In the CNS, myelination is accomplished by mature oligodendrocytes that arise from OPCs. During CNS development, a substantial pool of OPCs must be generated for production of mature oligodendrocytes in sufficient numbers for adequate myelination of axons. The adult CNS still contains large numbers of OPCs that differ somewhat from perinatal progenitors in their capability for motility and proliferation, yet respond to most of the same stimuli and express a similar set of phenotypic markers as their perinatal counterparts. Adult OPCs account for a large percentage of the proliferating cells in the mature CNS [7, 9] and are responsible for production of new oligodendrocytes to replace damaged cells. Newly-differentiated oligodendrocytes derived from adult OPCs, rather than pre-existing oligodendrocytes, are responsible for remyelination of axons that occurs following various types of demyelinating events [8, 10, 32–34]. Factors that influence OPC proliferation and differentiation are therefore of great importance for our understanding of both developmental myelination and myelin repair.
The NG2 proteoglycan contributes to the proliferation of OPCs during CNS development. In the NG2 null mouse, decreased OPC proliferation reduces the size of the OPC pool, leading to a delay in production of normal numbers of mature oligodendrocytes and to a corresponding delay in axon myelination . We have used lysolecithin-induced demyelination of the spinal cord to examine the possibility that ablation of NG2 also impedes repair of myelin damage in the adult CNS. Following microinjection into CNS white matter, lysolecithin replaces phospholipids and forms micelles in the membrane bilayer , rapidly inducing local myelin destruction , blood-brain barrier damage, and recruitment of macrophages and local microglial cells into the lesion site . This commonly-used demyelination model [4, 19, 35–37] has the advantage that the site and extent of the injury are well-defined and reproducible, facilitating data acquisition. In addition, lysolecithin-induced demyelination occurs as an acute event, such that all subsequent phenomena are associated with the regenerative response. This provides a useful means of separating events and mechanisms associated with the respective processes of demyelination and remyelination .
The regeneration of myelin following demyelination is a multifactorial process, due in part to the involvement of multiple cell types in the damage and repair mechanisms. In addition to neurons and OPCs, microglia, macrophages, and pericytes also contribute to these processes [38–41]. Our work shows that the NG2 proteoglycan is expressed by three cell types that invade demyelinated lesions: OPCs, pericytes, and macrophages/microglia. The differential contributions of these three cell types to the damage and repair processes, combined with differences in NG2 function in the respective cell types, are probably responsible for the complex patterns of demyelination and remyelination that we see in the global NG2 null mouse. Figure 2 shows that although the extent of initial demyelination is reduced in the NG2 null mouse, repair of this lesion nevertheless proceeds more slowly than repair of the larger lesion found in the wild type mouse. The impact of NG2 ablation on OPCs is likely confined to deficiencies seen during the repair process, since OPCs generate oligodendrocytes that carry out remyelination. Conversely, diminished involvement of macrophages/microglia probably provides the best explanation for the reduced extent of initial demyelination seen in the NG2 null mouse. However, macrophages/microglial cells also contribute to myelin repair by clearing myelin debris and by producing cytokines and growth factors that promote recruitment of OPCs and prime interactions between OPCs and axons. Thus, NG2-dependent deficits in macrophage/microglia function may also contribute to the reduced myelin repair seen in the NG2 null mouse. Similarly, it is possible that pericytes affect both myelin damage and repair. The recruitment of pericytes for revascularization of the lesion and repair of the blood brain barrier likely plays an important role in the healing process. However, vascularization also provides increased access to inflammatory cells and cytokines that contribute to myelin damage [40, 42–45]. Since many of the pericytes in lysolecithin-induced lesions are not associated with vascular endothelial cells, another consideration is the ability of pericytes to serve as mesenchymal stem cells [46, 47] with immunomodulatory properties that can promote myelin repair via their effects on the activities of inflammatory cells .
Our evidence suggests that promoting cell proliferation is a key functional role for NG2 in OPCs, pericytes, and macrophages/microglia. BrdU incorporation reveals significant reductions in mitotic index for all three cell types in demyelinated lesions in the NG2 null mouse. In the case of OPCs, this confirms a similar result obtained in our studies of developmental myelination: namely, that ablation of NG2 reduces the OPC mitotic index, with a corresponding decrease in the number of myelinating oligodendrocytes . Thus, NG2 is important for promoting the proliferation of both perinatal OPCs and adult OPCs. The BrdU results also confirm our report that ablation of NG2 diminishes pericyte proliferation during pathological retinal neovascularization, leading to decreased blood vessel formation in the retinas of NG2 null mice . This negative effect of NG2 ablation on cell proliferation may be a fairly general one, since we also observe diminished keratinocyte proliferation in the skin of newborn NG2 null mice . Our in vitro studies also support a role for NG2 in promoting cell proliferation. NG2 is able to enhance proliferation via two mechanisms: promotion of signaling by β1 integrins  and promotion of signaling by receptors for the growth factors PDGF and FGF [27, 51].
In vitro studies also indicate that NG2-dependent signaling by β1 integrins and growth factor receptors can promote cell motility as well as cell proliferation [27, 50, 52, 53]. In vivo, one indication of this effect is seen in our current studies on macrophage invasion into demyelinated lesions. BrdU tracking studies at day 5, one day after lysolecithin injection, show that 8 to 10% of the macrophages/microglia in dorsal column white matter are located outside demyelinated lesions. By 7 days post-injection in wild type mice, 90% of these peripherally-located cells have migrated into the lesion. By contrast, only 20% of extra-lesional cells have migrated into the lesion in NG2 null mice, indicative of the NG2 dependence of macrophage motility. Similar measurements were not possible in the case of OPCs or pericytes due to the rare occurrence of BrdU-labeled cells outside of demyelinated lesions.
Our finding of changes in cytokine expression following NG2 ablation may also be important in understanding changes in demyelination and remyelination in the NG2 null mouse. Although it remains to be determined whether changes in cytokine expression in the NG2 null mouse are associated with changes in macrophages as opposed to other inflammatory cell types, decreased levels of IFNγ and IL-1β coupled with increased levels of IL-4 and IL-10 suggest that NG2 ablation shifts a pro-inflammatory phenotype to an anti-inflammatory one. IFNγ provokes acute re-occurrence of demyelination in MS patients , and IL-1β is present in CNS-infiltrating myeloid cells in MS models . It therefore seems possible that decreased levels of IFNγ and IL-1β in spinal cord lesions in the NG2 null mouse (or altered activities of cells expressing these cytokines) are responsible for the reduced white matter damage seen in these mice. Moreover, decreased IL-4 production in the CNS exacerbates experimental autoimmune encephalitis, and is associated with increased infiltration of inflammatory cells , while increased IL-10 expression is associated with reduced inflammation . The possibility that NG2 null macrophages/microglia may exhibit less inflammatory properties than wild type cells is in line with the in vitro finding of a reduced inflammatory phenotype upon knockdown of NG2 in microglia . We speculate that diminished occurrence of myeloid cells in NG2 null spinal cord lesions, coupled with alterations in the intrinsic properties/functions of NG2-negative macrophages/microglial cells, can affect the progression of demyelination and remyelination in NG2 null mice.