RESUMO
Oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Identifying the signaling pathways involved in myelin repair through oligodendroglial progenitor maturation is essential for the development of new therapies. This article investigated the role of the Notch signaling pathway in CNS demyelination and apotransferrin-induced remyelination in a focal lysolecithin-induced demyelination model in rats. Notch was found activated in Nestin-expressing neural progenitor cells and in NG2-expressing oligodendroglial precursor cells in the subventricular zone and corpus callosum of lysolecithin-demyelinated rats. Notch activation seemed to be driven by Jagged1, which led to a high expression of downstream gene Hes5 in the subventricular zone of demyelinated rats. Apotransferrin injection induced remyelination, while the injection of the γ-secretase inhibitor reversed this effect. In addition, 24 h after apotransferrin injection, evidence showed Notch activation concomitantly with an increase in F3/contactin levels and the up-regulation of the myelin-associated glycoprotein gene in the subventricular zone and corpus callosum of demyelinated rats. Collected evidence supports the participation of both canonical and non-canonical Notch signaling pathways in demyelination/remyelination. Notch activation was found to trigger Hes5 expression as a consequence of focal demyelination, which might promote oligodendroglial precursor cell proliferation. During apotransferrin-induced remyelination, Notch activation seemed to be mediated by the expression of F3/contactin, which might induce apotransferrin-mediated oligodendroglial maturation. Evidence of the participation of Notch signaling in the demyelination/remyelination process will help further understand demyelinating disorders such as Multiple Sclerosis and the use of aTf should be taken into consideration as a possible therapeutic intervention.
Assuntos
Apoproteínas/farmacologia , Encéfalo/metabolismo , Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , Receptores Notch/metabolismo , Transferrina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Feminino , Lisofosfatidilcolinas , Masculino , Células-Tronco Neurais/patologia , Oligodendroglia/patologia , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Neural stem and progenitor cells (NSC/NPCs) are multipotent self-renewing cells that are able to generate neurons, astrocytes and oligodendrocytes (OLs) within the adult central nervous system. We cultured NSC/NPCs from the rat subventricular zone as neurospheres (NS) and studied apoTransferrin (aTf) effects on oligodendroglial specification and maturation. Our findings suggest that aTf acts at different stages during progression from NSC to mature oligodendrocytes. On the one hand, an early event associated with the activation of NSC/NPCs proliferation and commitment toward the oligodendroglial fate, as indicated by increased BrdU incorporation, larger neurospheres production, and higher ability to generate OL precursors (OPCs) from undifferentiated cultures. On the other hand, aTf exposure during differentiating conditions favours OL maturation from OPCs by promoting OL morphological development. This evidence supports a key role of Tf on the generation of OL from NSC/NPCs and highlights its potential in demyelinating disorder treatment.
Assuntos
Apoproteínas/metabolismo , Plexo Corióideo/metabolismo , Células-Tronco Neurais/metabolismo , Transferrina/metabolismo , Animais , Antígenos/metabolismo , Apoproteínas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Plexo Corióideo/citologia , Feminino , Masculino , Modelos Biológicos , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Cultura Primária de Células , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismo , Transferrina/farmacologiaRESUMO
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
We have used a model of iron deficiency in the rat to analyze the effects of a disruption in iron availability on oligodendroglial cell (OLGc) maturation and myelinogenesis and to explore the possible beneficial influence of an intracranial injection (ICI) of apotransferrin (aTf) at 3 days of age on this process. Studies carried out on postnatal days 17 and 24 showed that iron deficiency produced a decrease in myelin proteins and lipids at 24 days of age. Immunohistochemistry showed that in untreated iron-deficient (ID) rats, the immunoreactivity of anti-adenomatous polyposis coli (APC) and anti-MBP antibodies decreased markedly with reference to normal controls, whereas in ID rats treated with an ICI of aTf, the immunoreactivity of these markers increased. A similar situation occurred with the immunoreactivity of H-ferritin. In primary OLGc cultures from ID rats, there was a high number of cells positive to the antibody against the polysialylated form of the cell surface glycoprotein NCAM (PSA-NCAM) compared with in OLGc cultures prepared from normal controls or from ID animals treated with aTf. The number of MBP+ cells in cultures from ID rats increased after treatment with aTf. The presence of lipid rafts evaluated with a specific anti-protein prion cellular (PrPc) antibody showed a smaller number of PrPc-positive structures in ID rat cultures. Treatment of the ID animals with a single ICI of aTf stimulated myelination, producing a significant correction in the different biochemical parameters affected by ID.
Assuntos
Anemia Ferropriva/tratamento farmacológico , Anemia Ferropriva/patologia , Apoproteínas/uso terapêutico , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/patologia , Fibras Nervosas Mielinizadas/patologia , Transferrina/uso terapêutico , Anemia Ferropriva/sangue , Animais , Animais Recém-Nascidos , Apoproteínas/farmacologia , Células Cultivadas , Doenças Desmielinizantes/sangue , Modelos Animais de Doenças , Feminino , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Transferrina/farmacologiaRESUMO
In this work, we have immunohistochemically analyzed the effects of single injections of apotransferrin (aTf) on the expression of myelin (myelin basic proteins [MBPs]) and axonal (protein gene product 9.5 [PGP 9.5] and beta(III)-tubulin [beta(III)-tub]) proteins in colchicine-injected and crushed sciatic nerves of adult rats. A protein redistribution was seen in the distal stump of injured nerves, with the appearance of MBP- and PGP 9.5-immunoreactive (IR) clusters which occurred earlier in crushed nerves (3 days post-injury [PI]) as compared to colchicine-injected nerves (7 days PI). beta(III)-tub-IR clusters appeared at 1 day PI preceding the PGP 9.5- and MBP-IR clusters in colchicine-injected nerves. With image analysis, the peak of clustering formation was found at 14 days PI for MBP and at 3 days PI for beta(III)-tub in colchicine-injected nerves. At 28 days of survival, the protein distribution patterns were almost normal. The intraneural application of aTf, at different concentrations (0.0005 mg/ml, 0.005 mg/ml, 0.05 mg/ml, 0.5 mg/ml), prevented nerve degeneration produced by colchicine, with the appearance of only a small number of MBP- and beta(III)-tub-IR clusters. However, aTf was not able to prevent clustering formation when the nerve was crushed, a kind of injury that also involves necrosis and blood flow alterations. The results suggest that aTf could prevent the colchicine effects by stabilizing the cytoskeleton proteins of the nerve fibers, avoiding the disruption of the axonal transport and thus the myelin degeneration. Transferrin is proposed as a complementary therapeutic avenue for treatment of cytotoxic nerve injuries.
Assuntos
Apoproteínas/farmacologia , Axônios/efeitos dos fármacos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Neuropatia Ciática/tratamento farmacológico , Transferrina/farmacologia , Degeneração Walleriana/tratamento farmacológico , Degeneração Walleriana/prevenção & controle , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Axônios/metabolismo , Colchicina/antagonistas & inibidores , Colchicina/toxicidade , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/patologia , Proteína Básica da Mielina/efeitos dos fármacos , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/toxicidade , Ratos , Ratos Wistar , Neuropatia Ciática/metabolismo , Neuropatia Ciática/fisiopatologia , Resultado do Tratamento , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Ubiquitina Tiolesterase/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Degeneração Walleriana/fisiopatologiaRESUMO
In the CNS, transferrin (Tf) is expressed by the oligodendroglial cells (OLGcs) and is essential for their development. We have previously shown that apotransferrin (aTf) accelerates maturation of OLGcs in vivo as well as in vitro. The mechanisms involved in this action appear to be complex and have not been completely elucidated. The aim of this study was to investigate if Tf participates in the regulation of the cell cycle of oligodendroglial progenitor cells (OPcs). Primary cultures of OPcs were treated with aTf and/or with different combinations of mitogenic factors. Cell cycle progression was studied by BrdU incorporation, flow cytometry and by the expression of cell cycle regulatory proteins. Apotransferrin decreased the number of BrdU+ cells, increasing the cell cycle time and decreasing the number of cells in S phase. The cell cycle inhibitors p27kip1, p21cip1 and p53 were increased, and in agreement with these results, the activity of the complexes involved in G1-S progression (cyclin D/CDK4, cyclin E/CDK2), was dramatically decreased. Apotransferrin also inhibited the mitogenic effects of PDGF and PDGF/IGF on OPcs, but did not affect their proliferation rate in the presence of bFGF, bFGF/PDGF or bFGF/IGF. Our results indicate that inhibition of the progression of the cell cycle of OPcs by aTf, even in the presence of PDGF, leads to an early beginning of the differentiation program, evaluated by different maturation markers (O4, GC and MBP) and by morphological criteria. The modulation by aTf of the response of OPcs to PDGF supports the idea that this glycoprotein might act as a key regulator of the OLGc lineage progression.
Assuntos
Apoproteínas/farmacologia , Ciclo Celular/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/farmacologia , Células-Tronco/efeitos dos fármacos , Transferrina/farmacologia , Animais , Antimetabólitos , Western Blotting , Bromodesoxiuridina , Diferenciação Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , DNA/biossíntese , Depressão Química , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Fase G1 , Imuno-Histoquímica , Oligodendroglia/ultraestrutura , Ratos , Fase S , Células-Tronco/ultraestrutura , Sais de Tetrazólio , TiazóisRESUMO
Twenty-one-day-old Wistar rats were fed a diet containing 0.6% cuprizone for 2 weeks. Studies carried out after withdrawal of cuprizone showed histological evidences of marked demyelination in the corpus callosum. Biochemical studies of isolated myelin showed a marked decrease in myelin proteins, phospholipids, and galactocerebrosides as well as a marked decrease in myelin yield. Treatment of these animals with a single intracranial injection of 350 ng of apotransferrin at the time of withdrawal of cuprizone induced a marked increase in myelin deposition resulting in a significantly improved remyelination, evaluated by histological, immunocytochemical, and biochemical parameters, in comparison to what was observed in spontaneous recovery. Immunocytochemical studies of cryotome sections to analyze developmental parameters of the oligodendroglial cell population at the time of termination of cuprizone and at different times thereafter showed that in the untreated animals, there was a marked increase in the number of NG2-BrdU-positive precursor cells together with a marked decrease in MBP expression at the peak of cuprizone-induced demyelination. As expected, the amount of precursor cells decreased markedly during spontaneous remyelination and was accompanied by an increase in MBP reactivity. In the apotransferrin-treated animals, these phenomena occurred much faster, and remyelination was much more efficient than in the untreated controls. The results of this study suggest that apotransferrin is a very active promyelinating agent which could be important for the treatment of certain demyelinating conditions.
Assuntos
Apoproteínas/uso terapêutico , Cuprizona , Doenças Desmielinizantes/tratamento farmacológico , Recuperação de Função Fisiológica/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Transferrina/uso terapêutico , Análise de Variância , Animais , Animais Recém-Nascidos , Antígenos/metabolismo , Apoproteínas/farmacologia , Encéfalo/patologia , Bromodesoxiuridina/farmacocinética , Antígeno CD11b/metabolismo , Contagem de Células/métodos , Proteínas do Citoesqueleto/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/fisiopatologia , Interações Medicamentosas , Galactolipídeos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Indóis , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Regeneração/fisiologia , Fatores de Tempo , Transferrina/farmacologiaRESUMO
The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.
Assuntos
Apoproteínas/farmacologia , Desferroxamina/farmacologia , Monócitos/microbiologia , Paracoccidioides/efeitos dos fármacos , Sideróforos/farmacologia , Transferrina/farmacologia , Desferroxamina/antagonistas & inibidores , Humanos , Ferro/fisiologia , Paracoccidioides/fisiologia , Sideróforos/antagonistas & inibidoresRESUMO
Apotransferrin (aTf), has been shown to accelerate the differentiation of oligodendroglial cells (OLGcs) in primary cultures and to increase the expression of different components of the myelin cytoskeleton (CSK). We examined the incorporation and distribution of human aTf (aTfh) exogenously added to OLGcs cultures and its effects on the CSK of the OLGcs. When OLGcs treated with aTfh were extracted with a CSK-stabilizing buffer containing detergent, aTfh was found in the soluble fraction. In vitro experiments showed that purified tubulin was not altered by the addition of aTfh. In OLGc primary cultures treated with aTfh, this glycoprotein showed a punctate distribution pattern along the OLGc processes. Treatment of the cultures with colchicine, cytochalasin, or taxol induced a displacement of the immunoreactivity of aTfh toward the OLGc soma. Analysis of the effects of aTfh on the cell distribution of tyrosinated and detyrosinated tubulin and STOP (stable tubule only polypeptide), showed that aTfh added to OLGc cultures promoted changes suggesting a stabilizing effect on the microtubules (MT) at the tip of the processes. Kinesin and dynein were found to colocalize with the aTfh, indicating that these motors participate in the transport of the added glycoprotein. Moreover, after treatment with aTfh, clathrin immunoreactivity was displaced from the OLGc body toward the cell processes. These results indicate that although aTfh added to OLGcs does not interact directly with CSK components, it seems to be transported in clathrin coated vesicles from the cell body to the tips of the OLGc processes where it promotes their stabilization. This mechanism may be of importance in the increased formation of the myelin membrane induced by aTf.
Assuntos
Apoproteínas/farmacologia , Citoesqueleto/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Transferrina/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Células Cultivadas , Córtex Cerebral/citologia , Citocalasinas/farmacologia , Citoesqueleto/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica/métodos , Microscopia Confocal/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Receptores da Transferrina/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Os mecanismos utilizados pelo Paracoccidioides brasiliensis para sobreviver em células fagocitárias ainda não estão elucidados. O metabolismo celular férrico é muito importante para o crescimento de inúmeros patógenos intracelulares cuja capacidade de se multiplicarem em fagócitos mononucleares é dependente da disponibilidade intracelular do íon ferro. Assim, o objetivo deste trabalho foi investigar o papel do ferro intracelular sobre a capacidade do P. brasiliensis sobreviver em monócitos humanos. O tratamento de monócitos com deferoxamina, uma droga quelante, diminuiu a sobrevivência de leveduras do fungo de forma dose-dependente. O efeito inibidor da deferoxamina sobre a sobrevivência do P. brasiliensis foi revertido por transferrina saturada com ferro (holotransferrina) mas não por transferrina insaturada (apotransferrina). Estes resultados sugerem que a sobrevivência do P. brasiliensis em monócitos humanos é dependente do íon ferro.
Assuntos
Humanos , Apoproteínas/farmacologia , Desferroxamina/farmacologia , Monócitos/microbiologia , Paracoccidioides/efeitos dos fármacos , Sideróforos/farmacologia , Transferrina/farmacologia , Desferroxamina/antagonistas & inibidores , Ferro/fisiologia , Paracoccidioides/fisiologia , Sideróforos/antagonistas & inibidoresRESUMO
We have demonstrated previously that a single intracranial injection of apotransferrin (aTf) in neonatal rats increases myelination and accelerates differentiation of oligodendroglial cells (OLGc). In addition, we have shown through in vitro experiments that OLGc isolated from 4-day-old rats (OLGc-4) treated with aTf were more differentiated than were controls although aTf had no effect upon OLGc isolated from 10-day-old animals (OLGc-10). In the present work, we analyzed the role of second messengers in the effect of aTf upon the maturation of OLGc at different stages of development. We isolated OLGc-4 and OLGc-10 from rat brain using a Percoll density gradient and briefly treated the cells with a pulse of aTf or kept them in culture during 2 days in the presence or absence of aTf. In OLGc-4, after a short pulse of aTf, there was an increase in the levels of cyclic AMP (cAMP), in the phosphorylation of cAMP response element-binding protein (CREB) and in the DNA-binding capacity of cAMP-responsive transcription factors. Treatment of OLGc-4 with aTf diminished bromodeoxyuridine (BrdU) incorporation and changed levels of p27 and cyclin D1. This glycoprotein seemed to act on OLGc through the cAMP pathway only at early stages of development and on a certain sensitive cell population, accelerating their differentiation, probably as a consequence of premature withdrawal from the cell cycle.
Assuntos
Apoproteínas/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Oligodendroglia/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transferrina/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Bromodesoxiuridina/metabolismo , Células Cultivadas , Ciclina D1/metabolismo , Ciclina E/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Humanos , Imuno-Histoquímica/métodos , Proteína Básica da Mielina/metabolismo , Oligodendroglia/citologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
We have previously shown that addition of apotransferrin (aTf) accelerates maturation of oligodendroglial cells (OLGcs) in primary cultures. In this work, we examined the effect of aTf on two conditionally immortalized cell lines: N19 and N20.1. These cells proliferate at 34 degrees C and differentiate into mature OLGcs at 39 degrees C. In vitro addition of aTf to both cell lines at the differentiation temperature for 7 days showed increased expression of galactocerebroside, O4, and myelin basic protein (MBP) and a drop in the percentage of BrdU+ cells. The effect on MBP expression was particularly interesting in the less mature N19 cells. These cells do not express either MBP mRNAs or proteins, so aTf induced, rather than modulated, MBP expression in this cell line. In addition, even though MBP mRNAs for all four isoforms were induced, only the 17 and 21.5 kDa appeared to be translated. OLGc differentiation has been shown to be stimulated by the cAMP-CREB pathway. In N19 cells, following a pulse of aTf, there was a 10-fold increase in cAMP levels accompanied by elevated levels of pCREB. In the more mature N20.1 cells, there were no changes in cAMP levels. We conclude that addition of aTf to immature OLGc lines can enhance their expression of differentiated markers, such as MBP. The action of aTf on MBP gene expression in the least mature line is likely to be mediated by the cAMP pathway. In the N20.1 cells, it appears that different signals and/or mechanisms are involved in modulating myelin lipid and MBP expression. The results suggest that aTf can influence OLGc gene expression and differentiation through multiple mechanisms depending on the maturation of the cell.
Assuntos
Apoproteínas/farmacologia , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Transferrina/farmacologia , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Transformada , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Oligodendroglia/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , Transdução de Sinais/fisiologiaRESUMO
A novel role of ovorubin as a protection system against oxidative damage in eggs from Pomacea canaliculata was investigated. Carotenoid composition, and their antioxidant capacity, as well as the carotenoid-apoprotein interaction, were studied for this lipoglycocarotenoprotein. Carotenoid extracts from ovorubin were analysed by TLC and spectrophotometry. The major carotenoid was astaxanthin in its free (40%), monoester (24%), and diester (35%) forms, mainly esterified with 16:0 fatty acid. The antioxidant capacity of ovorubin carotenoids was studied by the inhibition of microsomal oxidation in a non-enzymatic system, showing strong protection against oxidative damage (IC50=3.9 nmol/mg protein). The carotenoid-apoprotein interaction was studied by spectrophotometry and electrophoresis using reconstituted ovorubin. Astaxanthin does not seem to affect the structural characteristics of ovorubin, however the carotenoid-protein association significantly protected astaxanthin against oxidation. Ovorubin therefore, besides its role in providing energy and structural precursors during embryogenesis, would be an antioxidant carrier, protecting at the same time this pigment from oxidation in the perivitellin fluid environment of the egg.
Assuntos
Antioxidantes/farmacologia , Proteínas do Ovo/farmacologia , Caramujos/química , Caramujos/fisiologia , beta Caroteno/análogos & derivados , Animais , Antioxidantes/química , Antioxidantes/fisiologia , Apoproteínas/análise , Apoproteínas/química , Apoproteínas/farmacologia , Carotenoides/análise , Carotenoides/química , Relação Dose-Resposta a Droga , Proteínas do Ovo/química , Proteínas do Ovo/fisiologia , Ácidos Graxos/análise , Ácidos Graxos/química , Feminino , Fluorescência , Microssomos Hepáticos/química , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos , Xantofilas , beta Caroteno/metabolismo , beta Caroteno/efeitos da radiaçãoRESUMO
In the central nervous system (CNS), apotransferrin (aTf) is produced by oligodendroglial cells (OLGcs), and aTf is essential for cell survival. We previously demonstrated that a single intracranial injection of aTf in 3-day-old rats accelerates differentiation of OLGc and that aTf acts at early stages of development on certain populations of OLGcs, promoting accelerated maturation, with no effect on late markers of cell differentiation. The objective of the present study was to analyze OLGc maturation at two different stages of rat development, 4 and 10 days of age, in OLGcs isolated from the brain after intracranial injection of aTf at 3 days of age, and to explore the in vitro effect of aTf added to cultures of OLGc isolated from aTf-injected and control brains. The maturational cell stages were identified by immunocytochemistry with different OLGc markers and by analysis of their morphological complexity. The OLGcs isolated from 4- and 10-day-old animals intracranially injected with aTf were more differentiated than control cells. Treatment with aTf of the cultures of OLGcs that were isolated from 4-day-old saline-injected control animals induced their differentiation, while a similar treatment of the cultures of OLGcs that were isolated from 10-day-old animals did not induce further maturation of the cells. The results presented in the present report demonstrate that the in vivo effects of aTf on OLGc maturation can be reproduced in cultures and that the effects of aTf occur early in development during a narrow, transient "temporal window" within which OLGcs are sensitive to its action.
Assuntos
Apoproteínas/farmacologia , Oligodendroglia/efeitos dos fármacos , Transferrina/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Biomarcadores , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Feminino , Masculino , Oligodendroglia/citologia , Ratos , Ratos WistarRESUMO
We have previously shown that a single intracranial injection of apotransferrin (aTf) in neonatal rats produces an accelerated mylinogenesis and increases the expression of certain myelin proteins such as myelin basic protein (MBP). In the present work, we studied the effects of aTf upon oligodendrocyte progenitor cell (Opc) cultures. In the presence of aTf, cells developed a multipolar morphology and showed an increased expression of O(4), MBP, O(1) and myelin-associated glycoprotein compared to controls. Migration studies using the agarose drop assay showed that aTf strongly inhibited OPc migration. This effect was not observed when an antibody against the transferrin receptor was added. The expression of two cell adhesion molecules, neural cell adhesion molecule (NCAM), N-cadherin and of polysialylated NCAM (PSA-NCAM) was evaluated by immunocytochemistry and by Western blot. Although NCAM expression did not change, there was a significant increase in N-cadherin expression and a decrease in PSA-NCAM in the aTf-treated cells. Time lapse studies of the expression of PSA-NCAM as an indicator of migration and of MBP as a marker of differentiation showed that in the cultures treated with aTf there is first a decrease in the percentage of cells expressing the former molecule which is followed by an increase in the percentage of cells expressing MBP. These results suggest that aTf added in vitro to cultured OPcs inhibits first their migration and then enhances their differentiation.
Assuntos
Apoproteínas/farmacologia , Oligodendroglia/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Transferrina/farmacologia , Animais , Caderinas/biossíntese , Caderinas/genética , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas da Mielina/biossíntese , Proteínas da Mielina/genética , Moléculas de Adesão de Célula Nervosa/biossíntese , Moléculas de Adesão de Célula Nervosa/genética , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
To determine whether neonatal intracranial injection of apotransferrin (aTf), which increases myelin deposition, has behavioral effects in rats, 3-day-old rats were intracranially injected with 350 ng of aTf and tested at 25 and 60 days of age. An anxiolytic-like behavior was observed in aTf-treated rats, evidenced by an increase in the exploration of open arms in the plus maze test without changes in the locomotor activity. This behavioral profile persists until adulthood. Intraperitoneal injection of 0.75 mg/kg of picrotoxin, a GABA(A) receptor channel antagonist, abolished this anxiolytic-like behavior, indicating that neonatal aTf induces a long-lasting increase in GABA(A) receptor functionality.
Assuntos
Apoproteínas/metabolismo , Apoproteínas/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Bainha de Mielina/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Transferrina/metabolismo , Transferrina/farmacologia , Animais , Animais Recém-Nascidos , Ansiedade/tratamento farmacológico , Ansiedade/fisiopatologia , Comportamento Animal/fisiologia , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Antagonistas GABAérgicos/farmacologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Bainha de Mielina/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Picrotoxina/farmacologia , Ratos , Ratos Wistar , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/metabolismoRESUMO
We have previously shown that in rat pups intracranially injected with a single dose of apotransferrin (aTf), there is an early oligodendroglial cell OLGc differentiation. The expression of the mRNAs of myelin basic proteins and of 2',3' cyclic nucleotide 3'-phosphodiesterase and the amount of the corresponding proteins, as well as myelin glycolipids and phospholipids, were significantly increased in these animals at 10 and 17 days of age. Microtubules and myelin basic proteins appear to be closely associated in OLGc and it has been shown that the mRNAs of myelin basic proteins are concentrated in the OLGc processes. The aim of this work was to clarify if the accelerated myelination produced by aTf could be linked to changes in certain cytoskeletal elements present in the myelin fraction such as tubulin, actin, and different microtubule-associated proteins (MAPs). A significant increase in the expression of the mRNA of tubulin and actin was observed in the brain of the aTf-treated animals. Several MAPs, particularly MAP 1B and stable tubule only peptide as well as actin and tubulin, were markedly increased in the Triton X-100 insoluble pellet obtained from the myelin fraction of these animals. The changes that we have previously described in the myelin of aTf intracranially injected rats, could be the consequence of its action on the cytoskeletal network of the OLGc. An enlargement of this structure would result in a more efficient and faster movement of the different components that are normally transported to the myelin by the cytoskeleton of this cell.
Assuntos
Apoproteínas/farmacologia , Proteínas do Citoesqueleto/genética , Citoesqueleto/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Básica da Mielina/genética , Oligodendroglia/fisiologia , Transferrina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Animais Recém-Nascidos , Apoproteínas/administração & dosagem , Citoesqueleto/metabolismo , Glicolipídeos/metabolismo , Microinjeções , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Fosfolipídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Ratos Wistar , Transcrição Gênica/efeitos dos fármacos , Transferrina/administração & dosagemRESUMO
In the present paper we first studied the brain distribution and the time and dose dependent effects of apotransferrin, after its intracranial injection into young rats and at different post-natal ages. Its action upon the transferrin receptor (TfR) and upon the expression of brain transferrin, as well as its effect on the proliferation and differentiation of oligodendroglial cells (OLGc) was one of the main objectives of our investigation. Total DNA and BrdU labeling, as an index of cellularity and proliferation, respectively, were the same in the control and experimental groups of rats. A significant increase in the MBP+ and CA II+ OLGc, and a decrease in the more immature (A2B5+) OLGc were found in the aTf injected rats. At 10 and 17 days of age, Tf-mRNA decreased to around 20% of the amount present in control animals. The TfR-mRNA in the animals receiving a single dose of aTf at 3 days of age showed an increase in its expression at 10 and 17 days of age, coincident with a higher immunoreactivity of the TfR itself of neurons, choroid plexus and brain capillaries in different brain areas. Although TfR+ OLGc were present up to 7 days of age in controls and in the Tf injected rats, no positive cells were observed at 17 days of age, even in the aTf injected rats. Our results give support to the hypothesis that aTf is an important factor necessary for the maturation of the OLGc, and that the effects that it produces in the OLGc-myelin unit after its intracranial injection in young rats are not due to an increase in proliferation, but to an accelerated differentiation of Tf-sensitive OLGc.
Assuntos
Apoproteínas/farmacologia , Encéfalo/metabolismo , Oligodendroglia/citologia , Transferrina/farmacologia , Animais , Apoproteínas/administração & dosagem , Apoproteínas/genética , Encéfalo/citologia , Diferenciação Celular , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Humanos , Injeções , Radioisótopos do Iodo/metabolismo , Masculino , Oligodendroglia/metabolismo , RNA Mensageiro , Ratos , Ratos Wistar , Receptores da Transferrina/genética , Crânio , Fatores de Tempo , Transferrina/administração & dosagem , Transferrina/genéticaRESUMO
Purified myelin obtained from 17 day old rats intracranially injected with aTf at 3 days of age was submitted to in vitro peroxidation using Fe + ascorbic acid (FeA) or Cu + H2O2 (CuH), to investigate the susceptibility of this membrane to in vitro metal catalyzed peroxidation. There was an increase in thiobarbituric acid-reactive-substances (TBARS) (60%) and in protein-associated carbonyls (PAC) (20%) in the myelin from aTf injected rats in comparison to myelin from controls, indicating a higher susceptibility to peroxidation. Desferoxamine (DFX) injected simultaneously with aTf did not change the response of myelin to aTf. CNS myelin is highly vulnerable to oxidative stress, and its susceptibility to peroxidation increases in myelin isolated from aTf injected rats. This increased liability to peroxidation as well as the previously reported aTf-dependent increment in certain myelin proteins and lipids and in the expression of specific myelin mRNAS, does not appear to be due to an increased amount of iron bound to the injected aTf. The changes in composition that we have previously reported could result in an abnormal myelin, allowing the peroxidative system to act upon the membrane more easily than in normal circumstances.
Assuntos
Apoproteínas/farmacologia , Peroxidação de Lipídeos , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Transferrina/farmacologia , Animais , Apoproteínas/administração & dosagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Química Encefálica , Quelantes/farmacologia , Desferroxamina/farmacologia , Feminino , Masculino , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Transferrina/administração & dosagemRESUMO
Transferrin (Tf) is a possible regulator of oligodendrocyte development in vitro (Espinosa de los Monteros et al., 1989). At least two different mechanisms may account for the effects of Tf on myelin synthesis. It may act as a trophic factor and enhance the formation of new myelin sheaths. Tf may also induce the synthesis of myelin proteins in the central nervous system. We recently demonstrated that a single intracranial injection of apotransferrin (aTf) in young rats induces an increased myelination (Escobar Cabrera et al., 1994). In the present study, we investigated the in vivo effect of aTf on the expression of mRNAs of specific myelin genes. Three-day-old rats were injected intracranially with aTf and killed at different ages after injection. Total brain RNA was isolated, and the expression of different mRNAs was analyzed by Northern blot. The amount of mRNAs of myelin basic protein and of 2'-3' cyclic nucleotide 3'-phosphohydrolase were markedly increased in the experimental animals, whereas myelin proteolipid protein mRNA did not show differences relative to controls. These results indicate that in the animals treated with aTf, there is a differential effect on the expression of certain specific myelin protein genes. They also suggest that aTf might exert its action at the posttranscriptional level and/or by direct transcriptional regulation of the genes.