P3‐278: Effect of gamma‐secretase modulating drugs on Abeta isoform production rates from cultured cells using stable isotope labeling kinetics and mass spectrometry

Several drugs have been proposed to modulate the cleavage site of gamma-secretase within amyloid precursor protein giving rise to production of different ratios and amounts of amyloid-beta (Abeta) isoforms with various C-termini. However, studies to date have focused on static measurements of Abeta isoform concentrations in biological fluids such as cell culture media or brain homogenates. We have developed a stable isotope labeling kinetic (SILK-Abeta isoform) methodology for measuring production of the C-terminally differentiated Abeta isoforms and have applied this methodology to the measurement of production rates of these isoforms from neuroblastoma cells over-expressing APP. Cells are grown in unlabeled media for 24 hours after which they are switched to media that contains 50% conditioned, unlabeled media and 50% unconditioned 13C6 labeled media. As cells produce Abeta the stable isotope is incorporated into newly synthesized Abeta. By measuring the ratio of labeled to unlabeled Abeta for each of the C-terminally differentiated Abeta isoforms at 4, 8, 12 and 24 hours after the media change we can estimate the production curves for each of these isoforms. Subsequently, we tested the effects that a variety of publicly available gamma-secretase modulating drugs have on the production curves of the Abeta isoforms. Our data show that the SILK-Abeta isoform methodology allows for precise quantification of the rate of production of different Abeta isoforms in cultured cells. This method has application for measuring the in vivo metabolism of Abeta isoforms in humans.