The identification of hereditary familial Alzheimer disease (FAD) mutations in the amyloid precursor protein (APP) and presenilin-1 (PS1) corroborated the causative role of amyloid-␤ peptides with 42 amino acid residues (A␤42) in the pathogenesis of AD. Although most FAD mutations are known to increase A␤42 levels, mutations within the APP GxxxG motif are known to lower A␤42 levels by attenuating transmembrane sequence dimerization. Here, we show that aberrant A␤42 levels of FAD mutations can be rescued by GxxxG mutations. The combination of the APP-GxxxG mutation G33A with APP-FAD mutations yielded a constant 60% decrease of A␤42 levels and a concomitant 3-fold increase of A␤38 levels compared with the Gly 33 wild-type as determined by ELISA. In the presence of PS1-FAD mutations, the effects of G33A were attenuated, apparently attributable to a different mechanism of PS1-FAD mutants compared with APP-FAD mutants. Our results contribute to a general understanding of the mechanism how APP is processed by the ␥-secretase module and strongly emphasize the potential of the GxxxG motif in the prevention of sporadic AD as well as FAD. APP2 and APLPs were conventionally thought to exist and to act as monomers. However, biochemical and structural data have accumulated over the past few years, indicating that APP and APLPs exist as functional dimers or even are present in higher oligomeric units (1-6). Interactions of APP and APLPs were reported to promote cell adhesion in a homo-and heterotypic manner (7,8). Among other mechanisms, the varying strength of APP dimerization mediated through N-terminal sites (5) or by the transmembrane sequence (TMS) (9) has been reported to influence APP processing.APP is first cleaved by the ␤-site APP cleaving enzyme and is then sequentially processed by the ␥-secretase complex to generate A␤ peptides of varying length (10, 11). ␥-Secretase cleavage specificity is modulated by the GxxxG ("G-triple-x-G") dimerization motif of the APP-TMS, and we showed previously that APP can be cleaved as a homodimer by ␤-and ␥-secretases (9). APP, APLP1, and APLP2 share similar interaction motifs and can form APP-APLP1 and APP-APLP2 complexes (7). Cotransfections of APP with APLP1 or APLP2 influenced APP processing into A␤ leading to decreased A␤40 and A␤42 levels likely through an influence on ␥-secretase cleavages (7).According to the amyloid hypothesis, A␤ peptides represent the main culprit of Alzheimer disease (AD). Based on this assumption is the appealing prediction that reducing A␤ levels would ameliorate Alzheimer symptoms (12,13). In the current model of A␤ generation, the initial cut at the ⑀-site is executed by the presenilins of the ␥-secretase complex, leading to formation of the APP intracellular domain and A␤49 or A␤48 peptides (10, 14). The latter two likely remain bound to the active site and are successively cleaved every three to four residues at the -site and at the ␥-sites (11, 15). Most likely, two product lines exist. In the product line encompassing A␤40, A␤49 is trimmed to A␤4...

The amyloid-β (Aβ) peptide is contained within the C-terminal fragment (β-CTF) of the amyloid precursor protein (APP) and is intimately linked to Alzheimer's disease. In vivo, Aβ is generated by sequential cleavage of β-CTF within the γ-secretase module. To investigate γ-secretase function, in vitro assays are in widespread use which require a recombinant β-CTF substrate expressed in bacteria and purified from inclusion bodies, termed C100. So far, little is known about the conformation of C100 under different conditions of purification and refolding. Since C100 dimerization influences the efficiency and specificity of γ-secretase cleavage, it is also of great interest to determine the secondary structure and the oligomeric state of the synthetic substrate as well as the binding properties of small molecules named γ-secretase modulators (GSMs) which we could previously show to modulate APP transmembrane sequence interactions [Richter et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14597-14602]. Here, we use circular dichroism and continuous-wave electron spin resonance measurements to show that C100 purified in a buffer containing SDS at micelle-forming concentrations adopts a highly stable α-helical conformation, in which it shows little tendency to aggregate or to form higher oligomers than dimers. By surface plasmon resonance analysis and molecular modeling we show that the GSM sulindac sulfide binds to C100 and has a preference for C100 dimers.

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