Abstract
Processing of the amyloid precursor protein (APP) by γ-secretase results in generation of Aβ peptides of different lengths ranging from 51 to 30 residues. Accumulation of Aβ and in particular Aβ42 is enhanced by familial Alzheimer disease (FAD) causing mutations in APP and is believed to play a pivotal role. The molecular mechanism underlying normal Aβ production, the impact of FAD mutations on this process and how anti-amyloidogenic γ-secretase modulators (GSMs) cause a selective decrease in Aβ40 and Aβ42 and an increase in shorter Aβ peptides, however, is poorly understood. By using a combined immuno- and LC-MS-based assay we identify several major intermediates, i.e. 3- and 4-peptides that line up head to head across the entire APP transmembrane sequence from Aβ51 to Aβ31/Aβ30 and from Aβ49 to Aβ30/31. FAD APP mutations displayed a relative increase in 3- and 4-peptides from Aβ48 to Aβ38 compared with Aβ49 to Aβ37. These findings correlate with an increase in the Aβ42/40 ratio. GSMs caused a decrease in Aβ40 and Aβ42 and an increase in Aβ37 and Aβ38 paralleled by an increase of the intermediates Aβ40-38 and Aβ42-39. Collectively, these data provide a thorough characterization of all intermediate steps in Aβ production in native cell membranes and provide key mechanistic insights to genetic and pharmacological modulation of Aβ generation.
Highlights
The 42 amino acid long amyloid beta peptide (A42) plays a pivotal role in Alzheimer disease
By using a combined immuno- and LC-MS-based assay we identify several major intermediates, i.e. 3- and 4-peptides that line up head to head across the entire amyloid precursor protein (APP) transmembrane sequence from A51 to A31/A30 and from A49 to A30/31
We established an assay combining an immuno-based electrochemiluminescence assay (Mesoscale discovery technology, MesoScale Discovery (MSD)), and liquid chromatography-mass spectrometry (LCMS) to detect longer A peptides and to identify all smaller (3, 4, 5, 6, and 7-) peptides derived from APP processing (Fig. 1 and 2)
Summary
The 42 amino acid long amyloid beta peptide (A42) plays a pivotal role in Alzheimer disease. Results: A novel assay was developed and enabled us to describe the process of 〈42 production and modulation. Accumulation of A and in particular A42 is enhanced by familial Alzheimer disease (FAD) causing mutations in APP and is believed to play a pivotal role. The molecular mechanism underlying normal A production, the impact of FAD mutations on this process and how antiamyloidogenic ␥-secretase modulators (GSMs) cause a selective decrease in A40 and A42 and an increase in shorter A peptides, is poorly understood. GSMs caused a decrease in A40 and A42 and an increase in A37 and A38 paralleled by an increase of the intermediates A40 –38 and A42–39 These data provide a thorough characterization of all intermediate steps in A production in native cell membranes and provide key mechanistic insights to genetic and pharmacological modulation of A generation
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