RESUMO
OBJECTIVES: Based on evidence that pregnant women with multiple sclerosis (MS) show a decline in the relapse rate during the third trimester and an increase during the first 3 months postpartum, the suggestion was made that high levels of circulating sex steroids are responsible for pregnancy-mediated neuroprotection. As both estradiol (E(2)) and progesterone exert neuroprotective and myelinating effects on the nervous system, the effects of sex steroids were studied in the experimental autoimmune encephalomyelitis (EAE) model of MS. METHODS: EAE was induced in female C57BL/6 mice by administration of a myelin oligodendrocyte protein (MOG(40-45)) peptide. Clinical signs of EAE, myelin protein expression and neuronal parameters were determined in mice with or without hormonal treatment. RESULTS: Progesterone given prior to EAE induction attenuated the clinical scores of the disease, slightly delayed disease onset and decreased demyelination foci, according to luxol fast blue staining (LFB), myelin basic protein (MBP) and proteolipid protein (PLP) and mRNA expression. Motoneuron expression of Na,K-ATPase mRNA was also enhanced by progesterone. In turn, combined E(2) plus progesterone therapy more effectively prevented neurological deficits, fully restored LFB staining, MBP and PLP immunoreactivity and avoided inflammatory cell infiltration. On the neuronal side, steroid biotherapy increased brain-derived neurotrophic factor (BDNF) mRNA. CONCLUSION: Early treatment with progesterone alone or more evidently in combination with E(2) showed a clinical benefit and produced myelinating and neuroprotective effects in mice with MOG(40-45)-induced EAE. Therefore, sex steroids should be considered as potential novel therapeutic strategies for MS.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Estradiol/farmacologia , Esclerose Múltipla/tratamento farmacológico , Sistemas Neurossecretores/imunologia , Progesterona/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Estradiol/metabolismo , Estradiol/uso terapêutico , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Esclerose Múltipla/fisiopatologia , Proteína Básica da Mielina/efeitos dos fármacos , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Proteína Proteolipídica de Mielina/efeitos dos fármacos , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/patologia , Progesterona/metabolismo , Progesterona/uso terapêutico , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
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
Schwann cells (SCs) in culture, without the presence of axons, become de-differentiated, reaching a condition similar to that of their precursor cells. The cytoplasmic accumulation of transferrin (Tf) in the myelinated peripheral nerve has been reported and data in the literature support a role for apoTf in myelination in the CNS. In the present report, we used SC cultures to evaluate the capacity of apoTf and holoTf to prevent cell de-differentiation promoted by fetal calf serum deprivation. SCs incubated in a serum-free medium showed a decrease in the expression of myelin basic protein (MBP) and P(0), markers of mature myelin-forming SCs, together with an increase in the levels of p75NTR and glial fibrillary acidic protein, markers of immature SCs. Treatment with holoTf prevented the decrease in expression of MBP and P(0) and the increase in p75NTR. ApoTf was unable to prevent these changes except when iron was added to the cultures. These results suggest a role for holoTf in the regulation of myelin formation by SCs.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Transferrina/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting , Diferenciação Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Proteína Glial Fibrilar Ácida/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Ferro/farmacologia , Proteína Básica da Mielina/efeitos dos fármacos , Proteína Básica da Mielina/metabolismo , Proteína P0 da Mielina/efeitos dos fármacos , Proteína P0 da Mielina/metabolismo , Ratos , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/efeitos dos fármacos , Receptores de Fator de Crescimento Neural/metabolismo , Células de Schwann/citologia , Células de Schwann/metabolismo , Transferrina/metabolismoRESUMO
Apotransferrin injected intracranially into young rats has been shown in our laboratories to induce an early differentiation of oligodendroglial cells and an increased deposition of myelin. The expression of some myelin-specific proteins such as myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and of their mRNAs were significantly increased in these animals. Also, in the cytoskeleton obtained from isolated myelin, it was found that several microtubule associated proteins (MAPs), particularly the stable tubule only peptide (STOP) and MAP 1B, as well as actin and tubulin were markedly increased. In the present paper, we compare the changes in expression of brain and myelin cytoskeletal proteins in a newly generated transferrin transgenic mouse (Tg), overexpressing the human transferrin gene, with the results obtained in aTf-injected rats. In the myelin cytoskeletal fraction of Tg mice there was a significant increase in the expression of MBP, tubulin, tau and STOP, similarly to what was previously found in the aTf-injected rats. Immunohistochemical studies showed that a variance with what occurs in the aTf-injected model, in which the above mentioned changes were limited to the corpus callosum, in the Tg mice the changes in expression of cytoskeletal proteins were observed in the various anatomical areas studied such as cerebral cortex, brain stem and cerebellum. There was also an increased expression of neurofilaments in the Tg animals, in contrast with results obtained in aTf-injected rats, suggesting that in the Tg mice, the continuous overexpression of Tf might also induce some neuronal changes. Changes in tau, total and acetylated tubulin and MAP 1B were observed in both neurons and OLGc. The increase in STOP was more significant in OLGc while the changes in MAP2 were exclusively found in neurons.
Assuntos
Encéfalo/metabolismo , Proteínas do Citoesqueleto/biossíntese , Bainha de Mielina/metabolismo , Transferrina/genética , Animais , Encéfalo/efeitos dos fármacos , Proteínas do Citoesqueleto/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/efeitos dos fármacos , Proteína Básica da Mielina/genética , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/genética , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Transferrina/farmacologiaRESUMO
Apotransferrin (aTf), intracranially administered into newborn rats, produces increased myelination with marked increases in the levels of myelin basic protein (MBP), phospholipids and galactolipids, and mRNAs of MBP and 2', 3' cyclic nucleotide 3'-phosphohydrolase (CNPase). Cytoskeletal proteins such as tubulin, actin, and microtubule-associated proteins are also increased after aTf injection. In contrast, almost no changes are observed in myelin proteolipid protein (PLP) or in its mRNA or cholesterol. In the present study, we used brain-tissue slices and cell cultures highly enriched for oligodendroglia to investigate signaling pathways involved in the action of aTf, and to find out whether cytoskeletal integrity and dynamics were essential for its action upon the neural expression of certain genes. Treatment of brain-tissue slices with aTf produced a marked increase in the expression of MBP, CNPase, and tubulin mRNAs. Colchicine, cytochalasin, and taxol severely reduced the effect of aTf. Addition to cultures of an antibody against transferrin receptor (TfR), protein kinase inhibitors, or a cyclic AMP (cAMP) analogue showed that a functionally intact TfR was necessary, and that tyrosine kinase, protein kinase C and A, as well as calcium-calmodulin-dependent kinase (Ca-CaMK) activities appeared to mediate aTf actions upon the expression of the above mentioned genes. Changes in the levels of phosphoinositides and cAMP induced by aTf in oligodendroglial cell (OLGc) cultures correlated with these results and coincide with an activation of the cyclic response element binding protein (CREB) and of mitogen activated protein kinases. The increased expression of certain myelin genes produced by aTf appear to be mediated by interaction of this glycoprotein with its receptor, by the cytoskeleton of the OLGc, and by a complex activation of protein kinases which lead to CREB phosphorylation.