Random Mutagenesis of Presenilin-1 Identifies Novel Mutants Exclusively Generating Long Amyloid β-Peptides

Abstract

Familial Alzheimer disease-causing mutations in the presenilins increase production of longer pathogenic amyloid ␤-peptides (A␤ 42/43 ) by altering ␥-secretase activity. The mechanism underlying this effect remains unknown, although it has been proposed that heteromeric macromolecular complexes containing presenilins mediate ␥-secretase cleavage of the amyloid ␤-precursor protein. Using a random mutagenesis screen of presenilin-1 (PS1) for PS1 endoproteolysis-impairing mutations, we identified five unique mutants, including R278I-PS1 and L435H-PS1, that exclusively generated a high level of A␤ 43 , but did not support physiological PS1 endoproteolysis or A␤ 40 generation. These mutants did not measurably alter the molecular size or subcellular localization of PS1 complexes. Pharmacological studies indicated that the up-regulation of activity for A␤ 43 generation by these mutations was not further enhanced by the difluoroketone inhibitor DFK167 and was refractory to inhibition by sulindac sulfide. These results suggest that PS1 mutations can lead to a wide spectrum of changes in the activity and specificity of ␥-secretase and that the effects of PS1 mutations and ␥-secretase inhibitors on the specificity are mediated through a common mechanism.

Potent amyloidogenicity and pathogenicity of Aβ43

“…In an effort to explore the role of Aβ43 in Aβ amyloidosis, we generated PS1-R278I knockin mice based on the knowledge that this mutation causes overproduction of Aβ43 in vitro 13 . We chose to use this PS mutation knockin paradigm rather than the overexpression strategy for the following reasons.…”

“…It is also noteworthy that Aβ43 appears to be equally as prone to aggregate in vitro as Aβ42 11 , leading to faster formation of oligomers than occurs in the case of Aβ40 12 . These observations imply that Aβ43 could be produced as a physiological or pathological metabolite of γ-secretase activity and may affect Aβ amyloidosis in the brain.In order to address whether Aβ43 contributes to Alzheimer's disease pathology, we decided to take advantage of the molecular phenotype of the presenilin-1 (PS1)-R278I mutation, as this mutation results in selective overt production of Aβ43 in vitro 13 , an effect which occurs to a much greater extent than in the case of other mutations such as R278K, R278S and R278T. The R278I mutation had independently been reported in a pedigree suffering from atypical Alzheimer's disease with language impairment 14 .…”

“…Furthermore, the increased Aβ43 levels accelerated Aβ pathology, thus contributing to the early onset of the disease. Therefore, we propose that Aβ43 be separately analyzed apart from Aβ42.In an effort to explore the role of Aβ43 in Aβ amyloidosis, we generated PS1-R278I knockin mice based on the knowledge that this mutation causes overproduction of Aβ43 in vitro 13 . We chose to use this PS mutation knockin paradigm rather than the overexpression strategy for the following reasons.…”

“…In order to address whether Aβ43 contributes to Alzheimer's disease pathology, we decided to take advantage of the molecular phenotype of the presenilin-1 (PS1)-R278I mutation, as this mutation results in selective overt production of Aβ43 in vitro 13 , an effect which occurs to a much greater extent than in the case of other mutations such as R278K, R278S and R278T. The R278I mutation had independently been reported in a pedigree suffering from atypical Alzheimer's disease with language impairment 14 .…”

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

“…Un factor importante que incide en los diferentes reportes es el método que se utiliza para cuantificar el β amiloide; autores como Ikeuche et al (35), usan técnicas cientos de veces más sensibles y específicas que la de ELISA, a tal punto que demuestran el incremento de β amiloide 1-40 y no sólo el incremento en la relación de β amiloide 1-42/1-40, como se reporta en la mayoría de las publicaciones (11,19,35,(37)(38)(39).…”