Increased NG2(+) glial cell proliferation and oligodendrocyte generation in the hypomyelinating mutant shiverer

Glial cells that express the NG2 proteoglycan (NG2(+) cells) are considered to be oligodendrocyte progenitors (OPCs) in the central nervous system (CNS), based on their ability to give rise to mature oligodendrocytes in vitro. To understand how dysmyelinated conditions influence OPC proliferation and differentiation, we studied proliferation and differentiation of NG2(+) OPCs in vivo in the shiverer mutant (shi), which do not form compact myelin due to a deletion in the myelin basic protein gene. Acute bromodeoxyuridine (BrdU) labeling studies revealed a 4- to 6-fold increase in NG2(+) cell proliferation in shi spinal cord between postnatal day18 (P18) and P60, and most BrdU(+) cells were NG2(+) after P18. The increased proliferation was accompanied by a 2-fold increase in the number of OPCs and oligodendrocytes. Survival studies following a single injection of BrdU at P18 revealed a decline in the number of BrdU(+)/NG2(+) cells with a concomitant increase in the number of BrdU(+) oligodendrocytes over time, suggesting that the proliferated NG2(+) cells had differentiated into oligodendrocytes. BrdU(+) oligodendrocytes were generated over a longer period of time in shi spinal cord and persisted longer in shi than in wild type spinal cord. These findings suggest that new oligodendrocytes continue to be generated in the dysmyelinated shi spinal cord by enhanced proliferation and differentiation of NG2(+) oligodendrocyte progenitor cells.     

Myelin status and oligodendrocyte lineage cells over time after spinal cord injury: What do we know and what still needs to be unwrapped?

Spinal cord injury (SCI) affects over 17,000 individuals in the United States per year, resulting in sudden motor, sensory and autonomic impairments below the level of injury. These deficits may be due at least in part to the loss of oligodendrocytes and demyelination of spared axons as it leads to slowed or blocked conduction through the lesion site. It has long been accepted that progenitor cells form new oligodendrocytes after SCI, resulting in the acute formation of new myelin on demyelinated axons. However, the chronicity of demyelination and the functional significance of remyelination remain contentious. Here we review work examining demyelination and remyelination after SCI as well as the current understanding of oligodendrocyte lineage cell responses to spinal trauma, including the surprisingly long-lasting response of NG2+ oligodendrocyte progenitor cells (OPCs) to proliferate and differentiate into new myelinating oligodendrocytes for months after SCI. OPCs are highly sensitive to microenvironmental changes, and therefore respond to the ever-changing post-SCI milieu, including influx of blood, monocytes and neutrophils; activation of microglia and macrophages; changes in cytokines, chemokines and growth factors such as ciliary neurotrophic factor and fibroblast growth factor-2; glutamate excitotoxicity; and axon degeneration and sprouting. We discuss how these changes relate to spontaneous oligodendrogenesis and remyelination, the evidence for and against demyelination being an important clinical problem and if remyelination contributes to motor recovery.     

Glial cell loss, proliferation and replacement in the contused murine spinal cord

Studies in the rat have shown that contusive spinal cord injury (SCI) results in devastating pathology, including significant loss of mature oligodendrocytes and astrocytes even in spared white matter. Subsequently, there is increased proliferation of endogenous NG2(+) cells, postulated to contribute to replacement of mature glia chronically, which is important for functional recovery. Studies of mechanisms that stimulate endogenous progenitor cells would be facilitated by using mouse models with naturally occurring and genetically engineered mutations. To determine whether the murine response is similar to that in the rat, we performed contusive SCI on adult female C57Bl/6 mice at the T8-9 level. Animals received bromodeoxyuridine injections in the first week following injury and were killed at 1, 3, 4, 7 or 28 days postinjury (DPI). The overall loss of macroglia and the temporal-spatial response of NG2(+) cells after SCI in the (C57Bl/6) mouse was very similar to that in the (Sprague-Dawley) rat. By 24 h after SCI nearly half of the macroglia in spared ventral white matter had been lost. Cell proliferation was increased at 1-7 DPI, peaking at 3-4 DPI. Dividing cells included NG2(+) cells and Cd11b(+) macrophages and microglia. Furthermore, cells dividing in the first week expressed markers of mature glia at 28 DPI. The similarities in endogenous progenitor cell response to SCI in the mouse and rat suggest that this is a fundamental injury response, and that transgenic mouse models may be used to further probe how this cellular response to SCI might be enhanced to improve recovery after SCI.     

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.     

Properties and fate of oligodendrocyte progenitor cells in the corpus callosum, motor cortex, and piriform cortex of the mouse

Oligodendrocyte progenitor cells (OPCs) in the postnatal mouse corpus callosum (CC) and motor cortex (Ctx) reportedly generate only oligodendrocytes (OLs), whereas those in the piriform cortex may also generate neurons. OPCs have also been subdivided based on their expression of voltage-gated ion channels, ability to respond to neuronal activity, and proliferative state. To determine whether OPCs in the piriform cortex have inherently different physiological properties from those in the CC and Ctx, we studied acute brain slices from postnatal transgenic mice in which GFP expression identifies OL lineage cells. We whole-cell patch clamped GFP-expressing (GFP(+)) cells within the CC, Ctx, and anterior piriform cortex (aPC) and used prelabeling with 5-ethynyl-2'-deoxyuridine (EdU) to assess cell proliferation. After recording, slices were immunolabeled and OPCs were defined by strong expression of NG2. NG2(+) OPCs in the white and gray matter proliferated and coexpressed PDGFRα and voltage-gated Na(+) channels (I(Na)). Approximately 70% of OPCs were capable of generating regenerative depolarizations. In addition to OLIG2(+) NG2(+) I(Na)(+) OPCs and OLIG2(+) NG2(neg) I(Na)(neg) OLs, we identified cells with low levels of NG2 limited to the soma or the base of some processes. These cells had a significantly reduced I(Na) and a reduced ability to incorporate EdU when compared with OPCs and probably correspond to early differentiating OLs. By combining EdU labeling and lineage tracing using Pdgfrα-CreER(T2) : R26R-YFP transgenic mice, we double labeled OPCs and traced their fate in the postnatal brain. These OPCs generated OLs but did not generate neurons in the aPC or elsewhere at any time that we examined.     

The tetraspanin protein,CD9, is expressed by progenitor cells committed to oligodendrogenesis and is linked to beta1 integrin,CD81, and Tspan-2

Previous studies identified the tetraspanin protein CD9 in myelinating oligodendrocytes. The present report extends these observations by identifying CD9 in a subpopulation of oligodendrocyte progenitor cells (OPCs) and in premyelinating oligodendrocytes in rodents. NG2-positive cells expressed CD9 in a temporal and spatial pattern during development that was consistent with CD9 expression in OPCs just prior to their differentiation into premyelinating oligodendrocytes. NG2-positive cells in mature brains were CD9-negative. CD9 expression during oligodendrocyte development in vitro supported this hypothesis, as all CD9-positive cells became O4-positive when switched to oligodendrocyte differentiating media. CD9 immunoreactivity was enriched in myelinating oligodendrocytes and their processes, and the outer aspects of myelin internodes. Immunoprecipitation of CD9 from postnatal rat cerebrum coprecipitated beta1 integrin, CD81, and Tspan-2, another tetraspanin protein recently identified in oligodendrocytes. Following surface biotinylation of oligodendrocytes in vitro, biotinylated beta1 integrin was identified in a CD9 immunoprecipitate. These data support a molecular link between surface integrins and a CD9, Tspan-2 molecular web during the differentiation of oligodendrocytes. Oligodendrocyte production and myelination appears to be normal in CD9-deficient mice. These data support the hypothesis that CD9 helps form the tetraspanin web beneath the plasma membranes of progenitor cells committed to oligodendrogenesis, but that CD9 is not essential for oligodendrogenesis and myelination.     

In vivo analysis of oligodendrocyte lineage development in postnatal FGF2 null mice

Analysis of fibroblast growth factor 2 null (FGF2-/-) and wild-type (FGF2+/+) mice was used to interpret the potential in vivo role of endogenous FGF2 on oligodendrocyte lineage cell (OLC) responses during oligodendrogenesis and myelination. In wild-type mouse spinal cord, FGF2 levels increased approximately threefold between the first and second postnatal weeks, a period corresponding with the peak of oligodendrogenesis. Absence of this developmental FGF2 elevation in FGF2-/- mice eliminated the transient overproduction of oligodendrocytes that is known to occur at the peak of oligodendrogenesis in wild-type mice. Absence of FGF2 did not affect oligodendrocyte progenitor (OP) density or proliferation, based on BrdU incorporation, and also did not alter survival, based on TUNEL analysis. To examine OLC differentiation in vivo, retrovirus encoding-enhanced green fluorescent protein (GFP) was injected into the spinal cord to heritably label endogenous cycling cells in the white matter at postnatal day 7 and then identify the generated cells at postnatal day 28. Phenotypes of cells expressing GFP were identified by morphology and immunolabeling, using CC1 for oligodendrocytes and NG2 combined with platelet-derived growth factor alpha receptor for OPs. Within the population of GFP-labeled cells, the proportion of oligodendrocytes was higher in FGF2-/- mice, indicating that endogenous FGF2 inhibited OLC differentiation in wild-type mice. Furthermore, in FGF2-/- mice fewer cells appeared to be generated from an initial retrovirus-labeled cell, consistent with more frequent differentiation into post-mitotic oligodendrocytes. This in vivo analysis demonstrates that the predominant role of endogenous FGF2 on OLCs in development is inhibition of differentiation.     

SOX17 is expressed in regenerating oligodendrocytes in experimental models of demyelination and in multiple sclerosis

We have previously demonstrated that Sox17 expression is prominent at developmental stages corresponding to oligodendrocyte progenitor cell (OPC) cycle exit and onset of differentiation, and that Sox17 promotes initiation of OPC differentiation. In this study, we examined Sox17 expression and regulation under pathological conditions, particularly in two animal models of demyelination/remyelination and in post‐mortem multiple sclerosis (MS) brain lesions. We found that the number of Sox17 expressing cells was significantly increased in lysolecithin (LPC)‐induced lesions of the mouse spinal cord between 7 and 30 days post‐injection, as compared with controls. Sox17 immunoreactivity was predominantly detected in Olig2⁺ and CC1⁺ oligodendrocytes and rarely in NG2⁺ OPCs. The highest density of Sox17⁺ oligodendrocytes was observed at 2 weeks after LPC injection, coinciding with OPC differentiation. Consistent with these findings, in cuprizone‐treated mice, Sox17 expression was highest in newly generated and in maturing CC1⁺ oligodendrocytes, but low in NG2⁺ OPCs during the demyelination and remyelination phases. In MS tissue, Sox17 was primarily detected in actively demyelinating lesions and periplaque white matter. Sox17 immunoreactivity was co‐localized with NOGO‐A+ post‐mitotic oligodendrocytes both in active MS lesions and periplaque white matter. Taken together, our data: (i) demonstrate that Sox17 expression is highest in newly generated oligodendrocytes under pathological conditions and could be used as a marker of oligodendrocyte regeneration, and (ii) are suggestive of Sox17 playing a critical role in oligodendrocyte differentiation and lesion repair. GLIA 2013;61:1659–1672     

Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice

We investigated the treatment of remitting-relapsing experimental autoimmune encephalomyelitis (EAE) in mice with human bone marrow stromal cells (hBMSCs). hBMSCs were injected intravenously into EAE mice upon onset of paresis. Neurological functional tests were scored daily by grading clinical signs (score 0-5). Immunohistochemistry was performed to measure the transplanted hBMSCs, cell proliferation (bromodeoxyuridine, BrdU), oligodendrocyte progenitor cells (NG2), oligodendrocytes (RIP), and brain-derived neurotrophic factor (BDNF). The maximum clinical score and the average clinical scores were significantly decreased in the hBMSC-transplanted mice compared to the phosphate-buffered-saline-treated EAE controls, indicating a significant improvement in function. Demyelination significantly decreased, and BrdU(+) and BDNF(+) cells significantly increased in the hBMSC-treated mice compared to controls. Some BrdU(+) cells were colocalized with NG2(+) and RIP(+) immunostaining. hBMSCs also significantly reduced the numbers of vessels containing inflammatory cell infiltration. These data indicate that hBMSC treatment improved functional recovery after EAE in mice, possibly, via reducing inflammatory infiltrates and demyelination areas, stimulating oligodendrogenesis, and by elevating BDNF expression.     

NG2-positive cells generate A2B5-positive oligodendrocyte precursor cells

Cellular specification of the oligodendrocyte lineage occurs through a series of stages identified by expression of distinct biochemical characteristics. The best characterized oligodendrocyte progenitor cell (OPC) in vitro is the bipotential O2-A progenitor, identified by labeling with monoclonal antibody A2B5, which proliferates predominantly in response to platelet derived growth factor (PDGF). The cellular ancestors of O2-A progenitor cells are currently unclear. In vivo OPCs can be identified by expression of the cell surface markers NG2 (a sulfated proteoglycan) and platelet derived growth factor receptor alphaR). Substantial evidence supports the generation of oligodendrocytes from NG2(+), PDGFalphaR(+) cells both in vivo and in vitro. The developmental relationship between NG2(+) cells and A2B5(-) positive cells is unknown and it is unclear whether they represent identical, partially overlapping or nonoverlapping populations of cells. Here we show that in cultures of developing brain NG2(+) and A2B5(+) cells arise from overlapping cell populations. NG2(+) cells appear prior to the expression of A2B5(+) cells and generate A2B5(+) cells. We propose that during development NG2(+)/A2B5(-) cells (pre-OPCs) represent the direct ancestor to A2B5(+) O2A progenitor cells (OPCs).     

Sema4D deficiency results in an increase in the number of oligodendrocytes in healthy and injured mouse brains

Semaphorins, a family of secreted and membrane‐bound proteins, are known to function as repulsive axon guidance molecules. Sema4D, a class 4 transmembrane‐type semaphorin, is expressed by oligodendrocytes in the central nervous system, but its role is unknown. In this study, the effects of Sema4D deficiency on oligodendrocytes were studied in intact and ischemic brains of adult mice. As observed in previous studies, Sema4D marked by β‐galactosidase in Sema4D mutant mice was localized exclusively on myelin‐associated glycoprotein (MAG)‐positive oligodendrocytes but not on NG2‐positive oligodendrocyte progenitor cells (OPCs). Although there was no difference in the number of the latter cells between Sema4D‐deficient and wild‐type mice, the number of MAG‐positive cells was significantly increased in the cerebral cortex of both nonischemic and postischemic brains of Sema4D‐deficient mice. Cell proliferation, observed by using bromodeoxyuridine incorporation, was evident in the MAG‐positive cells that developed after cerebral ischemia. These data indicate that Sema4D is involved in oligodendrogenesis during development and during recovery from ischemic injury. © 2009 Wiley‐Liss, Inc.     

NG2 colocalizes with axons and is expressed by a mixed cell population in spinal cord lesions

The NG2 proteoglycan is of general interest after spinal cord injury because it is expressed by oligodendrocyte progenitors (OPCs), which contribute to central nervous system remyelination; however, NG2 may inhibit axon regeneration. We and others have examined the spatiotemporal expression of NG2 after spinal cord injury (SCI). Here, we extend those observations and provide a comprehensive analysis of the distribution, phenotype, and colocalization of NG2 cells with axons in a clinically relevant model of spinal contusion. Because contusion models mimic the majority of human SCI, this information is important for understanding endogenous processes that promote and/or prevent repair. The data demonstrate that NG2 levels rise significantly between 3 and 7 days postinjury (dpi) and remain elevated chronically throughout the lesions. NG2 within the lesions could be derived from an array of infiltrating cells; thus, a panel of antibodies was used to investigate NG2 cell phenotypes. First, platelet-derived growth factor-alpha receptor (PDGFalphaR) colocalization was examined because OPCs normally express both markers. PDGFalphaR cells were present in lesions at all times examined. However, only 37% of NG2 cells coexpressed PDGFalphaR at 14 dpi, which dropped to <1% by 70 dpi. This contrasts with the nearly complete overlap in spared tissue surrounding the lesion. In contrast, 40% to 60% of NG2 cells expressed p75 and approximately 84% expressed Sox10, suggesting that many NG2 cells were nonmyelinating Schwann cells. Despite rising levels of NG2, we noted robust and sustained axon growth into the lesions, many of which were located along NG2 profiles. Thus, spinal contusion produces an NG2-rich environment into which axons grow and in which the source of NG2 appears considerably different from that in surrounding spared tissue.     

Iron is essential for oligodendrocyte genesis following intraspinal macrophage activation

Progenitor proliferation and differentiation are necessary for oligodendrocyte replacement. Previously, we showed that intraspinal activation of microglia and macrophages with the TLR4 agonist lipopolysaccharide (LPS) induced robust oligodendrocyte genesis. In this study we investigated whether this process involves iron since LPS can alter macrophage regulation of iron and its storage protein ferritin, and oligodendrocytes require iron for proper development and myelination. Further, activated macrophages can sequester and release iron and ferritin. We first examined whether iron or ferritin was present following LPS microinjection. Using Perl's stain, we noted a slight increase in iron at 1d, and peak iron levels 3d post-injection coincident with maximal macrophage activation. Ferritin+ cells were prevalent by 3d and included macrophages and NG2 cells (putative oligodendrocyte progenitors). At 7d, ferritin was mainly expressed by new oligodendrocytes prevalent throughout the lesions. Because of the timing and distribution of iron and ferritin after LPS, we next used an iron chelator to test whether free iron was necessary for maximal LPS-induced oligodendrocyte genesis. Chelating iron by Deferasirox (Exjade®) after LPS microinjection significantly reduced the number of proliferating NG2 cells and new oligodendrocytes. Of the remaining oligodendrocytes, there was a 2-fold decrease in those expressing ferritin, revealing that the number of oligodendrocytes with high iron stores was reduced. Collectively, these results establish that iron accumulates after intraspinal TLR4 activation and is required for maximal TLR4-induced oligodendrogenesis. Since TLR4 agonists are abundant in CNS injury/disease sites, these results suggest that iron may be essential for macrophage/oligodendrocyte communication and adult glial replacement.     

Ferritin stimulates oligodendrocyte genesis in the adult spinal cord and can be transferred from macrophages to NG2 cells in vivo

Injured CNS tissue often contains elevated iron and its storage protein ferritin, which may exacerbate tissue damage through pro-oxidative mechanisms. Therefore, therapeutic studies often target iron reduction as a neuroprotective strategy. However, iron may be crucial for oligodendrocyte replacement and remyelination. For instance, we previously showed that intraspinal toll-like receptor 4 macrophage activation induced the generation of new ferritin-positive oligodendrocytes, and that iron chelation significantly reduced this oligodendrogenic response. Since macrophages can secrete ferritin, we hypothesize that ferritin is a macrophage-derived signal that promotes oligodendrogenesis. To test this, we microinjected ferritin into intact adult rat spinal cords. Within 6 h, NG2+ progenitor cells proliferated and accumulated ferritin. By 3 d, many of these cells had differentiated into new oligodendrocytes. However, acute neuron and oligodendrocyte toxicity occurred in gray matter. Interestingly, ferritin-positive NG2 cells and macrophages accumulated in the area of cell loss, revealing that NG2 cells thrive in an environment that is toxic to other CNS cells. To test whether ferritin can be transferred from macrophages to NG2 cells in vivo, we loaded macrophages with fluorescent ferritin then transplanted them into intact spinal white matter. Within 3-6 d, proliferating NG2 cells migrated into the macrophage transplants and accumulated fluorescently labeled ferritin. These results show that activated macrophages can be an in vivo source of ferritin for NG2 cells, which induces their proliferation and differentiation into new oligodendrocytes. This work has relevance for conditions in which iron-mediated injury and/or repair likely occur, such as hemorrhage, stroke, spinal cord injury, aging, Parkinson's disease, and Alzheimer's disease.     

Progesterone attenuates astro- and microgliosis and enhances oligodendrocyte differentiation following spinal cord injury

Reactive gliosis, demyelination and proliferation of NG2+ oligodendrocyte precursor cells (OPC) are common responses to spinal cord injury (SCI). We previously reported that short-term progesterone treatment stimulates OPC proliferation whereas chronic treatment enhances OPC differentiation after SCI. Presently, we further studied the proliferation/differentiation of glial cells involved in inflammation and remyelination in male rats with SCI subjected to acute (3 days) or chronic (21 days) progesterone administration. Rats received several pulses of bromodeoyuridine (BrdU) 48 and 72 h post-SCI, and sacrificed 3 or 21 days post-SCI. Double colocalization of BrdU and specific cell markers showed that 3 days of SCI induced a strong proliferation of S100β+ astrocytes, OX-42+ microglia/macrophages and NG2+ cells. At this stage, the intense GFAP+ astrogliosis was BrdU negative. Twenty one days of SCI enhanced maturation of S100β+ cells into GFAP+ astrocytes, but decreased the number of CC1+ oligodendrocytes. Progesterone treatment inhibited astrocyte and microglia /macrophage proliferation and activation in the 3-day SCI group, and inhibited activation in the 21-day SCI group. BrdU/NG2 double labeled cells were increased by progesterone at 3 days, indicating a proliferation stimulus, but decreased them at 21 days. However, progesterone-enhancement of CC1+/BrdU+ oligodendrocyte density, suggest differentiation of OPC into mature oligondendrocytes. We conclude that progesterone effects after SCI involves: a) inhibition of astrocyte proliferation and activation; b) anti-inflammatory effects by preventing microglial activation and proliferation, and c) early proliferation of NG2+ progenitors and late remyelination. Thus, progesterone behaves as a glioactive factor favoring remyelination and inhibiting reactive gliosis.