The production ratios of AICDε51 and Aß42 by intramembrane proteolysis of ßAPP do not always change in parallel.

BACKGROUND: During intramembrane proteolysis of ß-amyloid protein precursor (ßAPP) by presenilin (PS)/γ-secretase, ε-cleavages at the membrane-cytoplasmic border precede γ-cleavages at the middle of the transmembrane domain. Generation ratios of Aß42, a critical molecule for Alzheimer's disease (AD) pathogenesis, and the major Aß40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aß42 production have been investigated by many pharmaceutical companies. Therefore, the ε-cleavages, rather than the γ-cleavage, might be more effective upstream targets for decreasing the relative generation of Aß42. Thus, one might evaluate compounds by analyzing the generation ratio of the ßAPP intracellular domain (AICD) species (ε-cleavage-derived), instead of that of Aß42. METHODS: Cell-free γ-secretase assays were carried out to observe de novo AICD production. Immunoprecipitation/MALDI-TOF MS analysis was carried out to detect the N-termini of AICD species. Aß and AICD species were measured by ELISA and immunoblotting techniques. RESULTS: Effects on the ε-cleavage by AD-associated pathological mutations around the ε-cleavage sites (i.e., ßAPP V642I, L648P and K649N) were analyzed. The V642I and L648P mutations caused an increase in the relative ratio of ε48 cleavage, as expected from previous reports. Cells expressing the K649N mutant, however, underwent a major ε-cleavage at the ε51 site. These results suggest that ε51, as well as ε48 cleavage, is associated with Aß42 production. Only AICDε51, though, and not Aß42 production, dramatically changed with modifications to the cell-free assay conditions. Interestingly, the increase in the relative ratio of the ε51 cleavage by the K649N mutation was not cancelled by these changes. CONCLUSION: Our current data show that the generation ratio of AICDε51 and Aß42 do not always change in parallel. Thus, to identify compounds that decrease the relative ratio of Aß42 generation, measurement of the relative level of Aß42-related AICD species (i.e., AICDε48 and AICDε51) might not be useful. Further studies to reveal how the ε-cleavage precision is decided are necessary before it will be possible to develop drugs targeting ε-cleavage as a means for decreasing Aß42 production.

The 28-amino acid form of an APLP1-derived Abeta-like peptide is a surrogate marker for Abeta42 production in the central nervous system.

Surrogate markers for the Alzheimer disease (AD)-associated 42-amino acid form of amyloid-beta (Abeta42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1-derived Abeta-like peptides (APL1beta) that are generated by beta- and gamma-cleavages at a concentration of approximately 4.5 nM. These novel peptides, APL1beta25, APL1beta27 and APL1beta28, were not deposited in AD brains. Interestingly, most gamma-secretase modulators (GSMs) and familial AD-associated presenilin1 mutants that up-regulate the relative production of Abeta42 cause a parallel increase in the production of APL1beta28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1beta28 levels are higher than in non-AD controls, while the relative Abeta42 levels are unchanged or lower. Most strikingly, the relative APL1beta28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non-AD controls. Based on these results, we propose the relative level of APL1beta28 in the CSF as a candidate surrogate marker for the relative level of Abeta42 production in the brain.