RESUMEN
γ-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-ß production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-ß production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-ß levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-ß generation. Likewise, purified recombinant GSAP had no effect on amyloid-ß generation in two distinct in vitro γ-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-ß levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-ß levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-ß generation are uncertain.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Regulación de la Expresión Génica , Piperazinas/farmacología , Proteínas/química , Pirimidinas/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Benzamidas , Encéfalo/metabolismo , Línea Celular Tumoral , Humanos , Mesilato de Imatinib , Masculino , Ratones , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismoRESUMEN
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders affecting humans and animals. At present, it is not possible to recognize individuals incubating the disease before the clinical symptoms appear. We investigated the effectiveness of the "Protein Misfolding Cyclic Amplification" (PMCA) technology to detect the protease-resistance disease-associated prion protein (PrP(res)) in pre-symptomatic stages. PMCA allowed detection of PrP(res) in the brain of pre-symptomatic hamsters, enabling a clear identification of infected animals as early as two weeks after inoculation. Furthermore, PMCA was able to amplify minute quantities of PrP(res) from a variety of experimental and natural TSEs. Finally, PMCA allowed the demonstration of PrP(res) in an experimentally infected cow 32 month post-inoculation, that did not show clinical signs and was negative by standard Western blot analysis. Our findings indicate that PMCA may be useful for the development of an ultra-sensitive diagnostic test to minimize the risk of further propagation of TSEs.
Asunto(s)
Priones/aislamiento & purificación , Pliegue de Proteína , Animales , Encéfalo/metabolismo , Cricetinae , Cabras , Humanos , Mesocricetus , Ratones , OvinosRESUMEN
In prion diseases, the normal prion protein (PrP(c)) undergoes a conformational change that results in the abnormal form, named scrapie prion protein (PrP(sc)). The visual system of rodents provides a relatively simple neuronal model in which the cell bodies of neurons are confined to the retina and the axons constitute the optic nerve. We investigated by Western blot the profile of PrP(c) in the optic nerve and retina of normal hamsters and mice. We found that in the optic nerve the amount of PrP(c) is significantly higher than in the retina. A less abundant non-glycosylated band was observed in retinas compared with the optic nerve and brain. Similar results were found in the gray and white matter from normal mice and hamsters. After stereotaxic injection of ME7 or 139A in the superior colliculus, a visual target area, the proportion and glycopattern of PrP changed in the retina and optic nerve throughout the course of the disease. Similar results were found in the gray and white matter at terminal stage of scrapie after injection of ME7 and 139A in the dorsal hippocampus. This is the first time that changes in the distribution and glycopattern of PrP have been described in an in vivo model of prion diseases.
Asunto(s)
Priones/química , Priones/metabolismo , Scrapie/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Cricetinae , Densitometría , Glicosilación , Hipocampo/metabolismo , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Nervio Óptico/metabolismo , Conformación Proteica , Retina/metabolismo , Factores de TiempoRESUMEN
Diverse human disorders, including the majority of neurodegenerative diseases, are thought to arise from the misfolding and aggregation of protein. We have recently described a novel technology to amplify cyclically misfolded proteins in vitro. This procedure, named protein misfolding cyclic amplification (PMCA), is conceptually analogous to DNA amplification by PCR and has tremendous implications for research and diagnosis. The PMCA concept has been proved on the amplification of prions implicated in the pathogenesis of transmissible spongiform encephalopathies. In this article we describe the rational behind PMCA and some of the many potential applications of this novel technology.