Fibroblast growth factor receptors (Fgfr) comprise a widely expressed family of developmental regulators implicated in oligodendrocyte (OL) maturation of the CNS. Fgfr2 is expressed by OLs in myelinated fiber tracks. In vitro , Fgfr2 is highly upregulated during OL terminal differentiation, and its activation leads to enhanced growth of OL processes and the formation of myelin-like membranes. To investigate the in vivo function of Fgfr2 signaling by myelinating glial cells, we inactivated the floxed Fgfr2 gene in mice that coexpress Cre recombinase ( cre ) as a knock-in gene into the OL-specific 2′,3′-cyclic nucleotide phosphodiesterase (Cnp1) locus. Surprisingly, no obvious defects were detected in brain development of these conditional mutants, including the number of OLs, the onset and extent of myelination, the ultrastructure of myelin, and the expression level of myelin proteins. However, unexpectedly, a subset of these conditional Fgfr2 knock-out mice that are homozygous for cre and therefore are also Cnp1 null, displayed a dramatic hyperactive behavior starting at ∼2 weeks of age. This hyperactivity was abolished by treatment with dopamine receptor antagonists or catecholamine biosynthesis inhibitors, suggesting that the symptoms involve a dysregulation of the dopaminergic system. Although the molecular mechanisms are presently unknown, this novel mouse model of hyperactivity demonstrates the potential involvement of OLs in neuropsychiatric disorders, as well as the nonpredictable role of genetic interactions in the behavioral phenotype of mice.

Paranodal axoglial junctions in myelinated nerve fibers are essential for efficient action potential conduction and ion channel clustering. We show here that, in the mature CNS, a fraction of the oligodendroglial 155 kDa isoform of neurofascin (NF-155), a major constituent of paranodal junctions, has key biochemical characteristics of a lipid raft-associated protein. However, despite its robust expression, NF-155 is detergent soluble before paranodes form and in purified oligodendrocyte cell cultures. Only during its progressive localization to paranodes is NF-155 (1) associated with detergent-insoluble complexes that float at increasingly lower densities of sucrose and (2) retained in situ after detergent treatment. Finally, mutant animals with disrupted paranodal junctions, including those lacking specific myelin lipids, have significantly reduced levels of raft-associated NF-155. Together, these results suggest that trans interactions between oligodendroglial NF-155 and axonal ligands result in cross-linking, stabilization, and formation of paranodal lipid raft assemblies.

Myelin-associated glycoprotein (MAG) has been implicated in inhibition of nerve regeneration in the CNS. This results from interactions between MAG and the Nogo receptor and gangliosides on the apposing axon, which generates intracellular inhibitory signals in the neuron. However, because myelin-axon signaling is bidirectional, we undertook an analysis of potential MAG-activated signaling in oligodendrocytes (OLs). In this study, we show that antibody cross-linking of MAG on the surface of OLs (to mimic axonal binding) leads to the redistribution of MAG into detergent (TX-100)-insoluble complexes, hyperphosphorylation of Fyn, dephosphorylation of serine and threonine residues in specific proteins, including lactate dehydrogenase and the beta subunit of the trimeric G-protein-complex, and cleavage of alpha-fodrin followed by a transient depolymerization of actin. We propose that these changes are part of a signaling cascade in OLs associated with MAG function as a mediator of axon-glial communication which might have implications for the mutual regulation of the formation and stability of axons and myelin.

The transient transfection of transgenes into oligodendrocytes offers an important tool for studying the function of proteins during myelin formation. Currently established procedures, however, have generally resulted in low survival rates and low levels of uptake of the transgene into primary oligodendrocyte progenitors. We describe an electroporation method which yields transient transfection of oligodendrocyte progenitors of up to 10–15% of the surviving cells, and provides approximately 104 surviving, transfected cells per electroporation reaction. In recent applications transgene expression persisted as the transfected progenitors progressed through subsequent stages of the oligodendrocyte lineage. This technique is expected to facilitate the study of the function of key proteins and lipids during the development of primary cultured oligodendrocytes.

Neutral glycolipids (NGL) were isolated and quantitated in 98 primary human brain tumors; 19 low grade astrocytomas (LGA), 12 anaplastic astrocytomas (AA), 37 high grade astrocytomas (HGA), 18 oligodendroglial tumors, and 12 primitive neuroectodermal tumors (PNET). In 38 of these, the nature of the hexose in the cerebroside was determined using immunothin-layer chromatographic techniques. Galactosylceramide (GalCer) was the major ceramide monohexoside (CMH), and glucosylcerebroside never comprised more than 6% of this fraction in any tumor type. Furthermore, there was no correlation between the proportion of glucosylcerebroside and histological diagnosis. AA had the most characteristic neutral glycolipid pattern, with high levels of total lipid, total neutral glycolipid, CMH, and ceramide dihexoside (CDH) but low water contents. Consistent with this glycolipid composition is the finding that AA usually had neither ceramide trihexoside (CTH) nor globoside. Oligodendrogliomas were somewhat similar to AA in having high levels of CMH and infrequently having CTH or globoside. However, oligodendrogliomas had low water and total lipid contents. PNET had low levels of total lipid, total NGL, and CMH, but frequently contained CTH and globoside. LGA had high water contents but low levels of total lipid and CMH. HGA tended to have intermediate levels of almost all constituents analyzed, probably reflecting the pronounced cellular heterogeneity of these tumors. The frequent presence of GalCer in astrocytomas raises the possibility that some of these contain a population of cells that are related to the oligodendroglial lineage. However, the low amounts of GalCer and infrequent presence of sulfatide in PNET is consistent with their lack of differentiation toward oligodendrocytes. It will be of interest to determine if the neutral glycolipid patterns reported here will correlate with patient survival and be of prognostic significance.

Oligodendrocytes synthesize dramatic amounts of myelin membrane. We hypothesized that this requires unique aspects of vesicular trafficking. Specific stages of the oligodendrocyte lineage were assayed for low molecular weight GTP‐binding proteins implicated in the regulation of vesicular transport pathway (two dimensional gel electrophoresis, [α‐ 32 P]GTP overlay). Consistent with the hypothesis, as oligodendrocytes differentiate from early progenitors to mature myelin‐producing cells, ≥≥ 12 small GTP‐binding proteins become up‐regulated. Myelin membrane also has a complex pattern of GTP‐binding proteins. Several of these proteins may be specific to oligodendrocytes, suggesting that oligodendrocytes may utilize cell‐type specific GTP‐binding proteins for biogenesis and maintenance of the myelin membrane.

The oligodendrocyte lineage in cerebrum is characterized by the expression of immunologically identified surface antigens resulting in the sequential appearance of three distinct phenotypes, A2B5+O4-, O4+GalC-, and O4+GalC+. In the present study we have placed O4+GalC- progenitors immunopanned from premyelinating rat cerebrum into a basal, defined medium that by itself does not support well either their proliferation or survival. The response of these progenitor cells to platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) was then examined. The results demonstrate that both PDGF and bFGF stimulated proliferation and short-term survival of newly cultured cells, but that their effect on the course of O4+GalC- differentiation was strikingly different. PDGF delayed postmitotic development by transiently reverting (ED50 = 3 ng/ml) O4+GalC- progenitors to A2B5+O4- preprogenitor-like cells that subsequently differentiated even in the continued presence of PDGF. bFGF restored mitogenic activity of the O4+GalC- progenitors to a saturable level at low doses (ED50 = 1 ng/ml); doses of bFGF > or = 10 ng/ml impaired differentiation of the progenitors into GalC+ cells and were also mitogenic for newly differentiated GalC+ oligodendrocytes. These data imply that bFGF supplants PDGF as a mitogen during lineage progression from A2B5+O4- to O4+GalC- progenitors. Lineage reversion of O4+GalC- cells in response to PDGF is suggested as a mechanism for facilitating remyelination by triggering the proliferative expansion of O4+GalC- progenitor-like cells persisting into adulthood.

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.

04+/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, 04+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 04+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.

The developmentally regulated appearance of surface immuno-reactivity of proligodendroblasts [oligodendrocyte progenitors reacting with monoclonal antibodies A007 and O4, but not anti-galactocerebroside (GalC), i.e., A007/O4+GalC-] to monoclonal antibodies R-mAb and O1 was studied both in culture and in vivo. In both cases staining with R-mAb shortly preceded that with O1; that is, a transient population of R-mAb+O1- cells was observed. R-mAb-O1+ cells were not detected. Differential staining with R-mAb and O1 was also noted at the subcellular level. In younger cultures in which R-mAb+ cells were first acquiring O1 immunoreactivity, many of these cells were stained by O1 only on the cell bodies and proximal portions of the processes, whereas in contrast R-mAb stained the whole cell, including the distal portions of the processes. Only in older, more mature R-mAb+ cells did O1 also stain the distal portions of processes. The expression of reactivity to R-mAb and O1 was compared to the proliferative capacity of the cells. Proliferation [assessed by bromodeoxyuridine (BrdU) incorporation] of both R-mAb+ and O1+ cells was negligible both in culture and in vivo. However, treatment of cells in culture with 10 ng/ml basic fibroblast growth factor resulted in an enhancement of proliferation of the R-mAb+ cells. Within the proliferating R-mAb+ BrdU+ population, 80% of the cells were O1- (i.e., anti-galactocerebroside negative). These events occur during a critical period of development when A007/O4+ proligodendroblasts begin to become post-mitotic and express surface galactocerebroside. The data demonstrate that the use of R-mAb as an "anti-GalC" must be interpreted with caution, and indicate the utility of dual staining with R-mAb and O1 to (1) further subdivide the oligodendrocyte lineage, thus identifying an additional, GalC- developmental compartment, and (2) observe the distribution of R-mAb and O1 immunoreactivity at a subcellular level.

We report the study of the in vivo morphology, differentiation, and proliferation of oligodendrocytes (OLs) and their progenitors identified by the antiglycolipid antibodies O4, R-mAb, and O1 in postnatal rat cerebellum, using a novel immunocytochemical staining protocol which allows the analysis of the expression of OL-specific glycolipids in live, unfixed brain slices. An analysis of the individual cells identified in double label immunocytochemistry indicated that the order of antigen expression in OLs during in vivo development is, first, antigens recognized by O4, second, antigens recognized R-mAb, and third, antigens recognized by O1. This order of antigen expression is correlated with increasing morphological complexity and is a pattern mimicked in many culture systems. In vivo O4 identified 3 distinct stages of the OL lineage: (1) morphologically simple proligodendrocyte antigen+ (POA+) R-mAb- blast cells localized at the leading edge of myelinogenesis; (2) morphologically more complex R-mAb+O1- cells; and (3) actively myelinating O1+ [i.e., galactocerebroside+ (GalC)] OLs residing within the white matter. Only the POA+R-mAb- cells incorporated BrdU in animals that were prelabeled 3 hr before immunocytochemistry. We have demonstrated in vivo the subdivision of pre-GalC+ OL progenitors into shorter, biologically noteworthy, stages of maturation. A spatial comparison of the cell populations identified by O4, R-mAb, and O1 demonstrated a progressive wave of OL maturation from the base of the cerebellum toward the folia. The data are consistent with the hypothesis that multiprocessed O4+GalC- progenitors are the most mature stage of the OL lineage with significant proliferative capacity and the first postmigratory stage in normal development.