Oligodendroglial Cell Line OLI-neu Research Articles

Bisphenol A Impairs Lipid Remodeling Accompanying Cell Differentiation in the Oligodendroglial Cell Line Oli-Neu.

In the central nervous system, the process of myelination involves oligodendrocytes that wrap myelin around axons. Myelin sheaths are mainly composed of lipids and ensure efficient conduction of action potentials. Oligodendrocyte differentiation is an essential preliminary step to myelination which, in turn, is a key event of neurodevelopment. Bisphenol A (BPA), a ubiquitous endocrine disruptor, is suspected to disrupt this developmental process and may, thus, contribute to several neurodevelopmental disorders. In this study, we assessed the effect of BPA on oligodendrocyte differentiation through a comprehensive analysis of cell lipidome by UHPLC-HRMS. For this purpose, we exposed the oligodendroglial cell line Oli-neu to several BPA concentrations for 72 h of proliferation and another 72 h of differentiation. In unexposed cells, significant changes occurred in lipid distribution during Oli-neu differentiation, including an increase in characteristic myelin lipids, sulfatides, and ethanolamine plasmalogens, and a marked remodeling of phospholipid subclasses and fatty acid contents. Moreover, BPA induced a decrease in sulfatide and phosphatidylinositol plasmalogen contents and modified monounsaturated/polyunsaturated fatty acid relative contents in phospholipids. These effects counteracted the lipid remodeling accompanying differentiation and were confirmed by gene expression changes. Altogether, our results suggest that BPA disrupts lipid remodeling accompanying early oligodendrocyte differentiation.

Interferon-γ Activates Nuclear Factor-κ B in Oligodendrocytes through a Process Mediated by the Unfolded Protein Response

Our previous studies have demonstrated that the effects of the immune cytokine interferon-γ (IFN-γ) in immune-mediated demyelinating diseases are mediated, at least in part, by the unfolded protein response (UPR) in oligodendrocytes. Data indicate that some biological effects of IFN-γ are elicited through activation of the transcription factor nuclear factor-κB (NF-κB). Interestingly, it has been shown that activation of the pancreatic endoplasmic reticulum kinase (PERK) branch of the UPR triggers NF-κB activation. In this study, we showed that IFN-γ-induced NF-κB activation was associated with activation of PERK signaling in the oligodendroglial cell line Oli-neu. We further demonstrated that blockage of PERK signaling diminished IFN-γ-induced NF-κB activation in Oli-neu cells. Importantly, we showed that NF-κB activation in oligodendrocytes correlated with activation of PERK signaling in transgenic mice that ectopically express IFN-γ in the central nervous system (CNS), and that enhancing IFN-γ-induced activation of PERK signaling further increased NF-κB activation in oligodendrocytes. Additionally, we showed that suppression of the NF-κB pathway rendered Oli-neu cells susceptible to the cytotoxicity of IFN-γ, reactive oxygen species, and reactive nitrogen species. Our results indicate that the UPR is involved in IFN-γ-induced NF-κB activation in oligodendrocytes and suggest that NF-κB activation by IFN-γ represents one mechanism by which IFN-γ exerts its effects on oligodendrocytes in immune-mediated demyelinating diseases.

MicroRNA expression in mouse oligodendrocytes and regulation of proteolipid protein gene expression

Overexpression of the major myelin proteolipid protein (PLP) is detrimental to brain development and function and is the most common cause of Pelizaeus-Merzbacher disease. microRNA (miRNA), small, noncoding RNAs, have been shown to play critical roles in oligodendrocyte lineage. In this study, we sought to investigate whether miRNAs control PLP abundance. To identify candidate miRNAs involved in this regulation, we have examined differentiation-induced changes in the expression of miRNAs in the oligodendroglial cell line Oli-neu and in enhanced green fluorescent protein positive oligodendrocytes ex vivo. We have identified 145 miRNAs that are expressed in oligodendrocyte cell lineage progression. Dicer1 expression decreases in differentiated oligodendrocytes, and knock down of Dicer1 results in changes in miRNAs similar to those associated with differentiation. To identify miRNAs that control the PLP expression, we have selected miRNAs whose expression is lower in differentiated vs. undifferentiated Oli-neu cells and that have one or more binding site(s) in the PLP 3'-untranslated region (3'UTR). The PLP 3'UTR fused to the luciferase gene reduces the activity of the reporter, suggesting that it negatively regulates message stability or translation. Such suppression is relieved by knock down of miR-20a. Overexpression of miR-20a decreases expression of the endogenous PLP in primary oligodendrocytes and of the reporter gene. Deletion or mutation of the putative binding site for miR-20a in the PLP 3'UTR abrogated such effects. Our data indicate that miRNA expression is regulated by Dicer1 levels in differentiated oligodendrocytes and that miR-20a, a component of the cluster that controls oligodendrocyte cell number, regulates PLP gene expression through its 3'UTR.

Thapsigargin‐induced Ca2+ increase inhibits TGFβ1‐mediated Smad2 transcriptional responses via Ca2+/calmodulin‐dependent protein kinase II

Transforming growth factor β (TGFβ) signalling plays important roles in a variety of tissues and cell types. Impaired TGFβ signalling contributes to several pathologies, including cancer, fibrosis as well as neurodegenerative diseases. TGFβ receptor type I-mediated phosphorylation of Smad2, the formation of the Smad2-Smad4 complex and translocation to the nucleus are critical steps of the TGFβ signalling pathway. Here, we demonstrate that thapsigargin-mediated increase of intracellular Ca(2+) concentrations inhibited TGFβ1-induced Smad2 transcriptional activity in the oligodendroglial cell line OLI-neu. We provide evidence that thapsigargin treatment dramatically reduced the nuclear translocation of Smad2 after TGFβ1 treatment but had no effect on its phosphorylation at Ser465/467. Moreover, using Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitors and a constitutively active CaMKII mutant, we provide evidence that the observed inhibition of TGFβ signalling in OLI-neu cells was strongly dependent on Ca(2+)-mediated CaMKII activation. In summary, this study clearly shows that the TGFβ1-induced Smad2 nuclear translocation is negatively regulated by intracellular Ca(2+) in OLI-neu cells and that increased intracellular Ca(2+) concentrations block Smad2-mediated transcription of TGFβ target genes. These results underline the importance of intracellular Ca(2+) for the regulation of TGFβ signalling.

Involvement of fractin in TGF‐β‐induced apoptosis in oligodendroglial progenitor cells

Transforming growth factor-beta (TGF-beta) induces apoptotic cell death during the development of the nervous system. We recently identified that TGF-beta induced apoptosis in oligodendroglial progenitor cells (primary cells as well as oligodendroglial cell line OLI-neu) is characterized by down-regulation of Bcl-xl. In this report, we now focused on mechanisms that mediate TGF-beta dependent Bcl-xl down-regulation in oligodendroglial cells. We showed that the caspase-specific cleavage product Fractin is produced in oligodendroglial cells during TGF-beta-mediated apoptosis, which represents an early event of the cascade. Cleavage of actin into Fractin was dependent on functional actin and caspases, and occurred simultaneously with a Fractin-Bcl-xl-interaction. This Fractin-Bcl-xl interaction indicated a connection between Bcl-xl down-regulation and Fractin appearance, since Bcl-xl regulation was also dependent on caspases and functional actin, and an overexpression of Fractin induced a Bcl-xl protein down-regulation. Further analysis of Fractin-Bcl-xl interaction in other culture systems confirmed these data. In conclusion, we show that Fractin is not only an apoptotic marker, but has indeed a functional role in apoptotic signaling in oligodendrocytes.

TGF-β superfamily members, ActivinA and TGF-β1, induce apoptosis in oligodendrocytes by different pathways

Activins and transforming growth factor (TGF)-betas, members of the TGF-beta superfamily, affect numerous physiological processes, including apoptosis, in a variety of organs and tissues. Apoptotic functions of TGF-betas, in contrast to those of the activins, are well documented in the developing and adult nervous system. TGF-betas operate in a context-dependent manner and cooperate with other cytokines in the regulation of apoptosis. In this study, we show, for the first time, an apoptotic function of ActivinA in the nervous system, i.e. in oligodendroglial progenitor cells. Using the oligodendroglial cell line OLI-neu, we show that ActivinA acts autonomously, without cooperating with TGF-beta. In contrast to the mechanism of TGF-beta-mediated apoptosis involving Bcl-xl down-regulation, Bcl-xl in ActivinA-induced apoptosis is classically sequestered by the BH3-only protein Puma. Puma expression is controlled by the transcription factor p53 as demonstrated by experiments with the p53 inhibitor Pifithrin-alpha. Furthermore, in the apoptotic TGF-beta pathway, caspase-3 is activated, whereas in the apoptotic ActivinA pathway, apoptosis-inducing factor is released to trigger DNA fragmentation. These data suggest that TGF-beta and ActivinA induce apoptosis in oligodendrocytes by different apoptotic pathways.

Tieg3/Klf11 induces apoptosis in OLI‐neu cells and enhances the TGF‐β signaling pathway by transcriptional repression of Smad7

TGF-beta signaling is indispensible for development of the nervous system since it regulates ontogenetic cell death. The recently identified TGF-beta-inducible zinc finger protein Tieg3/Klf11 belongs to the family of Sp1/Klf-like transcription factors and shares all structural and functional features with other Tieg proteins. Using the established TGF-beta-responsive oligodendroglial cell line OLI-neu, we analyzed the role of Tieg3/Klf11 in TGF-beta signaling. In this report, we show that Tieg3/Klf 11 mimics TGF-beta effects by inducing apoptotic cell death accompanied by activation of caspase-3. Moreover, we demonstrate that Tieg3/Klf11 enhances TGF-beta signaling by transcriptional repression of the inhibitory Smad7 and, thereby, disrupts the negative feedback loop of the TGF-beta signaling pathway. Loss of the N-terminal repression domains of Tieg3/Klf11 abrogates the pro-apoptotic nature of this transcription factor and abolishes the enhancement of Smad-mediated TGF-beta responses. In conclusion, we provide evidence that the recently identified transcription factor Tieg3/Klf11 is a downstream mediator of TGF-beta-induced apoptosis in the oligodendroglial cell line OLI-neu. Since other signaling molecules are able to initiate transcription of members of the Tieg family, the ability of Tieg3/Klf11 to modulate TGF-beta signaling by transcriptional inhibition of Smad7 might be an important clue for the understanding of the crosstalk between different signaling pathways.

Human TIEG2/KLF11 induces oligodendroglial cell death by downregulation of Bcl-XL expression

TGF-beta-induced apoptosis is essential for embryonic development and mainteanance of adult tissues. Impairment of the apoptotic pathway, regulated by TGF-beta, plays a center role in tumorigenesis and manifestations of different diseases. TIEG2/KLF11 is a recently identified human TGF-beta-inducible zinc finger protein belonging to the family of Sp1/KLF-like transcription factors. In human and murine tissues it has been shown that TIEG1 and TIEG2 induce apoptosis and inhibit cell growth. Since TGF-beta and Tieg1 are able to induce apoptosis in the oligodendroglial cell line OLI-neu, we analysed the ability of TIEG2 to mimic the effects observed after treatment with TGF-beta and overexpression of Tieg1. Herein we report that TIEG2 induces Caspase3-dependent apoptosis in murine OLI-neu cells. Furthermore, we could demonstrate that TIEG2 decreases the levels of the anti-apoptotic protein Bcl-X(L) and inhibits transcription driven by the Bcl-X(L) promoter. These data suggest that TIEG2 serves as a downstream mediator of TGF-beta, bridging TGF-beta-dependent signaling to the intracellular pathway of apoptosis.

Functional domains of the TGF‐β‐inducible transcription factor Tieg3 and detection of two putative nuclear localization signals within the zinc finger DNA‐binding domain

The recently identified TGF-beta-inducible early gene 3 (Tieg3) belongs to the gene family of Sp1/Klf-like transcription factors and is upregulated immediately after TGF-beta treatment. To explore the molecular mechanisms of Tieg3-mediated transcriptional control, GAL4-based luciferase assays were performed in order to determine regulatory domains within the Tieg3 protein. Using EGFP-fusion proteins, we monitored the intracellular localization and mapped putative nuclear localization signals (NLS). We provide evidence that the amino-terminus of Tieg3 is essential to repress the transcription and that the loss of the mSin3A interacting domain (SID) disrupts the repressive effects of Tieg3 in the oligodendroglial cell line OLI-neu. Herein we also demonstrate that the zinc finger containing DNA-binding domain (DBD) alone is able to activate the transcription of a reporter gene. Sequence analysis of the zinc finger region revealed no similarities to known activation domains. Analysis of the subcellular localization disclosed Tieg3 as a nuclear protein. Further, we identified the DBD as being essential for the nuclear localization of Tieg3. We detected two closely located putative bipartite NLS within the second and third zinc finger, which are conserved among the members of the Tieg family of proteins. Together these results may help to increase the understanding of Tieg3-mediated transcriptional control and to characterize this TGF-beta-induced Sp1/Klf-like transcription factor.