Mutations in APP have independent effects on Aβ and CTFγ generation
Introduction
Production of Aβ42 is believed to be a central event in the pathogenesis of Alzheimer's disease (AD) (Golde et al., 2000, Sambamurti et al., 2002, Selkoe, 2001). Almost all mutations in the three genes (β-amyloid precursor protein—APP, presenilin1—PS1 and presenilin 2—PS2) that have been found to cause familial Alzheimer's disease (FAD) increase the ratio of Aβ42 to Aβ40. However, it is still unclear how FAD mutations alter the cleavage specificity toward longer Aβ species or how under normal conditions the production of the shorter Aβ40 peptide is favored.
Aβ is generated from the β-amyloid precursor protein (APP) as a result of proteolytic processing through the β-secretase pathway (Esler and Wolfe, 2001). The N-terminus of Aβ is produced by β-secretase cleavage (BACE1) (Sinha et al., 1999, Vassar et al., 1999, Yan et al., 1999), while generation of the C-terminus of Aβ involves an unusual intramembranous cleavage by a multi-protein γ-secretase complex (De Strooper et al., 1998, Steiner et al., 1999). γ-Secretase cleavage seems not to be site restricted since it generates Aβ fragments of 37–43 amino acids in length (Wang et al., 1996). γ-Secretase cleavage depends critically on the presenilins (De Strooper et al., 1998, Steiner et al., 1999). Two aspartate residues in TM domains 6 and 7 of PS are essential for activity and could represent part of the γ-secretase catalytic center (Berezovska et al., 2000, Kimberly et al., 2000, Steiner et al., 1999, Wolfe et al., 1999). Other components of the γ-secretase complex have also been identified: nicastrin, Aph1, and Pen2 (De Strooper, 2003, Edbauer et al., 2003, Francis et al., 2002, Goutte et al., 2002, Levitan et al., 2001, Steiner et al., 2002, Yu et al., 2000). Indeed, recent in vitro studies have demonstrated that these four proteins are sufficient to reconstitute γ-secretase activity (Edbauer et al., 2003). While the β-secretase pathway is favored in neurons, processing of APP by the α-secretase pathway is predominant in all other cell types. α-Secretase cleaves APP within the Aβ sequence (between amino acids 16 and 17 of Aβ), this is followed by γ-secretase cleavage generating an N-terminally truncated non-amyloidogenic version of the Aβ peptide called p3. Recent in vivo and in vitro studies have identified a PS-dependent C-terminal fragment (CTFγ/AICD) that may mediate APP signaling in a manner analogous to the NICD fragment of Notch (Kimberly et al., 2001, Leissring et al., 2002). Indeed, it has been shown that CTFγ enters the nucleus when bound to Fe65 protein and that this complex can activate transcription of reporter genes (Cao and Sudhof, 2001). Thus, it seems that both Aβ and CTFγ production may be important for understanding APP biology.
Although CTFγ is a C-terminal product of γ-secretase cleavage, it is not a direct product of cleavage at the γ40/42 site(s) but is generated by an additional cleavage event within the TM domain of APP that resembles the S3 cleavage site of Notch1 (Figs. 1A and B) (Gu et al., 2001, Sastre et al., 2001, Yu et al., 2001). The S3-like cleavage site of APP (also termed γ49 or ɛ site) has a similar primary sequence to the S3 site in Notch1 (Val at the P1′ position of the cleavage), is located close to the cytoplasmic side of the membrane, is presenilin-dependent, and is sensitive to γ-secretase inhibitors (Sastre et al., 2001, Yu et al., 2001, Weidemann et al., 2002). The major CTFγ fragment contains 50 amino acids (CTFγ50) (Sastre et al., 2001, Weidemann et al., 2002), although minor fragments of CTFγ48/49/51 have also been reported (Gu et al., 2001, Yu et al., 2001). Intramembranous cleavage at two topologically distinct sites (one in the middle of the TM domain that generates Aβ-like peptides and the other close to the cytoplasmic border that liberates intracellular domains-ICDs) has also been reported for other γ-secretase substrates: Notch1 and CD44 (Fig. 1A) (Kimberly et al., 2003, Lammich et al., 2002, Okochi et al., 2002).
The generation of CTFγ50 presents a puzzle since neither Aβ49, the N-terminal species left after γ-secretase cleavage at the S3-like site, nor CTFγ57/59 fragments, that would be generated by cleavage at the γ40/42 sites, have been detected. Several alternative mechanisms of Aβ and CTFγ generation have been suggested: cleavage at the γ40/42 site followed by aminopeptidase activity to generate CTFγ50, cleavage at the S3-like site followed by carboxypeptidase activity to generate Aβ40/42, or cleavage by γ-secretase at both the γ40/42 and S3-like site. Alternatively, γ40/42 and S3-like cleavage could be mediated by different enzyme activities that are both presenilin-dependent.
Since the γ40/42 site(s) and the S3-like site lie within the transmembrane domain of APP, we tested whether these cleavage events could affect each other and whether there could be a relationship between Aβ and CTFγ production. In addition, since all FAD mutations in the TM domain of APP lie close to or between the γ42 and S3-like site (T714–L723P) (Kwok et al., 2000), we investigated whether these FAD mutations also affect CTFγ production. We show that some familial Alzheimer's disease (FAD) mutations in APP that lie between these two cleavage sites can directly affect both γ40/42 cleavage and S3-like cleavage. Furthermore, we demonstrate that formation of CTFγ and Aβ can be dissociated: total CTFγ levels do not correlate with either an increase or decrease in Aβ-peptides and inhibition of Aβ-peptide formation by β-secretase cleavage site mutation does not affect CTFγ generation. These results support the notion that the presenilin-dependent dual intramembranous cleavages of APP are two separate events, thus APP signaling and Aβ production are not tightly linked. Our findings have implications for understanding the mechanism and the role of the PS-dependent dual intramembranous cleavage of APP and other γ-secretase substrates.
Section snippets
Cell culture and transfection
Human embryonic kidney 293 (HEK 293) cells were obtained from the ATCC (Rockville, MD) and PS1−/−PS2−/− mouse embryonic fibroblasts were provided by Dr. Bart De Strooper. Cells were grown in DMEM (GibcoBRL, Grand Island, NY) containing 10% fetal bovine serum (GibcoBRL), 2 mM l-glutamine, 100 μg/ml penicillin and streptomycin under a 5% CO2 atmosphere. Cells were transiently transfected using Fugene™ 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) and harvested 40–48 h
In vivo CTFγ assay
To enable analysis of CTFγ production in vivo (in the cell), we designed APP constructs containing a 6myc tag at their C-terminus. The C99+6myc or APPsw+6myc constructs were transiently cotransfected in PS1/2 KO cells with either wild type PS1, PS1-ΔE9 (a functional FAD-PS1 variant that does not go endoproteolysis) or a loss of function mutant (PS1-D257A) that has previously been shown to abolish Aβ and NICD formation. Processing of 6myc-tagged C99 (Fig. 2A) and APPsw (not shown) was analyzed
Discussion
Presenilin-dependent dual intramembranous cleavage has been described for several γ-secretase substrates: APP, Notch1, and CD44 (Gu et al., 2001, Kimberly et al., 2003, Lammich et al., 2002, Okochi et al., 2002, Sastre et al., 2001). One cleavage (in the middle of the TM domain) results in secretion of a hydrophobic peptide (Aβ/Nβ/CD44β) while the second cut (close to the cytoplasmic border) liberates the intracellular domain-ICD (AICD/NICD/CD44-ICD) for putative nuclear signaling. This finding
Acknowledgments
We thank Dr. R. Kopan for the pCS2+6MT vector, and for critical review of the manuscript and Drs. D. Selkoe, P. Seubert and P. Mathews for antibodies. We thank Anne Brunkan, Yonghua Pan, and Ma. Xenia U. Garcia for technical advice and Dr. Krista Moulder and K. Alexander Smith for help in confocal microscopy. S.H. is supported by a Fulbright Postdoctoral fellowship, The John Douglas French Alzheimer's Foundation and the Daniel Weinstock Research Fund for Alzheimer disease. This work was
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