C-terminal maturation fragments of presenilin 1 and 2 control secretion of APPα and Aβ by human cells and are degraded by proteasome

Abstract

Background: Most early-onset forms of Alzheimer's disease are due to missense mutations located on two homologous proteins named presenilin 1 and 2 (PS 1 and PS2). Several lines of evidence indicate that PS 1 and PS2 undergo various post-transcriptional events including endoproteolytic cleavages, giving rise to 28-30 kD N-terminal (NTF) and 18-20 kD C-terminal (CTF) fragments 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 βAPP maturation by CTF-PS1/PS2 and the catabolic process of the latter proteins by the multicatalytic complex, proteasome. Materials and Methods: We transiently and stably transfected HEK293 cells with CTF-PS1 or CTF-PS2 cDNA. We examined these transfectants for their production of Aβ40, Aβ42, and APPα 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 βAPP fragments by CTF-expressing cells was assessed by combined immunoprecipitation and densitometric analyses. Results: We showed that transient and stable transfection of CTF-PS1 and CTF-PS2 cDNAs in human cells leads to increased secretion of APPα and Aβ, the maturation products of βAPP. Furthermore, we demonstrated that 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 APPα and Aβ elicited by CTF-PS1/PS2. Conclusion: Our data establish that the C-terminal products of PS 1 and PS2 maturation exhibit biological activity and in particular control βAPP maturation upstream to α-and β/γ-secretase cleavages. This function is directly controlled by the proteasome that modulates the intracellular concentration of CTFs.