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
It has been recently demonstrated that ubiquitin-proteasome-mediated proteolysis is required for long-term synaptic facilitation in Aplysia. Here we show that the hippocampal blockade of this proteolytic pathway is also required for the formation of long-term memory in the rat. Bilateral infusion of lactacystin, a specific proteasome inhibitor, to the CA1 region caused full retrograde amnesia for a one-trial inhibitory avoidance learning when given 1, 4 or 7h, but not 10 h, after training. Proteasome inhibitor I produced similar effects. In addition, inhibitory avoidance training resulted in an increased ubiquitination and 26S proteasome proteolytic activity and a decrease in the levels of IkappaB, a substrate of the ubiquitin-proteasome cascade, in hippocampus 4 h after training. Together, these findings indicate that the ubiquitin-proteasome cascade is crucial for the establishment of LTM in the behaving animal.
Assuntos
Cisteína Endopeptidases/metabolismo , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Complexos Multienzimáticos/metabolismo , Neurônios/metabolismo , Ratos Wistar/metabolismo , Ubiquitina/metabolismo , Amnésia Retrógrada/induzido quimicamente , Amnésia Retrógrada/metabolismo , Amnésia Retrógrada/fisiopatologia , Animais , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Complexos Multienzimáticos/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Peptídeo Hidrolases/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Ratos , Ratos Wistar/anatomia & histologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles (NFT), senile plaques, and cerebrovascular deposits of amyloid-beta. Ubiquitin has also been shown to be present in some of the inclusions characteristic of this disease. To obtain further insight into the role played by the ubiquitin pathway in AD, we investigated the capacity of postmortem samples of cerebral cortex from normal and AD patients to form high-molecular-weight ubiquitin-protein conjugates. Activity of the ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2) involved in the ubiquitin pathway was also determined. In normal samples, the amount of high-molecular-weight ubiquitin-protein conjugates (HMW-UbPC) in cytosol increased with incubation time, whereas, in samples of AD cases, these were almost undetectable. The addition of an adult rat fraction, enriched in ubiquitinating enzymes, restored the capacity of AD brain cytosolic fraction to form conjugates. The trypsin-like proteolytic activity of the 26S proteasome was found to be decreased in AD cytosol brain. Assay of the activity of E1 and E2 by thiol-ester formation revealed a significant decrease in AD samples. Moreover, Western blotting using a specific antibody against E1 showed a dramatic drop of this enzyme in the cytosolic fraction, whereas normal levels were found in the particulate fraction, suggesting a possible delocalization of the enzyme. Our results suggest that a failure in the ubiquitination enzymatic system in brain cytosol may contribute to fibrillar pathology in AD.