Characteristics of β‐secretase activity on APP‐V604M and APP‐V669L

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AbstractBackgroundSeveral mutations near the amyloid‐beta peptide (Aβ) of Amyloid Precursor Protein (APP) were reported to as pathogenic mutations in Alzheimer's disease (AD), especially in early‐onset Alzheimer's disease (EOAD). β‐Secretase would cleave APP and release sAPPβ and CTF99. Then, γ‐secretase would cleave CTF99 sequentially in generating Aβ. Previously, APP‐V604M and APP‐V669L mutations (APP Seoul mutation) were found from patients with EOAD and their mutant cell lines were established by CRISPR/Cas9. Their pathogenicity of APP‐V604M was demonstrated the mutant cells from observing the increased both of Aβ42/40 ratio and oligomers. For APP‐V669L, the decreased ratio Aβ42/40 was noticeable from the elevation of Aβ40. Still, increased Aβ oligomers were measured. Here, their pathogenicity was supported by measuring β‐secretase activity in above mutant cells.MethodPreviously, APP‐V604M and APP‐V669L cells were generated by CRISPR/Cas9. Cells were seeded and incubated for 48 h. After washing, conditioned media was added and incubated for 48 h. Afterwards, cytosolic proteins and solubilized membrane were extracted with the commercial kit. β‐secretase activities were measured with the substrate at ex/em 355/510 nm.ResultDifferences of β‐secretase activity were observed from above mutations in comparison with control. From the cytosolic protein fractions, the increased β‐secretase activity was observed from APP‐V604M, whereas the decreased activity was monitored from APP‐V669L. Interestingly, from the solubilized membrane fractions, the higher β‐secretase activities were seen than cytosolic protein tractions. The similar activities were measured between APP‐V604M and the control, whereas the decreased β‐secretase activities were measured in APP‐V669L than the control.ConclusionSince APP mutations would directly affect by β‐secretase in generating Aβ, it was important to monitor the β‐secretase activity for supporting the mutant. Here, differences in β‐secretase activities were quantified in APP‐V604M and APP‐V669L mutant cells. As APP Seoul mutation was located at the β‐secretase cleavage site, it would be detrimental to β‐secretase activity. Increased activity on APP‐V604M suggested the possible alteration of structural/cleavage β‐secretase activity from outside of Aβ region. Moreover, the higher β‐secretase activity was observed in the fraction from membrane than the cytosolic fraction. Our results emphasize the importance of measuring β‐secretase activity from the membrane fraction.