Inverse patterns of myelination and GAP-43 expression in the adult CNS: Neurite growth inhibitors as regulators of neuronal plasticity?

In the central nervous system (CNS) myelin is present not only in white matter, but also in varying amounts in many gray matter areas. In addition to the function of electrical insulation of axons, myelin and oligodendrocytes contain molecules that are powerful inhibitors of neurite growth. Nevertheless plastic changes involving sprouting of nerve terminals occur in several brain regions of adult animals after partial lesions. In this study we have tried to correlate the plastic potential of CNS regions with the degree of their myelination. The expression of the growth-associated protein GAP-43 was used as an indicator of the potential for plastic changes, and a histological myelin stain was used to assess myelination. We have found that myelination and GAP-43 expression have strikingly inverse expression patterns in the majority of CNS gray matter areas. Densely myelinated regions, that is, most brainstem nuclei, the tegmentum, and the inferior colliculus, are low in GAP-43. In contrast, unmyelinated or lightly myelinated areas, such as the substantia gelatinosa of the spinal cord, the nucleus of the solitary tract, or the septum, express high levels of GAP-43. Areas known to show lesion-induced sprouting are typically high in GAP-43 and only lightly myelinated. During postnatal development the myelination pattern precedes the GAP-43 pattern, a sequence that is consistent with a role of myelin and the associated neurite growth inhibitors in modifying GAP-43 expression. Our results support the hypothesis that myelin-associated neurite growth inhibitors are involved in regulating the stability of neural connections. © Wiley-Liss, Inc.     

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).     

Appearance of myelin proteins during development in the chick central nervous system

The time course of the appearance of myelin-specific markers was studied in the developing chick central nervous system (CNS). Chick CNS tissue was studied for the presence of both proteolipid and myelin basic protein by electroblotting and for 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) by enzyme assay. Four regions of chick spinal cord (cervical, brachial, thoracic and lumbar), brain stem, cerebellum, optic nerve and cortex were studied. In general, myelin basic protein appeared approximately 1 day earlier than proteolipid. In spinal cord and brain stem, myelin basic protein appeared at 13 days incubation. In cerebellum and optic nerve, it appeared at 17 days incubation and in cortex at hatching. CNPase activity increased in most CNS regions between 16 days incubation and hatching. These results suggest that myelination occurs earlier in the chick than in the rat and that it occurs over a shorter time period.     

Rumpshaker: an X-linked mutation causing hypomyelination: developmental differences in myelination and glial cells between the optic nerve and spinal cord.

The X-linked mutation rumpshaker (rsh), which is probably an allele of jimpy (jp), causes hypomyelination in the CNS of mice. This study examines the developmental expression of the morphology, glial cells, and immunostaining of myelin proteins in the optic nerve and spinal cord. The optic nerve contains varying numbers of amyelinated and myelinated fibres. The majority of such sheaths are of normal thickness whereas in the spinal cord most axons are associated with a disproportionately thin sheath which changes little in thickness during development. In the optic nerve glial cell numbers are elevated in mutants during early and peak myelination but then fall slightly below normal in adults. In contrast, the number of glial cells is consistently elevated after 16 days of age in the spinal cord. The majority of the alterations to total glial cells are due to corresponding changes in the oligodendrocyte population. Immunostaining intensity is somewhat reduced for myelin basic protein (MBP) and the C-terminal common to proteolipid protein (PLP) and DM-20 and profoundly decreased for the PLP-specific peptide. Glial fibrillary acidic protein (GFAP) is increased in rsh. It is probable that some of the variation in myelination between optic nerve and cord in rsh is related to the difference in axon diameter in the two locations, as there are adequate numbers of oligodendrocytes at the time of myelination. However, the effect of the mutation on cell development in the brain and the spinal cord may be different. The immunostaining indicates a marked deficiency in PLP in myelin but suggests that DM-20 levels may be relatively normal. rsh shows several major differences from jp and other X-linked myelin mutants, particularly in relation to oligodendrocyte numbers, and will be useful to elucidate the role of the PLP gene in influencing oligodendrocyte differentiation and survival.     

Dorsally-derived oligodendrocytes in the spinal cord contribute to axonal myelination during development and remyelination following focal demyelination

In the developing spinal cord, the majority of oligodendrocytes are derived from the ventral ventricular zone. Several recent studies suggested that a small number of oligodendrocyte precursor cells (OPCs) can also be generated in the dorsal spinal cord. However, it is not clear whether these dorsal oligodendrocyte precursor cells participate in myelination and remyelination. To investigate the fate and potential function of these dorsally-derived oligodendrocytes (dOLs) in the adult spinal cord, Cre-lox genetic fate mapping in transgenic mice was employed. We used the Pax3(Cre) knock-in mouse to drive Cre expression in the entire dorsal epithelium and the Rosa26-lacZ or Z/EG reporter line to trace their spatial distribution and population dynamics in the spinal cord. The dorsal OPCs generated from the Pax3-expressing domains migrate into all regions of spinal cord and subsequently undergo terminal differentiation and axonal myelination. In response to a focal demyelination injury, a large number of newly differentiated oligodendrocytes originated from dOLs, suggesting that dOLs may provide an important source of OPCs for axonal remyelination in multiple sclerosis or spinal cord injury.     

Increased Expression of the Growth-associated Protein GAP-43 in the Myelin-free Rat Spinal Cord

In the normal central nervous system (CNS) the regional expression of the growth-associated protein GAP43 is complementary to the pattern of myelination. This has led us to suspect that myelin-associated neurite growth inhibitors might contribute to the suppression of GAP-43 expression by suppressing sprouting and plastic changes of synaptic terminals in myelinated CNS areas. In order to study the relationship between myelination and GAP-43 expression more directly, we experimentally prevented myelination of the lumbar spinal cord of rats through neonatal X-irradiation. The GAP-43 protein expression in myelin-free spinal cords was analysed by immunohistochemistry and immunoblotting and compared to age-matched normal spinal cords. We found that in the absence of myelination, GAP-43 expression is strongly increased in the spinal cord of 4-week-old rats. GAP-43 was most strongly expressed in descending fibre tracts, where expression in the normal spinal cord is very low. In grey matter the typical regional pattern of GAP-43 expression did not develop; instead GAP-43 expression was high in all regions of the spinal cord. The overall pattern of myelination and GAP-43 expression in the myelin-free cord resembled that of early postnatal stages. This indicates that the regional down-regulation of GAP-43 expression during normal postnatal development did not occur in the myelin-free areas. Our results support the hypothesis that neurite growth inhibitors from oligodendrocytes and CNS myelin suppress sprouting and plastic changes of synaptic terminals in the normal CNS and are thereby involved in regulating the stability of neural connections.     

GlialCAM, an immunoglobulin-like cell adhesion molecule is expressed in glial cells of the central nervous system

Using structure based genome mining targeting vascular endothelial and platelet derived growth factor immunoglobulin (Ig) like folds, we have identified a sequence corresponding to a single transmembrane protein with two Ig domains, which we cloned from a human brain cDNA library. The cDNA is identical to hepatocyte cell adhesion molecule (hepaCAM), which was originally described as a tumor suppressor gene in liver. Here, we show that the protein is predominantly expressed in the mouse and human nervous system. In liver, the expression is very low in humans, and is not detected in mice. To identify the central nervous system (CNS) regions and cell types expressing the protein, we performed a LacZ reporter gene assay on heterozygous mice in which one copy of the gene encoding the novel protein had been replaced with beta-galactosidase. beta-galactosidase expression was prominent in white matter tracts of the CNS. Furthermore, expression was detected in ependymal cells of the brain ventricular zones and the central canal of the spinal cord. Double labeling experiments showed expression mainly in CNPase positive oligodendrocytes (OL). Since the protein is predominantly expressed in the CNS glial cells, we named the molecule glial cell adhesion molecule (GlialCAM). A potential role for GlialCAM in myelination was supported by its up-regulation during postnatal mouse brain development, where it was concomitantly expressed with myelin basic protein (MBP). In addition, in vitro, GlialCAM was observed in various developmental stages of OL and in astrocytes in processes and at cell contact sites. In A2B5 positive OL, GlialCAM colocalizes with GAP43 in OL growth cone like structures. Overall, the data presented here indicate a potential function for GlialCAM in glial cell biology.     

A mutation in the canine gene encoding folliculin‐interacting protein 2 (FNIP2) associated with a unique disruption in spinal cord myelination

Novel mutations in myelin and myelin‐associated genes have provided important information on oligodendrocytes and myelin and the effects of their disruption on the normal developmental process of myelination of the central nervous system (CNS). We report here a mutation in the folliculin‐interacting protein 2 (FNIP2) gene in the Weimaraner dog that results in hypomyelination of the brain and a tract‐specific myelin defect in the spinal cord. This myelination disruption results in a notable tremor syndrome from which affected dogs recover with time. In the peripheral tracts of the lateral and ventral columns of the spinal cord, there is a lack of mature oligodendrocytes. A genome‐wide association study of DNA from three groups of dogs mapped the gene to canine chromosome 15. Sequencing of all the genes in the candidate region identified a frameshift mutation in the FNIP2 gene that segregated with the phenotype. While the functional role of FNIP2 is not known, our data would suggest that production of truncated protein results in a delay or failure of maturation of a subpopulation of oligodendrocytes. GLIA 2014;62:39–51     

Oligodendrocyte proliferation and CNS myelination in cultures containing dissociated embryonic neuroglia and dorsal root ganglion neurons

In this report we have described several aspects of glial development in cultures containing dissociated DRG neurons and glial cells obtained from dissociated spinal cord at least a week prior to the onset of in vivo myelination. With time in culture the dissociated neurons and glia interact and become organized into 3-dimensional structures possessing many features characteristic of developing CNS in vivo. We have presented evidence that some of the glial cells proliferate and differentiate into galactocerebroside positive (GC+) cells and that some produce myelin sheaths. Thymidine was incorporated into precursors of GC+ cells, but not into cells which were already GC+. Nearly all the astrocytes in areas where neurons were present participated in the formation of large fascicles, and it was within such fascicles that most myelin was formed, suggesting a possible role for astrocytes in creating a favorable microenvironment for myelination. Both the formation of myelin sheaths and the morphological maturity of the oligodendrocytes within the fascicles indicated that oligodendrocyte differentiation proceeded practically to completion in these cultures. In conclusion we believe that the culture system herein described provides an excellent model for in vitro studies of CNS development, while retaining some of the advantages of dissociated cell cultures as well as the possibility of separating and re-uniting the various cell types of interest.     

PAK1 Positively Regulates Oligodendrocyte Morphology and Myelination

The actin cytoskeleton is crucial for oligodendrocyte differentiation and myelination. Here we show that p21-activated kinase 1 (PAK1), a well-known actin regulator, promotes oligodendrocyte morphologic change and myelin production in the CNS. A combination of in vitro and in vivo models demonstrated that PAK1 is expressed throughout the oligodendrocyte lineage with highest expression in differentiated oligodendrocytes. Inhibiting PAK1 early in oligodendrocyte development decreased oligodendrocyte morphologic complexity and altered F-actin spreading at the tips of oligodendrocyte progenitor cell processes. Constitutively activating AKT in oligodendrocytes in male and female mice, which leads to excessive myelin wrapping, increased PAK1 expression, suggesting an impact of PAK1 during active myelin wrapping. Furthermore, constitutively activating PAK1 in oligodendrocytes in zebrafish led to an increase in myelin internode length while inhibiting PAK1 during active myelination decreased internode length. As myelin parameters influence conduction velocity, these data suggest that PAK1 may influence communication within the CNS. These data support a model in which PAK1 is a positive regulator of CNS myelination.SIGNIFICANCE STATEMENT Myelin is a critical component of the CNS that provides metabolic support to neurons and also facilitates communication between cells in the CNS. Recent data demonstrate that actin dynamics drives myelin wrapping, but how actin is regulated during myelin wrapping is unknown. The authors investigate the role of the cytoskeletal modulator PAK1 during differentiation and myelination by oligodendrocytes, the myelinating cells of the CNS. They demonstrate that PAK1 promotes oligodendrocyte differentiation and myelination by modulating the cytoskeleton and thereby internode length, thus playing a critical role in the function of the CNS.     

Dysmyelination and reduced myelin basic protein gene expression by oligodendrocytes of SHP-1-deficient mice

We have shown previously that myelin-forming oligodendrocytes express the protein tyrosine phosphatase SHP-1 and that myelin formation was decreased in SHP-1-deficient motheaten mice compared to that in normal littermates. These studies suggested a potential importance for SHP-1 in oligodendrocyte and myelin development. To address further this possibility, we analyzed myelin formation by microscopy and myelin basic protein (MBP) gene expression in motheaten mice at ages when myelination occurs in the developing central nervous system (CNS). Furthermore, we correlate these findings with MBP gene expression in oligodendrocytes grown in vitro. We have found that CNS myelination was significantly reduced in SHP-1-deficient mice relative to their normal littermates at multiple times during the active period of myelination. Under electron microscopy, greater numbers of axons in spinal cords of motheaten mice were either unmyelinated or had thinner myelin sheathes compared to those in matched areas of normal littermates. Accordingly, MBP protein and mRNA levels were reduced in SHP-1-deficient mice compared to that in the CNS of normal littermates. In vitro, O1(+) oligodendrocytes from motheaten mice expressed much less MBP than O1(+) oligodendrocytes of normal littermates indicating an alteration in oligodendrocyte differentiation. The latter correlated with reduced MBP mRNA relative to cerebroside galactosyl transferase (CGT) gene mRNA in SHP-1-deficient oligodendrocytes in purified cultures. We propose that SHP-1 is a critical regulator of developmental signals leading to terminal differentiation and myelin sheath formation by oligodendrocytes.     

Myelination ofjp,jp andqk axons by normal glia in vitro: Ultrastructural and autoradiographic evidence

Normal optic nerve glia were ‘injected’ into hypomyelinated mutantjp,jp^msd, andqk cerebellum by co-culturing explants in direct physical contact. Quantitative light microscopic studies demonstrated that such glial injection significantly increased the number of myelin profiles counted in cultures, suggesting that axons in all 3 mutants can accept myelination from competent glia when they are made available. In each mutant, the observed increase in myelination was independent of the ages of donor optic nerves and recipient cultures, but absolutely required positioning of the optic nerve so that direct contact occurred with the mutant cerebellar explants. The additional myelin found near the zone of fusion with the optic nerve morphologically resembled normal, not mutant myelin. Autoradiographs made after [³H]thymidine-labeled normal optic nerve was injected intojp^msd cultures showed that labeled cells had colonized the nearby mutant tissue. Labeled cells identified as oligodendrocytes by ultrastructural criteria were found adjacent to myelin segments near the fusion zone, but direct continuity between processes of these oligodendrocytes and myelin sheaths was not demonstrated. The astrocytes and phagocytic cells which were also labeled had no obvious relationship to myelinated axons. These results provide experimental evidence that the primary abnormalities produced by the three mutationsjp,jp^msd, andqk are inherent in their glial cells, probably although not definitely in the oligodendrocytes.     

Growth factor control of CNS myelination.

The molecular signals required for initiating myelination and maintenance of the myelin internode are not known. Several growth factor families have been implicated in promoting oligodendrocyte survival or differentiation and may have consequences on formation of myelin. We developed a reliable assay for detecting ensheathment of neurites by oligodendrocytes in spinal cord explants. This system was used to assay the effect of selected growth factors on myelin internode formation. We examined the influence on myelination of the polypeptide growth factors neuregulin (NRG), platelet-derived growth factor (PDGF), leukemia inhibitory factor (LIF), and the thyroid hormone T(3). We found that NRG, PDGF, and T(3) treatments enhanced myelination while LIF treatment inhibited it. We furthermore found that the most potent combination of factors to enhance myelination was NRG and T(3). Our results demonstrate that the role of growth factors on CNS myelination can be reliably studied in a controlled in vitro environment and that the impact of individual or combinations of growth factors on myelination cannot be predicted by their known effects on oligodendrocyte survival, proliferation, or differentiation.     

Demyelination in the central nervous system mediated by an anti-oligodendrocyte antibody

The factors responsible for the major demyelinating disease of the central nervous system (CNS), multiple sclerosis, are poorly defined. Although T-cell-mediated immune responses play a pivotal role in establishing the inflammatory response, humoral factors also may be critical in disease progress. We have isolated a mouse monoclonal antibody (mAb 2B10) that recognizes a cell-surface molecule expressed exclusively by rat oligodendrocytes, the cells responsible for the formation and maintenance of CNS myelin. In cultures of neonatal rat spinal cord, mAb 2B10 specifically mediated oligodendrocyte cell death in the absence of complement. In the current study, mAb 2B10–producing hybridoma cells were implanted into adult rat brain ventricles, and the effect of mAb 2B10 on CNS cytoarchitecture was examined. In the optic nerves of mAb 2B10–treated animals, there was significant focal myelin degeneration near the optic chiasm. Axons in the myelin degenerate regions were largely healthy. There was no significant infiltration of hematopoietic-derived cells into the affected regions, but microglia were activated focally and phagocytosed the collapsed myelin. This study demonstrates that an antibody directed against myelin-forming cells induces CNS demyelination and supports the hypothesis that autoantibodies may play a role in CNS demyelinating diseases. J. Neurosci. Res. 54:158–168, 1998. © 1998 Wiley-Liss, Inc.     

Oligodendrocyte responses to buprenorphine uncover novel and opposing roles of μ-opioid- and nociceptin/orphanin FQ receptors in cell development: implications for drug addiction treatment during pregnancy

Although the classical function of myelin is the facilitation of saltatory conduction, this membrane and the oligodendrocytes, the cells that make myelin in the central nervous system (CNS), are now recognized as important regulators of plasticity and remodeling in the developing brain. As such, oligodendrocyte maturation and myelination are among the most vulnerable processes along CNS development. We have shown previously that rat brain myelination is significantly altered by buprenorphine, an opioid analogue currently used in clinical trials for managing pregnant opioid addicts. Perinatal exposure to low levels of this drug induced accelerated and increased expression of myelin basic proteins (MBPs), cellular and myelin components that are markers of mature oligodendrocytes. In contrast, supra-therapeutic drug doses delayed MBP brain expression and resulted in a decreased number of myelinated axons. We have now found that this biphasic-dose response to buprenorphine can be attributed to the participation of both the μ-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOP receptor) in the oligodendrocytes. This is particularly intriguing because the NOP receptor/nociceptin system has been primarily linked to behavior and pain regulation, but a role in CNS development or myelination has not been described before. Our findings suggest that balance between signaling mediated by (a) MOR activation and (b) a novel, yet unidentified pathway that includes the NOP receptor, plays a crucial role in the timing of oligodendrocyte maturation and myelin synthesis. Moreover, exposure to opioids could disrupt the normal interplay between these two systems altering the developmental pattern of brain myelination.     

The transition zone of the central nervous system-peripheral nervous system of the adult rat; ultrastructural and immunocytochemical studies: a new function of the astroglia?]

At the transition between central nervous system (CNS) and peripheral nervous system (PNS), the CNS compartment forms cone-shaped incursions into the peripheral part of the dorsal root. The ultrastructural study of the CNS-PNS transitional zone shows that this region is particularly rich in astrocyte processes. In an attempt to investigate the possible role of the CNS-PNS interface astrocytes in myelin formation, a photonic microscopy immunocytochemical study has been done with anti-GFAP and anti-MBP sera. The CNS glial expansion shows an important GFAP immunoreactivity with intimate association between astrocyte processes and myelinated axons. This may indicate that the transitional myelin originates from astrocytes. The same region is also MBP-positive. Two explanations are considered: some astrocytes form transitional myelin sheathes and express MBP epitopes, or oligodendrocytes, with cell bodies distant from the CNS-PNS interface, send myelinating cytoplasmic expansions which are not shown by the techniques we used.     

Development of transferrin-positive oligodendrocytes in the rat central nervous system

Transferrin is the second most abundant plasma protein and functions to transport iron. It is an essential constituent in culture media for virtually all cells. In a recent study, we reported that transferrin (Tf) is specifically located in oligodendrocytes in the rat nervous system. This investigation examines immunohistochemically the development of Tf in the cerebral cortex, corpus striatum, and spinal cord. Tf is first seen in oligodendrocytes in the spinal cord white matter at 5 days of age. The immunoreactivity is confined to the white matter in the periphery of the spinal cord between 5 and 8 days of age. By 10-12 days of age, the number of immunoreactive oligodendrocytes in the spinal cord white matter increases considerably, corresponding to the onset of myelination. Tf-positive oligodendrocytes are first found in the gray matter at 15 days of age. By 30 days of age, the number and distribution of Tf-positive oligodendrocytes in both the brain and spinal cord have reached the adult pattern. The results of this study demonstrate a spatial and temporal association between Tf development and myelinogenesis. This suggests that part of the process of differentiation of oligodendrocytes includes the accumulation of Tf, perhaps in order to support the metabolic demands associated with the production and maintenance of myelin.     

Schwann cells and oligodendrocytes read distinct signals in establishing myelin sheath thickness

Schwann cells and oligodendrocytes produce myelin sheaths of widely varying sizes. How these cells determine the size of myelin sheath for a particular axon is incompletely understood. Axonal diameter has long been suspected to be a signal in this process. We have analyzed myelin sheath thickness in L5 lumbar root and spinal cord white matter of a series of mouse mutants with diminished axonal calibers resulting from a deficiency of neurofilaments (NFs). In the PNS, average axonal diameters were reduced by 20-37% in the NF mutants. Remarkably, the average myelin sheath thickness remained unchanged from control values, and regression analysis showed sheaths abnormally thick for a given size of axon. These data show that a genetically induced reduction in axonal caliber does not cause a reduction in myelin sheath thickness in PNS and indicate that Schwann cells read some intrinsic signal on axons that can be uncoupled from axonal diameter. Interestingly, myelin sheaths in the spinal cord of these animals were not abnormally thick, arguing that axonal diameter may contribute directly to the regulation of myelination in the CNS and that oligodendrocytes and Schwann cells use different cues to set myelin sheath thickness.     

Studies on the control of myelinogenesis. 3. Signalling of oligodendrocyte myelination by regenerating peripheral axons

Continuing from earlier work which demonstrated the peripheral axonal regulation of Schwann cell myelination, this study has investigated the possibility that a peripheral axon can stimulate oligodendrocyte myelination. To test this hypothesis, regenerating PNS axons were allowed to interact with uncommitted oligodendrocytes by transecting a rat peroneal nerve and inserting a segment of the autologous optic nerve between the cut ends. Grafts were maintained for 4–28 weeks and then examined by light and electron microscopy. A few regenerating peripheral myelinated nerve fibers penetrated the optic nerve graft. Some axons penetrated the outer margin of the graft, were myelinated by Schwann cells, and surrounded by astrocyte processes bordered by basal lamina. More centrally in the optic nerve graft, regenerating peripheral axons displayed myelin of CNS type. The outer myelin lamella abutted directly on the plasmalemma surface of surrounding astrocytic processes and was expanded focally to form a glial tongue. These observations demonstrate the experimental induction of central myelination by regenerating peripheral axons and suggest the existence of a common neuronal mechanism to stimulate myelin formation by both the Schwann cell and the oligodendrocyte.