Oligodendrogenesis is differentially regulated in gray and white matter of jimpy mice

The factors that regulate oligodendrogenesis have been studied extensively in optic nerve, where oligodendrocyte production and myelination quickly follow colonization of the nerve by progenitor cells. In contrast, oligodendrocyte production in the cerebral cortex begins approximately 1 week after progenitor cell colonization and continues for 3-4 weeks. This and other observations raise the possibility that oligodendrogenesis is regulated by different mechanisms in white and gray matter. The present study examined oligodendrocyte production in the developing cerebral cortex of jimpy (jp) and jimpy(msd) (msd) mice, which exhibit hypomyelination and oligodendrocyte death due to mutations in and toxic accumulations of proteolipid protein, the major structural protein of CNS myelin. Proliferation of oligodendrocyte progenitors and production of myelinating oligodendrocytes was reduced in jp cerebral cortex when compared to wild-type (wt) and msd mice. The incidence of oligodendrocyte cell death was similar in jp and msd cortex, but total dying oligodendrocytes were greater in msd. We confirm previous reports of increased oligodendrocyte production in white matter of both jp and msd mice. The jp mutation, therefore, reduces oligodendrocyte production in cerebral cortex but not in white matter. These data provide additional evidence that oligodendrogenesis is differentially regulated in white matter and gray matter and implicate PLP/DM20 as a modulator of these differences.     

GFAP-positive and myelin marker-positive glia in normal and pathologic environments

The data herein demonstrate that in addition to the well-characterized myelin marker-positive, glial fibrillary acidic protein (GFAP)-negative, membrane sheet-bearing oligodendrocytes, another type of myelin marker-positive, process-bearing glia exists in normal and pathologic conditions. This second type of myelin marker-positive glia expresses GFAP, and therefore these cells have been referred to as mixed phenotype glia. Although mixed phenotype glia have been documented previously, their identity and function have remained a mystery. The goal of this immunocytochemical study was to further characterize these cells. Using the MBPlacZ transgenic mouse in which beta-galactosidase is under the control of the myelin basic protein (MBP) gene promoter, GFAP-positive/beta-galactosidase-positive and myelin/oligodendrocyte-specific protein (MOSP)-positive/beta-galactosidase-positive cells were detected in subcortical white matter and in perivascular locations within cerebral white and gray matter. In cultures prepared from highly enriched myelin marker-positive immature glia, mixed phenotype glia were detected that were GFAP-positive and either MOSP-, MBP-, O1-, and O4-positive. The expression of multiple myelin markers by mixed phenotype glia may suggest that these cells are of oligodendrocyte origin. Increased numbers of MOSP-positive/GFAP-positive mixed phenotype glia were detected in sections from adult hypomyelinated brain from shiverer, quaking, and PKU mice compared to myelinated control adult mouse brain. Similarly, cultures from control brain exposed to elevated pH for 2-3 weeks showed dramatically increased numbers of mixed phenotype glia (80%) compared to control (<10%). Increased numbers of mixed phenotype glia also were detected in shiverer cultures (40%). Since increases in the number of mixed phenotype glia occur in shiverer, quaking, and PKU mouse brain, these data suggest that mixed phenotype glia contribute to gliosis in pathologic white matter.     

From neural stem cells to myelinating oligodendrocytes

The potential to generate oligodendrocytes progenitors (OP) from neural stem cells (NSCs) exists throughout the developing CNS. Yet, in the embryonic spinal cord, the oligodendrocyte phenotype is induced by sonic hedgehog in a restricted anterior region. In addition, neuregulins are emerging as potent regulators of early and late OP development. The ability to isolate and grow NSCs as well as glial-restricted progenitors has revealed that FGF2 and thyroid hormone favor an oligodendrocyte fate. Analysis of genetically modified mice showed that PDGF controls the migration and production of oligodendrocytes in vivo. Interplay between mitogens, thyroid hormone, and neurotransmitters may maintain the undifferentiated stage or result in OP growth arrest. Notch signaling by axons inhibits oligodendrocyte differentiation until neuronal signals--linked to electrical activity-trigger initiation of myelination. To repair myelin in adult CNS, multipotential neural precursors, rather than slowly cycling OP, appear the cells of choice to rapidly generate myelin-forming cells.     

Stage specific, (O4+GalC-) isolated oligodendrocyte progenitors produce MBP+ myelin in vivo.

O4+/A007+GalC- proligodendroblasts represent a distinct stage of development in the oligodendrocyte lineage, occurring just prior to the appearance of postmitotic GalC+ oligodendrocytes. These cells, isolated directly from postnatal rat telencephalon by an immunopanning procedure, can terminally differentiate and myelinate axons when transplanted back into an in vivo environment. Specifically, after 30 days in the brain of newborn shiverer mouse hosts, O4+GalC- oligodendrocyte progenitors produced myelin basic protein positive (MBP+) patches. These MBP+ patches, examined by both light and confocal microscopy, contained oligodendrocyte cell bodies and ensheathed host shiverer axons morphologically similar to those found in normal rat brain at an analogous age. These results suggest that isolated O4+GalC- cells can become biochemically mature oligodendrocytes with the capacity to elaborate myelin sheaths, and further define the period of development during which oligodendrocytes retain their capacity to myelinate axons when given a receptive environment.     

Lysenin-sphingomyelin binding at the surface of oligodendrocyte lineage cells increases during differentiation in vitro

We have investigated the relationship between the developmental expression of sphingomyelin, a major component of myelin, and oligodendrocyte lineage. Using lysenin as a cytochemical probe for membrane sphingomyelin, we have now determined the distribution pattern of sphingomyelin on the plasma membrane of rat cultured oligodendrocytes. Although lysenin does not bind to A2B5(+)/NG2(+) bipolar oligodendrocyte progenitors, lysenin recognizes sphingomyelin on the cell bodies of multipolar A2B5(+) cells, but not on their processes. O4(+) and O1(+) immature and MBP(+) mature oligodendrocytes are strongly labeled by lysenin from cell bodies to the tips of processes. The content of sphingomyelin in immature and mature oligodendrocytes is approximately 2-fold higher than that in oligodendrocyte progenitors. These findings show that sphingomyelin increases during differentiation of cells in the oligodendrocyte lineage. In multipolar oligodendrocyte progenitors exposed to Triton X-100 at 4 degrees C, lysenin labels cell processes in addition to cell bodies. In contrast, Triton X-100 extraction does not alter the distribution of lysenin binding on O4(+), O1(+) and MBP(+) cells, although the immunocytochemical intensities of the lysenin bindings increase. Our data suggest that the alteration in sphingomyelin content and distribution in the oligodendrocyte lineage cells could have important consequences for cell recognition and downstream signaling events through sphingomyelin-rich domains.     

Bcl-2-expressing oligodendrocytes in multiple sclerosis lesions.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system leading to selective destruction of myelin sheaths and/or oligodendrocytes. The immunological mechanisms responsible for myelin destruction and the primary target of the immune response have not yet been identified. Prior studies have reported a variable degree of oligodendrocyte preservation in actively demyelinating lesions. We have previously demonstrated that oligodendrocyte survival is heterogenous and varies between individual MS patients. Bcl-2 belongs to the group of apoptosis-associated proteins that protects cells from cell death. The purpose of the present study was to determine whether bcl-2 expression is associated with oligodendrocyte preservation observed in some early MS lesions. Double immunocytochemistry was performed with antibodies against bcl-2 and myelin oligodendrocyte glycoprotein (MOG) to identify bcl-2-expressing oligodendrocytes within MS lesions from 43 patients. The number of bcl-2-positive oligodendrocytes was determined depending on the lesion demyelinating activity and the disease course of the patients. The number of bcl-2-expressing oligodendrocytes increased within demyelinating lesions compared to the periplaque white matter, with highest numbers in remyelinating lesions. There was a significant association between the presence of bcl-2-positive oligodendrocytes and the presence of remyelination. The highest proportion of bcl-2-positive oligodendrocytes was observed in a subgroup of patients with relapsing-remitting disease course. The expression of apoptosis-associated proteins may contribute to oligodendrocyte preservation or loss in MS lesions.     

Dynamic changes in myelin aberrations and oligodendrocyte generation in chronic amyloidosis in mice and men

Myelin loss is frequently observed in human Alzheimer's disease (AD) and may constitute to AD‐related cognitive decline. A potential source to repair myelin defects are the oligodendrocyte progenitor cells (OPCs) present in an adult brain. However, until now, little is known about the reaction of these cells toward amyloid plaque deposition neither in human AD patients nor in the appropriate mouse models. Therefore, we analyzed cells of the oligodendrocyte lineage in a mouse model with chronic plaque deposition (APPPS1 mice) and samples from human patients. In APPPS1 mice defects in myelin integrity and myelin amount were prevalent at 6 months of age but normalized to control levels in 9‐month‐old mice. Concomitantly, we observed an increase in the proliferation and differentiation of OPCs in the APPPS1 mice at this specific time window (6–8 months) implying that improvements in myelin aberrations may result from repair mechanisms mediated by OPCs. However, while we observed a higher number of cells of the oligodendrocyte lineage (Olig2+ cells) in APPPS1 mice, OLIG2+ cells were decreased in number in postmortem human AD cortex. Our data demonstrate that oligodendrocyte progenitors specifically react to amyloid plaque deposition in an AD‐related mouse model as well as in human AD pathology, although with distinct outcomes. Strikingly, possible repair mechanisms from newly generated oligodendrocytes are evident in APPPS1 mice, whereas a similar reaction of oligodendrocyte progenitors seems to be strongly limited in final stages of human AD pathology. © 2012 Wiley Periodicals, Inc.     

H-ferritin is the major source of iron for oligodendrocytes

There is a critical relationship between oligodendrocyte development, myelin production, and iron bioavailability. Iron deficiency leads to hypomyelination both in humans and animal models, and the neurological sequelae of hypomyelination are significant. Therefore, understanding molecular mechanisms of iron import into oligodendrocytes is necessary for devising effective strategies for iron supplementation. Although transferrin has been considered as an essential component of oligodendrocyte media in culture, oligodendrocytes in vivo lack transferrin receptors. We have established that receptors for H-ferritin (HF) exist on cells of oligodendroglial lineage and that uptake of extracellular HF by oligodendrocyte progenitors is via receptor mediated endocytosis. These data strongly argue that ferritin is a major source of iron for oligodendrocytes. In this study, we demonstrate that media deficient in transferrin results in loss of viability of oligodendrocyte progenitors in culture. Cell loss could be prevented by supplementing the media with HF. Moreover, the addition of extracellular HF stimulates development of oligodendrocyte progenitor cells (OPCs) by increasing expression of myelin basic protein (MBP) and olig2 proteins without increasing their proliferation. The effect of HF on the OPCs could be mimicked by addition of membrane permeable 3,5,5-trimethylhexanoyl ferrocene (TMH-Fe) as an iron source to the media, but not membrane-impermeable ferric ammonium citrate. Overall, therefore, our results demonstrate the importance of iron for OPCs viability and differentiation and identify extracellular HF as a critical source of iron for oligodendrocytes. Given that ferritin receptors, but not transferrin receptors can be demonstrated on oligodendrocytes in vivo, the delivery of iron to oligodendrocytes via ferritin may be the more biological relevant delivery system.     

Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors

Much current knowledge of oligodendrocyte biology, the myelin-forming cells in the central nervous system, comes from cell culture studies mainly from postnatal rat tissue but mouse cells have been much more difficult to produce in large quantities. We have developed a high yield protocol for production of oligodendrocyte precursor cells from mouse embryonic neural progenitors grown as neurospheres. Neurospheres can be maintained and expanded for long periods in culture in the presence of epidermal growth factor (EGF). When floating neurospheres were plated on substrate-coated dishes in media supplemented with platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), the spheres attached and generated migrating cells that were predominantly oligodendrocyte-lineage cells. Furthermore, cells in spheres could be shifted to the oligodendrocyte phenotype prior to plating on substrate, by incubation in suspension with PDGF/bFGF. Single cell suspensions plated after dissociation of either EGF-treated neurospheres or PDGF/bFGF-treated oligospheres had the bipolar, elongated morphology characteristic of oligodendrocyte precursor cells. mRNA and protein expression analysis of the cells generated by this method confirmed their oligodendrocyte lineage. Oligodendrocyte precursors generated by this method matured in response to ciliary neurotrophic factor treatment, producing cells with multiple processes and myelin-like membranes. The most important aspect of this protocol is the ability to generate very high numbers of relatively pure mouse oligodendrocyte progenitor cells, which can be easily transfected. These studies open up many kinds of investigations on transgenic and mutant mouse oligodendrocytes, thereby providing a valuable tool to study oligodendrocyte biology and development.     

Transient exposure to FGF2 enhances myelination in embryonic brain cell cocultures

The amount of myelination in vivo and in vitro depends on the number of oligodendrocyte progenitors, their differentiation, and on the neuron function. It has been shown that continuous administration of FGF2, a mitotic and neuroprotective factor, allows oligodendrocyte progenitors to proliferate, but prevents them from differentiating and myelinating. This study was designed to test the effect of transient exposure to FGF2 on myelination in an oligodendrocyte/neuron coculture system. At 2 days in vitro, cultures were treated with a single dose of 20 ng/ml FGF2. Cell proliferation was determined by BrdU uptake. The number of cells of the oligodendrocyte lineage was determined by immunocytology of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). The maturation of oligodendrocytes and myelination was followed by immunocytological analysis of MBP (myelin basic protein). Electron microscopy was used to study the ultrastructure of myelin. BrdU uptake procedure showed an increase in cell proliferation in FGF2-treated cultures after 48 h of treatment. At 15-18 days in vitro, CNPase(+) and MBP(+) cells were much more abundant in cultures treated with FGF2 than in control cultures. We observed differentiation and maturation of oligodendrocytes and a higher degree of myelination in FGF2-treated cultures compared to controls. Electron microscopy showed the presence of myelin structures in FGF2-treated cultures that did not differ morphologically from those observed in control cultures. Transient exposure of cultured brain cells to FGF2 increased myelination in vitro. Administration of FGF2 over a short period might thus enhance remyelination in demyelinating diseases in vivo.     

Myelin protein expression in the myelin-deficient rat brain and cultured oligodendrocytes

The myelin-deficient (MD) rat does not express the major protein of CNS myelin, proteolipid protein (PLP). Here we further analyze whether this defect is reflected at the level of mRNA and whether the expression of other myelin proteins is affected in oligodendrocytes in vivo and in vitro. Both myelin basic protein (MBP) and PLP message levels were reduced in MD rats to 10-20% of the normal littermate controls, while the level of expression of an astrocyte-specific gene, glial fibrillary acidic protein (GFAP), was normal. Although MBP and PLP mRNAs were equally depressed, only MBP was detected with immunolabeling of corpus callosum, while PLP was absent in oligodendrocytes both in vivo and in vitro. A reduced number of MD rat oligodendrocytes express MBP in vitro compared to controls. The MD rat optic nerve contains normal numbers of 0-2A progenitors, but they tend to differentiate into GC-positive oligodendrocytes faster than oligodendrocytes from control littermates. In conclusion, the absence of PLP and reduced levels of MBP in the MD rats point to similarities with the jimpy mouse lesion. Moreover, the defect influences the expression of other myelin proteins and the oligodendrocyte developmental pathway.     

Myelin degeneration induced by mutant superoxide dismutase 1 accumulation promotes amyotrophic lateral sclerosis

Myelin is a specialized membrane that wraps around nerve fibers and is essential for normal axonal conduction in neurons. In the central nervous system, oligodendrocytes are responsible for myelin formation. Recent studies have reported pathological abnormalities in oligodendrocytes in human patients with amyotrophic lateral sclerosis (ALS) and a mouse model of ALS expressing the G93A mutation of the human superoxide dismutase 1 (mtSOD1). However, it is unclear whether oligodendrocyte pathology in ALS represents the primary dysfunction induced by mtSOD1 and how mtSOD1 contributes to oligodendrocyte degeneration and ALS pathogenesis. We analyzed GAL4-VP16-UAS transgenic zebrafish selectively expressing mtSOD1 in mature oligodendrocytes. We observed that mtSOD1 directly induced oligodendrocyte degeneration by disrupting the myelin sheath and downregulating monocarboxylate transporter 1 (MCT1), thereby causing spinal motor neuron degeneration. Pathological changes observed in this transgenic zebrafish were similar to the pathology observed in the SOD1^G93A mouse model of ALS, which is characterized by expression of mtSOD1 in all cells. In addition, oligodendrocyte dysfunction induced by mtSOD1 was associated with anxiety-related behavioral abnormalities, learning impairments, and motor defects in the early symptomatic stage. We also found that treatment with potassium channel inhibitors rescued behavioral abnormalities without rescuing MCT1 expression, suggesting that myelin disruption induces behavioral abnormalities independently of MCT1. These results indicate that mtSOD1-induced dysfunction of mature oligodendrocytes is sufficient to induce motor neuron degeneration, thus informing future therapeutic strategies targeted at oligodendrocytes in ALS.     

Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination

Identifying a source of cells with the capacity to generate oligodendrocytes in the adult CNS would help in the development of strategies to promote remyelination. In the present study, we examined the ability of the precursor cells of the adult mouse subventricular zone (SVZ) to differentiate into remyelinating oligodendrocytes. After lysolecithin-induced demyelination of the corpus callosum, progenitors of the rostral SVZ (SVZa) and the rostral migratory pathway (RMS), expressing the embryonic polysialylated form of the neural cell adhesion molecule (PSA-NCAM), increased progressively with a maximal expansion occurring after 2 weeks. This observation correlated with an increase in the proliferation activity of the neural progenitors located in the SVZa and RMS. Moreover, polysialic acid (PSA)-NCAM-immunoreactive cells arizing from the SVZa were detected in the lesioned corpus callosum and within the lesion. Tracing of the constitutively cycling cells of the adult SVZ and RMS with 3H-thymidine labelling showed their migration toward the lesion and their differentiation into oligodendrocytes and astrocytes but not neurons. These data indicate that, in addition to the resident population of quiescent oligodendrocyte progenitors of the adult CNS, neural precursors from the adult SVZ constitute a source of oligodendrocytes for myelin repair.     

Glial and Neuronal Protein Tyrosine Phosphatase Alpha (PTPα) Regulate Oligodendrocyte Differentiation and Myelination

CNS myelination defects occur in mice deficient in receptor-like protein tyrosine phosphatase alpha (PTPα). Here, we investigated the role of PTPα in oligodendrocyte differentiation and myelination using cells and tissues from wild-type (WT) and PTPα knockout (KO) mice. PTPα promoted the timely differentiation of neural stem cell-derived oligodendrocyte progenitor cells (OPCs). Compared to WT OPCs, KO OPC cultures had more NG2+ progenitors, fewer myelin basic protein (MBP)+ oligodendrocytes, and reduced morphological complexity. In longer co-cultures with WT neurons, more KO than WT OPCs remained NG2+ and while equivalent MBP+ populations of WT and KO cells formed, the reduced area occupied by the MBP+ KO cells suggested that their morphological maturation was impeded. These defects were associated with reduced myelin formation in KO OPC/WT neuron co-cultures. Myelin formation was also impaired when WT OPCs were co-cultured with KO neurons, revealing a novel role for neuronal PTPα in myelination. Canonical Wnt/β-catenin signaling is an important regulator of OPC differentiation and myelination. Wnt signaling activity was not dysregulated in OPCs lacking PTPα, but suppression of Wnt signaling by the small molecule XAV939 remediated defects in KO oligodendrocyte differentiation and enhanced myelin formation by KO oligodendrocytes. However, the myelin segments that formed were significantly shorter than those produced by WT oligodendrocytes, raising the possibility of a role for glial PTPα in myelin extension distinct from its pro-differentiating actions. Altogether, this study reveals PTPα as a molecular coordinator of oligodendroglial and neuronal signals that controls multiple aspects of oligodendrocyte development and myelination.     

Caspase-3 activation in oligodendrocytes from the myelin-deficient rat

The myelin-deficient (MD) rat has a point mutation in its proteolipid protein (PLP) gene that causes severe dysmyelination and oligodendrocyte cell death. Using an in vitro model, we have shown that MD oligodendrocytes initially differentiate similarly to wild-type cells, expressing galactocerebroside, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin basic protein. However, at the time when PLP expression would normally begin, the MD oligodendrocytes die via an apoptotic pathway involving caspase activation. The active form of caspase-3 was detected, along with the cleavage products of poly-(ADP-ribose) polymerase (PARP) and spectrin, major targets of caspase-mediated proteolysis. A specific inhibitor of casapse-3, Ac-DEVD-CMK, reduced apoptosis in MD oligodendrocytes, but the rescued cells did not mature fully or express myelin-oligodendrocyte glycoprotein. These results suggest that mutant PLP affects not only cell death but also oligodendrocyte differentiation.     

Conservative amino acid substitution in the myelin proteolipid protein of jimpymsd mice.

The capacity for synthesizing and maintaining a compact myelin sheath is destroyed in a number of inborn errors of myelin metabolism. One class of hypomyelinating mutations, which displays an X-linked pattern of inheritance, is distinguished by marked disturbances in oligodendrocyte differentiation. We have defined the molecular defect in one such mutant that lacks mature oligodendrocytes, the X-linked jimpy myelin synthesis deficient (jpmsd) trait in mice. The structure of the gene encoding the most abundant myelin protein, proteolipid protein (PLP), was determined by mapping and partially sequencing genomic clones from jpmsd and wild-type mice. Jpmsd mice have a single base change in PLP, a C----T transition in exon 6 that would substitute a valine for alanine in both PLP and its alternatively spliced isoform, DM20. The mutation was confirmed by polymerase chain reaction-amplifying exon 6 from genomic DNA and then either sequencing the amplified DNA or directly probing exon 6 with oligonucleotides designed to detect a single base mismatch. The conservative amino acid replacement in PLP/DM20 of jpmsd mice results in a pleiotropic phenotype similar to that observed for the allelic mutation jimpy, in which a splicing defect has radically altered the PLP/DM20 protein. The accelerated turnover of oligodendrocytes in both mouse mutants suggests a function for PLP/DM20 in oligodendrocyte differentiation distinct from the role of these proteolipid proteins as structural components of the myelin sheath.     

Oligodendrocyte maturation is inhibited by bone morphogenetic protein

Mature oligodendrocytes myelinate axons in the CNS. The development of the myelin sheath is dependent on the proper maturation of oligodendrocytes from precursors cells, a spatially restricted process that is regulated by inductive and repressive cues. Several members of the bone morphogenetic protein family (BMP2 and 4) have been implicated as repressors of oligodendrocyte development in vitro by shifting oligodendrocyte precursors into the astrocyte lineage. We now report on a second role of BMPs in oligodendrocyte development, regulation of myelin protein expression in immature oligodendrocytes. Purified immature rodent oligodendrocytes treated with BMP4 maintained galactocerebroside (GalC) expression, whereas the expression of three key myelin proteins, proteolipid protein (PLP), myelin basic protein (MBP), and 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNP), was severely decreased. Paradoxically, BMP-treated oligodendrocytes show increased process extension and complexity, normally a feature of oligodendrocyte maturation. We also investigated whether BMP4 could inhibit myelin protein expression in an E 12.5 mouse explant culture of cervical spinal cord and hindbrain that maintains the in vivo cellular relationships and architecture. Beads soaked in BMP protein implanted into these explants inhibited the expression of myelin proteins, proteolipid protein, and myelin-associated glycoprotein (MAG), in the local area surrounding the bead. Since these explants also contained precursors cells, expression of galactocerebroside and O4, an oligodendrocyte marker, were also decreased by BMP treatment but to a much lesser degree than the myelin markers. Together, these data indicate that BMPs have multiple roles in oligodendrocyte development. At earlier stages, they affect cell lineage decisions and at later stages, they inhibit cell specialization.