The control of reactive oxygen species production by SHP‐1 in oligodendrocytes

We have previously described reduced myelination and corresponding myelin basic protein (MBP) expression in the central nervous system of Src homology 2 domain‐containing protein tyrosine phosphatase 1 (SHP‐1) deficient motheaten (me/me) mice compared with normal littermate controls. Deficiency in myelin and MBP expression in both brains and spinal cords of motheaten mice correlated with reduced MBP mRNA expression levels in vivo and in purified oligodendrocytes in vitro. Therefore, SHP‐1 activity seems to be a critical regulator of oligodendrocyte gene expression and function. Consistent with this role, this study demonstrates that oligodendrocytes of motheaten mice and SHP‐1‐depleted N20.1 cells produce higher levels of reactive oxygen species (ROS) and exhibit corresponding markers of increased oxidative stress. In agreement with these findings, we demonstrate that increased production of ROS coincides with ROS‐induced signaling pathways known to affect myelin gene expression in oligodendrocytes. Antioxidant treatment of SHP‐1‐deficient oligodendrocytes reversed the pathological changes in these cells, with increased myelin protein gene expression and decreased expression of nuclear factor (erythroid‐2)‐related factor 2 (Nrf2) responsive gene, heme oxygenase‐1 (HO‐1). Furthermore, we demonstrate that SHP‐1 is expressed in human white matter oligodendrocytes, and there is a subset of multiple sclerosis subjects that demonstrate a deficiency of SHP‐1 in normal‐appearing white matter. These studies reveal critical pathways controlled by SHP‐1 in oligodendrocytes that relate to susceptibility of SHP‐1‐deficient mice to both developmental defects in myelination and to inflammatory demyelinating diseases. GLIA 2015;63:1753–1771     

The thrombin receptor is a critical extracellular switch controlling myelination

Hemorrhagic white matter injuries in the perinatal period are a growing cause of cerebral palsy yet no neuroprotective strategies exist to prevent the devastating motor and cognitive deficits that ensue. We demonstrate that the thrombin receptor (protease‐activated receptor 1, PAR1) exhibits peak expression levels in the spinal cord at term and is a critical regulator of the myelination continuum from initiation to the final levels achieved. Specifically, PAR1 gene deletion resulted in earlier onset of spinal cord myelination, including substantially more Olig2‐positive oligodendrocytes, more myelinated axons, and higher proteolipid protein (PLP) levels at birth. In vitro, the highest levels of PAR1 were observed in oligodendrocyte progenitor cells (OPCs), being reduced with differentiation. In parallel, the expression of PLP and myelin basic protein (MBP), in addition to Olig2, were all significantly higher in cultures of PAR1?/? oligodendroglia. Moreover, application of a small molecule inhibitor of PAR1 (SCH79797) to OPCs in vitro increased PLP and MBP expression. Enhancements in myelination associated with PAR1 genetic deletion were also observed in adulthood as evidenced by higher amounts of MBP and thickened myelin sheaths across large, medium, and small diameter axons. Enriched spinal cord myelination in PAR1?/? mice was coupled to increases in extracellular‐signal‐regulated kinase 1/2 and AKT signaling developmentally. Nocturnal ambulation and rearing activity were also elevated in PAR1?/? mice. These studies identify the thrombin receptor as a powerful extracellular regulatory switch that could be readily targeted to improve myelin production in the face of white matter injury and disease. GLIA 2015;63:846–859     

In Vivo Immunological Suppression of Spinal Cord Myelin Development

The onset of myelination in the embryonic chick spinal cord begins on embryonic day (E) 12 or E13 of the 21 day in ovo developmental period. This event coincides with a loss of functional axonal regeneration following complete transection of the thoracic spinal cord. In this study, we have characterised an immunological method for delaying the developmental onset of myelination in vivo until later stages of development (developmental myelin-suppression). A single injection of heterologous or homologous serum complement proteins plus myelin-specific, complement-binding antibodies into the spinal cord prior to E13 delayed the onset of myelination until E17. The state of spinal cord myelin was assessed with immunohistochemical, histological and ultrastructural techniques. Northern blot analysis indicated that myelin basic protein mRNA was not downregulated in myelin-suppressed spinal cords, which suggests that oligodendrocytes survived developmental myelin-suppression. Glial fibrillary acidic protein immunostaining of normal and treated tissue indicated that myelin-suppression did not alter the resident astrocyte population of the spinal cord or elicit astrogliosis. Immunostaining with microtubule-associated protein-2 and thionine staining of normal and myelin-suppressed tissue further indicated that the neuronal architecture was unaffected by the immunological protocol.     

Human oligodendrocytes in remyelination research

Studies on myelination and oligodendrocyte development are inevitably linked with demyelinating conditions such as multiple sclerosis (MS), leukodystrophies or spinal cord injury (SCI). Chronic loss of myelin, subsequently leading to neurodegeneration, is the ultimate cause of severe and permanent disability. Thus, fast restoration of myelin (remyelination) is essential for circumventing demyelination‐caused pathologies. Implantation of exogenous remyelinating cells has been considered as a potential remyelination strategy. Researchers have examined a variety of cell types endowed with myelin‐forming capacity (oligodendrocytes, Schwann cells, olfactory ensheathing cells etc.) in vitro and in vivo for their potential application as myelin restoring cell grafts. This review gives a summary of studies on the generation and testing of pure suspensions of human oligodendrocytes as a clinically relevant, efficient cellular tool for treating myelin pathology. We start with a brief overview of the current knowledge on the development of human oligodendrocytes from the late stages of embryogenesis up to the early postnatal stage. Insight in the specific extrinsic and intrinsic factors regulating normal oligodendrogenesis is crucial in order to achieve and maintain a sufficient population of engraftable functional oligodendrocytes in vitro. We discuss potential sources of human oligodendrocytes, including novel oligodendrocyte generation strategies employing induced pluripotent stem cells (iPSCs) and direct conversion technology. Finally, we provide a systematic overview of (the outcome of) experimental studies, in which human oligodendrocytes were tested for their (re)myelination capacity and efficiency. GLIA 2015;63:513–530     

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.     

Dysmyelination in Chow Chow dogs: Further studies in older dogs

Summary The ultrastructure of myelin deficiency in Chow Chow dogs was studied in the spinal cord of a 15-month-old and a 3-year-old animal. It was found that myelination progresses with age in these dogs but is still deficient at the age of 3 years. The findings included axons with thin or uncompacted myelin sheaths, separated from each other by massive astrocytosis, and bizarre myelin formations. Normal numbers of morphologically normal oligodendrocytes were present in the myelin-deficient areas. The disease in these Chow Chow dogs consists of a strongly retarded myelination which is possibly due to a dysfunction or delay in glial maturation.Supported by the Schweizer Nationalfonds grant no. 3.805.79     

Dynamic expression of Cx47 in mouse brain development and in the cuprizone model of myelin plasticity

The study shows the dynamic expression of connexin47 (Cx47) in oligodendrocytes and myelin of mice, either in myelinogenesis occurring in early development or in an experimental model of new‐myelinogenesis of adult mice. Cx47 first appeared in the embryonic mouse brain at E10.5 successively the expression increased, principally in regions populated by developing oligodendrocytes. The expression declined postnatally toward adulthood and immunoreactivity was restricted to a few specific areas, such as the corpus callosum, the striatum, the cerebellum, and the spinal cord. Since the expression of Cx47 in developing oligodendrocytes preceded those of Cx32 and Cx29, a role of Cx47 in myelinogenesis was postulated. This hypothesis was tested in a model of re‐myelination, which principally involved the corpus callosum, occurring in adult mice by treatment with cuprizone. Cx47 was upregulated during demyelination and recovered during the remyelination phase. During demyelination, Cx47 was first over‐expressed in the corpus callosum and later, when the myelin virtually disappeared in the injured areas, Cx47 was expressed in astrocytes located inside and closely around the demyelinated areas. The remyelination of injured areas occurred after stopping the administration of cuprizone and continued to complete recovery. In this period the expression of Cx47 shifted from astrocytes to newly‐formed myelin. Thus, Cx47 exhibits in this model a transient and de novo expression in astrocytes with a topographic segregation in the injured areas, only when oligodendrocytes and the myelin were most severely affected. Taken as a whole the evidence suggests that Cx47 play a key role in myelination. © 2010 Wiley‐Liss, Inc.     

Oligodendrocyte gene expression in the human fetal spinal cord during the second trimester of gestation

A comprehensive evaluation of myelination during normal human development is essential to understand the pathology of congenital diseases of white matter. The present study establishes quantitative values for normal oligodendrocyte-specific gene expression during the early stages of myelination in the human fetal spinal cord. Complementary techniques of Northern and immunoblotting were used to determine relative amounts of oligodendrocyte-specific mRNAs and proteins between 12 and 24 gestational weeks. Values were determined for myelin basic protein, 2′,3′-cyclic nucleotide 3′-phosphodiesterase, and proteolipid protein. The relative amount of myelin-associated glycoprotein mRNA was also estimated. To compare gene expression between glial cell types, the relative amounts of mRNA and protein were determined for glial fibrillary acidic protein (GFAP), a cell-type specific marker for astrocytes. All oligodendrocyte-specific genes expressed similar developmental kinetics. Between 12 and 15 gestational weeks, less than a five-fold increase was detected in the expression of these genes and their protein products. Between 15 and 22 gestational weeks, the relative amounts of mRNA and protein for the myelin genes increased more than 80-fold. The kinetics of GFAP expression were similar to those of the myelin-associated genes. Absolute values for the increase in mass of the human fetal spinal cord were also obtained. These results provide data that may aid in the neuropathologic assessment and characterization of myelin disorders in the preterm, neonatal, and pediatric spinal cord. J. Neurosci. Res. 47:332–340, 1997. © 1997 Wiley-Liss, Inc.     

Identification of Tmem10/Opalin as an oligodendrocyte enriched gene using expression profiling combined with genetic cell ablation

Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, which allows efficient and rapid propagation of action potentials. However, little is known about the molecular mechanisms operating at the onset of myelination and during maintenance of the myelin sheath in the adult. Here we use a genetic cell ablation approach combined with Affymetrix GeneChip microarrays to identify a number of oligodendrocyte-enriched genes that may play a key role in myelination. One of the "oligogenes" we cloned using this approach is Tmem10/Opalin, which encodes for a novel transmembrane glycoprotein. In situ hybridization and RT-PCR analysis revealed that Tmem10 is selectively expressed by oligodendrocytes and that its expression is induced during their differentiation. Developmental immunofluorescence analysis demonstrated that Tmem10 starts to be expressed in the white matter tracks of the cerebellum and the corpus callosum at the onset of myelination after the appearance of other myelin genes such as MBP. In contrast to the spinal cord and brain, Tmem10 was not detected in myelinating Schwann cells, indicating that it is a CNS-specific myelin protein. In mature oligodendrocytes, Tmem10 was present at the cell soma and processes, as well as along myelinated internodes, where it was occasionally concentrated at the paranodes. In myelinating spinal cord cultures, Tmem10 was detected in MBP-positive cellular processes that were aligned with underlying axons before myelination commenced. These results suggest a possible role of Tmem10 in oligodendrocyte differentiation and CNS myelination.     

PLP overexpression perturbs myelin protein composition and myelination in a mouse model of Pelizaeus-Merzbacher disease

Duplication of PLP1, an X‐linked gene encoding the major myelin membrane protein of the human CNS, is the most frequent cause of Pelizaeus‐Merzbacher disease (PMD). Transgenic mice with extra copies of the wild type Plp1 gene, a valid model of PMD, also develop a dysmyelinating phenotype dependant on gene dosage. In this study we have examined the effect of increasing Plp1 gene dosage on levels of PLP/DM20 and on other representative myelin proteins. In cultured oligodendrocytes and early myelinating oligodendrocytes in vivo, increased gene dosage leads to elevated levels of PLP/DM20 in the cell body. During myelination, small increases in Plp1 gene dosage (mice hemizygous for the transgene) elevate the level of PLP/DM20 in oligodendrocyte soma but cause only minimal and transient effects on the protein composition and structure of myelin suggesting that cells can regulate the incorporation of proteins into myelin. However, larger increases in dosage (mice homozygous for the transgene) are not well tolerated, leading to hypomyelination and alteration in the cellular distribution of PLP/DM20. A disproportionate amount of PLP/DM20 is retained in the cell soma, probably in autophagic vacuoles and lysosomes whereas the level in myelin is reduced. Increased Plp1 gene dosage affects other myelin proteins, particularly MBP, which is transitorily reduced in hemizygous mice but consistently and markedly lower in homozygotes in both myelin and naïve or early myelinating oligodendrocytes. Whether the reduced MBP is implicated in the pathogenesis of dysmyelination is yet to be established. © 2006 Wiley‐Liss, Inc.     

Protease activated receptor 2 controls myelin development,resiliency and repair

Oligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated receptor 2 (PAR2), a unique protease activated G protein‐coupled receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss‐of‐function were accompanied by increased numbers of Olig2‐ and CC1‐positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.     

Gene expression and oligodendrocyte development in the myelin deficient rat

The proteolipid proteins play a major role in the structure of the CNS myelin sheath, but they have also been implicated in the oligodendrocyte development leading to myelination. Mutations in the PLP gene result in severe dysmyelination and a paucity of mature oligodendrocytes. The myelin deficient (md) rat, carrying a Thr75? Pro substitution present in both isoforms of proteolipid protein (PLP and DM20), is the most severely affected of the PLP mutants described to date. The expression of myelin associated genes was quantitated to determine the effect of the mutation on oligodendrocyte development in vivo. At 5 days postnatal, gene expression in the and rat approximated that in age-matched control rats, but as they matured, there was a progressive inhibition of gene expression in the and rats. The genes expressed late in the myelination program (PLP and MBP) were affected more dramatically than those expressed earlier in oligodendrocyte development (CNP and GPDH). The results indicate that the later stages of oligodendrocyte maturation and myelin elaboration are inhibited. © 1995 Wiley-Liss, Inc.     

The function of contactin‐2/TAG‐1 in oligodendrocytes in health and demyelinating pathology

The oligodendrocyte maturation process and the transition from the pre‐myelinating to the myelinating state are extremely important during development and in pathology. In the present study, we have investigated the role of the cell adhesion molecule CNTN2/TAG‐1 on oligodendrocyte proliferation, differentiation, myelination, and function during development and under pathological conditions. With the combination of in vivo, in vitro, ultrastructural, and electrophysiological methods, we have mapped the expression of CNTN2 protein in the oligodendrocyte lineage during the different stages of myelination and its involvement on oligodendrocyte maturation, branching, myelin‐gene expression, myelination, and axonal function. The cuprizone model of central nervous system demyelination was further used to assess CNTN2 in pathology. During development, CNTN2 can transiently affect the expression levels of myelin and myelin‐regulating genes, while its absence results in reduced oligodendrocyte branching, hypomyelination of fiber tracts and impaired axonal conduction. In pathology, CNTN2 absence does not affect the extent of de‐ and remyelination. However during remyelination, a novel, CNTN2‐independent mechanism is revealed that is able to recluster voltage gated potassium channels (VGKCs) resulting in the improvement of fiber conduction.     

Low density lipoprotein receptor-related protein-2/megalin is expressed in oligodendrocytes in the mouse spinal cord white matter

Lipoprotein receptor-related protein-2 (LRP2)/megalin is a member of the low density lipoprotein receptor (LDLR) family, and is essential in absorptive epithelia for endocytosis of lipoproteins, low molecular weight proteins, cholesterol and vitamins, as well as in cellular signaling. Previous studies have shown megalin expression in ependymal cells and choroid plexus. We have investigated megalin expression in the spinal cord of postnatal mice with immunohistochemistry and immunoblot. Antibodies recognizing either the cytoplasmic tail (MM6) or the extracellular domain (E11) of megalin labeled oligodendrocytes in the spinal cord white matter, in parallel with myelination. MM6 antibodies, predominantly labeled the nuclei, whereas E11 antibodies labeled the cytoplasm of these cells. MM6 antibodies labeled also nuclei of oligodendrocytes cultured from embryonic mouse spinal cord. Immunoblots of spinal cord showed intact megalin, as well as its carboxyterminal fragment, the part remaining after shedding of the extracellular domain of megalin. Megalin-immunoreactive oligodendrocytes also expressed presenilin 1, an enzyme responsible for gamma-secretase mediated endodomain cleavage. These findings show that spinal cord oligodendrocytes are phenotypically different from those in the brain, and indicate that megalin translocates signals from the cell membrane to the nucleus of oligodendrocytes during the formation and maintenance of myelin of long spinal cord pathways.