Human Molecular Genetics, 2001, Vol. 10, No. 16 1665-1671
© 2001 Oxford University Press
Pathogenic APP mutations near the 
-secretase cleavage site differentially affect Aß secretion and APP C-terminal fragment stability
Laboratory of Neurogenetics, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, University of Antwerp, Belgium, 1Laboratory of Neuronal Cell Biology, Department of Human Genetics, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Belgium and 2Institut de Pharmacologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Valbonne, France
Received April 5, 2001; Revised and Accepted June 5, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Release of amyloid ß (Aß) from the amyloid precursor protein (APP) requires cleavages by ß- and
-secretases and plays a crucial role in Alzheimer’s disease (AD) pathogenesis. Missense mutations in the APP gene causing familial AD are clustered around the ß-, 
- and particular -secretase cleavage sites. We systematically compare in primary neurons the effect on APP processing of a series of clinical APP mutations (two of which not characterized before) located in close proximity to the -secretase cleavage site. We confirm and extend previous observations showing that all these mutations (T714I, V715M, V715A, I716V, V717I and V717L) affect -secretase cleavage causing an increased relative ratio of Aß42 to Aß40. Taking advantage of these extended series of APP mutations we were able to demonstrate an inverse correlation between these ratios and the age at onset of the disease in the different families. In addition, a subset of mutations caused the accumulation of APP C-terminal fragments indicating that these mutations also influence the stability of APP C-terminal fragments. However, it is unlikely that these fragments contribute significantly to the disease process. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the amyloid precursor protein (APP) gene were the first to be recognized to cause early-onset autosomal-dominant Alzheimer’s disease (AD). Since the initial report of a pathogenic APP mutation (1), 10 different APP mutations have been identified in a limited number of early-onset AD families (2–10; C.Van Broeckhoven, unpublished data). All mutations cluster near the proteolytic cleavage sites in APP and were shown to affect the release of amyloidß (Aß) (6–8,11–18; C.Van Broeckhoven, unpublished data). Release of Aß involves successive cleavages by ß-secretase (BACE) and
-secretase. BACE cleaves APP between residues 671 and 672 (codon numbering of the APP770 isoform) or between residues 681 and 682 (19), leading to the secretion of the APP ectodomain (sAPPß) in the extracellular medium and to the retention of a 12 kDa cell-bound C-terminal fragment (CTFß or C99). Further cleavage of CTFß by -secretase, predominantly occurring between residues 711 and 712 (40-cleavage), and to a lesser extent between residues 713 and 714 (42-cleavage), results in the secretion of, respectively, a 40 and 42 amino acid Aß peptide (Aß40 and Aß42). The remaining 55–57 residue long CTF (CTF or C55/57, or AID:APP intracellular domain), is rapidly degraded (P.Cupers, W.Annaert and B.De Stooper, manuscript submitted for publication) and is only present at extremely low levels in vitro (20,21) and in vivo (22). The function of this fragment remains elusive. Alternatively, APP can be cleaved within the Aß sequence by -secretase, leading to the secretion of the APP ectodomain (sAPP) and the retention of a 10 kDa CTF (CTF or C83). Subsequent cleavage of CTF by -secretase leads to the production of a 3 kDa N-truncated amyloid peptide, named p3.
Presenilin (PS) proteins are required in -secretase-mediated cleavage of APP, since deletion of the PS1 gene inhibits Aß generation (23). Moreover, the finding that mutagenesis of critical aspartate residues (D257 and D385) in PS1 causes reduced Aß secretion and accumulation of APPCTFs led to the hypothesis that PS1 may exert an aspartyl protease activity and that it might be identical to -secretase (24). In addition to most APP mutations, PS interferes with -secretase activity in such a way that production of the highly amyloidogenic Aß42 is increased (25). Consequently, a leading hypothesis for AD etiology is ‘the amyloid cascade hypothesis’ (26) stating that increased Aß42 secretion represents a gain of misfunction.
However, it was reported recently that the APP V715M mutation affects Aß40 rather than Aß42 secretion (7). Moreover, the artifical putative active site D257A PS1 mutant causes accumulation of APPCTFs without a concomitant reduction in Aß secretion, indicating that accumulation of APPCTFs is not necessarily a direct consequence of decreased Aß production (27).
Recently, several novel mutations, clustered at the -secretase processing site of APP, have been identified (6–10). To compare their relative effects on -secretase cleavage, we expressed human APP695 with or without the clinical mutants T714I, V715M, V715A, I716V, V717I and V717L in primary cultures of cortical neurones and analysed the full scope of APP metabolites. We demonstrate that these mutations lead to significantly increased relative Aß42/Aß40 concentrations and that the level of increase is inversely correlated with the age at onset of AD. Furthermore, some but not all C-terminal APP mutations also influenced the turnover of APPCTFs, resulting in the accumulation of APPCTFs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Effect on Aß secretion
We systematically analysed the effect of a series of clinical mutations near the
-secretase cleavage site of APP. To this end, we expressed human wild-type APP or APP containing either the Austrian T714I (8), French V715M (7), German V715A (C.Van Broeckhoven, unpublished data), Florida I716V (6), Indiana V717L (9) or London V717I (1) mutation in primary cultures of neurons. We specifically immunoprecipitated Aß40 or Aß42 from the conditioned medium using antibody FCA3340 or antibody FCA3542, respectively (28). All APP C-terminal mutations with the exception of V715M, increased Aß1–42 secretion (Fig. 1). The increase in Aß1–42 ranged from 1.54-fold for T714I to 2.71-fold for V717L (Fig. 1C). This was accompanied by an increase in the N-truncated Aß-isoforms ending at residue 42 (Aßx-42). These isoforms run in the gels as doublet bands (Fig. 1B), and are presumably generated by alternative ß-secretase activity at amino acid residue 11 (19,29) or by -secretase activity at amino acid residue 17 (30) of the Aß sequence. Aß1–40, on the other hand, is decreased with most mutations. The T714I, V715M and V715A mutations reduce Aß1–40 secretion most drastically to 20, 30 and 55% of the wild-type levels, respectively. The V717I and V717L mutations affect Aß40 secretion to a lesser extent and the I716V mutation has apparently no effect on Aß40 secretion (Fig. 1C). The secretion of N-truncated Aß40 isoforms (Aßx-40) was decreased to an extent comparable to Aß1–40.
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Hence, when the Aß1–42/Aß1–40 ratio is compared to wild-type APP (arbitrarily set equal to 1) for each mutation, this ratio is increased in all APP mutations. This increase ranges from 1.89-fold for V717I to 8.20-fold for T714I (Fig. 2A). Moreover, the Aß1–42/Aß1–40 ratio correlates inversely (r = –0.84) with the mean age of onset for the different mutations (http://molgen-www.uia.ac.be/ADMutations/) (Fig. 2B).
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Effect on secretion of sAPP
We immunoprecipitated the secreted APP ectodomain from the conditioned medium of neurons transduced with SFV expressing wild-type or mutant APP using antibody 207. No significant differences in levels of sAPP between any of the mutants and wild-type APP were observed (data not shown), in agreement with the assumption that those mutations mainly affect
-secretase processing of APP.
Effect on the generation of APPCTFs
The CTFs (CTF and CTFß) that remain associated with the cells after -, or ß-secretase cleavage of APP were immunoprecipitated using antibody B11/4, directed against the last 20 amino acids of the cytoplasmic domain of APP. Mutations at codons 714 (T714I) and 717 (V717I and V717L) caused increased levels of APPCTFs (1.41–2.17-fold). The other mutations, at codons 715 (V715A and V715M) and 716 (I716V), displayed similar or even slightly decreased levels of APPCTFs compared with wild-type APP (Fig. 3).
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We further investigated whether the accumulation of APPCTFs observed with the T714I mutation was the consequence of reduced degradation or increased production. Therefore pulse-chase experiments were performed and the turnover rate of wild-type and mutant CTFs was assessed. As can be appreciated from Figure 4A and B, the turnover rate of the APP holoprotein is more or less identical for wild-type and mutant APP. In contrast, the turnover rates of wild-type APPCTFS or APPI716VCTFs is faster than the turnover rates of the APPCTFs containing the T714I or V717I mutation (Fig. 4B). Three hours after the pulse labeling, the level of APPCTFs is clearly decreasing with wild-type APP while APPCTFs continue to accumulate with the T714I or the V717I mutation. The degradation rate of I716V mutant APPCTFs was identical to the wild-type.
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DISCUSSION |
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The common feature of all APP C-terminal mutations tested in the current study, is the significant increase in the relative ratio of Aß42 to Aß40. By far the largest increase (8.20-fold) was observed with the T714I mutant, corroborating our previous data in HEK-293 cells (8). It is striking that this mutant is also clinically the most aggressive one and displays an unusually early age of onset of 34 years, compared to other APP mutations. It will be interesting to test this mutant in APP transgenic mice, anticipating the rapid development of amyloid plaques and disease in these animals. More importantly, by systematically analysing an extended series of C-terminal APP mutants in a single neuronal expression paradigm, we were able to demonstrate for the first time a clear inverse correlation between Aß42/Aß40 ratio and age of onset for the APP mutations, which strongly supports (a variant of) the ‘amyloid peptide hypothesis’. It should be mentioned that in APP V717I families age of onset is apparently modulated by the APOE genotype with APOE4 decreasing and APOE2 increasing the age of onset (31,32). The other APP mutations were found in single families and APOE data is too sparse to allow analysis of APOE effect on age of onset (http://molgen-www.uia.ac.be/ADMutations/). Our correlation data also contrast with the observation that in PS1 families, age of onset is not inversely correlated with Aß42 secretion (33–35). In these families, age of onset is also not influenced by APOE genotype (36). Remarkably, the observed correlation only holds true when the relative levels of Aß42 to Aß40 are considered. It is not completely clear why absolute amounts of Aß42 do not correlate with age at onset of disease. A ‘protective’ role of Aß40 seems unlikely since Aß40 levels by themselves are not correlated with age at onset of disease (data not shown).
At the level of -secretase processing, the different mutations apparently have slightly different effects. Mutations at codons 714 (T714I) and 715 (V715A and V715M) appear to primarily affect the 40-cleavage and cause a decreased secretion of Aß40. Mutations at codons 716 (I716V) and 717 (V717I and V717L) predominantly affect the 42-cleavage and increase Aß42 secretion. Hence, pathogenic APP mutations exert their largest effect on the cleavage site which is located two or three residues more N-terminal to the substitution (Fig. 5A). This suggests that the residues in the P3' and P4' positions are of particular importance for the exposure of the scissile bounds to the catalytic sites of the -secretase protease(s). Remarkably, the drastic decreases in Aß40 secretion observed with the mutations at codons 714 and 715 are not clearly accompanied by a large increase in Aß42 secretion. However, as we demonstrated previously for the APP T714I mutation (8), Aß isoforms ending at residues 39, 38 and to a lesser extent 37 may counterbalance the decrease in Aß40. Overall, it follows that the ratio of Aß42 to Aß40 is the best indicator of the severity of the disease in patients with APP mutations.
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Unexpectedly, APP C-terminal mutations also influence the levels of APPCTFs. The T714I, V717I and V717L mutations cause the accumulation of APPCTFs in neurons, whereas the V715A, V715M or I716V mutations have no significant effect on APPCTF levels. Both
- and ß-secretase-generated APPCTFs increase approximately to the same extent. This elevation is not clearly accompanied by increased secretion of the sAPP, indicating that the accumulation of APPCTFs is not the direct result of increased - or ß-secretase processing of APP. Increased levels of APPCTFs could also result from reduced -secretase activity (23). It could be envisaged that the presence of the C-terminal APP mutation reduces -secretase activity. Since Aß40 is the major Aß isoform produced, a specific reduction of the 40 cleavage could theoretically explain the accumulation of APPCTFs while maintaining Aß42 levels at a pathogenic level. However, the APP mutations that drastically decreased Aß40 secretion (V715A and V715M) did not display APPCTF accumulation, whereas the others (V717I and V717L) did not significantly affect Aß40 secretion but accumulated APPCTFs. Therefore, the effect of the latter mutations on APPCTF metabolism must occur independently from the -secretase cleavage step. Our pulse–chase data indicated a reduced turnover rate of these mutant CTFs, suggesting that these APP mutations not only affect -secretase processing, but also the catabolic pathways that degrade these fragments. Accumulation of APPCTFs was shown before with the Swedish and London APP mutants K670N/M671L, V717I, V717F and V717G (37), in agreement with our findings. McPhie et al. (37) proposed that APPCTFß is linked to a signalling transduction pathway. Accumulation of APPCTFß was speculated to disrupt this pathway, leading to cytoskeletal abnormalities and neurodegeneration. However, our data show that accumulation of APPCTFs is not a consistent feature of all APP mutants causing AD. In fact, mutations V717I and I716V result in a very similar mean age of onset, whereas APPCTFs accumulate with the former but not with the latter. Therefore, accumulation of APPCTFs is likely a less important factor in the overall pathogenesis of the disease. It would be of interest to investigate these different mutations at the pathological level in the brain of patients. Unfortunately, no material is yet available from patients with mutations that do not cause APPCTF accumulation, i.e. mutations at positions 715 or 716 of the APP sequence.
Finally, we propose a molecular model that integrates our observations. If one presumes an -helical conformation for the transmembraneous part of APP (38) (Fig. 5B), then the mutations at residues 714 and 715, which primarily affect 40-secretase cleavage, are located at one side of the helix, whereas mutations at residues 716 and 717, which predominantly affect 42-secretase cleavage, are located at the opposite side. It should be noted that the mutations at residues 714 and 717, leading to accumulation of APPCTFs, are located at one side of the helix, whereas mutations at residues 715 and 716, which do not lead to accumulation of APPCTFs are at the other side (Fig. 5B). Our recent unpublished data (W.Annaert and B.De Strooper) demonstrated that the region in APP spanning the currently investigated clinical mutations is involved in the direct binding of APP to PS1. We speculate, therefore, that the investigated clinical mutations cause subtle changes in the binding interaction between PS1 and APP, and therefore mainly affect exposure of the scissile bonds to the catalytic site of -secretase The abnormal accumulation of APPCTFs observed with some of the APP mutants could also fit this hypothesis, since the PS1 D257A catalytic site mutant leads to accumulation of APPCTFs without the concomitant reduction in Aß production (27).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Preparation of recombinant Semliki Forest virus
The Austrian T714I (8), German V715A (C.Van Broeckhoven, unpublished data), French V715M (7), Florida I716V (6) and Indiana V717L (9) mutations were introduced in human wild-type APP cDNA (695 isoform) by site-directed mutagenesis using appropriate oligonucleotides and the Quick-change mutagenesis system (Stratagene, La Jolla, CA). The mutant APP cDNA was subsequently cloned into the SmaI-site of pSFV-1 (Gibco BRL, Bethesda, MD). Recombinant Semliki Forest viruses were produced as described (39).
Primary neuronal cultures
Primary cortical neurons were isolated from E14 embryonic mice as described (39). Briefly, after dissection of the brain and dissociation of the cells by trypsinization, cells were plated on poly-lysine coated dishes and incubated in neurobasal medium + B27 supplement. Proliferation of non-neuronal cells was prevented by adding 5 µM cytosine arabinoside.
Transduction of primary neurons
Recombinant SFV was diluted 1:10 in conditioned culture medium and added to 3–5-day-old primary cortical neurons. After 1 h, medium was replaced with normal culture medium and transduction continued for 2 h. Medium was then replaced with methionine-free medium containing 100 µCi [35S]methionine (ICN, Irvine, CA). After 4 h of metabolic labelling, culture supernatants were collected, cells were washed in PBS and finally lysed in DIP buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS), containing protease inhibitors (5 mM EDTA, 100 U/ml trasylol, 1 µg/ml pepstatin).
Pulse–chase
Neurons were transduced with recombinant SFV as described above. Neurons were pulse-labeled with 100 µCi [35S]methionine for 30 min and chased for 60, 120 and 240 min. Cell extracts were processed as described below.
Immunoprecipitation and SDS–PAGE
Antibodies directed against Aß, 4G8 (1:200, Senetek, Napa, CA; recognizes total Aß), FCA3340 (1:200, specifically recognizes Aß40) (28) or FCA3542 (1:200, specifically recognizes Aß42) (28) or to the secreted ectodomain of APP (antibody 207, 1:200) (40) were added to the culture supernatants and immune complexes were recovered with protein G–Sepharose. Antibody B11/4 was used to precipitate and ß C-terminal fragments of APP from the cell extracts (41). Immunoprecipitated material was resolved on a 4–12% NuPAGE gel (Novex; Invitrogen, Carlsbad, CA) run with MES buffer. The intensity of the radioactive bands was quantified using a phosphoimager (Molecular Dynamics, Sunnyvale, CA) and the ImageQuant software package. All quantitative data were normalized to the signal obtained for the corresponding APP holoprotein to compensate for variations in expression between culture dishes or between the different APP mutants used.
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ACKNOWLEDGEMENTS |
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This work was supported by the Fund for Scientific Research-Flanders (FWO) and the Interuniversitary Attraction Poles (IUAP P4/17). C.D.J. and W.A.. are postdoctoral fellows of the FWO.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +32 16 346227; Fax: +32 16 347181; Email: ad@med.kuleuven.ac.be
To whom correspondence should be addressed. Tel: +32 16 346227; Fax: +32 16 347181; Email: ad@med.kuleuven.ac.be
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