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

ABSTRACT

Understanding how different species of Aß are generated by γ-secretase cleavage has broad therapeutic implications, because shifts in γ-secretase processing that increase the relative production of Aßx-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-Aß fusion protein expression constructs designed to generate peptides from Aß1-38 to Aß1-55 and C99 (CTFß) were transfected into cells, and Aß production was assessed. Secreted and cell-associated Aß were detected using ELISA and immunoprecipitation MALDI-TOF mass spectrometry. Aß peptides from 1-38 to 1-55 were readily detected in the cells and as soluble full-length Aß proteins in the media. Aß peptides longer than Aß1-48 were efficiently cleaved by γ-secretase and produced varying ratios of Aß1-40Aß1-42. γ-Secretase cleavage of Aß1-51 resulted in much higher levels of Aß1-42 than any other long Aß peptides, but the processing of Aß1-51 was heterogeneous with significant amounts of shorter Aßs, including Aß1-40, produced. Two PSEN1 variants altered Aß1-42 production from Aß1-51 but not Aß1-49. Unexpectedly, long Aß peptide substrates such as Aß1-49 showed reduced sensitivity to inhibition by γ-secretase inhibitors. In contrast, long Aß 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 Aß, as well as the use of truncated substrates for assessing relative potency of γ-secretase inhibitors.