Identity, distribution, and development of polydendrocytes: NG2-expressing glial cells

Cells that express the NG2 proteoglycan (NG2+ cells) comprise a unique population of glial cells in the central nervous system. While there is no question that some NG2+ cells differentiate into oligodendrocytes during development, the persistence of numerous NG2+ cells in the mature CNS has raised questions about their identity, relation to other CNS cell types, and functions besides their progenitor role. NG2+ cells also express the alpha receptor for platelet-derived growth factor (PDGF αR), a receptor that mediates oligodendrocyte progenitor proliferation during development. Antigenically, NG2+ cells are distinct from fibrous and protoplasmic astrocytes, resting microglia, and mature oligodendrocytes. Therefore, we propose the term polydendrocytesto refer to all NG2-expressing glial cells in the CNS parenchyma. This distinguishes them from the classical glial cell types and identifies them as the fourth major glial population in the CNS. Recent observations suggest that polydendrocytes are complex cells that physically and functionally interact with other cell types in the CNS. Committed oligodendrocyte progenitor cells arise from restricted foci in the ventral ventricular zone in both spinal cord and brain. It remains to be clarified whether there are multiple sources of oligodendrocytes, and if so whether polydendrocytes (NG2+ cells) represent progenitor cells of all oligodendrocyte lineages. Proliferation of NG2+ cells during early development appears to be dependent on PDGF, but the regulatory mechanisms that govern NG2+ cell proliferation in the mature CNS remain unknown. Pulse-chase labeling with bromodeoxyuridine indicates that polydendrocytes that proliferate in the postnatal spinal cord differentiate into oligodendrocytes. Novel experimental approaches are being developed to further elucidate the functional properties and differentiation potential of polydendrocytes.     

NG2+ CNS glial progenitors remain committed to the oligodendrocyte lineage in postnatal life and following neurodegeneration

The mammalian CNS contains a ubiquitous population of glial progenitors known as NG2+ cells that have the ability to develop into oligodendrocytes and undergo dramatic changes in response to injury and demyelination. Although it has been reported that NG2+ cells are multipotent, their fate in health and disease remains controversial. Here, we generated PDGFαR-CreER transgenic mice and followed their fate in vivo in the developing and adult CNS. These studies revealed that NG2+ cells in the postnatal CNS generate myelinating oligodendrocytes, but not astrocytes or neurons. In regions of neurodegeneration in the spinal cord of ALS mice, NG2+ cells exhibited enhanced proliferation and accelerated differentiation into oligodendrocytes but remained committed to the oligodendrocyte lineage. These results indicate that NG2+ cells in the normal CNS are oligodendrocyte precursors with restricted lineage potential and that cell loss and gliosis are not sufficient to alter the lineage potential of these progenitors.     

Generation of demyelination models by targeted ablation of oligodendrocytes in the zebrafish CNS

Demyelination is the pathological process by which myelin sheaths are lost from around axons, and is usually caused by a direct insult targeted at the oligodendrocytes in the vertebrate central nervous system (CNS). A demyelinated CNS is usually remyelinated by a population of oligodendrocyte progenitor cells, which are widely distributed throughout the adult CNS. However, myelin disruption and remyelination failure affect the normal function of the nervous system, causing human diseases such as multiple sclerosis. In spite of numerous studies aimed at understanding the remyelination process, many questions still remain unanswered. Therefore, to study remyelination mechanisms in vivo, a demyelination animal model was generated using a transgenic zebrafish system in which oligodendrocytes are conditionally ablated in the larval and adult CNS. In this transgenic system, bacterial nitroreductase enzyme (NTR), which converts the prodrug metronidazole (Mtz) into a cytotoxic DNA cross-linking agent, is expressed in oligodendrocyte lineage cells under the control of the mbp and sox10 promoter. Exposure of transgenic zebrafish to Mtz-containing media resulted in rapid ablation of oligodendrocytes and CNS demyelination within 48 h, but removal of Mtz medium led to efficient remyelination of the demyelinated CNS within 7 days. In addition, the demyelination and remyelination processes could be easily observed in living transgenic zebrafish by detecting the fluorescent protein, mCherry, indicating that this transgenic system can be used as a valuable animal model to study the remyelination process in vivo, and to conduct high-throughput primary screens for new drugs that facilitate remyelination.     

NG2 cells in the brain: a novel glial cell population.

There exists a significantly large population of glial cells in the mammalian central nervous system (CNS) that can be identified by the expression of the NG2 proteoglycan. Cells that express NG2 (NG2 cells) are found in the developing and mature CNS and are distinct from neurons, astrocytes, microglia, and mature oligodendrocytes. They are often referred to as oligodendrocyte progenitor cells because of their ability to differentiate into oligodendrocytes in culture. However, the observation that a large number of NG2 cells persist uniformly and ubiquitously in the adult CNS and display a differentiated morphology is not entirely consistent with the notion that NG2 cells are all oligodendrocyte progenitor cells. The role of NG2 cells in oligodendrocyte regeneration and their non-progenitor role in the mature CNS are discussed in this review.     

Cytology and lineage of NG2-positive glia

We present evidence that NG2+ glia are an integral part of an oligodendrocyte/synantocyte (OS) lineage stream the progenitors of which begin to produce both glial phenotypes at about birth. The NG2 CSPG is differentially distributed within the OS lineage, being expressed in progenitors and synantocytes but not in oligodendrocytes. All cells in the OS lineage, except the primordial stem cells, express O4. The oligodendrocyte line reacts with CD9, but synantocytes are CD9?. Nonetheless, synantocytes are morphologically complex and specialised glia which contact axolemma in myelinated fibres at nodes of Ranvier and synaptic terminals, and form >99% of all NG2+ glia in the adult CNS. Thus, the other NG2+ phenotype, the adult oligodendrocyte progenitor cell (AOPC), constitutes a small population of <1% of all NG2+ glia in the mature CNS. AOPC are a heterogeneous set of cells probably originating from multiple sources which, by definition, produce oligodendrocytes in the adult to replace loss after trauma, demyelination and normal ‘wear and tear’. The definitive functions of synantocytes remain undefined.     

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside⁺ (GalC) and O4⁺ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.     

Defining the NG2-expressing cell of the adult CNS

The NG2 proteoglycan is believed to be an in vivomarker for oligodendrocyte progenitors found in the developing brain. The prevalence of NG2-expressing cells that remain in the adult CNS following the end of gliogenesis is significant. Current research is focused on how this cell participates in the normal function of the adult CNS and whether it may be activated by injury and/or contribute to repair. Despite substantial evidence for a sub-population of NG2-expressing cells playing a glial progenitor role in the adult CNS, there is much to be learned. Specifically, the heterogeneity of this population has not been adequately addressed for the adult CNS and while NG2 cells continue to divide in the adult CNS it is not clear what function they serve once myelination is complete. Future studies should elucidate the functional importance of NG2 in a variety of cell functions and shed light on the role NG2-expressing cells play in the intact and diseasedCNS.     

Defective adult oligodendrocyte and Schwann cell development, pigment pattern, and craniofacial morphology in puma mutant zebrafish having an alpha tubulin mutation

The processes of myelination remain incompletely understood but are of profound biomedical importance owing to the several dysmyelinating and demyelinating disorders known in humans. Here, we analyze the zebrafish puma mutant, isolated originally for pigment pattern defects limited to the adult stage. We show that puma mutants also have late-arising defects in Schwann cells of the peripheral nervous system, locomotor abnormalities, and sex-biased defects in adult craniofacial morphology. Using methods of positional cloning, we identify a critical genetic interval harboring two alpha tubulin loci, and we identify a chemically induced missense mutation in one of these, tubulin alpha 8-like 3a (tuba8l3a). We demonstrate tuba8l3a expression in the central nervous system (CNS), leading us to search for defects in the development of oligodendrocytes, the myelinating cells of the CNS. We find gross reductions in CNS myelin and oligodendrocyte numbers in adult puma mutants, and these deficits are apparent already during the larval-to-adult transformation. By contrast, analyses of embryos and early larvae reveal a normal complement of oligodendrocytes that nevertheless fail to localize normal amounts of myelin basic protein (mbp) mRNA in cellular processes, and fail to organize these processes as in the wild-type. This study identifies the puma mutant as a valuable model for studying microtubule-dependent events of myelination, as well as strategies for remyelination in the adult.     

Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell.

Optic nerves of neonatal rats contain a bipotential glial progenitor cell which can be induced by tissue culture conditions to differentiate into either an oligodendrocyte (the myelin-forming cell of the CNS) or a type 2 astrocyte (an astrocyte population found only in the myelinated tracts of the CNS). In our previous studies most oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells differentiated within 3 days in vitro with relatively little division of the progenitors or their differentiated progeny. We have now found that the O-2A progenitors are stimulated to divide in culture by purified populations of type 1 astrocytes, another glial cell-type found in the rat optic nerve. This cell-cell interaction appears to be mediated by a soluble factor(s) and results in the production of large numbers of both progenitor cells and oligodendrocytes. As type 1 astrocytes are the major glial cell-type in the optic nerve when oligodendrocytes first begin to be produced in large numbers in vivo, our results suggest that this astrocyte subpopulation may play an important role in expanding the oligodendrocyte population during normal development.     

Radiation-induced apoptosis in the adult central nervous system is p53-dependent

Oligodendrocytes and subependymal cells in the adult CNS have been shown to undergo radiation-induced apoptosis. Here, we examined the role of p53 in radiation-induced apoptosis in the adult mouse CNS. In the spinal cord of p53+/+ mice, apoptotic glial cells were observed within 24 h after irradiation, and the apoptotic response peaked at 8 h. These apoptotic cells demonstrated the immunohistochemical phenotype of oligodendrocytes, and decreased oligodendrocyte density was observed at 24 h after 22 Gy. A similar time course of radiation-induced apoptosis was seen in subependymal cells in the adult mouse brain. Radiation-induced apoptosis was preceded by an increase in nuclear p53 expression in glial cells of the spinal cord and subependymal cells of the brain. There was no evidence of radiation-induced apoptosis in the spinal cord and subependymal region of p53-/- animals. We conclude that the p53 pathway may be a mechanism through which DNA damage induces apoptosis in the adult CNS.     

Contact with central nervous system myelin inhibits oligodendrocyte progenitor maturation

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated during development through the proliferation and differentiation of a distinct progenitor population. Not all oligodendrocyte progenitors generated during development differentiate, however, and large numbers of oligodendrocyte progenitors are present in the adult CNS, particularly in white matter. These "adult progenitors" can be identified through expression of the NG2 proteoglycan. Adult oligodendrocyte progenitors are thought to develop from the original pool of progenitors and in vitro are capable of differentiating into oligodendrocytes. Why these cells fail to differentiate in the intact CNS is currently unclear. Here we show that contact with CNS myelin inhibits the maturation of immature oligodendrocyte progenitors. The inhibition of oligodendrocyte progenitor maturation is a characteristic of CNS myelin that is not shared by several other membrane preparations including adult and neonatal neural membrane fractions, PNS myelin, or liver. This inhibition is concentration dependent, is reversible, and appears not to be mediated by either myelin basic protein or basic fibroblast growth factor. Myelin-induced inhibition of oligodendrocyte progenitor maturation provides a mechanism to explain the generation of a residual pool of immature oligodendrocyte progenitors in the mature CNS.     

The MEK/ERK pathway is the primary conduit for Borrelia burgdorferi-induced inflammation and P53-mediated apoptosis in oligodendrocytes

Lyme neuroborreliosis (LNB) affects both the central and peripheral nervous systems. In a rhesus macaque model of LNB we had previously shown that brains of rhesus macaques inoculated with Borrelia burgdorferi release inflammatory mediators, and undergo oligodendrocyte and neuronal cell death. In vitro analysis of this phenomenon indicated that while B. burgdorferi can induce inflammation and apoptosis of oligodendrocytes per se, microglia are required for neuronal apoptosis. We hypothesized that the inflammatory milieu elicited by the bacterium in microglia or oligodendrocytes contributes to the apoptosis of neurons and glial cells, respectively, and that downstream signaling events in NFkB and/or MAPK pathways play a role in these phenotypes. To test these hypotheses in oligodendrocytes, several pathway inhibitors were used to determine their effect on inflammation and apoptosis, as induced by B. burgdorferi. In a human oligodendrocyte cell line (MO3.13), inhibition of the ERK pathway in the presence of B. burgdorferi markedly reduced inflammation, followed by the JNK, p38 and NFkB pathway inhibition. In addition to eliciting inflammation, B. burgdorferi also increased total p53 protein levels, and suppression of the ERK pathway mitigated this effect. While inhibition of p53 had a minimal effect in reducing inflammation, suppression of the ERK pathway or p53 reduced apoptosis as measured by active caspase-3 activity and the TUNEL assay. A similar result was seen in primary human oligodendrocytes wherein suppression of ERK or p53 reduced apoptosis. It is possible that inflammation and apoptosis in oligodendrocytes are divergent arms of MAPK pathways, particularly the MEK/ERK pathway.     

A Sublethal Dose of TNFα Potentiates Kainate-Induced Excitotoxicity in Optic Nerve Oligodendrocytes

Glutamate receptor-induced cell death, known as excitotoxicity in both neurons and oligodendrocytes, has been implicated as a common pathway of cell death in numerous central nervous system (CNS) diseases and trauma. Research in both neuronal and oligodendrocyte excitotoxicity has examined glutamate’s receptor-mediated effects on CNS cells, and explored strategies to protect cells exposed to the elevated glutamate levels that occur in CNS trauma and disease. Proinflammatory cytokines are also elevated in the injured CNS, and have also been implicated in CNS cell death. Recently, several laboratories have examined cytokines’ effects on neuronal and glial excitotoxicity. Here, we review literature concerning the dynamic susceptibility of both neurons and oligodendrocytes to excitotoxicity, and present new data from our laboratory showing that the susceptibility of oligodendrocytes to excitotoxicity is acutely potentiated by the proinflammatory cytokine TNFα.     

Clonal analysis for elucidating the lineage potential of embryonic NG2+ cells

Background aimsThe widespread NG2-expressing neural progenitors in the central nervous system (CNS) are considered to be multifunctional cells with lineage plasticity, thereby possessing the potential for treating CNS diseases. Their lineages and functional characteristics have not been completely unraveled. The present study aimed to disclose the lineage potential of clonal NG2⁺ populations in vitro and in vivo.MethodsTwenty-four clones from embryonic cerebral cortex-derived NG2⁺ cells were induced for oligodendrocyte, astrocyte, neuronal and chondrocyte differentiation. The expression profiles of neural progenitor markers chondroitin sulfate proteoglycan 4 (NG2), platelet-derived growth factor-α receptor (PDGFαR); nestin and neuronal cell surface antigen (A2B5) were subsequently sorted on cells with distinct differentiation capacity. Transplantation of these NG2⁺ clones into the spinal cord was used to examine their lineage potential in vivo.ResultsIn vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and seven of them were bipotent (oligodendrocytes and astrocytes). Amazingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal–glial lineages but also chondrocytes. These distinct subtypes were further found to exhibit phenotypic heterogeneity based on the examination of a spectrum of neural progenitor markers. Transplanted clones survived, migrated extensively and differentiated into oligodendrocytes, astrocytes or even neurons to integrate with the host spinal cord environmentConclusionsThese results suggest that NG2⁺ cells contain heterogeneous progenitors with distinct differentiation capacities, and the immortalized clonal NG2⁺ cell lines might provide a cell source for treating spinal cord disorders.     

Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

A key aim of therapy for multiple sclerosis (MS) is to promote the regeneration of oligodendrocytes and remyelination in the central nervous system (CNS). The present study provides evidence that the vitamin K-dependent protein growth arrest specific 6 (Gas6) promotes such repair in in vitro cultures of mouse optic nerve and cerebellum. We first determined expression of Gas6 and TAM (Tyro3, Axl, Mer) receptors in the mouse CNS, with all three TAM receptors increasing in expression through postnatal development, reaching maximal levels in the adult. Treatment of cultured mouse optic nerves with Gas6 resulted in significant increases in oligodendrocyte numbers as well as expression of myelin basic protein (MBP). Gas6 stimulation also resulted in activation of STAT3 in optic nerves as well as downregulation of multiple genes involved in MS development, including matrix metalloproteinase-9 (MMP9), which may decrease the integrity of the blood–brain barrier and is found upregulated in MS lesions. The cytoprotective effects of Gas6 were examined in in vitro mouse cerebellar slice cultures, where lysolecithin was used to induce demyelination. Cotreatment of cerebellar slices with Gas6 significantly attenuated demyelination as determined by MBP immunostaining, and Gas6 activated Tyro3 receptor through its phosphorylation. In conclusion, these results demonstrate that Gas6/TAM signaling stimulates the generation of oligodendrocytes and increased myelin production via Tyro3 receptor in the adult CNS, including repair after demyelinating injury. Furthermore, the effects of Gas6 on STAT3 signaling and matrix MMP9 downregulation indicate potential glial cell repair and immunoregulatory roles for Gas6, indicating that Gas6-TAM signaling could be a potential therapeutic target in MS and other neuropathologies.     

NG2-glia as multipotent neural stem cells: fact or fantasy?

Cycling glial precursors-"NG2-glia"-are abundant in the developing and mature central nervous system (CNS). During development, they generate oligodendrocytes. In culture, they can revert to a multipotent state, suggesting that they might have latent stem cell potential that could be harnessed to treat neurodegenerative disease. This hope has been subdued recently by a series of fate-mapping studies that cast NG2-glia as dedicated oligodendrocyte precursors in the healthy adult CNS-though rare, neuron production in the piriform cortex remains a possibility. Following CNS damage, the repertoire of NG2-glia expands to include Schwann cells and possibly astrocytes-but so far not neurons. This reaffirms the central role of NG2-glia in myelin repair. The realization that oligodendrocyte generation continues throughout normal adulthood has seeded the idea that myelin genesis might also be involved in neural plasticity. We review these developments, highlighting areas of current interest, contention, and speculation.     

The cellular and molecular events of central nervous system remyelination

Central nervous system (CNS)* regeneration is a subject of great interest, particularly in diseases causing a dramatic loss of neurons. However, some CNS diseases do not affect neurons but damage other cells, such as the myelin-forming cells--called oligodendrocytes--which are also crucial to the harmonious function of the nervous system. Diseases in which oligodendrocytes and myelin are attacked can cause devastating neurological dysfunction which is sometimes followed by recovery and myelin repair or remyelination. The question of the regeneration potential of oligodendrocytes in experimental and human demyelinating diseases such as multiple sclerosis has been debated for a long time. Present evidence suggests that oligodendrocyte precursor cells persist in the adult CNS and that oligodendrocyte regeneration can occur but may be limited by ongoing disease processes. Here we will briefly review recent advances which have broadened our understanding of the cellular and molecular events of CNS remyelination.