identified Cthrc1 (Collagen triple helix repeat containing 1) as a gene upregulated in Schwann cells upon interaction with the axon. Cthrc1 encodes a secreted protein previously shown to be involved in migration and proliferation in different cell types. We performed a functional analysis of Cthrc1 in Schwann cells by loss-of- and gain-of-function approaches using RNA interference knockdown in cell culture and a transgenic mouse line that overexpresses the gene. This work establishes that Cthrc1 enhances Schwann cell proliferation but prevents myelination. In particular, time-course analysis of myelin formation intransgenic animals reveals that overexpression of Cthrc1 in Schwann cells leads to a delay in myelin formation with cells maintaining a proliferative state. Our data, therefore, demonstrate that Cthrc1 plays a negative regulatory role, fine-tuning the onset of peripheral myelination.     

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

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

Specific inhibition of secreted NRG1 types I–II by heparin enhances Schwann Cell myelination

Primary cultures of mixed neuron and Schwann cells prepared from dorsal root ganglia (DRG) are extensively used as a model to study myelination. These dissociated DRG cultures have the particular advantage of bypassing the difficulty in purifying mouse Schwann cells, which is often required when using mutant mice. However, the drawback of this experimental system is that it yields low amounts of myelin. Here we report a simple and efficient method to enhance myelination in vitro. We show that the addition of heparin or low molecular weight heparin to mixed DRG cultures markedly increases Schwann cells myelination. The myelin promoting activity of heparin results from specific inhibition of the soluble immunoglobulin (Ig)‐containing isoforms of neuregulin 1 (i.e., NRG1 types I and II) that negatively regulates myelination. Heparin supplement provides a robust and reproducible method to increase myelination in a simple and commonly used culture system. GLIA 2016;64:1227–1234     

Schwann cell proliferation and migration during paranodal demyelination

This study examined Schwann cell behavior during paranodal demyelination induced by beta,beta'-iminodipropionitrile (IDPN). The stimuli for Schwann cell proliferation, extensively studied in vitro, are less well understood in vivo. Most in vivo systems previously used to examine Schwann cell proliferation in disease are dominated by loss of internodal myelin sheaths. As used in this study, IDPN administration produces neurofilamentous axonal swellings and paranodal demyelination, without segmental demyelination or fiber degeneration. We asked whether Schwann cells would proliferate following the restricted paranodal demyelination that accompanies the axonal swellings, and if so what the sources and distributions of new Schwann cells might be. IDPN was given as a single large dose (2 ml/kg) to 21-d-old rats. Neurofilamentous axonal swellings formed in the proximal regions of motor axons, reaching their greatest enlargement in the root exit zone 8 d after IDPN administration. These swellings subsequently migrated distally down the nerves at rates approaching 1 mm/d. The axonal enlargement was consistently associated with displacement of the myelin sheath attachment sites into internodal regions, and consequent paranodal demyelination. This stage was associated with perikaryal changes, including nucleolar enlargement, "girdling" of the perikaryon, and formation of attenuated stalks separating the perinuclear region from the external cytoplasmic collar. Schwann cells proliferated abundantly during this stage. Daughter Schwann cells migrated within the endoneurial space (outside the nerve fiber basal laminae) to overlie the demyelinated paranodes of swollen nerve fibers. In these regions, local proliferation of Schwann cells continued, resulting in large paranodal clusters of Schwann cells. As the axonal calibers subsequently returned to normal, the outermost myelin lamellae of the original internodes returned to their paranodal attachment sites and the supernumerary Schwann cells disappeared. Formation of short internodes, segmental demyelination, and nerve fiber loss were rare phenomena. These results indicate that paranodal demyelination is a sufficient stimulus to excite abundant Schwann cell proliferation; neither internodal demyelination nor myelin breakdown is a necessary stimulus for mitosis. The 3H-thymidine incorporation studies indicated that the sources of new Schwann cells included markedly increased division of the Schwann cells of unmyelinated fibers and, as they formed, supernumerary Schwann cells. In addition, there were rare examples of 3H-thymidine incorporation by Schwann cells associated with myelinated nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regeneration of axons from adult human retina in vitro

In an effort to establish an in vitro model of regenerating adult human central nervous system (CNS) neurons, we have investigated the potential for neurite growth from explants prepared from normal adult human retina. Eyes (donated for corneal transplantation) were removed within 2.0 h postmortem and stored on ice for 1.5 to 7.0 days. Retinal explants (1 mm2) were prepared and cultured at 37 degrees C on cellular or acellular substrata in an oxygen-rich, humidified atmosphere. Neurite outgrowth, visualized by neurofilament immunofluorescence, was observed only in the presence of Schwann cells, after a quiescent period of approximately 6 days in vitro. Of 50 explants cultured for 7 days or more on substantia containing Schwann cells, 43 showed evidence of viability in vitro and 28 extended neurites onto Schwann cell surfaces. Estimated rates of neurite growth on Schwann cell substrata reached a maximum of 0.22 mm/day. Neurites did not grow beyond the explant border onto culture substrata composed of either polylysine, laminin, type-I collagen, or monolayers of adult human retinal glia. These results demonstrate that under selected conditions, explants prepared from adult human retina harbor viable neurons and that Schwann cells promote and support regeneration of neurites from these neurons in vitro, allowing systematic analysis of conditions favorable to axonal regeneration from adult human CNS neurons.     

The myelin protein MAL affects peripheral nerve myelination: a new player influencing p75 neurotrophin receptor expression

The myelin and lymphocyte protein (MAL) is a raft-associated membrane protein predominantly expressed by oligodendrocytes and Schwann cells. Here we show that MAL regulates myelination in the peripheral nervous system. In mice overexpressing MAL, myelination was retarded and fibers were hypomyelinated, whereas myelination in MAL knockout mice was accelerated. This was not due to impaired Schwann cell proliferation, differentiation or axonal sorting. We found that the expression level of p75 neurotrophin receptor mRNA and protein was strongly reduced in developing sciatic nerves in MAL-overexpressing mice. This reduction is well correlated with the observed alterations in myelination initiation, speed of myelination and alterations in Remak bundle development. Our results suggest a functional role for MAL in peripheral myelination by influencing the expression of membrane components that mediate axon-glia interaction during ensheathment and myelin wrapping.     

Nerve growth factor modulates myelin-associatedglycoprotein binding to sensory neurons

Myelin-associated glycoprotein (MAG) is a molecule expressed by myelinating cells at the myelin/axon interface, which binds to an as yet unidentified molecule on neurons. We have used a MAG-immunoglobulin Fc fusion protein to examine the expression and regulation of the MAG binding molecule on sensory neurons in culture. Binding of the MAG-Fc reached a maximum at 24-48 h and was higher on neurons which expressed high levels of neurofilament. Nerve growth factor (NGF) upregulated expression of the MAG binding molecule in a dose dependent manner. Schwann cells co-cultured with sensory neurons in serum-free medium stimulated maximal expression of the MAG binding molecule, which was decreased by addition of anti-NGF to the co-cultures. This indicated that Schwann cells can modulate expression of the MAG binding molecule via production of NGF and may represent a physiological mechanism for regulation of MAG-MAG binding molecule interactions during myelination and remyelination.     

Increased expression of pp60c-src protein-tyrosine kinase during peripheral nerve regeneration.

Since little is known about the intracellular changes that take place in response to Schwann cell-neuron interactions that occur during neurite outgrowth and myelination, we investigated the expression of a protein-tyrosine kinase, pp60c-src, during peripheral nerve regeneration through a silicone tube. Segments of regenerated nerve, extracted at various times following nerve-transection, showed an induction of in vitro c-src kinase activity as measured by autophosphorylation of immunoprecipitated pp60c-src. This activity occurred at 7 days following nerve transection coincident with the onset of neurite outgrowth in vivo. This kinase activity, which peaked out between 21 and 35 days and decreased thereafter, appeared to be associated with axonal growth and myelination, but not mitogenesis in the tube. Analysis of c-src proteins levels by Western blot showed a similar expression profile as that of the kinase activity. Qualitatively, the expression of an immunoreactive c-src band, migrating slightly slower than pp60, was detected in extracts of regenerating nerve segments as well as in the corresponding L4 and L5 dorsal root ganglia. This protein may be the CNS neuronal-specific form (pp60+) of the c-src protein. In situ hybridization revealed that Schwann cells and sensory and motor neurons associated with the regenerated sciatic nerve were positive for c-src mRNA during regeneration possibly accounting for the increased src protein expression during regeneration. Since the increased expression of pp60c-src in regenerated nerve segments coincides with both axonal sprouting and myelination, our findings suggest that the c-src protein may play a role in Schwann cell-neuron interactions which facilitate the occurrence of these events during regeneration. In addition, although pp60+ is generally not detectable in the mature PNS, our findings show that this protein may be induced during conditions of PNS differentiation which promote neurite outgrowth.     

Axonal regeneration, but not myelination, is partially dependent on local cholesterol reutilization in regenerating nerve

A recycling pathway in peripheral nerve permits cholesterol from degenerating myelin to be salvaged by macrophages and resupplied to myelinating Schwann cells by locally produced lipoproteins. A similar reutilization of cholesterol by regenerating axons has been proposed but not demonstrated. Neurites in culture, however, do take up cholesterol and cholesterol-containing lipoproteins, where these molecules are found to promote neurite extension. To test the requirement for cholesterol reutilization in axon regeneration and myelination, we examined 2 models of blocked intracellular cholesterol transport: 1) bone marrow transplants from Niemann-Pick C mice into wild-type recipient mice, and 2) imipramine treatment. Following nerve crush in these models, we found that unusually large, debris-filled macrophages appeared and persisted for many weeks. A morphometric analysis of regenerating nerves revealed that myelination proceeded at a normal rate (normal g-ratios), but that axon growth was retarded (decreased fiber numbers and diameters) in these animals. Cholesterol synthesis was elevated in these nerves, indicating that Schwann cells compensated for the decreased exogenous supply of cholesterol by up-regulating de novo synthesis to support myelination. These data indicate that Schwann cells are not dependent on cholesterol reutilization to support myelination, but that optimal axonal regeneration is dependent on a local supply of cholesterol.     

Synthesis of progesterone in Schwann cells: regulation by sensory neurons

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3 beta-hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3 beta-hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3 beta-hydroxysteroid dehydrogenase were induced. Regulation of 3 beta-hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3 beta-hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re-expressed. After nerve transection, which does not allow axonal regeneration, 3 beta-hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3 beta-hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.     

Transplantation of Nogo-66 receptor gene-silenced cells in a poly(D,L-lactic-co-glycolic acid) scaffold for the treatment of spinal cord injury

Inhibition of neurite growth,which is in large part mediated by the Nogo-66 receptor,affects neural regeneration following bone marrow mesenchymal stem cell transplantation.The tissue engineering scaffold poly(D,L-lactide-co-glycolic acid) has good histocompatibility and can promote the growth of regenerating nerve fibers.The present study used small interfering RNA to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells and Schwann cells,which were subsequently transplanted with poly(D,L-lactide-co-glycolic acid) into the spinal cord lesion regions in rats.Simultaneously,rats treated with scaffold only were taken as the control group.Hematoxylin-eosin staining and immunohistochemistry revealed that at 4 weeks after transplantation,rats had good motor function of the hind limb after treatment with Nogo-66 receptor gene-silenced cells plus the poly(D,L-lactide-co-glycolic acid) scaffold compared with rats treated with scaffold only,and the number of bone marrow mesenchymal stem cells and neuron-like cells was also increased.At 8 weeks after transplantation,horseradish peroxidase tracing and transmission electron microscopy showed a large number of unmyelinated and myelinated nerve fibers,as well as intact regenerating axonal myelin sheath following spinal cord hemisection injury.These experimental findings indicate that transplantation of Nogo-66 receptor gene-silenced bone marrow mesenchymal stem cells and Schwann cells plus a poly(D,L-lactide-co-glycolic acid) scaffold can significantly enhance axonal regeneration of spinal cord neurons and improve motor function of the extremities in rats following spinal cord injury.