Oligodendroglia Confer Neuroprotection to NSC-34 Motor Neuronal Cells Against the Toxic Insults of Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder with multifactorial pathomechanisms affecting not only motor neurons but also glia. Both astrocytes and microglia get activated and contribute significantly to neurodegeneration. The role of oligodendroglia in such a situation remains obscure, especially in the sporadic form of ALS (SALS), which contributes to 90% of cases. Here, we have investigated the role of oligodendroglia in SALS pathophysiology using a human oligodendroglial cell line, MO3.13, by exposing the cells to cerebrospinal fluid from SALS patients (ALS-CSF; 10% v/v for 48 h). ALS-CSF significantly reduced the viability of MO3.13 cells and down-regulated the expression of oligodendroglia-specific proteins, namely, CNPase and Olig2. Furthermore, to investigate the effect of the observed oligodendroglial changes on motor neurons, NSC-34 motor neuronal cells were co-cultured/supplemented with conditioned/spent medium of MO3.13 cells upon exposure to ALS-CSF. Live cell imaging experiments revealed protection to NSC-34 cells against ALS-CSF toxicity upon co-culture with MO3.13 cells. This was evidenced by the absence of neuronal cytoplasmic vacuolation and beading of neurites, which instead resulted in better neuronal differentiation. Enhanced lactate levels and increased expression of its transporter, MCT-1, with sustained expression of trophic factors, namely, GDNF and BDNF, by MO3.13 cells hint towards metabolic and trophic support provided by the surviving oligodendroglia. Similar metabolic changes were seen in the lumbar spinal cord oligodendroglia of rat neonates intrathecally injected with ALS-CSF. The findings indicate that oligodendroglia are indeed rescuer to the degenerating motor neurons when the astrocytes and microglia turn topsy-turvy.

RNA-Seq Transcriptome Analysis of Differentiated Human Oligodendrocytic MO3.13 Cells Shows Upregulation of Genes Involved in Myogenesis.

In this work, we examined the differentiation of oligodendrocytic MO3.13 cells and changes in their gene expression after treatment with phorbol 12-myristate 13-acetate, PMA, or with RNA polymerase I (Pol I) inhibitor, CX-5461. We found that MO3.13 cells changed their morphology when treated with both agents. Interestingly, CX-5461, but not PMA, induced noticeable changes in the integrity of the nucleoli. Then, we analyzed the p53 transcriptional activity in MO3.13 cells and found that it was increased in both cell populations, but particularly in cells treated with PMA. Interestingly, this high p53 transcriptional activity in PMA-treated cells coincided with a lower level of an unmodified (non-phosphorylated) form of this protein. Since morphological changes in MO3.13 cells after PMA and CX-5461 treatment were evident, suggesting that cells were induced to differentiate, we performed RNA-seq analysis of PMA-treated cells, to reveal the direction of alterations in gene expression. The analysis showed that the largest group of upregulated genes consisted of those involved in myogenesis and K-RAS signaling, rather than those associated with oligodendrocyte lineage progression.

Mechanoreceptor Piezo1 Is Downregulated in Multiple Sclerosis Brain and Is Involved in the Maturation and Migration of Oligodendrocytes in vitro.

Mechanical properties of the brain such as intracranial pressure or stiffness of the matrix play an important role in the brain's normal physiology and pathophysiology. The physical properties are sensed by the cells through mechanoreceptors and translated into ion currents which activate multiple biochemical cascades allowing the cells to adapt and respond to changes in their microenvironment. Piezo1 is one of the first identified mechanoreceptors. It modulates various central nervous system functions such as axonal growth or activation of astrocytes. Piezo1 signaling was also shown to play a role in the pathophysiology of Alzheimer's disease. Here, we explore the expression of the mechanoreceptor Piezo1 in human MO3.13 oligodendrocytes and human MS/non-MS patients' brains and investigate its putative effects on oligodendrocyte proliferation, maturation, and migration. We found that Piezo1 is expressed in human oligodendrocytes and oligodendrocyte progenitor cells in the human brain and that its inhibition with GsMTx4 leads to an increment in proliferation and migration of MO3.13 oligodendrocytes. Activation of Piezo1 with Yoda-1 induced opposite effects. Further, we observed that expression of Piezo1 decreased with MO3.13 maturation in vitro. Differences in expression were also observed between healthy and multiple sclerosis brains. Remarkably, the data showed significantly lower expression of Piezo1 in the white matter in multiple sclerosis brains compared to its expression in the white matter in healthy controls. There were no differences in Piezo1 expression between the white matter plaque and healthy-appearing white matter in the multiple sclerosis brain. Taken together, we here show that Piezo1-induced signaling can be used to modulate oligodendrocyte function and that it may be an important player in the pathophysiology of multiple sclerosis.

Corrigendum: Cannabidiol Displays Proteomic Similarities to Antipsychotics in Cuprizone-Exposed Human Oligodendrocytic Cell Line MO3.13.

[This corrects the article DOI: 10.3389/fnmol.2021.673144.].

A glimpse on the architecture of hnRNP C1/C2 interaction network in cultured oligodendrocytes.

hnRNP represent a large family of RNA-binding proteins related to regulation of transcriptional and translational processes. More specifically, hnRNPs play pivotal roles in the myelination of the central nervous system. The regulation of these proteins are associated with neurodegenerative and psychiatric disorders, including schizophrenia. hnRNPs were shown differentially regulated on schizophrenia postmortem brain tissue as well as in cultured oligodendrocytes treated with clozapine, a common antipsychotic used in schizophrenia treatment. Here we employed co-immunoprecipitation of hnRNP C1/C2 to investigate for the first time in a large-scale manner its interaction partners on cultured oligodendrocytes (MO3.13). Even preliminarily, results bring a more comprehensive description of hnRNP C1/C2 interaction network, and therefore insights regarding the potential role of this protein in the central nervous system in health and disease, warranting further investigation.

Cannabidiol Displays Proteomic Similarities to Antipsychotics in Cuprizone-Exposed Human Oligodendrocytic Cell Line MO3.13.

Cannabidiol, a compound of Cannabis sativa, has been proposed as an alternative treatment of schizophrenia. Preclinical and clinical data have suggested that cannabidiol shares more similarity with atypical antipsychotics than typical, both of which are customarily used to manage schizophrenia symptoms. While oligodendrocytes are known to be relevant targets of antipsychotics, the biochemical knowledge in this regard is still limited. Here we evaluated the molecular pathways modulated by cannabidiol compared to the antipsychotics clozapine (atypical) and haloperidol (typical), additionally evaluating the effects of benztropine, a muscarinic receptor antagonist that displays a protective effect in oligodendrocytes and myelination. For this purpose, we employed nano-chromatography coupled with mass spectrometry to investigate the proteomic response to these drugs both in healthy oligodendrocytic cells and in a cuprizone-based toxicity model, using the human oligodendrocyte precursor cell line MO3.13. Cannabidiol shares similarities of biochemical pathways with clozapine and benztropine, in agreement with other studies that indicated an atypical antipsychotic profile. All drugs tested affected metabolic and gene expression pathways and cannabidiol, benztropine, and clozapine modulated cell proliferation and apoptosis when administered after cuprizone-induced toxicity. These general pathways are associated with cuprizone-induced cytotoxicity in MO3.13 cells, indicating a possible proteomic approach when acting against the toxic effects of cuprizone. In conclusion, although modeling oligodendrocytic cytotoxicity with cuprizone does not represent the entirety of the pathophysiology of oligodendrocyte impairments, these results provide insight into the mechanisms associated with the effects of cannabidiol and antipsychotics against cuprizone toxicity, offering new directions of study for myelin-related processes and deficits.

EBI2 Is Temporarily Upregulated in MO3.13 Oligodendrocytes during Maturation and Regulates Remyelination in the Organotypic Cerebellar Slice Model.

The EBI2 receptor regulates the immune system and is expressed in various immune cells including B and T lymphocytes. It is also expressed in astrocytes in the central nervous system (CNS) where it regulates pro-inflammatory cytokine release, cell migration and protects from chemically induced demyelination. Its signaling and expression are implicated in various diseases including multiple sclerosis, where its expression is increased in infiltrating immune cells in the white matter lesions. Here, for the first time, the EBI2 protein in the CNS cells in the human brain was examined. The function of the receptor in MO3.13 oligodendrocytes, as well as its role in remyelination in organotypic cerebellar slices, were investigated. Human brain sections were co-stained for EBI2 receptor and various markers of CNS-specific cells and the human oligodendrocyte cell line MO3.13 was used to investigate changes in EBI2 expression and cellular migration. Organotypic cerebellar slices prepared from wild-type and cholesterol 25-hydroxylase knock-out mice were used to study remyelination following lysophosphatidylcholine (LPC)-induced demyelination. The data showed that EBI2 receptor is present in OPCs but not in myelinating oligodendrocytes in the human brain and that EBI2 expression is temporarily upregulated in maturing MO3.13 oligodendrocytes. Moreover, we show that migration of MO3.13 cells is directly regulated by EBI2 and that its signaling is necessary for remyelination in cerebellar slices post-LPC-induced demyelination. The work reported here provides new information on the expression and role of EBI2 in oligodendrocytes and myelination and provides new tools for modulation of oligodendrocyte biology and therapeutic approaches for demyelinating diseases.

Susceptibility to erastin-induced ferroptosis decreases during maturation in a human oligodendrocyte cell line.

Ferroptosis, a form of iron-dependent cell death caused by lipid peroxidation, has been implicated in neurological and other disorders. However, the mechanism of ferroptosis in oligodendrocytes is unclear. We tested the susceptibility of MO3.13 cells, an oligodendrocyte line, to ferroptosis after erastin treatment. Immature MO3.13 cells were more susceptible to erastin-induced ferroptosis than chemically differentiated mature MO3.13 cells. Increased expression of solute carrier family 7 member 11 (SLC7A11), which encodes a cystine/glutamate transporter, and greater glutathione concentrations were observed in mature compared with immature MO3.13 cells, linking glutathione to the resistance of mature MO3.13 cells to erastin-induced ferroptosis. These findings highlight the usefulness of immature MO3.13 cells in studies of ferroptosis and investigations into neuropathologies that involve oligodendrocytes.

Reappraisal of Human HOG and MO3.13 Cell Lines as a Model to Study Oligodendrocyte Functioning.

Myelination of neuronal axons is essential for proper brain functioning and requires mature myelinating oligodendrocytes (myOLs). The human OL cell lines HOG and MO3.13 have been widely used as in vitro models to study OL (dys) functioning. Here we applied a number of protocols aimed at differentiating HOG and MO3.13 cells into myOLs. However, none of the differentiation protocols led to increased expression of terminal OL differentiation or myelin-sheath formation markers. Surprisingly, the applied protocols did cause changes in the expression of markers for early OLs, neurons, astrocytes and Schwann cells. Furthermore, we noticed that mRNA expression levels in HOG and MO3.13 cells may be affected by the density of the cultured cells. Finally, HOG and MO3.13 co-cultured with human neuronal SH-SY5Y cells did not show myelin formation under several pro-OL-differentiation and pro-myelinating conditions. Together, our results illustrate the difficulty of inducing maturation of HOG and MO3.13 cells into myOLs, implying that these oligodendrocytic cell lines may not represent an appropriate model to study the (dys)functioning of human (my)OLs and OL-linked disease mechanisms.

Maturation of a Human Oligodendrocyte Cell Line.

Techniques such as the maturation and differentiation of cell lines and progenitor cells are important for the improvement and development of representative and relevant in vitro models. In this context, the following chapter proposes a maturation model of the MO3.13 cell line, aiming to contribute to a more robust and credible in vitro model of human oligodendrocytes. This may prove to be an important tool in the study of diseases related to dysfunctions in oligodendrocytes and demyelination, including schizophrenia and multiple sclerosis.

Conditioned medium from relapsing-remitting multiple sclerosis patients reduces the expression and release of inflammatory cytokines induced by LPS-gingivalis in THP-1 and MO3.13 cell lines.

The present research was aimed at evaluating the effect of the conditioned medium (CM) from human periodontal ligament stem cells (hPDLSCs) obtained from healthy donors (hPDLSCs-CM) and from Relapsing-Remitting Multiple Sclerosis patients (RR-MS-CM) on inflammatory response induced by Porphyromonas gingivalis lipopolysaccharide (LPS-G) in a monocytoid human cell line (THP-1) and human oligodendrocyte cell line (MO3.13). Human periodontal ligament biopsies were carried out from control donor patients and selected RR-MS donors. Sample tissues were obtained from premolar teeth during root scaling and subsequently cultured. The effect of hPDLSCs-CM and RR-MS-CM on cell viability in PMA differentiated THP-1 (as a model of microglia) was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. The same experiments were performed in undifferentiated and differentiated MO3.13 cells used as models of progenitor cells and oligodendrocytes, respectively. The expression of tumor necrosis factor alpha (TNF)-α, interleukin (IL)-1ß and IL-6 was evaluated by Real-Time Polymerase Chain Reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA). The expression level of the Toll-like receptor 4 (TLR-4), for which LPS-G is a ligand, was evaluated by Western blot analysis. The results were analyzed by ANOVA using Graph Pad Prism software. LPS-G significantly increased TNFα, IL-1ß and IL-6 mRNA expression and protein levels in the differentiated THP-1 cells and oligodendrocyte MO3.13 progenitor cells. Treatment with hPDLSCs-CM or with RR-MS-CM significantly attenuated the LPS-induced expression and production of these pro-inflammatory cytokines. The CM from both healthy donors and RR-MS patients also reduced the LPS-G stimulated protein levels of TLR-4 in differentiated THP-1 cells. On the whole our data add new evidence on the anti-inflammatory effects of these peculiar stem cells even when derived from RR-MS patients and open novel perspectives in the therapeutic use of autologous periodontal stem cells in neuroinflammatory/neurodegenerative diseases including MS.

MK-801-Treated Oligodendrocytes as a Cellular Model to Study Schizophrenia.

Glutamate is the most important excitatory neurotransmitter in the brain. The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is found both in neurons and glial cells such as oligodendrocytes, which have been shown to be dysfunctional in schizophrenia. For this reasons, the oligodendrocyte MO3.13 cell line has been used to study glutamatergic dysfunction as a model of schizophrenia using the NMDA receptor antagonists such as MK-801 to block receptor function. Here, we describe a comprehensive protocol for culturing and carrying out proteomic analyses of MK-801-treated MO3.13 cells as a means of identifying potential new biomarkers and targets for drug discovery in schizophrenia research.

Lithium improves cell viability in psychosine-treated MO3.13 human oligodendrocyte cell line via autophagy activation.

Globoid cell leukodystrophy (GLD) is a rare, rapidly progressing childhood leukodystrophy triggered by deficit of the lysosomal enzyme galactosylceramidase (GALC) and characterized by the accumulation of galactosylsphingosine (psychosine; PSY) in the nervous system. PSY is a cytotoxic sphingolipid, which leads to widespread degeneration of oligodendrocytes and Schwann cells, causing demyelination. Here we report on autophagy in the human oligodendrocyte cell line MO3.13 treated with PSY and exploitation of Li as an autophagy modulator to rescue cell viability. We demonstrate that PSY causes upregulation of the autophagic flux at the level of autophagosome and autolysosome formation and LC3-II expression. We show that pretreatment with Li, a drug clinically used to treat bipolar disorders, can further stimulate autophagy, improving cell tolerance to PSY. This Li protective effect is found not to be linked to reduction of PSY-induced oxidative stress and might not stem from a reduction of PSY accumulation. These data provide novel information on the intracellular pathways activated during PSY-induced toxicity and suggest the autophagy pathway as a promising novel therapeutic target for ameliorating the GLD phenotype. © 2016 Wiley Periodicals, Inc.

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).