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Comparative imaging study of the trafficking and processing of gamma-secretase substrates

Final Activity Report Summary - ITP-GAMMAS (Comparative imaging study of the trafficking and processing of gamma-secretase substrates)

Regulated intramembrane proteolysis (RIP) is an unusual proteolytic event that can abrogate or initiate downstream signalling by cleaving substrate proteins within their transmembrane domains. One such mechanism is governed by gamma-secretase that catalyses intramembrane cleavage of over 30 type I transmembrane proteins, including the amyloid precursor protein (APP), Notch, E-Cadherin, p75NTR and syndecans. Its activity is confined to a complex consisting of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2. The existence of two PS homologues (PS1 and PS2) and several Aph-1 isoforms suggest the presence of distinct complexes and this heterogeneity opens new avenues for selective inhibition.

The spatial and temporal localisation of substrates versus gamma-secretase is of major importance for the regulation of intramembrane proteolysis. Therefore, we proposed an imaging approach to compare the trafficking route of diverse substrates versus the gamma-secrestase complexes.

Confocal analysis of PS1 or PS2 knockout MEFs stably rescued with their respective fluorescently tagged human orthologues (EGFP-PS1 and EGFP-PS2) demonstrated significant differences on the relative distribution of PS1 vs PS2. PS1 is broadly distributed along the secretory and endocytic pathway whereas PS2 is essentially restricted to late endosomal/lysosomal compartments. Only limited levels of PS2, relative to PS1 and NCT, are present at the plasma membrane based on cell surface biotinylation and analysis of isolated plasma membrane sheets. Furthermore, PS2 when present at the plasma membrane does not colocalise with PS1 supporting the idea that they indeed associate with different microdomains. Together, our findings strengthen the hypothesis that the distinct subcellular distribution of gamma-secretase complexes can provide a basis for substrate and/or cleavage specificity. Secondly, we identified independent internalization routes for APP versus its processing enzymes BACE1 and gamma-secretase. As they likely meet downstream in early to late endosomes, we provide a mechanism to interfere with APP processing through modulating the specific entry routes of the secretases.

Both research lines are currently further explored using superresolution nanoscopy with the purpose of obtaining stoichiometric information on the composition and nanodomain association of gamma-secretase complexes.