RESUMO
Mechanisms that regulate oligodendroglial cell (OLGc) differentiation are the focus of intensive research in the field of cellular and molecular neurobiology. We have previously shown that the addition of apotransferrin (aTf) to primary OLGc cultures accelerates their differentiation and induces an increase in the expression of different components of the myelin cytoskeleton (CSK) such as actin, tubulin, and some of the microtubule-associated proteins, particularly the stable tubulin only peptide (STOP). Fyn protein-tyrosine kinase (Fyn kinase), a member of the Src family, participates in signalling pathways that regulate OLGs/myelin cytoskeletal reorganization. It is essential for myelin development in the central nervous system (CNS), and its absence results in hypomyelination. In the present study, we used both primary cell and N19 cell line cultures to investigate further the mechanisms of action involved in the accelerated differentiation of OLGcs induced by aTf. In particular, we were interested in studying the participation of Fyn kinase in the different pathways involved in the reorganization of the OLGc/myelin cytoskeleton. In agreement with results already published, we found that in OLGcs, Fyn kinase is associated with Tau and tubulin. Using a dominant-negative of Tau in which the Fyn-Tau-microtubules (MTs) interaction is blocked, we found that aTf was unable to induce OLGc morphological differentiation. It was also observed that aTf decreases the activated RhoA content in coincidence with a redistribution of actin immunoreactivity. These results give support to our hypothesis that Fyn kinase plays a key role in the differentiation process of OLGcs promoted by aTf.
Assuntos
Apoproteínas/farmacologia , Citoesqueleto/metabolismo , Oligodendroglia/enzimologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Células-Tronco/enzimologia , Transferrina/farmacologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Apoproteínas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/enzimologia , Bainha de Mielina/ultraestrutura , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Wistar , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Transferrina/metabolismo , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas tau/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
Peroxisome proliferator activated receptors (PPARs, alpha, beta/delta, gamma) control lipid homeostasis and differentiation in various tissues and tumor cells. PPARbeta and PPARgamma increase oligodendrocyte maturation in glial mixed populations and spinal cord oligodendrocytes, respectively, and PPARbeta is known to modulate the activity of other PPARs. To assess a possible interaction between PPARs in glial cell differentiation we used the undifferentiated C6 glioma cell line as model. These cells express all three PPARs, but only PPARgamma shows transcriptional activity in agonist-based reporter gene assay. Agonist-activated PPARgamma up-regulates oligodendrocyte markers, down-regulates an astrocyte marker, and increases alkyl-dihydroxyacetone phosphate synthase, enzyme involved in the synthesis of myelin-rich plasmalogens. Similar effects are induced in PPARgamma overexpressing cells, which in addition show PPARbeta up-regulation. PPARbeta or PPARalpha agonists show no effect. Nevertheless, PPARbeta overexpression up-regulates PPARgamma and commits C6 cells to oligodendrocytes; effect that is abrogated by a PPARgamma antagonist or PPARgamma interference RNA. Moreover, PPARbeta overexpression also induces PPARalpha and its target genes, including acyl-CoA oxidase, enzyme involved in very long chain fatty acid recycling, and in the synthesis of myelin components such as docosahexaenoic acid. These results indicate for the first time, that PPARs concertedly cooperate in C6 glioma cell differentiation to oligodendrocytes. Further, they suggest that active PPARbeta might be essential for increasing oligodendrocyte distinctive markers and enzymes required for myelin synthesis in C6 glioma cells through up-regulation of PPARgamma and PPARalpha.
Assuntos
Diferenciação Celular , Linhagem da Célula , Glioma/metabolismo , Metabolismo dos Lipídeos , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Receptor Cross-Talk , Alquil e Aril Transferases/biossíntese , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Indução Enzimática , Glioma/enzimologia , Glioma/patologia , Metabolismo dos Lipídeos/genética , Camundongos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/enzimologia , Oligodendroglia/patologia , PPAR alfa/metabolismo , PPAR gama/metabolismo , PPAR beta/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Receptores Ativados por Proliferador de Peroxissomo/genética , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Rosiglitazona , Tiazóis/farmacologia , Tiazolidinedionas/farmacologia , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
In the central nervous system, transferrin (Tf) is produced by oligodendroglial cells (OLGcs) and is essential for their development. Recently, using the complete cDNA of the human Tf gene, we obtained clones overexpressing Tf in two OLGc lines, N19 and N20.1, which represent different stages of differentiation. We showed that the overexpression of this glycoprotein promotes the maturation and myelinogenic capacity of both cell lines. In this work, using cDNA array technology, we examined changes induced by Tf in 1,176 genes. We found 41 genes differentially expressed in both cell lines, all of them involved in OLGc development. In the less mature cells (N19) overexpressing Tf, there was a significant increase in key enzymes of neurosteroid metabolism, such as cholesterol side chain cleavage cytochrome P450, 3beta-hydroxysteroid dehydrogenase and 5alpha-reductase type 1. In the more mature cell line (N20.1), Tf overexpression produced an induction of several mRNAs of the GABA(A) receptor subunits, of thyroid hormone receptors and of proteins involved in axon-glia interactions such as F3/contactin. In addition, in both cell lines, Tf overexpression induced an increase in the expression of different isoforms of transforming growth factor beta receptors and in several genes related to mitochondrial function and to complex lipid metabolism, crucial steps in myelin synthesis. Differentiation produced by Tf in both cell lines seems to occur by modulation of different genes depending on the maturational stage of the cells. Our findings provide new insights into the molecular basis of OLGc differentiation and on the role played by Tf in this process.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oligodendroglia/metabolismo , Transferrina/fisiologia , 3-Hidroxiesteroide Desidrogenases/genética , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Senescência Celular/genética , Colestenona 5 alfa-Redutase/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Humanos , Isoenzimas/genética , Metabolismo dos Lipídeos , Proteínas Mitocondriais/genética , Bainha de Mielina/fisiologia , Oligodendroglia/citologia , Oligodendroglia/enzimologia , Oligodendroglia/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Esteroides/metabolismo , Transferrina/genéticaRESUMO
The olfactory bulb (OB) presents a unique pattern of permanent acquisition of primary afferents and interneurons, but not much detail is known about the differentiation of its oligodendroglial cells. We studied the expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a protein related to axonal ensheathment by myelinating cells. Expression of CNPase in OB follows a general caudorostral gradient, with the exception of the glomerular layer (GL). At postnatal day 5-6 (P5-P6), the first CNPase(+) profiles appeared in the dorsal lateral olfactory tract adjacent to the accessory OB (AOB), followed by rare cell bodies and processes in AOB internal plexiform layer at P7. At P9, the main OB (MOB) granular cell layer (GrCL) already showed intensely stained CNPase(+) processes. From P5 to P12, small numbers of CNPase(+) cells were found in the subventricular zone (SVZ), throughout its rostral extension (SVZ-RE), and in the intrabulbar subependymal layer. The appearance of CNPase(+) profiles delimiting glomeruli started in the GL rostralmost region at P12, extending to all GL levels, but glomeruli remained open caudally at P15. At P18, oligodendroglial glomeruli were evident throughout OB, but the adult pattern was established only after P30. There was no age-related loss of CNPase immunoreactivity in glial cell bodies, possibly indicating de novo ensheathment of neurites. Our results show an earlier onset of oligodendroglial differentiation in OB than previously reported and a rostrocaudal gradient of formation of oligodendroglial glomeruli. They also raise the possibility that a minor fraction of OB oligodendrocytes might derive from the SVZ-RE.
Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Bulbo Olfatório/enzimologia , Mucosa Olfatória/enzimologia , Oligodendroglia/enzimologia , Envelhecimento , Animais , Animais Recém-Nascidos , Feminino , Imuno-Histoquímica , Masculino , Neurônios/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/crescimento & desenvolvimento , Bulbo Olfatório/metabolismo , Mucosa Olfatória/crescimento & desenvolvimento , Mucosa Olfatória/metabolismo , Nervo Olfatório/metabolismo , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
We have used lactacystin, a specific inhibitor of the 26S proteasome, in oligodendroglial cell (OLGc) primary cultures to explore the possible participation of the proteasome-ubiquitin-dependent pathway in the decision of the OLGcs to arrest their proliferation and start differentiation. Addition of lactacystin at various concentrations to cultures containing a majority of OLGc was found to produce their withdrawal from the cell cycle and to induce their biochemical and morphological differentiation, with the appearance of extensive myelin-like sheets. The three classic proteolytic activities of the proteasome were significantly decreased in the lactacystin-treated cultures, and the immunocytochemical analysis showed an increase in the number of O4-, O1-, myelin basic protein-, and myelin proteolipid protein-positive cells and a decrease in A2B5-reacting cells. Quantitative immunochemical evaluation of the expression of certain proteins controlling the cell cycle showed an increase in p27kip1-, cyclin D-, and cdk4-positive cells, with a decrease in cyclin E- and cdk2-positive cells. In the lactacystin-treated OLGcs, there was a dose-dependent decrease in the number of cells incorporating bromodeoxyuridine and in the activity of the complexes cyclin D-cdk4 and cyclin E-cdk2. Furthermore, increased levels of expression of several STAT factors were found, suggesting that proteasome inhibition in OLGcs could stabilize signals of survival and differentiation that might be processed through the JAK/STAT signaling cascade.
Assuntos
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/enzimologia , Peptídeo Hidrolases/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma , Animais , Bromodesoxiuridina , Proteínas de Ciclo Celular/biossíntese , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quinases Ciclina-Dependentes/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Leupeptinas/farmacologia , Substâncias Macromoleculares , Oligodendroglia/citologia , Peptídeo Hidrolases/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Mature oligodendrocytes emit numerous myelinating processes. Force generating molecules are required for process outgrowth and spreading. We have analyzed the effect of the myosin II light chain kinase inhibitors ML-7 and ML-9 in cultured oligodendrocytes. Both drugs affect oligodendrocyte cell shape, provoking a retraction of high order processes. Our results suggest that the adhesion of the myelinating processes to the substrate depends on MLC phosphorylation, thus likely implicating myosin IIA.
Assuntos
Azepinas/farmacologia , Inibidores Enzimáticos/farmacologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Naftalenos/farmacologia , Oligodendroglia/efeitos dos fármacos , Animais , Quinase de Cadeia Leve de Miosina/metabolismo , Oligodendroglia/citologia , Oligodendroglia/enzimologia , Fosforilação , Ratos , Ratos WistarRESUMO
The effects of hypothyroidism on oligodendroglial differentiation and myelination are for the first time studied by immunohistochemical localization of an early oligodendroglial marker, the 2'3'cyclic nucleotide 3'phosphodiesterase (E.C. 3.1.4.37-CNPase), in developing rats. Two groups received methimazol; one during gestation (H) and another postnatally (PN). One H sub-group received thyroxine after birth (T). We observed a delay in CNPase expression followed by a decrease in the number of CNPase immunoreactive fibers in both H and PN groups. The T sub-group was not different from controls. Furthermore, the immunoreactive fibers, in mature hypothyroid animals, showed a continuous pattern of staining in contrast with a discontinuous one in controls. Myelinogenesis is a highly regulated timed event. CNPase links myelin related proteins to the cytoskeleton also interacting with membrane lipids during extension and wrapping of the oligodendroglial process around the axon (ensheathment phase). In mature myelinated fiber the CNPase is absent from compact myelin sheath, being located only in the inner and outer loops and in paranodal loops. Thus, our data suggest a disorder in myelin compaction and point once more to the post-natal period as critical for the mechanisms that are thyroid hormone regulated in myelinogenesis.
Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/análise , Encéfalo/anormalidades , Encéfalo/enzimologia , Hipotireoidismo Congênito , Hipotireoidismo/metabolismo , Bainha de Mielina/enzimologia , Animais , Antitireóideos , Encéfalo/patologia , Feminino , Hipotireoidismo/induzido quimicamente , Hipotireoidismo/patologia , Imuno-Histoquímica , Metimazol , Bainha de Mielina/patologia , Oligodendroglia/enzimologia , Oligodendroglia/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Ratos Wistar , Tiroxina/farmacologiaRESUMO
The differentiation of oligodendrocytes in the forebrain of the opossum (Didelphis marsupialis) has been studied by the immunohistochemical identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and by the autoradiographic detection of the uptake of 3H-thymidine. CNPase is expressed early in oligodendroglia somata and fibre sheaths (myelin) in the forebrain and its persistence in the cell bodies is regionally heterogeneous, being ephemeral in cells within the optic pathway, supraoptic decussation, and posterior commissure, of intermediate duration in the mamillo-thalamic fascicle, and stria medullaris, and long-lasting in other diencephalic and in telencephalic tracts. In the cerebral cortex, most CNPase+ cells have small somata and multiple processes (types I and II). CNPase-expressing oligodendrocytes are also regionally heterogeneous in terms of proliferative capability, which could not be detected in forebrain tracts or diencephalon, but has appeared in a small proportion of cells in the neocortical white matter and in the fimbria. Our findings provide additional evidence in favour of the heterogeneity of oligodendrocytes.
Assuntos
Diferenciação Celular , Oligodendroglia/citologia , Gambás/crescimento & desenvolvimento , Prosencéfalo/crescimento & desenvolvimento , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/análise , Envelhecimento , Animais , Autorradiografia , DNA/biossíntese , Diencéfalo/citologia , Diencéfalo/enzimologia , Imuno-Histoquímica , Oligodendroglia/enzimologia , Gambás/anatomia & histologia , Prosencéfalo/anatomia & histologia , Telencéfalo/citologia , Telencéfalo/enzimologiaRESUMO
Immunoreactivity to 2',3' cyclic nucleotide 3' phosphohydrolase (CNPase; EC 3.1.4.37) was studied in the developing opossum brain stem and cerebellum. Regional differences were found in oligodendrocytes concerning the time of appearance (early: medical longitudinal fascicle [mlf]; intermediate: inferior colliculus [IC], deep layers of the superior colliculus [SC] and white matter of cerebellar folia; late: optic layer of SC) and duration of immunoreactivity (short: optic layer of SC; intermediate: mlf; long: cerebellar folia, etc). The results suggest that regional heterogeneities in CNPase expression are linked to intrinsic properties of local and afferent axons.