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.     

Cellular reaction to an acute demyelinating/remyelinating lesion of the rat brain stem: Localisation of GD3 ganglioside immunoreactivity

We describe a simple and reproducible acute demyelinating lesion of the rat brain stem induced by injection of ethidium bromide into the cisterna magna of young adult rats. Using immunofluorescence with a panel of antibodies to cell-specific antigens we have studied the changes in cell populations that occur at various stages during lesion progression and repair. In particular we localized the expression of ganglioside G_D3 immunoreactivity, a marker for oligodendroglial progenitors in developing brain. Both astroglia (GFAP⁺) and oligodendroglia (CNP⁺) were destroyed during the early response to the ethidium bromide although axons were spared. Splitting of myelin lamellae occurred as early as 4 days post-injection (DPI), with extensive demyelination of the inferior cerebellar peduncle following by 6 DPI. Large numbers of ED1⁺ and OX-42⁺ macrophages were present in the lesion site at this stage. Astrogliosis occurred around the perimeter of the lesions. Two populations of G_D3⁺ cells appeared within and around the lesion sites during the demyelination. One population was identified by the phenotype G_D3⁺ ED1⁺ and thus probably belonged to the macrophage/microglial lineage. In these cells both antigens appeared cytoplasmic. The second population of G_D3⁺ cells exhibited cell membrane G_D3 immunoreactivity but did not express the ED1 antigen. These cells are suggested to be oligodendroglial progenitors generated in response to the demyelination. No such cells were seen in control tissue. G_D3⁺ cells were present within the lesion sites from 6 DPI until 10–12. Following the clearance of myelin debris from the lesions, remyelination was a relatively rapid event with thin MBP⁺ myelin sheaths first seen at 11–12 DPI. Remyelination, which was extensive by 25 DPI, was predominantly oligodendroglial in origin (MBP⁺Po^? myelin) with only small pockets of peripheral myelin (MBP⁺Po⁺ myelin) observed. The present study, in addition to identifying putative glial progenitors within a demyelinated lesion, also demonstrates the difficulties in unambiguously identifying such cells in the normal and damaged adult CNS. © 1993 Wiley-Liss, Inc.     

Transient reversion of O4+ Galc− oligodendrocyte progenitor development in response to the phorbol ester TPA

The physiological importance of protein kinase C during oligodendrocyte progenitor maturation was investigated by analyzing the effects of the protein kinase C activator phorbol 12-myristate 13-acetate (TPA) on the morphology, proliferation, and differentiation of oligodendrocytes at sequential stages of development. Monoclonal antibodies A2B5 and O4 were used to identify the A2B5⁺ O4^? and the A2B5⁺ O4⁺ galactocerebroside^? progenitor stages. Anti-galactocerebroside and anti-myelin basic protein were used to identify mature, post-mitotic oligodendrocytes. Proliferation was measured by bromodeoxyuridine incorporation. Within 24 hr after addition, TPA induced a down-regulation of the O4 antigen in OL progenitors, and an increase of expression of the intermediate filament protein vimentin, leading to a phenotypic reversion from the vimentin^? A2B5⁺ O4⁺ phenotype to the less mature vimentin⁺ A2B5⁺ O4^? stage. Concomitantly, TPA induced an increase in the number of bromodeoxyuridine-labeled oligodendrocyte progenitors and extensive process elongation. The response of O4⁺ progenitors was transient. Even with continued exposure to TPA, by 4 days after TPA addition the reverted cells ceased proliferation, reacquired O4 immunoreactivity, became vimentin-negative, and began to express galactocerebroside and myelin basic protein, and to display the complex, highly branched morphology characteristic of terminally differentiated oligodendrocytes. These results indicate that modulation of protein kinase C activity by TPA induces a transient reversion of O4⁺ progenitors to less mature O4^? cells, causing a transient inhibition of terminal differentiation. The relationship of these data to similar responses of the OL lineage to specific growth factors and implications for remyelination after pathologic injury are discussed. © 1993 Wiley-Liss, Inc.     

Trophic effects of basic fibroblast growth factor (bFGF) on differentiated oligodendroglia: A mechanism for regeneration of the oligodendroglial lineage

We have investigated the effect of basic fibroblast growth factor (bFGF) on the proliferation and phenotype of differentiated oligodendroglia. Using primary cell cultures enriched in oligodendrocytes but containing few O2A-oligodendrocyte progenitor cells, we demonstrate that bFGF treatment greatly increases the proportion of 02A cells while decreasing the proportion of galactocerebroside ⁺(GalC⁺), myelin basic protein ⁺(MBP⁺) oligodendrocytes, and the steady state levels of MPB mRNA. Complement mediated cell lysis experiments using the A2B5 antibody to deplete existing O2A cells or the R-Mab antibody to deplete existing oligodendroglia show that bFGF elicits a rapid increse in the number of O2A cells in cultures previously depleted of O2A cells, but does not cause an early incrase in O2A cells in cultures from which oligodendroglia had been removed, indication that the oligodendrocytes are the source of the newly recruited O2A cells. This bFGF-mediated transition from oligodendrocyte to O2A cells occurs with a time course similar to the bFGF-induced incrase of the proliferation rate of the GalC⁺ oligo-dendrocvtes. Studies with purified, passaged cells of the oligodendroglial lineage show that bFGF augmenta oligodendroglial dedifferntiation and proliferation in chronologically adult oligodendrocytes and in the virtual absence of other cell types. We have thusdemonstrated that mature oligodendrocytes are induced by bFGF to dedifferentiate and proliferate, suggesting a mechanism for regeneration of the oligodendroglial lineage following demyelinating disease. © 1993 Wiley-Liss, Inc.     

Neurofilament‐tubulin binding site peptide NFL‐TBS.40–63 increases the differentiation of oligodendrocytes in vitro and partially prevents them from lysophosphatidyl choline toxiciy

During multiple sclerosis (MS), the main axon cystoskeleton proteins, neurofilaments (NF), are altered, and their release into the cerebrospinal fluid correlates with disease severity. The role of NF in the extraaxonal location is unknown. Therefore, we tested whether synthetic peptides corresponding to the tubulin‐binding site (TBS) sequence identified on light NF chain (NFL‐TBS.40–63) and keratin (KER‐TBS.1–24), which could be released during MS, modulate remyelination in vitro. Biotinylated NFL‐TBS.40–63, NFL‐Scramble2, and KER‐TBS.1–54 (1–100 μM, 24 hr) were added to rat oligodendrocyte (OL) and astrocyte (AS) cultures, grown in chemically defined medium. Proliferation and differentiation were characterized by using specific antibodies (A2B5, CNP, MBP, GFAP) and compared with untreated cultures. Lysophosphatidyl choline (LPC; 2 × 10^–5 M) was used to induce OL death and to test the effects of TBS peptides under these conditions. NFL‐TBS.40–63 significantly increased OL differentiation and maturation, with more CNP⁺ and MBP⁺ cells characterized by numerous ramified processes, along with myelin balls. When OL were challenged with LPC, concomitant treatment with NFL‐TBS.40–63 rescued more than 50% of OL compared with cultures treated with LPC only. Proliferation of OL progenitors was not affected, nor were AS proliferation and differentiation. NFL‐TBS.40–63 peptide induces specific effects in vitro, increasing OL differentiation and maturation without altering AS fate. In addition, it partially protects OL from demyelinating injury. Thus release of NFL‐TBS.40–63 caused by axonal damage in vivo could improve repair through increased OL differentiation, which is a prerequisite for remyelination. © 2013 Wiley Periodicals, Inc.     

Transient expression of 3,5,3′-triiodothyronine nuclear receptors in rat oligodendrocytes: In vivo and in vitro immunocytochemical studies

It is generally accepted that the action of thyroid hormones is mediated through specific nuclear receptors. Recent studies have demonstrated the homology of the thyroid receptor with the cellular product of the oncogen v-erbA. So far, two genes have been identified and classified as α and β subtypes. In this study, the expression of nuclear triiodothyronine (T₃) receptors (NT₃RS) was examined in secondary cultures containing 85–90% oligodendrocytes (OL) prepared from newborn rat brain primary cultures enriched in OL. These cultures, which are able to produce myelin membranes, were examined by double immunolabelling with a monoclonal antibody (2B3) raised against purified rat liver NT₃Rs and with antibodies against two maturation markers of OL: an early marker, galactocerebroside (GC), and myelin basic protein (MBP), which is expressed later than GC. 2B3 recognized three nuclear proteins with the same molecular weights as β, α1, and α2 sybtypes with different capacities for binding T₃. In 5-day -old OL secondary cultures (25 days, total time in culture), 2B3-NT₃R immunoreactivity was located in 77% of morphologically immature OL (GC)⁺ cells, whereas only 44% of morphologically mature OL were immunoreactive. Only 35% of the MBP⁺ cells co-expressed NT₃Rs. In the corpus callosum of devloping rat brain, at all ages studied from 7–60 days postnatal, the total absence of NT₃Rs in dark OL (morphologically mature), confirmed by ultrastructural immunocytochemistry, indicates an even more dramatic decrease during maturation. Furthermore, the percentage of medium OL (less mature) stained by 2B3 is reduced by approximately half in 60- compared to 20-day-old rat brain. It is of interest to note that the in vitro observation with maturation markers mirrors the in vivo decrease of NT₃R expression during development. It is intersting that NT₃Rs are absent in vivo before the critical period of active myelination. These data indicate the presence of a nuclear T₃ binding protein in the nuclei of OL at the time of myelination both in vitro and in vivo. The transient expression of these NT₃Rs during active myelination argues in favour of a direct effect of thyroid hormones on OL. © 1994 Wiley-Liss, Inc.     

Oligodendroglial progenitors labeled with the O4 antibody persist in the adult rat cerebral cortex in vivo

The monoclonal antibody O4 has been used to define a biologically distinct stage of the oligodendroglial lineage in vitro. Furthermore, O4⁺ oligodendroglial progenitors have been found in cell cultures derived from mature tissue, leading to speculation about the presence of oligodendroglial progenitors in the adult central nervous system (CNS). However, the existence of adult oligodendroglial progenitors has yet to be conclusively demonstrated in the intact animal. We have investigated the expression of O4 immunoreactivity in the developing and mature rat forebrain and the relationship of these cells to cells expressing the early oligodendroglial progenitor markers G_D3 ganglioside and NG₂ chondroitin sulfate proteoglycan, and to differentiated galactocerebroside expressing oligodendroglia. By the day of birth O4⁺ cells were already widely distributed throughout the formative corpus callosum and increased in number in the white matter and cortical gray matter over the first 2 postnatal weeks. In contrast to cell culture observations, most O4⁺ cells seen over this period failed to express G_D3, although the majority did express NG₂. Beginning at postnatal day 4, NG₂⁺/O4⁻ progenitors in the corpus callosum and cerebral cortical gray matter underwent a wave of differentiation into NG₂⁺/O4⁺ cells and then into galactocerebroside-positive oligodendroglia. Interestingly, not all cells underwent this progression: a population remained as O4⁺/NG₂⁺ progenitors. Furthermore, this O4⁺/NG₂⁺ population persisted into adulthood and failed to express either G_D3, galactocerebroside, RIP, or myelin basic protein (MBP). They were also distinguishable from glial fibrillary acidic protein⁺ and glutamine synthetase⁺ astrocytes and OX-42⁺ microglia. We therefore propose that these O4⁺/NG₂⁺ cells represent adult oligodendroglial progenitors hitherto only described in cell culture. © 1997 Wiley-Liss Inc.     

Transplanted Transgenically Marked Oligodendrocytes Survive, Migrate and Myelinate in the Normal Mouse Brain as They Do in the Shiverer Mouse Brain

The dye Hoechst 33342 was combined with an immunodetectable transgene product (chloramphenicol acetyltransferase, CAT) expressed in differentiated oligodendrocytes to trace their fate after transplantation in the normal and the shiverer mouse brain. In the shiverer brain, the technique allowed us to visualize grafted cells inside myelin basic protein-positive myelin patches. Most of these cells were CAT-positive/Hoechst 33342-negative, reinforcing our hypothesis that cell division probably follows migration of grafted oligodendrocytes. Correlation of their morphology and distribution with their location in the host CNS suggested a local effect on the cell division and morphogenesis of the grafted material. When compared with transplantation of fragments of normal newborn donor tissue into the newborn shiverer brain, no difference could be seen between the behaviour of normal and transgenic oligodendrocytes. In the normal brain, transgenic oligodendrocytes survived at least 150 days and successfully myelinated the host axons. The timing of differentiation of grafted cells was similar in both types of recipient brains. Migration occurred rostrally and caudally. Although migrating cells could be observed along the meninges and the blood vessels, migration occurred preferentially along white matter tracts. The extent of migration was influenced by the site of implantation, and grafted cells could be found up to 6 mm from the grafting point. No differences in the timing of differentiation or the pattern or extent of migration could thus be demonstrated when transgenic oligodendrocytes were transplanted in the normal or the shiverer brain. This validates our previous studies using the newborn shiverer mouse as recipient.     

Tracing transplanted oligodendrocytes during migration and maturation in the shiverer mouse brain

Fragments of neural tissue from normal newborn mouse were stained with Hoechst 33342 dye before transplantation into the newborn shiverer mouse brain. Combination of this technique with immunohistochemistry demonstrated that, after transplantation, these cells are able to survive as long as unstained cells and to myelinate in the shiverer mouse host brain. Stained cells express the normal sequence of differentiation in terms of chronology of differentiation marker expression [04, galactocerebroside (GalC), myelin basic protein (MBP)], as normal cell do in situ. It has thus been possible by this technique to show the migration pathways of transplanted cells and to correlate them with the expression of specific markers: long distance migration along white matter axonal pathways occurs when cells are o4-positive, GalC-negative. By contrast, only GalC-positive cells are able to migrate across the grey matter in the absence of radial glia. Finally, it has been possible to propose a migration and differentiation sequence of these cells, suggesting that MBP-positive oligodendrocytes divide after migration in the target zone.     

Two mitogenic regions of myelin basic protein interact with different receptors to induce Schwann cell proliferation in a cAMP dependent process

Previous studies have shown that myelin basic protein (MBP) is mitogenic for Schwann cells (SCs) in the presence of elevated intracellular cAMP. Two mitogenic regions of MBP have been identified: one mitogenic region within the first 44 residues of the aminoterminus (1–44) and the other mitogenic region within the terminal 15 residues of the carboxyl end of the molecule (152–167). Unlike the mitogenic effect of a myelin enriched fraction (MEF), the mitogenic effect of MBP was not reduced by the addition of the lysosomal inhibitor, ammonium chloride. These data indicate that MBP causes SC proliferation by direct interaction of MBP with a surface receptor. Using Scatchard analysis of the binding of MBP to SCs, we report that treatment with forskolin does not cause the upregulation of receptors for MBP. Moreover, MBP blocks the cross-linking of ¹²⁵I-bFGF with two fibroblast growth factor (FGF) receptors having apparent molecular weights of 140 kDa and 120 kDa, respectively. Since neither TGF-β nor PDGFBB displaced cell surface bound ¹²⁵I-MBP, we conclude that MBP binds to the FGF receptor rather than other growth factor receptors. Furthermore, only MBP_1–44 interacted with ganglioside GM1, whereas MBP_152–167 did not interact with this ganglioside. These results are consistent with the view that ganglioside GM1 mediates the mitogenic effects of MBP_1–44, while the FGF receptor mediates the mitogenic effect of MBP_152–67. Intracellular cAMP of SCs was transiently increased after the addition of macrophage conditioned medium, suggesting that macrophages may produce factors in vivo which can transiently elevate intracellular cAMP levels, allowing a wave of SC proliferation in response to MBP-related mitogens. ©1995 Wiley-Liss, Inc.     

Transplantations of newborn CNS fragments into the brain of shiverer mutant mice: extensive myelination by transplanted oligodendrocytes. II. Electron microscopic study

As already demonstrated by immunohistochemistry, oligodendrocytes from newborn normal mice are able to survive, migrate and myelinate when transplanted into the newborn shiverer (shi/shi) mouse brain. The survival of the grafted cells and their interaction with the host brain were studied at different times after transplantation. Normal myelin was found in the host parenchyma basing our observation on the morphological difference between normal and shiverer myelin: the shiverer myelin deprived of major dense line appears uncompacted as compared to normal myelin. Myelin formed by transplanted oligodendrocytes was detected around the graft and, after immunohistochemical prelocalization, at considerable distance from the site of implantation. Normal and shiverer myelin were detected around axons adjacent to each other or around the same axon. These results confirm and extend at the ultrastructural level our previous data obtained by immunohistochemistry.     

Transplantation of CNS fragments into the brain of shiverer mutant mice: extensive myelination by implanted oligodendrocytes. I. Immunohistochemical studies

Solid fragments of olfactory bulb from new-born normal (B6CBA and C57BL6) mice were implanted into new-born shiverer (shi/shi) brains. The shiverer mouse being characterized by the absence of myelin basic protein (MBP), myelination due to implanted oligodendrocytes can be detected in the shiverer brain using an antiserum anti-MBP. Observation of sagittal sections of the host brains revealed very extensive areas of normal myelination from the level of the graft (rostral thalamus) up to the caudal brain (diencephalon, cerebellum, pons). Thus, oligodendrocytes contained in the implant migrate out of the graft over long distances in the host brain, before they differentiate and synthesize myelin. These results raise the question of the behaviour of oligodendrocytes in normal development.     

Hydrogels Containing Peptide or Aminosugar Sequences Implanted into the Rat Brain: Influence on Cellular Migration and Axonal Growth

Biocompatible polymer matrices for implantation into lesion sites in the brain were synthesized by incorporating peptide or aminosugar sequences intoN-(2-hydroxypropyl)methacrylamide (HPMA) hydrogels. RGD peptide sequences were chemically linked to the hydrogel backbone via a glycylglycine spacer; aminosugars were glucosamine (NHGlc) orN-acetylglucosamine residues. Unmodified or sequence containing HPMA hydrogels were implanted into the lesioned optic tract or cerebral cortex of juvenile (17- to 19-day-old) or adult rat brains, respectively. After 10–12 months host animals were perfused and the brains were processed for immunohistochemistry using antibodies to neurofilaments (RT97), laminin, glial fibrillary acidic protein (GFAP), carbonic anhydrase II (CAII), S100 protein, macrophages (ED1), and myelin basic protein (MBP). Unmodified (control) HPMA hydrogels contained no cellular infiltration or axonal growth. Peptide (RGD)- and aminosugar-modified hydrogels showed increased adhesion properties with host neural tissue, were vascularized, and were infiltrated by host nonneuronal cells. Astrocytes (GFAP⁺) and macrophages (ED1⁺) were the major cell types seen within modified HPMA hydrogels, the largest numbers being found in RGD-containing polymers. CAII⁺oligodendroglia were not seen within any of the hydrogel matrices. RT97⁺/MBP⁻axons grew into both the RGD and NHGlc hydrogel matrices for small distances. The number of axons was greatest in hydrogels implanted into cerebral cortex but in both cortex and optic tract implants the highest density of axons was seen in polymers containing RGD. The findings of this study are discussed in the context of CNS tissue replacement and the construction of bioactive scaffolds to promote regenerative axonal growth across areas of injury in the brain and spinal cord.     

Human fetal radial glia cells generate oligodendrocytes in vitro

Limited knowledge about human oligodendrogenesis prompted us to explore the lineage relationship between cortical radial glia (RG) cells and oligodendrocytes (OLs) in the human fetal forebrain. RG cells were isolated from cortical ventricular/subventricular zone and their progeny was followed in vitro. One portion of RG cells differentiated into cells of OL lineage identified by cell-type specific antibodies, including platelet-derived growth factor receptor-alpha (PDGFRalpha), NG2, O4, myelin basic protein, and myelin oligodendrocyte glycoprotein. Moreover, using Cre Lox fate mapping (brain lipid binding protein-Cre/Floxed-yellow fluorescent protein) we established a direct link between RG cells and OL progenitors. In vitro generation of RG-derived O4(+) OL progenitors was enhanced by addition of sonic hedgehog (SHH) and reduced by the SHH inhibitor, cyclopamine, suggesting the role of SHH signaling in this process. In summary, our in vitro experiments revealed that a portion of cortical RG cells isolated from human forebrain at the second trimester of gestation generates OL progenitors and this suggests a role of SHH in this process.     

Xeno-free culture for generation of forebrain oligodendrocyte precursor cells from human pluripotent stem cells

Oligodendrocytes (OLs) show heterogeneous properties that depend on their location in the central nervous system (CNS). In this regard, the investigation of oligodendrocyte precursor cells (OPCs) derived from human pluripotent stem cells (hPSCs) should be reconsidered, particularly in cases of brain-predominant disorders for which brain-derived OPCs are more appropriate than spinal cord-derived OPCs. Furthermore, animal-derived components are responsible for culture variability in the derivation and complicate clinical translation. In the present study, we established a xeno-free system to induce forebrain OPCs from hPSCs. We induced human forebrain neural stem cells (NSCs) on Laminin 511-E8 and directed the differentiation to the developmental pathway for forebrain OLs with SHH and FGF signaling. OPCs were characterized by the expression of OLIG2, NKX2.2, SOX10, and PDGFRA, and subsequent maturation into O4⁺ cells. In vitro characterization showed that >85% of the forebrain OPCs (O4⁺) underwent maturation into OLs (MBP⁺) 3 weeks after mitogen removal. Upon intracranial transplantation, the OPCs survived, dispersed in the corpus callosum, and matured into (GSTπ⁺) OLs in the host brains 3 months after transplantation. These findings suggest our xeno-free induction of forebrain OPCs from hPSCs could accelerate clinical translation for brain-specific disorders.     

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.     

Arrested oligodendrocyte lineage progression during human cerebral white matter development: dissociation between the timing of progenitor differentiation and myelinogenesis

Immature oligodendrocytes (OLs) derive from a large pool of late OL progenitors that populate human cerebral white matter throughout the latter half of gestation. We recently reported that a minor population of immature OLs are present in human cerebral white matter for at least 3 months before these cells commit to myelinogenesis around 30 wk postconceptional age. Since this finding supports dissociation between the events that regulate human immature OL maturation and their commitment to myelinogenesis, we characterized here the cellular sequence of events that characterize immature OLs during the transition from a premyelinating to a myelinating state. Commitment of immature OLs to myelinogenesis in human cerebral white matter correlated with the longitudinal extension of specialized processes, designated "pioneer processes," that made multiple types of apparent contacts with axons. This event coincided with the appearance of 3 distinct populations of sheaths that varied in their labeling for myelin basic protein (MBP). Since few axons initially labeled for MBP, this supported the occurrence in vivo of O4-negative, O1-positive premyelin sheaths that precede MBP-positive compacted myelin. These observations identify 3 sequential stages of early myelinogenesis: 1) the initial ensheathment of axons by premyelin sheaths generated by immature OLs; 2) the initial insertion of MBP into transitional sheaths; and 3) the generation of MBP-rich mature myelin.     

Selenium is required for normal upregulation of myelin genes in differentiating oligodendrocytes

The purpose of this study was to characterize the selenium requirement for the normal differentiation of oligodendrocyte lineage cells. In primary mixed glial cultures prepared from newborn rat brains, the overall growth of cultures, as seen from the total RNA yield, was not significantly affected by selenium. However, 30 nM selenium was required for the normal upregulation the proteolipid protein, basic protein, and myelin-associated glycoprotein gene expression assessed by Northern blot analysis. Selenium deprivation during initial, rapid phase of the gene upregulation irreversibly suppressed the genes, indicating the existence of a critical period in oligodendrocyte differentiation. In purified oligodendrocyte cultures prepared by mechanical dislodging of progenitor (O-2A) cells from mixed glial cultures, total cell number and total RNA yield were virtually unaffected by selenium deprivation; however, the developmental upregulation of the myelin genes was profoundly attenuated. Immunocytochemical analysis confirmed the suppressive effect of selenium deficiency on the differentiation of oligodendrocyte lineage cells, as seen from a significant decrease in the population of GalC⁺ and O4⁺ cells. Because the number of GC⁺ cells was more reduced than the number of O4⁺ cells, the results indicate that selenium deficiency may specifically inhibit the progression from immature to mature oligodendrocytes. J. Neurosci. Res. 47:626–635, 1997. © 1997 Wiley-Liss, Inc.     

Transplantation of bulk-separated oligodendrocytes into the brains of shiverer mutant mice: Immunohistochemical and electron microscopic studies on the myelination

Normal oligodendrocytes were separated from 7-day-old mouse (BALB/c) brains by the Percoll gradient method. Immunohistochemical staining with an anti-galactocerebroside serum revealed that about 85% of the separated cells were oligodendrocytes. The oligodendrocytes were transplanted into the corpus striatum of 4-week-old shiverer mutant mice which are characterized by the lack of myelin basic protein (MBP). Myelination by the implanted oligodendrocytes was investigated immunohistochemically and electron microscopically 6 weeks after operation. Certain areas in the corpus striatum were intensely stained with antiserum to MBP. Electron microscopic examination showed that some axons were surrounded by normal type myelin sheaths with major dense lines. These results clearly indicate that matured oligodendrocytes are able to survive and myelinate the host axons even in the adult brain.     

Abnormal brain MRI signal in 18q-syndrome not due to dysmyelination

Background

18q-Syndrome is a chromosomal disorder exhibiting various symptoms arising from the central nervous system. Brain magnetic resonance imaging (MRI) of patients with this syndrome usually demonstrates abnormal white matter intensities. This is widely believed to be due to impaired myelin formation because this syndrome involves the deletion of the myelin basic protein (MBP) gene in 18q23. However, this hypothesis has not been confirmed by actual pathology because early death is unusual and autopsy rarely performed.

Patient

A 6-year-old boy with ring chromosome 18 syndrome was examined by genetic analysis for the MBP gene, brain MRI, and autopsy.

Results

Haploinsufficiency of the MBP gene was confirmed. T₂-weighted MRI revealed diffuse high intensities throughout the cerebral white matter. Pathological examination showed the cerebral white matter to be uniformly stained by Klüver–Barrera and MBP immunohistochemical staining. Oligodendrocytes were immunoreactive for proteolipid protein and ferritin but not MBP. Electron microscopy revealed clusters of axons wrapped in compact myelin sheaths with distinct major dense lines. Holzer and immunohistochemical staining for glial fibrillary acidic protein showed extensive staining of the white matter and an increased number of glial filaments.

Conclusions

This pathological study demonstrated that in this disorder, the brain was well myelinated, contrary to established hypotheses about this disorder. The MRI signal abnormalities in 18q-syndrome could be attributed to gliosis and not to dysmyelination.