RESUMO
Changes of neurosteroids may be involved in the pathophysiology of multiple sclerosis (MS). The present study investigated whether changes of neurosteroidogenesis also occurred in the grey and white matter regions of the brain in mice subjected to cuprizone-induced demyelination. Accordingly, we compared the expression of neurosteroidogenic proteins, including steroidogenic acute regulatory protein (StAR), voltage-dependent anion channel (VDAC) and 18 kDa translocator protein (TSPO), as well as neurosteroidogenic enzymes, including the side chain cleavage enzyme (P450scc), 3ß-hydroxysteroid dehydrogenase/isomerase and 5α-reductase (5α-R), during the demyelination and remyelination periods. Using immunohistochemistry and a quantitative polymerase chain reaction, we demonstrated a decreased expression of StAR, P450scc and 5α-R with respect to an increase astrocytic and microglial reaction and elevated levels of tumor necrosis factor (TNF)α during the cuprizone demyelination period in the hippocampus, cortex and corpus callosum. These parameters, as well as the glial reaction, were normalised after 2 weeks of spontaneous remyelination in regions containing grey matter. Conversely, persistent elevated levels of TNFα and low levels of StAR and P450scc were observed during remyelination in corpus callosum white matter. We conclude that neurosteroidogenesis/myelination status and glial reactivity are inversely related in the hippocampus and neocortex. Establishing a cause and effect relationship for the measured variables remains a future challenge for understanding the pathophysiology of MS.
Assuntos
Encéfalo/enzimologia , Encéfalo/metabolismo , Bainha de Mielina/enzimologia , Bainha de Mielina/metabolismo , Remielinização , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Colestenona 5 alfa-Redutase/metabolismo , Cuprizona/administração & dosagem , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Esclerose Múltipla/induzido quimicamente , Esclerose Múltipla/enzimologia , Esclerose Múltipla/metabolismo , Bainha de Mielina/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neuroglia/enzimologia , Neuroglia/metabolismo , Fosfoproteínas/metabolismo , Receptores de GABA/metabolismo , Remielinização/efeitos dos fármacos , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
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
Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na(+) and K(+) gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250-300g were excised, divided into two groups, and incubated with and without substrate, the para-nitrophenyl phosphate (pNPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CM 100 electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity.
Assuntos
Bainha de Mielina/enzimologia , Nervo Isquiático/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Masculino , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/ultraestrutura , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/enzimologia , Fibras Nervosas/ultraestrutura , Potássio/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/ultraestrutura , ATPase Trocadora de Sódio-Potássio/ultraestruturaRESUMO
We have used passive transfer of myelin-reactive lymphocytes in the Wistar rat model of experimental autoimmune encephalomyelitis (EAE) to investigate the nature of the central nervous system immunopathological alterations induced by these cells. Mononuclear cells from lymph nodes or spleen from sick myelin/complete Freund's adjuvant-immunized donors did not transfer clinical disease. However, depending on the previous treatment of the transferred cells, recipients develop central nervous system biochemical and histological alterations. Fresh cells from lymph nodes immediately transferred after procurement from the sick EAE donor rat were capable of inducing the most significant diminution in the content of myelin basic protein, sulfatides, and 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, with concomitant inflammatory infiltrations of white matter, principally in spinal cord and cerebellar lobules. Similar alterations were observed when animals were injected with spleen mononuclear cells activated in the presence of a nonspecific mitogen as concanavalin A. However, antigen-specific activated spleen cells generated by culturing in the presence of bovine myelin induced alterations to a lesser degree. Results point to a dissociation of the clinical disease from the central nervous system biochemical and histopathological lesions occurring in the EAE-transferred Wistar rats and indicate that these alterations in EAE are induced principally by T cells activated in vivo rather than by cells activated in vitro by myelin antigens. Therefore, these findings suggest a possible participation of lymphocytes unlike the encephalitogenic T cells in the induction of the described alterations and provide a useful model to explore further the subclinical responses to this experimental disease.
Assuntos
Sistema Nervoso Central/química , Encefalomielite Autoimune Experimental/imunologia , Proteína Básica da Mielina/imunologia , Linfócitos T/transplante , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/análise , Animais , Autoanticorpos/sangue , Bovinos , Células Cultivadas , Sistema Nervoso Central/imunologia , Concanavalina A , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Masculino , Proteína Básica da Mielina/farmacologia , Bainha de Mielina/enzimologia , Bainha de Mielina/imunologia , Ratos , Ratos Wistar , Linfócitos T/citologia , Linfócitos T/enzimologiaRESUMO
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 possibility that cerebroside sulfates and myelin proteolipid (PLP) could be simultaneously located in transport vesicles destined to be assembled in myelin was investigated in the brain of 20 day old rats. The brain was homogenized and fractionated according to Burkart et al. (J Biol Chem 257:3151-3156, 1982) to obtain a microsomal fraction that was further subfractionated in a linear sucrose density gradient following the procedure of Siegrist et al. (J Neurochem 33:497-504, 1979) to obtain a vesicular fraction which has been shown to transport cerebroside sulfates (Burkart et al., as above). This fraction was associated with acid hydrolase activity and had a lipid composition different from that of myelin and microsomal fractions. Studied by slab gel electrophoresis, dot blot, and Western blot analysis, using a highly specific anti-PLP antibody, it was found to contain myelin PLP. In view of previous findings of several laboratories including our own, the presence of myelin proteolipid in a vesicular fraction which is related to the transport of cerebroside sulfates gives further support to the hypothesis that the delivery of both constituents to the myelin membrane could be associated.