Platelet-derived growth factor regulates oligodendrocyte progenitor numbers in adult CNS and their response following CNS demyelination

To design therapies for demyelinating diseases such as multiple sclerosis, it will be important to understand the mechanisms that control oligodendrocyte progenitor cell (OPC) numbers in the adult central nervous system (CNS). During development, OPC numbers are limited by the supply of platelet-derived growth factor-A (PDGF-A). Here, we examine the role of PDGF-A in regulating OPC numbers in normal and demyelinated adult CNS using transgenic mice that overexpress PDGF-A in astrocytes under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-PDGF-A mice). In adult GFAP-PDGF-A mice, there was a marked increase in OPC density, particularly in white matter tracts, indicating that the PDGF-A supply controls OPC numbers in the adult CNS as well as during development. To discover whether increasing PDGF expression increases the number of OPCs following demyelination and whether this enhances the efficiency of remyelination, we induced demyelination in GFAP-PDGF-A transgenic mice by intraspinal injection of lysolecithin or dietary administration of cuprizone. In both demyelinating models, OPC density within lesions was significantly increased compared to wild-type mice. However, morphological analysis of lysolecithin lesions did not reveal any difference in the time course or extent of remyelination between GFAP-PDGF-A and wild-type mice. We conclude that the availability of OPCs is not rate limiting for remyelination of focal demyelinated lesions in the mouse. Nevertheless, our experiments show that it is possible to increase OPC population density in demyelinated areas by artificially increasing the supply of PDGF.     

Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord

Elucidation of the response of oligodendrocyte progenitor cell populations to demyelination in the adult central nervous system (CNS) is critical to understanding why remyelination fails in multiple sclerosis. Using the anti-NG2 monoclonal antibody to identify oligodendrocyte progenitor cells, we have documented their response to antibody-induced demyelination in the dorsal column of the adult rat spinal cord. The number of NG2+ cells in the vicinity of demyelinated lesions increased by 72% over the course of 3 days following the onset of demyelination. This increase in NG2+ cell numbers did not reflect a nonspecific staining of reactive cells, as GFAP, OX-42, and Rip antibodies did not co-localise with NG2+ cells in double immunostained tissue sections. NG2+ cells incorporated BrdU 48–72 h following the onset of demyelination. After the onset of remyelination (10–14 days), the number of NG2+ cells decreased to 46% of control levels and remained consistently low for 2 months. When spinal cords were exposed to 40 Grays of x-irradiation prior to demyelination, the number of NG2+ cells decreased to 48% of control levels by 3 days following the onset of demyelination and remained unchanged at 3 weeks. Since 40 Grays of x-irradiation kills dividing cells, these studies illustrate a responsive and nonresponsive NG2+ cell population following demyelination in the adult spinal cord and suggest that the responsive NG2+ cell population does not renew itself. GLIA 22:161–170, 1998. © 1998 Wiley-Liss, Inc.     

Thyroid hormones promote differentiation of oligodendrocyte progenitor cells and improve remyelination after cuprizone-induced demyelination

In the present work we analyzed the capacity of thyroid hormones (THs) to improve remyelination using a rat model of cuprizone-induced demyelination previously described in our laboratories. Twenty one days old Wistar rats were fed a diet containing 0.6% cuprizone for two weeks to induce demyelination. After cuprizone withdrawal, rats were injected with triiodothyronine (T3). Histological studies carried out in these animals revealed that remyelination in the corpus callosum (CC) of T3-treated rats improved markedly when compared to saline treated animals. The cellular events occurring in the CC and in the subventricular zone (SVZ) during the first week of remyelination were analyzed using specific oligodendroglial cell (OLGc) markers. In the CC of saline treated demyelinated animals, mature OLGcs decreased and oligodendroglial precursor cells (OPCs) increased after one week of spontaneous remyelination. Furthermore, the SVZ of these animals showed an increase in early progenitor cell numbers, dispersion of OPCs and inhibition of Olig and Shh expression compared to non-demyelinated animals. The changes triggered by demyelination were reverted after T3 administration, suggesting that THs could be regulating the emergence of remyelinating oligodendrocytes from the pool of proliferating cells residing in the SVZ. Our results also suggest that THs receptor β mediates T3 effects on remyelination. These results support a potential role for THs in the remyelination process that could be used to develop new therapeutic approaches for demyelinating diseases.     

NG2-positive oligodendrocyte progenitor cells in adult human brain and multiple sclerosis lesions.

Multiple sclerosis (MS) is characterized by multifocal loss of myelin, oligodendrocytes, and axons. Potential MS therapies include enhancement of remyelination by transplantation or manipulation of endogenous oligodendrocyte progenitor cells. Characteristics of endogenous oligodendrocyte progenitors in normal human brain and in MS lesions have not been studied extensively. This report describes the distribution of cells in sections from normal adult human brain and MS lesions by using antibodies directed against NG2, an integral membrane chondroitin sulfate proteoglycan expressed by oligodendrocyte progenitor cells. Stellate-shaped NG2-positive cells were detected in the white and gray matter of normal adult human brain and appeared as abundant as, but distinct from, astrocytes, oligodendrocytes, and microglia. Stellate-shaped or elongated NG2-positive cells also were detected in chronic MS lesions. A subpopulation of the elongated NG2-positive cells expressed the putative apoptotic signaling molecule p75(NTR). TUNEL-positive cells in three active, nine chronic active, and four chronic inactive lesions, however, were p75(NTR)-negative. These studies identify cells with phenotypic markers of endogenous oligodendrocyte progenitors in the mature human CNS and suggest that functional subpopulations of NG2-positive cells exist in MS lesions. Endogenous oligodendrocyte progenitor cells may represent a viable target for future therapies intended to enhance remyelination in MS patients.     

Astrocyte TNFR2 is required for CXCL12-mediated regulation of oligodendrocyte progenitor proliferation and differentiation within the adult CNS

Multiple sclerosis (MS) is characterized by episodes of inflammatory demyelination with progressive failure of remyelination. Prior studies using murine models of MS indicate that remyelination within the adult central nervous system (CNS) requires the expression and activity of TNFR2 and CXCR4 by oligodendrocyte progenitor cells (OPCs), promoting their proliferation and differentiation into mature oligodendrocytes. Here, we extend these studies by examining the role of TNFR2 in the expression of the CXCR4 ligand, CXCL12, within the corpus callosum (CC) during cuprizone (CPZ) intoxication and by demonstrating that lentiviral-mediated gene delivery of CXCL12 to the demyelinated CC improves OPC proliferation and myelin expression during remyelination. Activated astrocytes and microglia express both TNFR1 and TNFR2 within the demyelinated CC. However, CPZ intoxicated TNFR2?/? mice exhibit loss of up-regulation of CXCL12 in astrocytes with concomitant decreases in numbers of CXCR4+ NG2+ OPCs within the CC. While CXCR4 antagonism does not affect OPC migration from subventricular zones into the CC, it decreases their proliferation and differentiation within the CC. Stereotactic delivery of lentivirus expressing CXCL12 protein into the CC of acutely demyelinated TNFR2?/? mice increases OPC proliferation and expression of myelin. In contrast, chronically demyelinated wild-type mice, which exhibit significant loss of astrocytes and OPCs, are unable to be rescued via CXCL12 lentivirus alone but instead required engraftment of CXCL12-expressing astrocytes for increased myelin expression. Our results show that TNFR2 activation induces CXCL12 expression in the demyelinated CC via autocrine signaling specifically within astrocytes, which promotes OPC proliferation and differentiation. In addition, gene delivery of critical pro-myelinating proteins might be a feasible approach for the treatment of remyelination failure in MS.     

NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS

Glial progenitor cells of the developing CNS committed to the oligodendrocyte lineage (OPCs) express the chondroitin sulfate proteoglycan, NG2. A proportion of OPCs fail to differentiate past the stage at which they express NG2 and the lipid antigen O4 and persist in the adult CNS in a phenotypically immature form. However, the physiological function of NG2(+) cells in the adult CNS is unknown. Using antibodies against NG2 we show that NG2 is expressed by a distinct cell population in the mature CNS with the homogeneous antigenic phenotype of oligodendrocyte progenitors. The morphology of NG2(+) OPCs varies from region to region, reflecting the different structural environments, but they appear to represent a homogeneous population within any one gray or white matter region. A study of nine CNS regions showed that NG2(+) OPCs are numerous throughout the CNS and numbers in the white matter are only 1.5 times that in the gray. Whereas the ratio of OPCs to myelinating oligodendrocytes in the spinal cord gray and white matter approximates 1:4, gray matter regions of the forebrain have a 1:1 ratio, a phenomenon that will have consequences for oligodendrocyte replacement following demyelination. BrdU incorporation experiments showed that NG2(+) cells are the major dividing cell population of the adult rat CNS. Since very little apoptosis was detected and BrdU became increasingly present in oligodendrocytes after a 10-day pulse chase, with a concomitant decrease in NG2(+) BrdU incorporating cells, we suggest that the size of the oligodendrocyte population may actually increase during adult life.     

CNS Schwann cells display oligodendrocyte precursor-like potassium channel activation and antigenic expression in vitro

Central nervous system (CNS) injury triggers production of myelinating Schwann cells from endogenous oligodendrocyte precursors (OLPs). These CNS Schwann cells may be attractive candidates for novel therapeutic strategies aiming to promote endogenous CNS repair. However, CNS Schwann cells have been so far mainly characterized in situ regarding morphology and marker expression, and it has remained enigmatic whether they display functional properties distinct from peripheral nervous system (PNS) Schwann cells. Potassium channels (K⁺) have been implicated in progenitor and glial cell proliferation after injury and may, therefore, represent a suitable pharmacological target. In the present study, we focused on the function and expression of voltage-gated K⁺ channels K_v1–12 and accessory β-subunits in purified adult canine CNS and PNS Schwann cell cultures using electrophysiology and microarray analysis and characterized their antigenic phenotype. We show here that K⁺ channels differed significantly in both cell types. While CNS Schwann cells displayed prominent K _D-mediated K⁺ currents, PNS Schwann cells elicited K _D- and K_A-type K⁺ currents. Inhibition of K⁺ currents by TEA and Ba²⁺ was more effective in CNS Schwann cells. These functional differences were not paralleled by differential mRNA expression of K_v1–12 and accessory β-subunits. However, O4/A2B5 and GFAP expressions were significantly higher and lower, respectively, in CNS than in PNS Schwann cells. Taken together, this is the first evidence that CNS Schwann cells display specific properties not shared by their peripheral counterpart. Both K_v currents and increased O4/A2B5 expression were reminiscent of OLPs suggesting that CNS Schwann cells retain OLP features during maturation.     

Interleukin-2 inhibition of oligodendrocyte progenitor cell proliferation depends on expression of the TAC receptor

Interleukin-2 (IL-2) has been shown to inhibit oligodendrocyte progenitor cell proliferation. Within the immune system, IL-2 biological action is dependent strictly on the expression of the IL-2 receptor. The antibody TAC, which specifically binds the lymphocyte IL-2 receptor, has been shown to also bind oligodendrocyte progenitor cells cultured in a serumless, chemically defined medium. The expression of the TAC antigen was found necessary for IL-2 inhibition of oligodendrocyte progenitor cell proliferation. After IL-2 induced down-regulation of the TAC antigen, the progenitor cell was unresponsive to IL-2, even 72 hr after IL-2 withdrawal. During this unresponsive period, the oligodendrocyte progenitor cell was immunocytochemically negative for the TAC antigen. Thus, in contrast to IL-2 receptors on T-cells, IL-2 does not up-regulate its receptor on oligodendrocyte progenitor cells. However, upon interleukin 1 (IL-1) addition both IL-2 responsiveness and TAC immunocytochemical staining reappeared. These data suggest that IL-2 inhibition of progenitor cell proliferation depends on the expression of the TAC antigen, which can be regulated by IL-1.     

Macrophage-depletion induced impairment of experimental CNS remyelination is associated with a reduced oligodendrocyte progenitor cell response and altered growth factor expression

Although macrophages are mediators of CNS demyelination, they are also implicated in remyelination. To examine the role of macrophages in CNS remyelination, adult rats were depleted of monocytes using clodronate liposomes and demyelination induced in the spinal cord white matter using lysolecithin. In situ hybridization for scavenger receptor-B and myelin basic protein (MBP) revealed a transiently impaired macrophage response associated with delayed remyelination in liposome-treated animals. Macrophage reduction corresponded with delayed recruitment of PDGFRalpha+ oligodendrocyte progenitor cells (OPCs), which preceded changes in myelin phagocytosis, indicating a macrophage effect on OPCs independent of myelin debris clearance. Macrophage-depletion induced changes in the mRNA expression of insulin-like growth factor-1 and transforming growth factor beta1, but not platelet-derived growth factor-A and fibroblast growth factor-2. These data suggest that the macrophage response to toxin-induced demyelination influences the growth factor environment, thereby affecting the behavior of OPCs and hence the efficiency of remyelination.     

Role of glial cells in innate immunity and their role in CNS demyelination

The adaptive and innate arms of the immune system are the two pillars of host defense against environmental pathogens. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS which is considered to be autoimmune and is thought to result from breakdown in the usual checks and balances of the adaptive immune response. The major pathological outcome of the disease is "the MS plaque" a unique feature of CNS demyelination characterized by the destruction of oligodendrocytes with loss of myelin and underlying axons. The MS plaque is not seen in other inflammatory disorders of the CNS. The prevailing opinion suggests that MS is mediated by the activation of an adaptive immune response which targets neural antigens. Currently, the role of an innate immune in the development of the lesions in MS has remained unclear. We explore the potential cellular elements of the innate immune system and in particular glial cells, which are likely candidates in inducing the specific pathological picture that is evident in MS. Activated microglia and the release of molecules which are detrimental to oligodendrocyte have been suggested as mechanisms by which innate immunity causes demyelination in MS. However a microglia/macrophage centric model does not explain the specificity of lesion development in MS. We propose that activation pathways of receptors of the innate immune system present on oligodendrocytes and astrocytes rather than microglia are central to the pathogenesis of demyelination seen in MS.     

The remyelinating potential and in vitro differentiation of MOG-expressing oligodendrocyte precursors isolated from the adult rat CNS

There is a long-standing controversy as to whether oligodendrocytes may be capable of cell division and thus contribute to remyelination. We recently published evidence that a subpopulation of myelin oligodendrocyte glycoprotein (MOG)-expressing cells in the adult rat spinal cord co-expressed molecules previously considered to be restricted to oligodendrocyte progenitors [G. Li et al. (2002) Brain Pathol., 12, 463-471]. To further investigate the properties of MOG-expressing cells, anti-MOG-immunosorted cells were grown in culture and transplanted into acute demyelinating lesions. The immunosorting protocol yielded a cell preparation in which over 98% of the viable cells showed anti-MOG- and O1-immunoreactivity; 12-15% of the anti-MOG-immunosorted cells co-expressed platelet-derived growth factor alpha receptor (PDGFRalpha) or the A2B5-epitope. When cultured in serum-free medium containing EGF and FGF-2, 15-18% of the anti-MOG-immunosorted cells lost anti-MOG- and O1-immunoreactivity and underwent cell division. On removal of these growth factors, cells differentiated into oligodendrocytes, or astrocytes and Schwann cells when the differentiation medium contained BMPs. Transplantation of anti-MOG-immunosorted cells into areas of acute demyelination immediately after isolation resulted in the generation of remyelinating oligodendrocytes and Schwann cells. Our studies indicate that the adult rat CNS contains a significant number of oligodendrocyte precursors that express MOG and galactocerebroside, molecules previously considered restricted to mature oligodendrocytes. This may explain why myelin-bearing oligodendrocytes were considered capable of generating remyelinating cells. Our study also provides evidence that the adult oligodendrocyte progenitor can be considered as a source of the Schwann cells that remyelinate demyelinated CNS axons following concurrent destruction of oligodendrocytes and astrocytes.     

Inducible expression of FGF2 by a rat oligodendrocyte precursor cell line promotes CNS myelination in vitro

Transplantation of glial cells into the central nervous system (CNS) may be a promising approach for the treatment of myelin disorders such as multiple sclerosis (MS). Myelination by transplantation of oligodendrocyte precursors has been obtained in different animal models of demyelination. A strategy to favor CNS remyelination is to enrich the lesioned areas in growth factors to stimulate the quiescent population of oligodendrocyte precursors. In this context, we have developed a genetically modified CG4 cell line (CG4-FGF2), which are able to release significant amounts of fibroblast growth factor 2 (FGF2) in a controlable fashion in vitro. The data presented here demonstrate that upon induction with Dox, CG4-FGF2 cells retain their capacity to differentiate in vitro. Additionally, we provide evidence that FGF2 release by engineered cells enhance proliferation and migration of cells of the oligodendrocyte lineage without preventing them to differentiate and myelinate axons in vitro.     

Nuclear expression of S100B in oligodendrocyte progenitor cells correlates with differentiation toward the oligodendroglial lineage and modulates oligodendrocytes maturation

The S100B protein belongs to the S100 family of EF-hand calcium binding proteins implicated in cell growth and differentiation. Here, we show that in the developing and the adult mouse brain, S100B is expressed in oligodendroglial progenitor cells (OPC) committed to differentiate into the oligodendrocyte (OL) lineage. Nuclear S100B accumulation in OPC correlates with the transition from the fast dividing multipotent stage to the morphological differentiated, slow proliferating, pro-OL differentiation stage. In the adult, S100B expression is down-regulated in mature OLs that have established contacts with their axonal targets, suggesting a nuclear S100B function during oligodendroglial cells maturation. In vitro, the morphological transformation and maturation of pro-OL cells are delayed in the absence of S100B. Moreover, mice lacking S100B show an apparent delay in OPC maturation in response to demyelinating insult. We propose that nuclear S100B participates in the regulation of oligodendroglial cell maturation.     

An efficient and economical culture approach for the enrichment of purified oligodendrocyte progenitor cells

Oligodendrocyte progenitor cell (OPC) culture has provided a powerful approach to mechanistically investigate the proliferation and differentiation of oligodendroglia. However, existing culture methods (including the traditional shake-off method) have limitations, particularly their low productivities. Therefore, we developed a simplified and highly efficient method to produce a large yield of OPCs with low expense by using specialised modified media, in which B104-conditioned medium (B104-CM) instead of growth factors was used as a mitogenic source for OPC propagation, while a modified OPC isolation-medium was applied to improve the isolation of OPCs. First, we withdrew foetal bovine serum when primary mixed glial cultures were 65-75% confluent and substituted with modified oligodendrocyte growth medium to enrich OPCs. Second, we employed a chemical-based method to isolate and purify OPCs from mixed glial cultures using a modified oligodendrocyte isolation medium. As a result, our approach produced a high yield of purified OPCs, approximately 90-fold higher than that produced via the traditional shake-off method. Importantly, the purified OPCs produced via our modified approach maintained typical capacities of proliferation and differentiation observed in oligodendrocyte lineage cells. Together, our modified method provides a highly efficient approach to OPC culture for oligodendrocyte research.