P2‐317: Impact of different γ‐secretase modulators on Notch‐1 Abeta‐like peptide (Nβ) formation

Processing of amyloid ß-precursor protein (APP) into the Alzheimer related amyloid ß-peptide (Aß) is a proteolytic event mediated by the γ-secretase complex. γ-Secretase cleaves APP in two different sites, γ-site and the ε-site, generating Aß-peptides of different lengths and the APP intracellular domain. The γ-secretase complex processes several type I membrane bound proteins and among them is the Notch receptor. Notch is an important signalling molecule in many tissues and organs during development but also in adulthood. γ-Secretase processes Notch in a similar pattern as APP, generating an Nß peptide (S4-cleavage) and the Notch intracellular domain (S3-cleavage), which mediates the Notch signal. Since γ-secretase mediates cleavage of many substrates, such as the Notch receptor, studies in vivo have pointed to the importance of sustaining this pathway while inhibiting the Aß-secretion. A way of avoiding Notch inhibition is to use γ-secretase modulators (GSMs) instead of γ-secretase inhibitors. GSMs are generally considered to be compounds that do not affect the overall rate of Notch and APP processing (ε and S3-cleavage), but to change the proportions of the different Aß peptides that are formed. The pharmacology of these substances has been thoroughly studied regarding Aß generation but less is known about their effect on Nß. In this project we have set out to investigate how different structural classes of GSMs affect the γ-secretase processing of APP and Notch with respect to γ/S4 cleavage. Cultured medium from HEK-293 cells stably expressing APP or Notch are used to monitor the profile of different Nß or Aß peptides under the influence of various GSMs. To examine the peptides, antibody pull down followed by mass-spectrometry (MALDI-TOF) are used. We are in the process of investigating the impact of different classes of GSMs on Nß formation as well as the Aß-profile. The results from these experiments will be presented. By identifying how different structural classes of GSMs affect Nß-and Aß-profile modulation, we will get more insights into the mode of action of this new class of promising γ-secretase modulating compounds.