Analysis of gamma secretase regulation by phosphatidylethanolamine using mammalian and Drosophila melanogaster in vitro and in vivo model systems

A?-amyloid peptide (A?), that plays a central role in the pathogenesis of Alzheimer’s disease is derived by sequential proteolytic processing from the amyloid precursor protein (APP) by ?- and ?-secretase. APP is additionally processed through a non-amyloidogenic pathway by ?-secretase. Recent work suggests that amyloidogenesis is highly dependent on the levels of cholesterol within plasma membrane/early endosomes’ microdomains termed “rafts”. Indeed APP cleaving machinery, required for A? generation has been shown to reside in lipid rafts and the secretase activity on APP to depend on membrane cholesterol levels. Counterintuitive to the localization of cleavage machinery, the substratum protein APP localizes, at constitutive levels of expression, in membrane microdomains enriched in phospholipids (PL), so-called non-raft domains. From these two series of results it arises that not only cholesterol-rich rafts but also cholesterol-poor/PL-rich non-rafts could be important modulators of AD implicated APP processing. In this work, I have addressed the question of how changes in the lipidic content of non-raft domains, where APP concentrates, affect proteolytic processing of this protein. As phosphatidylethanolamine (PE), an important regulator of diverse cell processes, accounts for the majority of PL I focused on the regulation of APP proteolyisis by this particular PL. For this purpose I utilized Drosophila melanogaster and mammalian model systems. Confirming previous work, APP was found in the non-raft domains of either insect or mammalian cells, excluded from cholesterol/ergosterol/Flotilin that enrich in rafts. The activity of ?-secretase on APP that is the crucial step in A? generation was assayed in Drosophila in vivo system using fly strains transgenic for human APP-C-terminal fragment, fused to the GAL4-VP16 (GV) transcription factor. Membrane PE levels in these ??secretase reporter flies were depleted by introducing the easPC80 mutation, that affects ethanolamine kinase (ETNK) an enzyme involved in PE synthesis pathway. A strong downregulation of hAPP-Ct processing by ?-secretase, readout by GV triggered cell lethal GRIM and GFP reporter genes expression was observed in low membrane PE flies compared to the ?-reporter flies with wild-type membrane PE levels. The effect of PE on APP proteolysis was additionally observed in mammalian HEK 293 cells stably expressing hAPP. In these cells membrane PE levels were altered by the treatment with RNAi directed against diverse PE synthesis enzymes including ETNK. Membrane PE level decrease, caused by RNAi treatment was shown to correlate with a downregulated ?- and ?-secretase processing of APP and correspondingly an elevated ?- secretase activity on APP. In the present study I could show that besides cholesterol/raft microdomains, PL (in particular PE), which are the major lipids in APP surrounding non-raft microdomains, appear to be involved in the regulation of APP proteolysis. From all the above I conclude that APP cleavage efficiency is highly dependent on the levels of the lipidic environment of non-raft domains, either because of affecting the degree of accessibility of the responsible cleaving enzymes to APP or by affecting the capacity of these enzymes to cleave the substratum. These are in my view important venues for future investigation, opened by this work