Expression patterns of Group III metabotropic glutamate receptors mGluR4 and mGluR8 in multiple sclerosis lesions.

Recent evidence supports a role for metabotropic glutamate receptors (mGluRs) in neuroinflammatory diseases. In the present study, we have investigated whether the group III mGluR subtypes mGluR4 and mGluR8 are expressed in MS lesions at various stages of evolution. In control patient tissue and in normal-appearing MS white matter (NAWM), no microglial or astrocyte staining was detected. In contrast, in active lesions, mGluR8 immunoreactivity (IR) was detected in cells of the microglia/macrophage lineage. Fewer macrophage-like cells were positive for mGluR8 in chronic active and inactive lesions. No mGluR4 IR was detected in cells of the microglia/macrophage lineage in the MS lesions studied. In chronic active lesions, however, a population of reactive astrocytes localized in the rim of the lesions expressed both mGluR4 and mGluR8. Our results suggest a role for these receptor subtypes in the inflammatory response in MS that involves both astrocytes and cells of the microglia/macrophage lineage.

Altered expression patterns of group I and II metabotropic glutamate receptors in multiple sclerosis.

Recent evidence supports a role for glutamate receptors in the pathophysiology of multiple sclerosis. In the present study, we have focused specifically on the expression of metabotropic glutamate receptors (mGluRs) in multiple sclerosis brain tissue. The expression of group I (mGluR1alpha and mGluR5) and group II (mGluR2/3) mGluRs was studied using immunohistochemistry in tissue from 12 multiple sclerosis cases and seven non-neurological controls. The expression patterns of both group I and II mGluRs in multiple sclerosis tissue differed significantly from those in control tissue. Strong mGluR1alpha immunoreactivity was observed in axons of the subcortical white matter, particularly in the centre of actively demyelinating lesions and in the borders of chronic active lesions. mGluR1alpha axonal immunopositivity was also found in normal appearing multiple sclerosis white matter, but axons in control white matter were generally negative. mGluR1alpha axonal labelling was associated with the presence of non-phosphorylated neurofilaments and beta-amyloid precursor protein, which are sensitive markers for axonal injury and disturbed axonal transport. Changes in mGluR immunoreactivity were also observed in glia. A diffuse increase in the expression of mGluR5 and mGluR2/3 was detected in reactive astrocytes in multiple sclerosis lesions. However, only a subpopulation of reactive astroglial cells expressed mGluR1alpha. In addition, labelling with antibodies to mGluR2/3 and, to a lesser extent labelling with antibodies to mGluR1alpha, was detected in a population of cells of the microglial/macrophage lineage that displayed a macrophage-like morphology. Our data suggest that mGluRs, like ionotropic glutamate receptors, play a role in the complex processes that are associated with the progressive brain damage in multiple sclerosis, including both glial activation and pathological changes in axons.

Oligodendrocyte survival, loss and birth in lesions of chronic-stage multiple sclerosis.

One of the hallmarks of the human demyelinating disease multiple sclerosis is the inability to compensate adequately for the loss of myelin and of oligodendrocytes, the myelin-forming cells of the CNS. Oligodendrocyte precursor cells, a potential source of oligodendrocytes, have been identified in lesions of chronic multiple sclerosis, but it is not known whether they develop into new, fully differentiated oligodendrocytes, capable of remyelination. Sections of post-mortem multiple sclerosis tissue were therefore immunolabelled with antibodies to galactocerebroside (GalC), the first oligodendrocyte-specific molecule to be expressed by differentiating oligodendrocyte precursor cells, and myelin oligodendrocyte glycoprotein (MOG), a marker for mature oligodendrocytes. In total, 23 lesions from 15 subjects with chronic progressive multiple sclerosis were analysed. The immunolabelling revealed that chronic multiple sclerosis lesions contain only small numbers of immature, process-bearing, GalC-positive oligodendrocytes (0-2 cells/mm(2) in 10 micrometer thick sections); they had a relatively large, pale nucleus (maximum diameter: 9.9 +/- 0.9 micrometer). Although they appeared to make contact with surrounding demyelinated axons, most immature oligodendrocytes appeared not to be engaged in myelination. These findings suggest that oligodendrocyte differentiation of precursor cells is a rare event in chronic multiple sclerosis, which is consistent with the general failure of myelin repair during the later stages of this disease. The lesions in the collection, in particular those with recent demyelinating activity, contained another distinct population of oligodendrocytes. It consisted of small, round cells with a small, dense nucleus (maximum diameter: 6.8 +/- 0.8 micrometer) that expressed both GalC and MOG but lacked processes, suggesting that these cells were mature oligodendrocytes that had survived the loss of their myelin sheaths, i.e. they were demyelinated oligodendrocytes. In the most recent lesions in the collection, the demyelinated oligodendrocytes were found in large numbers throughout the centre of the lesion (up to 700 cells/mm(2)), while in the older lesions they were found only at the edges. Moreover, when the borders of these older lesions still contained numerous macrophages, they tended to contain more demyelinated oligodendrocytes than those lacking macrophages. These findings suggest that mature, demyelinated oligodendrocytes gradually disappear from lesion areas with increasing age of the lesion. The present study thus suggests that the failure of myelin repair in at least some cases of chronic multiple sclerosis is due to (i) the loss of demyelinated oligodendrocytes from lesion areas and (ii) the failure of the oligodendrocyte precursor population to expand and generate new oligodendrocytes. Gaining further insight into these processes may prove crucial for the development of remyelination promoting strategies.

Chronic stage multiple sclerosis lesions contain a relatively quiescent population of oligodendrocyte precursor cells.

In the past decade, considerable progress has been made in the understanding of the biology of rodent oligodendrocyte precursor cells and their role in the generation of oligodendrocytes in the developing and adult rodent CNS. Much less is known about human oligodendrocyte lineage cells and about the reasons for the failure of the regeneration of the oligodendrocyte population during chronic stages of multiple sclerosis (MS). In particular, the fate of the oligodendrocyte precursor population in MS has remained elusive. The present study examined the possibility that oligodendrocyte regeneration ultimately fails because of the local destruction of both oligodendrocytes and their precursor cells. Analysis of chronic stage MS tissue suggested that this is not the case, because all chronic MS lesions studied contained significant numbers of oligodendrocyte precursor cells, identified as process-bearing cells that bound the O4 antibody but not antibodies to GalC and GFAP. The oligodendrocyte precursor cells appeared, however, to be relatively quiescent, because none expressed the nuclear proliferation antigen recognized by the Ki-67 antibody, and because most lesions lacked myelinating oligodendrocytes in their centers. Thus, it appears that the regeneration of the oligodendrocyte population fails during chronic stages of MS because of the inability of oligodendrocyte precursor cells to proliferate and differentiate rather than because of the local destruction of all oligodendrocyte lineage cells. The identification of ways of stimulating the endogenous oligodendrocyte precursor population to expand and generate remyelinating cells may represent an alternative to transplantation of oligodendrocyte lineage cells to promote myelin repair in MS.

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells derived from adult rat spinal cord: in vitro characteristics and response to PDGF, bFGF and NT-3.

We have analysed in detail the properties of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells derived from the spinal cords of adult rats to gain further insights into the mechanisms that control the generation of oligodendrocytes in the healthy and demyelinated adult central nervous systems (CNS). When O-2A progenitor cells from adult spinal cord are exposed in vitro to the AA homodimeric form of platelet-derived growth factor (PDGF-AA), they express a unipolar morphology, an O4-positive, vimentin-negative antigenic phenotype, divide at slow rates, and appear to generate oligodendrocytes by asymmetric division and differentiation. Furthermore, exposure of these cells to PDGF-AA is sufficient to stimulate their proliferation at clonal density. When adult spinal cord O-2A progenitor cells are exposed simultaneously to PDGF-AA and basic fibroblast growth factor (PDGF/bFGF), they are almost completely inhibited from differentiating into oligodendrocytes, divide more rapidly than cells treated with PDGF-AA, and express a bipolar morphology and an O4-negative, vimentin-positive antigenic phenotype. These findings indicate that adult spinal cord O-2A progenitor cells resemble in many aspects their well-characterised adult optic nerve counterparts. In addition, evidence is presented to indicate that neurotrophin-3 (NT-3) is not mitogenic for adult spinal cord O-2A progenitor cells and that it does not enhance their proliferative response to PDGF-AA or PDGF/bFGF. Since relatively large numbers of O-2A progenitor cells can be obtained from adult spinal cord, it should facilitate the further characterisation of these cells.

Adult rat optic nerve oligodendrocyte progenitor cells express a distinct repertoire of voltage- and ligand-gated ion channels.

Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole-cell patch-clamp technique and the fura-2-based Ca++ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K+ currents, both delayed outward and inward rectifying. The inwardly rectifying K+ currents were often as large as the outward delayed rectifying K+ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4-aminopyridine, but not by 2 or 5 mM BaCl2. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A-type K+ currents. Both Ca++ and Na+ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABAA receptors and kainate receptors that are Ca(++)-permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes.

Strongly GD3+ cells in the developing and adult rat cerebellum belong to the microglial lineage rather than to the oligodendrocyte lineage.

A recent study has shown that ramified microglia in the adult rat optic nerve express the ganglioside GD3 [Wolswijk Glia 10:244-249, 1994], thereby raising the possibility that some GD3+ in the developing rat central nervous system (CNS) belong to the microglial lineage rather than to the oligodendrocyte lineage, as previously thought. To examine this possibility, sections of postnatal and adult cerebellum were double-labelled with markers for rat microglia [the B4 isolectin derived from Griffonia simplicifolia (GSI-B4), the ED1 monoclonal antibody (mAb), and the OX-42 mAb] and anti-GD3 mAbs (the mAbs R24 and LB1). These immunolabellings showed that ramified microglia as well as amoeboid microglia are strongly GD3+ in vivo. Moreover, most, if not all, cells that express high levels of GD3 in sections of developing cerebellum appear to belong to the microglial lineage. These observations contradict previous suggestions that the strongly GD3+ cells in the putative white matter regions of the developing brain are oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells; the cells that give rise to oligodendrocytes in the CNS. The present study did, however, confirm that some O-2A progenitor cells in sections of postnatal cerebellum are weakly GD3+ in vivo. Amoeboid microglia are present in areas of the developing cerebellum where newly generated oligodendrocytes are found, suggesting that these cells play a role in the phagocytosis of the large numbers of oligodendrocytes that die as part of CNS development.

GD3+ cells in the adult rat optic nerve are ramified microglia rather than O-2Aadult progenitor cells.

The adult central nervous system (CNS) contains a population of adult oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells (O-2Aadult progenitor cells). These cells may provide a source of the new oligodendrocytes that are needed to repair demyelinated lesions. In order to examine the role of O-2Aadult progenitor cells in the regeneration of the oligodendrocyte population following demyelinating damage, it is essential to be able to identify such cells unambiguously in sections of adult CNS tissue. The present study examined whether antibodies to the ganglioside GD3 specifically label O-2Aadult progenitor cells in cultures and sections of adult optic nerve, since previous studies on the developing CNS had suggested that O-2Aperinatal progenitor cells were GD3+ in vitro and in vivo. Evidence is presented indicating that, although O-2Aadult progenitor cells in vitro were labelled with the R24 mAb (an anti-GD3 mAb), all GD3+ cells in sections of adult optic nerve bound the OX-42 mAb and the B4 isolectin derived from Griffonia Simplicifolia, and thus were not O-2Aadult progenitor cells, but ramified microglia. The data suggest that O-2Aadult progenitor cells become GD3+ when placed in culture and that ramified microglia lose GD3-expression in vitro.

The O-2A(adult) progenitor cell: a glial stem cell of the adult central nervous system.

Systematic comparison of the properties of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells derived from optic nerves of perinatal and adult rats has revealed that these two populations differ in many fundamental properties. In particular, O-2A(perinatal) progenitor cells are rapidly dividing cells capable of generating large numbers of oligodendrocytes over a relatively short time span. Oligodendrocyte differentiation generally occurs synchronously in all members of a clone, thus leading to elimination of that clone from the pool of dividing cells. However, some O-2A(perinatal) progenitors are also capable of giving rise to O-2A(adult) progenitors. These latter cells express many of the characteristics of stem cells of adult animals, including the capacity to undergo asymmetric division and differentiation. We suggest that precursors which function during early development give rise to terminally differentiated end-stage cells and to a second generation of precursors with properties more appropriate for later developmental stages. It is this second generation of precursors which express the properties of stem cells in adult animals, and we therefore further suggest that our work offers novel insights into the possible developmental origin of stem cells.

Development and regeneration in the O-2A lineage: studies in vitro and in vivo.

This brief review discusses selected aspects of our studies on the control of division and differentiation of the glial precursor cells which give rise to oligodendrocytes. For more extensive reviews on this topic, the reader is referred to recent reviews by Raff (1989), Richardson et al. (1991), Noble (1991) and Noble et al. (1991).

Cooperation between PDGF and FGF converts slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with characteristics of O-2Aperinatal progenitor cells.

We have shown previously that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerves can be distinguished in vitro from their perinatal counterparts on the basis of their much slower rates of division, differentiation, and migration when grown in the presence of cortical astrocytes or PDGF. This behavior is consistent with in vivo observations that there is only a modest production of oligodendrocytes in the adult CNS. As such a behavior is inconsistent with the likely need for a rapid generation of oligodendrocytes following demyelinating damage to the mature CNS, we have been concerned with identifying in vitro conditions that allow O-2Aadult progenitor cells to generate rapidly large numbers of progeny cells. We now provide evidence that many slowly dividing O-2Aadult progenitor cells can be converted to rapidly dividing cells by exposing adult optic nerve cultures to both PDGF and bFGF. In addition, these O-2Aadult progenitor cells appear to acquire other properties of O-2Aperinatal progenitor cells, such as bipolar morphology and high rate of migration. Although many O-2Aadult progenitor cells in cultures exposed to bFGF alone also divide rapidly, these cells are multipolar and migrate little in vitro. Oligodendrocytic differentiation of O-2Aadult progenitor cells, which express receptors for bFGF in vitro, is almost completely inhibited in cultures exposed to bFGF or bFGF plus PDGF. As bFGF and PDGF appear to be upregulated and/or released after injury to the adult brain, this particular in vitro response of O-2Aadult progenitor cells to PDGF and bFGF may be of importance in the generation of large numbers of new oligodendrocytes in vivo following demyelination.

In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells.

We have been studying the differing characteristics of oligodendrocyte-type-2 astrocyte (O-2A) progenitors isolated from optic nerves of perinatal and adult rats. These two cell types display striking differences in their in vitro phenotypes. In addition, the O-2Aperinatal progenitor population appears to have a limited life-span in vivo, while O-2Aadult progenitors appear to be maintained throughout life. O-2Aperinatal progenitors seem to have largely disappeared from the optic nerve by 1 mo after birth, and are not detectable in cultures derived from optic nerves of adult rats. In contrast, O-2Aadult progenitors can first be isolated from optic nerves of 7-d-old rats and are still present in optic nerves of 1-yr-old rats. These observations raise two questions: (a) From what source do O-2Aadult progenitors originate; and (b) how is the O-2Aadult progenitor population maintained in the nerve throughout life? We now provide in vitro evidence indicating that O-2Aadult progenitors are derived directly from a subpopulation of O-2Aperinatal progenitors. We also provide evidence indicating that O-2Aadult progenitors are capable of prolonged self renewal in vitro. In addition, our data suggests that the in vitro generation of oligodendrocytes from O-2Aadult progenitors occurs primarily through asymmetric division and differentiation, in contrast with the self-extinguishing pattern of symmetric division and differentiation displayed by O-2Aperinatal progenitors in vitro. We suggest that O-2Aadult progenitors express at least some properties of stem cells and thus may be able to support the generation of both differentiated progeny cells as well as their own continued replenishment throughout adult life.

Platelet-derived growth factor is mitogenic for O-2Aadult progenitor cells.

We report that platelet-derived growth factor (PDGF) is a potent mitogen for oligodendrocyte type-2 astrocyte (O-2A) progenitor cells derived from the optic nerves of adult rats. Moreover, O-2Aadult progenitors cultured in PDGF express the range of properties we have described previously for O-2Aadult progenitors cultured in the presence of type-1 astrocytes. Similarly, previous studies have demonstrated that PDGF is able to mimic the influence of type-1 astrocytes on O-2Aperinatal progenitors. Specifically, O-2Aadult progenitors and O-2Aperinatal progenitors exposed to PDGF express differences in average cell cycle time (59 +/- 5 h for O-2Aadult progenitors versus 20 +/- 6 h for O-2Aperinatal progenitors), average rate of migration (4.1 +/- 0.6 microns h-1 versus 24.6 +/- 5.4 microns h-1), morphology (unipolar versus bipolar), and antigenic phenotype (04+ vimentin- versus 04- vimentin+). Thus, our present results indicate that a single signalling molecule secreted by type-1 astrocytes produces markedly different cellular behaviours in two related O-2A progenitor populations.

Coexistence of perinatal and adult forms of a glial progenitor cell during development of the rat optic nerve.

We have studied the developmental appearance of the O-2A(adult) progenitor cell, a specific type of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell that we have identified previously in cultures prepared from the optic nerves of adult rats. O-2A(adult) progenitors differ from their counterparts in perinatal animals (O-2A perinatal progenitor cells) in antigenic phenotype, morphology, cell cycle time, rate of migration, time course of differentiation into oligodendrocytes or type-2 astrocytes and sensitivity to the lytic effects of complement in vitro. In the present study, we have found that O-2A(adult) progenitor-like cells first appear in the developing optic nerve approximately 7 days after birth and that by 1 month after birth these cells appear to be the dominant progenitor population in the nerve. However, the perinatal-to-adult transition in progenitor populations is a gradual one and O-2A(adult) and O-2A perinatal progenitors coexist in the optic nerve for 3 weeks or more. In addition, cells derived from optic nerves of P21 rats express characteristic features of O-2adult and O-2A perinatal progenitors for extended periods of growth in the same tissue culture dish. Our results thus indicate that the properties that distinguish these two types of O-2A progenitors from each other are expressed in apparently identical environments. Thus, these cells must either respond to different signals present in the environment, or must respond with markedly different behaviours to the binding of identical signalling molecules.

Development and regeneration in the central nervous system.

As part of our attempts to understand principles that underly organism development, we have been studying the development of the rat optic nerve. This simple tissue is composed of three glial cell types derived from two distinct cellular lineages. Type-1 astrocytes appear to be derived from a monopotential neuroepithelial precursor, whereas type-2 astrocytes and oligodendrocytes are derived from a common oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell. Type-1 astrocytes modulate division and differentiation of O-2A progenitor cells through secretion of platelet-derived growth factor, and can themselves be stimulated to divide by peptide mitogens and through stimulation of neurotransmitter receptors. In vitro analysis indicates that many dividing O-2A progenitors derived from optic nerves of perinatal rats differentiate symmetrically and clonally to give rise to oligodendrocytes, or can be induced to differentiate into type-2 astrocytes. O-2Aperinatal progenitors can also differentiate to form a further O-2A lineage cell, the O-2Aadult progenitor, which has properties specialized for the physiological requirements of the adult nervous system. In particular, O-2Aadult progenitors have many of the features of stem cells, in that they divide slowly and asymmetrically and appear to have the capacity for extended self-renewal. The apparent derivation of a slowly and asymmetrically dividing cell, with properties appropriate for homeostatic maintenance of existing populations in the mature animal, from a rapidly dividing cell with properties suitable for the rapid population and myelination of central nervous system (CNS) axon tracts during early development, offers novel and unexpected insights into the possible origin of self-renewing stem cells and also into the role that generation of stem cells may play in helping to terminate the explosive growth of embryogenesis. Moreover, the properties of O-2Aadult progenitor cells are consistent with, and may explain, the failure of successful myelin repair in conditions such as multiple sclerosis, and thus seem to provide a cellular biological basis for understanding one of the key features of an important human disease.

Control of division and differentiation in oligodendrocyte-type-2 astrocyte progenitor cells.

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells give rise to oligodendrocytes and type-2 astrocytes in cultures of rat optic nerve. These progenitors are one of the few cell types in which most aspects of proliferation and differentiation can be manipulated in a defined in vitro environment. When exposed to platelet-derived growth factor (PDGF), O-2A progenitors divide a limited number of times before clonally related cells differentiate into oligodendrocytes with a timing similar to that seen in vivo. In contrast, O-2A progenitors grown in the absence of mitogen do not divide but differentiate prematurely into oligodendrocytes, and progenitors exposed to appropriate inducing factors differentiate into type-2 astrocytes. O-2A progenitors can become immortalized through at least two different mechanisms. First, when O-2A progenitors are exposed to a combination of PDGF and basic fibroblast growth factor (bFGF) these cells undergo continuous self-renewal in the absence of differentiation. In contrast, the application of bFGF alone is associated with premature oligodendrocytic differentiation of dividing O-2A lineage cells. Thus, cooperation between growth factors can modulate O-2A progenitor self-renewal in a defined chemical environment by eliciting a novel programme of division and differentiation which cannot be predicted from the effects of either factor examined in isolation. A further mechanism which allows prolonged self-renewal in the O-2A lineage is the generation of a stem cell. O-2A progenitors isolated from optic nerves of perinatal rats also have the capacity to give rise to a population of cells called O-2Aadult progenitors, which differ from their perinatal counterparts in many characteristics. Most importantly, O-2Aadult progenitors have a slow cell cycle, divide and differentiate asymmetrically and appear to have the capacity for prolonged self-renewal. Thus, immortalization in this lineage can also be achieved by the generation of a cell with stem cell-like characteristics from a rapidly dividing progenitor population.