Persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229E

Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after approximately 130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus's apparent genomic stability.     

NCAM1 is the Target of miRNA-572: Validation in the Human Oligodendroglial Cell Line

The neural cell adhesion molecule 1 (NCAM1) is a fundamental protein in cell–cell interaction and in cellular developmental processes, and its dysregulation is involved in a number of diseases including multiple sclerosis. Studies in rats suggest that the modulation of NCAM1 expression is regulated by miRNA-572, but no data are available confirming such interaction in the human system. We analyzed whether this is the case using a human oligodendroglial cell line (MO3.13). MO3.13 cells were transfected with miRNA-572 mimic and inhibitor separately; NCAM1 mRNA and protein expression levels were analyzed at different time points after transfection. Results indicated that NCAM1 expression is increased after transfection with miRNA-572 inhibitor, whereas it is decreased after transfection with the mimic (p < 0.005). The interaction between NCAM1 and miRNA-572 was subsequently confirmed in a Vero cell line that does not express NCAM1, by luciferase assay after transfection with NCAM1. These results confirm that miRNA-572 regulates NCAM1 and for the first time demonstrate that this interaction regulates NCAM1 expression in human cells. Data herein also support the hypothesis that miRNA-572 is involved in diseases associated with NCAM1 deregulation, suggesting its possible use as a biomarker in these diseases.     

Protective effects of estradiol on TRAIL-induced apoptosis in a human oligodendrocytic cell line: evidence for multiple sites of interactions

Demyelinating diseases are high impact neurological disorders. Steroids are regarded as protective molecules in the susceptibility to these diseases. Here, we studied the interactions between tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent proapoptotic molecule toxic to oligodendrocytes, and 17-beta-estradiol (E-17-beta), in human oligodendrocytic MO3.13 cells. Exposure of cells to TRAIL resulted in the upregulation of both death receptors DR4 and DR5 and apoptosis, as well as the activation of caspase-8 and -3, increased phosphorylation of Jun-N-terminal kinase and p38 kinase, and the reduction of bcl-2 and bcl-xL proteins. TRAIL-mediated MO3.13 cell apoptosis was abrogated by the dominant-negative form of the adaptor protein FADD and by caspase inhibitors. Preincubation with E-17-beta completely prevented both TRAIL-induced DR4 and DR5 upregulation and apoptosis. Estrogen-induced cytoprotection was time and concentration dependent and reverted by antiestrogens. Estrogen treatment per se reduced kinase phosphorylation, and upregulated bcl-2 and bcl-xL proteins. In conclusion, our data show that the detrimental role of TRAIL on oligodendrocytes can be effectively counteracted by estrogens, thus suggesting that the underlying molecular interactions can be of potential relevance in characterizing novel targets for therapy of demyelinating disorders.     

The human oligodendrocyte proteome

Myelination of the CNS is performed by oligodendrocytes (OLs), which have been implicated in brain disorders, such as multiple sclerosis and schizophrenia. We have used the human oligodendroglial cell line MO3.13 to establish an OL reference proteome database. Proteins were prefractionationated by SDS‐PAGE and after in‐gel digestion subjected to nanoflow LC‐MS analysis. Approximately 11 600 unique peptides were identified and, after stringent filtering, resulted in 2290 proteins representing nine distinct biological processes and various molecular classes and functions. OL‐specific proteins, such as myelin basic protein (MBP) and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), as well as other proteins involved in multiple sclerosis and schizophrenia were also identified and are discussed. Proteins of this dataset have also been classified according to their chromosomal origin for providing useful data to the Chromosome‐centric Human Proteome Project (C‐HPP). Given the importance of OLs in the etiology of demyelinating and oligodendrogial disorders, the MO3.13 proteome database is a valuable data resource. The MS proteomics data have been deposited to the ProteomeXchange with identifier PXD000263 ( http://proteomecentral.proteomexchange.org/dataset/PXD000263 ).     

Prolyl oligopeptidase inhibition reduces alpha-synuclein aggregation in a cellular model of multiple system atrophy

Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25α to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25α. Similar to earlier studies, p25α increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25α transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25α did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 µM KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells.     

A human glial hybrid cell line differentially expressing genes subserving oligodendrocyte and astrocyte phenotype

We have developed a series of immortal human-human hybrid cell lines that express phenotypic characteristics of primary oligodendrocytes, by fusing a 6-thioguanine–resistant mutant of the human rhabdomyosarcoma RD with adult human oligodendrocytes by a lectin-enhanced polyethylene glycol procedure. Hybrids were selected in an aminopterin-containing media. In contrast to the tumor parent cells, a hybrid clone M03.13 expressed surface immunoreactivity for galactosyl cerebroside and intracellular immunoreactivity for myelin basic protein (MBP), proteolipid protein (PLP), and glial fibrillary acidic protein (GFAP). Serum deprivation or chronic treatment with a protein kinase C activator 4-β-phorbol 12-myristate 13-acetate (PMA), but not dibutyl cyclic adenosine monophosphate induced coordinate up-regulation or de novo induction of oligodendrocyte phenotypic markers with concomitant down-regulation of GFAP expression. Consistent with immunohistochemical studies, northern blot analysis demonstrated that both MBP and PLP mRNA were up-regulated in MO3.13 cells by PMA treatment. M03.13 cells provide an immortalized clonal model system suitable for study of gene expression subserving oligodendrocyte and astrocyte phenotypes. © 1995 John Wiley & Sons, Inc.     

AMPA glutamate receptor-mediated calcium signaling is transiently enhanced during development of oligodendrocytes

Cells of the oligodendroglial lineage express Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-preferring glutamate receptors (AMPA-GluR) during development. Prolonged activation of their AMPA-GluR causes Ca2+ overload, resulting in excitotoxic death. Prior studies have shown that oligodendroglial progenitors and immature oligodendrocytes are susceptible to excitotoxicity, whereas mature oligodendrocytes are resistant. An unresolved issue has been why Ca2+-permeability of AMPA-GluR varies so markedly with oligodendroglial development, although the level of expression of edited GluR2, an AMPA-GluR subunit which blocks Ca2+ entry, is relatively constant. To address this question, we performed Ca2+ imaging, molecular and electrophysiological analyses using purified cultures of the rat oligodendroglial lineage. We demonstrate that transient up-regulation of expression of GluR3 and GluR4 subunits in oligodendroglial progenitors and immature oligodendrocytes results in the assembly by these cells, but not by oligodendroglial pre-progenitors or mature oligodendrocytes, of a population of AMPA-GluR which lack GluR2. This stage-specific up-regulation of edited GluR2-free, and hence Ca2+-permeable, AMPA-GluR explains the selective susceptibility to excitotoxicity of cells at these stages of oligodendroglial differentiation, and is likely to be important to these cells in the trans-synaptic Ca2+-signaling from glutamatergic neurons, which occurs in hippocampus     

nNOS mediated mitochondrial injury in LPS stimulated oligodendrocytes

Products of inflammation and the activation of nitric oxide synthase have been proposed as a mechanism of oligodendrocyte injury in CNS inflammation. There are currently three well described and known isoforms of NOS. Of these, neuronal NOS (nNOS) was initially discovered in neurons, endothelial NOS (eNOS) in vascular endothelium, while the inducible form of NOS (iNOS) is known to be activated in oligodendrocytes, astrocytes and microglia. We examined the activation of nNOS and the down stream effects of NO production in oligodendrocyte precursor cells (OPC) and MO3.13 cell line following culture with LPS. Our studies show that both MO3.13 cells and OPC are susceptible to the cellular injury resulting from LPS mediated activation and NO production. Activation of the TLR4 receptor with LPS led to decrease in cell viability that was associated with loss of mitochondrial membrane potential and impaired enzymatic activity of complex I and complex IV protein of the respiratory chain. 7-NI, a known inhibitor of nNOS was able to rescue of cells from LPS mediated mitochondrial damage. Loss of mitochondrial function was associated with translocation of cytochrome C and apoptosis inducing factor to the cytosol, setting the stage for apoptosis. Phosphorylation of PI3K and Akt was required for optimal activation of NOS. These studies provide a biochemical basis for nNOS mediated oligodendrocyte injury and suggest similar mechanisms may play a role in diseases characterized by oligodendrocyte loss and demyelination.     

Ion Mobility‐Enhanced Data‐Independent Acquisitions Enable a Deep Proteomic Landscape of Oligodendrocytes

Oligodendrocytes are a type of neuroglia that provide trophic support and insulation to axons in the central nervous system. The genesis and maturation of oligodendrocytes are essential processes for myelination and the course of CNS development. Using ion mobility‐enhanced, data‐independent acquisitions and 2D‐nanoUPLC fractionation operating at nanoscale flow rates, we established a comprehensive data set of proteins expressed by the human oligodendroglia cell line MO3.13. The final dataset incorporating all fractions comprised 223 531 identified peptides assigned to 10 390 protein hits, an improvement of 4.5 times on identified proteins described previously by our group using the same cell line. Identified proteins play pivotal roles in many biological processes such as cell growth and development and energy metabolism, providing a rich resource for future studies on oligodendrocyte development, myelination, axonal support, and the regulation of such process. Our results can help further studies that use MO3.13 cells as a tool of investigation, not only in relation to oligodendrocyte maturation, but also to diseases that have oligodendrocytes as key players. All MS data have been deposited in the ProteomeXchange with identifier PXD004696.     

Expression of biochemical properties of oligodendrocytes in oligodendrocyte x glioma cell hybrids proliferating in vitro

In order to generate cell lines that grow continuously in tissue culture and that express the biochemical properties of oligodendrocytes (the cells which produce myelin in the central nervous system), we isolated oligodendrocytes from calf brain and fused them with C₆ rat glioma cells. Of the 60 hybrid clones tested, several expressed oligodendroglial properties at levels comparable to isolated oligodendrocytes. In particular, hybrid clone CO-13-7 showed a high level of expression of all six oligodendroglial properties tested: 2′ : 3′-cyclic nucleotide 3′-phosphohydroiase, glycero-3-phosphate dehydrogenase, and induction of GPDH by hydrocortisone, all of which were also found in C₆ cells and in oligodendrocytes; and galactocerebroside, sulfatide, and myelin basic protein, which were found in normal oligodendrocytes but not in C₆ glioma cells. Therefore, the hybrids express a spectrum of oligodendrocyte biochemical properties that is not found in any other cell line that can be maintained continuously in tissue culture.     

CD8+ T cell-mediated HLA-A*0201-restricted cytotoxicity to transaldolase peptide 168-176 in patients with multiple sclerosis

Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes and targeted by autoreactive T cells of patients with multiple sclerosis (MS). Among 14 TAL peptides with predicted HLA-A2 binding, TAL 168-176 (LLFSFAQAV, TALpep) exhibited high affinity for HLA-A2. Prevalence of HLA-A2-restricted CD8+ T cells specific for TALpep was increased in PBMC of HLA-A2+ MS patients, as compared with HLA-A2- MS patients, HLA-A2+ other neurological disease patients, and HLA-A2+ healthy donors. HLA-A*0201/TALpep tetramers detected increased frequency of TAL-specific CD8+ T cells, and precursor frequency of TAL-specific IFN-gamma-producing T cells was increased in each of seven HLA-A2+ MS patients tested. Stimulation by TALpep or rTAL of PBMC from HLA-A2+ MS patients elicited killing of TALpep-pulsed HLA-A2-transfected HmyA2.1 lymphoma cells, but not HLA-A3-transfected control HmyA3.1 targets. Without peptide pulsing of targets, HLA-A2-transfected, but not control MO3.13 oligodendroglial cells, expressing high levels of endogenous TAL, were also killed by CD8+ CTL of MS patients, indicating recognition of endogenously processed TAL. TCR Vbeta repertoire analysis revealed use of the TCR Vbeta14 gene by T cell lines (TCL) of MS patients generated via stimulation by TAL- or TALpep-pulsed APCs. All TAL-specific TCL-binding HLA-A*0201/TALpep tetramers expressed TCR Vbeta14 on the cell surface. Moreover, Ab to TCR Vbeta14 abrogated cytotoxicity by HLA-A2-restricted TAL-specific TCL. Therefore, TAL-specific CTL may serve as a novel target for therapeutic intervention in patients with MS.     

Acute and persistent infection of human neural cell lines by human coronavirus OC43

Human coronaviruses (HuCV) are recognized respiratory pathogens. Data accumulated by different laboratories suggest their neurotropic potential. For example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the OC43 strain of HuCV (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). We speculate that the neurotropism of HuCV will lead to persistence within the central nervous system, as was observed for murine coronaviruses. As a first step in the verification of our hypothesis, we have characterized the susceptibility of various human neural cell lines to infection by HuCV-OC43. Viral antigen, infectious virus progeny, and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, oligodendrocytic MO3.13, and the CHME-5 immortalized fetal microglial cell lines, were all susceptible to an acute infection by HuCV-OC43. Viral antigen and RNA and release of infectious virions were observed during persistent HuCV-OC43 infections ( approximately 130 days of culture) of U-87 MG, U-373 MG, MO3.13, and H4 cell lines. Nucleotide sequences of RNA encoding the putatively hypervariable viral S1 gene fragment obtained after 130 days of culture were compared to that of initial virus input. Point mutations leading to amino acid changes were observed in all persistently infected cell lines. Moreover, an in-frame deletion was also observed in persistently infected H4 cells. Some point mutations were observed in some molecular clones but not all, suggesting evolution of the viral population and the emergence of viral quasispecies during persistent infection of H4, U-87 MG, and MO3.13 cell lines. These results are consistent with the potential persistence of HuCV-OC43 in cells of the human nervous system, accompanied by the production of infectious virions and molecular variation of viral genomic RNA.     

Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line

This study delineates the molecular mechanism underlying psychosine-induced oligodendroglial cell death. An immortalized human oligodendroglial cell line, MO3.13, was treated with exogenous psychosine (beta-galactosylsphingosine), a toxic metabolite that accumulates in the tissues of patients with Krabbe's disease. The mode of cell death induced by psychosine was found to be apoptotic, as revealed by different apoptotic markers viz., TUNEL, DNA fragmentation and caspase cleavage/activation. The action of psychosine was redox sensitive, as measured by changes in mitochondrial membrane potential (psidelta), and this effect of psychosine could be reversed by pre-treatment with the antioxidant molecules N-acetyl-l-cysteine or pro-cysteine. Psychosine directly affects the mitochondria as revealed by the activation of caspase 9 but not caspase 8. Up-regulation of the c-jun/c-jun N-terminal kinase pathway by psychosine leads to the induction of AP-1 and, at the same time, psychosine also down-regulates the lipopolysaccharide-induced NF-kappaB transactivation. These observations indicate that the mechanism of action of psychosine is, through the up-regulation of AP-1, a pro-apoptotic pathway as well as, through the down-regulation of the NF-kappaB pathway, an antiapoptotic pathway.     

Frataxin deficiency induces Schwann cell inflammation and death

Mutations in the frataxin gene cause dorsal root ganglion demyelination and neurodegeneration, which leads to Friedreich's ataxia. However the consequences of frataxin depletion have not been measured in dorsal root ganglia or Schwann cells. We knocked down frataxin in several neural cell lines, including two dorsal root ganglia neural lines, 2 neuronal lines, a human oligodendroglial line (HOG) and multiple Schwann cell lines and measured cell death and proliferation. Only Schwann cells demonstrated a significant decrease in viability. In addition to the death of Schwann cells, frataxin decreased proliferation in Schwann, oligodendroglia, and slightly in one neural cell line. Thus the most severe effects of frataxin deficiency were on Schwann cells, which enwrap dorsal root ganglia neurons. Microarray of frataxin-deficient Schwann cells demonstrated strong activations of inflammatory and cell death genes including interleukin-6 and Tumor Necrosis Factor which were confirmed at the mRNA and protein levels. Frataxin knockdown in Schwann cells also specifically induced inflammatory arachidonate metabolites. Anti-inflammatory and anti-apoptotic drugs significantly rescued frataxin-dependent Schwann cell toxicity. Thus, frataxin deficiency triggers inflammatory changes and death of Schwann cells that is inhibitable by inflammatory and anti-apoptotic drugs.     

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.     

Induction of hepatocyte growth factor/scatter factor by interferon-γ in human leukemia cells

Induction of hepatocyte growth factor/scatter factor (HGF/SF) may be one of the critical steps in organ regeneration, wound healing, and embryogenesis. We previously reported the production of HGF/SF from various human leukemia cell lines and a high level of the growth factor in blood and bone marrow plasma from patients with various types of leukemia. We determined here the effects of hematopoietic cytokines on HGF/SF production in human leukemia cell lines, KG-1, a myeloid cell line, and RPMI-8226, a B cell line. Interferon (IFN)-γ remarkably stimulated HGF/SF production in both cell lines at concentrations of more than 0.1 or 1 IU/ml. IFN-α and IFN-β were as effective as IFN-γ in RPMI-8226 cells, but less than IFN-γ in KG-1 cells. HGF/SF gene expression in KG-1 cells was also up-regulated by IFN-γ. Granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-5 and IL-6 had no effect on HGF/SF production in the 2 leukemia cell lines. We also determined the effects of HGF/SF inducers known for human fibroblasts on the growth factor production in leukemia cells. Out of phorbol 12-myristate 13-acetate (PMA), cholera toxin, IL-1β, and tumor necrosis factor (TNF)-α, the former three were as effective as IFN-γ in KG-1 cells, but only TNF-α stimulated HGF/SF production in RPMI-8226 cells, whose effect was less than those of IFN-α, IFN-β, and IFN-γ. The effect of IFN-γ in KG-1 cells was synergistic with that of PMA. In contrast with the effect in leukemia cells, HGF/SF induction by IFN-γ in human skin fibroblasts was much less than that by PMA or cholera toxin. These results indicated that IFN-γ is a potent inducer of HGF/SF in human leukemia cells. This finding suggests the presence of a homeostatic control mechanism in liver regeneration and repair: hepatic injury, DNA synthesis inhibition, or apoptosis caused by IFN-γ is subsequently overcome by cytokine-induced HGF/SF, a potent promoter of liver DNA synthesis. J. Cell. Physiol. 174:107–114, 1998. © 1998 Wiley-Liss, Inc.