γ-Secretase processing and effects of γ-secretase inhibitors and modulators on long Aβ peptides in cells.

Understanding how different species of Aβ are generated by γ-secretase cleavage has broad therapeutic implications, because shifts in γ-secretase processing that increase the relative production of Aβx-42/43 can initiate a pathological cascade, resulting in Alzheimer disease. We have explored the sequential stepwise γ-secretase cleavage model in cells. Eighteen BRI2-Aβ fusion protein expression constructs designed to generate peptides from Aβ1-38 to Aβ1-55 and C99 (CTFβ) were transfected into cells, and Aβ production was assessed. Secreted and cell-associated Aβ were detected using ELISA and immunoprecipitation MALDI-TOF mass spectrometry. Aβ peptides from 1-38 to 1-55 were readily detected in the cells and as soluble full-length Aβ proteins in the media. Aβ peptides longer than Aβ1-48 were efficiently cleaved by γ-secretase and produced varying ratios of Aβ1-40:Aβ1-42. γ-Secretase cleavage of Aβ1-51 resulted in much higher levels of Aβ1-42 than any other long Aβ peptides, but the processing of Aβ1-51 was heterogeneous with significant amounts of shorter Aβs, including Aβ1-40, produced. Two PSEN1 variants altered Aβ1-42 production from Aβ1-51 but not Aβ1-49. Unexpectedly, long Aβ peptide substrates such as Aβ1-49 showed reduced sensitivity to inhibition by γ-secretase inhibitors. In contrast, long Aβ substrates showed little differential sensitivity to multiple γ-secretase modulators. Although these studies further support the sequential γ-secretase cleavage model, they confirm that in cells the initial γ-secretase cleavage does not precisely define subsequent product lines. These studies also raise interesting issues about the solubility and detection of long Aβ, as well as the use of truncated substrates for assessing relative potency of γ-secretase inhibitors.