RESUMO
Presenilins (PSs) are part of the gamma-secretase complex that produces the amyloid beta-peptide (Abeta) from its precursor [beta-amyloid precursor protein (betaAPP)]. Mutations in PS that cause familial Alzheimer's disease (FAD) increase Abeta production and trigger p53-dependent cell death. We demonstrate that PS deficiency, catalytically inactive PS mutants, gamma-secretase inhibitors, and betaAPP or amyloid precursor protein-like protein 2 (APLP2) depletion all reduce the expression and activity of p53 and lower the transactivation of its promoter and mRNA expression. p53 expression also is diminished in the brains of PS- or betaAPP-deficient mice. The gamma- and epsilon-secretase-derived amyloid intracellular C-terminal domain (AICD) fragments (AICDC59 and AICDC50, respectively) of betaAPP trigger p53-dependent cell death and increase p53 activity and mRNA. Finally, PS1 mutations enhance p53 activity in human embryonic kidney 293 cells and p53 expression in FAD-affected brains. Thus our study shows that AICDs control p53 at a transcriptional level, in vitro and in vivo, and that FAD mutations increase p53 expression and activity in cells and human brains.
Assuntos
Doença de Alzheimer/fisiopatologia , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/deficiência , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases , Encéfalo/metabolismo , Estudos de Casos e Controles , Caspase 3 , Caspases/metabolismo , Morte Celular , Linhagem Celular , Ativação Enzimática , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação , Proteínas do Tecido Nervoso/deficiência , Fragmentos de Peptídeos/metabolismo , Presenilina-1 , Presenilina-2 , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Transcrição Gênica , Ativação Transcricional , Proteína Supressora de Tumor p53/genéticaRESUMO
Amyloid beta-peptide (Abeta), which plays a central role in Alzheimer Disease, is generated by presenilin-dependent and presenilin-independent gamma-secretase cleavages of beta-amyloid precursor protein (betaAPP). We report that the presenilins (PS1 and PS2) also regulate p53-associated cell death. Thus, we established that PS deficiency, catalytically inactive PS mutants, gamma-secretase inhibitors and betaAPP or APLP2 depletion reduced the expression and activity of p53, and lowered the transactivation of its promoter and mRNA levels. p53 expression was also reduced in the brains or betaAPP-deficient mice or in brains where both PS had been invalidated by double conditional knock out. AICDC59 and AICDC50, the gamma- and epsilon-secretase-derived C-terminal fragments of betaAPP, respectively, trigger the activation of caspase-3, p53-dependent cell death, and increase p53 activity and mRNA. Finally, HEK293 cells expressing PS1 harboring familial AD (FAD) mutations or FAD-affected brains, all display enhanced p53 activity and p53 expression. Our studies demonstrate that AICDs control p53 at a transcriptional level, in vitro and in vivo and unravel a still unknown function for presenilins.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Peptídeos beta-Amiloides/química , Animais , Humanos , Modelos Biológicos , Estrutura Terciária de ProteínaRESUMO
A beta peptide accumulates in the extracellular space during Alzheimer's disease. It is the cleavage product of APP (Amyloid Precursor Protein), a large transmembrane protein. After ultracentrifugation, APP is found in a low-density fraction, enriched in cholesterol. These properties are characteristic of lipid rafts, which are microdomains that "float" like rafts on the plasma membrane. We have confirmed the presence of cholesterol in the core of the senile plaque, using the fluorescent probe filipin. In addition, we have shown that flotillin-1, a marker of rafts, accumulated in lysosomes of neurons in Alzheimer's disease. In most cases (76% of the flotillin-1 positive neurons), the accumulation was associated with the presence of neurofibrillary tangles. Our data suggest that the A beta peptide, which is poorly soluble in water, is actually linked with cholesterol, possibly from cellular membranes, in the extracellular space.
Assuntos
Doença de Alzheimer/patologia , Proteínas de Membrana/análise , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Córtex Cerebral/patologia , Humanos , Lisossomos/patologia , Lisossomos/ultraestrutura , Microdomínios da Membrana/patologia , Microdomínios da Membrana/ultraestrutura , Neurônios/patologiaRESUMO
In transgenic mice expressing human mutant beta-amyloid precursor protein (APP) and mutant presenilin-1 (PS1), Abeta antibodies labeled granules, about 1 microm in diameter, in the perikaryon of neurons clustered in the isocortex, hippocampus, amygdala, thalamus, and brainstem. The granules were present before the onset of Abeta deposits; their number increased up to 9 months and decreased in 15-month-old animals. They were immunostained by antibodies against Abeta 40, Abeta 42, and APP C-terminal region. In double immunofluorescence experiments, the intracellular Abeta co-localized with lysosome markers and less frequently with MG160, a Golgi marker. Abeta accumulation correlated with an increased volume of lysosomes and Golgi apparatus, while the volume of endoplasmic reticulum and early endosomes did not change. Some granules were immunolabeled with an antibody against flotillin-1, a raft marker. At electron microscopy, Abeta, APP-C terminal, cathepsin D, and flotillin-1 epitopes were found in the lumen of multivesicular bodies. This study shows that Abeta peptide and APP C-terminal region accumulate in multivesicular bodies containing lysosomal enzymes, while APP N-terminus is excluded from them. Multivesicular bodies could secondarily liberate their content in the extracellular space as suggested by the association of cathepsin D with Abeta peptide in the extracellular space.