Killing of rat glial cells by complement: Deficiency of the rat analogue of CD59 is the cause of oligodendrocyte susceptibility to lysis

In an effort to understand the mechanisms of complement-mediated injury of the myelin/oligodendrocyte complex in demyelinating disease, we have examined the lytic susceptibility of rat glial cells in culture. It is known that rat oligodendrocytes are extremely sensitive to the lytic action of autologous complement, whereas other cells in the same culture system, including type II astrocytes which derive from the same progenitor cell, are relatively insensitive. Here we demonstrate that the complement sensitivity of oligodendrocytes is associated with a lack of expression of a complement-regulatory protein, the rat homologue of human CD59, and that complement resistance can be restored by the incorporation of purified rat CD59 into the cell membrane. Furthermore, neutralisation of rat CD59 on complement-resistant astrocytes renders them susceptible to lysis. Immature oligodendrocytes were resistant to complement attack yet did not express CD59, suggesting that a complement-activating factor appears on the membrane during oligodendrocyte maturation.     

Bcl-2-expressing oligodendrocytes in multiple sclerosis lesions.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system leading to selective destruction of myelin sheaths and/or oligodendrocytes. The immunological mechanisms responsible for myelin destruction and the primary target of the immune response have not yet been identified. Prior studies have reported a variable degree of oligodendrocyte preservation in actively demyelinating lesions. We have previously demonstrated that oligodendrocyte survival is heterogenous and varies between individual MS patients. Bcl-2 belongs to the group of apoptosis-associated proteins that protects cells from cell death. The purpose of the present study was to determine whether bcl-2 expression is associated with oligodendrocyte preservation observed in some early MS lesions. Double immunocytochemistry was performed with antibodies against bcl-2 and myelin oligodendrocyte glycoprotein (MOG) to identify bcl-2-expressing oligodendrocytes within MS lesions from 43 patients. The number of bcl-2-positive oligodendrocytes was determined depending on the lesion demyelinating activity and the disease course of the patients. The number of bcl-2-expressing oligodendrocytes increased within demyelinating lesions compared to the periplaque white matter, with highest numbers in remyelinating lesions. There was a significant association between the presence of bcl-2-positive oligodendrocytes and the presence of remyelination. The highest proportion of bcl-2-positive oligodendrocytes was observed in a subgroup of patients with relapsing-remitting disease course. The expression of apoptosis-associated proteins may contribute to oligodendrocyte preservation or loss in MS lesions.     

Early loss of oligodendrocytes in human and experimental neuromyelitis optica lesions

Neuromyelitis optica (NMO) is a chronic, mostly relapsing inflammatory demyelinating disease of the CNS characterized by serum anti-aquaporin 4 (AQP4) antibodies in the majority of patients. Anti-AQP4 antibodies derived from NMO patients target and deplete astrocytes in experimental models when co-injected with complement. However, the time course and mechanisms of oligodendrocyte loss and demyelination and the fate of oligodendrocyte precursor cells (OPC) have not been examined in detail. Also, no studies regarding astrocyte repopulation of experimental NMO lesions have been reported. We utilized two rat models using either systemic transfer or focal intracerebral injection of recombinant human anti-AQP4 antibodies to generate NMO-like lesions. Time-course experiments were performed to examine oligodendroglial and astroglial damage and repair. In addition, oligodendrocyte pathology was studied in early human NMO lesions. Apart from early complement-mediated astrocyte destruction, we observed a prominent, very early loss of oligodendrocytes and oligodendrocyte precursor cells (OPCs) as well as a delayed loss of myelin. Astrocyte repopulation of focal NMO lesions was already substantial after 1 week. Olig2-positive OPCs reappeared before NogoA-positive, mature oligodendrocytes. Thus, using two experimental models that closely mimic the human disease, our study demonstrates that oligodendrocyte and OPC loss is an extremely early feature in the formation of human and experimental NMO lesions and leads to subsequent, delayed demyelination, highlighting an important difference in the pathogenesis of MS and NMO.     

Normal rat serum cytotoxicity against syngeneic oligodendrocytes. Complement activation and attack in the absence of anti-myelin antibodies

The role of complement in mediating oligodendrocyte and myelin injury has been investigated by studying the effects of normal adult rat serum on syngeneic cultured neonatal glial cells. Rat serum has cytotoxic activity directed against oligodendrocytes but not astrocytes, the potency of which increases with cell maturation. The effects of heat inactivation, decomplemented rat serum, EGTA treatment, removal of any possible anti-myelin antibody by absorption using syngeneic myelin and absence of surface staining for immunoglobulins on serum-treated oligodendrocytes, C9 depletion and reconstitution, and oligodendrocyte staining for surface C9 demonstrate that this cytotoxicity is mediated by complement via antibody independent activation of the classical pathway and is membrane attack complex dependent. These findings significantly extend the previous demonstration of complement activation by extracted myelin, and may have significance for the pathogenesis of demyelinating diseases.     

CD44 overexpression by oligodendrocytes: a novel mouse model of inflammation-independent demyelination and dysmyelination

The CD44 transmembrane glycoprotein family has been implicated in cell-cell adhesion and cell signaling in response to components of the extracellular matrix but its role in the nervous system is not understood. CD44 proteins are elevated in Schwann cells and oligodendrocytes following nervous system insults, in inflammatory demyelinating lesions, and in tumors. Here, we tested the hypothesis that elevated CD44 expression influences Schwann cell and oligodendrocyte functions by generating transgenic mice that express CD44 under the control of the 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) promoter. These mice failed to develop peripheral nerve or CNS tumors. However, they did develop severe tremors that were associated with CNS dysmyelination and progressive demyelination. Loss of CNS myelin was not due to alterations in early oligodendrocyte precursor differentiation, proliferation, or survival. Myelination in the PNS appeared normal. In no instance was there any evidence of an inflammatory response that could account for the loss of CNS myelin. These findings suggest that CNPase-CD44 mice are a novel model for noninflammatory progressive demyelinating disease and support a potential role for CD44 proteins expressed by glial cells in promoting demyelination.     

Activating transcription factor 6α deficiency exacerbates oligodendrocyte death and myelin damage in immune‐mediated demyelinating diseases

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) play a critical role in immune‐mediated demyelinating diseases, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), by regulating the viability of oligodendrocytes. Our previous studies show that activation of the PERK branch of the UPR protects myelinating oligodendrocytes against ER stress in young, developing mice that express IFN‐γ, a key pro‐inflammatory cytokine in MS and EAE, in the CNS. Several studies also demonstrate that PERK activation preserves oligodendrocyte viability and function, protecting mice against EAE. While evidence suggests activation of the ATF6α branch of the UPR in oligodendrocytes under normal and disease conditions, the effects of ATF6α activation on oligodendrocytes in immune‐mediated demyelinating diseases remain unknown. Herein, we showed that ATF6α deficiency had no effect on oligodendrocytes under normal conditions. Interestingly, we showed that ATF6α deficiency exacerbated ER stressed‐induced myelinating oligodendrocyte death and subsequent myelin loss in the developing CNS of IFN‐γ‐expressing mice. Moreover, we found that ATF6α deficiency increased EAE severity and aggravated EAE‐induced oligodendrocyte loss and demyelination, without affecting inflammation. Thus, these data suggest the protective effects of ATF6α activation on oligodendrocytes in immune‐mediated demyelinating diseases.     

The origin of remyelinating cells in the central nervous system.

A clear understanding of the cellular events underlying successful remyelination of demyelinating lesions is a necessary prerequisite for an understanding of the failure of remyelination in multiple sclerosis (MS). The potential for remyelination of the adult central nervous system (CNS) has been well-established. However, there is still some dispute whether remyelinating oligodendrocytes arise from dedifferentiation and/or proliferation of mature oligodendrocytes, or are generated solely from proliferation and differentiation of glial progenitor cells. This review focuses on studies carried out on remyelinating lesions in the adult rat spinal cord produced by injection of antibodies to galactocerebroside and serum complement that show: (1) oligodendrocytes which survive within an area of demyelination do not contribute to remyelination, (2) remyelination is carried out by oligodendrocyte progenitor cells, (3) recruitment of oligodendrocyte progenitors to an area of demyelination is a local response, and (4) division of oligodendrocyte progenitors is symmetrical, resulting in chronic depletion of the oligodendrocyte progenitor population in the normal white matter around an area of remyelination. Such results suggest that repeated episodes of demyelination could lead to a failure of remyelination due to a depletion of oligodendrocyte progenitors.     

Morphology of oligodendrocytes during demyelination in optic nerves of mice infected with Semliki Forest virus

Multiple sclerosis (MS) is a demyelinating disease which affects oligodendrocytes, the myelinating cells of the CNS. Demyelination is known to occur in the optic nerves of Balb/c mice infected with the avirulent A7(74) strain of Semliki Forest virus (SFV), and many of the changes are similar to those of patients with MS. The aim of the present study was to determine how demyelination proceeds in individual oligodendrocytes in SFV infection, to help in understanding the pathology of demyelination and remyelination in MS. The whole-cell morphology of individual oligodendrocyte units (defined as the oligo-dendrocyte, its processes and the internodal myelin segments of the axons it ensheaths) was characterized using intracellular dye injection in isolated intact optic nerves. In untreated control mice, oligodendrocytes had a relatively uniform morphology and each cell on average provided 20 or so nearby axons with single myelin sheaths with internodal lengths of ～ 150 μm. In SFV infected mice, during the peak of demyelination at post inoculation days 14–21, 55% of oligodendrocytes displayed a range of morphological abnormalities, which most likely represented sequential changes in oligodendrocytes during demyelination. Thus, at the earliest stage of demyelination oligodendrocytes developed swellings or vacuolations along their internodal myelin sheaths, which became gradually attenuated and were completely lost in extreme cases. The results show that whole oligodendrocyte units were affected during SFV-induced demyelination and this is the basis of the focal nature of lesions in this viral model of MS. Individual oligodendrocyte units which had lost their full complement of myelin sheaths had the appearance of immature oligodendrocytes, suggesting they had undergone de-differentiation. We concluded that these cells may not be destroyed during demyelination and it is possible they are capable of remyelination which is a feature of SFV infection in mice and MS in humans.     

Multiple sclerosis: From a myelin point of view

Multiple sclerosis (MS) is a demyelinating disease during which an autoimmune reaction is directed against oligodendroyctes. Alterations of normal myelin structure or oligodendrocyte metabolism may be primary events that influence the susceptibility to MS. Once triggered, the immune system attacks and destroys myelin and the myelin forming cell. Evidence is presented that the oligodendrocyte responds to the attack by immune cells and their secreted products through modulation of its metabolism and gene expression. Cytokines, immunoglobulins, and complement complexes may elicit a survival response in the oligodendrocytes, involving the induction of heat shock proteins and other protective molecules. The possibility of manipulating these complex glial cell functions and controlling their pathologic interactions with immune cells will illuminate how myelin damage can be contained and how the injured tissue can be repaired. © 1996 Wiley-Liss, Inc.     

Role of transmembrane semaphorin Sema6A in oligodendrocyte differentiation and myelination

Myelination is regulated by extracellular proteins, which control interactions between oligodendrocytes and axons. Semaphorins are repulsive axon guidance molecules, which control the migration of oligodendrocyte precursors during normal development and possibly in demyelinating diseases. We show here that the transmembrane semaphorin 6A (Sema6A) is highly expressed by myelinating oligodendrocytes in the postnatal mouse brain. In adult mice, Sema6A expression is upregulated in demyelinating lesions in cuprizone‐treated mice. The analysis of the optic nerve and anterior commissure of Sema6A‐deficient mice revealed a marked delay of oligodendrocyte differentiation. Accordingly, the development of the nodes of Ranvier is also transiently delayed. We also observed an arrest in the in vitro differentiation of purified oligodendrocytes lacking Sema6A, with a reduction of the expression level of Myelin Basic Protein. Their morphology is also abnormal, with less complex and ramified processes than wild‐type oligodendrocytes. In myelinating co‐cultures of dorsal root ganglion neurons and purified oligodendrocytes we found that myelination is perturbed in absence of Sema6A. These results suggest that Sema6A might have a role in myelination by controlling oligodendrocyte differentiation. © 2012 Wiley Periodicals, Inc     

Preferential loss of myelin-associated glycoprotein reflects hypoxia-like white matter damage in stroke and inflammatory brain diseases

Destruction of myelin and oligodendrocytes leading to the formation of large demyelinated plaques is the hallmark of multiple sclerosis (MS) pathology. In a subset of MS patients termed pattern III, actively demyelinating lesions show preferential loss of myelin-associated glycoprotein (MAG) and apoptotic-like oligodendrocyte destruction, whereas other myelin proteins remain well preserved. MAG is located in the most distal periaxonal oligodendrocyte processes and primary "dying back" oligodendrogliopathy may be the initial step of myelin degeneration in pattern III lesions. In the present study, various human white matter pathologies, including acute and chronic white matter stroke, virus encephalitis, metabolic encephalopathy, and MS were studied. In addition to a subset of MS cases, a similar pattern of demyelination was found in some cases of virus encephalitis as well as in all lesions of acute white matter stroke. Brain white matter lesions presenting with MAG loss and apoptotic-like oligodendrocyte destruction, irrespective of their primary disease cause, revealed a prominent nuclear expression of hypoxia inducible factor-1alpha in various cell types, including oligodendrocytes. Our data suggest that a hypoxia-like tissue injury may play a pathogenetic role in a subset of inflammatory demyelinating brain lesions.     

Myelin-oligodendrocyte glycoprotein (MOG) is a surface marker of oligodendrocyte maturation

The myelin-oligodendrocyte glycoprotein (MOG) is a minor component of central nervous system myelin. Using neonatal rat optic nerve oligodendrocyte cultures we have compared the development in vitro of MOG with galactocerebroside, myelin basic protein and 2' ,3'-cyclic-nucleotide 3'-phosphodiesterase. MOG appears on the surface of oligodendrocytes 1-2 days later than these other oligodendrocyte markers, suggesting that MOG may be a useful indicator of oligodendrocyte maturation. The relevance of these findings for investigating mechanisms of myelin injury in vitro and the role of oligodendrocyte damage in demyelinating disease is discussed.     

Oligodendroglia are protected from antibody-mediated complement injury by normal immunoglobulins ("IVIg")

High-dose intravenous immunoglobulin (IVIg) treatment has become a promising immune therapy that can modulate the immune system at several levels, including the complement cascade. In relation to inflammatory demyelinating disease, there is some clinical evidence for the suppression of disease activity by IVIg, while a role in promoting remyelination after experimental myelin damage has been described. Antibody and complement deposition have been implicated in the immune attack in some cases of multiple sclerosis (MS), and to investigate the mechanisms of action of IVIg, we studied the effect of IVIg using the model of complement-mediated cell injury on oligodendroglia in vitro. There was no effect on direct complement lysis of the oligodendroglial cell line CG4, but antibody-dependent complement damage was inhibited in a dose-dependent manner by IVIg. These results were confirmed with primary cultures of oligodendrocyte precursor cells (OPC) and oligodendrocytes. The addition of excess C1, C3, and C4 did not influence the inhibitory effect of IVIg, implying that binding of these complement components does not play a role, in contrast to other experimental models of complement damage. F(ab')2 immunoglobulin fragments were at least partially responsible for the effect. We conclude that IVIg may be protective in antibody-mediated complement injury of oligodendrocytes and their progenitors, and that this effect is likely to be mediated via antibody binding, rather than interference with complement activation. Inhibition of inflammatory mechanisms, as opposed to a direct effect on remyelinating cells, may underlie the role of IVIg in promoting myelin repair in experimental models.     

Endogenous leukemia inhibitory factor production limits autoimmune demyelination and oligodendrocyte loss

Autoimmune injury to oligodendrocytes evokes an endogenous response in the central nervous system, which initially limits the acute injury to oligodendrocytes and myelin, and subsequently promotes remyelination. The key molecular and cellular events responsible for this beneficial outcome are incompletely understood. In this article, we utilize murine autoimmune encephalomyelitis (EAE) to focus on the effect of endogenously produced leukemia inhibitory factor (LIF) upon mature oligodendrocyte survival after demyelinating injury. We show that the mRNA for LIF is markedly upregulated in the spinal cord in the context of acute inflammatory demyelination. After clinical disease onset, administration of neutralizing anti-LIF antibodies over a four day period significantly worsens disease severity in two different murine EAE models. We also show that administration of neutralizing antibodies results in reduced activation of the cognate LIF receptor components in the spinal cord. Histologically, anti-LIF antibody administration increases the extent of acute demyelination (P < 0.01) and doubles the oligodendrocyte loss already induced by EAE (P < 0.05), without altering the extent of inflammatory infiltration into the spinal cord. Although acute EAE induces a rapid, three-fold increase in the proliferation of NG2 positive oligodendrocyte progenitors (P < 0.001), this response is not diminished by antagonism of endogenous LIF. We conclude that endogenous LIF is induced in response to autoimmune demyelination in the spinal cord and protects mature oligodendrocytes from demyelinating injury and cell death, thereby resulting in attenuation of clinical disease severity.     

Live imaging of remyelination after antibody-mediated demyelination in an ex-vivo model for immune mediated CNS damage

Mononuclear cell infiltrates, deposits of immunoglobulin and complement as well as demyelination and axonal damage are neuropathological hallmarks of Multiple Sclerosis (MS) lesions. An involvement of antibodies is further suggested by the presence of oligoclonal immunoglobulins in the cerebrospinal fluid of almost all MS patients. However, which mechanisms are most relevant for de- and remyelination and axonal loss in MS lesions is poorly understood.To characterize the regenerative abilities of demyelinated CNS tissue, we utilized murine organotypic cerebellar slice cultures expressing GFP in oligodendrocytes. The addition of a demyelinating monoclonal antibody specific for myelin oligodendrocyte glycoprotein and complement induced complete myelin destruction and oligodendrocyte loss, as demonstrated by confocal live imaging and staining for different myelin proteins. After removal of antibodies and complement we visualized the stages of remyelination, presumably originating from proliferating oligodendrocyte precursor cells and guided by morphologically intact appearing axons.Allowing for the detailed live imaging of de- and remyelination in an ex vivo situation closely resembling the three dimensional cytoarchitecture of the CNS, we provide a useful experimental system for the evaluation of new therapeutic strategies to enhance remyelination and repair in MS.     

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.     

Evidence for a “dying-back” gliopathy in demyelinating disease

Recurent demyelination was produced in mice by Cuprizone administration. During the second course of Cuprizone, the animals showed greater resistance to the toxin and demyelination occurred slowly and was complete only after prolonged periods. The earliest changes in oligodendrocytes occurred in the most distal processes, the inner cytoplasmic tongues, which showed degenerative changes 3 to 4 weeks before degeneration of the oligodendrocyte cell bodies or demyelination occurred. The results show for the first time that in demyelinating disease, a “dying-back” process similar to that described in axonal disease can affect the oligodendrocyte.     

Contrasting potential of nitric oxide and peroxynitrite to mediate oligodendrocyte injury in multiple sclerosis

Nitric oxide (NO) and peroxynitrite (ONOO(-)) are potential mediators of the injury and cytotoxicity occurring over time to oligodendrocytes in multiple sclerosis (MS) lesions. Our in vitro results indicate that human adult CNS-derived oligodendrocytes are relatively resistant to NO-mediated damage. In contrast, human oligodendrocytes are highly susceptible to peroxynitrite-mediated injury. In situ, we found that inducible nitric oxide synthase (iNOS) was expressed in astrocytes and macrophages in all active demyelinating and remyelinating MS lesions examined, yet no correlation was found between numbers of glial cells expressing iNOS and the extent of oligodendrocyte cell death. Nitrotyrosine groups, indicative of the presence of peroxynitrite in vivo, could be detected on astrocytes, macrophages, and oligodendrocytes in MS lesions. High numbers of nitrotyrosine-positive oligodendrocytes were found in one MS case that featured extensive oligodendrocyte cell death. Our results indicate that NO alone is unlikely to induce oligodendrocyte injury, whereas its more potent byproduct peroxynitrite is a potential mediator of injury to oligodendrocytes in MS.