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.     

Expression of janusin (J1–160/180) in the retina and optic nerve of the developing and adult mouse

We have analyzed the expression of the oligodendrocyte-derived extracellular matrix molecule janusin (previously termed J1–160/180) in the retina and optic nerve ofdeveloping and adult mice using indirect light and electron microscopic immunocytochemistry, immunoblot analysis, and enzyme-linked immunosorbent assay. In the optic nerve, janusin is not detectable in neonatal and only weakly detectable in 7-day-old animals. Expression is at a peak in 2- or 3-week-old animals and subsequently decreases with inceasing age. In the retina, expression inceases until the third postnatal week and then remains at a constant level. In immunocytochemical investigations at the light microscopic level, janusin was found in the myelinated regions of the nerve with spots of increased immunoreactivity possibly corresponding to an accumulation of the light microscopic level, janusin was found in the myelinated regions of the nerve with sports of increased immunoreactivity possibly corresponding to an accumulation of the molecule at the nodes of Ranvier. At the electron microscopic level, contact sites between unmyelinated axons, between axons, and glial cells, and between axons and processes of myelinating oligodendrocytes were immunoreactive. Cell surfaces of astrocytes at the periphery of the nerve and forming the glial-limiting membrane, in contrast, were only weakly immunopositive or negative. In cell cultures of young postnatal mouse or rat optic nerves, oligodendrocytes and type-2 astrocytes, but not type-1 astrocytes were stained by janusin antibodies. In the oligodendrocyte-free retina, janusin was detectable in association with neuronal cell surfaces, but not with cell surfaces of Muller cells or retinal astrocytes. Our observations indicate that expression of janusin in the optic nerve and in the retina is developmentally differentially regulated and that other cell types, in addition to oligodendrocytes, express the molecule. Since the time course of janusin expression in the optic nerve coincides with the appearance of oligodendrocytes and myelin and since janusin is associated with cell surfaces of oligodendrocytes and outer aspects of myelin sheaths and is concentrated at nodes of Ranvier, we suggest that janusin is functionally involved in the process of myelination. © 1993 Wiley-Liss, Inc.     

Oligodendroglial development in human fetal cerebrum

We have successfully established mixed glial cell primary cultures prepared from individual fetal human brains (15–18 weeks' gestation in age). Cultures were maintained for as long as 3 months in either 10% fetal calf serum (FCS) or serum-free chemically defined medium (CDM). By morphological and immunohistochemical criteria, the precursor cell for human oligodendrocytes (O-2A cell) was identified. This cell exhibited the bipolar morphology and A2B5-positive (A2B5⁺) immunoreactivity typical of the O-2A precursor cell. With time in culture, cells possessing a stellate morphology appeared, some of which stained with the O4 antibody, indicative of cell differentiation in the oligodendroglial lineage. At yet older culture age, arborized cells bearing the O1 (galactocerebroside, GC) immunohistochemical marker and displaying the morphological characteristics typical of more mature oligodendrocytes were found, confirming their oligodendroglial identity. Oligodendroglial differentiation was supported best by CDM rather than FCS. To complement these observations, double immunofluorescent studies were performed on parietal sections from human fetal brains at 20 to 22 weeks of gestation. Bipolar A2B5⁺, multipolar A2B5⁺/O4⁺, and arborized A2B5^?/O1⁺ cells were found, thus confirming the presence of oligodendrocytes in human fetal brain at this stage of prenatal development and consistent with the observations made in cell culture.     

Changes in ion channel expression during in vitro differentiation of trout oligodendrocyte precursor cells

Voltage-gated ionic currents were studied in cultured trout oligodendrocyte precursor cells derived from larval trout brain with the whole-cell mode of the patch-clamp technique. These bipolar cells which carry the ganglioside epitope A2B5 on their surface differentiated in vitro into immature multipolar oligodendrocytes expressing the myelin glycoprotein IP2, which signifies the initial step of oligodendroglial development in fish CNS. Depolarization above -40 mV activated a fast transient sodium inward current that was eliminated by substituting Na⁺ for choline and blocked in the presence of 1 μM TTX. The kinetics and the voltage-dependence of inactivation (half-maximal inactivation at -68 mV) resembled those of sodium currents described in mammalian oligodendrocyte precursor cells and CNS neurons. The expression of Na⁺ channels was developmentally regulated, since high amplitudes were measured only in A2B5⁺ cells with a characteristic bipolar morphology of glial progenitors. Depolarizing voltage steps, additionally elicited outward potassium currents that were sensitive to external 4-AP. In a subpopulation of cells this outward current consisted of a sustained and a transient component. The amplitude of both components were dependent on the prepulse potential. © 1995 Wiley-Liss, Inc.     

Complexity analysis of oligodendroglial processes expressing myelin-associated glycoprotein

Oligodendroglia synthesize myelin in the mammalian central nervous system. Mature oligodendroglia have been identified in culture by two criteria: the expression of molecules characteristic of myelin, such as galactocerebroside (galC) and myelin-associated glycoprotein (MAG), and the elaboration of complex processes. Myelin gene expression can be documented by the binding of specific antibodies and antisera to the myelin-specific molecules; process complexity can be described by the fractal dimension, D. In this study, anti-MAG antisera was used to document MAG expression in the processes of oligodendroglia. Eighty percent of the galC⁺ oligodendroglia bound anti-MAG antiserum. With time in culture, MAG immunoreactivity seemed to extend from the cell soma into the oligodendroglial processes. To quantify this observation, fractal dimensions were calculated using either galC or MAG immunoreactivity to visualize oligodendroglial processes. A fractal dimension of 1.5 was calculated for O1⁺ processes by day 4 of culture; this value for D remained constant over the course of 1 month in culture. The fractal dimension calculated for MAG⁺ processes increased from 1.2 to 1.5 over the course of 28 days in culture. This change in fractal dimension confirms our visual impression that galC-containing processes acquire MAG slowly over the course of several weeks in culture. © 1996 Wiley-Liss, Inc.     

Differentiation-regulated loss of the polysialylated embryonic form and expression of the different polypeptides of the neural cell adhesion molecule by cultured oligodendrocytes and myelin

The expression of the neural cell adhesion molecule (N-CAM) on cultured murine oligodendrocytes, their precursors, and myelin was examined by indirect immunofluorescence, biosynthetic radiolabeling followed by immunoprecipitation and Western blot analysis, using antibodies specific for various forms of the molecule. In all culture systems studied, whether the oligodendrocytes were cultured as an enriched fraction containing precursor cells or in the presence of astrocytes and neurons, a similar differentiation-stage-related expression of N-CAM was seen. At early developmental stages many tetanus toxin receptor- and A2B5 antigen-positive putative oligodendrocyte precursors with bipolar morphology were seen and found to express N-CAM in its embryonic form. Of the 04 antigen-positive immature oligodendrocytes with few slender processes most expressed N-CAM, but few the embryonic form of N-CAM. The more mature 01 or 010 antigen-positive oligodendrocytes were found to express exclusively the adult form of N-CAM. Oligodendrocytes synthesized the 120 and 140 kD forms of N-CAM (N-CAM 120 and N-CAM 140), but not N-CAM 180, although with differentiation, N-CAM 120 predominated in oligodendrocytes and also in pure myelin. N-CAM 120 could be released from oligodendrocytes and myelin by phosphatidylinositol-specific phospholipase C, suggesting that in both oligodendrocytes and myelin N-CAM 120 is inserted into the membrane by covalent linkage to phosphatidylinositol.     

Expression of the immunoglobulin superfamily cell adhesion molecule F3 by oligodendrocyte-lineage cells

We have analysed the expression of glycosylphosphatidylinositol (GPI)-anchored proteins by oligodendrocyte-lineage cells. Biosynthetic labeling of mouse oligodendroglial primary cultures and an oligodendroglial precursor cell line demonstrated that these cells synthesis a variety of different GPI-anchored proteins. GPI-anchored proteins were isolated as a bulk preparation from the precursor cell line, and the individual proteins separated by 2D gell electrophoresis and analysed by microsequencing after tryptic digestion of the separated components. One of the most prominent GPI-anchored proteins synthesized by the cell line was identified as the cell adhesion molecule F3, previously thought to be exclusively expressed by neurons. Western blotting and immunoprecipitation with several polyclonal sera confirmed the expression of F3 by oligodendrocyte-lineage cells and demonstrated the presence of F3 in myelin. Double staining with a panel of oligodendrocyte-specific antibodies and anti-F3 antibodies of cerebellar cultures, as well as oligodendrocytes isolated by panning, showed a colocalization of F3 with oligodendrocyte markers. Oligodendrocyte F3 is shown to be susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage, similar to neuronal F3. Northern blots demonstrated that the oligodendroglial F3 mRNA is the same size as the neuronal message; however, no F3 mRNA could be detected in cortical astrocytes and an astrocytic cell line. Thus, in addition to the expression by neurons, the cell-type specificity of F3 expression must be extended to oligodendroglial cells, underscoring the importance of this Ig superfamily member in the nervous system. GLIA 19:199–212, 1997. © 1997 Wiley-Liss, Inc.     

Cystine/glutamate antiporter blockage induces myelin degeneration

The cystine/glutamate antiporter is a membrane transport system responsible for the uptake of extracellular cystine and release of intracellular glutamate. It is the major source of cystine in most cells, and a key regulator of extrasynaptic glutamate in the CNS. Because cystine is the limiting factor in the biosynthesis of glutathione, and glutamate is the most abundant neurotransmitter, the cystine/glutamate antiporter is a central player both in antioxidant defense and glutamatergic signaling, two events critical to brain function. However, distribution of cystine/glutamate antiporter in CNS has not been well characterized. Here, we analyzed expression of the catalytic subunit of the cystine/glutamate antiporter, xCT, by immunohistochemistry in histological sections of the forebrain and spinal cord. We detected labeling in neurons, oligodendrocytes, microglia, and oligodendrocyte precursor cells, but not in GFAP⁺ astrocytes. In addition, we examined xCT expression and function by qPCR and cystine uptake in primary rat cultures of CNS, detecting higher levels of antiporter expression in neurons and oligodendrocytes. Chronic inhibition of cystine/glutamate antiporter caused high toxicity to cultured oligodendrocytes. In accordance, chronic blockage of cystine/glutamate antiporter as well as glutathione depletion caused myelin disruption in organotypic cerebellar slices. Finally, mice chronically treated with sulfasalazine, a cystine/glutamate antiporter inhibitor, showed a reduction in the levels of myelin and an increase in the myelinated fiber g‐ratio. Together, these results reveal that cystine/glutamate antiporter is expressed in oligodendrocytes, where it is a key factor to the maintenance of cell homeostasis. GLIA 2016. GLIA 2016;64:1381–1395     

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.     

Tracing human oligodendroglial development in vitro

Human neural precursor cell cultures (neurospheres) were established from fetal brain tissues of 15-20 gestation weeks and propagated for over a year in the presence of epidermal growth factor, basic fibroblast growth factor and leukemia inhibitory factor. Neurospheres were differentiated without the presence of above growth factors to follow the development of oligodendroglia. Oligodendroglial progenitors, identified by their bipolar morphology and expression of platelet-derived growth factor receptor-alpha (PDGFRalpha), emerged from spheres as early as 1 DIV; O4+ cells with bipolar to multipolar processes were observed at 3 DIV whereas O1+ multiprocess-bearing oligodendroglia did not appear until 5-7 DIV. They further differentiated to myelin basic protein-expressing oligodendrocytes after 2-3 weeks in culture. Thus, human oligodendroglial maturation in vitro follows the same pathway as rat cells but takes twice as long as their rodent counterparts. Bromodeoxyuridine incorporation indicated that PDGFRalpha-expressing cells but not O4+ oligodendroglia proliferated. More oligodendroglial progenitors incorporated BrdU and more O4+ cells survived when they were in contact with neurons and astrocytes than when they developed beyond the astrocyte layer. In addition, oligodendroglia on astrocytes had a complex process branching whereas those growing beyond astrocyte layer often formed membrane sheaths. Thus the survival, proliferation and maturation of oligodendroglia are influenced by other cell types.     

Culture of oligodendrocyte precursor cells (NG2(+)/O1(-)) and oligodendrocytes (NG2(-)/O1(+)) from embryonic rat cerebrum

I have established a novel culture technique of oligodendrocyte precursor cells (OPC, NG2(+)/O1(-)) and oligodendrocytes (OL, NG2(-)/O1(+)) from embryonic day 16 (E16) rat cerebrum distinguished by morphological and immunocytochemical analyses. This novel protocol does not require immunopanning techniques using specific antibodies. The OPC were isolated by two passages every 7 days and culturing them on Petri dishes in different culture medium at each step to eliminate neurons and astrocytes. The yield of pure OPC and OL was relatively large compared with immunopanning techniques. In addition, to examine myelination processes in vitro, the OL from different stages were co-cultured with primary neurons from E17 rat cerebrum on a feeder layer of rat cerebrum astrocytes. This co-culture system resulted in successful formation of myelin in the presence or absence of astrocytes. This culture system was useful for studying OL lineage and initiation of myelination.     

Pluripotent stem cell‐derived radial glia‐like cells as stable intermediate for efficient generation of human oligodendrocytes

Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor‐mediated expansion with pre‐exposure to the differentiation‐inducing agent retinoic acid and subsequent immunoisolation of CD133‐positive cells. This protocol yields an adherent and self‐renewing population of hindbrain/spinal cord radial glia (RG)‐like neural precursor cells (RGL‐NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL‐NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate‐determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL‐NPCs efficiently convert into NG2‐positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL‐NPCs expedite the generation of PSC‐derived oligodendrocytes with O4‐, 4860‐, and myelin basic protein (MBP)‐positive cells that already appear within 7 weeks following growth factor withdrawal‐induced differentiation. Thus, RGL‐NPCs may serve as robust tool for time‐efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. GLIA 2015;63:2152–2167     

The Extracellular Matrix Molecule Janusin Regulates Neuronal Morphology in a Substrate- and Culture Time-dependent Manner

Janusin is an extracellular matrix glycoprotein with structural homology to tenascin. In search of extracellular matrix components which govern the differentiation of neurons in the central nervous system, we have investigated the influence of janusin on the differentiation of hippocampal neurons in vitro. Janusin coated onto nitrocellulose was a good substrate for attachment of cell bodies and neurite outgrowth after 21 h of culture. Most cells exhibited a polarized morphology with one long major neurite and one or two short minor neurites. When janusin was coated onto a polyornithine-conditioned plastic surface, it increased the polarity of neurons in that the length of major neurites was increased and the length and number of minor neurites were decreased when compared with the control polyornithine-conditioned plastic without janusin. As we have shown before for tenascin, laminin and fibronectin, polarization was preceded by an increase in the number and length of all neurites during the first hours after cell plating. This study therefore adds janusin to the increasing number of extracellular matrix glycoproteins which promote axonal but not dendritic growth.     

Down-regulation of mu-opioid receptor expression in rat oligodendrocytes during their development in vitro

In the central nervous system, opioid receptors are found in neurons and also in glial cells. To gain more information on their presence and possibly on their function, we investigated the expression of mu-opioid receptors (MOR) during oligodendroglial cell development in two culture systems. In these models, during the first days, the cells are O-2A bipotential progenitor cells (also called OPCs; oligodendrocyte precursor cells), and then they differentiate into oligodendrocytes, which mature. In the first system, oligodendroglial cells, derived from newborn rat brain hemispheres, are grown in primary culture in the presence of a confluent layer of astrocytes, and they differentiate slowly. In the second, cells are specifically detached from the mixed cultures of the first system and are grown thereafter alone in secondary culture, a condition allowing a rapid cell differentiation. Under both conditions OPCs and immature oligodendrocytes were found to express a high level of MOR mRNA, whereas mature oligodendrocytes did not express it at all. The decrease of MOR expression during oligodendrocyte maturation was progressive, suggesting that it was not a primary effect of differentiation but an indirect secondary effect. Our study also shows that basic fibroblast growth factor (bFGF), which has been claimed by some authors to induce a dedifferentiation of the mature oligodendrocytes, and retinoic acid (RA), which had not been tested before, were not able to restore MOR expression in mature oligodendrocytes. These results indicate that bFGF and RA neither reverse the maturation process nor dedifferentiate the cells. However, RA was found to inhibit almost completely the expression of the myelin basic protein. The main result of this study is that MOR is expressed in progenitors and in immature oligodendrocytes, but not in mature oligodendrocytes. This suggests that MOR could be involved in some developmental process of the cells of the oligodendroglial lineage.     

Distribution and differentiation of A2B5+ glial precursors in the developing rat spinal cord

In many regions of the rat central nervous system, oligodendrocytes develop from migratory A2B5⁺ precursor cells. In the rat spinal cord, during early embryonic development the capacity for oligodendrogenesis appears to be restricted to ventral regions of the spinal cord, while cultures of postnatal rat spinal cord contain a distinct population of A2B5⁺ astrocyte precursors. To determine if, as in other regions of the CNS, spinal cord A2B5⁺ cells give rise directly to oligodendrocytes and astrocytes, the initial distribution, and subsequent dispersion, proliferation, and differentiation of spinal cord A2B5⁺ cells have been examined in both explant and dissociated cell cultures. Spinal cord oligodendrocytes develop from A2B5⁺ cells. At E14, A2B5⁺ cells are restricted to ventral regions of the spinal cord and as development proceeds they become more uniformly distributed throughout the spinal cord. In explant cultures, greater than 95% of the explants that contain oligodendrocytes also contain A2B5⁺ cells and a proportion of mature oligodendrocytes retain detectable A2B5 immunoreactivity briefly on their surface. The maturation of spinal cord oligodendrocyte precursors occurs in a number of distinct stages characterized by the expression of O4 immunoreactivity, which first appears at E16, and GC immunoreactivity, which first appears at E18. As spinal cord oligodendrocyte precursors acquire O4 immunoreactivity they appear to lose the ability to proliferate in response to PDGF but retain the ability to proliferate in response to bFGF, suggesting that the control of proliferation of oligodendrocyte precursors is, in part, dependent on their maturational state. In the presence of high serum, spinal cord A2B5⁺ cells fail to develop in isolated E14 dorsal spinal cord cultures, while in ventral cultures they subsequently differentiate into A2B5⁺ astrocytes suggesting that A2B5⁺ astrocyte precursors are also initially ventrally located. Unlike oligodendrocyte differentiation, however, the differentiation of spinal cord A2B5⁺ cells into astrocytes is delayed in early embryonic-derived cultures compared to those from older animals. These observations suggest that local influences may regulate the timing of spinal cord A2B5⁺ astrocyte development, but not spinal cord oligodendrocyte development. © 1994 Wiley-Liss, Inc.     

Interaction with antigen-specific T cells regulates expression of the lactate transporter MCT1 in primary rat astrocytes: specific link between immunity and homeostasis

Monocarboxylates like lactate are provided by astrocytes and can be used as fuel by neurons and oligodendrocytes. In an autoimmune inflammatory environment, homeostatic functions of astrocytes are incompletely understood. In primary Lewis rat astrocytes, co-culture with MHC class II-restricted myelin basic protein (MBP)-specific T cells in the presence of MBP resulted in a marked upregulation of the astrocytic lactate transporter MCT1 that is to export lactate into the extracellular space. It was evident that the increase in MCT1 was triggered by T cells in an antigen-dependent manner. The glial isoform of the glucose transporter GLUT1 was not regulated under these conditions. T-cell blasts that had been pre-activated by antigen and splenic antigen-presenting cells (APCs) beforehand also led to an increase in the expression of astrocytic MCT1 after co-culture. Resting T cells did not induce a relevant upregulation of MCT1 in astrocytes. However, resting T cells stimulated the expression of MCT1 when anti-MHC class II antibodies, but not when anti-MHC class I antibodies, were added to the co-culture. Therefore, even in the presence of inactive T cells, complexation of MHC class II molecules on astrocytes might lead to the regulation of certain astrocytic transport proteins. Consistent with the in vitro experiments, an upregulation of MCT1 was observed in the spinal cord of autoimmune encephalitic rats while GLUT1 expression appeared to be unchanged. This T-cell-mediated regulation of MCT1 might contribute to a compensatory or protective mechanism in order to guarantee substrate pools for neurons and oligodendrocytes under inflammatory conditions.     

Expression of the anaphylatoxin C5a receptor in the oligodendrocyte lineage

Expression of the C5a receptor in the central nervous system has been demonstrated on microglia, astrocytes and neurons. In the present study, we demonstrate C5aR expression in vitro by rat and murine O2-A progenitor cells and oligodendrocytes. We also observed that in vitro differentiation of O2-A progenitors into mature oligodendrocytes is accompanied by down-regulation of C5aR mRNA expression. These results suggest that the C5aR may be a marker for oligodendroglial differentiation and play a role in oligodendrocyte function.     

Collateral neuronal apoptosis in CNS gray matter during an oligodendrocyte‐directed CD8+ T cell attack

Demyelination and death of oligodendrocytes accompanied by transection of neurites and neuronal apoptosis are pathological hallmarks of cortical and subcortical gray matter lesions in demyelinating viral and autoimmune inflammatory CNS disorders. In these disorders, leukocortical lesions, containing the perikarya of most efferent neurons, display pronounced infiltration by CD8⁺ T cells of putative specificity for oligodendrocyte‐ and myelin‐related antigens. Hence, neuronal apoptosis in gray matter lesions may be a collateral effect of an oligodendrocyte‐directed attack by CD8⁺ T cells. To challenge this hypothesis, we transferred activated antigen‐specific CD8⁺ T cells (OT‐I T cells) into acute coronal brain slices from mice selectively expressing ovalbumin as a cytosolic neo‐self‐antigen in oligodendrocytes (ODC‐OVA mice). We studied mechanisms and kinetics of oligodendroglial and neuronal apoptosis in the neocortex and hippocampus, using multicolor staining for different cell types and activated caspase‐3. Within the gray matter, a single OT‐I T cell caused simultaneous caspase‐3 activation in about 30 ODCs and 10 neurons within 6 h in a strictly antigen‐dependent manner. Experiments with OT‐I T cells genetically deficient for perforin or the granzyme B‐cluster and with blocking anti‐FasL antibodies as well as proinflammatory cytokines revealed, that collateral apoptosis of neurons was likely due to a spillover of perforin and granzyme(s) from the OT‐I T cell itself or the immunological synapse that it selectively formed with antigen‐presenting oligodendrocytes. Collateral neuronal apoptosis could contribute to substantial neuronal loss in gray matter lesions and cause persistent neurological impairment in both acute and chronic gray matter lesions in various inflammatory CNS disorders. © 2009 Wiley‐Liss, Inc.