RESUMO
Beta-amyloid peptide (Abeta) plays a central role in mediating neurotoxicity and in the formation of senile plaques in Alzheimer's disease (AD). The investigation of the roles of ubiquitin (Ub) in the process underlying the association of abnormal protein with the inclusion bodies that characterize AD is of great importance for the further understanding of this disorder. We have used primary cultures of cortical neurons and astrocytes to investigate the participation of the Ub-proteasome pathway in the degradation of Abeta and the effect of Abeta(1-42) and of the fragment Abeta(25-35) upon neural cells. We have found that Abeta(25-35) and Abeta(1-42) produce a significant increase in Ub-protein conjugates and in the expression of the Ub-activating enzyme E1. On the other hand, beta peptides inhibited the proteolytic activities of the 26S proteasome. When the proteolytic activity of the 26S proteasome was inhibited with lactacystin, there was a marked decrease in Abeta(1-42) degradation, suggesting that the peptide, in both astrocytes and neurons, could be a possible substrate of this enzymatic complex. Treatment of the cultures with lactacystin prior to the exposure to Abeta produced a significant decrease in cell viability, possibly as a consequence of the inhibition of Abeta degradation leading to a persistent exposure of the cells to the amyloidogenic peptide which results in cell death. Alterations in the Ub-proteasome pathway in AD could affect the normal proteolytic removal of Abeta, leading to an abnormal accumulation of Abeta(1-42).
Assuntos
Acetilcisteína/análogos & derivados , Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismo , Neurônios/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Acetilcisteína/farmacologia , Peptídeos beta-Amiloides/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Imuno-Histoquímica , Ligases/metabolismo , Substâncias Macromoleculares , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeo Hidrolases/efeitos dos fármacos , Ratos , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Although the participation of the ubiquitin-dependent pathway and of the proteasome in apoptosis has been proposed, its role in this process is not yet clearly defined. In previous studies, we have shown that in the central nervous system of the rat, programmed cell death and the ubiquitin-dependent proteolytic pathway are closely related to each other and that different types of neurons and of glial cells, shown different types of correlation between the two phenomena. In this work, we have used lactacystin, a highly specific inhibitor of the proteasome, to explore in Schwann cell cultures the relationship between the activity of the Ub-dependent pathway and apoptosis. Apoptosis was explored analyzing changes in nuclear morphology, using the Annexin V assay and by flow cytometry. Activity of caspase-3 was also measured. Changes in the levels of ubiquitin-protein conjugates and of the ubiquitin activating enzymes, E1, as well as expression of proteins that instruct the cells to apoptosis (p53, NFkappaB-IkappaB, Bcl2), or that participate in the control and regulation of the cell cycle, were also examined. Our results indicate that the decrease in the activity of the proteasome induced by lactacystin in Schwann cells, induces apoptotic cell death through changes in the concentration of certain key proteins that are involved in the apoptosis-signaling pathways.
Assuntos
Apoptose , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Células de Schwann/citologia , Ubiquitina/metabolismo , Animais , Células Cultivadas , Citometria de Fluxo , Hidrólise , Imuno-Histoquímica , Complexo de Endopeptidases do Proteassoma , Ratos , Ratos Wistar , Células de Schwann/metabolismoRESUMO
The multicatalytic protease complex or proteasome is a fundamental nonlysosomal tool that the cell uses to process or degrade proteins at a fast rate through the ubiquitin and ATP-dependent proteolytic pathway. Examples of these important proteins include the tumor suppressor protein p53, various cyclins, the cyclin-dependent kinase inhibitor p27, NFkappaB, IkappaB, c-fos, and c-jun. The activation of proteolytic enzymes, including certain cystein-proteases of the ced-3/ICE (interleukin-1beta-converting enzyme) family, is a characteristic feature of the apoptotic program. However, the role of the multicatalytic protease complex in apoptosis is not well known. In order to obtain further information regarding the participation of the ubiquitin-mediated pathway in the decision of the cell to execute the cell death program, we have used a specific inhibitor of the multicatalytic protease complex, lactacystin, in cultured cerebellar granule cells. Cells were obtained from the cerebellum of 6- to 8-day-old Wistar rats and cultured in Neurobasal medium supplemented with B-27. Addition of lactacystin to the cultures induced apoptosis of the granule cells in a time-dependent fashion. The morphological changes produced by the proteasome inhibitor included nuclear condensation and DNA fragmentation measured by the diphenylamine test, as well as a positive labeling by the TUNEL (terminal deoxynucleotidyltransferase mediated-dUTP nick end labeling) assay, all of them typical features of apoptosis. Concomitant with apoptosis, there were changes in the expression of the ubiquitin mRNA, a progressive depletion in the free ubiquitin pool, and an increase in the high molecular weight ubiquitin-protein conjugates. Caspase-3, a member of the ced-3/ICE family of cystein-proteases, showed a marked increase in activity in the lactacystin-treated cells. In flow cytometry studies, the amount of cells in the S phase of the cell cycle was smaller in the lactacystin-treated cells than in controls, suggesting that apoptosis could be due, in part, to an alteration of the cell cycle.
Assuntos
Acetilcisteína/análogos & derivados , Apoptose , Caspases/metabolismo , Cerebelo/fisiologia , Cisteína Endopeptidases/efeitos dos fármacos , Complexos Multienzimáticos/efeitos dos fármacos , Neurônios/fisiologia , Acetilcisteína/farmacologia , Animais , Caspase 3 , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/enzimologia , Cisteína Endopeptidases/fisiologia , Ativação Enzimática , Expressão Gênica/efeitos dos fármacos , Complexos Multienzimáticos/fisiologia , Neurônios/citologia , Neurônios/enzimologia , Complexo de Endopeptidases do Proteassoma , Ratos , Ratos Wistar , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
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.
Assuntos
Química Encefálica/fisiologia , Bainha de Mielina/metabolismo , Proteolipídeos/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Animais , Western Blotting , Encéfalo/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Feminino , Immunoblotting , Masculino , Microssomos/enzimologia , Microssomos/metabolismo , Bainha de Mielina/enzimologia , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismoRESUMO
Brain slices obtained from young rats were incubated with different radioactive precursors, in the presence and absence of L-cycloserine (an inhibitor of the synthesis of sphingosine) in order to explore the possibility that transport of proteolipids--and specifically of the major myelin proteolipid PLP--to the myelin membrane could be coupled to the transport of cerebrosides or sulfatides. At a concentration of 0.15 mM L-cycloserine, the incorporation of [3H] glycine into total proteins, proteolipid apoproteins (APL), PLP, and myelin basic proteins (MBP) of the total homogenate was unaffected by the presence of the inhibitor, whereas the incorporation of [3H] serine into glycosphingolipids decreased markedly. Under similar incubation conditions, the entry of labeled APL and of PLP into the myelin membranes in the presence of L-cycloserine decreased markedly (50%) in comparison to controls. Entry of MBP was not affected by the inhibitor. These results indicate that when synthesis of glycosphingolipids is inhibited by L-cycloserine, thus decreasing the availability of cerebrosides and sulfatides, the translocation of PLP to myelin is disrupted, suggesting that its transport through the oligodendroglial cell could be coupled to the transport of glycosphingolipids and, most probably, of sulfatides.
Assuntos
Transporte Axonal/efeitos dos fármacos , Encéfalo/metabolismo , Ciclosserina/farmacologia , Glicoesfingolipídeos/metabolismo , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Feminino , Masculino , Proteína Proteolipídica de Mielina , Ratos , Ratos EndogâmicosRESUMO
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.