Letteratura scientifica selezionata sul tema "Primary oligodendrocyte culture"

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.

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.

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.

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.

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.

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.

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.

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.

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.

Dans le système nerveux central (SNC), les oligodendrocytes (OL) enveloppent les axones de leurs prolongements membranaires formant ainsi les gaines de myéline. La mort des OL et la perte de myéline (démyélinisation) surviennent dans les maladies démyélinisantes telles que la sclérose en plaques, pour lesquelles il n'existe pas de traitement efficace. Notre objectif est de stimuler des processus de réparation endogène via la voie ADAM10/sAPPa. Le fragment soluble sAPPa est un peptide neuroprotecteur issu du clivage de la protéine précurseur de l'amyloïde (APP) par les a-secrétases de la famille ADAM (A Desintegrin And Metalloprotease). Nous étudions plus particulièrement ADAM10 car il s'agit de l'a-sécrétase principale dans le SNC. Nos résultats antérieurs montrent que l'activation pharmacologique des a-sécrétases stimule la différenciation des OL in vitro, la protection de la myéline contre la démyélinisation et la remyélinisation ex vivo et in vivo et améliore la fonction locomotrice. L'objectif de ma thèse était donc d'approfondir le rôle d'ADAM10 dans les OL, dans la formation et la maintenance de la myéline. Trois objectifs ont été menés. Le premier objectif était d'étudier l'expression régionale et cellulaire d'ADAM10 dans le SNC par immunomarquage de la protéine dans le SNC de souris adultes et dans des cultures neuronales et gliales primaires. Nous avons observé une large expression de l'enzyme dans le cerveau, le cervelet et la moelle épinière, plus forte dans l'hippocampe et le cortex piriforme. Bien que l'expression d'ADAM10 soit majoritairement neuronale dans les tissus, nous avons pu détecter ADAM10 in vitro dans les OL, les astrocytes et la microglie. Le second objectif était d'étudier le rôle de l'ADAM10 oligodendrocytaire dans la myélinisation. Nous avons donc généré une nouvelle lignée de souris (KOOLA10) permettant la délétion d'ADAM10 dans les OL à des moments spécifiques du développement des OL et de la myéline. Dans cette lignée, l'exon 3 du gène Adam10 flanqué de deux séquences loxP est excisé lors de l'induction par le tamoxifène de la recombinase Cre, exprimée sous le contrôle du promoteur PLP (Proteolipid Protein). Lorsque la déficience est induite à la naissance pendant l'oligodendrogenèse, des défauts d'exploration sont observés à P21, compensés par la suite. Lorsque la déficience est induite à l'âge adulte lors de la maintenance de la myéline, ces défauts s'aggravent avec le temps. Des analyses supplémentaires sont nécessaires pour expliquer ces déficits comportementaux. De manière inattendue, les niveaux de protéine MBP (Myelin Basic Protein) ne montrent pas de changement apparent chez les KO. Le troisième objectif était d'étudier le rôle d'ADAM10 dans le développement et la fonctionnalité des OL. Les résultats de l'invalidation d'ADAM10 à l'aide de siRNA dans la lignée d'OL 158N montrent une diminution de l'expression des gènes de la myéline sans différences dans la morphologie, la prolifération ou la migration des OL. Pour valider ces résultats, nous avons mis en place un protocole d'isolement et de culture primaires d'OL. Les données préliminaires indiquent une diminution de l'expression des gènes de la myéline dans les OL issus de souris KO. Enfin, des cultures organotypiques de cervelet de souris KO montrent une diminution significative du nombre d'axones myélinisés après une démyélinisation induite par lysolécithine, suggérant un effet protecteur d'OL ADAM10 dans la protection ou la réparation de la myéline. En conclusion, j'ai établi la cartographie d'ADAM10 dans le SNC, j'ai généré la lignée de souris KOOLA10 et mis en place différents outils permettant d'étudier le rôle de l'ADAM10 oligodendrocytaire dans la myélinisation. Mes données indiquent un rôle important de l'ADAM10 dans les OL, révélant même des conséquences comportementales. Des études complémentaires sont nécessaires pour mieux comprendre le rôle de cette enzyme dans la maintenance de la myéline et la re/myélinisation du SNC
In the central nervous system (CNS), oligodendrocytes (OL) envelop the axons with their membrane extensions, forming the myelin sheath. The OL death and the loss of myelin (demyelination) occur in demyelinating diseases such as multiple sclerosis, for which there is no specific cure nowadays. Our goal is to enhance an endogenous repair process via the ADAM10/sAPPa pathway. The Amyloid Precursor Protein (APP) can be cleaved by a-secretases, members of the ADAM (A Desintegrin And Metalloprotease) family such as ADAM10, the main a-secretase in the CNS. The enzymatic cleavage of APP generates a neuroprotective soluble peptide called sAPPa. Our previous results showed that the pharmacological activation of a-secretases was able to enhance OL differentiation in vitro, to promote myelin protection from demyelination, to enhance remyelination ex vivo and in vivo and to improve the locomotor function. The aim of my thesis was, thus, to further investigate the role of oligodendroglial ADAM10 in myelin formation and maintenance. Three lines of investigation have been pursued. The first aim was to investigate the regional and cellular expression of ADAM10 in the CNS by immunolabeling of ADAM10 protein in adult mice and in primary neuronal and glial cultures. ADAM10 was widely expressed in brain, cerebellum and spinal cord with high expression in the hippocampus and piriform cortex. Neurons expressed much more ADAM10 than glial cells in CNS tissues and in vitro we were able to detect ADAM10 in neurons, OL, astrocytes and microglia. The second aim was to investigate the role of oligodendroglial ADAM10 in myelination. Therefore, we have created a novel mouse strain (KOOLA10) that allows the deletion of OL ADAM10 at specific time points related to the process of oligodendrogenesis and myelination. In this mouse strain, the deficiency is induced by the excision of the exon 3 of Adam10 gene flanked by 2 loxP sequences by the Cre recombinase, which is under the control of the PLP (Proteolipid Protein) promoter. When ADAM10 deficiency was induced at birth during oligodendrogenesis, an impairment in exploratory activity was observed at P21 but it was compensated later on. When ADAM10 deficiency was induced during myelin maintenance in adult mice, the aforementioned behavior worsened over time. Further analysis is still required to explain the behavioral changes observed in KO mice. Surprisingly, the level of MBP (Myelin Basic Protein), assessed by western blot and immunohistological studies, did not show an apparent change in KO mice. The third aim was to investigate the role of ADAM10 in OL development and functionality. The ADAM10 knock-down using siRNA in the 158N OL cell line did not modify cell morphology, proliferation or migration but it induced a decrease in myelin gene expression. To validate these results, we set up a new OL primary cell isolation and culture protocol. Preliminary results also pointed to a reduction of myelin genes expression in ADAM10-deficient OL. Finally, we used organotypic culture of cerebellum, highly rich in myelin, to address the effect of ADAM10 deficiency. We set up a transfection protocol to knock down ADAM10 in cerebellar slices and further focused on the study of myelination in KOOLA10. A significant decrease in the number of myelinated axons was observed in cerebellar slices from KO mice after demyelination, suggesting a beneficial effect of OL ADAM10 in myelin protection or repair. In conclusion, I have shown the distribution of ADAM10 in the CNS, generated the KOOLA10 mouse strain and set up different protocols and tools that allow the investigation of the role of oligodendroglial ADAM10 in myelination. I have obtained evidence suggesting that OL ADAM10 affects exploratory behavior and myelin and is necessary for myelin protection and/or repair. Further investigation is required to better decipher the role of OL ADAM10 in myelin maintenance, and CNS re/myelination

Le ph intracellulaire (ph#i) et les mecanismes responsables de sa regulation ont ete etudies par microfluorimetrie de la sonde bcecf au cours des stades successifs de la differenciation en culture primaire des oligodendrocytes du cervelet de rat. Nous avons montre qu'une augmentation de ph#i d'environ 0,15 unite intervient au debut de la differenciation des cellules progenitrices 0-2a (a2b5#+, gd3#+, 04#-) vers la voie oligodendrocytaire. Nos resultats permettent en particulier d'etablir que trois mecanismes distincts assurent la regulation de ph#i des cellules o-2a : un echangeur ci#-/hco#3#-, un echangeur na#+/h#+ et un co-transporteur na#+-hco#3#-. Ces deux derniers mecanismes sont retrouves aux stades ulterieurs de la differenciation : pre-oligodendrocytes (o4#+, galc#-) et oligodendrocytes matures (galc#+). Par contre, ces deux stades n'expriment plus aucune activite d'echange ci#-/hco#3#-. La presence de cet echangeur uniquement dans les cellules o-2a a pour consequence d'y maintenir une valeur moyenne de ph#i de 6,9, significativement plus basse que la valeur de 7,1 mesuree dans les memes conditions experimentales dans les cellules plus matures. Nous avons pu preciser que les cellules progenitrices perdent cet echangeur ct/hco#3#- a un stade de differenciation tres voisin de celui ou elles cessent d'exprimer les marqueurs a2b5 et gd3. Ce stade correspond, par ailleurs, a celui ou les cellules cessent d'incorporer la bromo-deoxy-uridine et cessent donc de se diviser. Le role que peuvent jouer la perte d'expression de l'echangeur ci#-/hco#3#- et l'alcalinisation qui en resulte dans l'arret de la division et dans la stimulation de la differenciation des oligodendrocytes reste a identifier.