Oligodendrocyte Cell Line Research Articles

Expression study of Krabbe Disease GALC missense variants - Insights from quantification profiles of residual enzyme activity, secretion and psychosine levels.

Krabbe disease (KD) is an autosomal recessive lysosomal storage disorder caused by loss-of-function mutations in the GALC gene, which encodes for the enzyme galactosylceramidase (GALC). GALC is crucial for myelin metabolism. Functional deficiency of GALC leads to toxic accumulation of psychosine, dysfunction and death of oligodendrocytes, and eventual brain demyelination. To date, 46 clinically-relevant, pathogenic GALC missense mutations (MMs) have been identified in KD patients. These MMs are present in ∼70% of KD cases reported over 8 published studies between 1996 - 2019. However, the mechanisms by which these MMs lead to GALC functional deficiency and their correlations with clinical phenotype remain poorly understood. To address this, we generated a GALC -knockout human oligodendrocytic cell line (MO3.13/ GALC -KO) using CRISPR-Cas9 method to assess GALC function and GALC secretion. We evaluated 5 polymorphic and 31 clinically-relevant MM variants (MMVs) using transient expression assays. Our results showed that 26 MMVs, including 10 co-variants with p.I562T, reduced GALC activity by 92% - 100% compared to wild-type GALC (WT-GALC). MMVs from infantile-onset KD patients produced < 2% of WT activity, whereas those associated with juvenile- and adult-onset cases retained up to 7% of WT activity. Residual GALC activity was correlated with mature, lysosomal GALC protein levels (Pearson r = 0.93, P<0.0001). Many low-activity MMVs did not correspondingly impair GALC secretion. Twenty-one of the 26 low-activity MMVs showed a 21% - 100% reduction in sec-GALC levels, indicating varying degrees of GALC mis-trafficking among these variants. Importantly, GALC activity among MMVs strongly correlates with clinical disease severity, based on the age of symptom onset in patients with either homozygous MM (Pearson r = 0.98, P<0.0001, n = 7) or compound heterozygous (Pearson r = 0.94, P<0.0001, n = 12) MM-null mutation genotypes. Thus, our data suggests that GALC activity could serve as a prognostic disease indicator under specific experimental conditions. We further investigated the impact of pathogenic MMVs on psychosine accumulation, a key biomarker for KD. Psychosine levels were 21-fold higher in mock control cells compared to WT-GALC transfected cells (mock = 0.349 pmol/mg, WT-GALC = 0.016 pmol/mg), but negatively correlated with GALC activity among pathogenic MMVs (Pearson r = -0.63, P < 0.01, n = 15). Although psychosine levels were higher in most MMVs associated with infantile-onset KD, no significant correlations with clinical onset were detected. Overall, our study provides a comprehensive quantitative analysis of the functional deficits and mis-trafficking associated with clinically-relevant GALC MMVs, enhancing our understanding of the molecular genetics and genotype-phenotype correlations of the GALC gene in Krabbe disease.

Monomeric Amyloid Peptide-induced Toxicity in Human Oligodendrocyte Cell Line and Mouse Brain Primary Mixed-glial Cell Cultures: Evidence for a Neuroprotective Effect of Neurosteroid 3α-O-allyl-allopregnanolone.

Amyloid-peptide (Aβ) monomeric forms (ABM) occurring in presymptomatic Alzheimer's disease (AD) brain are thought to be devoid of neurotoxicity while the transition/aggregation of ABM into oligomers is determinant for Aβ-induced toxicity since Aβ is predominantly monomeric up to 3µM and aggregates over this concentration. However, recent imaging and/or histopathological investigations revealed alterations of myelin in prodromal AD brain in absence of aggregated Aβ oligomers, suggesting that ABM may induce toxicity in myelin-producing cells in early AD-stages. To check this hypothesis, here we studied ABM effects on the viability of the Human oligodendrocyte cell line (HOG), a reliable oligodendrocyte model producing myelin proteins. Furthermore, to mimic closely interactions between oligodendrocytes and other glial cells regulating myelination, we investigated also ABM effects on mouse brain primary mixed-glial cell cultures. Various methods were combined to show that ABM concentrations (600nM-1µM), extremely lower than 3µM, significantly decreased HOG cell and mouse brain primary mixed-glial cell survival. Interestingly, flow-cytometry studies using specific cell-type markers demonstrated that oligodendrocytes represent the most vulnerable glial cell population affected by ABM toxicity. Our work also shows that the neurosteroid 3α-O-allyl-allopregnanolone BR351 (250 and 500nM) efficiently prevented ABM-induced HOG and brain primary glial cell toxicity. Bicuculline (50-100nM), the GABA-A-receptor antagonist, was unable to block/reduce BR351 effect against ABM-induced HOG and primary glial cell toxicity, suggesting that BR351-evoked neuroprotection of these cells may not depend on GABA-A-receptor allosterically modulated by neurosteroids. Altogether, our results suggest that further exploration of BR351 therapeutic potential may offer interesting perspectives to develop effective neuroprotective strategies.

Ring finger protein 216 loss-of-function induces white matter hyperintensities by inhibiting oligodendroglia proliferation.

White matter hyperintensities(WMHs) refer to a group of diseases with numerous etiologies while oligodendrocytes remain the centerpiece in the pathogenesis of WMHs. Ring Finger Protein 216 (RNF216) encodes a ubiquitin ligase, and its mutation begets WMHs, ataxia, and cognitive decline in patients. Yet no study has revealed the function of RNF216 in oligodendroglia and WHIs before. In this study, we summarized the phenotypes of RNF216-mutation cases and explored the normal distribution of RNF216 in distinct brain regions and neuronal cells by bioinformatic analysis. Furthermore, MO3.13, a human oligodendrocyte cell line, was applied to study the function alteration after RNF216 knockdown. As a result, WMHs were the most common symptom in RNF216-mutated diseases, and RNF216 was indeed relatively enriched in corpus callosum and oligodendroglia in humans. The downregulation of RNF216 in oligodendroglia remarkably hampered cell proliferation by inhibiting the Akt pathway while having no significant effect on cell injury and oligodendrocyte maturation. Combining clinical, bioinformatical, and experimental evidence, our study implied the pivotal role of RNF216 in WMHs which might serve as a potent target in the therapy of WMHs.

Immortalization and Characterization of GFAP-expressing Glial Cells from the Adult Mouse Hypothalamus, Cortex, and Brain Stem

The generation of astrocyte cell lines from the hypothalamus is key to study glial involvement in hypothalamic physiology, including energy homeostasis. As such, we immortalized astrocytes from the hypothalamus of an adult male CD-1 mouse using SV40 T-antigen to generate the mHypoA-Ast1 cell line. A comparative approach was taken with two other murine GFAP-expressing cell lines that were also generated in this study: a mixed glial cell line from the cortex (mCortA-G1) and an oligodendrocyte cell line from the brainstem (mBstA-Olig1), as well as an established microglial cell line (IMG). mHypoA-Ast1 cells express GFAP, alongside other astrocytic markers such as Aldh1l1, Aqp4, Glt1 and S100b, and express low levels of microglial, ependymal and oligodendrocyte markers. 100 ng/mL lipopolysaccharide (LPS) elevated mRNA levels of Il6, Il1b, Tnfα and Cxcl5 in mHypoA-Ast1 cells after 4 h, while 50 μM palmitate increased Il6 and Chop mRNA, demonstrating the ability of these cells to respond to inflammatory and nutrient signals. Interestingly, co-culture of mHypoA-Ast1 cells with mHypoE-N46 hypothalamic neuronal cells prevented the palmitate-mediated increase in orexigenic neuropeptide Agrp mRNA in mHypoE-N46 cells, suggesting that this cell line can alter neuronal responses to nutrients. In conclusion, we report mHypoA-Ast1 cells representing a functional astrocyte cell line from the adult mouse brain that can be used to study the complex interactions of hypothalamic cells, as well as dysregulation that may occur in disease states, providing a key tool for neuroendocrine research.

Attenuation of Immunogenicity in MOG-Induced Oligodendrocytes by the Probiotic Bacterium Lactococcus Sp. PO3.

Background and Objectives: Milk is healthy and includes several vital nutrients and microbiomes. Probiotics in milk and their derivatives modulate the immune system, fight inflammation, and protect against numerous diseases. The present study aimed to isolate novel bacterial species with probiotic potential for neuroinflammation. Materials and Methods: Six milk samples were collected from lactating dairy cows. Bacterial isolates were obtained using standard methods and were evaluated based on probiotic characteristics such as the catalase test, hemolysis, acid/bile tolerance, cell adhesion, and hydrophobicity, as well as in vitro screening. Results: Nine morphologically diverse bacterial isolates were found in six different types of cow's milk. Among the isolates, PO3 displayed probiotic characteristics. PO3 was a Gram-positive rod cell that grew in an acidic (pH-2) salty medium containing bile salt and salinity (8% NaCl). PO3 also exhibited substantial hydrophobicity and cell adhesion. The sequencing comparison of the 16S rRNA genes revealed that PO3 was Lactococcus raffinolactis with a similarity score of 99.3%. Furthermore, PO3 was assessed for its neuroanti-inflammatory activity on human oligodendrocyte (HOG) cell lines using four different neuroimmune markers: signal transducer and activator of transcription (STAT-3), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and GLAC in HOG cell lines induced by MOG. Unlike the rest of the evaluated neuroimmune markers, STAT-3 levels were elevated in the MOG-treated HOG cell lines compared to the untreated ones. The expression level of STAT-3 was attenuated in both PO3-MOG-treated and only PO3-treated cell lines. On the contrary, in PO3-treated cell lines, MBP, GFAP, and GLAC were significantly expressed at higher levels when compared with the MOG-treated cell lines. Conclusions: The findings reported in this article are to be used as a foundation for further in vivo research in order to pave the way for the possible use of probiotics in the treatment of neuroinflammatory diseases, including multiple sclerosis.

Cover Image, Volume 140, Issue 20

The cover image by Senay Ustunel features a co-culturing system wherein neuroblastoma (SH-SY5Y) and oligodendrocyte (MO3.13) cell lines grow within a 3D liquid crystal elastomer (LCE) scaffold. LCE scaffolds can effectively impact cellular function, differentiation, and myelination of co-cultured cell lines, allowing the mimicry of a more endogenous environment and providing support for long term studies to manipulate cellular crosstalk, like in this case to study myelination. DOI: 10.1002/app.53883

3D Co‐culturing of human neuroblastoma and human oligodendrocytes, emulating native tissue using 3D porous biodegradable liquid crystal elastomers

AbstractBiomaterials cover and are an integral part of tissue engineering and regenerative medicine fields. They are crucial to mimic endogenous tissue responses and help to eliminate high research costs and ethical concerns that come with 2D systems and animal models. In this study, we present a co‐culturing system wherein neuroblastoma (SH‐SY5Y) and oligodendrocyte (MO3.13) cell lines grow within both a caprolactone‐based 3D elastomer and liquid crystal elastomer (LCE) scaffolding. We also demonstrate that LCE scaffolds promote maturation and differentiation of both cell lines alone, and in co‐culturing fashion as they provide a suitable 3D in vitro environment to study myelination. Additionally, we validate that these scaffolds are suitable for primary cell growth and proliferation using neurons, oligodendrocyte progenitor cells (OPCs) and astrocytes. Our data suggest that responsive LCE scaffolds can effectively impact cellular function, differentiation, and myelination of co‐cultured cell lines, allowing the mimicry of a more endogenous environment and providing support for long term studies to manipulate cellular crosstalk.

Oligodendrocyte Cell Line OLP6 Successfully Differentiates on Decellularized Brain Tissue

Oligodendrocyte Cell Line OLP6 Successfully Differentiates on Decellularized Brain Tissue

Total astragalosides promote oligodendrocyte precursor cell differentiation and enhance remyelination in cuprizone-induced mice through suppression of Wnt/β-catenin signaling pathway

Ethnopharmacological relevanceRadix Astragali is a traditional Chinese medicine with various pharmacological effects. Total astragalosides (TA), the main effective ingredients in Radix Astragali, exert properties including anti-oxidative stress, anti-neuroinflammation, and neuroprotection. We previously found that TA alleviated experimental autoimmune encephalomyelitis (EAE) progression, a widely used animal model of multiple sclerosis (MS). As a chronic demyelination disease, MS generally manifests myelin loss and fails to myelin regeneration. Regulation of oligodendrocyte progenitor cells (OPCs) differentiation and remyelination is the fundamental strategy for MS treatment. However, whether TA could directly promote OPCs differentiation and remyelination is still unknown. Aims of the studyThis study was aimed to investigate pro-differentiation and myelin regeneration effects of TA on OPCs and Cuprizone (CPZ)-induced demyelination mice, an animal model of MS, and to explore mechanism underlying from regulation of OPCs differentiation and maturation. Materials and methodsMice were orally given CPZ (400 mg/kg) daily for 4 weeks to induce myelin loss, and then treated with TA (25 and 50 mg/kg) daily for 1 week. Cell proliferation assay, Western blot, RT-PCR, immunocytochemistry and immunohistochemistry were performed to explore the mechanisms. The role of TA in oligodendrocyte differentiation and maturation was evaluated using MO3.13, a human oligodendrocytic hybrid cell line. ResultsTA was shown to mitigate behavioral impairment in CPZ-induced mice. It markedly ameliorated myelin loss and enhanced remyelination in the corpus callosum of mice, evidenced by increased expression of myelin basic protein (MBP) and the number of CC1+ newly generated oligodendrocytes (OLs). TA also enhanced the expression of MBP at both mRNA and protein levels in MO3.13 cells. In CPZ-induced mice and MO3.13 cells, TA remarkably promoted the activation of GSK3β, repressed the phosphorylation of β-catenin, reduced the expression of transcription factor 4 and inhibitor of DNA binding 2. The agonist of β-catenin, SKL2001, partially abolished the pro-differentiation effect of TA in MO3.13 cells. ConclusionsTaken together, we clarified that TA could effectively enhance the differentiation and maturation of OPCs and accelerate remyelination in CPZ-induced mice through inhibition of Wnt/β-catenin signaling pathway. This study provides new insight into the beneficial effect of TA in the treatment of MS.

Historical origin and meaning of the term „glial tumor“

In everyday neurosurgical practice, the term „glial tumor“ is associated with astrocytomas, glioblastomas, and oligodendrogliomas, although historically this has not always been the case. The term „glial tumor“ was first given by Virchow in the 19th century as a term initially combining all primary brain tumors under this name. It derives from the name of the group of „supporting“ nerve cells - glia or neuroglia (from the Greek glia - glue), a group which for many years was wrongly ascribed only a cohesive or supporting function. In 1926, in their classification of glial tumors - A Classification of the Tumors of the Glioma Group on a Histogenetic Basis with a Correlated Study of Prognosis, one of the founding fathers of neuropathology Percival Bailey and the founding father of modern neurosurgery – Harvey Cushing ascribed several different tumors in this group: in addition to neuroepithelioma, spongioblastoma multiforme, astrocytoma and ependymoma, they also add medulloblastoma, astroblastoma, oligodendroglioma and unipolar spongioblastoma. Since then, the classification of glial tumors has undergone many changes to its current form. In the latest classification of brain tumors published in 2021, glial tumors are united in a common group together with glioneuronal and neuronal tumors. Their extensive group includes tumors with different prognosis, age presentation, molecular profile and therapeutic response. From a neurosurgical point of view, the term „glial tumor“ does not carry a prognostic value, but only determines the belonging of the tumor to the astrocyte, oligodendrocyte cell line or their precursor cells. In relation to that an interesting question arises- why the remaining tumors originating from glial cells other than astrocytic, such as ependymomas, lost their belonging to the group of glial tumors, or such as intracranial schwannomas, are not included in it at all.

Cannabinoids modulate proliferation, differentiation, and migration signaling pathways in oligodendrocytes.

Cannabinoid signaling, mainly via CB1 and CB2 receptors, plays an essential role in oligodendrocyte health and functions. However, the specific molecular signals associated with the activation or blockade of CB1 and CB2 receptors in this glial cell have yet to be elucidated. Mass spectrometry-based shotgunproteomics and in silico biology tools were used to determine which signaling pathways and molecular mechanisms are triggered in a human oligodendrocytic cell line (MO3.13) by several pharmacological stimuli: the phytocannabinoid cannabidiol (CBD); CB1 and CB2 agonists ACEA, HU308, and WIN55, 212-2; CB1 and CB2 antagonists AM251 and AM630; and endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The modulation of cannabinoid signaling in MO3.13 was found to affect pathways linked to cell proliferation, migration, and differentiation of oligodendrocyte progenitor cells. Additionally, we found that carbohydrate and lipid metabolism, as well as mitochondrial function, were modulated by these compounds. Comparing the proteome changes and upstream regulators among treatments, the highest overlap was between the CB1 and CB2 antagonists, followed by overlaps between AEA and 2-AG. Our study opens new windows of opportunities, suggesting that cannabinoid signaling in oligodendrocytes might be relevant in the context of demyelinating and neurodegenerative diseases.Proteomics data are available at ProteomeXchange (PXD031923).

Contact-Dependent Granzyme B-Mediated Cytotoxicity of Th17-Polarized Cells Toward Human Oligodendrocytes

Multiple sclerosis (MS) is characterized by the loss of myelin and of myelin-producing oligodendrocytes (OLs) in the central nervous system (CNS). Pro-inflammatory CD4+ Th17 cells are considered pathogenic in MS and are harmful to OLs. We investigated the mechanisms driving human CD4+ T cell-mediated OL cell death. Using fluorescent and brightfield in vitro live imaging, we found that compared to Th2-polarized cells, Th17-polarized cells show greater interactions with primary human OLs and human oligodendrocytic cell line MO3.13, displaying longer duration of contact, lower mean speed, and higher rate of vesicle-like structure formation at the sites of contact. Using single-cell RNA sequencing, we assessed the transcriptomic profile of primary human OLs and Th17-polarized cells in direct contact or separated by an insert. We showed that upon close interaction, OLs upregulate the expression of mRNA coding for chemokines and antioxidant/anti-apoptotic molecules, while Th17-polarized cells upregulate the expression of mRNA coding for chemokines and pro-inflammatory cytokines such as IL-17A, IFN-γ, and granzyme B. We found that secretion of CCL3, CXCL10, IFN-γ, TNFα, and granzyme B is induced upon direct contact in cocultures of human Th17-polarized cells with human OLs. In addition, we validated by flow cytometry and immunofluorescence that granzyme B levels are upregulated in Th17-polarized compared to Th2-polarized cells and are even higher in Th17-polarized cells upon direct contact with OLs or MO3.13 cells compared to Th17-polarized cells separated from OLs by an insert. Moreover, granzyme B is detected in OLs and MO3.13 cells following direct contact with Th17-polarized cells, suggesting the release of granzyme B from Th17-polarized cells into OLs/MO3.13 cells. To confirm granzyme B–mediated cytotoxicity toward OLs, we showed that recombinant human granzyme B can induce OLs and MO3.13 cell death. Furthermore, pretreatment of Th17-polarized cells with a reversible granzyme B blocker (Ac-IEPD-CHO) or a natural granzyme B blocker (serpina3N) improved survival of MO3.13 cells upon coculture with Th17 cells. In conclusion, we showed that human Th17-polarized cells form biologically significant contacts with human OLs and exert direct toxicity by releasing granzyme B.

Neuropathogenicity of non-viable Borrelia burgdorferi ex vivo

Even after appropriate treatment, a proportion of Lyme disease patients suffer from a constellation of symptoms, collectively called Post-Treatment Lyme Disease Syndrome (PTLDS). Brain PET scan of patients with PTLDS have demonstrated likely glial activation indicating persistent neuroinflammatory processes. It is possible that unresolved bacterial remnants can continue to cause neuroinflammation. In previous studies, we have shown that non-viable Borrelia burgdorferi can induce neuroinflammation and apoptosis in an oligodendrocyte cell line. In this follow-up study, we analyze the effect of sonicated remnants of B. burgdorferi on primary rhesus frontal cortex (FC) and dorsal root ganglion (DRG) explants. Five FC and three DRG tissue fragments from rhesus macaques were exposed to sonicated B. burgdorferi and analyzed for 26 inflammatory mediators. Live bacteria and medium alone served as positive and negative control, respectively. Tissues were also analyzed for cell types mediating inflammation and overall apoptotic changes. Non-viable B. burgdorferi induced significant levels of several inflammatory mediators in both FC and DRG, similar to live bacteria. However, the levels induced by non-viable B. burgdorferi was often (several fold) higher than those induced by live ones, especially for IL-6, CXCL8 and CCL2. This effect was also more profound in the FC than in the DRG. Although the levels often differed, both live and dead fragments induced the same mediators, with significant overlap between FC and DRG. In the FC, immunohistochemical staining for several inflammatory mediators showed the presence of multiple mediators in astrocytes, followed by microglia and oligodendrocytes, in response to bacterial remnants. Staining was also seen in endothelial cells. In the DRG, chemokine/cytokine staining was predominantly seen in S100 positive (glial) cells. B. burgdorferi remnants also induced significant levels of apoptosis in both the FC and DRG. Apoptosis was confined to S100 + cells in the DRG while distinct neuronal apoptosis was also detected in most FC tissues in response to sonicated bacteria. Non-viable B. burgdorferi can continue to be neuropathogenic to both CNS and PNS tissues with effects likely more profound in the former. Persistence of remnant-induced neuroinflammatory processes can lead to long term health consequences.

The Synergistic Anti-Apoptosis Effects of Amniotic Epithelial Stem Cell Conditioned Medium and Ponesimod on the Oligodendrocyte Cells.

Multiple sclerosis is a chronic inflammatory and neurodegenerative disease of the central nervous system. The current treatment of Multiple sclerosis is based on anti-inflammatory disease-modifying treatments, which can not regenerate myelin and eventually neurons. So, we need new approaches for axonal protection and remyelination. Amniotic epithelial stem cells amniotic epithelial cells, as a neuroprotective and neurogenic agent, are a proper source in tissue engineering and regenerative medicine. Due to differentiation capability and secretion of growth factors, AECs can be a candidate for the treatment of MS. Moreover, sphingosine-1-phosphate (S1P) receptor modulators were recently approved by FDA for MS. Ponesimod is an S1P receptor-1 modulator that acts selectively as an anti-inflammatory agent and provides a suitable microenvironment for the function of the other neuroprotective agents. In this study, due to the characteristics of AECs, they are considered a treatment option in MS. The conditioned medium of AECs concurrently with ponesimod was used to evaluate the viability of the oligodendrocyte cell line after induction of cell death by cuprizone. Cell viability after treatment by conditioned medium and ponesimod was increased compared to untreated groups. Also, the results showed that combination therapy with CM and ponesimod had a synergistic anti-apoptotic effect on oligodendrocyte cells. The combination treatment with CM and ponesimod reduced the expression of caspase-3, caspase-8, Bax, and Annexin V proteins and increased the relative BCL-2/Bax ratio, indicating inhibition of apoptosis as a possible mechanism of action. Based on these promising results, combination therapy with amniotic stem cells and ponesimode could be a proper alternative for multiple sclerosis treatment.

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.

SIRT1 decelerates morphological processing of oligodendrocyte cell lines and regulates the expression of cytoskeleton-related oligodendrocyte proteins

SIRT1 is involved in the regulation of a variety of biological processes such as metabolism, stress response, autophagy and differentiation. Although progenitor cells of oligodendrocytes (OPCs) express high level of SIRT1, its function on differentiation is unknown. Because we have shown that SIRT1 plays a pivotal role in differentiation of neural precursor cells, we hypothesized that SIRT1 may also participate in the differentiation of oligodendrocytes (OLGs). We examined whether SIRT1 was expressed in two human oligodendrocyte cell lines: KG-1-C and MO 3.13 OLG. Transfection of cell lines with SIRT1-siRNA and SIRT2-siRNA promoted the extension of cellular processes. SIRT1-siRNA and SIRT2-siRNA increased acetyl-α-tubulin level, conversely, over expression of SIRTs resulted in decreased the ratio of acetyl-α-tubulin to α-tubulin. We also found knockdown of SIRT1 and SIRT2 induced overexpression of βIV-tubulin and tubulin polymerization promoting protein (TPPP) (OLG-specific cytoskeleton-related molecules) that distributed widely in cell bodies. Taken together, SIRT1 may play a role in oligodenroglial differentiation and myelinogenesis.

Suppressors of cytokine signaling 1 protein in a regenerative model of the Gekko japonicus spinal cord.

Demyelination is one of the pathological outcomes that occur immediately following spinal cord injury. Protection of oligodendrocytes against death/apoptosis proves to be beneficial for the preservation of neurological functions. Suppressors of cytokine signaling 1 protein inhibit the harmful effects of several inflammatory cytokines on oligodendrocytes, but its roles in spinal cord injury (SCI) induced apoptosis of oligodendrocytes remain unclear. We cloned suppressors of cytokine signaling 1 cDNA from Gekko japonicus (Japanese gecko) and analyzed the protein structure revealing the conserved domains contained in other vertebrate suppressors of cytokine signaling 1 proteins. The gecko suppressors of cytokine signaling 1 protein expression were increased in the injured spinal cord following gecko tail amputation and displayed colocalization with oligodendrocytes. The enforced expression of gecko suppressors of cytokine signaling 1 by adenovirus in the Gsn3 gecko oligodendrocyte cell line demonstrated that gecko suppressors of cytokine signaling 1 significantly suppressed cell apoptosis-induced by glucose deprivation. Determination of apoptosis-related proteins revealed that gecko suppressors of cytokine signaling 1 was able to activate extracellular regulated protein kinases (ERK) and serine/threonine protein kinases (Akt). The results presented a distinct protective role of gecko suppressors of cytokine signaling 1 in the regenerative model of the spinal cord, which may provide new cues for central nervous system repair in mammals.

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.

Hsv-1 Endocytic Entry into a Human Oligodendrocytic Cell Line is Mediated by Clathrin and Dynamin but Not Caveolin.

Endocytosis is a pathway used by viruses to enter cells that can be classified based on the proteins involved, such as dynamin, clathrin or caveolin. Although the entry of herpes simplex type 1 (HSV-1) by endocytosis has been documented in different cell types, its dependence on clathrin has not been described whereas its dependence on dynamin has been shown according to the cell line used. The present work shows how clathrin-mediated endocytosis (CME) is one way that HSV-1 infects the human oligodendroglial (HOG) cell line. Partial dynamin inhibition using dynasore revealed a relationship between decrease of infection and dynamin inhibition, measured by viral titration and immunoblot. Co-localization between dynamin and HSV-1 was verified by immunofluorescence at the moment of viral entry into the cell. Inhibition by chlorpromazine revealed that viral progeny also decreased when clathrin was partially inhibited in our cell line. RT-qPCR of immediately early viral genes, specific entry assays and electron microscopy all confirmed clathrin’s participation in HSV-1 entry into HOG cells. In contrast, caveolin entry assays showed no effect on the entry of this virus. Therefore, our results suggest the participation of dynamin and clathrin during endocytosis of HSV-1 in HOG cells.

A Novel Function of Sphingosine Kinase 2 in the Metabolism of Sphinga-4,14-Diene Lipids

The number, position, and configuration of double bonds in lipids affect membrane fluidity and the recruitment of signaling proteins. Studies on mammalian sphingolipids have focused on those with a saturated sphinganine or mono-unsaturated sphingosine long chain base. Using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), we observed a marked accumulation of lipids containing a di-unsaturated sphingadiene base in the hippocampus of mice lacking the metabolic enzyme sphingosine kinase 2 (SphK2). The double bonds were localized to positions C4–C5 and C14–C15 of sphingadiene using ultraviolet photodissociation-tandem mass spectrometry (UVPD-MS/MS). Phosphorylation of sphingoid bases by sphingosine kinase 1 (SphK1) or SphK2 forms the penultimate step in the lysosomal catabolism of all sphingolipids. Both SphK1 and SphK2 phosphorylated sphinga-4,14-diene as efficiently as sphingosine, however deuterated tracer experiments in an oligodendrocyte cell line demonstrated that ceramides with a sphingosine base are more rapidly metabolized than those with a sphingadiene base. Since SphK2 is the dominant sphingosine kinase in brain, we propose that the accumulation of sphingadiene-based lipids in SphK2-deficient brains results from the slower catabolism of these lipids, combined with a bottleneck in the catabolic pathway created by the absence of SphK2. We have therefore uncovered a previously unappreciated role for SphK2 in lipid quality control.