Robert P. Skoff

PPAR δ agonists stimulate oligodendrocyte differentiation in tissue culture

Peroxisome proliferator–activated receptors (PPARs) are ligand‐activated transcription factors of the nuclear hormone receptor superfamily that have been described as master genes that switch cells from an undifferentiated phenotype to a differentiated phenotype. In the present investigation, we examined the possibility that ligands for PPARs are potent activators of oligodendrocyte (OL) differentiation and/or proliferation. Primary glial cultures and enriched OL cultures of neonatal mouse cerebra were treated with three different PPAR agonists: a PPAR γ–selective agonist, a PPAR δ–selective agonist, and a pan agonist selective for both PPAR γ and δ. Treatment with PPAR γ agonist does not have an effect on the differentiation of OLs; however, PPAR δ agonist and the pan agonist treatment accelerates the differentiation of OLs within 24 h of application in mixed glial cultures. The number of OLs with processes and huge membrane sheets increases two‐ to threefold in both groups. The increase in the size of the sheets is also mirrored by changes in the intensity and distribution of myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs. As compared to controls, the PPAR δ agonist–treated groups contain more OLs that have MBP and PLP mRNA extending into distal processes. These results indicate that PPAR δ plays a significant role in the maturation of OLs and regulates the size of OL sheets. BrdU immunostaining reveals that these agonists do not significantly stimulate proliferation of OLs expressing glycolipids. The studies in enriched OL cultures reproduce the effects of the PPAR agonists seen in the mixed glial cultures, indicating that the effect of the PPAR agonists is directly on the OLs and not via astrocytes. In the enriched cultures, the total number of OLs increases significantly in the PPAR δ agonist–treated groups, but BrdU immunostaining does not show an increased proliferation of cells. These findings suggest that PPAR δ increases the survival of cells and/or prevents cell death in enriched cultures. Although PPAR δ is expressed in various cell types, its role as a factor in the transcriptional regulation of OL differentiation has not been explored. We show for the first time that a ligand that serves as an agonist for PPAR δ activates the program of OL differentiation in primary and enriched OL cultures. GLIA 33:191–204, 2001.

Cultured oligodendrocytes mimic in vivo phenotypic characteristics: Cell shape, expression of myelin-specific antigens, and membrane production

Primary cultures of neonatal mouse cerebra were maintained for up to 4 weeks in the absence of neurons. Oligodendrocytes in these cultures pass through a sequence of cytoarchitectural change and antigen expression which mimics the differentiation of oligodendrocytes in vivo. The cell bodies and processes of oligodendrocytes first express the myelin-specific antigen galactocerebroside (GC) by 2 days in vitro. Myelin basic protein (MBP) appears several days later. The majority of oligodendrocytes then proceed to elaborate large sheets of membranous material from the tips and lengths of cell processes. These membranous sheets, which contain GC and MBP, are reminiscent of unwrapped myelin profiles in vivo. As with the cell bodies and processes, GC is inserted into the sheets several days before MBP. Our results establish that oligodendrocytes cultured without neurons are able to produce extensive membranes containing myelin-specific antigens. They also suggest that oligodendrocyte shape and membrane production are, in part, regulated from within the oligodendrocyte itself.

Gliogenesis in rat optic nerve: Astrocytes are generated in a single wave before oligodendrocytes☆

The time of origin for astrocytes in the rat optic nerve was investigated to determine whether this cell type is generated in two waves, a first wave which occurs before the formation of oligodendrocytes and a second wave which occurs after the peak period of oligodendrocyte formation. To answer this question, multiple injections of radioactive thymidine were administered to rats after the peak period of oligodendrocyte production in the optic nerve and the animals were sacrificed several weeks after the first injection. Thymidine-labeled cells in the optic nerve were identified with the electron microscope. Of the labeled cells, greater than 80% are oligodendrocytes, 4% are microglia, 2% are astrocytes, and the remainder are unclassifiable. The thymidine-labeled cells in the nerve were not immunostained for glial fibrillary acidic protein (GFAP), a marker characteristic of astrocytes. The number of thymidine-labeled glia generated after the second postnatal week is a small fraction of the total number of glia generated neonatally. No evidence exists for a second wave of astrocyte formation in the rat optic nerve as has been suggested in a study by Miller et al. (1985, Dev. Biol. 111, 35-41); rather, the vast majority of astrocytes are generated during the first 2 postnatal weeks and these data are in keeping with classical studies of gliogenesis. The question of whether astrocytes in the rat optic nerve arise directly from division of an undifferentiated, common progenitor cell or from a cell committed to the astrocyte lineage was addressed by combining thymidine autoradiography with GFAP immunocytochemistry. Rats were sacrificed 1 hr after an injection of thymidine and their nerves were processed for GFAP immunocytochemistry and autoradiography. During the first postnatal week, many thymidine-labeled cells are immunostained for GFAP. These observations demonstrate that cells committed to the astrocyte lineage divide neonatally and give rise to additional astrocytes.

Increased proliferation of oligodendrocytes in the hypomyelinated mouse mutant-jimpy

Previous studies of the hypomyelinated mouse mutant jimpy have shown that the number of oligodendrocytes are reduced about 50%. To determine the cause of the cellular reduction, light and electron microscopy were combined with thymidine autoradiographic techniques. The number of neuroglial cells which incorporate radioactive thymidine in the mutants is increased severalfold over control values. Electron microscopic autoradiograms indicate the majority of the labeled cells are oligodendroblasts. However, the total number of glia in the white matter of jimpy and control animals is the same during development and even up to the time of the animals death. The presence of mitotic cells suggest that the oligodendrocytes undergo division but the abundance of dying cells suggests that they die sometime afterwards. The results of the quantitative autoradiographic studies in combination with our other data strongly suggest that the immediate failure of these cells to form myelin sheaths is due to a shortened life span and/or continued cell proliferation.

The Pathobiology of Myelin Mutants Reveal Novel Biological Functions of the MBP and PLP Genes

Substantial biological data indicate that the myelin basic protein (MBP) and myelin proteolipid protein (PLP/DM20) genes produce products with functions beyond that of serving as myelin structural proteins. Much of this evidence comes from studies on naturally‐occurring and man‐made mutations of these genes in mice and other species. This review focuses upon recent evidence showing the existence of other products of these genes that may account for some of these other functions, and recent studies providing evidence for alternative biological functions of PLP/DM20. The MBP and PLP/DM20 genes each encode the classic MBP and PLP isoforms, as well as a second family of proteins that are not involved in myelin structure. The biological roles of these other products of the genes are becoming clarified. The non‐classic MBP gene products appear to be components of transcriptional complexes in the nucleus, and they also may be involved in signaling pathways in T‐cells and in neural cells. The non‐classic PLP/DM20 gene products appear to be components of intracellular transport vesicles in oligodendrocytes. There is evidence for other functions of the classic PLP/DM20 proteins, including a role in neural cell death mechanisms, autocrine and paracrine regulation of oligodendrocytes and neurons, intracellular transport and oligodendrocyte migration.

Proliferation and death of oligodendrocytes and myelin proteins are differentially regulated in male and female rodents.

Sexual dimorphism of neurons and astrocytes has been demonstrated in different centers of the brain, but sexual dimorphism of oligodendrocytes and myelin has not been examined. We show, using immunocytochemistry and in situ hybridization, that the density of oligodendrocytes in corpus callosum, fornix, and spinal cord is 20–40% greater in males compared with females. These differences are present in young and aged rodents and are independent of strain and species. Proteolipid protein and carbonic anhydrase-II transcripts, measured by real-time PCR, are approximately two to three times greater in males. Myelin basic protein and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase, measured by Western blots, are 20–160% greater in males compared with females. Surprisingly, both generation of new glia and apoptosis of glia, including oligodendrocytes, are approximately two times greater in female corpus callosum. These results indicate that the lifespan of oligodendrocytes is shorter in females than in males. Castration of males produces a female phenotype characterized by fewer oligodendrocytes and increased generation of new glia. These findings indicate that exogenous androgens differentially affect the lifespan of male and female oligodendrocytes, and they can override the endogenous production of neurosteroids. The data imply that turnover of myelin is greater in females than in males. μ-Calpain, a protease upregulated in degeneration of myelin, is dramatically increased at both transcriptional and translational levels in females compared with males. These morphological, molecular, and biochemical data show surprisingly large differences in turnover of oligodendrocytes and myelin between sexes. We discuss the potential significance of these differences to multiple sclerosis, a sexually dimorphic disease, whose progression is altered by exogenous hormones.

Hypoxic-ischemic injury results in acute disruption of myelin gene expression and death of oligodendroglial precursors in neonatal mice

Studies of ischemic brain injury in neonatal rodents have focused upon the pathophysiology of neuronal damage. Much less consideration has been given to white matter injury, even though it is a major contributor to chronic neurological dysfunction in children. In the human neonate, particularly in those born prematurely, periventricular white matter is highly susceptible to hypoxic–ischemic (H–I) injury. To understand the basis for this selective vulnerability, we examined myelin gene expression and cell death in the subventricular layer and the surrounding white matter of neonatal mice following H–I insult. Using an in situ hybridization technique that gives high resolution and is very sensitive, we examined myelin basic protein and proteolipid protein gene expression three and twenty‐four hours after a H–I insult. To elicit unilateral forebrain hypoxic and ischemic injury, 9–10‐day‐old mice underwent right carotid artery ligation followed by timed (40–70 min) exposure to 10% oxygen. Twenty‐four hours following H–I, myelin basic protein and proteolipid protein transcripts were markedly reduced in striatum, external capsule, fornix, and corpus callosum in the injured side. Three hours after lesioning (ligation+70 min hypoxic exposure) myelin basic protein gene transcripts were visibly reduced in the ipsilateral white matter tracts. Interestingly, some cells in the subventricular layer expressed proteolipid protein transcripts, and 3 h after a H–I insult they were degenerating in the injured but not contralateral side. TUNEL staining showed an increase in the number of positive cells in the injured subventricular layer and corpus callosum but the adjacent striatum did not show a corresponding change in the number of TUNEL labeled cells. Ultrastructural studies of the subventricular zone and corpus callosum 3 h after H–I revealed that many subventricular cells, glial cells in the corpus callosum, and callosal axons in the injured side had already degenerated. However, the subventricular cells, glia and axons in the contralateral corpus callosum were spared. Many cells in the injured corpus callosum exhibited a apoptotic morphology; yet more mature oligodendrocytes in this region appeared normal. Our results show that a H–I insult causes a surprisingly swift and dramatic degenerative response in the subventricular layer and adjacent white matter. Within 3 h after H–I, the programmed cell death cascade was initiated; internucleosomal DNA degradation took place in subventricular and glial cells; oligodendrocyte progenitors died and axonal degeneration in the ipsilateral corpus callosum was extensive. The swiftness of the subventricular and glial cell degeneration suggests the H–I insult directly targets glia, as well as neurons, and raises the provocative question of whether glia exert damaging effects upon neurons and axons. Since the severity of the H–I insult can be modulated by varying the duration of hypoxia, the model is ideal to study whether oligodendrocyte progenitors are more susceptible to death than mature oligodendrocytes, whether mature oligodendrocytes de‐differentiate and then are induced to remyelinate surviving axons, and/or whether oligodendrocyte progenitors in the subventricular layer can be stimulated to proliferate, migrate, and remyelinate the surviving axons.

Member of the peroxisome proliferator-activated receptor family of transcription factors is differentially expressed by oligodendrocytes.

Peroxisome proliferator–activated receptors (PPARs) are ligand‐activated transcription factors that form a subfamily within the steroid hormone receptor group. Recent work has shown that one member of this group, PPARγ, plays a central role in adipocyte differentiation. As oligodendrocytes are major lipid‐producing cells, we investigated whether members of the PPAR family were present in oligodendrocytes and whether known PPAR activators affect oligodendrocyte differentiation. Polymerase chain reaction and nuclease protection analyses demonstrated that the principal PPAR present in optic nerve and sciatic nerve is PPARδ, whereas adipose tissue expresses mainly PPARγ. In situ hybridization of primary glial cultures revealed PPARδ message in oligodendrocytes but not in astrocytes. PPARδ message was strongly expressed in immature oligodendrocytes, suggesting a role in oligodendrocyte differentiation. Glial cultures containing immature oligodendrocytes were treated with CP 68,722 and bromopalmitate, compounds known to activate PPARs in adipocytes. These agents increased the number of oligodendrocytes with membrane sheets three‐ to fourfold, accelerated the rate of formation of membrane sheets, and increased the size of the membrane sheets. The abundant expression of PPARδ in oligodendrocytes in vivo and in vitro suggests that this PPAR plays a critical role in oligodendrocyte development and that PPAR activators can be used to manipulate oligodendrocyte maturation in tissue culture. J. Neurosci. Res. 51:563–573, 1998.

New oligodendrocytes are generated after neonatal hypoxic‐ischemic brain injury in rodents

Neonatal hypoxic‐ischemic (HI) white matter injury is a major contributor to chronic neurological dysfunction. Immature oligodendrocytes (OLGs) are highly vulnerable to HI injury. As little is known about in vivo OLG repair mechanisms in neonates, we studied whether new OLGs are generated after HI injury in P7 rats. Rats received daily BrdU injections at P12–14 or P21–22 and sacrificed at P14 to study the level of cell proliferation or at P35 to permit dividing OLG precursors to differentiate. In P14 HI‐injured animals, the number of BrdU+ cells in the injured hemisphere is consistently greater than controls. At P35, sections were double‐labeled for BrdU and markers for OLGs, astrocytes, and microglia. Double‐labeled BrdU+/myelin basic protein+ and BrdU+/carbonic anhydrase+ OLGs are abundant in the injured striatum, corpus callosum, and the infarct core. Quantitative studies show four times as many OLGs are generated from P21–35 in HI corpora callosa than controls. Surprisingly, the infarct core contains many newly generated OLGs in addition to hypertrophied astrocytes and activated microglia. These glia and non‐CNS cells may stimulate OLG progenitor proliferation or induce their migration. At P35, astrogliosis and microgliosis are dramatic ipsilaterally but only a few microglia and some astrocytes are BrdU+. This finding indicates microglial and astrocytic hyperplasia occurs shortly after HI but before the P21 BrdU injections. Although the neonatal brain undergoes massive cell death and atrophy the first week after injury, it retains the potential to generate new OLGs up to 4 weeks after injury within and surrounding the infarct.

Expression of galactocerebroside in developing normal and jimpy oligodendrocytes in situ

SummaryIntense and specific immunostaining of oligodendrocytesin vivo has been obtained for the first time using antibodies to galactocerebroside. We have examined the differentiation of oligodendrocytes in normal mice and then compared their differentiation to the myelin-deficient mouse jimpy, using immunoperoxidase, immunogold and immunofluorescence labelling techniques. We also compared staining for galactocerebroside with staining obtained using antibodies to myelin basic protein, carbonic anhydrase II, 2′, 3′-cyclic nucleotide 3′-phosphohydrolase and proteolipid protein. The results of this comparative study confirm previous tissue culture studies and show that galactocerebroside is specific for oligodendrocytesin situ. As in tissue culture, galactocerebroside is one of the earliest oligodendrocyte markers to be expressed, making it an important marker for studying the differentiation of this cell type.The shape of oligodendrocytesin situ changes distinctly with time, shifting from an early stellate form with numerous spidery processes to a cell with a few processes radiating from the perikaryon. These morphological changes are observed for both normal and jimpy mice and they parallel those describedin vitro. Oligodendrocytes in jimpy mice express most myelin markers, but the staining within the cells is generally less intense than in normal oligodendrocytes and the antigens are restricted to the cell body and processes without being incorporated into myelin sheaths. Quantification of the number of oligodendrocytes stained for galactocerebroside in normal and jimpy mice show that their number is not reduced in the corpus callosum and cerebellum during the first 2 weeks postnatal. This finding shows that many cells in jimpy mice which were considered to be unclassifiable by the application of morphological criteria have, in fact, differentiated to the stage where they are galactocerebroside-positive.