OLN-93 Cells Research Articles

Tri-block copolymer nanoparticles modified with folic acid for temozolomide delivery in glioblastoma.

In the present study, Folic Acid (FA) ligand was used to functionalize Temozolomide-loaded Poly (ethylene Glycol)-Poly (Butylene Adipate)-Poly (ethylene Glycol)-coated magnetite nanoparticles (TMZ-SPION-PEG-PBA-PEG) for targeted chemotherapy of glioma cells. Four types of nanoparticles were synthesized with the hydrodynamic diameters of 24-49 nm. Using MTT, Prussian blue, and ICP-OES assays, the cytotoxicity effect and cellular uptake of nanoparticles were evaluated in C6 cancer cells and OLN-93 normal cells. Moreover, in vitro anti-tumor efficacy of nanoparticles was evaluated through colony formation, quantitative real-time PCR, and flow cytometry analysis. As compared to OLN-93 cells TMZ-SPION-PEG-PBA-PEG-FA nanoparticles showed an increase in the cytotoxicity of the loaded TMZ in C6 cells within 24 and 48 h treatment (P < 0.0001), while such effect was not observed in the case of non-targeting nanoparticles. The colony formation, flow cytometry, and real-time PCR assays showed that TMZ-SPION-PEG-PBA-PEG-FA led to the enhancement of inhibitory effects to C6 cells compared to TMZ alone (P < 0.05). These results suggested that TMZ-SPION-PEG-PBA-PEG-FA could effectively slow down cell proliferation, due to the targeting effect and the high accumulation of TMZ in C6 cells via an FA-receptor mediated endocytosis. In conclusion, TMZ-loaded magnetite FA-conjugated PEG-PBA-PEG NPs could be used as a targeted drug delivery system for targeted therapy of brain glioma.

Formulation of temozolomide by folic acid-conjugated tri-block copolymer nanoparticles for targeted drug delivery

Introduction: Glioblastoma multiforme (GBM) is the most frequent primary malignant tumor of the brain. But, the treatment of GBM is one of the most problems in cancer therapy because of poor drug penetration across the blood-brain barrier (BBB). Targeting drug delivery system and conjugating targeting moieties was recognized to overcome the poor penetration of chemotherapy drugs into tumor cells. In the present study, folic acid- conjugated magnetite tri-block copolymer was utilized to targeted chemotherapy of the glioma cells. Materials and Methods: The characterization and morphology of NPs (SPION-PEG-PBA- PEG, SPION-PEG-PBA-PEG-FA, TMZ-SPION-PEG-PBA-PEG, and TMZ-SPION-PEG-PBA-PEG- FA) were determined by Dynamic Light Scattering (DLS) analysis and transmission electron microscope (TEM). The in vitro release behaviors of TMZ from NPs were evaluated with an equilibrium dialysis bag diffusion method. Additionally, to identify the targeting effect of FA- conjugated NPs, C6 glioblastoma cells and OLN-93 glial cells were used. The cytotoxicity effect of NPs was determined by the MTT assays in both cell lines. Results: DLS analysis showed that all nanoparticles had mean diameters of 24-49 nm. In our study, TMZ entrapment efficiency of TMZ-SPION-PEG-PBA-PEG-FA and TMZ-SPION-PEG- PBA-PEG nanoparticles were 52.8 and 50%, respectively, with the drug loading capacity of 6.65 and 6.3%, respectively. It was found that nearly 90% of TMZ in stock solution was released within the first 2 h. However, TMZ-loaded NPs generated only 75% leakage within the 48 h and revealed a sustained release feature, which might be described by that drug was gradually released with the dissolution of polymers. The results from the MTT assay indicated that TMZ-SPION-PEG-PBA-PEG-FA NPs revealed the highest anti-proliferation effect on the C6 cells compared with OLN-93 cells (P < 0.0001). Also, compared with unmodified NPs, conjugation with FA-ligand could further elevate the cytotoxicity effect on C6 cells (P < 0.0001) which demonstrated a satisfactory drug delivery system. Conclusion: TMZ-SPION-PEG-PBA-PEG-FA NPs served as a potential system for the transport of TMZ across the GBM cells through receptor-mediated endocytosis. The results revealed that proposed system could be exploited as potential carrier for delivery of drugs to the brain.

Safranal Attenuates Excitotoxin-Induced Oxidative OLN-93 Cells Injury.

Researches have been shown that glutamic acid (GA) or quinolinic acid (QA) can play role in neuroinflammatory and demyelinating diseases including multiple sclerosis (MS), mainly via oligodendrocytes activation and extreme free radicals generation. Recent studies have demonstrated that safranal, an active constituent of Crocus sativus, has several pharmacological effects such as antioxidant, anti-inflammatory and neuroprotective properties. Since there is no data about the impact of safranal on MS, this study was designed to investigate the protective effect of safranal on OLN-93 oligodendrocytes injury induced by GA or QA. At first, the potential toxic effect of safranal on OLN-93 viability was evaluated. Also, the cells were pretreated with safranal (0.1, 1, 10, 50, 100 and 200 μM) for 2 h and then subjected to GA (16 mM) or QA (8 mM) toxicity for 24 h, in which the same treatments were applied. The cell viability and parameters of redox status such as the levels of intracellular reactive oxygen species (ROS) and lipid peroxidation were measured. Safranal at concentration ranges of 1-800 μM had no toxic effect on cell viability (p>0.05). Treatment with safranal significantly increased cell viability following GA or QA insults at concentrations higher than 1 μM (p<0.01). The cytoprotective potential of safranal was also accompanied by decreased ROS accumulation (p<0.001) and malondialdehyde level (p<0.001) following GA or QA insults. The data suggests that safranal exhibits oligoprotection potential by means of inhibiting oxidative stress parameters.

Sulfasalazine alters microglia phenotype by competing endogenous RNA effect of miR-136-5p and long non-coding RNA HOTAIR in cuprizone-induced demyelination

Sulfasalazine (SF) promotes remyelination and improves the outcome of multiple sclerosis (MS) patients. However, the underlining mechanism remains elusive. Here, we examined whether SF blocks microglia switching to a pro-inflammatory M1-like phenotype through a competing endogenous RNA (ceRNA) effects in cuprizone-induced demyelination. The microglia reprogramming effects of SF in the mice model of cuprizone-induced demyelination was measured by histological, immunohistochemical and molecular biological methods. We also measured the effects of the condition media from SF-treated microglia on the differentiation of OLN-93 cells. Insights of the mechanism of ceRNAs of miR-136-5p and long non-coding RNA (lncRNA) HOTAIR were gained from bioinformatic analysis, luciferase assays and RNA binding protein immunoprecipitation. Microglia switched to a pro-inflammatory M1-like phenotype in cuprizone induced-demyelination. Conversely, SF inhibited the M1-like polarization with the increased remyelination which was attenuated by microglia depletion. SF inhibited production of M1-like factors TNF-α and INF-γ in microglia, and thereby promoted the differentiation of OLN-93 oligodendrocytes. SF down-regulated lncRNA HOTAIR but up-regulated miR-136-5p, and thus inactivated AKT2-NF-κB in cuprizone-treated microglia. Importantly, lncRNA HOTAIR overexpression reversed the increased miR-136-5p expression by SF and thereby attenuated the inhibition of AKT2-mediated NF-κB activation. Mimic of miR-136-5p inhibited cuprizone-induced activation of AKT2-NF-κB in the microglia. In summary, SF blocks microglia switching to a pro-inflammatory M1-like phenotype by ceRNA effect of miR-136-5p and lncRNA HOTAIR in cuprizone-induced demyelination. Our findings show the therapeutic potential of SF for human MS probably by targeting epigenetic regulation in microglia.

Chemical hypoxia-induced integrated stress response activation in oligodendrocytes is mediated by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2).

The extent of remyelination in multiple sclerosis lesions is often incomplete. Injury to oligodendrocyte progenitor cells can be a contributing factor for such incomplete remyelination. The precise mechanisms underlying insufficient repair remain to be defined, but oxidative stress appears to be involved. Here, we used immortalized oligodendrocyte cell lines as model systems to investigate a causal relation of oxidative stress and endoplasmic reticulum stress signaling cascades. OLN93 and OliNeu cells were subjected to chemical hypoxia by blocking the respiratory chain at various levels. Mitochondrial membrane potential and oxidative stress levels were quantified by flow cytometry. Endoplasmic reticulum stress was monitored by the expression induction of activating transcription factor 3 and 4 (Atf3, Atf4), DNA damage-inducible transcript 3 protein (Ddit3), and glucose-regulated protein 94. Lentiviral silencing of nuclear factor (erythroid-derived 2)-like 2 or kelch-like ECH-associated protein 1 was applied to study the relevance of NRF2 for endoplasmic reticulum stress responses. We demonstrate that inhibition of the respiratory chain induces oxidative stress in cultured oligodendrocytes which is paralleled by the expression induction of distinct mediators of the endoplasmic reticulum stress response, namely Atf3, Atf4, and Ddit3. Atf3 and Ddit3 expression induction is potentiated in kelch-like ECH-associated protein 1-deficient cells and absent in cells lacking the oxidative stress-related transcription factor NRF2. This study provides strong evidence that oxidative stress in oligodendrocytes activates endoplasmic reticulum stress response in a NRF2-dependent manner and, in consequence, might regulate oligodendrocyte degeneration in multiple sclerosis and other neurological disorders.

Terminalia chebula attenuates quinolinate-induced oxidative PC12 and OLN-93 cell death

BackgroundQuinolinic acid (QA) is a product of tryptophan degradation and its pathologic accumulation has been found to induce neuroinflammatory and demyelinating diseases such as multiple sclerosis via excessive free radicals generation. Recent studies showed that Terminalia chebula has several pharmacological effects such as antioxidant, anti-inflammatory and neuroprotective properties. The aim of this study was evaluation of the protective effect of T. chebula alcoholic extract (TCAE) on oxidative PC12 and OLN-93 cells death induced by QA. MethodsThe cells were pretreated with TCAE (6.25–50μg/mL) for 2h and then subjected to QA (8mM) for 24h. Cell viability and the parameters of redox status including the levels of intracellular reactive oxygen species (ROS), lipid peroxidation and oxidative DNA damage were measured using 2-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium (MTT), 2,7-dicholorofluorecin diacetate (DCF-DA), thiobarbituric acid and comet assays, respectively. ResultsBased on Folin-Ciocalteu method, the total phenolic compounds in TCAE were estimated about 1.18%. TCAE at concentration ranges of 6.25–50μg/mL had no toxic effect on cell viability (p>0.05). Treatment with TCAE significantly increased cell viability following QA insult at concentrations above 25μg/mL (p<0.01). Cytoprotective potential of TCAE also ameliorated ROS accumulation, lipid peroxidation and DNA damage induced by QA. ConclusionThese data suggest that TCAE exhibits neuroprotection and oligoprotection potential by means of alleviating oxidative stress parameters.

Oxygen impairs oligodendroglial development via oxidative stress and reduced expression of HIF-1\u03b1

The premature increase of oxygen tension may contribute to oligodendroglial precursor cell (OPC) damage in preterm infants. Fetal OPCs are exposed to low oxygen tissue tensions not matched when cells are cultured in room air. Maturation (A2B5, O4, O1, MBP, CNP, arborization), oxidative stress (nitrotyrosine Western blot, NRF2 and SOD2 expression), apoptosis (TUNEL), proliferation (Ki67), and expression of transcription factors regulated by Hypoxia-Inducible-Factor-1-alpha (Hif-1α) expressed in OPCs (Olig1, Olig2, Sox9, Sox10) were assessed in rat OPCs and OLN93 cells cultured at 5% O2 and 21% O2. Influences of Hif-1α were investigated by Hif-1α luciferase reporter assays and Hif-1α-knockdown experiments. At 21% O2, cell proliferation was decreased and process arborization of OPCs was reduced. Expression of MBP, CNP, Olig1, Sox9 and Sox10 was lower at 21% O2, while Nrf2, SOD2, nitrotyrosine were increased. Apoptosis was unchanged. Luciferease reporter assay in OLN93 cells indicated increased Hif-1α activity at 5% O2. In OLN93 cells at 5% O2, Hif-1α knockdown decreased the expression of MBP and CNP, similar to that observed at 21% O2. These data indicate that culturing OPCs at 21% O2 negatively affects development and maturation. Both enhanced oxidative stress and reduced expression of Hif-1α-regulated genes contribute to these hyperoxia-induced changes.

FTY720 (Fingolimod) reverses α-synuclein-induced downregulation of brain-derived neurotrophic factor mRNA in OLN-93 oligodendroglial cells

Multiple system atrophy (MSA) is a demyelinating neurodegenerative disorder characterized by accumulation of aggregated α-synuclein (aSyn) inside oligodendrocyte precursors, mature oligodendroglia, and neurons. MSA dysfunction is associated with loss of trophic factor production by glial and neuronal cells. Here, we report that recombinant wild type human aSyn uptake by OLN-93, an oligodendroglia cell-line, reduced brain-derived neurotrophic factor (BDNF) expression. Furthermore, OLN-93 cells stably transfected with human wild type or an MSA-associated mutant aSyn, A53E that produces neuronal and glial inclusions, reduced BDNF mRNA to nearly unmeasurable qPCR levels. Curiously, another MSA-associated aSyn mutant, G51D that also produces neuronal and glial inclusions, caused only a trend toward BDNF mRNA reduction in transfected OLN-93 cells. This suggests that oligodendrocyte-associated BDNF loss occurs in response to specific aSyn types. Treating OLN-93 cells with 160 nM FTY720 (Fingolimod, Gilenya®), a Food and Drug Administration (FDA) approved therapeutic for multiple sclerosis, counteracted BDNF downregulation in all aSyn OLN-93 cells. FTY720 also restored BDNF mRNA in OLN-93 cells treated with recombinant aSyn, as measured by qPCR or semiquantitatively on agarose gels. Immunoblots confirmed that FTY720 increased histone 3 acetylation in OLN-93, and chromatin immunoprecipitation assays showed increased acetylated histone 3 at BDNF promoter 1 after FTY720. Moreover, OLN-93 cells treated with valproic acid, a classic histone deacetylase inhibitor, confirmed that increasing acetylated histone 3 levels increases BDNF expression. Cumulatively, the data suggest that FTY720-associated histone deacetylase inhibition stimulates BDNF expression in oligodendroglial cells, raising the possibility that MSA patients may also benefit by treatment with FTY720.

Protandim Protects Oligodendrocytes against an Oxidative Insult.

Oligodendrocyte damage and loss are key features of multiple sclerosis (MS) pathology. Oligodendrocytes appear to be particularly vulnerable to reactive oxygen species (ROS) and cytokines, such as tumor necrosis factor-α (TNF), which induce cell death and prevent the differentiation of oligodendrocyte progenitor cells (OPCs). Here, we investigated the efficacy of sulforaphane (SFN), monomethyl fumarate (MMF) and Protandim to induce Nrf2-regulated antioxidant enzyme expression, and protect oligodendrocytes against ROS-induced cell death and ROS-and TNF-mediated inhibition of OPC differentiation. OLN-93 cells and primary rat oligodendrocytes were treated with SFN, MMF or Protandim resulting in significant induction of Nrf2-driven (antioxidant) proteins heme oygenase-1, nicotinamide adenine dinucleotide phosphate (NADPH): quinone oxidoreductase-1 and p62/SQSTM1, as analysed by Western blotting. After incubation with the compounds, oligodendrocytes were exposed to hydrogen peroxide. Protandim most potently promoted oligodendrocyte cell survival as measured by live/death viability assay. Moreover, OPCs were treated with Protandim or vehicle control prior to exposing them to TNF or hydrogen peroxide for five days, which inhibited OPC differentiation. Protandim significantly promoted OPC differentiation under influence of ROS, but not TNF. Protandim, a combination of five herbal ingredients, potently induces antioxidants in oligodendrocytes and is able to protect oligodendrocytes against oxidative stress by preventing ROS-induced cell death and promoting OPC differentiation.

Kynurenic Acid Induces Impairment of Oligodendrocyte Viability: On the Role of Glutamatergic Mechanisms

Kynurenic acid (KYNA) is an end stage product of tryptophan metabolism with a variety of functions in the human body, both in the central nervous system (CNS) and in other organs. Although its activity in the human brain has been widely studied and effects on neural cells were emphasized, the effect of KYNA on oligodendroglial cells remains unknown. Present study aims at describing the activity of high concentration of KYNA in OLN-93 cells. The inhibition of OLN-93 oligodendrocytes viability by KYNA in a medium with reduced serum concentration has been demonstrated. Although decreased metabolic activity of KYNA treated OLN-93 cells was shown, the cells proliferation was not altered. KYNA treatment did not alter morphology as well as expression level of cell cycle and proliferation regulating proteins. Furthermore, glutamate receptor antagonists and agonists did not alter the inhibitory effect of KYNA on viability of OLN-93 oligodendrocytes. This study contributes to the elucidation of effects of KYNA on oligodendrocytes in vitro, yet further analyses are necessary to explain the mechanisms behind the damage and loss of myelin sheaths.

Dimethylsulfoniopropionate Promotes Process Outgrowth in Neural Cells and Exerts Protective Effects against Tropodithietic Acid.

The marine environment harbors a plethora of bioactive substances, including drug candidates of potential value in the field of neuroscience. The present study was undertaken to investigate the effects of dimethylsulfoniopropionate (DMSP), produced by several algae, corals and higher plants, on cells of the mammalian nervous system, i.e., neuronal N2a and OLN-93 cells as model system for nerve cells and glia, respectively. Additionally, the protective capabilities of DMSP were assessed in cells treated with tropodithietic acid (TDA), a marine metabolite produced by several Roseobacter clade bacteria. Both cell lines, N2a and OLN-93, have previously been shown to be a sensitive target for the action of TDA, and cytotoxic effects of TDA have been connected to the induction of oxidative stress. Our data shows that DMSP promotes process outgrowth and microtubule reorganization and bundling, accompanied by an increase in alpha-tubulin acetylation. Furthermore, DMSP was able to prevent the cytotoxic effects exerted by TDA, including the breakdown of the mitochondrial membrane potential, upregulation of heat shock protein Hsp32 and activation of the extracellular signal-regulated kinases 1/2 (ERK1/2). Our study points to the conclusion that DMSP provides an antioxidant defense, not only in algae but also in mammalian neural cells.

A Blue-Light-Emitting BODIPY Probe for Lipid Membranes.

Here we describe a new BODIPY-based membrane probe (1) that provides an alternative to dialkylcarbocyanine dyes, such as DiI-C18, that can be excited in the blue spectral region. Compound 1 has unbranched octadecyl chains at the 3,5-positions and a meso-amino function. In organic solvents, the absorption and emission maxima of 1 are determined mainly by solvent acidity and dipolarity. The fluorescence quantum yield is high and reaches 0.93 in 2-propanol. The fluorescence decays are well fitted with a single-exponential in pure solvents and in small and giant unilamellar vesicles (GUV) with a lifetime of ca. 4 ns. Probe 1 partitions in the same lipid phase as DiI-C18(5) for lipid mixtures containing sphingomyelin and for binary mixtures of dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC). The lipid phase has no effect on the fluorescence lifetime but influences the fluorescence anisotropy. The translational diffusion coefficients of 1 in GUVs and OLN-93 cells are of the same order as those reported for DiI-C18. The directions of the absorption and emission transition dipole moments of 1 are calculated to be parallel. This is reflected in the high steady-state fluorescence anisotropy of 1 in high ordered lipid phases. Molecular dynamic simulations of 1 in a model of the DOPC bilayer indicate that the average angle of the transition moments with respect to membrane normal is ca. 70°, which is comparable with the value reported for DiI-C18.

Noscapine protects OLN-93 oligodendrocytes from ischemia-reperfusion damage: Calcium and nitric oxide involvement.

This study was carried out to evaluate the effects of noscapine, a benzylisoquinoline alkaloid from opium poppy, on oligodendrocyte during ischemia/reperfusion-induced excitotoxic injury. Changes in intracellular calcium levels due to chemical ischemia and nitric oxide (NO) production during ischemia/reperfusion were evaluated as the hallmarks of ischemia-derived excitotoxic event. OLN-93 cell line (a permanent immature rat oligodendrocyte) was used as a model of oligodendrocyte. 30- or 60-minute-oxygen-glucose deprivation/24 hours reperfusion were used to induce excitotoxicity. MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay was used to evaluate cell viability. Ratiometric fluorescence microscopy using Ca(2+)-sensitive indicator Fura-2/AM was utilized to assess intracellular calcium levels. NO production was evaluated by Griess method. Noscapine (4 μM) significantly attenuated intracellular Ca(2+) elevation (P < 0.001). Also, noscapine significantly decreased NO production during a 30-minute oxygen-glucose deprivation/reperfusion (P < 0.01). The inhibitory effect of noscapine (4 μM) on intracellular Ca(2+) was greater than ionotropic glutamate receptors antagonists. Noscapine is protective against ischemia/reperfusion-induced excitotoxic injury in OLN-93 oligodendrocyte. This protective effect seems to be related to attenuation of intracellular Ca(2+) overload and NO production.

Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin.

The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3–0.5 µg/mL (1.4–2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca2+-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death.

Neuroinflammatory signals enhance the immunomodulatory and neuroprotective properties of multipotent adult progenitor cells.

IntroductionStem cell-based therapies are currently widely explored as a tool to treat neuroimmune diseases. Multipotent adult progenitor cells (MAPC) have been suggested to have strong immunomodulatory and neuroprotective properties in several experimental models. In this study, we investigate whether MAPC are of therapeutic interest for neuroinflammatory disorders such as multiple sclerosis by evaluating their capacities to modulate crucial pathological features and gain insights into the molecular pathways involved.MethodsRat MAPC were treated with combinations of pro-inflammatory cytokines that are closely associated with neuroinflammatory conditions, a process called licensing. mRNA expression of immunomodulatory molecules, chemokines and chemokine receptors was investigated. The migratory potential of licensed rat MAPC towards a broad spectrum of chemokines was tested in a Transwell assay. Furthermore, the effect of licensing on the ability of rat MAPC to attract and suppress the proliferation of encephalitogenic T cells was assessed. Finally, neuroprotective properties of rat MAPC were determined in the context of protection from oxidative stress of oligodendrocytes. Therefore, rat MAPC were incubated with conditioned medium of OLN93 cells subjected to sublethal doses of hydrogen peroxide and the gene expression of neurotrophic factors was assessed.ResultsAfter licensing, a wide variety of immunomodulatory molecules and chemokines, including inducible nitric oxide synthase and fractalkine, were upregulated by rat MAPC. The migratory properties of rat MAPC towards various chemokines were also altered. In addition, rat MAPC were found to inhibit antigen-specific T-cell proliferation and this suppressive effect was further enhanced after pro-inflammatory treatment. This phenomenon was partially mediated through inducible nitric oxide synthase or cyclooxygenase-2. Activated rat MAPC secreted factors that led to attraction of myelin-specific T cells. Finally, exposure of rat MAPC to an in vitro simulated neurodegenerative environment induced the upregulation of mRNA levels of vascular endothelial growth factor and ciliary neurotrophic factor. Factors secreted by rat MAPC in response to this environment partially protected OLN93 cells from hydrogen peroxide-induced cell death.ConclusionsRat MAPC possess immune modulatory and neuroprotective properties which are enhanced in response to neuroinflammatory signals. These findings thereby warrant further research to evaluate MAPC transplantation as a therapeutic approach in diseases with an immunological and neurodegenerative component such as multiple sclerosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0169-z) contains supplementary material, which is available to authorized users.

ADNP/ADNP2 expression in oligodendrocytes: implication for myelin-related neurodevelopment.

Oligodendrocytes, the myelin-forming cells of the central nervous system, play important roles in brain development and maintenance. Activity-dependent neuroprotective protein (ADNP), an early marker essential for brain formation, interacts with microtubule end-binding proteins (EB1, EB2, and EB3). EB1 and EB3 are highly expressed in neurons (axons and dendritic spines, respectively) and EB1 enhancement of neurite outgrowth is attenuated by EB2. ADNP/EB presence in oligodendrocytes has not been studied so far. Here, we measured messenger RNA (mRNA) levels of ADNP and EB1-EB3 in rat brain oligodendrocytes during culture maturation and in rat brains during development (1, 35, and 75days) in comparison with rat astrocytes, dorsal root ganglion (DRG) neurons, and the oligodendroglia cell lines (OLN-93 cell line, not expressing the microtubule-associated protein (MAP) tau, and OLN-93 cells stably transfected to express various forms of tau). Results showed that all transcripts studied were expressed in oligodendrocytes. ADNP and EB2 mRNA transcript content peaked at the time of oligodendrocyte maturation (5days in vitro) and was highest in newborn rat brains compared with mature brains. ADNP2 (the only family member of ADNP), and EB1, although expressed in lower quantities, essentially paralleled ADNP and EB2 expression patterns, respectively. EB3 mRNA, peaking upon oligodendrocyte maturation, showed an apparent second peak of expression (10days in vitro) and increased in the mature rat brain compared with the newborn brain. DRG cells expressed the highest levels of EB3, when compared with oligodendrocyte precursors and with astrocytes but not when compared with mature oligodendrocytes. Mature oligodendrocytes and oligodendrocyte precursors expressed ~30-40-fold more EB2 vs. EB3, and ~4-7-fold vs. ADNP. DRGs expressed ~5-fold more EB2 vs. EB3 and astrocytes showed an in-between (~20-fold) ratio. Only DRGs expressed similar EB1 and EB3 transcript levels, contrasting with oligodendrocyte and astrocytes (~10-30-fold more EB1). Astrocytes expressed more ADNP than DRGs and oligodendrocyte precursor cells (~2-fold) but not compared with mature oligodendrocytes. EB1 and EB3 were previously found to be associated with tau. Immortalized oligodendrocytes showed an intermediate phenotype of mRNA expression compared with oligodendrocyte precursor cells and mature oligodendrocytes with tau transfection reducing overall ADNP and EB expression. In summary, ADNPs and EBs are highly expressed in oligodendrocytes suggesting an impact on myelin formation in health and disease.

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.

Effects of nitric oxide synthase deficiency on a disintegrin and metalloproteinase domain-containing protein 12 expression in mouse brain samples.

A disintegrin and metalloproteinase domain-containing protein 12 (ADAM12) belongs to the ADAM family of transmembrane proteins. Via proteolysis, cell adhesion, cell-cell fusion, cell-matrix interaction and membrane protein shedding, ADAM proteins are involved in normal brain development, and also in cancer genesis and progression, and in inflammation. Therefore, neurobiological research focusing on this protein is increasing. Nitric oxide (NO), which is endogenously produced by NO synthases (NOS), is associated with glial tumors. However, knock-out of NOS produces only limited antitumor effects. The present study analyzed the expression of ADAM12 in the cortex and hippocampus of C57/BL6 wild-type mice, and endothelial NOS-, neuronal NOS-(nNOS) or inducible NOS (iNOS)-deficient (-/-) mice, at different stages of development. Expression of ADAM12 was quantified using immunoblot analysis of cortical and hippocampal tissue samples from fetal, neonatal (5 days postnatal), adult (12 weeks old) or >1 year old mice. Using reverse transcription-quantitative polymerase chain reaction, ADAM12 expression was analyzed in cultured N9, OLN93, C6 and PC12 cells, representing the four main cell types in the brain, following NOS inhibition. ADAM12 expression was low in all mouse genotypes and regions of the brain, and in fetal and neonatal mice, an increase in expression was observed with increasing age. The highest levels of expression were observed in the cortex of adult mice, iNOS(-/-) mice of >1 year and wild-type mice, and in the hippocampus of adult and iNOS(-/-) mice of >1 year. By contrast, ADAM12 expression was lowest in adult nNOS(-/-) mice. Inhibition of NOS using N(ω)-Nitro-L-arginine methyl ester hydrochloride, induced ADAM12 mRNA expression in N9 and PC12 cell lines. Inhibition of NOS using L-N(6)-(1-Iminoethyl)lysine dihydrochloride, induced ADAM12 mRNA expression in N9 and C6 cell lines. No change in ADAM12 expression was observed in OLN93 cells following NOS inhibition. ADAM12 expression in mouse hippocampus and cortex samples demonstrated considerable variation during development, with a marked increase observed in adult and >1 year old mice, compared with that in fetal and neonatal mice.

Inhibition of HDAC6 modifies tau inclusion body formation and impairs autophagic clearance.

Proteinaceous inclusions in nerve cells and glia are a defining neuropathological hallmark in a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Their occurrence may be related to malfunctions of the proteolytic degradation systems. In cultured oligodendrocytes, proteasomal inhibition leads to protein aggregate formation resembling coiled bodies, which are characteristic for PSP and CBD. Large protein aggregates are excluded from the proteasome and can only be degraded by autophagy, a lysosomal pathway. Autophagy is a highly selective process, which requires a variety of receptor proteins for ubiquitinated proteins, such as p62 and histone deacetylase 6 (HDAC6). HDAC6 is mainly localized in the cytoplasm, and alpha-tubulin is its major substrate. HDAC6 is considered as a sensor of proteasomal stress; it is involved in the autophagosomal pathway and can mediate the retrograde transport of ubiquitinated proteins along the microtubules. As we have shown recently, HDAC6 is present in oligodendrocytes and its inhibition leads to morphological alterations, microtubule bundling, modulation of acetylation, and phosphorylation of the microtubule-associated protein tau. The present study was undertaken to investigate whether HDAC6 is involved in protein aggregate formation in oligodendrocytes and whether its inhibition modifies the consequences of MG-132-induced inhibition of the ubiquitin proteasome system (UPS). The data show that HDAC6 and acetylated tau are recruited to protein aggregates after proteasomal inhibition. Pharmacological inhibition of HDAC6 by the selective inhibitor tubastatin A (TST) and its small hairpin RNA (shRNA)-mediated downregulation alters the assembly of MG-132-induced compact protein aggregates. After TST treatment, they appear more diffusely dispersed throughout the cytoplasm. This is not a protective means but promotes the onset of apoptotic cell death. Furthermore, the heat shock response is altered, and TST suppresses the MG-132-stimulated induction of HSP70. To test whether the alteration of protein aggregate formation is related to the influence of HDAC6 on the autophagic degradation system, an oligodendroglial cell line, i.e., OLN-93 cells stably expressing green fluorescent protein (GFP)-microtubule associated protein light chain 3 (LC3) and tau, was used. During autophagosome formation, endogenous LC3 is processed to LC3-I, which is then converted to LC3-II. An increase of LC3-II is used as a reliable marker for autophagosome formation and abundance. It is demonstrated that inhibition of HDAC6 leads to the accumulation of LC3-positive autophagosomal vacuoles and an increase in LC3-II immunoreactivity, but the autophagic flux is rather impaired. Hence, the inhibition or dysregulation of HDAC6 contributes to stress responses and pathological processes in oligodendrocytes.

Effect of berberine on nitric oxide production during oxygen-glucose deprivation/reperfusion in OLN-93 oligodendrocytes.

In this study, the effect of berberine, an isoquinoline alkaloid isolated from Coptidis rhizoma, on Nitric Oxide (NO) production, as a possible involved factor, during excitotoxic injury in oligodendroglial cells were evaluated. The overactivation of ionotropic glutamate receptors which is known as the excitotoxicity, is an important phenomenon because of the contribution in acute injury to the central nervous system, chronic neurodegenerative disorders, oligodendrocyte loss and demyelinating diseases as Multiple Sclerosis (MS). Intracellular Ca2+ overload, have a key role during excitotoxic injury and such increase in cytoplasmic Ca2+ triggers a series of events such as production of NO that end to cell death. Previous report showed the protective effects of berberine on ischemic-induced excitotoxic insult in oligodendrocytes. Hereby, we intended to know if the NO production could be associated with oxygen-glucose deprivation/reperfusion-induced excitotoxic damage in oligodendrocyte; moreover, the alteration of NO production could be considered as an involved mechanism for protective effect of berberine in such condition. Therefore, the effect of berberine (2 μM) on NO production during oxygen-glucose deprivation/24 h reperfusion in oligodendrocytes were examined. The OLN-93 cell line (a permanent immature rat oligodendrocyte) was used as a model of oligodendrocyte. Thirty minutes-oxygen-glucose deprivation/24 h reperfusion was used to induce excitotoxicity. NO production was evaluated by Griess method. Our results demonstrated that berberine (2 μM) significantly decreased NO production during 30 min oxygen-glucose deprivation/reperfusion. It seems that blockade of NO production by berberine may also participate in oligodendroglial cell protection against oxygen-glucose deprivation/reperfusion-induced insult.