RESUMO

We have previously reported that in the distal stump of ligated sciatic nerves, there is a change in the distribution of myelin basic protein (MBP) and P0 protein immunoreactivities. These results agreed with the studies of myelin isolated from the distal stump of animals submitted to ligation of the sciatic nerve, showing a gradual increase in a 14 kDa band with an electrophoretic mobility similar to that of an MBP isoform, among other changes. This band, which was resolved into two bands of 14 and 15 kDa using a 16% gel, was found to contain a mixture of MBP fragments and peptides with great homology with alpha- and beta-globins. In agreement with these results, we have demonstrated that the mRNA of alpha-globin is present in the proximal and distal stumps of the ligated nerve. It is also detected at very low levels in Schwann cells isolated from normal nerves. These results could be due to the presence of alpha- and/or beta-globin arising from immature cells of the erythroid series. Also, they could be present in macrophages, which spontaneously migrate to the injured nerve to promote the degradation of myelin proteins. Cells isolated from normal adult rat bone marrow which were injected intraortically were found to migrate to the injured area. These cells could contribute to the remyelination of the damaged area participating in the removal of myelin debris, through their transdifferentiation into Schwann cells or through their fusion with preexisting Schwann cells in the distal stump of the injured sciatic nerve.

Assuntos

Células da Medula Óssea/fisiologia , Globinas/biossíntese , Degeneração Neural/patologia , Regeneração Nervosa/fisiologia , RNA Mensageiro/biossíntese , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Animais , Western Blotting , Movimento Celular/fisiologia , Eletroforese em Gel de Poliacrilamida , Feminino , Imuno-Histoquímica , Masculino , Proteína Básica da Mielina/metabolismo , Ensaios de Proteção de Nucleases , Peptídeos/química , Nervos Periféricos/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Schwann/fisiologia , Nervo Isquiático/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina

RESUMO

The presence of calcium dependent, cobalt sensitive steps in the transport of proteins to myelin was studied using slices obtained from the brains of 20 day old Wistar rats. When 0.18 mM cobalt chloride was added to the incubation medium, although protein synthesis in the total homogenate was not affected, the entry of labeled PLP into myelin and fraction SN(4) (a myelin related membrane), decreased to 20% of control values. Transport of basic and Wolfgram proteins was not affected by cobalt ions. Similar results were obtained when slices were incubated in a calcium-free medium or in a calcium free medium containing cobalt chloride. The entry of fucose labeled glycoproteins into myelin, which followed a pattern similar to that of PLP, was also inhibited by the presence of cobalt in the incubation medium. These results indicate that the delivery of PLP and glycoproteins on the one hand and of the other myelin proteins on the other is regulated by different mechanisms and that calcium-dependent, cobalt-sensitive steps are involved in the transfer of the former. Acylation of myelin PLP, assayed by the incorporation of palmitic acid, was not influenced by the presence of cobalt chloride in the incubation medium, suggesting that this posttranscriptional event ocurrs close to myelin or in the myelin membrane itself.

RESUMO

The present study was carried out in order to obtain further information regarding the mechanism of transport and assembly of myelin proteins in different subcellular fractions isolated from brain slices incubated in vitro with radioactive amino acids under different experimental conditions. It was found that proteolipid protein (PLP) showed a lag in the entry into the myelin membrane, while basic and Wolfgram proteins appeared to be inserted in this structure immediately after their synthesis. Addition of 500 microM colchicine to the incubation medium blocked the transport of PLP, while the entry of the other proteins was not affected. Pulse-chase experiments using cycloheximide suggest that a precursor-product relationship between microsomes, fraction SN4 and myelin exists only for PLP. The results obtained allow us to draw the following conclusions: The delay in the entry of PLP into myelin membrane is probably due to the time required for its transport towards the final site of assembly; the microtubular network of the oligodendroglial cell is directly involved in the transport of PLP; basic and probably Wolfgram proteins follow a route which clearly differs from that of PLP; delivery of myelin proteins from the site of synthesis towards their site of deposition depends, at least, on two different mechanisms of intracellular transport.

Assuntos

Encéfalo/metabolismo , Colchicina/farmacologia , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Proteolipídeos/metabolismo , Animais , Feminino , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Frações Subcelulares/metabolismo

RESUMO

Rats of 20-days of age were injected intracranially with radioactive palmitic acid to study its incorporation into proteolipid protein (PLP) of myelin and myelin subfractions. At short times (120 min), the radioactivity present in PLP was shown to be due to palmitic acid bound to the protein by ester linkages. The specific radioactivity of palmitic acid labeled PLP was identical in all the myelin subfractions except the myelin-like fraction, in which it was lower, suggesting that the entry of the fatty acid into PLP of the different subfractions occurs simultaneously. Experiments using time staggered injections of (14)C- and (3)H-labeled palmitic acid also showed that entry of the fatty acid into PLP of the various subfractions was simultaneous. These results seem to indicate that the acylation of PLP occurs in the myelin membrane and that synthesis and transport of this protein are events unrelated to the acylation process.

Assuntos

Masculino , Feminino , Animais , Ratos , Cérebro , Colchicina , Técnicas In Vitro , Proteínas da Mielina , Bainha de Mielina , Proteolipídeos

RESUMO

The present study was carried out in order to obtain further information regarding the mechanism of transport and assembly of myelin proteins in different subcellular fractions isolated from brain slices incubated in vitro with radioactive amino acids under different experimental conditions. It was found that proteolipid protein (PLP) showed a lag in the entry into the myelin membrane, while basic and Wolfgram proteins appeared to be inserted in this structure immediately after their synthesis. Addition of 500 microM colchicine to the incubation medium blocked the transport of PLP, while the entry of the other proteins was not affected. Pulse-chase experiments using cycloheximide suggest that a precursor-product relationship between microsomes, fraction SN4 and myelin exists only for PLP. The results obtained allow us to draw the following conclusions: The delay in the entry of PLP into myelin membrane is probably due to the time required for its transport towards the final site of assembly; the microtubular network of the oligodendroglial cell is directly involved in the transport of PLP; basic and probably Wolfgram proteins follow a route which clearly differs from that of PLP; delivery of myelin proteins from the site of synthesis towards their site of deposition depends, at least, on two different mechanisms of intracellular transport.

Assuntos

Masculino , Feminino , Animais , Ratos , Técnicas In Vitro , Bainha de Mielina , Cérebro , Colchicina , Proteínas da Mielina , Proteolipídeos

RESUMO

Brain slices prepared from 20-day old rats were incubated with [(3)H]palmitic acid to study its incorporation into myelin proteins. After separation by SDS-PAGE, most of the label was found to be associated with the major proteolipid protein (PLP) and with the intermediate protein (I). The radioactivity measured in PLP at short incubation times was shown to be due to palmitic acid bound to the protein by ester linkages. Time-course incorporation of [(3)H]palmitic acid into PLP of fraction SN(4) (a myelin like membrane) and of purified myelin showed that the former was poorly labeled and no relationship of the type 'precursor-product' between these fractions could be detected. Incorporation of the fatty acid into PLP was not affected by inhibition of the synthesis or transport of myelin PLP with cycloheximide or colchicine, indicating that the pool of PLP that can be acylated must be larger than the extramyelin pool. Addition of unlabeled palmitic acid to the incubation medium, 30 min after the addition of [(3)H]palmitate, stopped the appearance of label in myelin PLP almost immediately, indicating that there is no significant extramyelin pool of PLP destined for transport into myelin. The results presented in this paper strongly suggest that esterification of PLP takes place in the myelin membrane or at a site very close to it.

RESUMO

The lipid and protein composition as well as the activity of 2'3' cyclic nucleotide 3' phosphohydrolase (CNPH) and the distribution of individual proteins separated by SDS-PAGE were studied in myelin and in a fraction closely related to myelin or assumed to be a precursor membrane of mature myelin (fraction SN4) isolated from 20-day-old rats made hypothyroid at birth or submitted to early malnutrition. In both experimental conditions lipid and protein components were found to be reduced in myelin when data were expressed as mg/g fresh tissue, but the results were close to those obtained in normal controls when data were expressed as mg/mg total protein of each fraction. CNPH activity was normal in myelin but markedly reduced in fraction SN4. Although the results appear to suggest that both experimental conditions produce a reduction in the amount of myelin but no qualitative changes, the data obtained with SDS-PAGE show that the distribution of the various types of proteins present in this fraction and fraction SN4 was abnormal. Myelin and fraction SN4 isolated from malnourished animals displayed a protein profile which was quite similar to that found in fraction SN4 isolated from normal rats, indicating a delay in the process of myelin maturation. The changes in protein composition of myelin and fraction SN4 produced by neonatal hypothyroidism on the other hand differed clearly from those produced by early malnutrition; the ratio small basic protein: large basic protein (SBP:LBP) was found to be reduced in both membrane fractions in the former condition and the protein patterns of myelin and that of fraction SN4 were different, at variance with what was found in the case of malnourished animals. Our findings appear to suggest that the effects of early malnutrition and neonatal hypothyroidism upon myelin and myelin-related membranes are different, and that myelination is more affected in the latter condition.