Cell Line OLN-93 Research Articles

Identification of key regulatory genes involved in myelination after spinal cord injury by GSEA analysis

Multilayer dense myelin tissue provides insulating space and nutritional support for axons in healthy spinal cord tissue. Oligodendrocyte precursor cells (OPCs) are the main glial cells that complement myelin loss in the central nervous system and play an important role in the repair of spinal cord injury (SCI). However, the regulation of axonal remyelination after SCI is still insufficient. In this study, we focused on the changes in genes related to myelin repair after rat hemisection SCI by gene set enrichment analysis (GSEA). Key genes proteolipid protein 1 (Plp1), hexosaminidase subunit alpha (Hexa), and hexosaminidase subunit beta (Hexb) during remyelination after SCI were found. Through quantitative real-time polymerase chain reaction (qPCR) experiments, we confirmed that within 28 days after rat hemisection SCI, the mRNA expression of gene Plp1 gradually decreased, while the expressions of gene Hexa and Hexb gradually increased, which was consistent with RNA sequencing results. In vitro, we performed EdU proliferation assays on OPC cell line OLN-93 and primary rat OPCs. We found that interference of Plp1 promoted OPC proliferation, while interference of Hexa and Hexb inhibited OPC proliferation. In addition, we performed in vitro differentiation experiments on primary rat OPCs. By measuring myelin sheath branch outgrowth and the fluorescence intensity of the mature myelin sheath marker myelin basic protein (MBP), we found that interference of Hexa or Hexb promoted OPC differentiation and maturation, but interference of Plp1 inhibited this process. Finally, we injected Hexb siRNA in vivo and found that interfering Hexb could improve motor movements and myelin regeneration after SCI in rats. Our results provide new target genes that can selectively regulate the proliferation and differentiation of endogenous OPCs, providing new ideas for promoting remyelination and functional recovery after SCI.

Nimodipine Exerts Beneficial Effects on the Rat Oligodendrocyte Cell Line OLN-93.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS). Therapy is currently limited to drugs that interfere with the immune system; treatment options that primarily mediate neuroprotection and prevent neurodegeneration are not available. Here, we studied the effects of nimodipine on the rat cell line OLN-93, which resembles young mature oligodendrocytes. Nimodipine is a dihydropyridine that blocks the voltage-gated L-type calcium channel family members Cav1.2 and Cav1.3. Our data show that the treatment of OLN-93 cells with nimodipine induced the upregulation of myelin genes, in particular of proteolipid protein 1 (Plp1), which was confirmed by a significantly greater expression of PLP1 in immunofluorescence analysis and the presence of myelin structures in the cytoplasm at the ultrastructural level. Whole-genome RNA sequencing additionally revealed the upregulation of genes that are involved in neuroprotection, remyelination, and antioxidation pathways. Interestingly, the observed effects were independent of Cav1.2 and Cav1.3 because OLN-93 cells do not express these channels, and there was no measurable response pattern in patch-clamp analysis. Taking into consideration previous studies that demonstrated a beneficial effect of nimodipine on microglia, our data support the notion that nimodipine is an interesting drug candidate for the treatment of MS and other demyelinating diseases.

Expression of Ectonucleoside Triphosphate Diphosphohydrolase 2 (NTPDase2) Is Negatively Regulated Under Neuroinflammatory Conditions In Vivo and In Vitro.

Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, which is the ligand for P2Y1,12,13 receptors. The present study describes the distribution of NTPDase2 in adult rat brains in physiological conditions, and in hippocampal neurodegeneration induced by trimethyltin (TMT). The study also describes the regulation of NTPDase2 by inflammatory mediators in primary astrocytes and oligodendroglial cell line OLN93. In physiological conditions, NTPDase2 protein was most abundant in the hippocampus, where it was found in fibrous astrocytes and synaptic endings in the synaptic-rich hippocampal layers. In TMT-induced neurodegeneration, NTPDase2-mRNA acutely decreased at 2-dpi and then gradually recovered to the control level at 7-dpi and 21-dpi. As determined by immunohistochemistry and double immunofluorescence, the decrease was most pronounced in the dentate gyrus (DG), where NTPDase2 withdrew from the synaptic boutons in the polymorphic layer of DG, whereas the recovery of the expression was most profound in the subgranular layer. Concerning the regulation of NTPDase2 gene expression, proinflammatory cytokines IL-6, IL-1β, TNFα, and IFNγ negatively regulated the expression of NTPDase2 in OLN93 cells, while did not altering the expression in primary astrocytes. Different cell-intrinsic stressors, such as depletion of intracellular energy store, oxidative stress, endoplasmic reticulum stress, and activation of protein kinase C, also massively disturbed the expression of the NTPDase2 gene. Together, our results suggest that the expression and the activity of NTPDase2 transiently cease in neurodegeneration and brain injury, most likely as a part of the acute adaptive response designed to promote cell defense, survival, and recovery.

Neuroprotective arylpiperazine dopaminergic/serotonergic ligands suppress experimental autoimmune encephalomyelitis in rats

Arylpiperazine-based dopaminergic/serotonergic ligands exert neuroprotective activity. We examined the effect of arylpiperazine D2 /5-HT1A ligands, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl}-phenyl]-picolinamide (6a) and N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), in experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation. Both compounds (10mg/kg i.p.) reduced EAE clinical signs in spinal cord homogenate-immunized Dark Agouti rats. Compound 6b was more efficient in delaying the disease onset and reducing the maximal clinical score, which correlated with its higher affinity for D2 and 5-HT1A receptors. The protection was retained if treatment was limited to the effector (from day 8 onwards), but not the induction phase (day 0-7) of EAE. Compound 6b reduced CNS immune infiltration and expression of mRNA encoding the proinflammatory cytokines tumor necrosis factor, IL-6, IL-1, and GM-CSF, TH 1 cytokine IFN-γ, TH 17 cytokine IL-17, as well as the signature transcription factors of TH 1 (T-bet) and TH 17 (RORγt) cells. Arylpiperazine treatment reduced apoptosis and increased the activation of anti-apoptotic mediators Akt and p70S6 kinase in the CNS of EAE animals. The invitro treatment with 6b protected oligodendrocyte cell line OLN-93 and neuronal cell line PC12 from mitogen-activated normal T cells or myelin basic protein-activated encephalitogenic T cells. In conclusion, arylpiperazine dopaminergic/serotonergic ligands suppress EAE through a direct neuroprotective action and decrease in CNS inflammation. Arylpiperazine dopaminergic/serotonergic ligands reduce neurological symptoms of acute autoimmune encephalomyelitis in rats without affecting the activation of autoreactive immune response, through mechanisms involving a decrease in CNS immune infiltration, as well as direct protection of CNS from immune-mediated damage. These data indicate potential usefulness of arylpiperazine-based compounds in the treatment of neuroinflammatory disorders such as multiple sclerosis.

XAV939, a small molecular inhibitor, provides neuroprotective effects on oligodentrocytes.

White matter tracts are composed of axons and myelinating oligodendrocytes. Oligodendrocytes are the myelinating cells in the central nervous system that allow formation of myelin and saltatory nerve conduction. Cerebral white matter is highly vulnerable to ischemic injury in adults and neonates. White matter injury in newborn brains results in cerebral palsy and cognitive disability. In this study, we found that XAV939, a small-molecular inhibitor that stimulated β-catenin degradation by stabilizing axin, protected against serum and glucose deprivation (SGD)-induced cell death in oligodentrocyte cell line OLN-93 cells in a concentration-dependent manner. We further showed that XAV939 reduced caspase-3 and caspase-8 levels and increased the expression of phosphorylated Akt in SGD-induced OLN-93 cells. Our data demonstrate that XAV939 protects against neonatal hypoxic/ischemic injury. In summary, our results demonstrate that XAV939 confers neuroprotection against SGD-induced injury in OLN-93 cells via its antiapoptotic activity and the loss of oligodendrocytes and neurons in neonatal hypoxic/ischemic injury.

4.1G promotes arborization and tight junction formation of oligodendrocyte cell line OLN-93

4.1G belongs to the membrane-associated band 4.1 protein family, which plays important roles in establishing and maintaining the links between transmembrane proteins and the cytoskeleton. Till date, expression and functions of 4.1G in the central nervous system (CNS) have not been fully elucidated. We investigated expression, cellular/subcellular distribution, and biological roles of 4.1G in the rat CNS and in cultured oligodendrocyte cell line OLN-93. Immunoblotting (IB) and immunoprecipitation revealed CNS 4.1G protein isoforms with molecular weights ranging from ∼80 to ∼180 kDa. In subconfluent OLN-93 cell culture, overexpression of full-length 4.1G and C-terminal-domain-deleted 4.1G, but not the FERM-domain-deleted 4.1G, promoted cellular arborization. In confluent cells, endogenous 4.1G was upregulated and clustered in the cytoplasmic periphery together with tight junction protein ZO-1. FERM domain seemed essential for this recruitment of 4.1G to OLN-93 cell periphery. Calcium switch experiment demonstrated that overexpressed 4.1G promoted tight junction reassembly, whereas siRNA knockdown of endogenous 4.1G inhibited tight junction formation among confluent OLN-93 cells. Together, these results suggest functional roles of 4.1G in cellular arborization and tight junction formation. In the CNS, 4.1G might be involved in maturation of host cells as well as in interaction among neurons/neuroglia.

Effects of Iron Chelators, Iron Salts, and Iron Oxide Nanoparticles on the Proliferation and the Iron Content of Oligodendroglial OLN-93 Cells

The oligodendroglial cell line OLN-93 was used as model system to investigate the consequences of iron deprivation or iron excess on cell proliferation. Presence of ferric or ferrous iron chelators inhibited the proliferation of OLN-93 cells in a time and concentration dependent manner, while the application of a molar excess of ferric ammonium citrate (FAC) prevented the inhibition of proliferation by the chelator deferoxamine. Proliferation of OLN-93 cells was not affected by incubation with 300 microM iron that was applied in the form of FAC, FeCl(2), ferrous ammonium sulfate or iron oxide nanoparticles, although the cells efficiently accumulated iron during exposure to each of these iron sources. The highest specific iron content was observed for cells that were exposed to the nanoparticles. These data demonstrate that the proliferation of OLN-93 cells depends strongly on the availability of iron and that these cells efficiently accumulate iron from various extracellular iron sources.

Fumaric acid dialkyl esters deprive cultured rat oligodendroglial cells of glutathione and upregulate the expression of heme oxygenase 1

Fumaric acid esters (FAE) are currently tested in clinical studies for their potential to treat multiple sclerosis. Since cellular glutathione is involved in the detoxification of xenobiotics and has been reported to form conjugates with FAE, we have analysed the consequences of an application of various FAE to oligodendroglial cells, using the oligodendroglial cell line OLN-93 and oligodendroglia-rich secondary cultures as model systems. In a concentration of 100 microM, dimethylfumarate (DMF) and diethylfumarate (DEF), but not fumarate nor the monoalkyl esters monomethylfumarate or monoethylfumarate, almost completely deprived both OLN-93 cells and secondary oligodendroglial cultures within 60 min of their cellular glutathione. None of the FAE applied compromised cell viability, nor did a treatment with DMF or DEF cause any extracellular accumulation of glutathione. Half-maximal effects on the cellular glutathione content of OLN-93 cells after 60 min of incubation were observed in the presence of 10 microM DMF or DEF. In contrast, fumaric acid monoalkyl esters had to be applied in millimolar concentrations to decrease the cellular glutathione content significantly within 60 min. After removal of DMF, OLN-93 cells completely restored their cellular glutathione content and strongly upregulated heme oxygenase 1. Thus, alterations in glutathione and heme oxygenase metabolism have to be considered when oligodendrocytes are exposed to fumaric acid dialkyl esters.

Effects of Methylprednisolone and Glatiramer Acetate on Nitric Oxide Formation of Cytokine-Stimulated Cells from the Rat Oligodendroglial Cell Line OLN-93

Objectives: In multiple sclerosis, an autoimmune inflammatory disease, oligodendroglia are primarily affected and play an important role in the onset and process of the degeneration of neuronal axons. High-dose therapy with glucocorticoids like 6α-methylprednisolone (MP) as well as the application of immunomodulatory agents like glatiramer acetate (GA) are commonly used in the treatment of MS. The purpose of our study was to examine, in an adequate cell culture model, the effect of MP and GA on oligodendroglial activation induced by pro-inflammatory stimuli. Methods: In the present study, we measured the mRNA (real-time RT-PCR) and protein (Western blot) expression of inducible nitric oxide synthase (iNOS) and the release of nitric oxide (NO; Griess reagent) of rat oligodendroglial progenitor cell line OLN-93 after pro-inflammatory stimulation, and searched for influences of MP and GA on these parameters. OLN-93 cells were treated either with a combination of TNF-α and IFN-γ alone, or additionally with MP or GA. Cell viability and cell protein contents were determined in parallel. Results: Our results show that TNF-α and IFN-γ increased iNOS mRNA and protein expression and the NO production in OLN-93 cells. The elevated production of NO and iNOS protein was reduced in the presence of MP, whereas under treatment with GA, the cytokine-induced overproduction of NO did not change significantly. Conclusions: The presented data suggest an active role of oligodendroglial cells in inflammatory processes like multiple sclerosis and indicate different properties of MP and GA regarding immunosuppression.

α-Synuclein Aggregation and Ser-129 Phosphorylation-dependent Cell Death in Oligodendroglial Cells

Multiple system atrophy is a neurodegenerative disorder characterized by accumulation of aggregated Ser-129-phosphorylated alpha-synuclein in oligodendrocytes. p25alpha is an oligodendroglial protein that potently stimulates alpha-synuclein aggregation in vitro. To model multiple system atrophy, we coexpressed human p25alpha and alpha-synuclein in the rat oligodendroglial cell line OLN-93 and observed a cellular response characterized by a fast retraction of microtubules from the cellular processes to the perinuclear region followed by a protracted development of apoptosis. This response was dependent on phosphorylation at Ser-129 in alpha-synuclein as demonstrated by site-directed mutagenesis. Treatment of the cells with the kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole that targets kinases like casein kinase 2, and polo-like kinases abrogated the toxicity. The polo-like kinase inhibitor BI 2536 caused apoptosis in the model. Ser-129 phosphorylation was linked to the formation of phosphorylated oligomers detectable by immunoblotting, and their formation was inhibited by 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole. The process of microtubule retraction was also dependent on aggregation as demonstrated by the protective effect of treating the cells with the specific peptide inhibitor of alpha-synuclein aggregation ASI1D and the non-selective inhibitors Congo Red and baicalein. The fast microtubule retraction was followed by the development of the apoptotic markers: activated caspase-3, phosphatidylserine externalization, nuclear condensation, and fragmentation. These markers could all be blocked by the inhibitors of phosphorylation, aggregation, and caspase-3. Hence, the model predicts that both Ser-129 phosphorylation and aggregation control the toxic alpha-syn pathway in oligodendroglial cells and may represent therapeutic intervention points in multiple system atrophy.

Toxicity of Tungsten Carbide and Cobalt-Doped Tungsten Carbide Nanoparticles in Mammalian Cells in Vitro

BackgroundTungsten carbide nanoparticles are being explored for their use in the manufacture of hard metals. To develop nanoparticles for broad applications, potential risks to human health and the environment should be evaluated and taken into consideration.ObjectiveWe aimed to assess the toxicity of well-characterized tungsten carbide (WC) and cobaltdoped tungsten carbide (WC-Co) nanoparticle suspensions in an array of mammalian cells.MethodsWe examined acute toxicity of WC and of WC-Co (10% weight content Co) nanoparticles in different human cell lines (lung, skin, and colon) as well as in rat neuronal and glial cells (i.e., primary neuronal and astroglial cultures and the oligodendro cyte precursor cell line OLN-93). Furthermore, using electron microscopy, we assessed whether nanoparticles can be taken up by living cells. We chose these in vitro systems in order to evaluate for potential toxicity of the nanoparticles in different mammalian organs (i.e., lung, skin, intestine, and brain).ResultsChemical–physical characterization confirmed that WC as well as WC-Co nanoparticles with a mean particle size of 145 nm form stable suspensions in serum-containing cell culture media. WC nanoparticles were not acutely toxic to the studied cell lines. However, cytotoxicity became apparent when particles were doped with Co. The most sensitive were astrocytes and colon epithelial cells. Cytotoxicity of WC-Co nanoparticles was higher than expected based on the ionic Co content of the particles. Analysis by electron microscopy demonstrated presence of WC nanoparticles within mammalian cells.ConclusionsOur findings demonstrate that doping of WC nanoparticles with Co markedly increases their cytotoxic effect and that the presence of WC-Co in particulate form is essential to elicit this combinatorial effect.

Ammonia at pathophysiologically relevant concentrations activates kynurenic acid synthesis in cultured astrocytes and neurons

The synthesis of the NMDA receptor glycine site antagonist kynurenic acid (KYNA) from exogenously added kynurenine was measured in cultured cerebral cortical neurons, cortical astrocytes and an oligodendroglial cell line OLN-93, incubated for 2 h in the presence or absence of ammonium acetate (“ammonia”) at 0.1–10 mM concentration. In neurons ammonia stimulated KYNA synthesis to ∼160–170% of control at 0.1–1.0 mM, did not produce any effect at 2.5 and 5 mM, and inhibited the synthesis to ∼80% of control at 10 mM concentration. In astrocytes, a stimulation to ∼200% of control occurred in the whole ammonia concentration range. No effect of ammonia was noted in OLN-93 cells. Increased KYNA synthesis may represent a cytoprotective response of astrocytes and neurons against the NMDA receptor-mediated cytotoxic activity of ammonia.

Diffusion of sphingomyelin and myelin oligodendrocyte glycoprotein in the membrane of OLN-93 oligodendroglial cells studied by fluorescence correlation spectroscopy

Evidence has been accumulated that the plasma membrane of various mammalian cell types is heterogeneous in structure and may contain lipid microdomains (lipid rafts). This study focuses on the membrane organization of living oligodendrocytes, which are the myelin-producing cells of the central nervous system. Fluorescence correlation spectroscopy (FCS) was used to monitor the lateral diffusion of a lipid and of a protein in the oligodendroglial cell line OLN-93. The lipid was fluorescently labelled sphingomyelin (Bodipy FL-C5 SM). The protein was the myelin oligodendrocyte glycoprotein (MOG). In order to monitor the lateral diffusion of MOG, OLN-93 cells were transfected with a MOG–EGFP (enhanced green fluorescent protein) fusion plasmid. The measurements were performed at room temperature. FCS data were analyzed for two-dimensional (2D) diffusion according to three models which all included a triplet fraction: (a) 2D 1 component (2D1C), (b) 2D anomalous diffusion (2D1Cα), and (c) 2D 2 components (2D2C). Preliminary results indicate that for the lipid case, the best fits are obtained with 2D2C. In the case of MOG–EGFP, 2D2C and 2D1Cα give fits of similar quality. The parameter estimates obtained with 2D1Cα, however, have a lower standard deviation. The anomaly parameter for MOG–EGFP is 0.59 ± 0.01 . To cite this article: E. Gielen et al., C. R. Biologies 328 (2005).

Expression of protease-activated receptors (PARs) in OLN-93 oligodendroglial cells and mechanism of PAR-1-induced calcium signaling

Protease-activated receptors (PARs) are a group of four members of the superfamily of G protein-coupled receptors that transduce cell signaling by proteolytic activity of extracellular serine proteases, such as thrombin. Possible expression and functions of PARs in oligodendrocytes, the myelin forming cells of the CNS, are still unclear. Here, the oligodendrocyte cell line OLN-93 was used to investigate the signaling of PARs. By reverse transcription-polymerase chain reaction (RT-PCR), immunostaining and Ca2+ imaging studies, we demonstrate that OLN-93 cells functionally express PAR-1. PAR-3 seems to be expressed without apparent activity, and PAR-2 and PAR-4 cannot be detected. Short-term stimulation of the OLN-93 cells with PAR-1 agonists, such as thrombin, trypsin and PAR-1 activating peptide, dose-dependently induced a transient rise of [Ca2+]i. Concentration-effect curves display a sigmoidal concentration dependence. Elevation of [Ca2+]i induced by PAR-1 mainly resulted from Ca2+ release from intracellular stores. Studies on the effects of pertussis toxin (PTX), phospholipase C antagonist and 2-APB, showed that in OLN-93 cells (i) the calcium signaling cascade from PAR-1 was mediated through PTX-insensitive G proteins, (ii) activation of phospholipase C and liberation of InsP3 were events upstream of the Ca2+ release from the stores. In addition, the present study analyzed PAR-1 desensitization caused by exposure to thrombin, trypsin, and PAR-1 activating peptide, elucidated the influence of the protease cathepsin G on PAR-1 activation, and also characterized PAR-1 desensitization. This is the first study, which shows that OLN-93 oligodendrocytes functionally express PAR-1, and identifies the receptor coupling to mobilization of intracellular calcium. Moreover, the expression of PAR-1 was demonstrated by RT-PCR in primary oligodendrocytes from rat brain.

Expression of protease-activated receptors (PARs) in OLN-93 oligodendroglial cells and mechanism of PAR-1-induced calcium signaling

Protease-activated receptors (PARs) are a group of four members of the superfamily of G protein-coupled receptors that transduce cell signaling by proteolytic activity of extracellular serine proteases, such as thrombin. Possible expression and functions of PARs in oligodendrocytes, the myelin forming cells of the CNS, are still unclear. Here, the oligodendrocyte cell line OLN-93 was used to investigate the signaling of PARs. By reverse transcription-polymerase chain reaction (RT-PCR), immunostaining and Ca 2+ imaging studies, we demonstrate that OLN-93 cells functionally express PAR-1. PAR-3 seems to be expressed without apparent activity, and PAR-2 and PAR-4 cannot be detected. Short-term stimulation of the OLN-93 cells with PAR-1 agonists, such as thrombin, trypsin and PAR-1 activating peptide, dose-dependently induced a transient rise of [Ca 2+] i. Concentration-effect curves display a sigmoidal concentration dependence. Elevation of [Ca 2+] i induced by PAR-1 mainly resulted from Ca 2+ release from intracellular stores. Studies on the effects of pertussis toxin (PTX), phospholipase C antagonist and 2-APB, showed that in OLN-93 cells (i) the calcium signaling cascade from PAR-1 was mediated through PTX-insensitive G proteins, (ii) activation of phospholipase C and liberation of InsP 3 were events upstream of the Ca 2+ release from the stores. In addition, the present study analyzed PAR-1 desensitization caused by exposure to thrombin, trypsin, and PAR-1 activating peptide, elucidated the influence of the protease cathepsin G on PAR-1 activation, and also characterized PAR-1 desensitization. This is the first study, which shows that OLN-93 oligodendrocytes functionally express PAR-1, and identifies the receptor coupling to mobilization of intracellular calcium. Moreover, the expression of PAR-1 was demonstrated by RT-PCR in primary oligodendrocytes from rat brain.

Association of increased bcl-2 expression with rescue from tumor necrosis factor-alpha-induced cell death in the oligodendrocyte cell line OLN-93.

: The present study investigated the effects of flupirtine(Katadolon) on tumor necrosis factor (TNF)‐α‐mediated cell death andBcl‐2 expression in the permanent rat oligodendrocyte cell line OLN‐93 (OLNcells). TNF‐α (500 U/ml) induced apoptosis of OLN cells, which wasconfirmed by DNA fragmentation using an in situ end‐labeling technique andultrastructural analysis. Flupirtine significantly reduced the rate ofspontaneous cell death of OLN cells already at low concentrations;TNF‐α‐mediated apoptosis was suppressed only with higher concentrationsof flupirtine (100 μM). Expression of Bcl‐2 protein and mRNA inOLN cells was detected by immunocytochemistry, western blot, and RT‐PCR.Quantitative analysis of western blots revealed an ∼2.5‐fold up‐regulationof Bcl‐2 protein during TNF‐α treatment. Furthermore, addition of 10 or100 μM flupirtine before incubation with TNF‐α led to anapproximately threefold increase of Bcl‐2 expression. Exposure of OLN cells toflupirtine alone moderately augmented the expression of Bcl‐2 protein. Ourdata demonstrate that flupirtine up‐regulates the expression of Bcl‐2 proteinin OLN cells; this Bcl‐2 induction is associated with a reduced rate ofTNF‐α‐induced cell death.

Regulation of the rat oligodendroglia cell line OLN-93 by antisense transfection of butyrylcholinesterase

Butyrylcholinesterase (BChE) is a glial cell marker with unknown function. For neuroepithelial cells, BChE has been shown to regulate cell division and expression of the postmitotic marker acetylcholinesterase (AChE), while similar studies are lacking for glial cells. By transducing an antisense-5'BChE cDNA expression vector via calcium phosphate precipitation, we have analyzed the effect of BChE inhibition on proliferation and differentiation of rat oligodendroglia-derived OLN-93 cells. OLN-93 cells were chosen because they are highly proliferative, while expressing markers of differentiated oligodendrocytes (Richter-Landsberg and Heinrich, 1996). First, we established that OLN-93 cells do express BChE protein, albeit chiefly in an inactive state, and that BChE was decreased by antisense-5'BChE transfection. Cell proliferation was also strongly diminished, protein kinase C (PKCalpha) was upregulated, and expression of cytoskeletal and cell surface proteins was altered. In particular, immunoreactivities of the intermediate filament proteins vimentin and the cell adhesion protein F11 were detected, indicating that BChE-inhibited OLN-93 cells have shifted toward an astrocytic phenotype. These data support a role of the glia marker BChE in CNS glial cell proliferation and differentiation, achieved via a nonenzymatic mechanism. The possible biomedical impact of BChE protein, e.g., on CNS nerve regeneration, is briefly discussed.