Joel M. Levine

The oligodendrocyte precursor cell in health and disease

Adult oligodendrocyte precursor cells (OPCs) make up around 5-8% of the glial cell population in the CNS. Their function in the undamaged CNS is largely unknown, but their processes are in contact with nodes of Ranvier and synapses, suggesting a regulatory role at these structures. The cells divide slowly, and constitute approximately 70% of cells labelled following a pulse injection of bromodeoxyuridine. In the injured CNS the cells form a reactive glial population that undergoes hypertrophy and mitosis, probably driven by a variety of growth factors and cytokines. In response to demyelination they divide and are thought to differentiate to provide new oligodendrocytes to replace those that have been lost. However, remyelination fails during the later stages of multiple sclerosis, and it is not clear whether this is as a result of a depletion of adult OPCs, inhibition within the glial scar, or damage to the axons that prevents myelination. Adult OPCs are also activated and proliferate following other forms of CNS damage, such as mechanical injury, excitotoxicity and viral infection. The cells produce several of the chondroitin sulphate proteoglycans that might inhibit axon regeneration.

Increased expression of the NG2 chondroitin-sulfate proteoglycan after brain injury

Injury to the adult mammalian CNS results in reactive changes among the glial cells surrounding the site of damage. Recently, an unusual class of glial cells has been identified within the intact adult rat cerebellum on the basis of the expression of the NG2 chondroitin- sulfate proteoglycan (Levine and Card, 1987). To determine whether the cells that express the NG2 proteoglycan show reactive changes after injury, small puncture lesions were made into the cerebelli of adult rats, and changes among astrocytes, microglia and NG2-positive cells were examined using immunohistochemical staining with cell type- specific marker antibodies. Beginning at 24 hr after lesion, NG2- positive cells immediately adjacent to the lesion site bound the anti- NG2 antibodies more heavily than cells within the undamaged areas of the cerebellum. This increase in anti-NG2 immunoreactivity was transient, reaching a maximum at 7 d postlesion and declining slowly thereafter. The increase in anti-NG2 immunoreactivity was accompanied by an increase in the levels of mRNA encoding the NG2 core protein as demonstrated by in situ hybridization. NG2-positive cells adjacent to the lesion site incorporated 3H-thymidine into their nuclei beginning at 24 hr postlesion and increased in number. Concurrent with these changes, microglia became activated and increased in number, monocytes invaded the damaged tissue, and an astrocytic scar formed. These observations demonstrate that the cells that express the NG2 proteoglycan are a reactive cell type that responds to brain injury. The increased expression of the NG2 chondroitin-sulfate proteoglycan may contribute to the failure of damaged CNS axons to regenerate successfully.

NG2-expressing cells in the central nervous system: are they oligodendroglial progenitors?

Antibodies against the chondroitin sulphate proteoglycan, NG2, are increasingly being used to identify the widespread population of oligodendrocyte progenitor cells in the adult mammalian CNS. However, the specificity of this marker and the role of NG2‐expressing cells in CNS function are still open to question. In this review we consider the evidence that NG2+ cells in the CNS are part of the oligodendrocyte lineage and whether they can give rise to new oligodendrocytes following demyelination. In both the developing and mature rodent CNS, NG2+ cells express the established oligodendrocyte lineage marker PDGF‐αR and from P7, the late progenitor antigen O4, which persists in immature oligodendrocytes. They do not express markers of other CNS populations, such as OX42 or GFAP, at any developmental age. NG2+ cells represent the major cycling cell population in the normal adult rat CNS, suggesting they have stem cell‐like properties. NG2 immunoreactivity is upregulated as a result of physical, viral, excitotoxic and inflammatory insults to the CNS. Following demyelination NG2+ cell number increases in the immediate vicinity of the lesion and rapid remyelination ensues. NG2 expression has also been investigated in human tissue. Multi‐process bearing cells, which morphologically resemble those identified with antibodies against O4, persist in chronically demyelinated multiple sclerosis lesions. J. Neurosci. Res. 61:471–479, 2000.

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.

Activation and Proliferation of Endogenous Oligodendrocyte Precursor Cells during Ethidium Bromide-Induced Demyelination

The adult brain contains a large population of glial cells with the properties of oligodendrocyte precursor cells (OPCs). The functions of this newly recognized class of glial cells in normal animals are unknown. Here, we analyzed the reactions of OPCs to a transient demyelination of the rat brainstem induced by the injection of ethidium bromide (EB) into the fourth ventricle. Within 22 h after EB injection, there is a 21% decrease in the number of OPCs within affected fiber tracts such as the spinal tract of the trigeminal nerve, most likely reflecting the toxic actions of EB. The surviving OPCs had enlarged cell bodies with fewer long processes and many membrane blebs. By 2 days after EB injection, these reactive OPCs had incorporated BrdU and increased in number. The increase in OPC cell number reached a maximum between 6-10 days after EB injection, at which time demyelination was complete. Myelin-specific marker antigens reappeared beginning at 12 days postinjection and the remyelination continued for up to 40 days. During remyelination, OPCs displayed a normal stellate morphology with an increased number of thin processes, many of which were closely associated with neurofilament-positive axonal profiles. The transient increase in the number of reactive OPCs within the demyelinated tissue and subsequent decrease in OPC number during remyelination demonstrates that the endogenous oligodendrocyte precursor population responds rapidly to the pathophysiological state of the brain. Demyelination generates a sufficient number of OPCs to participate in the repair of the demyelinated lesions.

Cells expressing the NG2 antigen contact nodes of Ranvier in adult CNS white matter.

The NG2 antibody, which recognises an integral membrane chondroitin sulphate, labels a significant population of cells in adult CNS white matter tracts of the rat optic nerve and anterior medullary velum (AMV). Adult NG2+ cells are highly complex with multiple branching processes and we show by EM immunocytochemistry that they extend perinodal processes, which contact nodes of Ranvier. NG2+ cells do not react to conventional immunohistochemical markers for adult glia and so we reservedly term them NG2P cells. In vitro, NG2 labels oligodendrocyte‐type‐2 astrocyte (O‐2A) progenitors that can give rise to oligodendrocytes or type‐2 astrocytes, depending on the culture medium. Thus, it is possible that NG2P cells may be derived from the same stem cells as oligodendrocytes. Interestingly, NG2+ cells identified previously in adult CNS displayed phenotypic characteristics of O‐2Aadult progenitors and it is possible that, like them, NG2P cells might retain the capacity of generating oligodendrocytes in the adult CNS. This may be an important role of NG2P cells in demyelinating diseases such as multiple sclerosis. It is significant therefore that the perinodal processes of NG2P cells contact the only sites of exposed axolemma in myelinated axons, so that NG2P cells are ideally situated to detect and respond to changes in axonal function during demyelination. A further implication of our finding is that NG2P cells may perform functions at nodes of Ranvier previously attributed to perinodal astrocytes, including the clustering and maintenance of sodium channels in the axon membrane at nodes, during development and following demyelination. GLIA 26:84–91, 1999.

Generation of oligodendroglial progenitors in acute inflammatory demyelinating lesions of the rat brain stem is associated with demyelination rather than inflammation.

Remyelination is an extremely efficient process in the adult rodent central nervous system yet the source of new oligodendroglia that appear following primary demyelination is still subject to much debate. Using a reliable marker for oligodendroglial progenitor cells in vivo, the NG2 chondroitin sulphate proteoglycan, we have evaluated the response of endogenous NG2+ cells in the adult rat brain stem and cerebellum to inflammatory demyelinating lesions in an experimentally induced animal model of multiple sclerosis (MS), antibody augmented experimental allergic encephalomyelitis (ADEAE). We have manipulated T-cell mediated EAE in Lewis rats by injecting in addition, either anti-myelin/oligodendroglial glycoprotein (MOG) antibodies to induce inflammatory demyelination, or non-specific mouse immunoglobulins to induce an inflammatory response without demyelination. We have examined the relationship of NG2+ progenitor cells to microglia (OX-42+), astrocytes (GFAP+) and mature oligodendroglia (CNP+), in the normal and demyelinated CNS. In the normal CNS NG2-expressing cells are closely intermingled with other glia but represent a distinct cell population. A prominent inflammatory response, identified by the presence of large perivascular and periventricular accumulations of reactive OX42+ macrophages/microglia, occurred in animals with ADEAE at 7–9 days post injection (DPI), coinciding with severe clinical symptoms. In animals injected with anti-MOG antibodies inflammation was followed by the appearance of large areas of demyelination at 11–14 DPI, at which point the animals had recovered clinically. The response of NG2+ cells was different depending on whether the inflammation was accompanied by demyelination. In the presence of inflammation, NG2+ cells responded by an increase in immunoreactivity and an alteration in their morphology, exhibiting enlarged cell bodies and an increased number of intensely stained processes. In areas of demyelination NG2+ cells had fewer intensely stained processes reminiscent of progenitor cells seen during development. Quantitative analysis revealed a 3-fold increase in the number of NG2+ cells in demyelinated lesions at 11 DPI, whereas no change was observed in areas of inflammation in the absence of demyelination. Mitotic figures were only seen in NG2+ cells in areas of demyelination. NG2+ cell numbers appeared to return to control levels following remyelination. These results suggest that endogenous oligodendroglial progenitors divide and/or migrate, in response to signals triggered by demyelinating rather than inflammatory events, to generate a large progenitor population sufficient to promote the rapid and successful remyelination observed in this model.

The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS.

Remyelination of primary demyelinated lesions is a common feature of experimental models of multiple sclerosis (MS) and is also suggested to be the normal response to demyelination during the early stages of MS itself. Many lines of evidence have shown that remyelination is preceded by the division of endogenous oligodendrocyte precursor cells (OPCs) in the lesion and its borders. It is suggested that this rapid response of OPCs to repopulate the lesion site and their subsequent differentiation into new oligodendrocytes is the key to the rapid remyelination. Antibodies to the NG2 chondroitin sulphate proteoglycan have proved exceedingly useful in following and quantitating the response of endogenous OPCs to demyelination. Here we review the literature on the response of NG2-expressing OPCs to demyelination and provide some new evidence on their response to the chronic inflammatory demyelinating environment seen in recombinant myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) in the DA rat. NG2-expressing OPCs responded to the inflammatory demyelination in this model by becoming reactive and increasing in number in a very focal manner. Evidence of NG2+OPCs in lesioned areas beginning to express the oligodendrocyte marker CNP was also seen. The response of OPCs appeared to occur following successive relapses but did not always lead to remyelination, with areas of chronic demyelination observed in the spinal cord. The presence of OPCs in the adult human CNS is clearly of vital importance for repair in multiple sclerosis (MS). As in rat tissue, the antibody labels an evenly distributed cell population present in both white and grey matter, distinct from HLA-DR+microglia. NG2+cells are sparsely distributed in the centre of chronic MS lesions. These cells apparently survive demyelination and exhibit a multi-processed or bipolar morphology in the very hypocellular environment of the lesion.

p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal

Differentiation of most cell types requires both establishment of G1 arrest and the induction of a program related to achieving quiescence. We have chosen to study the differentiation of oligodendrocyte cells to determine the role of p27 and p21 in this process. Here we report that both p27 and p21 are required for the appropriate differentiation of these cells. p27 is required for proper withdrawal from the cell cycle, p21 is not. Instead, p21 is required for the establishment of the differentiation program following growth arrest. Similar observations were made in vivo. We show that p21−/− cells withdraw from the cell cycle similar to wild‐type cells; however, early in animal life, the brain is hypomyelinated, inferring that the loss of p21 delayed myelination in the cerebellum. We found that we could complement or bypass the differentiation failure in p21−/− cells with either PD98059, an inhibitor of Mek1, or by transducing them with a tat–p16Ink4a protein. We concluded that the two cdk inhibitors serve non‐redundant roles in this program of differentiation, with p27 being responsible for arrest and p21 having a function in differentiation independent of its ability to control exit from the cell cycle.

AN2/NG2 protein-expressing glial progenitor cells in the murine CNS: isolation, differentiation, and association with radial glia.

During early neural development, the lineage specification of initially pluripotent progenitor cells is associated with proliferation, differentiation, and migration. Oligodendroglial progenitor cells migrate from their sites of origin to reach the axons that they will myelinate. We have described a cell‐surface protein, AN2, expressed by oligodendroglial progenitor cells in vitro and showed that antibodies against AN2 inhibited the migration of cultured primary oligodendroglial progenitor cells, suggesting that the AN2 antigen plays a role in their migration. Recently, results from MALDI mass spectroscopy showed that AN2 is the mouse homologue of the rat NG2 protein. In this study, we have analyzed cells staining with AN2 antibodies during development and in the adult murine central nervous system (CNS), carried out double stainings with antibodies against NG2, and investigated the differentiation potential of cells in vitro after isolation from early postnatal brain using AN2 antibodies. AN2 and NG2 antibodies stained totally overlapping populations of cells in the CNS. AN2/NG2 expressing cells in embryonic and postnatal brain expressed the PDGF‐α‐receptor and in postnatal brain exhibited electrophysiological properties typical of glial progenitor cells. Cells isolated from early postnatal brain using AN2 monoclonal antibody developed into oligodendrocytes in low serum medium or into astrocytes in the presence of fetal calf serum. In the embryonic spinal cord, cells staining with AN2 antibodies were found closely apposed to radial glial cells, suggesting that glial precursors, like neurons, may use radial glia as scaffolds for migration. GLIA 34:213–228, 2001.