Familial Alzheimer Disease-linked Presenilin 1 Variants Enhance Production of Both Aβ1–40 and Aβ1–42 Peptides That Are Only Partially Sensitive to a Potent Aspartyl Protease Transition State Inhibitor of “γ-Secretase”

Abstract

Presenilin 1 (PS1) plays an essential role in intramembranous "␥-secretase" processing of several type I membrane proteins, including the ␤-amyloid precursor proteins (APP) and Notch1. In this report, we examine the activity of two familial Alzheimer's disease-linked PS1 variants on the production of secreted A␤ peptides and the effects of L-685,458, a potent ␥-secretase inhibitor, on inhibition of A␤ peptides from cells expressing these PS1 variants. We now report that PS1 variants enhance the production and secretion of both A␤1-42 and A␤1-40 peptides. More surprisingly, whereas the IC 50 for inhibition of A␤1-40 peptide production from cells expressing wild-type PS1 is ϳ1.5 M, cells expressing the PS1⌬E9 mutant PS1 exhibit an IC 50 of ϳ4 M. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry reveal that the levels of A␤1-43 peptides are elevated in medium of PS1⌬E9 cells treated with higher concentrations of inhibitor. The differential effects of wild-type and mutant PS1 on ␥-secretase production of A␤ peptides and the disparity in sensitivity of these peptides to a potent ␥-secretase suggest that PS may be necessary, but not sufficient, to catalyze hydrolysis at the scissile bonds that generate the termini of A␤1-40 and A␤1-42 peptides.Presenilin 1 and 2 (PS1 and PS2) 1 are polytopic membrane proteins that are mutated in the majority of pedigrees with early onset familial Alzheimer's disease (FAD) (1-3). It is now established that PS play an essential role in intramembranous "␥-secretase" processing of type I membrane proteins, including the ␤-amyloid precursor proteins (APP) (4, 5), the developmental signaling receptor, Notch1 (6 -8), the tyrosine kinase receptor ErbB4 (9, 10), and N-and E-cadherins (11). For APP, ␥-secretase catalyzes proteolysis of a set of membrane-tethered APP derivatives, termed APP-CTFs, resulting in the production and secretion of ␤-amyloid (A␤) peptides. On the other hand, ␥-secretase-mediated processing of the membrane-tethered Notch1 derivative, termed S2/NEXT, releases the intracellular domain (S3/NICD) that subsequently translocates to the nucleus and activates transcription of target genes (7,8). The observation that A␤ and S3/NICD production are completely eliminated in cells derived from mouse blastocysts with compound deletions of PS1 and PS2, lends convincing support to the notion that PS are critical for intramembranous cleavage of APP and Notch1 (12, 13).Although the mechanism(s) by which PS facilitates ␥-secretase processing of APP and Notch1 have not been fully elucidated, the generation of A␤ (14 -16) and S3/NICD (17) have been show to be inhibited by highly potent and selective aspartyl protease transition state inhibitors that bind specifically to PS1 and PS2 (14,18). These data, taken with the description of a family of signal peptide peptidases with limited homology to PS (19), have led to the conclusion that PS are the elusive ␥-secretases (20).While appealing, the "PS is ␥-secretase" model has several weaknesses. F...

Potent amyloidogenicity and pathogenicity of Aβ43

“…Thus, inhibition of this Aβ43-to-Aβ40 conversion could account for the increase in Aβ43 and the concomitant decrease in Aβ40 in the knockin MEFs. Interestingly, treatment of PS1-ΔE9-expressing cells with L-685,458 resulted in elevated Aβ43 production 36 , consistent with the notion that multiple processes are involved in the generation of various Aβ species and that a partial loss of γ-secretase activity might give rise to a particular Aβ species. However, in vitro γ-secretase activity of heterozygous and homozygous PS1-R278I was drastically reduced in a gene-dose-dependent manner, while there were no significant differences of the steady state levels of total MEF-produced Aβ compared to wild type MEFs.…”

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

“…Previous reports have shown that FAD-linked mutations attenuate the effect of ␥-secretase inhibitors (32)(33)(34); however, these data were obtained with cells expressing both WT-PS1 and mutant PS1. We therefore reevaluated the effects of ␥-secretase inhibitors using MEFs expressing a single genotype of PS1.…”

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

“…El incremento de producción de β amiloide excretado en líneas transfectadas con mutaciones en presenilina, se ha reportado como un incremento de β amiloide 1-42 y 1-40 (35), mientras que otros autores indican que el incremento sólo se encuentra en el β amiloide 1-42 (10) o que el incremento en la producción de β amiloide 1-42 se produce a expensas (reduciendo la frecuencia) de especies más cortas de β amiloide (36).…”

“…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).…”

“…Otros autores frecuentemente prueban las mutaciones de presenilina 1 con células que poseen la proteína precursora de amiloide con la mutación sueca (KM595/596NL) (11,35,(40)(41)(42), que aceleran notablemente la producción de β amiloide 1-42 (43). En cambio, en nuestro trabajo, las células CHO-7W contienen la proteína precursora de amiloide silvestre.…”