RESUMO
Spine pathology has been implicated in the early onset of Alzheimer's disease (AD), where Aß-Oligomers (AßOs) cause synaptic dysfunction and loss. Previously, we described that pharmacological inhibition of c-Abl prevents AßOs-induced synaptic alterations. Hence, this kinase seems to be a key element in AD progression. Here, we studied the role of c-Abl on dendritic spine morphological changes induced by AßOs using c-Abl null neurons (c-Abl-KO). First, we characterized the effect of c-Abl deficiency on dendritic spine density and found that its absence increases dendritic spine density. While AßOs-treatment reduces the spine number in both wild-type (WT) and c-Abl-KO neurons, AßOs-driven spine density loss was not affected by c-Abl. We then characterized AßOs-induced morphological changes in dendritic spines of c-Abl-KO neurons. AßOs induced a decrease in the number of mushroom spines in c-Abl-KO neurons while preserving the populations of immature stubby, thin, and filopodia spines. Furthermore, synaptic contacts evaluated by PSD95/Piccolo clustering and cell viability were preserved in AßOs-exposed c-Abl-KO neurons. In conclusion, our results indicate that in the presence of AßOs c-Abl participates in synaptic contact removal, increasing susceptibility to AßOs damage. Its deficiency increases the immature spine population reducing AßOs-induced synapse elimination. Therefore, c-Abl signaling could be a relevant actor in the early stages of AD.
RESUMO
The early stages of Alzheimer's disease are characterised by impaired synaptic plasticity and synapse loss. Here, we show that amyloid-ß oligomers (AßOs) activate the c-Abl kinase in dendritic spines of cultured hippocampal neurons and that c-Abl kinase activity is required for AßOs-induced synaptic loss. We also show that the EphA4 receptor tyrosine kinase is upstream of c-Abl activation by AßOs. EphA4 tyrosine phosphorylation (activation) is increased in cultured neurons and synaptoneurosomes exposed to AßOs, and in Alzheimer-transgenic mice brain. We do not detect c-Abl activation in EphA4-knockout neurons exposed to AßOs. More interestingly, we demonstrate EphA4/c-Abl activation is a key-signalling event that mediates the synaptic damage induced by AßOs. According to this results, the EphA4 antagonistic peptide KYL and c-Abl inhibitor STI prevented i) dendritic spine reduction, ii) the blocking of LTP induction and iii) neuronal apoptosis caused by AßOs. Moreover, EphA4-/- neurons or sh-EphA4-transfected neurons showed reduced synaptotoxicity by AßOs. Our results are consistent with EphA4 being a novel receptor that mediates synaptic damage induced by AßOs. EphA4/c-Abl signalling could be a relevant pathway involved in the early cognitive decline observed in Alzheimer's disease patients.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/metabolismo , Receptor EphA4/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/fisiologia , Animais , Células Cultivadas , Espinhas Dendríticas/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Ratos Sprague-Dawley , Sinapses/patologiaRESUMO
Niemann-Pick type C (NPC) is a neurodegenerative disease characterized by the intralysosomal accumulation of cholesterol leading to neuronal apoptosis. We have previously reported the activation of the c-Abl/p73 proapoptotic pathway in the cerebellum of NPC mice; however, upstream signals underlying the engagement of this pathway remain unknown. Here, we investigate the possible role of oxidative stress in the activation of c-Abl/p73 using different in vitro and in vivo NPC models. Our results indicate a close temporal correlation between the appearance of nitrotyrosine (N-Tyr; a post-translational tyrosine modification caused by oxidative stress) and the activation of c-Abl/p73 in NPC models. To test the functional role of oxidative stress in NPC, we have treated NPC neurons with the antioxidant NAC and observed a dramatic decrease of c-Abl/p73 activation and a reduction in the levels of apoptosis in NPC models. In conclusion, our data suggest that oxidative stress is the main upstream stimulus activating the c-Abl/p73 pathway and neuronal apoptosis in NPC neurons.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/fisiologia , Neurônios/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Proteínas Nucleares/fisiologia , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-abl/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Regulação para Cima/fisiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/patologia , Doença de Niemann-Pick Tipo C/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
APP intracellular domain (AICD) has been proposed as a transcriptional inductor that moves to the nucleus with the adaptor protein Fe65 and regulates transcription. The two proteins, APP and Fe65, can be phosphorylated by c-Abl kinase. Neprilysin has been proposed as a target gene for AICD. We found that AICD expression is decreased by treatment with STI-571, a c-Abl inhibitor, suggesting a modulation of AICD transcription by c-Abl kinase. We observed interaction between c-Abl kinase, the AICD fragment and the Fe65 adaptor protein. In addition, STI-571 reduces apoptosis in APPSw, and the apoptotic response induced by Fe65 over-expression was inhibited by with the expression of a kinase dead (KD) c-Abl and enhanced by over-expression of WT-c-Abl. However, in the APPSw cells, the ability of the KD-c-Abl to protect against Fe65 was reduced. Finally, in APPSw clone, we detected higher trans-activation of the pro-apoptotic p73 isoform, TAp73 promoter. Our results show that c-Abl modulates AICD dependent cellular responses, transcriptional induction as well as the apoptotic response, which could participate in the onset and progression of the neurodegenerative pathology, observed in Alzheimer's disease (AD).
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Apoptose , Proteínas Proto-Oncogênicas c-abl/metabolismo , Receptores de Superfície Celular/metabolismo , Ativação Transcricional , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzamidas , Linhagem Celular Tumoral , Sobrevivência Celular , Genes Reporter , Humanos , Peróxido de Hidrogênio/farmacologia , Mesilato de Imatinib , Camundongos , Mutação , Neprilisina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Oxidantes/farmacologia , Fosforilação , Piperazinas/farmacologia , Regiões Promotoras Genéticas , Nexinas de Proteases , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/genética , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
Niemann-Pick type C (NPC) disease is a fatal autosomal recessive disorder characterized by the accumulation of free cholesterol and glycosphingolipids in the endosomal-lysosomal system. Patients with NPC disease have markedly progressive neuronal loss, mainly of cerebellar Purkinje neurons. There is strong evidence indicating that cholesterol accumulation and trafficking defects activate apoptosis in NPC brains. The purpose of this study was to analyze the relevance of apoptosis and particularly the proapoptotic c-Abl/p73 system in cerebellar neuron degeneration in NPC disease. We used the NPC1 mouse model to evaluate c-Abl/p73 expression and activation in the cerebellum and the effect of therapy with the c-Abl-specific inhibitor imatinib. The proapoptotic c-Abl/p73 system and the p73 target genes are expressed in the cerebellums of NPC mice. Furthermore, inhibition of c-Abl with imatinib preserved Purkinje neurons and reduced general cell apoptosis in the cerebellum, improved neurological symptoms, and increased the survival of NPC mice. Moreover, this prosurvival effect correlated with reduced mRNA levels of p73 proapoptotic target genes. Our results suggest that the c-Abl/p73 pathway is involved in NPC neurodegeneration and show that treatment with c-Abl inhibitors is useful in delaying progressive neurodegeneration, supporting the use of imatinib for clinical treatment of patients with NPC disease.