Transplantation of human olfactory ensheathing cells elicits remyelination of demyelinated rat spinal cord

Human olfactory ensheathing cells (OECs) were prepared from adult human olfactory nerves, which were removed during surgery for frontal base tumors, and were transplanted into the demyelinated spinal cord of immunosuppressed adult rats. Extensive remyelination was observed in the lesion site: In situ hybridization using a human DNA probe (COT-1) indicated a similar number of COT-1-positive cells and OEC nuclei within the repaired lesion. The myelination was of a peripheral type with large nuclei and cytoplasmic regions surrounding the axons, characteristic of Schwann cell and OEC remyelination. These results provide evidence that adult human OECs are able to produce Schwann cell-like myelin sheaths around demyelinated axons in the adult mammalian CNS in vivo.     

Cultures of rat olfactory ensheathing cells are contaminated with Schwann cells

Implantation of cultured olfactory ensheathing cells into the damaged spinal cord of adult rats has been reported to remyelinate central axons. This observation is curious because olfactory ensheathing cells do not myelinate axons in their native environment. We have recently determined that calponin is the first definitive phenotypic marker for olfactory ensheathing cells. Primary cultures of adult rat olfactory mucosa and olfactory bulb were immunostained for p75 neurotrophin receptor and calponin. Our results reveal that two populations of p75 neurotrophin receptor-positive cells exist in primary cultures of the olfactory mucosa and bulb: calponin-positive olfactory ensheathing cells and calponin-negative Schwann cells. As olfactory tissues likely yield a mixed glial population, the idea that olfactory ensheathing cells are capable of de novo myelin synthesis after intraspinal implantation should be re-evaluated.     

Transplanted Schwann cells, not olfactory ensheathing cells, myelinate optic nerve fibres

In a previous study we found that olfactory ensheathing cells transplanted into complete retrobulbar transections of the rat optic nerve mediated regeneration of severed retinal ganglion cell axons through the graft region. Although the regenerating axons were ensheathed by the transplanted cells, none of the regenerating axons became myelinated by either central or peripheral type myelin. In the present study we used the same operative procedure but transplanted Schwann cells instead of olfactory ensheathing cells. As with the olfactory ensheathing cell transplants the Schwann cells transplants also induced regeneration of the severed retinal ganglion cell axons into the graft region. In contrast to the situation with the olfactory ensheathing cell transplants, however, a considerable number of the regenerating axons became myelinated by peripheral type myelin produced by the transplanted Schwann cells. This observation identifies a further distinction between these two cell types which are phenotypically similar in many ways, but which have been shown to have major functional differences with regard to regeneration in spinal cord lesions.     

Insulin-like growth factor-I promotes myelination of peripheral sensory axons

Insulin-like growth factor-I (IGF-I) in vivo or in the presence of other permissive factors can promote myelination in the central nervous system. In the current study, we examine the role of IGF-I in the myelination of peripheral nerves. In rat cocultures of dorsal root ganglia (DRG) and Schwann cells (SC) grown in serum- and insulin-free defined medium, IGF-I induces a dose dependent upregulation in myelin proteins such as P0, corresponding to maximal SC ensheathment. Furthermore, IGF-I is essential in promoting a dose-dependent, long-term myelination of DRG sensory axons. In the absence of IGF-I, axons and SC survive, but fail to myelinate. In the presence of 10 nM IGF-I, 59% of axons are myelinated at 21 days, whereas in the absence of IGF-I myelination fails to occur. Maximum SC ensheathment occurs 48 hours after addition of IGF-I. If IGF-I is withdrawn at 48 hours, axon segregation by SC persists, however, most axons and SC do not exhibit a one-to-one relationship and little myelination is observed. IGF-I is important in myelination and is critical not only for initial SC ensheathment of the axon and upregulation of myelin proteins, but also for sustained myelination. Furthermore, IGF-I associated axonal size is not the sole determinant for myelination.     

Myelination by mature ovine oligodendrocytes in vivo and in vitro: Evidence that different steps in the myelination process are independently controlled

The ability of isolated mature post-myelination ovine oligodendrocytes to myelinate was investigated in tissue culture and in vivo. In culture, although the cells adhered preferentially to rat dorsal root ganglia (DRG) axons, sent out processes that encircled and wrapped them, proliferated, and synthesised myelin proteins (MBP), no myelination was found. This failure to find myelination occurred despite the fact that the oligodendrocytes both in the present experiments and in previous studies elaborated membranous structures that have been shown chemically and structurally to be similar to normal central nervous system myelin. These findings contrasted with those seen when neonatal rodent glial cells were added to similar DRG neuron cultures, in which myelination readily occurred. When the same adult ovine oligodendrocytes were transplanted into the brains of Shiverer mice, normal compact myelin was formed, proving that the cells were capable of myelination and suggesting that cross-species incompatibility was probably not a major factor in the lack of myelination in vitro. It is possible that the failure of ovine oligodendrocytes to myelinate DRG axons is due either to the relatively low number of supporting glial cells, such as astrocytes or microglia which may be necessary for satisfactory myelination, or that some other factor in the microenvironment is lacking; in any event, these results point to the complexity of oligodendrocyte-axon interactions. It is clear that each of the events, from adherence to proliferation to wrapping and the myelin compaction may be under the control of a different signal and may operate through a distinct mechanism, even though each process is dependent on the other. The results also point to the potential usefulness of this model system for deciphering such signals and mechanisms. © 1993 Wiley-Liss, Inc.     

Astrocytes, but not olfactory ensheathing cells or Schwann cells, promote myelination of CNS axons in vitro

We have examined the interaction between olfactory ensheathing cells (OECs), Schwann cells (SC), oligodendrocytes, and CNS axons using cultures generated from embryonic rat spinal cord. Oligodendrocyte process extension and myelination in these cultures was poor if the cells were plated on OECs or SCs. Myelin internodes and nodes of Ranvier formed frequently if these cultures were plated onto monolayers of neurosphere-derived astrocytes (NsAs). In the myelinated fibers generated on NsAs, Nav channels, caspr, and neurofascin molecules were correctly assembled at the nodes of Ranvier. The density of neurites, survival, and antigenic differentiation of oligodendrocytes was similar on OEC and NsAs monolayers. However, on OEC monolayers, despite a transient increase in the number of endogenous oligodendrocytes, there was a decrease in oligodendrocyte process extension and axonal ensheathment when compared with cultures plated on NsAs monolayers. To determine if these changes were due to axonal or glial factors, spinal cord oligodendrocytes were plated onto monolayers of OECs, NsAs, and poly-L-lysine in the absence of neurons. In these cultures, process extension and myelin-like membrane formation by oligodendrocytes was improved on monolayers of OEC. This suggests that inhibition of process extension is mediated via cross-talk between OECs and neurites. In cultures containing axons plated on OEC monolayers, oligodendrocyte process formation, axonal ensheathment, and myelination occurred albeit lower if the cultures were supplemented with NsAs conditioned medium. These data suggest OECs can permit neurite extension and oligodendrocyte proliferation, but lack secreted factor(s) and possible cell-cell contact that is necessary for oligodendrocyte process extension and 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.     

Type III neuregulin-1 promotes oligodendrocyte myelination

The axonal signals that regulate oligodendrocyte myelination during development of the central nervous system (CNS) have not been established. In this study, we have examined the regulation of oligodendrocyte myelination by the type III isoform of neuregulin-1 (NRG1), a neuronal signal essential for Schwann cell differentiation and myelination. In contrast to Schwann cells, primary oligodendrocytes differentiate normally when cocultured with dorsal root ganglia (DRG) neurons deficient in type III NRG1. However, they myelinate type III NRG1-deficient neurites poorly in comparison to wild type cultures. Type III NRG1 is not sufficient to drive oligodendrocyte myelination as sympathetic neurons are not myelinated even with lentiviral-mediated expression of NRG1. Mice haploinsufficient for type III NRG1 are hypomyelinated in the brain, as evidenced by reduced amounts of myelin proteins and lipids and thinner myelin sheaths. In contrast, the optic nerve and spinal cord of heterozygotes are myelinated normally. Together, these results implicate type III NRG1 as a significant determinant of the extent of myelination in the brain and demonstrate important regional differences in the control of CNS myelination. They also indicate that oligodendrocyte myelination, but not differentiation, is promoted by axonal NRG1, underscoring important differences in the control of myelination in the CNS and peripheral nervous system (PNS).     

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.     

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     

Excitable Axonal Domains Adapt to Sensory Deprivation in the Olfactory System

The axon initial segment (AIS), nodes of Ranvier, and the oligodendrocyte-derived myelin sheath have significant influence on the firing patterns of neurons and the faithful, coordinated transmission of action potentials (APs) to downstream brain regions. In the olfactory bulb (OB), olfactory discrimination tasks lead to adaptive changes in cell firing patterns, and the output signals must reliably travel large distances to other brain regions along highly myelinated tracts. Whether myelinated axons adapt to facilitate olfactory sensory processing is unknown. Here, we investigate the morphology and physiology of mitral cell (MC) axons in the olfactory system of adult male and female mice and show that unilateral sensory deprivation causes system-wide adaptations in axonal morphology and myelin thickness. MC spiking patterns and APs also adapted to sensory deprivation. Strikingly, myelination and MC physiology were altered on both the deprived and nondeprived sides, indicating system level adaptations to reduced sensory input. Our work demonstrates a previously unstudied mechanism of plasticity in the olfactory system.SIGNIFICANCE STATEMENT Successful transmission of information from the olfactory bulb (OB) to piriform cortex through the lateral olfactory tract (LOT) relies on synchronized arrival of action potentials (APs). The coincident arrival of APs is dependent on reliable generation of APs in the axon initial segment (AIS) and fast conduction mediated by axon myelination. Here, we studied changes in mitral cell (MC) firing and AIS structure as well as changes in myelination of the LOT on unilateral olfactory deprivation in the adult mouse. Strikingly, myelination and MC physiology were altered on both the deprived and nondeprived sides, indicating system level adaptations to reduced sensory input. Our work demonstrates a previously unstudied mechanism of plasticity in the olfactory system.     

MAG-deficient schwann cells myelinate dorsal root ganglion neurons in culture

The myelin-associated glycoprotein (MAG) has been postulated to play a crucial role during myelin formation. Evidence supporting this hypothesis was provided by infecting rat Schwann cells with a retrovirus expressing MAG antisense RNA; these Schwann cells showed reduced levels of MAG expression and failed to myelinate DRG neurons in vitro. However, when MAG expression was disrupted by generating MAG-deficient mice, normal myelin sheaths were formed in peripheral nerves in vivo. In the present study we investigated whether myelination is compromised in MAG-deficient Schwann cells in vitro, i.e., under similar conditions where Schwann cells expressing MAG antisense RNA failed to myelinate. We show that MAG-deficient Schwann cells do myelinate DRG neurons in vitro and express the myelin-specific glycolipid galactocerebroside (Gal-C) and the myelin proteins P0 and MBP. Furthermore, myelin sheaths appear morphologically normal with both compacted and uncompacted aspects when investigated by electron microscopy. Quantitative analysis revealed that the number of myelin sheaths was similar in cultures from MAG-deficient and wild-type mice. These findings support the view that MAG is not essential for myelin formation in the PNS. GLIA 22:213–220, 1998. © 1998 Wiley-Liss, Inc.     

In Vitro Enfolding of Olfactory Neurites by p75 NGF Receptor Positive Ensheathing Cells from Adult Rat Olfactory Bulb

Secondary cultures of adult rat olfactory bulb (OB) contained three different types of cell: (i) process-bearing cells; (ii) macrophage-like cells and (iii) fusiform cells. The immunohistochemical properties of process-bearing cells closely corresponded to those described for ensheathing glia in vivo. The most distinctive feature of these cells was their immunoreactivity for low affinity nerve growth factor receptor (NGFR). Process-bearing cells also shared the ultrastructural properties of ensheathing glia in vivo , as well as the ability to ensheath olfactory axons. In contrast, macrophage-like cells had the immunostaining properties of microglia, and fusiform cells were likely capillary endothelial cells.
Neurites outgrowing from olfactory epithelium explants, when co-cultured with adult OB cells, grew preferentially over NGFR positive cells. Olfactory neurites exhibited NGFR immunoreactivity and were enfolded by NGFR positive cells. After ensheathment, this immunoreactivity decreased from the neurite and disappeared from the glial membrane in contact with the neurite. However, NGFR immunoreactivity was maintained in the portion of the glial membrane not involved in ensheathing. In summary, ensheathing cells in vitro retained both the ultrastructure shown in vivo and the ability to ensheath olfactory neurites. The Schwann cell-like properties of ensheathing glia, could partially explain the permissibility of adult OB to axonal growth.     

Two novel monoclonal antibodies (1.9.E and 4.11.C) against olfactory bulb ensheathing glia

We produced and characterized two monoclonal antibodies, termed 1.9.E and 4.11.C, that specifically recognize olfactory bulb ensheathing glia. Both antibodies were generated using the olfactory nerve layer (ONL) of newborn rat olfactory bulbs (P0, P1) as immunogens. The specificity of these antibodies was tested by immunofluorescence techniques on tissue sections and cultures of adult and neonatal rat olfactory bulbs, and by Western blot analysis. 1.9.E labeled the ONL and glomerular layer of the olfactory bulb (OB) of adult rats. In newborn rats, 1.9.E immunostained ensheathing cells from the ONL and peripheral olfactory fascicles. Furthermore, 1.9.E reacted with some processes of the radial glia in the periventricular germinal layer of the newborn rat. Although 4.11.C also specifically labeled ensheathing cells in the adult OB, it did not stain any cell type in the ONL of newborn rats. The lack of double labeling with either 1.9.E or 4.11.C and anti-olfactory marker protein (OMP) antibody, a specific marker for olfactory axons, indicated that none of the monoclonals recognized olfactory axons. Double immunostaining of adult OB cultures with 1.9.E or 4.11.C and anti-p75-nerve growth factor receptor revealed that both antibodies specifically recognized ensheathing glia in those cultures. Filaments were strongly labeled throughout the entire cytoplasm of ensheathing cells, suggesting that 1.9.E and 4.11.C immunoreacted with ensheathing glia cytoskeleton. 4.11.C stained a few Schwann cells in adult sciatic nerve sections. Moreover, 4.11.C immunostained cortical astrocyte cultures from newborn rats (P1). In Western blot analysis both antibodies recognized a major component, migrating with an apparent molecular weight of 60 kDa, from olfactory nerve and glomerular layer (ONGL) extracts of adult and neonatal rats. The pattern of immunoreactivity of 1.9.E and 4.11.C antibodies suggest that both antibodies are specific markers for olfactory ensheathing glia in the adult rat central nervous system (CNS). GLIA 24:352–364, 1998. © 1998 Wiley-Liss, Inc.     

Proteomic evaluation reveals that olfactory ensheathing cells but not Schwann cells express calponin

Human clinical trials have begun worldwide that use olfactory ensheathing cells (OECs) to ameliorate the functional deficits following spinal cord injury. These trials have been initiated largely because numerous studies have reported that OECs transform into Schwann Cell (SC)-like cells that myelinate axons and support new growth in adult rats with spinal injury. This phenomenon is remarkable because OECs do not myelinate olfactory axons in their native environment. Furthermore, these myelinating OECs are morphologically identical to SCs, which can invade the spinal cord after injury. One factor that has contributed to a possible confusion in the identification of these cells is the lack of phenotypic markers to distinguish unequivocally between OECs and SCs. Such markers are required to first assess the degree of SC contamination in OEC cultures before intraspinal implantation, and then to accurately identify grafted OECs and invading SCs in the injured spinal cord. Using two-dimensional gel electrophoresis, we have identified calponin, an actin binding protein, as the first definitive phenotypic marker that distinguishes between OECs and SCs in vitro and in vivo. We have also provided ultrastructural evidence that calponin-immunopositive OECs do not transform into myelinating SC-like cells after intraspinal implantation. Rather, the grafted OECs retain their morphological and neurochemical features. These data yield new insight into the phenotypic characteristics of OECs, which together with invading SCs can enhance regeneration of the injured spinal cord.     

Hereditary hypertrophic neuropathy in the Trembler mouse: Part 2. Histopathological studies: Electron microscopy

The Trembler mouse suffers from a dominantly inherited hypertrophic neuropathy. Electron microscopy, including a quantitative analysis of myelination was performed on the nerves of Trembler mice from birth to senility and compared with the findings in control mice. Axons in adult Trembler nerves were thinly myelinated and were surrounded by very few myelin lamellae which in turn were often uncompact circumferentially and longitudinally. Schwann cell cytoplasm was copious and had a normal content of organelles. Well-developed “onion-bulb” formations which consisted of thinly myelinated axons surrounded by empty membrane configurations were frequently seen.

The initiation of myelination was studied. The diameter distribution of promyelin fibres of control and Trembler sciatic nerve at ages day 2, 4, and 7 was calculated Myelination in Trembler nerves commenced on axons of larger diameters than controls.

The effectiveness of myelination was studied by relating the number of turns of myelin to the axon area of control and Trembler sciatic nerves from age 2 days to adult mice. At all ages Trembler axons were less well myelinated than controls and the difference was more pronounced with age.

Schwann cell activity was examined by relating the area of the Schwann cell cytoplasm to the area of the axon it invests. The relative amount of Schwann cell cytoplasm decreased progressively in control axons with age and as the axon became better myelinated. By contrast, that of Tremblers did not undergo a similar reduction as the animal matured and the relative amount of Schwann cell cytoplasm was markedly increased in adult Tremblers when compared with controls.

The periodicity of control and Trembler compact myelin was compared. The myelin period of Trembler mouse was significantly greater than that of controls. The defect in Trembler peripheral nerves was considered to be that of dysmyelinogenesis. The Schwann cell was active but ineffective in the synthesis, compaction and maintenance of myelin.     

The olfactory bulb and olfactory mucosa obtained from human cadaver donors as a source of olfactory ensheathing cells

During the last decade, olfactory ensheathing cells (OECs) have been successfully applied in multiple experimental approaches aimed to repair damaged mammalian spinal cord. Some of these experiments have consequently been translated into clinical trials. Finding a reliable source of human OECs that is easily accessible and can ensure a sufficient number of cells is a major prerequisite for conducting studies on OEC-mediated spinal cord regeneration. Here, we present a procedure for obtaining olfactory bulbs (OBs) and olfactory mucosa (OM) simultaneously from adult cadaver heart-beating donors for OEC isolation and analyze some of the factors that may condition successful OEC culture. We show that the results of OEC culture from OBs (10 cases) correlated significantly with warm ischemia time (WIT) as well as the initial viability of the isolated cells. Efficient OEC culture was possible when the WIT for the OB was up to 20 min. Brain damage, assessed by determination of S100B serum level, was not related to the success of OEC culture from the OB. Cadaver OM (7 cases) was shown to be a more reliable source of human OECs than the OB. In most of the examined cases the efficacy of culturing OECs from cadaver OM obtained even 180 min after cardiac arrest was comparable to that of living patients. The method of obtaining OBs and OM from cadavers enables the use of an alternative source of primary adult human OECs for further preclinical and clinical studies on their neurotrophic properties.     

Myelination of the rat retina by transplantation of oligodendrocytes into 4-day-old hosts

Oligodendrocyte transplantation into the retina enables us to investigate the early events in myelin formation in a new in vivo system. The axons of rat retinal ganglion cells are unmyelinated in the eye but should express a myelination initiation signal since they acquire myelin posterior to the globe. The lamina cribrosa may block the migration of oligodendrocytes from the optic nerve into the retina. Animals that lack a lamina cribosa such as the rabbit have myelinated retinas. We have bypassed the lamina cribrosa by using transplantation techniques and inserted freshly isolated syngeneic 3-week-old rat oligodendrocytes into the unmyelinated 4-day-old rat retina during the period of active optic nerve myelination. The animals are sacrificed at 1-week intervals for 8 weeks. The retinas are examined immunocytochemically for myelin with an antibody to myelin basic protein (MBP). MBP-positive cells are seen extending processes at 1 and 2 weeks. Three and four week retinas show the formation of thicker and longer myelin sheaths oriented along the same radial path as the retinal ganglion axons with maximal MBP staining intensity seen by 5 weeks. Transplanted retinas are negative when stained for P0, a Schwann cell antigen, ruling out Schwann cell myelination of our retinas. We have shown that rat cerebral oligodendrocytes survive, mature, and express a myelin-specific protein in the retinal environment in a pattern consistent with myelination of ganglion cell axons. Retinal transplantation provides a new in vivo model to study oligodendrocyte development and axonal-glial interactions, free from the difficulties inherent in culture systems.     

Insights into olfactory ensheathing cell development from a laser-microdissection and transcriptome-profiling approach

Olfactory ensheathing cells (OECs) are neural crest-derived glia that ensheath bundles of olfactory axons from their peripheral origins in the olfactory epithelium to their central targets in the olfactory bulb. We took an unbiased laser microdissection and differential RNA-seq approach, validated by in situ hybridization, to identify candidate molecular mechanisms underlying mouse OEC development and differences with the neural crest-derived Schwann cells developing on other peripheral nerves. We identified 25 novel markers for developing OECs in the olfactory mucosa and/or the olfactory nerve layer surrounding the olfactory bulb, of which 15 were OEC-specific (that is, not expressed by Schwann cells). One pan-OEC-specific gene, Ptprz1, encodes a receptor-like tyrosine phosphatase that blocks oligodendrocyte differentiation. Mutant analysis suggests Ptprz1 may also act as a brake on OEC differentiation, and that its loss disrupts olfactory axon targeting. Overall, our results provide new insights into OEC development and the diversification of neural crest-derived glia.     

Calponin is expressed by fibroblasts and meningeal cells but not olfactory ensheathing cells in the adult peripheral olfactory system

Olfactory ensheathing cells (OECs), the principal glial cells of the peripheral olfactory system, have many phenotypic similarities with Schwann cells of the peripheral nervous system. This makes reliably distinguishing these two cells types difficult, especially following transplantation into areas of injury in the central nervous system. In an attempt to identify markers by which these two cells types can be distinguished, a recent proteomic analysis of fetal OECs and adult Schwann cells identified the actin-binding protein calponin as a potential marker expressed by OECs but not Schwann cells. Since many studies designed with the translational goal of autologous transplantation in mind have used adult OECs, this study examined the expression of calponin by adult OECs, both in vivo within the peripheral olfactory system and in vitro. Calponin colocalized with strongly fibronectin positive fibroblasts in the olfactory mucosa (OM) and meningeal cells in the olfactory bulb (OB) but not with S100beta or neuropeptide-Y positive OECs. In tissue culture, calponin was strongly expressed by fibronectin-expressing fibroblasts from OM, sciatic nerve and skin and by meningeal cells from the OB, but not by p75(NTR)- and S100beta-expressing OECs. These data, supported by Western blotting, indicate that calponin can not be used to distinguish adult OECs and Schwann cells.