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Schuster, Kristen H., Alexandra F. Putka, and Hayley S. McLoughlin. "Pathogenetic Mechanisms Underlying Spinocerebellar Ataxia Type 3 Are Altered in Primary Oligodendrocyte Culture." Cells 11, no. 16 (August 22, 2022): 2615. http://dx.doi.org/10.3390/cells11162615.
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Emerging evidence has implicated non-neuronal cells, particularly oligodendrocytes, in the pathophysiology of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease and Spinocerebellar ataxia type 3 (SCA3). We recently demonstrated that cell-autonomous dysfunction of oligodendrocyte maturation is one of the of the earliest and most robust changes in vulnerable regions of the SCA3 mouse brain. However, the cell- and disease-specific mechanisms that underlie oligodendrocyte dysfunction remain poorly understood and are difficult to isolate in vivo. In this study, we used primary oligodendrocyte cultures to determine how known pathogenic SCA3 mechanisms affect this cell type. We isolated oligodendrocyte progenitor cells from 5- to 7-day-old mice that overexpress human mutant ATXN3 or lack mouse ATXN3 and differentiated them for up to 5 days in vitro. Utilizing immunocytochemistry, we characterized the contributions of ATXN3 toxic gain-of-function and loss-of-function in oligodendrocyte maturation, protein quality pathways, DNA damage signaling, and methylation status. We illustrate the utility of primary oligodendrocyte culture for elucidating cell-specific pathway dysregulation relevant to SCA3. Given recent work demonstrating disease-associated oligodendrocyte signatures in other neurodegenerative diseases, this novel model has broad applicability in revealing mechanistic insights of oligodendrocyte contribution to pathogenesis.
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Malek-Hedayat, S., and LH Rome. "Expression of a beta 1-related integrin by oligodendroglia in primary culture: evidence for a functional role in myelination." Journal of Cell Biology 124, no. 6 (March 15, 1994): 1039–46. http://dx.doi.org/10.1083/jcb.124.6.1039.
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We have investigated the expression of integrins by rat oligodendroglia grown in primary culture and the functional role of these proteins in myelinogenesis. Immunochemical analysis, using antibodies to a number of alpha and beta integrin subunits, revealed that oligodendrocytes express only one detectable integrin receptor complex (alpha OL beta OL). This complex is immunoprecipitated by a polyclonal anti-human beta 1 integrin subunit antibody. In contrast, astrocytes, the other major glial cell type in brain, express multiple integrins including alpha 1 beta 1, alpha 3 beta 1, and alpha 5 beta 1 complexes that are immunologically and electrophoretically indistinguishable from integrins expressed by rat fibroblasts. The beta subunit of the oligodendrocyte integrin (beta OL) and rat fibroblast beta 1 have different electrophoretic mobilities in SDS-PAGE. However, the two beta subunits appear to be highly related based on immunological cross-reactivity and one-dimensional peptide mapping. After removal of N-linked carbohydrate chains, beta OL and beta 1 comigrated in SDS-PAGE and peptide maps of the two deglycosylated subunits were identical, suggesting differential glycosylation of beta 1 and beta OL accounts entirely for their size differences. The oligodendrocyte alpha subunit, alpha OL, was not immunoprecipitated by antibodies against well characterized alpha chains which are known to associate with beta 1 (alpha 3, alpha 4, and alpha 5). However, an antibody to alpha 8, a more recently identified integrin subunit, did precipitate two integrin subunits with electrophoretic mobilities in SDS-PAGE identical to alpha OL and beta OL. Functional studies indicated that disruption of oligodendrocyte adhesion to a glial-derived matrix by an RGD-containing synthetic peptide resulted in a substantial decrease in the level of mRNAs for several myelin components including myelin basic protein (MBP), proteolipid protein (PLP), and cyclic nucleotide phosphodiesterase (CNP). These results suggest that integrin-mediated adhesion of oligodendrocytes may trigger signal(s) that induce the expression of myelin genes and thus influence oligodendrocyte differentiation.
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Maiuolo, Jessica, Roberta Macrì, Irene Bava, Micaela Gliozzi, Vincenzo Musolino, Saverio Nucera, Cristina Carresi, et al. "Myelin Disturbances Produced by Sub-Toxic Concentration of Heavy Metals: The Role of Oligodendrocyte Dysfunction." International Journal of Molecular Sciences 20, no. 18 (September 14, 2019): 4554. http://dx.doi.org/10.3390/ijms20184554.
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Evidence has been accumulated demonstrating that heavy metals may accumulate in various organs, leading to tissue damage and toxic effects in mammals. In particular, the Central Nervous System (CNS) seems to be particularly vulnerable to cumulative concentrations of heavy metals, though the pathophysiological mechanisms is still to be clarified. In particular, the potential role of oligodendrocyte dysfunction and myelin production after exposure to subtoxic concentration I confirmed. It is ok of heavy metals is to be better assessed. Here we investigated on the effect of sub-toxic concentration of several essential (Cu2 +, Cr3 +, Ni2 +, Co2+) and non-essential (Pb2 +, Cd2+, Al3+) heavy metals on human oligodendrocyte MO3.13 and human neuronal SHSY5Y cell lines (grown individually or in co-culture). MO3.13 cells are an immortal human–human hybrid cell line with the phenotypic characteristics of primary oligodendrocytes but following the differentiation assume the morphological and biochemical features of mature oligodendrocytes. For this reason, we decided to use differentiated MO3.13 cell line. In particular, exposure of both cell lines to heavy metals produced a reduced cell viability of co-cultured cell lines compared to cells grown separately. This effect was more pronounced in neurons that were more sensitive to metals than oligodendrocytes when the cells were grown in co-culture. On the other hand, a significant reduction of lipid component in cells occurred after their exposure to heavy metals, an effect accompanied by substantial reduction of the main protein that makes up myelin (MBP) in co-cultured cells. Finally, the effect of heavy metals in oligodendrocytes were associated to imbalanced intracellular calcium ion concentration as measured through the fluorescent Rhod-2 probe, thus confirming that heavy metals, even used at subtoxic concentrations, lead to dysfunctional oligodendrocytes. In conclusion, our data show, for the first time, that sub-toxic concentrations of several heavy metals lead to dysfunctional oligodendrocytes, an effect highlighted when these cells are co-cultured with neurons. The pathophysiological mechanism(s) underlying this effect is to be better clarified. However, imbalanced intracellular calcium ion regulation, altered lipid formation and, finally, imbalanced myelin formation seem to play a major role in early stages of heavy metal-related oligodendrocyte dysfunction.
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Liu, Yin, Yingyun Cai, and Xuming Zhang. "Induction of Caspase-Dependent Apoptosis in Cultured Rat Oligodendrocytes by Murine Coronavirus Is Mediated during Cell Entry and Does Not Require Virus Replication." Journal of Virology 77, no. 22 (November 15, 2003): 11952–63. http://dx.doi.org/10.1128/jvi.77.22.11952-11963.2003.
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ABSTRACT Murine coronavirus mouse hepatitis virus (MHV) causes demyelination of the central nervous system (CNS) in rats and mice. Apoptotic oligodendrocytes have been detected in the vicinity of the CNS demyelinating lesions in these animals. However, whether MHV can directly induce oligodendrocyte apoptosis has not been documented. Here, we established a rat oligodendrocyte culture that is morphologically and phenotypically indistinguishable from the primary rat oligodendrocytes. Using this culture, we showed that mature rat oligodendrocytes were permissive to MHV infection but did not support productive virus replication. Significantly, oligodendrocytes infected with both live and ultraviolet light-inactivated viruses underwent apoptosis to a similar extent, which was readily detectable at 24 h postinfection as revealed by apoptotic bodies and DNA fragmentation, indicating that MHV-induced apoptosis is mediated during the early stages of the virus life cycle and does not require virus replication. Prior treatment of cells with the lysosomotropic agents NH4Cl and chloroquine as well as the vacuolar proton pump-ATPase inhibitor bafilomycin A1, all of which block the acidification of the endosome, prevented oligodendrocytes from succumbing to apoptosis induced by MHV mutant OBLV60, which enters cells via endocytosis, indicating that fusion between the viral envelope and cell membranes triggers the apoptotic cascade. Treatment with the pan-caspase inhibitor Z-VAD-fmk blocked MHV-induced apoptosis, suggesting an involvement of the caspase-dependent pathway. Our results, thus, for the first time provide unequivocal evidence that infection of oligodendrocytes with MHV directly results in apoptosis. This finding provides an explanation for the destruction of oligodendrocytes and the damage of myelin sheath in MHV-infected CNS and suggests that oligodendrocyte apoptosis may be one of the underlying mechanisms for the pathogenesis of MHV-induced demyelinating diseases in animals.
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Villaverde, Marcela, Elsa Hincapié Arias, Martin Merenzon, Alejandro Mazzon, Eduardo Seoane, Denise Belgorosky, and Ana Maria Eiján. "TMET-26. PRIMARY OLIGODENDROGLIOMA CELL CULTURE VIABILITY: AN IN VITRO STUDY WITH METABOLIC MODULATORS." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii267. http://dx.doi.org/10.1093/neuonc/noac209.1031.
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Abstract Oligodendrogliomas are tumors that develop from oligodendrocytes, the myelinating cells of the central nervous system. Oligodendrocytes are highly metabolically active cells that synthesize and transfer metabolites to neighboring cells. Given its intimate metabolic relation with neurons, we aim to investigate oligodendrocyte metabolism as an antitumoral target. A recurred oligodendroglioma WHO grade III was surgically removed from a 50-year patient after 10 years of progression-free disease. The tumor sample was mechanically digested and cultured at 37oC, 5% CO2, in DMEM: F12, 10 % FBS and antibiotics. Primary oligodendroglioma cells were trypsinized and seeded in a 96-well plate. After 24 hs, cells were treated with metabolic modulators: metformin (MET, mitochondrial complex II inhibitor, 5 mM), 2 deoxyglucose (2DG, hexokinase inhibitor, 1 mM), 6-aminonicotinamide (6AN, pentose phosphate pathway inhibitor, 50 µM) and/or 1400W and S-methylisothiourea (both iNOS inhibitors, 5 µM, and 50 µM respectively). Standard treatment with temozolomide (TMZ, 200 µM) was also performed. After 5 days of treatment, cells were stained with violet crystal. Two weeks after tumor sample digestion, a primary oligodendroglioma culture was successfully established. In vitro, proliferating cells appeared mostly undifferentiated with reduced branching complexity. Hexokinase inhibition by 2DG notoriously affected the viability of oligodendroglioma cells. Similar results were obtained with standard TMZ treatment. On the other hand, the inhibition of the pentose phosphate pathway by 6AN did not affect the cell monolayer. However, 6AN was able to increase the effect of MET as monotherapy. Both, MET and 2DG altered oligodendrocyte morphology inducing a more fusiform shape. Finally, iNOS inhibition modestly disrupted cell's monolayers and this effect did not seem to be improved by combinatory therapies. Glycolytic inhibitor 2DG resulted effective against oligodendroglioma cells. Whereas further studies are needed to validate these results, a better understanding of metabolic susceptibility could allow the development of better-targeted and more-effective therapeutic approaches.
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Boussouf, Abdelhamid, and St�phane Gaillard. "Intracellular pH changes during oligodendrocyte differentiation in primary culture." Journal of Neuroscience Research 59, no. 6 (March 15, 2000): 731–39. http://dx.doi.org/10.1002/(sici)1097-4547(20000315)59:6<731::aid-jnr5>3.0.co;2-g.
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Xu, Li, Arjun Saha, Roberta Parrott, Shane O’Neil, Joanne Kurtzberg, and Anthony Filiano. "Abstract 5 Human Umbilical Cord Blood-Derived Cell Therapy Product, DUOC-01, Promotes Remyelination by Driving the Differentiation of Oligodendrocyte Progenitor Cells." Stem Cells Translational Medicine 11, Supplement_1 (September 1, 2022): S7. http://dx.doi.org/10.1093/stcltm/szac057.005.
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Abstract Introduction DUOC-01 is a macrophage-like cell therapy product manufactured by culturing banked human umbilical cord blood cells under GMP conditions. Currently, the safety of DUOC-01 is being tested as a bridging therapy in children with demyelinating leukodystrophies undergoing unrelated donor umbilical cord blood transplantation after myeloablative conditioning. DUOC-01 protects against loss of function in several preclinical models with demyelinating conditions of the central nervous system, making it an attractive therapy for patients with multiple sclerosis (MS) who experience destruction of myelin sheaths as pathology of their disease. The mechanism by which DUOC-01 promotes remyelination and if it directly influences oligodendrocyte lineage cells is untested. Objective Using multiple systems (primary oligodendrocyte precursor cell [OPC] cultures, in vitro cerebellar slice cultures, and experimental autoimmune encephalomyelitis [EAE], a mouse model of MS), we examined how DUOC-01 influences numerous steps of pathology and recovery. Methods Using a brain slice culture, we added DUOC-01 to the lysophosphatidylcholine (LPC)-treated slices. We quantified myelinated axons by assessing percent co-localization of myelin basic protein and neurofilament in the control, LPC, and LPC+DUOC-01 groups. To test the DUOC-01 effect in the EAE model, we immunized C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide (MOG35-55) in complete Freund’s adjuvant. To match clinical protocols, we incubated DUOC-01 in Ringer’s lactate with hydrocortisone (HC) for 2 hours at room temperature. At the onset of EAE disease symptoms, we injected DUOC-01 into the cerebrospinal fluid by a single intra-cisterna magna injection, then recorded clinical scores daily for 2 weeks. To test if DUOC-01 could directly affect OPCs, we set up a primary OPC culture isolated from neonatal mice and added DUOC-01 treatment to the culture. Results In the cerebellar slice model, we demonstrated a higher number of myelinated neuron fibers in the DUOC-treated group compared with the LPC-treated group. In the EAE model, compared with mice injected with Ringer’s or HC+Ringer’s, mice injected with DUOC-01 derived clinical benefit with lower clinical scores. In the primary OPC culture, the DUOC-01 treatment drove the maturation of OPC to become myelin producing oligodendrocytes. Discussion Our data suggest that DUOC-01 could be beneficial in treating MS and other diverse neurological demyelinating conditions.
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Peppard, Jane V., Catherine A. Rugg, Matthew A. Smicker, Elaine Powers, Erica Harnish, Joy Prisco, Dragan Cirovic, Paul S. Wright, Paul R. August, and Karen J. Chandross. "High-Content Phenotypic Screening and Triaging Strategy to Identify Small Molecules Driving Oligodendrocyte Progenitor Cell Differentiation." Journal of Biomolecular Screening 20, no. 3 (November 13, 2014): 382–90. http://dx.doi.org/10.1177/1087057114559490.
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Multiple Sclerosis is a demyelinating disease of the CNS and the primary cause of neurological disability in young adults. Loss of myelinating oligodendrocytes leads to neuronal dysfunction and death and is an important contributing factor to this disease. Endogenous oligodendrocyte precursor cells (OPCs), which on differentiation are responsible for replacing myelin, are present in the adult CNS. As such, therapeutic agents that can stimulate OPCs to differentiate and remyelinate demyelinated axons under pathologic conditions may improve neuronal function and clinical outcome. We describe the details of an automated, cell-based, morphometric-based, high-content screen that is used to identify small molecules eliciting the differentiation of OPCs after 3 days. Primary screening was performed using rat CG-4 cells maintained in culture conditions that normally support a progenitor cell–like state. From a library of 73,000 diverse small molecules within the Sanofi collection, 342 compounds were identified that increased OPC morphological complexity as an indicator of oligodendrocyte maturation. Subsequent to the primary high-content screen, a suite of cellular assays was established that identified 22 nontoxic compounds that selectively stimulated primary rat OPCs but not C2C12 muscle cell differentiation. This rigorous triaging yielded several chemical series for further expansion and bio- or cheminformatics studies, and their compelling biological activity merits further investigation.
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Allinquant, B., S. M. Staugaitis, D. D'Urso, and D. R. Colman. "The ectopic expression of myelin basic protein isoforms in Shiverer oligodendrocytes: implications for myelinogenesis." Journal of Cell Biology 113, no. 2 (April 15, 1991): 393–403. http://dx.doi.org/10.1083/jcb.113.2.393.
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The myelin basic proteins (MBPs) are a set of peripheral membrane polypeptides that are required for the compaction of the major dense line of central nervous system myelin. We have used primary cultures of oligodendrocytes from MBP-deficient shiverer mice as host cells for the expression by cDNA transfection of each of the four major MBP isoforms. The distributions of the encoded polypeptides were studied by immunofluorescence and confocal microscopy and compared with patterns of MBP expression in normal mouse oligodendrocytes in situ and in culture. The exon II-containing 21.5- or 17-kD MBPs were distributed diffusely in the cytoplasm and in the nucleus of the transfectants, closely resembling the patterns obtained in myelinating oligodendrocytes in 9-d-old normal mouse brains. By contrast, the distribution of the 14- and 18.5-kD MBPs in the transfectants was confined to the plasma membrane and mimicked the distribution of MBP in cultures of normal adult oligodendrocytes. Our results strongly suggest that the exon II-containing MBPs are expressed first and exclusively during oligodendrocyte maturation, where they may play a role in the early phase of implementation of the myelination program. In contrast, the 14- and 18.5-kD MBPs that possess strong affinity for the plasma membrane are likely to be the principle inducers of myelin compaction at the major dense line.
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Masaki, Katsuhisa, Yoshifumi Sonobe, Ghanashyam Ghadge, Peter Pytel, Paula Lépine, Florian Pernin, Qiao-Ling Cui, et al. "RNA-binding protein altered expression and mislocalization in MS." Neurology - Neuroimmunology Neuroinflammation 7, no. 3 (March 26, 2020): e704. http://dx.doi.org/10.1212/nxi.0000000000000704.
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ObjectiveTo determine whether there are nuclear depletion and cellular mislocalization of RNA-binding proteins (RBPs) transactivation response DNA-binding protein of 43 kDa (TDP-43), fused in sarcoma (FUS), and polypyrimidine tract–binding protein (PTB) in MS, as is the case in amyotrophic lateral sclerosis (ALS) and oligodendrocytes infected with Theiler murine encephalomyelitis virus (TMEV), we examined MS lesions and in vitro cultured primary human brain–derived oligodendrocytes.MethodsNuclear depletion and mislocalization of TDP-43, FUS, and PTB are thought to contribute to the pathogenesis of ALS and TMEV demyelination. The latter findings prompted us to investigate these RBPs in the demyelinated lesions of MS and in in vitro cultured human brain–derived oligodendrocytes under metabolic stress conditions.ResultsWe found (1) mislocalized TDP-43 in oligodendrocytes in active lesions in some patients with MS; (2) decreased PTB1 expression in oligodendrocytes in mixed active/inactive demyelinating lesions; (3) decreased nuclear expression of PTB2 in neurons in cortical demyelinating lesions; and (4) nuclear depletion of TDP-43 in oligodendrocytes under metabolic stress induced by low glucose/low nutrient conditions compared with optimal culture conditions.ConclusionTDP-43 has been found to have a key role in oligodendrocyte function and viability, whereas PTB is important in neuronal differentiation, suggesting that altered expression and mislocalization of these RBPs in MS lesions may contribute to the pathogenesis of demyelination and neurodegeneration. Our findings also identify nucleocytoplasmic transport as a target for treatment.
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Kaji, S., T. Maki, N. Uemura, and R. Takahashi. "Elucidating alpha-synuclein pathology of multiple system atrophy using primary oligodendrocyte culture." Journal of the Neurological Sciences 381 (October 2017): 66–67. http://dx.doi.org/10.1016/j.jns.2017.08.243.
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Giri, Shailendra, Hamid Suhail, Jaspreet Singh, Ashok Kumar, and Ramandeep Rattan. "Early burst of glycolysis in microglia regulates mitochondrial dysfunction in oligodendrocytes under neuro-inflammation." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 49.16. http://dx.doi.org/10.4049/jimmunol.200.supp.49.16.
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Abstract Brain resident microglia plays a pathological role in the loss of oligodendrocyte’s function under neuroinflammation. The precise signaling mechanism by which microglial driven factor attenuates the myelin gene expression under such condition is poorly defined. Here we used bioenergetic approach to understand the microglia-oligodendrocyte crosstalk. We found that mixed glial cells under inflammatory stimulation using lipopolysaccharide plus interferon gamma (LI) had significantly enhanced glycolysis with drastic attenuation of mitochondrial respiration and myelin gene expression. Dissecting the mixed glial culture into pure microglia, astrocyte and oligodendrocytes (OLs) revealed that primary microglia was the main source of higher glycolysis, inflammatory cytokines, and nitric oxide (NO). An early burst of glycolysis in microglia in response to LI treatment caused the production of inflammatory cytokines and NO, which is mediated by PDK1-Akt signaling. Culturing of OLs with LI-conditioned microglial media (mCM) resulted in complete failure of mitochondrial respiration in OLsalong with decreased expression of myelin genes. We identified that nitric oxide produced by microglia is the key player in the loss of mitochondrial respiration in OLs and targeting either glycolysis, PDK1 or Akt in microglia resulted in significant recovery of mitochondrial function and myelin genes expression in OLs. In summary, we identified a potential therapeutic signaling cascade in microglia; an early burst of glycolysis mediated by PDK1 and Akt, responsible for hyper-production of NO, ensuing in the failure of mitochondrial respiration in oligodendrocytes, and leading in reduction of myelin genes expression.
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Kim, Ji-Young, Ju-Young Yoon, Yuki Sugiura, Soo-Kyoung Lee, Jae-Don Park, Gyun-Jee Song, and Hyun-Jeong Yang. "Dendropanax morbiferus leaf extract facilitates oligodendrocyte development." Royal Society Open Science 6, no. 6 (June 2019): 190266. http://dx.doi.org/10.1098/rsos.190266.
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Treatment of multiple sclerosis is effective when anti-inflammatory, neuroprotective and regenerative strategies are combined. Dendropanax morbiferus ( DM ) has anti-inflammatory, anti-oxidative properties, which may be beneficial for multiple sclerosis. However, there have been no reports on the effects of DM on myelination, which is critical for regenerative processes. To know whether DM benefits myelination, we checked differentiation and myelination of oligodendrocytes (OLs) in various primary culture systems treated with DM leaf EtOH extracts or control. DM extracts increased the OL membrane size in the mixed glial and pure OL precursor cell (OPC) cultures and changed OL-lineage gene expression patterns in the OPC cultures. Western blot analysis of DM -treated OPC cultures showed upregulation of MBP and phosphorylation of ERK1/2. In myelinating cocultures, DM extracts enhanced OL differentiation, followed by increased axonal contacts and myelin gene upregulations such as Myrf, CNP and PLP. Phytochemical analysis by LC-MS/MS identified multiple components from DM extracts, containing bioactive molecules such as quercetin, cannabidiol, etc. Our results suggest DM extracts enhance OL differentiation, followed by an increase in membrane size and axonal contacts, thereby indicating enhanced myelination. In addition, we found that DM extracts contain multiple bioactive components, warranting further studies in relation to finding effective components for enhancing myelination.
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Hayakawa, Kazuhide, Loc-Duyen D. Pham, Ji Hae Seo, Nobukazu Miyamoto, Takakuni Maki, Yasukazu Terasaki, Sava Sakadžić, et al. "CD200 restrains macrophage attack on oligodendrocyte precursors via toll-like receptor 4 downregulation." Journal of Cerebral Blood Flow & Metabolism 36, no. 4 (September 30, 2015): 781–93. http://dx.doi.org/10.1177/0271678x15606148.
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There are numerous barriers to white matter repair after central nervous system injury and the underlying mechanisms remain to be fully understood. In this study, we propose the hypothesis that inflammatory macrophages in damaged white matter attack oligodendrocyte precursor cells via toll-like receptor 4 signaling thus interfering with this endogenous progenitor recovery mechanism. Primary cell culture experiments demonstrate that peritoneal macrophages can attack and digest oligodendrocyte precursor cells via toll-like receptor 4 signaling, and this phagocytosis of oligodendrocyte precursor cells can be inhibited by using CD200-Fc to downregulate toll-like receptor 4. In an in vivo model of white matter ischemia induced by endothelin-1, treatment with CD200-Fc suppressed toll-like receptor 4 expression in peripherally circulating macrophages, thus restraining macrophage phagocytosis of oligodendrocyte precursor cells and leading to improved myelination. Taken together, these findings suggest that deleterious macrophage effects may occur after white matter ischemia, whereby macrophages attack oligodendrocyte precursor cells and interfere with endogenous recovery responses. Targeting this pathway with CD200 may offer a novel therapeutic approach to amplify endogenous oligodendrocyte precursor cell-mediated repair of white matter damage in mammalian brain.
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Golub, Mari S., Wei Zhang, Carl L. Keen, and Tzipora Goldkorn. "Cellular actions of Al at low (1.25 μM) concentrations in primary oligodendrocyte culture." Brain Research 941, no. 1-2 (June 2002): 82–90. http://dx.doi.org/10.1016/s0006-8993(02)02597-0.
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Fetisova, Elena K., Maria S. Muntyan, Konstantin G. Lyamzaev, and Boris V. Chernyak. "Therapeutic Effect of the Mitochondria-Targeted Antioxidant SkQ1 on the Culture Model of Multiple Sclerosis." Oxidative Medicine and Cellular Longevity 2019 (July 1, 2019): 1–10. http://dx.doi.org/10.1155/2019/2082561.
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Multiple sclerosis (MS) is a heterogeneous autoimmune disease of unknown etiology characterized by inflammation, demyelination, and axonal degeneration that affects both the white and gray matter of CNS. Recent large-scale epidemiological and genomic studies identified several genetic and environmental risk factors for the disease. Among them are environmental factors of infectious origin, possibly causing MS, which include Epstein-Barr virus infection, reactivation of some endogenous retrovirus groups, and infection by pathogenic bacteria (mycobacteria, Chlamydia pneumoniae, and Helicobacter pylori). However, the nature of the events leading to the activation of immune cells in MS is mostly unknown and there is no effective therapy against the disease. Amazingly, whatever the cause of the disease, signs of damage to the nerve tissue with MS lesions were the same as with infectious leprosy, while in the latter case nitrozooxidative stress was suggested as the main cause of the nerve damage. With this in mind and following the hypothesis that excessive production of mitochondrial reactive oxygen species critically contributes to MS pathogenesis, we studied the effect of mitochondria-targeted antioxidant SkQ1 in an in vitro MS model of the primary oligodendrocyte culture of the cerebellum, challenged with lipopolysaccharide (LPS). SkQ1 was found to accumulate in the mitochondria of oligodendrocytes and microglial cells, and it was also found to prevent LPS-induced inhibition of myelin production in oligodendrocytes. The results implicate that mitochondria-targeted antioxidants could be promising candidates as components of a combined therapy for MS and related neurological disorders.
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Xu, Li, Shane O'Neil, Joanne Kurtzberg, and Anthony Filiano. "Abstract 30 Cord Blood Derived Macrophages Promote Remyelination." Stem Cells Translational Medicine 12, Supplement_1 (September 1, 2023): S32. http://dx.doi.org/10.1093/stcltm/szad047.031.
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Abstract Introduction Autoimmune diseases of the central nervous system, like multiple sclerosis (MS), result from autoreactive immune cells attacking myelin. Although numerous therapies targeting the immune system are effective for slowing the progression of MS, there are no therapies to promote remyelination. Therefore, we developed DUOC-01, a first-generation macrophage cell-therapy product manufactured from banked cord blood to promote remyelination. Using mouse models of demyelination, we determined that DUOC-01 blocked progression of disease and enhanced remyelination. Objectives Although preclinical data using DUOC-01 are promising, shortfalls in manufacturing and an unknown mechanism of action limits their use for MS. Here, we examined the effects of DUOC-01 on oligodendrocyte precursor cells (OPCs) to develop a second generation product that promotes the maturation of OPCs. Methods We first developed an ex vivo system, using organotypic brain slices treated with lysophosphatidylcholine (LPC), to measure remyelination by imaging the co-localization of myelin basic protein (MBP), a marker for myelin, and neurofilament, a marker for neuronal axons. Next we developed a primary OPC differentiation assay and measured maturation through markers of proliferation and differentiation. Using these assays, we set out to determine the ability of DUOC-01 and a second generation product to promote remyelination. Results In culture, our brain slices maintained well-preserved cytoarchitecture and myelinated axons, indicating a health three-dimensional system. Treating the slices with LPC caused massive demyelination of neuron processes three days post exposure. After washing out LPC, we treated cultures with vehicle or DUOC-01 and determined that DUOC-01 promoted remyelination when compared to controls. Using primary OPC cultures, we determined that DUOC-01 skewed OPC cultures to a more mature phenotype, demonstrated by the increased expression of the mature oligodendrocyte marker MBP. When treating OPC cultures with DUOC-01 conditioned media or separating DUOC-01 from OPCs using a transwell, OPC cultures still upregulated expression of MBP, indicating that bioactivity was due to factors released by DUOC-01. Discussion Overall, our data demonstrated that DUOC-01 promoted remyelination, and the factors released by DUOC-01 drive the maturation of oligodendrocytes. Thus, these remyelination factors released by DUOC-01 have the potential to work as a cell-free therapeutic for treating MS and other diverse neurological demyelinating diseases.
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Liu, Chang, Xu Hu, Yawen Li, Wenjie Lu, Wenlin Li, Nan Cao, Saiyong Zhu, Jinke Cheng, Sheng Ding, and Mingliang Zhang. "Conversion of mouse fibroblasts into oligodendrocyte progenitor-like cells through a chemical approach." Journal of Molecular Cell Biology 11, no. 6 (January 10, 2019): 489–95. http://dx.doi.org/10.1093/jmcb/mjy088.
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Abstract Transplantation of oligodendrocyte progenitor cells (OPCs) is a promising way for treating demyelinating diseases. However, generation of scalable and autologous sources of OPCs has proven difficult. We previously established a chemical condition M9 that could specifically initiate neural program in mouse embryonic fibroblasts. Here we found that M9 could induce the formation of colonies that undergo mesenchymal-to-epithelial transition at the early stage of reprogramming. These colonies may represent unstable and neural lineage-restricted intermediates that have not established a neural stem cell identity. By modulating the culture signaling recapitulating the principle of OPC development, these intermediate cells could be reprogrammed towards OPC fate. The chemical-induced OPC-like cells (ciOPLCs) resemble primary neural stem cell-derived OPCs in terms of their morphology, gene expression, and the ability of self-renewal. Upon differentiation, ciOPLCs could produce functional oligodendrocytes and myelinate the neuron axons in vitro, validating their OPC identity molecularly and functionally. Therefore, our study provides a non-integrating approach to OPC reprogramming that may ultimately provide an avenue to patient-specific cell-based or in situ regenerative therapy.
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Dasgupta, Somsankar, and Swapan K. Ray. "Ceramide and Sphingosine Regulation of Myelinogenesis: Targeting Serine Palmitoyltransferase Using microRNA in Multiple Sclerosis." International Journal of Molecular Sciences 20, no. 20 (October 11, 2019): 5031. http://dx.doi.org/10.3390/ijms20205031.
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Ceramide and sphingosine display a unique profile during brain development, indicating their critical role in myelinogenesis. Employing advanced technology such as gas chromatography–mass spectrometry, high performance liquid chromatography, and immunocytochemistry, along with cell culture and molecular biology, we have found an accumulation of sphingosine in brain tissues of patients with multiple sclerosis (MS) and in the spinal cord of rats induced with experimental autoimmune encephalomyelitis. The elevated sphingosine leads to oligodendrocyte death and fosters demyelination. Ceramide elevation by serine palmitoyltransferse (SPT) activation was the primary source of the sphingosine elevation as myriocin, an inhibitor of SPT, prevented sphingosine elevation and protected oligodendrocytes. Supporting this view, fingolimod, a drug used for MS therapy, reduced ceramide generation, thus offering partial protection to oligodendrocytes. Sphingolipid synthesis and degradation in normal development is regulated by a series of microRNAs (miRNAs), and hence, accumulation of sphingosine in MS may be prevented by employing miRNA technology. This review will discuss the current knowledge of ceramide and sphingosine metabolism (synthesis and breakdown), and how their biosynthesis can be regulated by miRNA, which can be used as a therapeutic approach for MS.
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Ainger, Kevin, Daniela Avossa, Amy S. Diana, Christopher Barry, Elisa Barbarese, and John H. Carson. "Transport and Localization Elements in Myelin Basic Protein mRNA." Journal of Cell Biology 138, no. 5 (September 8, 1997): 1077–87. http://dx.doi.org/10.1083/jcb.138.5.1077.
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Myelin basic protein (MBP) mRNA is localized to myelin produced by oligodendrocytes of the central nervous system. MBP mRNA microinjected into oligodendrocytes in primary culture is assembled into granules in the perikaryon, transported along the processes, and localized to the myelin compartment. In this work, microinjection of various deleted and chimeric RNAs was used to delineate regions in MBP mRNA that are required for transport and localization in oligodendrocytes. The results indicate that transport requires a 21-nucleotide sequence, termed the RNA transport signal (RTS), in the 3′ UTR of MBP mRNA. Homologous sequences are present in several other localized mRNAs, suggesting that the RTS represents a general transport signal in a variety of different cell types. Insertion of the RTS from MBP mRNA into nontransported mRNAs, causes the RNA to be transported to the oligodendrocyte processes. Localization of mRNA to the myelin compartment requires an additional element, termed the RNA localization region (RLR), contained between nucleotide 1,130 and 1,473 in the 3′ UTR of MBP mRNA. Computer analysis predicts that this region contains a stable secondary structure. If the coding region of the mRNA is deleted, the RLR is no longer required for localization, and the region between nucleotide 667 and 953, containing the RTS, is sufficient for both RNA transport and localization. Thus, localization of coding RNA is RLR dependent, and localization of noncoding RNA is RLR independent, suggesting that they are localized by different pathways.
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Ray, Swapan K., Timothy J. Neuberger, Gail Deadwyler, Gloria Wilford, George H. DeVries, and Naren L. Banik. "Calpain and calpastatin expression in primary oligodendrocyte culture: Preferential localization of membrane calpain in cell processes." Journal of Neuroscience Research 70, no. 4 (October 25, 2002): 561–69. http://dx.doi.org/10.1002/jnr.10414.
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Gard, Anthony L., Steven E. Pfeiffer, and Wesley C. Williams II. "Immunopanning and Developmental Stage-Specific Primary Culture of Oligodendrocyte Progenitors (O4+GalC−) Directly from Postnatal Rodent Cerebrum." Neuroprotocols 2, no. 3 (June 1993): 209–18. http://dx.doi.org/10.1006/ncmn.1993.1027.
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Verploegh, I. S. C., A. Conidi, R. W. W. Brouwer, W. F. J. van IJcken, M. Lamfers, S. Leenstra, and D. Huylebroeck. "P04.02 Single-cell transcriptomic analysis reveals shifts in glioblastoma cell composition in different BMP4-treated primary tumor cultures." Neuro-Oncology 23, Supplement_2 (September 1, 2021): ii18. http://dx.doi.org/10.1093/neuonc/noab180.059.
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Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive primary brain tumor. The well-known cellular heterogeneity of this cancer, which includes glioma tumor-initiating cells with stem cell characteristics (GSCs), (co)influences therapy resistance and tumor recurrence. Bone Morphogenetic Protein-4 (BMP4) promotes differentiation of GSCs towards astroglial lineage while suppressing oligodendrocyte maturation. Treatment with BMP4 is associated with increased survival in mice. BMPs exert effects in cell-type and context dependent fashion, but also generate subtle heterogeneity in transcriptional response among similar cells. We studied first the cell viability of BMP4-treated primary GBM cultures followed by single-cell RNA-sequencing (scRNA-seq) on two differently responding cultures, and found correlation between their responses and therapy sensitivity. MATERIAL AND METHODS Cell viability, proliferation and apoptosis were assessed in 17 patient-derived BMP4-treated GBM cell cultures. We selected one culture in which this treatment induced high in vitro therapeutic efficacy, and one in which the treatment was ineffective, for analysis by scRNA-seq and then compared the results on the initial panel of 17 cultures. RESULTS After 7 days of treatment with BMP4, cell viability ranged from 28% (referred to as highest in vitro therapeutic efficacy) to 132% compared to untreated cells. scRNA-seq of the previously mentioned cultures in passage 7 showed that all neural cell types that are usually found in freshly resected GBM, were also present in our cultures. In the culture where BMP4 induced high in vitro therapeutic efficacy, BMP4 induces the formation of a large new cell population displaying decreased cell proliferation, increased migration and cell death, while the pro-inflammatory cells were depleted. RNA-velocity analysis revealed that, the cycling of cells was greatly diminished in the culture where therapy with BMP4 was efficacious, whereas this was enhanced in the GBM culture with the lowest cell viability after treatment. Astroglial differentiation was induced in all BMP4-treated cultures, while neuronal differentiation was reduced most in the cultures in which BMP4 induced lower or no in vitro therapeutic efficacy. In the culture without therapeutic efficacy of BMP4 cell cycle arrest was not induced anymore. In addition, OLIG1/2 mRNA and protein levels seemed predictive for BMP4-therapy efficacy, while activation of translation-associated genes (RPL27A, RPS27) was a suitable, immediate post-therapeutic marker for this. CONCLUSION scRNA-seq of in vitro GBM cultures provides advanced insights into the mechanism underlying therapy efficacy of BMP4. Neural differentiation status is distinctive for therapeutic efficacy of BMP4 in vitro before and after therapy.
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Nagata, Satoshi, Ryo Yamasaki, Ezgi Ozdemir Takase, Kotaro Iida, Mitsuru Watanabe, Katsuhisa Masaki, Marion Heleen Cathérine Wijering, Hiroo Yamaguchi, Jun-ichi Kira, and Noriko Isobe. "Iguratimod Ameliorates the Severity of Secondary Progressive Multiple Sclerosis in Model Mice by Directly Inhibiting IL-6 Production and Th17 Cell Migration via Mitigation of Glial Inflammation." Biology 12, no. 9 (September 7, 2023): 1217. http://dx.doi.org/10.3390/biology12091217.
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We previously reported a novel secondary progressive multiple sclerosis (SPMS) model, progressive experimental autoimmune encephalomyelitis (pEAE), in oligodendroglia-specific Cx47-inducible conditional knockout (Cx47 icKO) mice. Based on our prior study showing the efficacy of iguratimod (IGU), an antirheumatic drug, for acute EAE treatment, we aimed to elucidate the effect of IGU on the SPMS animal model. We induced pEAE by immunizing Cx47 icKO mice with myelin oligodendrocyte glycoprotein peptide 35–55. IGU was orally administered from 17 to 50 days post-immunization. We also prepared a primary mixed glial cell culture and measured cytokine levels in the culture supernatant after stimulation with designated cytokines (IL-1α, C1q, TNF-α) and lipopolysaccharide. A migration assay was performed to evaluate the effect of IGU on the migration ability of T cells toward mixed glial cell cultures. IGU treatment ameliorated the clinical signs of pEAE, decreased the demyelinated area, and attenuated glial inflammation on immunohistochemical analysis. Additionally, IGU decreased the intrathecal IL-6 level and infiltrating Th17 cells. The migration assay revealed reduced Th17 cell migration and IL-6 levels in the culture supernatant after IGU treatment. Collectively, IGU successfully mitigated the clinical signs of pEAE by suppressing Th17 migration through inhibition of IL-6 production by proinflammatory-activated glial cells.
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Casadomé-Perales, Álvaro, Sara Naya, Elisa Fernández-Martínez, Bea G. Mille, Marta Guerrero-Valero, Héctor Peinado, Francesc X. Guix, Carlos G. Dotti, and Ernest Palomer. "Neuronal Prosurvival Role of Ceramide Synthase 2 by Olidogendrocyte-to-Neuron Extracellular Vesicle Transfer." International Journal of Molecular Sciences 24, no. 6 (March 22, 2023): 5986. http://dx.doi.org/10.3390/ijms24065986.
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Ageing is associated with notorious alterations in neurons, i.e., in gene expression, mitochondrial function, membrane degradation or intercellular communication. However, neurons live for the entire lifespan of the individual. One of the reasons why neurons remain functional in elderly people is survival mechanisms prevail over death mechanisms. While many signals are either pro-survival or pro-death, others can play both roles. Extracellular vesicles (EVs) can signal both pro-toxicity and survival. We used young and old animals, primary neuronal and oligodendrocyte cultures and neuroblastoma and oligodendrocytic lines. We analysed our samples using a combination of proteomics and artificial neural networks, biochemistry and immunofluorescence approaches. We found an age-dependent increase in ceramide synthase 2 (CerS2) in cortical EVs, expressed by oligodendrocytes. In addition, we show that CerS2 is present in neurons via the uptake of oligodendrocyte-derived EVs. Finally, we show that age-associated inflammation and metabolic stress favour CerS2 expression and that oligodendrocyte-derived EVs loaded with CerS2 lead to the expression of the antiapoptotic factor Bcl2 in inflammatory conditions. Our study shows that intercellular communication is altered in the ageing brain, which favours neuronal survival through the transfer of oligodendrocyte-derived EVs containing CerS2.
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Chen, Yan, Wenjie Guo, Liangzhi Xu, Wenjuan Li, Meng Cheng, Ying Hu, and Wenming Xu. "17β-Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model." BioMed Research International 2016 (2016): 1–13. http://dx.doi.org/10.1155/2016/7891202.
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Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17β-estradiol (E2) on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG) explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague–Dawley rats. Immunostaining for myelin basic protein (MBP) expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the “nerve bridge” region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2′-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation.
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Bayram, Keziban Korkmaz, Juliette Fitremann, Arslan Bayram, Zeynep Yılmaz, Ecmel Mehmetbeyoğlu, Yusuf Özkul, and Minoo Rassoulzadegan. "Gene Expression of Mouse Hippocampal Stem Cells Grown in a Galactose-Derived Molecular Gel Compared to In Vivo and Neurospheres." Processes 9, no. 4 (April 18, 2021): 716. http://dx.doi.org/10.3390/pr9040716.
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Background: N-heptyl-D-galactonamide (GalC7) is a small synthetic carbohydrate derivative that forms a biocompatible supramolecular hydrogel. In this study, the objective was to analyze more in-depth how neural cells differentiate in contact with GalC7. Method: Direct (ex vivo) cells of the fresh hippocampus and culture (In vitro) of the primary cells were investigated. In vitro, investigation performed under three conditions: on culture in neurospheres for 19 days, on culture in GalC7 gel for 7 days, and on culture in both neurospheres and GalC7 gel. Total RNA was isolated with TRIzol from each group, Sox8, Sox9, Sox10, Dcx, and Neurod1 expression levels were measured by qPCR. Result: Sox8 and Sox10, oligodendrocyte markers, and Sox9, an astrocyte marker, were expressed at a much higher level after 7 days of culture in GalC7 hydrogel compared to all other conditions. Dcx, a marker of neurogenesis, and Neurod1, a marker of neuronal differentiation, were expressed at better levels in the GalC7 gel culture compared to the neurosphere. Conclusions: These results show that the GalC7 hydrogel brings different and interesting conditions for inducing the differentiation and maturation of neural progenitor cells compared with polymer-based scaffolds or cell-only conditions. The differences observed open new perspectives in tissue engineering, induction, and transcript analysis.
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LoVerso, Peter R., Christopher M. Wachter, and Feng Cui. "Cross-species Transcriptomic Comparison of In Vitro and In Vivo Mammalian Neural Cells." Bioinformatics and Biology Insights 9 (January 2015): BBI.S33124. http://dx.doi.org/10.4137/bbi.s33124.
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The mammalian brain is characterized by distinct classes of cells that differ in morphology, structure, signaling, and function. Dysregulation of gene expression in these cell populations leads to various neurological disorders. Neural cells often need to be acutely purified from animal brains for research, which requires complicated procedure and specific expertise. Primary culture of these cells in vitro is a viable alternative, but the differences in gene expression of cells grown in vitro and in vivo remain unclear. Here, we cultured three major neural cell classes of rat brain (ie, neurons, astrocytes, and oligodendrocyte precursor cells [OPCs]) obtained from commercial sources. We measured transcript abundance of these cell types by RNA sequencing (RNA-seq) and compared with their counterparts acutely purified from mouse brains. Cross-species RNA-seq data analysis revealed hundreds of genes that are differentially expressed between the cultured and acutely purified cells. Astrocytes have more such genes compared to neurons and OPCs, indicating that signaling pathways are greatly perturbed in cultured astrocytes. This dataset provides a powerful resource to demonstrate the similarities and differences of biological processes in mammalian neural cells grown in vitro and in vivo at the molecular level.
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Niu, Jianqin, Feng Mei, Nan Li, Hanzhi Wang, Xinmin Li, Jiming Kong, and Lan Xiao. "Haloperidol promotes proliferation but inhibits differentiation in rat oligodendrocyte progenitor cell culturesThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process." Biochemistry and Cell Biology 88, no. 4 (August 2010): 611–20. http://dx.doi.org/10.1139/o09-178.
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Haloperidol is a commonly used, typical, antipsychotic drug (APD) that acts strongly against positive symptoms, but has fewer therapeutic effects on, or may even aggravate, negative symptoms and cognitive deficits in patients with schizophrenia. Loss of oligodendrocytes has been suggested as a factor associated with the negative symptoms of schizophrenia. Recent study shows that chronic haloperidol treatment induced down-regulation of oligodendrocyte-related genes in certain brain regions of mouse. In this study, we used primary oligodendrocyte progenitor cell cultures from 1- to 3-day-postnatal rats to investigate the direct effects of haloperidol on the proliferation and differentiation of oligodendrocyte progenitor cells. Our results showed that (i) haloperidol (0–10.0 µmol·L–1) facilitated the proliferation of oligodendrocyte progenitor cells, (ii) chronic haloperidol (0.5 µmol·L–1) treatment decreased the number of myelin basic protein positive oligodendrocytes and reduced the oligodendrocytes cells possessing myelin-like membranes, resulting in inhibition of the terminal differentiation of oligodendrocytes, and (iii) D3 receptor mRNA was detected in oligodendrocyte progenitor cells, and haloperidol treatment induced a down-regulation of D3 receptor mRNA. These results suggest that the typical antipsychotic drug haloperidol affects the development of oligodendrocyte progenitor cells, and that D3 receptor down regulation may be involved. Our observations provide new insight into possible cellular mechanisms responsible for the side effects of typical antipsychotic drugs and support the concept that abnormality of oligodendrocytes may be involved in the pathogenesis of schizophrenia.
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El-Badri (Dajani), Nagwa S., Samuel Saporta, Xiaomei Liang, Cyndy D. Sanberg, and Paul R. Sanberg. "Cord Blood Mesenchymal Stem Cells Are Enriched for Neural and Glial Progenitors." Blood 104, no. 11 (November 16, 2004): 4180. http://dx.doi.org/10.1182/blood.v104.11.4180.4180.
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Abstract Human bone marrow stroma is the primary source for mesenchymal stem cells (MSCs), a mixture of multipotent progenitors that differentiate into fibrous, cartilage, adipose, bone and reticular tissue cells. Recent reports suggest that human umbilical cord blood (UCB) is a feasible source for MSCs. In this study, we examined whether MSCs from umbilical cord blood contain progenitors that differentiate into cells of neural and glial lineages. UCB mononuclear cell fraction was cultured in Iscove’s MDM culture medium supplemented with 20% fetal bovine serum and L-glutamine. After 2 weeks in culture, adherent cells formed colony forming unit fibroblast (CFU-F)-like colonies. When adherent cells reached 70% confluency, they were further subcultured in the same medium for three more weeks before replacing IMDM with neurogenic differentiation medium. Cord blood MSCs were devoid of hematopoietic activity since they failed to produce CFU-GM, CFU-E, or CFU-GEMM hematopoietic colonies when cultured in methyl cellulose medium supplemented with the appropriate hematopoietic growth factors. Flow cytometric analysis showed a population of cells devoid of hematopoietic phenotype (CD34−, CD45−, CD38−, CD3−, CD19−). After 7 days incubation in serum free supplemented Neurobasal™ medium, cultured cord blood MSCs expressed neuron cell surface antigen A2B5, neurofilament polypeptide NF200, oligodendrocyte marker 04, and intermediate filament proteins, nestin and vimentin. Strong immunoreactivity was observed with astrocyte marker GFAP. A proportion of cultured MSCs developed into neuron-like cells that were positive for TUJ-1 neural progenitor marker. These results indicate that human UCB-derived MSCs are enriched for neuro-glial progenitors, and could be further developed for the treatment of stroke, spinal cord injury, and neurodegenerative diseases such as like Alzheimer’s, Parkinson’s and Multiple Sclerosis.
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Augustus, Meera, Donovan Pineau, Franck Aimond, Safa Azar, Davide Lecca, Frédérique Scamps, Sophie Muxel, et al. "Identification of CRYAB+ KCNN3+ SOX9+ Astrocyte-Like and EGFR+ PDGFRA+ OLIG1+ Oligodendrocyte-Like Tumoral Cells in Diffuse IDH1-Mutant Gliomas and Implication of NOTCH1 Signalling in Their Genesis." Cancers 13, no. 9 (April 27, 2021): 2107. http://dx.doi.org/10.3390/cancers13092107.
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Diffuse grade II IDH-mutant gliomas are slow-growing brain tumors that progress into high-grade gliomas. They present intratumoral cell heterogeneity, and no reliable markers are available to distinguish the different cell subtypes. The molecular mechanisms underlying the formation of this cell diversity is also ill-defined. Here, we report that SOX9 and OLIG1 transcription factors, which specifically label astrocytes and oligodendrocytes in the normal brain, revealed the presence of two largely nonoverlapping tumoral populations in IDH1-mutant oligodendrogliomas and astrocytomas. Astrocyte-like SOX9+ cells additionally stained for APOE, CRYAB, ID4, KCNN3, while oligodendrocyte-like OLIG1+ cells stained for ASCL1, EGFR, IDH1, PDGFRA, PTPRZ1, SOX4, and SOX8. GPR17, an oligodendrocytic marker, was expressed by both cells. These two subpopulations appear to have distinct BMP, NOTCH1, and MAPK active pathways as stainings for BMP4, HEY1, HEY2, p-SMAD1/5 and p-ERK were higher in SOX9+ cells. We used primary cultures and a new cell line to explore the influence of NOTCH1 activation and BMP treatment on the IDH1-mutant glioma cell phenotype. This revealed that NOTCH1 globally reduced oligodendrocytic markers and IDH1 expression while upregulating APOE, CRYAB, HEY1/2, and an electrophysiologically-active Ca2+-activated apamin-sensitive K+ channel (KCNN3/SK3). This was accompanied by a reduction in proliferation. Similar effects of NOTCH1 activation were observed in nontumoral human oligodendrocytic cells, which additionally induced strong SOX9 expression. BMP treatment reduced OLIG1/2 expression and strongly upregulated CRYAB and NOGGIN, a negative regulator of BMP. The presence of astrocyte-like SOX9+ and oligodendrocyte-like OLIG1+ cells in grade II IDH1-mutant gliomas raises new questions about their role in the pathology.
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Egawa, Naohiro, Gen Hamanaka, Kelly K. Chung, Hidehiro Ishikawa, Akihiro Shindo, Takakuni Maki, Ryosuke Takahashi, Haruhisa Inoue, Eng H. Lo, and Ken Arai. "High Mobility Group A1 Regulates Transcription Levels of Oligodendrocyte Marker Genes in Cultured Oligodendrocyte Precursor Cells." International Journal of Molecular Sciences 23, no. 4 (February 17, 2022): 2236. http://dx.doi.org/10.3390/ijms23042236.
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Oligodendrocyte precursor cells (OPCs) serve as progenitor cells of terminally differentiated oligodendrocytes. Past studies have confirmed the importance of epigenetic system in OPC differentiation to oligodendrocytes. High mobility group A1 (HMGA1) is a small non-histone nuclear protein that binds DNA and modifies the chromatin conformational state. However, it is still completely unknown about the roles of HMGA1 in the process of OPC differentiation. In this study, we prepared primary OPC cultures from the neonatal rat cortex and examined whether the loss- and gain-of-function of HMGA1 would change the mRNA levels of oligodendrocyte markers, such as Cnp, Mbp, Myrf and Plp during the process of OPC differentiation. In our system, the mRNA levels of Cnp, Mbp, Myrf and Plp increased depending on the oligodendrocyte maturation step, but the level of Hmga1 mRNA decreased. When HMGA1 was knocked down by a siRNA approach, the mRNA levels of Cnp, Mbp, Myrf and Plp were smaller in OPCs with Hmga1 siRNA compared to the ones in the control OPCs. On the contrary, when HMGA1 expression was increased by transfection of the Hmga1 plasmid, the mRNA levels of Cnp, Mbp, Myrf and Plp were slightly larger compared to the ones in the control OPCs. These data may suggest that HMGA1 participates in the process of OPC differentiation by regulating the mRNA expression level of myelin-related genes.
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Bossu, Jean Louis, Laetitia Wioland, Frédéric Doussau, Philippe Isope, Michel R. Popoff, and Bernard Poulain. "Epsilon Toxin from Clostridium perfringens Causes Inhibition of Potassium inward Rectifier (Kir) Channels in Oligodendrocytes." Toxins 12, no. 1 (January 6, 2020): 36. http://dx.doi.org/10.3390/toxins12010036.
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Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, causes serious neurological disorders in animals. ETX can bind to the white matter of the brain and the oligodendrocytes, which are the cells forming the myelin sheath around neuron axons in the white matter of the central nervous system. After binding to oligodendrocytes, ETX causes demyelination in rat cerebellar slices. We further investigated the effects of ETX on cerebellar oligodendrocytes and found that ETX induced small transmembrane depolarization (by ~ +6.4 mV) in rat oligodendrocytes primary cultures. This was due to partial inhibition of the transmembrane inward rectifier potassium current (Kir). Of the two distinct types of Kir channel conductances (~25 pS and ~8.5 pS) recorded in rat oligodendrocytes, we found that ETX inhibited the large-conductance one. This inhibition did not require direct binding of ETX to a Kir channel. Most likely, the binding of ETX to its membrane receptor activates intracellular pathways that block the large conductance Kir channel activity in oligodendrocyte. Altogether, these findings and previous observations pinpoint oligodendrocytes as a major target for ETX. This supports the proposal that ETX might be a cause for Multiple Sclerosis, a disease characterized by myelin damage.
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Gard, A. L., M. R. Burrell, S. E. Pfeiffer, J. S. Rudge, and W. C. Williams. "Astroglial control of oligodendrocyte survival mediated by PDGF and leukemia inhibitory factor-like protein." Development 121, no. 7 (July 1, 1995): 2187–97. http://dx.doi.org/10.1242/dev.121.7.2187.
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Programmed death and the identification of growth factors delaying this process in the oligodendrocyte lineage suggest that other cell types provide oligodendrogliotrophins. To determine their source, primary cultures of oligodendroblasts immunopurified from postnatal rat cerebrum were used to screen other cultured neural and non-neural cell types for the release of survival factors into a defined insulin-containing medium. In non-conditioned medium, oligodendroblasts died 1–2 days after undergoing terminal differentiation into oligodendrocytes, as defined by the onset of expression of galactocerebroside. In medium conditioned by astrocytes, unlike the other tested cell types, differentiated oligodendrocytes survived for weeks in a mature myelinogenic state. Survival was partially reduced by immunoabsorption of the medium with antibodies to platelet-derived growth factor and abolished by immunoabsorption with antibodies to leukemia inhibitory factor. By the same criterion, survival activity was not attributed to other astrocytic products, ciliary neurotrophic factor and basic fibroblast growth factor. Membrane ultrafiltration analysis indicated the activity corresponded to heat-labile protein smaller (M(r) = 10(−30) × 10(3)) than native rat leukemia inhibitory factor (M(r) = 43 × 10(3)). The astrocytic stimulus was > 4-fold more efficacious than other known oligodendrogliotrophic cytokines, including ciliary neurotrophic factor, neurotrophin-3 and leukemia inhibitory factor itself, tested singly or in combination, and promoted survival additively with these agents. These findings suggest that astrocytes function as paracrine regulators of oligodendroblast and oligodendrocyte survival and that their effect is mediated initially by platelet-derived growth factor and thereafter by a powerful cytokine related to leukemia inhibitory factor.
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Wedel, Miriam, Franziska Fröb, Olga Elsesser, Marie-Theres Wittmann, D. Chichung Lie, André Reis, and Michael Wegner. "Transcription factor Tcf4 is the preferred heterodimerization partner for Olig2 in oligodendrocytes and required for differentiation." Nucleic Acids Research 48, no. 9 (April 8, 2020): 4839–57. http://dx.doi.org/10.1093/nar/gkaa218.
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Abstract Development of oligodendrocytes and myelin formation in the vertebrate central nervous system is under control of several basic helix-loop-helix transcription factors such as Olig2, Ascl1, Hes5 and the Id proteins. The class I basic helix-loop-helix proteins Tcf3, Tcf4 and Tcf12 represent potential heterodimerization partners and functional modulators for all, but have not been investigated in oligodendrocytes so far. Using mouse mutants, organotypic slice and primary cell cultures we here show that Tcf4 is required in a cell-autonomous manner for proper terminal differentiation and myelination in vivo and ex vivo. Partial compensation is provided by the paralogous Tcf3, but not Tcf12. On the mechanistic level Tcf4 was identified as the preferred heterodimerization partner of the central regulator of oligodendrocyte development Olig2. Both genetic studies in the mouse as well as functional studies on enhancer regions of myelin genes confirmed the relevance of this physical interaction for oligodendrocyte differentiation. Considering that alterations in TCF4 are associated with syndromic and non-syndromic forms of intellectual disability, schizophrenia and autism in humans, our findings point to the possibility of an oligodendroglial contribution to these disorders.
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Reccia, Mafalda Giovanna, Floriana Volpicelli, Eirkiur Benedikz, Åsa Fex Svenningsen, and Luca Colucci-D’Amato. "Generation of High-Yield, Functional Oligodendrocytes from a c-myc Immortalized Neural Cell Line, Endowed with Staminal Properties." International Journal of Molecular Sciences 22, no. 3 (January 23, 2021): 1124. http://dx.doi.org/10.3390/ijms22031124.
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Neural stem cells represent a powerful tool to study molecules involved in pathophysiology of Nervous System and to discover new drugs. Although they can be cultured and expanded in vitro as a primary culture, their use is hampered by their heterogeneity and by the cost and time needed for their preparation. Here we report that mes-c-myc A1 cells (A1), a neural cell line, is endowed with staminal properties. Undifferentiated/proliferating and differentiated/non-proliferating A1 cells are able to generate neurospheres (Ns) in which gene expression parallels the original differentiation status. In fact, Ns derived from undifferentiated A1 cells express higher levels of Nestin, Kruppel-like factor 4 (Klf4) and glial fibrillary protein (GFAP), markers of stemness, while those obtained from differentiated A1 cells show higher levels of the neuronal marker beta III tubulin. Interestingly, Ns differentiation, by Epidermal Growth Factors (EGF) and Fibroblast Growth Factor 2 (bFGF) withdrawal, generates oligodendrocytes at high-yield as shown by the expression of markers, Galactosylceramidase (Gal-C) Neuron-Glial antigen 2 (NG2), Receptor-Interacting Protein (RIP) and Myelin Basic Protein (MBP). Finally, upon co-culture, Ns-A1-derived oligodendrocytes cause a redistribution of contactin-associated protein (Caspr/paranodin) protein on neuronal cells, as primary oligodendrocytes cultures, suggesting that they are able to form compact myelin. Thus, Ns-A1-derived oligodendrocytes may represent a time-saving and low-cost tool to study the pathophysiology of oligodendrocytes and to test new drugs.
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Sandarage, RV, A. Galuta, and EC Tsai. "P.145 Uncovering differences in oligodendrogenesis between human and rodent spinal cord stem cells." Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques 50, s2 (June 2023): S96. http://dx.doi.org/10.1017/cjn.2023.233.
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Background: Spinal cord regeneration in pre-clinical rodent studies is feasible by promoting oligodendrocyte regrowth, which is necessary for axon myelination. It is uncertain whether human neural stem/progenitor cells (NSPCs) are capable of differentiation into oligodendrocytes, similar to rat. In this study, we compare the functional and transcriptional features of primary spinal cord NSPCs from adult humans and rats. Methods: Oligodendrogenesis between human & rat NSPCs from adult donors and rats was cultured using the Neurosphere assay. NSPCs were exposed to 1% FBS to trigger differentiation & PDGF-AA to promote oligodendrocyte formation. Immunocytochemistry & RNA sequencing compared transcriptomes and analyzing differentially expressed genes. Results: Human NSPCs showed a reduced potential for oligodendrocyte generation compared to rat NSPCs (0.013±0.01% and 0.029±0.01% O4+ after one and two weeks in humans; 4.9±0.4% and 6.3±0.6% O4+ after one and two weeks in rats). PDGF-AA treatment at 40 ng/µL for one week was able to effectively promote oligodendrocyte differentiation in rat NSPCs, but had a minimal effect on human NSPCs (8.5±1.4 fold increase in O4+). OLIG1/2, SOX10, and CNP were enriched in rat NSPCs. Conclusions: We compared oligodendrogenesis potential between human and rat NSPCs and found significantly lower capacity in human NSPCs, possibly hindering successful myelinating techniques.
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Deloulme, J. C., T. Janet, D. Au, D. R. Storm, M. Sensenbrenner, and J. Baudier. "Neuromodulin (GAP43): a neuronal protein kinase C substrate is also present in 0-2A glial cell lineage. Characterization of neuromodulin in secondary cultures of oligodendrocytes and comparison with the neuronal antigen." Journal of Cell Biology 111, no. 4 (October 1, 1990): 1559–69. http://dx.doi.org/10.1083/jcb.111.4.1559.
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Neuromodulin (also called GAP43, G50, F1, pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellate-shaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of neuromodulin in secondary cultures of newborn rat oligodendrocytes and its absence in type 1 astrocytes was confirmed by Northern blot analysis of isolated total RNA from these different types of cells using a cDNA probe for the neuromodulin mRNA and by Western blot analysis of the cell extracts using polyclonal antibodies against neuromodulin. The properties of the neuromodulin protein in cultured oligodendrocytes and neuronal cells have been compared. Although neuromodulin in oligodendrocytes is soluble in 2.5% perchloric acid like the neuronal counterpart it migrates essentially as a single protein spot on two-dimensional gel electrophoresis whereas the neuronal antigen can be resolved into at least three distinct protein spots. To obtain precise alignments of the different neuromodulin spots from these two cell types, oligodendrocyte and neuronal cell extracts were mixed together and run on the same two-dimensional gel electrophoresis system. Oligodendroglial neuromodulin migrates with a pI identical to the basic forms of the neuronal protein in isoelectric focusing gel. However, the glial neuromodulin shows a slightly lower mobility in the second dimensional lithium dodecyl sulfate-PAGE than its neuronal counterpart. As measured by 32Pi incorporation, neuromodulin phosphorylation in oligodendrocytes is dramatically increased after short-term phorbol ester treatments, which activate protein kinase C, and is totally inhibited by long-term phorbol ester treatments, which downregulates protein kinase C, thus confirming its probable specific in vivo phosphorylation by protein kinase C. In primary cultures of neuronal cells, two of the three neuromodulin spots were observed to be phosphorylated with an apparent preferential phosphorylation of the more acid forms.
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Anand, Sumyuktha V., Alexander G. Skorput, Allan T. Gulledge, Isabella B. Fox, Damian A. Bonnin, Alison L. Young, and Matthew C. Havrda. "Abstract 905: Targeting muscarinic acetylcholine receptors in glioma stem like cells." Cancer Research 82, no. 12_Supplement (June 15, 2022): 905. http://dx.doi.org/10.1158/1538-7445.am2022-905.
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Abstract Primary gliomas arising within the brain remain the deadliest form of brain cancer and account for 78% of all malignant brain tumors. Glioblastoma patients have a 5 percent five-year survival rate and drug-resistant tumors often recur following surgical resection and treatment with radiation or chemotherapy. The cancer stem cell hypothesis suggests the presence of a subset of undifferentiated cells, namely glioma stem cells (GSCs), in the heterogenous tumor mass that are likely responsible for tumor initiation and recurrence of tumors post resection and give rise to drug resistant tumors. Determining a way to suppress these malignant characteristics and/or depleting the GSC population could improve current cancer treatments and the survival of glioma patients. Some GSCs are similar to oligodendrocyte precursor cells (OPCs), found during neural development and also residing in the adult brain. OPCs are prone to malignant transformation and believed to be a cellular origin for glioma. Recent findings indicate that the well-characterized neurotransmitter acetylcholine (ACh) maintains the primitive state of normal OPCs via muscarinic ACh receptors (mAChRs) preventing maturation and cell cycle exit. We investigated the functional characterization of ACh and mAChRs in modulating malignancy in OPC-like cells. We studied cultures of primary mouse OPC-like GSCs. We used the primary cells to culture three-dimensional tumor organoids in vitro to better represent tumor heterogeneity. We also used patient derived xenografts (PDX) of glioma to grow flank xenografts in NSG mice for an in vivo model. Publicly available data and studies in our lab show high levels of expression of CHRM3 (M3mAChR) in glioma patients and in primary OPC like GSCs. A drug screen conducted in the context of multiple sclerosis determined that benztropine (BZT), an anti muscarinic drug targeting M3mAChR, causes normal OPCs to exit the cell cycle, lose stem cell like characteristics, and differentiate. In our lab, electrophysiological studies demonstrated that activation of mAChRs in OPC-like cells from mouse and patient tumors generates rapid (< 1 second) increases in cytosolic calcium. Pharmacologic studies indicated that treatment with FDA approved anti muscarinic benztropine suppresses proliferation in cultured glioma cells. We also observed that serially passaging flank tumors treated with BZT into new host mice slowed down recurrence of new tumors. In order to determine key receptors mediating cholinergic responses in OPC-like cells we performed protein quantification, which displayed lowered phosphorylation of ERK. Electrophysiological studies are being conducted to dissect the mechanisms by which ACh evokes calcium release. These studies improve understanding of how cholinergic microenvironment influences stem-like glioma cells, providing a platform for repositioning available small molecule modifiers for treatment of glioma. Citation Format: Sumyuktha V. Anand, Alexander G. Skorput, Allan T. Gulledge, Isabella B. Fox, Damian A. Bonnin, Alison L. Young, Matthew C. Havrda. Targeting muscarinic acetylcholine receptors in glioma stem like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 905.
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Minami, Jenna, Nicholas Bayley, Christopher Tse, Danielle Morrow, Henan Zhu, William Yong, Linda Liau, Timothy Cloughesy, Thomas Graeber, and David Nathanson. "TAMI-26. INVESTIGATING TUMOR MICROENVIRONMENT METABOLIC DEPENDENCIES IN GLIOBLASTOMA." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi203. http://dx.doi.org/10.1093/neuonc/noab196.810.
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Abstract Metabolic reprogramming is a hallmark of cancer. Malignant cells must acquire metabolic adaptations in response to a multitude of intrinsic and extrinsic factors to fuel neoplastic progression. Mutations or changes in metabolic gene expression can impose nutrient dependencies in tumors, and even in the absence of metabolic defects, cancer cells can become auxotrophic for particular nutrients or metabolic byproducts generated by other cells in the tumor microenvironment (TME). Altered metabolism in GBM is becoming an increasingly promising area of research to identify novel therapeutic targets and biomarkers, as metabolic rewiring can occur across numerous genotypes. The unique features of the brain TME pose a difficult challenge when studying GBM and other primary brain cancers – currently, the availability of nutrients in the brain, as well as how they influence or are influenced by tumor metabolism, are not well understood. Our group has identified a subgroup of gliomas, hereafter termed TME-dependent, which can only form tumors in the brain TME. While genetically heterogeneous, these tumors share transcriptional identities linked to oligodendrocyte precursor cell (OPC) and neuronal lineages. Systematic molecular profiling of over 75 patient tumors and their matched cell culture and brain orthotopic xenograft derived models revealed that TME-dependent tumors display lipid metabolic signatures linked to signaling and interactions with surrounding neurons and glial cells. Collectively, these data emphasize the metabolic heterogeneity within GBM, and reveal a subset of gliomas that lack metabolic plasticity in fatty acid biosynthetic programs, indicating a potential brain-microenvironment specific metabolic dependency linked to transcriptional identity that can be targeted for therapy.
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Nmezi, Bruce, Laura L. Vollmer, Tong Ying Shun, Albert Gough, Harshvardhan Rolyan, Fang Liu, Yumeng Jia, Quasar S. Padiath, and Andreas Vogt. "Development and Optimization of a High-Content Analysis Platform to Identify Suppressors of Lamin B1 Overexpression as a Therapeutic Strategy for Autosomal Dominant Leukodystrophy." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 8 (April 30, 2020): 939–49. http://dx.doi.org/10.1177/2472555220915821.
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Autosomal dominant leukodystrophy (ADLD) is a fatal, progressive adult-onset disease characterized by widespread central nervous system (CNS) demyelination and significant morbidity. The late age of onset together with the relatively slow disease progression provides a large therapeutic window for the disorder. However, no treatment exists for ADLD, representing an urgent and unmet clinical need. We have previously shown that ADLD is caused by duplications of the lamin B1 gene causing increased expression of the lamin B1 protein, a major constituent of the nuclear lamina, and demonstrated that transgenic mice with oligodendrocyte-specific overexpression of lamin B1 exhibit temporal and histopathological features reminiscent of the human disease. As increased levels of lamin B1 are the causative event triggering ADLD, approaches aimed at reducing lamin B1 levels and associated functional consequences represent a promising strategy for discovery of small-molecule ADLD therapeutics. To this end, we have created an inducible cell culture model of lamin B1 overexpression and developed high-content analysis in connection with multivariate analysis to define, analyze, and quantify lamin B1 expression and its associated abnormal nuclear phenotype in mouse embryonic fibroblasts (MEFs). The assay has been optimized to meet high-throughput screening (HTS) criteria in multiday variability studies. To control for batch-to-batch variation in the primary MEFs, we have implemented a screening strategy that employs sentinel cells to avoid costly losses during HTS. We posit the assay will identify bona fide suppressors of lamin B1 pathophysiology as candidates for development into potential therapies for ADLD.
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Hudson, L. D., V. L. Friedrich, T. Behar, M. Dubois-Dalcq, and R. A. Lazzarini. "The initial events in myelin synthesis: orientation of proteolipid protein in the plasma membrane of cultured oligodendrocytes." Journal of Cell Biology 109, no. 2 (August 1, 1989): 717–27. http://dx.doi.org/10.1083/jcb.109.2.717.
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Proteolipid protein (PLP) is the most abundant transmembrane protein in myelin of the central nervous system. Conflicting models of PLP topology have been generated by computer predictions based on its primary sequence and experiments with purified myelin. We have examined the initial events in myelin synthesis, including the insertion and orientation of PLP in the plasma membrane, in rat oligodendrocytes which express PLP and the other myelin-specific proteins when cultured without neurons (Dubois-Dalcq, M., T. Behar, L. Hudson, and R. A. Lazzarini. 1986. J. Cell Biol. 102:384-392). These cells, identified by the presence of surface galactocerebroside, the major myelin glycolipid, were stained with six anti-peptide antibodies directed against hydrophilic or short hydrophobic sequences of PLP. Five of these anti-peptide antibodies specifically stained living oligodendrocytes. Staining was only seen approximately 10 d after PLP was first detected in the cytoplasm of fixed and permeabilized cells, suggesting that PLP is slowly transported from the RER to the cell surface. The presence of PLP domains on the extracellular surface was also confirmed by cleavage of such domains with proteases and by antibody-dependent complement-mediated lysis of living oligodendrocytes. Our results indicate that PLP has only two transmembrane domains and that the great majority of the protein, including its amino and carboxy termini, is located on the extracellular face of the oligodendrocyte plasma membrane. This disposition of the PLP molecule suggests that homophilic interactions between PLP molecules of apposed extracellular faces may mediate compaction of adjacent bilayers in the myelin sheath.
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Dauth, Stephanie, Maike M. Schmidt, Maren Rehders, Frank Dietz, Sørge Kelm, Ralf Dringen, and Klaudia Brix. "Characterisation and metabolism of astroglia-rich primary cultures from cathepsin K-deficient mice." Biological Chemistry 393, no. 9 (September 1, 2012): 959–70. http://dx.doi.org/10.1515/hsz-2012-0145.
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Abstract Cathepsin K is important for the brain, because its deficiency in mice is associated with a marked decrease in differentiated astrocytes and changes in neuronal patterning in the hippocampus as well as with learning and memory deficits. As cathepsin K activity is most prominent in hippocampal regions of wild type animals, we hypothesised alterations in astrocyte-mediated support of neurons as a potential mechanism underlying the impaired brain functions in cathepsin K-deficient mice. To address this hypothesis, we have generated and characterised astroglia-rich primary cell cultures from cathepsin K-deficient and wild type mice and compared these cultures for possible changes in metabolic support functions and cell composition. Interestingly, cells expressing the oligodendrocytic markers myelin-associated glycoprotein and myelin basic protein were more frequent in astroglia-rich cultures from cathepsin K-deficient mice. However, cell cultures from both genotypes were morphologically comparable and similar with respect to glucose metabolism. In addition, specific glutathione content, glutathione export and γ-glutamyl-transpeptidase activity remained unchanged, whereas the specific activities of glutathione reductase and glutathione-S-transferase were increased by around 50% in cathepsin K-deficient cultures. Thus, lack of cathepsin K in astroglia-rich cultures appears not to affect metabolic supply functions of astrocytes but to facilitate the maturation of oligodendrocytes.
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Oh, Y. J., G. J. Markelonis, and T. H. Oh. "Immunocytochemical localization of mitochondrial malate dehydrogenase in primary cultures of rat astrocytes and oligodendrocytes." Journal of Histochemistry & Cytochemistry 39, no. 5 (May 1991): 681–88. http://dx.doi.org/10.1177/39.5.1707906.
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To assess the oxidative metabolism of glial cells, we visualized mitochondrial malate dehydrogenase (mMDH) in purified cultures of neonatal rat polygonal and process-bearing astrocytes as well as in oligodendrocytes, using indirect immunofluorescence. Double immunofluorescent localization of rabbit anti-mMDH and either mouse monoclonal antiglial fibrillary acidic protein or anti-myelin basic protein demonstrated that both process-bearing astrocytes and oligodendrocytes showed uniformly intense anti-mMDH immunoreactivity in their cell bodies. However, immunoreactivity to mMDH among polygonal astrocytes varied from very weakly positive to intensely positive. Experiments with rhodamine 123, a mitochondrion-specific fluorochrome, indicated that polygonal astrocytes contain relatively similar numbers of mitochondria; this suggested that the variable intensities of anti-mMDH immunoreactivity observed did not result from differences in mitochondrial numbers. In cultures of polygonal astrocytes maintained in a chemically defined medium containing growth factors and hormones, or in complete culture medium containing 1mM N6, O2-dibutyryl adenosine 3',5'-cyclic phosphate, the resultant stellate astrocytes still showed their original variable levels of anti-mMDH immunoreactivity. This suggested that the mMDH distribution pattern did not depend on the degree of morphological differentiation. Furthermore, cultures of polygonal astrocytes isolated from four specific regions of neonatal rat brain showed variable but reproducible profiles of anti-mMDH immunoreactivity. Our results suggest that there may be an appreciable range in the level of oxidative metabolism among individual polygonal astrocytes in culture.
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Yu, Zhenwei, Min Shi, Tessandra Stewart, Pierre-Olivier Fernagut, Yang Huang, Chen Tian, Benjamin Dehay, et al. "Reduced oligodendrocyte exosome secretion in multiple system atrophy involves SNARE dysfunction." Brain 143, no. 6 (May 18, 2020): 1780–97. http://dx.doi.org/10.1093/brain/awaa110.
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Abstract Transportation of key proteins via extracellular vesicles has been recently implicated in various neurodegenerative disorders, including Parkinson’s disease, as a new mechanism of disease spreading and a new source of biomarkers. Extracellular vesicles likely to be derived from the brain can be isolated from peripheral blood and have been reported to contain higher levels of α-synuclein (α-syn) in Parkinson’s disease patients. However, very little is known about extracellular vesicles in multiple system atrophy, a disease that, like Parkinson’s disease, involves pathological α-syn aggregation, though the process is centred around oligodendrocytes in multiple system atrophy. In this study, a novel immunocapture technology was developed to isolate blood CNPase-positive, oligodendrocyte-derived enriched microvesicles (OEMVs), followed by fluorescent nanoparticle tracking analysis and assessment of α-syn levels contained within the OEMVs. The results demonstrated that the concentrations of OEMVs were significantly lower in multiple system atrophy patients, compared to Parkinson’s disease patients and healthy control subjects. It is also noted that the population of OEMVs involved was mainly in the size range closer to that of exosomes, and that the average α-syn concentrations (per vesicle) contained in these OEMVs were not significantly different among the three groups. The phenomenon of reduced OEMVs was again observed in a transgenic mouse model of multiple system atrophy and in primary oligodendrocyte cultures, and the mechanism involved was likely related, at least in part, to an α-syn-mediated interference in the interaction between syntaxin 4 and VAMP2, leading to the dysfunction of the SNARE complex. These results suggest that reduced OEMVs could be an important mechanism related to pathological α-syn aggregation in oligodendrocytes, and the OEMVs found in peripheral blood could be further explored for their potential as multiple system atrophy biomarkers.
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Enz, Lukas S., Thomas Zeis, Annalisa Hauck, Christopher Linington, and Nicole Schaeren-Wiemers. "Combinatory Multifactor Treatment Effects on Primary Nanofiber Oligodendrocyte Cultures." Cells 8, no. 11 (November 12, 2019): 1422. http://dx.doi.org/10.3390/cells8111422.
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Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system. Neurological deficits are attributed to inflammatory demyelination, which compromises axonal function and survival. These are mitigated in experimental models by rapid and often complete remyelination of affected axons, but in MS this endogenous repair mechanism frequently fails, leaving axons increasingly vulnerable to the detrimental effects of inflammatory and metabolic stress. Understanding the molecular basis of remyelination and remyelination failure is essential to develop improved therapies for this devastating disease. However, recent studies suggest that this is not due to a single dominant mechanism, but rather represents the biological outcome of multiple changes in the lesion microenvironment that combine to disrupt oligodendrocyte differentiation. This identifies a pressing need to develop technical platforms to investigate combinatory and/or synergistic effects of factors differentially expressed in MS lesions on oligodendrocyte proliferation and differentiation. Here we describe protocols using primary oligodendrocyte cultures from Bl6 mice on 384-well nanofiber plates to model changes affecting oligodendrogenesis and differentiation in the complex signaling environment associated with multiple sclerosis lesions. Using platelet-derived growth factor (PDGF–AA), fibroblast growth factor 2 (FGF2), bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein 4 (BMP4) as representative targets, we demonstrate that we can assess their combinatory effects across a wide range of concentrations in a single experiment. This in vitro model is ideal for assessing the combinatory effects of changes in availability of multiple factors, thus more closely modelling the situation in vivo and furthering high-throughput screening possibilities.
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de los Monteros, A. Espinosa, G. Roussel, C. Gensburger, J. L. Nussbaum, and G. Labourdette. "Precursor cells of oligodendrocytes in rat primary cultures." Developmental Biology 108, no. 2 (April 1985): 474–80. http://dx.doi.org/10.1016/0012-1606(85)90050-8.
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Muñoz-Esquivel, Jonathan, Peter Göttle, Lucinda Aguirre-Cruz, José Flores-Rivera, Teresa Corona, Gustavo Reyes-Terán, Patrick Küry, and Klintsy J. Torres. "Sildenafil Inhibits Myelin Expression and Myelination of Oligodendroglial Precursor Cells." ASN Neuro 11 (January 2019): 175909141983244. http://dx.doi.org/10.1177/1759091419832444.
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Phosphodiesterases (PDEs) have previously been implicated in oligodendrocyte maturation and myelination of central nervous system axons. Sildenafil citrate is a phosphodiesterase inhibitor known to block PDE5, which also reduces inflammation in the experimental autoimmune encephalomyelitis demyelinating model. To find out whether this inhibitor might exert beneficial effects on central nervous system myelin repair activities, we investigated to what degree sildenafil modulates differentiation and maturation of cultured primary rat oligodendroglial precursor cells (OPCs). To this end, gene and protein expression of 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, myelin basic protein, and myelin oligodendrocyte glycoprotein, as well as of negative regulators of myelin expression (Hes1, Hes5, Id2, Id4, Rock2, and p57Kip2) were measured in OPCs treated with sildenafil. Moreover, the subcellular distribution of the p57kip2 protein was determined after sildenafil treatment, as this revealed to be an early predictor of the oligodendroglial differentiation capacity. In vitro myelination assays were done to measure the myelination capacity of oligodendrocytes treated with sildenafil. We found that sildenafil significantly diminished myelin gene expression and protein expression. Moreover, sildenafil also increased the expression of Id2 and Id4 negative transcriptional regulators, and the degree of OPCs with cytoplasmic p57kip2 protein localization was reduced, providing evidence that the PDE blocker impaired the differentiation capacity. Finally, sildenafil also interfered with the establishment of internodes as revealed by in vitro myelination assays. We therefore conclude that blocking PDE5 activities exerts a negative impact on intrinsic oligodendroglial differentiation processes.
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Iacobas, Dumitru A., Sanda Iacobas, Randy F. Stout, and David C. Spray. "Cellular Environment Remodels the Genomic Fabrics of Functional Pathways in Astrocytes." Genes 11, no. 5 (May 7, 2020): 520. http://dx.doi.org/10.3390/genes11050520.
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We profiled the transcriptomes of primary mouse cortical astrocytes cultured alone or co-cultured with immortalized precursor oligodendrocytes (Oli-neu cells). Filters between the cell types prevented formation of hetero-cellular gap junction channels but allowed for free exchange of the two culture media. We previously reported that major functional pathways in the Oli-neu cells are remodeled by the proximity of non-touching astrocytes and that astrocytes and oligodendrocytes form a panglial transcriptomic syncytium in the brain. Here, we present evidence that the astrocyte transcriptome likewise changes significantly in the proximity of non-touching Oli-neu cells. Our results indicate that the cellular environment strongly modulates the transcriptome of each cell type and that integration in a heterocellular tissue changes not only the expression profile but also the expression control and networking of the genes in each cell phenotype. The significant decrease of the overall transcription control suggests that in the co-culture astrocytes are closer to their normal conditions from the brain. The Oli-neu secretome regulates astrocyte genes known to modulate neuronal synaptic transmission and remodels calcium, chemokine, NOD-like receptor, PI3K-Akt, and thyroid hormone signaling, as well as actin-cytoskeleton, autophagy, cell cycle, and circadian rhythm pathways. Moreover, the co-culture significantly changes the gene hierarchy in the astrocytes.
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Bhan, Arunoday Kuldeep, Khairul I. Ansari, Clara Chen, and Rahul Jandial. "Abstract P1-21-05: GM-CSF is an autocrine driver of HER2+ breast leptomeningeal carcinomatosis." Cancer Research 82, no. 4_Supplement (February 15, 2022): P1–21–05—P1–21–05. http://dx.doi.org/10.1158/1538-7445.sabcs21-p1-21-05.
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Abstract Leptomeningeal carcinomatosis (LC) occurs when tumor cells spread to the cerebrospinal fluid containing leptomeninges surrounding the brain and spinal cord. LC is an ominous complication of cancer with a dire prognosis. Although any malignancy can spread to the leptomeninges, breast cancer, particularly the HER2+ subtype, is its most common origin. HER2+ breast LC (HER2+ LC) remains incurable, with few treatment options, and the molecular mechanisms underlying proliferation of HER2+ breast cancer cells in the acellular, protein, and cytokine-poor leptomeningeal environment remain elusive. Therefore, we sought to characterize signaling pathways that drive HER2+ LC development as well as those that restrict its growth to leptomeninges. Primary HER2+ LC patient-derived ("Lepto") cell lines in co-culture with various central nervous system (CNS) cell types revealed that oligodendrocyte progenitor cells (OPC), the largest population of dividing cells in the CNS, inhibited HER2+ LC growth in vitro and in vivo, thereby limiting the spread of HER2+ LC beyond the leptomeninges. Cytokine array-based analyses identified Lepto cell-secreted granulocyte-macrophage colony-stimulating factor (GM-CSF) as an oncogenic autocrine driver of HER2+ LC growth. Liquid chromatography-tandem mass spectrometry-based analyses revealed that the OPC-derived protein TPP1 proteolytically degrades GM-CSF, decreasing GM-CSF signaling and leading to suppression of HER2+ LC growth and limiting its spread. Lastly, intrathecal delivery of neutralizing anti-GM-CSF antibodies and a pan-Aurora kinase inhibitor (CCT137690) synergistically inhibited GM-CSF and suppressed activity of GM-CSF effectors, reducing HER2+ LC growth in vivo. Thus, OPC suppress GM-CSF-driven growth of HER2+ LC in the leptomeningeal environment, providing a potential targetable axis. Citation Format: Arunoday Kuldeep Bhan, Khairul I Ansari, Clara Chen, Rahul Jandial. GM-CSF is an autocrine driver of HER2+ breast leptomeningeal carcinomatosis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-21-05.