Familial Alzheimer's disease mutations inhibit γ‐secretase‐mediated liberation of β‐amyloid precursor protein carboxy‐terminal fragment

Abstract

Cleavage of the b-secretase processed b-amyloid precursor protein by c-secretase leads to the extracellular release of Ab42, the more amyloidogenic form of the b-amyloid peptide, which subsequently forms the amyloid-plaques diagnostic of Alzheimer's disease. Mutations in b-amyloid precursor protein (APP), presenilin-1 and presenilin-2 associated with familial Alzheimer's disease (FAD) increase release of Ab42, suggesting that FAD may directly result from increased c-secretase activity. Here, we show that familial Alzheimer's disease mutations clustered near the sites of c-secretase cleavage actually decrease c-secretase-mediated release of the intracellular fragment of APP (CTFc). Concordantly, presenilin-1 mutations that result in Alzheimer's disease also decrease the release of CTFc. Mutagenesis of the epsilon cleavage site in APP mimicked the effects of the FAD mutations, both decreasing CTFc release and increasing Ab42 production, suggesting that perturbation of this site may account for the observed decrement in c-secretase-mediated proteolysis of APP. As CTFc has been implicated in transcriptional activation, these data indicate that decreased signaling and transcriptional regulation resulting from FAD mutations in bamyloid precursor protein and presenilin-1 may contribute to the pathology of Alzheimer's disease. Familial Alzheimer's disease (FAD) is an autosomal dominant disorder caused by missense mutations in b-amyloid precursor protein (APP), presenilin-1 (PS1) and presenilin-2 (PS2) (Selkoe 2001). APP is a type I transmembrane protein that is sequentially proteolytically processed by a-, b-, and c-secretase. APP cleavage by a-secretase results in the release of the large extracellular domain and the generation of the C83 membrane-resident fragment. b-secretase cleavage of APP, mediated by the BACE atypical aspartyl protease (Vassar et al. 1999), also liberates the extracellular domain and generates the C99 membrane-resident fragment. Subsequently, C83 and C99 are processed by the recently characterized heterotetrameric c-secretase complex consisting of Nicastrin, Aph-1, Pen-2, and PS1/2 (Francis et al. 2002;Edbauer et al. 2003;Kimberly et al. 2003;Takasugi et al. 2003). c-Secretase cleavage of C83 and C99 produces the innocuous p3 peptide and the pathogenic Ab peptide, respectively, and releases the intracellular fragment of APP from the membrane (referred to in this work as CTFc). PS1 and PS2 form the catalytic core for the c-secretase processing of APP (Schroeter et al. 2003;Cervantes et al. 2004). Generation of FAD pathology by mutations in either the APP substrate or the PS1/2 cleaving enzyme indicates that the etiology of the disorder resides in some alteration in the enzyme-substrate relationship.Multiple alternative sites of cleavage of APP by c-secretase generate a variety of Ab peptides, of which Ab40 and Ab42 are the most studied. The FAD mutations in

The presenilin hypothesis of Alzheimer's disease: Evidence for a loss-of-function pathogenic mechanism

“…Interestingly, the vast majority of PS mutations that impair NICD production also impair AICD production, indicating a general impairment of ␥-secretasedependent function that is not limited to a single substrate (Table 1) (11)(12)(13)(14)(15). This correspondence of the effects of mutations on liberation of NICD and AICD may reflect mechanistic similarities between the S3 and cleavages of Notch and APP, respectively, which occur at similar intramembranous positions near the cytoplasmic face of the plasma membrane.…”

Fe65 Stimulates Proteolytic Liberation of the β-Amyloid Precursor Protein Intracellular Domain

“…The original APP-Gal4VP16 (APPGV16) vector generation has already been described (17), and all of the subsequent vectors used the same strategy. 5Ј primers were generated that contain a HindIII restriction site and a start codon in front of the coding portion of the cDNA representing the 5Ј end of C99, C83, or C50.…”

“…In experiments also overexpressing PS1 isoforms, 150 ng/well of the PS1 construct was used. Normalization was done using 50 ng/well of the EF2-␤Gal construct previously described (17). For experiments unaccompanied by transactivation assays, cells were grown in 6-well plates, and 1000 ng/well of the APPderived constructs and 4000 ng/well of the Fe65 vectors were used to maintain the 1:4 APP:Fe65 vector ratio.…”

“…The APPGV16 682 YENPTY 687 point mutants were generated by the method employed previously (17). Briefly, mutagenic primers were designed with changes in the coding sequence to alter either Tyr 682 or Tyr 687 to alanine and insert a silent restriction site using the standard QuikChange site-directed mutagenesis approach (Stratagene).…”

Evaluación de la producción de b-amiloide por la mutación E280A en el gen de la presenilina 1

“…El incremento en la generación del péptido β amiloide 1-42 por mutaciones en presenilina 1, se ha demostrado, tanto en ratones como en cultivo de células neuroblastoides, y se ha encontrado que sólo unas pocas mutaciones en la presenilina 1 incrementan también el péptido β amiloide 1-40 (11,19,20). El incremento en la producción de β amiloide 1-42 en los portadores de un alelo mutado de presenilina 1, lleva a pensar en una ganancia de función (o disfunción) del alelo mutante sobre el alelo silvestre, también llamado efecto dominante negativo (21).…”