Search tips
Search criteria 


Logo of jbcThe Journal of Biological Chemistry
J Biol Chem. 2010 June 4; 285(23): 17798–17810.
Published online 2010 March 26. doi:  10.1074/jbc.M110.103283
PMCID: PMC2878544

β-Amyloid Precursor Protein Mutants Respond to γ-Secretase Modulators*


Pathogenic generation of the 42-amino acid variant of the amyloid β-peptide (Aβ) by β- and γ-secretase cleavage of the β-amyloid precursor protein (APP) is believed to be causative for Alzheimer disease (AD). Lowering of Aβ42 production by γ-secretase modulators (GSMs) is a hopeful approach toward AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the Aβ domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of γ-secretase, we analyzed mutations located at different positions of the APP transmembrane domain around or within the Aβ domain regarding their response to GSMs. We found that Aβ42-increasing familial AD mutations of the γ-secretase cleavage site domain responded robustly to Aβ42-lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of Aβ42 produced. We thus expect that familial AD patients carrying mutations at the γ-secretase cleavage sites of APP should respond to GSM-based therapeutic approaches. Systematic phenylalanine-scanning mutagenesis of this region revealed a high permissiveness to GSM-1 and demonstrated a complex mechanism of GSM action as other Aβ species (Aβ41, Aβ39) could also be lowered besides Aβ42. Moreover, certain mutations simultaneously increased Aβ42 and the shorter peptide Aβ38, arguing that the proposed precursor-product relationship of these Aβ species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in Aβ42 generation (Gly-29, Gly-33) and potentially in GSM-binding (Lys-28) were also responsive to GSMs, a finding that may question APP substrate targeting of GSMs.

Keywords: Alzheimer Disease, Amyloid, Aspartic Protease, Intramembrane Proteolysis, Secretases, GXXXG Motif, NSAID, Amyloid Precursor Protein (APP), Gamma-Secretase, Gamma-Secretase Modulator (GSM)


Alzheimer disease (AD)3 is the most common neurodegenerative disorder worldwide. The β-amyloid precursor protein (APP), a type I membrane protein, plays a central role in the pathogenesis of the disease (1). Sequential cleavage of APP by β- and γ-secretase generates the amyloid-β (Aβ) peptide, which deposits as plaques in the brain of affected patients and represents one of the principal pathological hallmarks of the disease (1). γ-Secretase is an intramembrane-cleaving protease complex, which cleaves the APP transmembrane domain (TMD) in a progressive, stepwise manner via cleavages at the ϵ-, ζ-, and γ-sites until it is sufficiently shortened to allow the release of Aβ from the membrane (2,4). Aβ peptides generated by γ-secretase cleavage differ in their C termini. The major product released is Aβ40, whereas Aβ38 and Aβ42 represent minor species (1). The highly aggregation-prone, neurotoxic Aβ42 is believed to be causative for AD by initiating a cascade of pathogenic events, which ultimately causes neurodegeneration and dementia (1). Increased production of Aβ42 underlies the vast majority of mutations associated with familial AD (FAD), which manifests with a very early disease onset. The majority of FAD mutations have been found in PS1, the catalytic subunit of γ-secretase (5), whereas only a few mutations were found in its homolog PS2. Few FAD-associated mutations were also found in APP, and those that affect γ-secretase cleavage toward an increased Aβ42 production localize to the C terminus of the APP TMD in the vicinity of the γ-secretase cleavage sites. Fluorescence resonance energy transfer-based studies suggest that changes in the generation of Aβ42 are due to alterations in the conformation of PS (6,10).

Inhibition of Aβ42 production is a major approach to therapeutically target AD. Selective Aβ42-lowering drugs, so-called γ-secretase modulators (GSMs) such as certain non-steroidal anti-inflammatory drugs (NSAIDs), have been identified as promising and attractive alternatives to inhibitors of γ-secretase, which target the active site and thus affect the processing of other physiologically important γ-secretase substrates, such as Notch1 (11). GSMs inhibit Aβ42 production with little effect on Aβ40 generation and the processing of other important γ-secretase substrates (12). Inhibition of Aβ42 by these compounds is accompanied by an increased production of Aβ38 (13). Because inverse modulators have also been identified (14), it was initially believed that the production of these peptides is interdependent, pointing to the possibility that Aβ42 might represent the precursor of Aβ38. Evidence has been presented that Aβ40 and Aβ42 derive from two different product lines by stepwise cleavage roughly every three residues at positions ϵ49–ζ46–γ43–γ40 and ϵ48–ζ45–γ42 of the Aβ domain, from which cleavages occur further downstream to generate Aβ39, Aβ38, and Aβ37 (15, 16), with Aβ38 primarily originating from the Aβ42-generating product line (16). In addition, dimerization of the APP TMD mediated by its central GXXXG helix-interaction motif has been suggested to affect the formation of Aβ42 (17). Substitution of the glycine residues to reduce APP TMD dimerization was shown to lower the production of Aβ42, whereas increasing that of Aβ38 (17). Mechanistically, it was suggested that dimerization via the GXXXG motif imposes a sterical hindrance for γ-secretase to proceed with stepwise processing such that Aβ40 and Aβ42 are normally released as final products. Decreasing dimerization strength would resolve this sterical constraint, now allowing γ-secretase to efficiently proceed to more N-terminal cleavage sites, thus generating shorter Aβ species, mostly Aβ38 (17). Interestingly, the GXXXG sequence is part of a GSM-binding site mapped to residues Aβ29–36 (GAIIGLMV) in the APP TMD (18). This region is known to be critical for Aβ aggregation (19,21), and aggregation inhibitors interacting with this region also act as GSMs (18). However, a recent study demonstrated that dimerization per se might not be a factor that determines γ-secretase cleavage specificity (22), and whether GXXXG mutants inhibit dimerization is controversial (23). In addition, the lack of a consistent response to GSMs regarding an inversely correlated production of Aβ42 and Aβ38 by PS FAD mutants observed earlier argued against a strict precursor-product relationship between Aβ42 and Aβ38 (24, 25). Moreover, while this manuscript was in preparation, a biophysical study failed to demonstrate GSM-binding to the APP substrate, however, and suggested that the reported GSM-APP interaction (18) was unspecific (26).

Interestingly, lowering of Aβ42 by GSMs is not effective for the majority of the PS FAD mutants investigated so far. In particular, aggressive FAD mutants that manifest with a very early disease onset due to their strongly increased Aβ42 production do not respond at all, whereas increased Aβ38 production was nevertheless still observed (24, 25). Whether or not APP FAD mutants respond to GSMs has not been conclusively studied yet, however (27). To address this question specifically as well as to gain a greater understanding of the mode of action of GSMs, in particular in light of the controversial data regarding the mechanism and binding site, we decided to study the action of GSMs on APP cleavage by γ-secretase in more detail. Analysis of FAD-associated APP mutations in the γ-secretase cleavage site region together with systematic phenylalanine-scanning mutagenesis of this region showed that all mutants respond robustly to the potent γ-secretase modulator GSM-1 (24), which was used as the principal GSM in this study, independent of the amount of Aβ42 produced by the mutant. Thus, unlike the majority of FAD mutants in PS1, APP TMD mutants at the γ-secretase cleavage sites are susceptible to GSMs, suggesting that the respective APP FAD mutant carriers should positively respond to GSM-based AD therapies. However, Aβ42 and Aβ38 generation was not strictly coupled, further arguing against a general mechanistic relationship for the generation of these Aβ species. Furthermore, GSM treatment was also found to lower Aβ39 and Aβ41 generation, demonstrating considerable imprecision and flexibility in the modulation of γ-secretase cleavage specificity in response to GSMs. Finally, we also investigated the impact of mutants close to or within the proposed GSM-binding site in the APP TMD. Because such mutants give rise to mutant Aβ species, these were also investigated in cell-free assays using purified γ-secretase and APP substrate (28) to circumvent potentially altered cellular metabolic fates of the mutant peptides in cultured cells. In both cell-based and cell-free assays, we found that GSM treatment could still effect changes in the ratios of Aβ species generated, a finding that may question GSM-binding within the Aβ domain.



Monoclonal antibody 2D8 against Aβ1–16 and polyclonal antibody 3552 to Aβ1–40 were described previously (29, 30). Monoclonal antibody 4G8 against Aβ17–24 was obtained from Covance. The C-terminal specific anti-Aβ38 antibody was obtained from Meso Scale Discovery, and C-terminal specific anti-Aβ40 (BAP24) and anti-Aβ42 (BAP15) antibodies were a kind gift of Dr. Manfred Brockhaus (Roche).

cDNA Constructs

cDNA encoding APPsw-6myc (31) was recloned into pcDNA3.1/zeo(+) (Invitrogen). The given mutations were generated in this and in the C99-6myc construct (31) by QuikChange mutagenesis (Stratagene) using oligonucleotide primers encoding the respective point mutations. Likewise, QuikChange mutagenesis was used to generate C100-His6 mutant substrate constructs in pQE60::C100-His6. These were expressed in Escherichia coli and purified as described (32).

Cell Culture and cDNA Transfections

Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin on poly-l-lysine-coated plates. Cells were plated at a density of 200,000 cells/24-well plate or 1,000,000 cells/6-well plate, and the following day, cells were transiently transfected with the indicated APP cDNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Suspension-adapted HEK293S cells were cultivated in instrumented 10-liter bioreactors using a Roche Applied Science proprietary serum-free hydrolysate-containing culture medium.

Analysis of Secreted Aβ from Cultured Cells

Following transfection, HEK293 cells were incubated for 24 h before media change and overnight incubation for 16 h in the presence of sulindac sulfide, flurbiprofen, or fenofibrate (all Sigma), GSM-1 (kind gift of Dr. Karlheinz Baumann, Roche), or vehicle control (dimethyl sulfoxide (DMSO)). Conditioned media were then collected and immediately analyzed by sandwich immunoassay to quantify Aβ species, or following immunoprecipitation, subjected to Tris-Bicine urea SDS-PAGE or mass spectrometry analysis. For Aβ quantitation, drug treatments were performed in triplicate, and all media samples were measured in duplicate for Aβ38, Aβ40, and Aβ42. For the analysis of modulation, data are always plotted as the percentage of change in the concentration of Aβ species from vehicle-treated cells, which are normalized to 100% for each cell line.

γ-Secretase in Vitro Assays

γ-Secretase was purified as described previously (28) except that HEK293S cells (kind gift of Georg Schmid and Elvira da Silva, Roche) were used as an enzyme source. In vitro γ-secretase activity was assessed as described using purified γ-secretase (Q-Sepharose eluate) and purified WT and mutant C100-His6 substrates in the presence or absence of GSM-1 or fenofibrate (28).

Quantification of Aβ

Secreted Aβ peptides in conditioned medium were quantified by a sandwich immunoassay using the Meso Scale Discovery SECTOR Imager 2400 as described previously (24). Quantification of total Aβ used essentially the same procedure except that for detection, 4G8 mouse monoclonal and ruthenylated anti-mouse antibodies were used in combination. For more sensitive detection of Aβ species (for the Phe mutants), the Meso Scale Discovery Aβ triplex sandwich immunoassay was used. Here, Meso Scale Discovery C-terminal specific antibodies were instead prespotted into each well, and a ruthenylated 6E10 antibody was used as the detection antibody. Meso Scale Discovery Aβ peptide standards were used for the Meso Scale Discovery triplex immunoassay. Aβ peptides generated by γ-secretase in vitro assays were quantified using the Meso Scale Discovery sandwich immunoassay as described (28).

SDS-PAGE and Mass Spectrometry Analysis of Aβ

Secreted Aβ was analyzed from medium conditioned overnight for 16 h by combined immunoprecipitation/immunoblotting using antibodies 3552/2D8 followed by Tris-Bicine urea SDS-PAGE (33). To analyze Aβ by mass spectrometry, Aβ species were immunoprecipitated from conditioned media or from γ-secretase in vitro assays using antibody 4G8 and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis as described previously (24, 28).


FAD Mutants in the γ-Secretase Cleavage Site Region Respond to GSMs

To address the question whether APP FAD mutants respond to GSM treatment, we introduced the Austrian T43I (T714I), Florida I45V (I716V), and London V46I (V717I) (34,36) mutations into APPsw-6myc, a well characterized and frequently used APP substrate (31), which was used as backbone for these and all other mutants of the APP TMD (Fig. 1A) analyzed in this study. The cDNA constructs were transiently transfected into HEK293 cells, and levels of secreted Aβ species generated by WT APP and the APP FAD mutants were analyzed by a highly sensitive specific Aβ immunoassay, which allows the detection and quantitation of Aβ38, Aβ40, and Aβ42 species (24, 29). As expected, the ratios of Aβ42 to total Aβ (i.e. the sum of Aβ38, Aβ40, and Aβ42) were increased for all mutants (Fig. 1B). The strongest increase was observed for the T43I mutant. Unexpectedly, this mutant also showed a strongly increased Aβ38/Aβtotal ratio, whereas that of Aβ40/Aβtotal was reduced. This behavior was not observed for the I45V and V46I FAD mutants and appeared to be characteristic for the T43I mutant. We thus conclude that certain FAD mutants occurring in the APP TMD can increase Aβ38 and Aβ42 in parallel.

Effect of GSM-1 on Aβ species of APP FAD mutants. A, schematic of the APP amino acid sequence encompassing the Aβ peptide and the transmembrane region of APP (underlined). Amino acids are numbered from position 1 of the Aβ peptide, ...

We next screened various GSMs to see whether the increased levels of Aβ42 produced from these mutants could be lowered. As shown in Fig. 1C, all GSMs were effective. GSM-1, a previously described GSM that is effective in the low micromolar range (24, 28), was overall the most potent compound with regard to modulation of the APP mutants. This GSM was capable of strongly reducing Aβ42 production from WT APP as well as from all three FAD mutants. The reduction of Aβ42 levels observed was ~90% as compared with the untreated controls. Consistent with previous results (24), GSM-1 increased the levels of Aβ38 produced from WT APP. Likewise, a robust increase of Aβ38 was also observed for the T43I, I45V, and V46I mutants. The NSAIDs sulindac sulfide and flurbiprofen were capable of reducing Aβ42 production for these mutants to a similar extent to WT APP, although flurbiprofen was more potent in this regard. Sulindac sulfide was more potent than flurbiprofen with regard to the modulation of Aβ38 levels, although all mutants responded by increasing Aβ38 upon treatment with either compound (Fig. 1C). As GSM-1 was the most effective modulator, we focused on this compound for the analysis of Aβ species by Tris-Bicine urea SDS-PAGE, which allows an effective separation of Aβ species. The modulatory effects were confirmed, and in addition, this analysis showed that the production of Aβ39 was reduced by GSM-1 treatment as well (Fig. 1D). Taken together, these data show that FAD mutants within the APP TMD that change the specificity of γ-secretase cleavage and thereby increase the production of Aβ42 are susceptible to different GSMs. GSM-1-mediated inhibition of Aβ42 generation is accompanied by an increased production of Aβ38, a strong inhibition of Aβ39, but has little effect on Aβ40. Since GSM-1 elicited the most potent effects with regard to both Aβ42 and Aβ38 modulation, this compound was used as the principal GSM for all subsequent modulation experiments in this study.

Phenylalanine Mutants of the γ-Secretase Cleavage Site Region Respond to GSM-1

To investigate whether mutations of the γ-secretase cleavage site region of APP are generally susceptible to GSM-1 as shown above for a subset of FAD mutants, we next analyzed previously described phenylalanine mutants, which span the region between the γ- and ϵ-cleavage sites and thus allow a systematic analysis of the GSM-1 response to mutants within this region (37, 38). These well characterized Phe mutants cover the sequence from Aβ43–51 of the Aβ domain and include two other FAD mutants, the Spanish I45F (I716F) (39) and the V46F (V717F) Indiana mutation (40). Analysis of the profile of the Aβ species generated by these mutants by Tris-Bicine urea SDS-PAGE was entirely consistent with that of the previous reports (37, 38) (Fig. 2A). Each mutant affected the profile of Aβ species produced by γ-secretase in an individual and characteristic manner. In agreement with the previous results (37, 38), the most striking changes were observed for the V44F, I45F, I47F, and V50F mutants. The I45F mutant produced the lowest amount of Aβ40 and the highest amount of Aβ42, whereas the V50F mutant produced almost exclusively Aβ40. Similarly, the V44F and I47F mutants produced very little Aβ42, but interestingly, both gave rise to the production of an alternative Aβ species, which migrated somewhat slower than Aβ42 and apparently represented Aβ41 (37, 38). Consistent with the previous reports (37, 38), the T43F–V46F mutants produced higher levels of the shorter Aβ variants Aβ38 and Aβ39 than the T48F–V50F mutants. The highest level of Aβ38 was observed for the V44F mutant, which did not generate detectable levels of Aβ39. Only small amounts of Aβ37 were produced for WT APP and all of the Phe mutants.

Effect of GSM-1 on Aβ species of APP phenylalanine mutants. A, Tris-Bicine urea SDS-PAGE immunoblot of Aβ species from cells expressing WT or the indicated phenylalanine mutants of APP. For Phe substitutions from positions 43–46, ...

Having confirmed the characteristic Aβ profile for each mutant, we next investigated whether and how the mutants would respond to GSM-1 treatment. Following drug treatment, changes in Aβ42 and Aβ38 were assessed by the Aβ immunoassay. As shown in Fig. 2B, as compared with the untreated controls, GSM-1 potently lowered the levels of Aβ42 of WT APP and all the Phe mutants by ~70–80%, even for the V44F mutant, which produced only extremely small amounts of Aβ42. Strikingly, the Phe mutants behaved differently in their response to GSM-1 with regard to its potency in increasing the levels of Aβ38 (Fig. 2B). Although WT APP showed the expected robust increase of Aβ38, the T43F, V44F, and I45F mutants were less responsive to GSM-1 treatment with respect to Aβ38. The FAD-associated V46F as well as the M51F mutant responded to GSM-1 similarly to WT APP. In contrast, GSM-1 treatment induced the production of considerably higher levels of Aβ38 in the I47F, T48F, L49F, and V50F mutants, the latter mutant showing the maximal increase of Aβ38 among these mutants (~6-fold increase as compared with control). Interestingly, Tris-Bicine urea SDS-PAGE analysis revealed that GSM-1 effected a strong decrease of Aβ41 for the V44F and of Aβ39 for the V46F mutant, which were selected to analyze the modulation of alternative Aβ species (Fig. 2C). Consistent with the results above, Aβ38 was increased for both mutants in response to GSM-1, and Aβ42 was decreased by GSM-1 treatment for the V46F mutant (Fig. 2C). Mass spectrometry analysis confirmed these results (Fig. 2D). Taken together, all APP Phe mutants responded robustly to the Aβ42-lowering capacity of GSM-1, further supporting our notion that Aβ42 produced from APP mutant carriers harboring Aβ42-increasing mutations in the γ-secretase cleavage site domain can be expected to be targetable by GSMs. Furthermore, these data show that additional γ-secretase cleavages can be modulated, suggesting substantial flexibility in the modulation of γ-secretase cleavage specificity.

Mutants of the GXXXG Motif in the APP TMD Respond to GSMs

We next investigated whether mutation of the glycine residues of the GXXXG motif, which had been implicated in the production of Aβ38 and Aβ42 (17) and which lie within the proposed GSM-binding site of APP (18), would affect GSM-induced changes of γ-secretase cleavage specificity. APPsw-6myc constructs containing the G29A, the G33A, or the stronger G33I substitution described previously (17) were transiently transfected into HEK293 cells, and the ratios of the secreted Aβ38, Aβ40, and Aβ42 species to total Aβ were examined by the Aβ immunoassay to assess changes in γ-secretase cleavage specificity. As shown in Fig. 3A, the G29A mutant showed a normal Aβ40/Aβtotal ratio but an increased Aβ38/Aβtotal ratio and a decreased Aβ42/Aβtotal ratio. The G33A mutant displayed an even higher Aβ38/Aβtotal ratio, but unlike the G29A mutant, it showed a normal Aβ42/Aβtotal ratio similar to that of the WT control. In further contrast to this mutant, a decreased Aβ40/Aβtotal ratio was immediately apparent for the G33A mutant, and this effect was observed even more strongly for the G33I mutant. For this mutant, Aβ38 became the principal species representing ~90% of the total Aβ measurable as compared with Aβ40, which represented only ~10% of total Aβ, whereas Aβ42 was almost undetectable. Thus, the G33I mutant showed a strong change in γ-secretase cleavage specificity, apparently at the expense of the normally major Aβ40 species. These results were confirmed using the C99-6myc substrate (31) to preclude any effects of dimerization of full-length APP via its ectodomain that may have affected its downstream processing and to rule out that the Swedish mutation at the β-secretase cleavage site of APPsw-6myc may have influenced the results (Fig. 3A). We next investigated whether the Gly mutants might also affect other Aβ species, which were not measured by our Aβ immunoassay. Tris-Bicine urea SDS-PAGE analysis was, however, hampered by an aberrant electrophoretic migration behavior of the mutant peptides, which precluded a clear assessment of the identity of the bands observed and revealed altered biochemical properties of the mutant peptides as compared with WT Aβ (data not shown). We therefore analyzed the profile of Aβ species by mass spectrometry, which confirmed the Aβ profiles of WT and the Gly mutants and additionally revealed substantial levels of Aβ37 and even shorter species for the G33I mutant (Fig. 3B) in full agreement with previous results (17). Thus, although overall the Gly mutants favor the production of shorter species, mostly Aβ38, they do not consistently change the cleavage specificity of γ-secretase toward a reduced production of Aβ42 but rather affect that of Aβ40. This was also reflected by the average amounts of the individual Aβ species measured in additional experiments (Table 1).

Profile of Aβ peptides generated in cells from APP GXXXG mutants and their response to GSMs. A, sandwich immunoassay of Aβ38, Aβ40, and Aβ42 species that were isolated from conditioned media of cells overexpressing WT and ...
Levels of the individual Aβ peptides (Aβ38, Aβ40, Aβ42) measured for mutants of the proposed GSM-binding site

Having determined the changes in γ-secretase cleavage specificity for the Gly mutants, we next asked whether they would show a GSM response. Aβ42 levels could be robustly lowered for the G29A and G33A mutants by GSM-1 treatment (Fig. 3C). Concomitantly, GSM-1 treatment caused an increase of Aβ38 levels for the G29A and G33A mutants. Probably due to the higher Aβ38 starting levels of the mutants, the increase was attenuated as compared with WT APP. Given its favored production of shorter Aβ species such as Aβ38, the G33I mutant was tested for its ability to respond to the inverse GSM fenofibrate (Fig. 3D). This showed that fenofibrate treatment caused a dramatic increase of Aβ42 (Fig. 3D), which had almost been undetectable for this mutant at baseline (see above). Furthermore, Aβ38 could still be lowered, despite its high baseline level for this mutant. Surprisingly, Aβ40 was increased in response to fenofibrate treatment as well (Fig. 3D), a modulation that, to our knowledge has so far not been observed for inverse GSMs. Thus, these data show that substitution of the glycine residues of the GXXXG motif does not interfere with the modulation of Aβ levels by GSMs.

APP TMD Mutants Respond to GSMs in a Cell-free γ-Secretase Assay Using Purified Components

A potential complication in the analysis of Aβ produced from Gly mutant APP is that these particular Aβ peptides are mutated within the Aβ domain itself and that their altered biochemical properties might potentially have an impact on the levels of Gly mutant Aβ peptides detectable in cultured cells downstream of production (e.g. altered secretion, enhanced degradation, or aggregation). In particular, the G33I mutant Aβ42 has been described as a highly aggregation-prone peptide (21). We therefore also carried out cell-free assays using purified recombinant APP C-terminal fragment-based C100-His6 substrates containing selected Phe mutants, the G29A, G33A, or G33I mutants, and purified lipid-reconstituted γ-secretase (28). We first monitored the production of Aβ peptides from C100-His6 substrates containing the I45F or V50F mutants, i.e. substrates that should generate high amounts of both Aβ38 and Aβ42 and low amounts of Aβ40 (I45F) or almost exclusively Aβ40 (V50F). Importantly, in contrast to the Gly mutants within the Aβ domain, Aβ species generated from the Phe mutants are WT in sequence. Therefore, altered effects with respect to the cellular metabolism of Aβ do not apply to these mutations. As shown in Fig. 4A, the production of Aβ38, Aβ40, and Aβ42 peptides as assessed by Aβ immunoassay from WT, I45F, and V50F C100-His6 substrates was consistent with the results obtained for these mutants in cultured cells and the previous reports by others (37, 38). We next monitored whether the I45F and V50F mutants would respond to GSM-1 treatment, which indeed proved to be the case (Fig. 4B). The responses of the two mutants to GSM-1 with respect to Aβ42 and Aβ38 generation were very similar to those observed in cultured cells, thus confirming the results described above and validating the cell-free assay for the analysis of APP TMD mutant substrates.

Validation of cell-free assay system concerning the cleavage specificity of purified γ-secretase on mutant APP C-terminal fragment based C100-His6 substrates. A, sandwich immunoassay of Aβ38, Aβ40, and Aβ42 species that ...

Analysis of the Aβ species generated in the in vitro assay revealed that the Gly mutants caused an increased relative production not only of Aβ38, but surprisingly also of Aβ42, whereas concomitantly reducing that of Aβ40 (Fig. 5A), although we noticed a greater variability of G33I mutant Aβ42 in this assay (Table 1). Mass spectrometry analysis of mutant Aβ peptides confirmed this unexpected result and revealed that G29A, and in particular the G33I mutant, additionally generated Aβ37. Although Aβ42 was robustly produced by γ-secretase for the G29A, G33A, and G33I mutants, it was less well detectable by mass spectrometry for the latter mutant. This is most likely because of very low ionization efficiency due to the increased hydrophobicity of this mutant peptide (Fig. 5B). Thus, although increased generation of Aβ38 from Gly mutants was a consistent observation in both cultured cells and the cell-free in vitro assay using purified components, the Aβ42 levels detected differed between these two systems. Assessment of Aβ production in the latter assay system shows that γ-secretase can in fact generate substantial amounts of Aβ42 from the Gly mutant substrates as solely Aβ production is assessed in this system.

Profile of Aβ peptides generated in vitro from purified C100-His6 GXXXG mutants and their response to GSMs. A, sandwich immunoassay of Aβ38, Aβ40, and Aβ42 species that were generated in cell-free assays (28) from WT and ...

We next assessed the response of the Gly mutants to GSM-1. Consistent with previous results (28), WT APP showed a clear response with lowered Aβ42 and increased amounts of Aβ38. Again, GSM-1 effected a change in the Aβ species produced by the Gly mutants by lowering the production of Aβ42 and increasing that of Aβ38 (Fig. 5C). This was clearly observed for the G29A mutant and in attenuated form for the G33A mutant (Fig. 5C) but unexpectedly, not observed for the G33I mutant, which instead showed an ~20% reduction of Aβ40 in response to GSM-1 (data not shown). Although Aβ42 production could not be increased further by fenofibrate treatment for the G33I mutant, it responded effectively to this inverse GSM with respect to both Aβ38 and Aβ40 generation (Fig. 5D), similar to the results obtained for this mutant in cultured cells. Taken together, these data show that the Gly mutants are susceptible to pharmacological modulation of γ-secretase cleavage specificity both in cultured cells as well as in the cell-free system, despite differences in the relative amounts of each Aβ species generated.

The K28E APP Mutant Responds to GSMs

It has been speculated that GSMs may shift the position of the substrate in the membrane plane relative to the γ-secretase active site. Aβ42-raising GSMs and Aβ42-lowering GSMs would either pull the substrate out from the membrane or, respectively, sink it into the membrane (18). Interestingly, all so far described Aβ42-lowering GSMs have a carboxyl group, whereas Aβ42-raising GSMs lack this group. The positively charged lysine 28 residue of the Aβ domain located directly at the extracellular border of the membrane could potentially form a salt bridge with the carboxyl group of Aβ42-lowering GSMs, which as a consequence might position the γ42 site away from the γ-secretase active site. To address such a potential mechanistic contribution of Lys-28 in GSM binding, we substituted this residue by the negatively charged glutamate. As shown in Fig. 6A, assessment of the Aβ ratios revealed that Aβ38 was the major species detected for the K28E mutant (~60% of the total Aβ). The Aβ40/Aβtotal ratio was strongly reduced, as was the Aβ42/Aβtotal ratio, indicating a strong preference for γ-secretase cleavage at the γ38 site (see Table 1 for average Aβ levels measured in additional experiments). Mass spectrometry confirmed this result and additionally revealed a substantial increase of the shorter Aβ species Aβ37 and Aβ33 (Fig. 6B). Despite the already high Aβ38 levels, they could still be significantly increased by GSM-1 treatment (Fig. 6C). The levels of Aβ42 were too low such that changes were at the limit of detection, and no significant changes were observed for the levels of Aβ40 (data not shown). These data suggest that the proposed electrostatic interaction does not play an essential role for the mechanism of γ-secretase modulation. Finally, treatment of the K28E mutant with fenofibrate showed that Aβ42 could be tremendously increased, whereas Aβ38 could still be substantially lowered. Interestingly, fenofibrate caused an increase of Aβ40 as well (Fig. 6D). This result shows that the K28E mutant responds to GSMs of two different classes.

Profile of Aβ peptides generated in cells from the APP K28E mutant and its response to GSMs. A, sandwich immunoassay of Aβ38, Aβ40, and Aβ42 species that were isolated from conditioned media of cells overexpressing WT and ...

As above, to investigate modulation of γ-secretase cleavage by an independent assay, which assesses production in the absence of cellular metabolism, we also investigated the K28E mutant in the in vitro system using a C100-His6 K28E mutant substrate. In the cell-free assay, a slightly increased Aβ42/Aβtotal ratio and a decreased Aβ40/Aβtotal ratio were observed for this mutant, whereas the Aβ38/Aβtotal ratio was similar to that of the WT substrate (Fig. 7A; see Table 1 for average Aβ levels of all experiments performed). Mass spectrometry confirmed this Aβ profile (Fig. 7B). Thus, unlike in cultured cells, the K28E mutant behaved as a comparably normal γ-secretase substrate in the cell-free assay. GSM-1 behaved as a modulator on the K28E mutant, lowering Aβ42 and increasing Aβ38 production. As compared with WT, the response of the K28E mutant to GSM-1 was attenuated, however (Fig. 7C). As shown in Fig. 7D, we also observed inverse modulation for the K28E mutant with fenofibrate. Taken together, despite the observed differences in the amounts of Aβ species detected in cultured cells versus the cell-free system, the K28E mutant was clearly responsive to GSMs both in cultured cells and in vitro using purified components, suggesting that Lys-28 of the Aβ domain is unlikely to play a major role for the action of GSMs.

Profile of Aβ peptides generated in vitro from the C100-His6 K28E mutant and its response to GSMs. A, sandwich immunoassay of Aβ38, Aβ40, and Aβ42 species that were generated in cell-free assays (28) from C100-His6 WT and ...


In this study, we have investigated the impact of GSMs on a variety of APP mutants to better understand their mode of action as well as the cleavage mechanism of γ-secretase. A summary of our principal findings is shown in Table 2. We establish that naturally occurring pathogenic mutations of the γ-secretase cleavage site region in the APP TMD, which affect the precision of γ-secretase cleavage toward an increased production of Aβ42, respond to GSM treatment. These mutants even respond as strongly as WT APP following treatment with GSM-1, a well characterized potent GSM (24), which was selected as the principal GSM in this study. FAD patients with mutations in APP that affect γ-secretase cleavage should therefore be susceptible to GSM treatment. In addition, phenylalanine-scanning mutagenesis analysis of the γ-secretase cleavage site region revealed responsiveness to GSM-1 for all mutants. We found that even very strong mutants among those, such as the I45F mutant that produces high amounts of Aβ42, responded to GSM-1. The I45F mutant represents the most aggressive APP FAD mutation identified so far with an extremely early disease onset of 31 years (39). Based on our data, we therefore conclude that treatment with GSMs might provide a successful therapeutic option also for these mutant carriers and others of the γ-secretase cleavage site domain with pathogenic Aβ42 production that may be identified in the future. Interestingly, our results contrast with those obtained recently for PS mutations (24, 25). Most of the PS FAD mutations investigated were not responsive to NSAIDs that act as Aβ42-lowering GSMs, such as sulindac sulfide. In particular, very strong and aggressive mutations producing high levels of Aβ42, such as PS1 L166P, and others, were not susceptible to the Aβ42-lowering capacity of sulindac sulfide. This mutant was also not responsive to the more potent GSM-1 (24). Thus, our data indicate that the mode of action of GSMs is different for FAD mutants of APP and PS, i.e. different for substrate and protease. We also note that AD mouse models expressing human APP FAD mutants as transgene, such as some of the ones investigated here (e.g. V46I or V46F), should be more suitable for in vivo validation of GSMs than models that additionally express strong PS FAD mutations.

Summary of mutations, their effects on Aβ generation, and their responsiveness to modulation

Recent data suggested that GSMs target the substrate rather than the protease by binding to the Aβ domain at residues 29–36 (18). Thus, it appeared possible that the modulatory capacity of GSMs could be different from that observed for the protease if they were targeting the substrate. Moreover, the GXXXG motif lying in this region was shown to determine Aβ42 and Aβ38 production in an inverse and interdependent manner via dimerization of the TMD. Our data do not support a mechanistic coupling of Aβ38 and Aβ42 production, however. The T43I and I45F (FAD) mutants both simultaneously increased Aβ38 and Aβ42 production and lowered that of Aβ40, suggesting that the production of Aβ38 and Aβ42 is not necessarily coupled in an inverse manner. Furthermore, the analysis of Phe mutants showed that although Aβ42 production was lowered for all mutants upon GSM-1 treatment, the concomitant increase in Aβ38 production was attenuated for T43F, V44F, and I45F mutants, indicating uncoupling effects. Interestingly, for the T43I, I45V, V46I, and V46F FAD mutants, the increased production of Aβ38 induced by GSM-1 was accompanied by a reduced production of Aβ39 in addition to Aβ42, which might indicate a (precursor-product) relationship between these two peptides. Moreover, as evident for the V44F mutant, which produced a high amount of Aβ41, we found that also, this Aβ species could be lowered by GSM-1 treatment. Thus, not only Aβ42 production, but also Aβ39 and Aβ41 production, can be lowered in response to a GSM. Taken together, these data are difficult to reconcile with the model that production of Aβ42 and Aβ38 is interdependent. The relationship between Aβ42 and Aβ38 is apparently more complex, and production of Aβ38 from Aβ42 (16) may in fact occur only in the WT situation.

Although our results obtained for mutants of the GXXXG dimerization motif are to a large extent consistent with previously reported findings (17), we noted some differences. Although we observed an increase in Aβ38 for all mutants, we did not detect a consistent concomitant change in Aβ42 with respect to both Aβ42/Aβtotal ratios and absolute levels of this species. As particularly evident for the G33A and the G33I mutants, the increase in Aβ38 rather correlated with reduced Aβ40 levels. The decrease of Aβ40 may be explained for the G33I mutant by the concomitant increase of Aβ37, which could be derived from the Aβ40 product line but not for the G33A mutant, where the Aβ37 increase was not observed. The Gly mutants were responsive to GSM-1, which effectively lowered Aβ42 for G29A and G33A, while increasing Aβ38 levels. Likewise, the G33I mutant, which showed the highest Aβ38 levels among the Gly mutants investigated, responded effectively to the inverse GSM fenofibrate, which lowered Aβ38 and increased Aβ42 to detectable levels for this Aβ38-biased mutant. Interestingly, fenofibrate also effected a strong increase of Aβ40 for this mutant. Such a property has to our knowledge not yet been observed for an inverse GSM. These observations suggest that the glycine residues in the proposed GSM-binding site of APP do not play an essential role for potential GSM binding at this site. In addition, these data suggest that pharmacological modulation of γ-secretase cleavage specificity appears not to be linked in an essential manner with GXXXG-dependent APP TMD interactions, whether involving dimerization or not (17, 23).

Because the GXXXG mutant substrates give rise to mutated Aβ peptides, whose altered biochemical properties may affect the levels detectable in cultured cells downstream of production, we also analyzed these using our recently described validated cell-free in vitro system consisting of purified γ-secretase and purified APP substrate (28). In this assay system, solely the production of Aβ is analyzed independent of e.g. altered secretion or degradation. As compared with cultured cells, similar results were obtained regarding the production of Aβ38 and Aβ40 in this system. In contrast, however, the rather substantial Aβ42 production observed for all mutants in the cell-free system using purified components shows that γ-secretase can per se generate Aβ42 from Gly mutant substrates. It is important to note that the cell-free assay system was fully validated by the I45F and V50F mutant substrates. These mutants represent two extremes of APP mutants regarding the production of Aβ38 and Aβ42 and, in contrast to the Gly mutants, generate Aβ38, Aβ40, and Aβ42 peptides without internal mutations. Both mutants behaved exactly as in cultured cells, proving that the γ-secretase enzyme itself is in the correct conformation in the in vitro assay and thus further validating the cell-free assay used. It remains possible that the altered biochemical properties of Gly mutant Aβ42 as compared with WT Aβ peptides may differentially affect the fate of this peptide in the two systems and thus account for the observed differences. Alternatively, it is also possible that slight conformational alterations may occur for substrates carrying mutations within the Aβ domain regarding the γ42 site cleavage, such as that of certain Gly mutant substrates, in the in vitro system. In agreement with the modulation results from cultured cells, responsiveness to GSM-1 was also observed for the Gly mutants in the cell-free system, although differences regarding the respective GSM response were noticed for the G33I mutant.

An interesting residue that might contribute to GSM binding in the APP TMD is lysine 28. This residue, which lies directly adjacent to the GSM-binding site in the APP TMD, might form a salt bridge between the positively charged ϵ-amino group of the lysine side chain and the negatively charged carboxyl group of GSMs, which is essential for their Aβ42-lowering activity (41). This ionic interaction might change the position of the APP TMD relative to the active site of γ-secretase, thus mediating a change in its cleavage specificity. However, our data show that the K28E mutant was susceptible to GSM-1 treatment, suggesting that an ionic interaction mediated by Lys-28 does not contribute to a potential GSM-APP interaction. The K28E mutant was also responsive to the inverse GSM fenofibrate, which lacks the carboxyl group and thus is apparently effective in the absence of an ionic interaction. Interestingly, mass spectrometric analysis revealed Aβ33 and Aβ37 as major Aβ species (species that are not detected by our Aβ immunoassay), possibly indicating that the K28E mutant Aβ might be turned over to shorter Aβ peptides in cultured cells. Analysis of the K28E mutant substrate in the cell-free system, however, i.e. in the absence of cellular metabolism, revealed that this mutant is normally processed by purified γ-secretase with only minor changes in the profile of Aβ species as compared with the WT APP control. Importantly, GSM-1 was also effective on the K28E mutant in this system, further suggesting that the membrane-flanking lysine residue does not play a major role for the mode of action of carboxyl group-containing GSMs.

With respect to the mechanism of γ-secretase cleavage, our data show an uncoupling of Aβ38 and Aβ42 generation for APP mutations located at different sites in the APP TMD, including mutations at or within the proposed GSM-binding site. All mutants allow a change of γ-secretase cleavage specificity with respect to the generation of Aβ38 and Aβ42 in response to GSMs. GSM-mediated modulation of γ-secretase cleavage specificity was shown to occur largely independent of the glycine residues of the GXXXG motif within the proposed GSM-binding site of APP, which were implicated in the generation of Aβ42 (17). Although these data do not entirely exclude GSM binding to this site, they suggest that the glycine residues are unlikely to play an essential mechanistic role for the mode of action of GSMs, irrespective of the current controversy regarding the GSM-APP interaction (18, 26). The GSM response of APP TMD mutants shown here, irrespective of their site and the amounts of Aβ42 generated, may favor a substrate-independent targeting mechanism of GSMs. Binding studies with more potent high affinity GSMs rather than the currently existing low affinity compounds (18) will provide important answers by clarifying whether GSMs target the enzyme, which was initially suggested by several previous studies (6, 27, 42,44). Mechanistically, NSAIDs that lower Aβ42 were suggested to allosterically alter the conformation of PS (6, 10, 44), and conformational changes of PS opposite to that induced by such NSAIDs were also observed for PS FAD mutants (6, 7, 10). It is thus conceivable that many aggressive PS FAD mutations are locked in a conformation that makes the PS-substrate interaction refractory to the Aβ42-lowering capacity of GSMs (24, 25). Clearly, as shown in this study, mutations in the APP substrate are permissive to GSMs, suggesting that the substrate is conformationally more flexible than the γ-secretase enzyme, allowing APP substrate positioning such that the γ42 site is less exposed to the active site of γ-secretase. Thus, unlike the situation for PS FAD mutants, AD mouse models carrying APP FAD mutant transgenes should be useful for the in vivo evaluation of GSMs, and APP FAD mutant carriers are expected to be susceptible to GSM-based therapeutic strategies for AD treatment.

Supplementary Material

Author profile:


We thank Alison Goate for APPsw-6myc and C99-6myc constructs, Karlheinz Baumann for GSM-1, Manfred Brockhaus for the Aβ40 and Aβ42 specific antibodies, Georg Schmid and Elvira da Silva for HEK293S cells, and Gabriele Basset for technical assistance. We also thank Stefan Lichtenthaler for critical reading of the manuscript and helpful discussion.

*This work was supported by the Deutsche Forschungsgemeinschaft (Collaborative Research Center (SFB596) “Molecular Mechanisms of Neurodegeneration” (to H. S. and C. H.)) and the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (“Degenerative Dementias: Target identification, Validation and Translation into Treatment Strategies” (to C. H. and H. S.)), the Alzheimer Research Award of the Hans and Ilse Breuer Foundation (to H. S.), and the Center for Integrated Protein Science Munich.

This article was selected as a Paper of the Week.

3The abbreviations used are:

Alzheimer disease
familial AD
amyloid β-peptide
β-amyloid precursor protein
γ-secretase modulator
non-steroidal anti-inflammatory drug
transmembrane domain
wild type
human embryonic kidney


1. Haass C., Selkoe D. J. (2007) Nat. Rev. Mol. Cell. Biol. 8, 101–112 [PubMed]
2. Steiner H., Fluhrer R., Haass C. (2008) J. Biol. Chem. 283, 29627–29631 [PMC free article] [PubMed]
3. Tolia A., De Strooper B. (2009) Semin. Cell. Dev. Biol. 20, 211–218 [PubMed]
4. Wolfe M. S. (2009) Semin. Cell. Dev. Biol. 20, 219–224 [PubMed]
5. Steiner H. (2008) Curr. Alzheimer Res. 5, 147–157 [PubMed]
6. Lleó A., Berezovska O., Herl L., Raju S., Deng A., Bacskai B. J., Frosch M. P., Irizarry M., Hyman B. T. (2004) Nat. Med. 10, 1065–1066 [PubMed]
7. Berezovska O., Lleo A., Herl L. D., Frosch M. P., Stern E. A., Bacskai B. J., Hyman B. T. (2005) J. Neurosci. 25, 3009–3017 [PubMed]
8. Tesco G., Ginestroni A., Hiltunen M., Kim M., Dolios G., Hyman B. T., Wang R., Berezovska O., Tanzi R. E. (2005) J. Neurochem. 95, 446–456 [PubMed]
9. Herl L., Thomas A. V., Lill C. M., Banks M., Deng A., Jones P. B., Spoelgen R., Hyman B. T., Berezovska O. (2009) Mol. Cell. Neurosci. 41, 166–174 [PMC free article] [PubMed]
10. Uemura K., Lill C. M., Li X., Peters J. A., Ivanov A., Fan Z., DeStrooper B., Bacskai B. J., Hyman B. T., Berezovska O. (2009) PLoS One 4, e7893. [PMC free article] [PubMed]
11. Tomita T. (2009) Expert Rev. Neurother. 9, 661–679 [PubMed]
12. Weggen S., Eriksen J. L., Sagi S. A., Pietrzik C. U., Golde T. E., Koo E. H. (2003) J. Biol. Chem. 278, 30748–30754 [PubMed]
13. Weggen S., Eriksen J. L., Das P., Sagi S. A., Wang R., Pietrzik C. U., Findlay K. A., Smith T. E., Murphy M. P., Bulter T., Kang D. E., Marquez-Sterling N., Golde T. E., Koo E. H. (2001) Nature 414, 212–216 [PubMed]
14. Kukar T., Murphy M. P., Eriksen J. L., Sagi S. A., Weggen S., Smith T. E., Ladd T., Khan M. A., Kache R., Beard J., Dodson M., Merit S., Ozols V. V., Anastasiadis P. Z., Das P., Fauq A., Koo E. H., Golde T. E. (2005) Nat. Med. 11, 545–550 [PubMed]
15. Qi-Takahara Y., Morishima-Kawashima M., Tanimura Y., Dolios G., Hirotani N., Horikoshi Y., Kametani F., Maeda M., Saido T. C., Wang R., Ihara Y. (2005) J. Neurosci. 25, 436–445 [PubMed]
16. Takami M., Nagashima Y., Sano Y., Ishihara S., Morishima-Kawashima M., Funamoto S., Ihara Y. (2009) J. Neurosci. 29, 13042–13052 [PubMed]
17. Munter L. M., Voigt P., Harmeier A., Kaden D., Gottschalk K. E., Weise C., Pipkorn R., Schaefer M., Langosch D., Multhaup G. (2007) EMBO J. 26, 1702–1712 [PubMed]
18. Kukar T. L., Ladd T. B., Bann M. A., Fraering P. C., Narlawar R., Maharvi G. M., Healy B., Chapman R., Welzel A. T., Price R. W., Moore B., Rangachari V., Cusack B., Eriksen J., Jansen-West K., Verbeeck C., Yager D., Eckman C., Ye W., Sagi S., Cottrell B. A., Torpey J., Rosenberry T. L., Fauq A., Wolfe M. S., Schmidt B., Walsh D. M., Koo E. H., Golde T. E. (2008) Nature 453, 925–929 [PMC free article] [PubMed]
19. Sato T., Kienlen-Campard P., Ahmed M., Liu W., Li H., Elliott J. I., Aimoto S., Constantinescu S. N., Octave J. N., Smith S. O. (2006) Biochemistry 45, 5503–5516 [PMC free article] [PubMed]
20. Hung L. W., Ciccotosto G. D., Giannakis E., Tew D. J., Perez K., Masters C. L., Cappai R., Wade J. D., Barnham K. J. (2008) J. Neurosci. 28, 11950–11958 [PubMed]
21. Harmeier A., Wozny C., Rost B. R., Munter L. M., Hua H., Georgiev O., Beyermann M., Hildebrand P. W., Weise C., Schaffner W., Schmitz D., Multhaup G. (2009) J. Neurosci. 29, 7582–7590 [PubMed]
22. Eggert S., Midthune B., Cottrell B., Koo E. H. (2009) J. Biol. Chem. 284, 28943–28952 [PMC free article] [PubMed]
23. Kienlen-Campard P., Tasiaux B., Van Hees J., Li M., Huysseune S., Sato T., Fei J. Z., Aimoto S., Courtoy P. J., Smith S. O., Constantinescu S. N., Octave J. N. (2008) J. Biol. Chem. 283, 7733–7744 [PMC free article] [PubMed]
24. Page R. M., Baumann K., Tomioka M., Pérez-Revuelta B. I., Fukumori A., Jacobsen H., Flohr A., Luebbers T., Ozmen L., Steiner H., Haass C. (2008) J. Biol. Chem. 283, 677–683 [PubMed]
25. Czirr E., Cottrell B. A., Leuchtenberger S., Kukar T., Ladd T. B., Esselmann H., Paul S., Schubenel R., Torpey J. W., Pietrzik C. U., Golde T. E., Wiltfang J., Baumann K., Koo E. H., Weggen S. (2008) J. Biol. Chem. 283, 17049–17054 [PubMed]
26. Beel A. J., Barrett P., Schnier P. D., Hitchcock S. A., Bagal D., Sanders C. R., Jordan J. B. (2009) Biochemistry 48, 11837–11839 [PMC free article] [PubMed]
27. Weggen S., Eriksen J. L., Sagi S. A., Pietrzik C. U., Ozols V., Fauq A., Golde T. E., Koo E. H. (2003) J. Biol. Chem. 278, 31831–31837 [PubMed]
28. Winkler E., Hobson S., Fukumori A., Dümpelfeld B., Luebbers T., Baumann K., Haass C., Hopf C., Steiner H. (2009) Biochemistry 48, 1183–1197 [PubMed]
29. Shirotani K., Tomioka M., Kremmer E., Haass C., Steiner H. (2007) Neurobiol. Dis. 27, 102–107 [PubMed]
30. Yamasaki A., Eimer S., Okochi M., Smialowska A., Kaether C., Baumeister R., Haass C., Steiner H. (2006) J. Neurosci. 26, 3821–3828 [PubMed]
31. Wang J., Brunkan A. L., Hecimovic S., Walker E., Goate A. (2004) Neurobiol. Dis. 15, 654–666 [PubMed]
32. Edbauer D., Winkler E., Regula J. T., Pesold B., Steiner H., Haass C. (2003) Nat. Cell. Biol. 5, 486–488 [PubMed]
33. Wiltfang J., Smirnov A., Schnierstein B., Kelemen G., Matthies U., Klafki H. W., Staufenbiel M., Hüther G., Rüther E., Kornhuber J. (1997) Electrophoresis 18, 527–532 [PubMed]
34. Kumar-Singh S., De Jonghe C., Cruts M., Kleinert R., Wang R., Mercken M., De Strooper B., Vanderstichele H., Löfgren A., Vanderhoeven I., Backhovens H., Vanmechelen E., Kroisel P. M., Van Broeckhoven C. (2000) Hum. Mol. Genet. 9, 2589–2598 [PubMed]
35. Eckman C. B., Mehta N. D., Crook R., Perez-tur J., Prihar G., Pfeiffer E., Graff-Radford N., Hinder P., Yager D., Zenk B., Refolo L. M., Prada C. M., Younkin S. G., Hutton M., Hardy J. (1997) Hum. Mol. Genet. 6, 2087–2089 [PubMed]
36. Goate A., Chartier-Harlin M. C., Mullan M., Brown J., Crawford F., Fidani L., Giuffra L., Haynes A., Irving N., James L., et al. (1991) Nature 349, 704–706 [PubMed]
37. Lichtenthaler S. F., Wang R., Grimm H., Uljon S. N., Masters C. L., Beyreuther K. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3053–3058 [PubMed]
38. Tan J., Mao G., Cui M. Z., Kang S. C., Lamb B., Wong B. S., Sy M. S., Xu X. (2008) J. Neurochem. 107, 722–733 [PMC free article] [PubMed]
39. Guardia-Laguarta C., Pera M., Clarimón J., Molinuevo J. L., Sánchez-Valle R., Lladó A., Coma M., Gómez-Isla T., Blesa R., Ferrer I., Lleó A. (2010) J. Neuropathol. Exp. Neurol. 69, 53–59 [PubMed]
40. Murrell J., Farlow M., Ghetti B., Benson M. D. (1991) Science 254, 97–99 [PubMed]
41. Narlawar R., Baumann K., Czech C., Schmidt B. (2007) Bioorg. Med. Chem. Lett. 17, 5428–5431 [PubMed]
42. Eriksen J. L., Sagi S. A., Smith T. E., Weggen S., Das P., McLendon D. C., Ozols V. V., Jessing K. W., Zavitz K. H., Koo E. H., Golde T. E. (2003) J. Clin. Invest. 112, 440–449 [PMC free article] [PubMed]
43. Takahashi Y., Hayashi I., Tominari Y., Rikimaru K., Morohashi Y., Kan T., Natsugari H., Fukuyama T., Tomita T., Iwatsubo T. (2003) J. Biol. Chem. 278, 18664–18670 [PubMed]
44. Beher D., Clarke E. E., Wrigley J. D., Martin A. C., Nadin A., Churcher I., Shearman M. S. (2004) J. Biol. Chem. 279, 43419–43426 [PubMed]
45. Grant M. A., Lazo N. D., Lomakin A., Condron M. M., Arai H., Yamin G., Rigby A. C., Teplow D. B. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 16522–16527 [PubMed]
46. Ren Z., Schenk D., Basi G. S., Shapiro I. P. (2007) J. Biol. Chem. 282, 35350–35360 [PubMed]
47. Scheuermann S., Hambsch B., Hesse L., Stumm J., Schmidt C., Beher D., Bayer T. A., Beyreuther K., Multhaup G. (2001) J. Biol. Chem. 276, 33923–33929 [PubMed]

Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry and Molecular Biology