Mutations in the transmembrane domain of APP altering γ-secretase specificity

Alzheimer's disease (AD) β-amyloid peptide (Aβ and βA4) is derived from the amyloid precursor protein (APP) by the subsequent action of the so- far unidentified β- and γ-secretases. γ-secretase, which generates the C- terminus of Aβ, cleaves within the transmembrane domain of APP, preferentially after Aβ-residue 40 (Aβ 40) but also after residue 42 (Aβ 42). This Aβ 42 represents the major subunit of the plaques in AD. Since the position of γ-secretase cleavage is crucial for understanding the pathogenic pathway, we investigated the effect of different point mutations at Thr43 on γ-secretase specificity in SPA4CT (SPC99)-expressing COS7 cells. These constructs only require γ-cleavage for Aβ release. We observed that all Thr43 mutations altered the specificity of γ-secretase. Small hydrophobic residues favored the generation of Aβ 42, leading to an increase in the 42/40 ratio of Aβ (1.6-2.8-fold). The increase was even stronger (5.6-5.8- fold) when combined with the familial mutation Va146Phe. Thus, these constructs might be highly valuable for the generation of animal models for AD. Processing of full-length APP or SPA4CT yielded the same 42/40 ratio of Aβ (4.7%). Both constructs, bearing the familial AD mutation Va146Phe, led to a similar increase in the 42/40 ratio (3.3- versus 3.6-fold). The p3 fragment, produced by α- and γ-secretase, showed 42/40 ratios similar to Aβ when derived from wild-type and mutant proteins. These results suggest that the different Aβ- and p3-species are generated by γ-cleavage activities with a similar enzymatic mechanism.