Literatura académica sobre el tema "158N oligodendroglial cell line"

Recent discoveries suggest links between abnormalities in cell morphogenesis in the brain and the functional deficiency of molecules controlling signal transduction in glial cells such as oligodendroglia. Rnd2 is one such molecule and one of the Rho family monomeric GTP-binding proteins. Despite the currently known functions of Rnd2, its precise roles as it relates to cell morphogenesis and disease state remain to be elucidated. First, we showed that signaling through the loss of function of the rnd2 gene affected the regulation of oligodendroglial cell-like morphological differentiation using the FBD-102b cell line, which is often utilized as a differentiation model. The knockdown of Rnd2 using the clustered regularly interspaced palindromic repeats (CRISPR)/CasRx system or RNA interference was shown to slow morphological differentiation. Second, the knockdown of Prag1 or Fyn kinase, a signaling molecule acting downstream of Rnd2, slowed differentiation. Rnd2 or Prag1 knockdown also decreased Fyn phosphorylation, which is critical for its activation and for oligodendroglial cell differentiation and myelination. Of note, hesperetin, a citrus flavonoid with protective effects on oligodendroglial cells and neurons, can recover differentiation states induced by the knockdown of Rnd2/Prag1/Fyn. Here, we showed that signaling through Rnd2/Prag1/Fyn is involved in the regulation of oligodendroglial cell-like morphological differentiation. The effects of knocking down the signaling cascade molecule can be recovered by hesperetin, highlighting an important molecular structure involved in morphological differentiation.

POLR3B and POLR3A are the major subunits of RNA polymerase III, which synthesizes non-coding RNAs such as tRNAs and rRNAs. Nucleotide mutations of the RNA polymerase 3 subunit b (polr3b) gene are responsible for hypomyelinating leukodystrophy 8 (HLD8), which is an autosomal recessive oligodendroglial cell disease. Despite the important association between POLR3B mutation and HLD8, it remains unclear how mutated POLR3B proteins cause oligodendroglial cell abnormalities. Herein, we show that a severe HLD8-associated nonsense mutation (Arg550-to-Ter (R550X)) primarily localizes POLR3B proteins as protein aggregates into lysosomes in the FBD-102b cell line as an oligodendroglial precursor cell model. Conversely, wild type POLR3B proteins were not localized in lysosomes. Additionally, the expression of proteins with the R550X mutation in cells decreased lysosome-related signaling through the mechanistic target of rapamycin (mTOR). Cells harboring the mutant constructs did not exhibit oligodendroglial cell differentiated phenotypes, which have widespread membranes that extend from their cell body. However, cells harboring the wild type constructs exhibited differentiated phenotypes. Ibuprofen, which is a non-steroidal anti-inflammatory drug (NSAID), improved the defects in their differentiation phenotypes and signaling through mTOR. These results indicate that the HLD8-associated POLR3B proteins with the R550X mutation are localized in lysosomes, decrease mTOR signaling, and inhibit oligodendroglial cell morphological differentiation, and ibuprofen improves these cellular pathological effects. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD8 and their amelioration.

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

Hypomyelinating leukodystrophy 10 (HLD10) is an autosomal recessive disease related to myelin sheaths in the central nervous system (CNS). In the CNS, myelin sheaths are derived from differentiated plasma membranes of oligodendrocytes (oligodendroglial cells) and surround neuronal axons to achieve neuronal functions. Nucleotide mutations of the pyrroline-5-carboxylate reductase 2 (PYCR2) gene are associated with HLD10, likely due to PYCR2’s loss-of-function. PYCR2 is a mitochondrial residential protein and catalyzes pyrroline-5-carboxylate to an amino acid proline. Here, we describe how each of the HLD10-associated missense mutations, Arg119-to-Cys [R119C] and Arg251-to-Cys [R251C], lead to forming large size mitochondria in the FBD-102b cell line, which is used as an oligodendroglial cell differentiation model. In contrast, the wild type proteins did not participate in the formation of large size mitochondria. Expression of each of the mutated R119C and R251C proteins in cells increased the fusion abilities in mitochondria and decreased their fission abilities relatively. The respective mutant proteins, but not wild type proteins also decreased the activities of mitochondria. While cells expressing the wild type proteins exhibited differentiated phenotypes with widespread membranes and increased expression levels of differentiation marker proteins following the induction of differentiation, cells harboring each of the mutant proteins did not. Taken together, these results indicate that an HLD10-associated PYCR2 mutation leads to the formation of large mitochondria with decreased activities, inhibiting oligodendroglial cell morphological differentiation. These results may reveal some of the pathological mechanisms in oligodendroglial cells underlying HLD10 at the molecular and cellular levels.

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