C-terminal maturation fragments of presenilin 1 and 2 control secretion ofAPP alpha and A beta by human cells and are degraded by proteasome

Background: Most early-onset forms of Alzheimer's disease are due to missen se mutations located on two homologous proteins named presenilin 1 and 2 (P S1 and PS2). Several lines of evidence indicate that PS 1 and PS2 undergo v arious post-transcriptional events including endoproteolytic cleavages, giv ing rise to 28-30 kD N-terminal (NTF) and 18-20 kD C-terminal (CTF) fragmen ts that accumulate in vivo. Whether the biological activity of presenilins is borne by the processed fragments or their holoprotein precursor remains in question. We have examined the putative control of beta APP maturation b y CTF-PS1/PS2 and the catabolic process of the latter proteins by the multi catalytic complex, proteasome. Materials and Methods: We transiently and stably transfected HEK293 cells w ith CTF:PS1 or CTF-PS2 cDNA. We examined these transfectants for their prod uction of A beta 40, A beta 42, and APP alpha by immunoprecipitation using specific polyclonals. The effect of a series of proteases inhibitors on the immunoreactivity of CTF-PS1/PS2 was examined by Western blot. Finally, the influence of proteasome inhibitors on the generation of beta APP fragments by CTF-expressing cells was assessed by combined immunoprecipitation and d ensitometric analyses. Results: We showed that transient and stable transfection of CTF-PS1 and CT F-PS2 cDNAs in human cells leads to increased secretion of APP alpha and A beta, the maturation products of beta APP. Furthermore, we demonstrated tha t two proteasome inhibitors, lactacystin and Z-IE(Ot-Bu)A-Leucinal, prevent the degradation of both CTFs. Accordingly, we established that proteasome inhibitors drastically potentiate the phenotypic increased production of AP P alpha and A beta elicited by CTP-PS1/PS2. Conclusion: Our data establish that the C-terminal products of PS1 and PS2 maturation exhibit biological activity and in particular control beta APP m aturation upstream to alpha-and beta/gamma-secretase cleavages. This functi on is directly controlled by the proteasome that modulates the intracellula r concentration of CTFs.