Limits...
Amyloid precursor protein mutation E682K at the alternative β-secretase cleavage β'-site increases Aβ generation.

Zhou L, Brouwers N, Benilova I, Vandersteen A, Mercken M, Van Laere K, Van Damme P, Demedts D, Van Leuven F, Sleegers K, Broersen K, Van Broeckhoven C, Vandenberghe R, De Strooper B - EMBO Mol Med (2011)

Bottom Line: Increasing exon- and exome-based sequencing efforts will identify many more putative pathogenic mutations without conclusive segregation-based evidence in a single family.Our study shows how functional analysis of such mutations allows to determine the potential pathogenic nature of these mutations.We propose to classify the E682K mutation as probable pathogenic awaiting further independent confirmation of its association with AD in other patients.

View Article: PubMed Central - PubMed

Affiliation: Department for Developmental and Molecular Genetics, VIB, Leuven, Belgium.

Show MeSH

Related in: MedlinePlus

Effects of E682K mutation on APP processing in cultured neuronsA. Primary cultured neurons were transduced with SFV expressing WT or mutant APP. Cell lysates were analysed by Western blotting using APP C-terminal antibody B63. Conditioned medium was analysed by Western blotting to determine secreted total sAPP (using 22C11 antibody) and sAPPβ (using anti-sAPPβ antibody).B, C. Semi-quantification of sAPPβ and C99 levels from Western blotting, data were normalized to APP levels (**p < 0.01; ns, statistically not significant; mean ± SEM; n = 4).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3377078&req=5

fig02: Effects of E682K mutation on APP processing in cultured neuronsA. Primary cultured neurons were transduced with SFV expressing WT or mutant APP. Cell lysates were analysed by Western blotting using APP C-terminal antibody B63. Conditioned medium was analysed by Western blotting to determine secreted total sAPP (using 22C11 antibody) and sAPPβ (using anti-sAPPβ antibody).B, C. Semi-quantification of sAPPβ and C99 levels from Western blotting, data were normalized to APP levels (**p < 0.01; ns, statistically not significant; mean ± SEM; n = 4).

Mentions: We next analysed the effects of the E682K mutation on APP processing in further detail. In primary neuronal cultures, this mutation increased C99 and sAPPβ levels two- to three-fold (Fig 2), which correlates well with the overall increases in Aβ levels as measured by ELISAs. Similar effects were observed in transiently transfected CHO cells, in which the E682K mutation caused a two- to three-fold increase in C99 and sAPPβ levels (Fig S1 of Supporting Information). These data show that the E682K mutation increased Aβ generation by favouring the β-site cleavage of APP. In contrast to the E682K mutation, the ‘Flemish’ A692G mutation did not significantly affect the β-secretase processing (as measured by C99 and sAPPβ generation), confirming that the increased Aβ generation with this mutant is caused by a different mechanism. It has indeed been shown that the ‘Flemish’ mutation affects an inhibitory domain in the APP sequence that modulates γ-secretase activity (Tian et al, 2010).


Amyloid precursor protein mutation E682K at the alternative β-secretase cleavage β'-site increases Aβ generation.

Zhou L, Brouwers N, Benilova I, Vandersteen A, Mercken M, Van Laere K, Van Damme P, Demedts D, Van Leuven F, Sleegers K, Broersen K, Van Broeckhoven C, Vandenberghe R, De Strooper B - EMBO Mol Med (2011)

Effects of E682K mutation on APP processing in cultured neuronsA. Primary cultured neurons were transduced with SFV expressing WT or mutant APP. Cell lysates were analysed by Western blotting using APP C-terminal antibody B63. Conditioned medium was analysed by Western blotting to determine secreted total sAPP (using 22C11 antibody) and sAPPβ (using anti-sAPPβ antibody).B, C. Semi-quantification of sAPPβ and C99 levels from Western blotting, data were normalized to APP levels (**p < 0.01; ns, statistically not significant; mean ± SEM; n = 4).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3377078&req=5

fig02: Effects of E682K mutation on APP processing in cultured neuronsA. Primary cultured neurons were transduced with SFV expressing WT or mutant APP. Cell lysates were analysed by Western blotting using APP C-terminal antibody B63. Conditioned medium was analysed by Western blotting to determine secreted total sAPP (using 22C11 antibody) and sAPPβ (using anti-sAPPβ antibody).B, C. Semi-quantification of sAPPβ and C99 levels from Western blotting, data were normalized to APP levels (**p < 0.01; ns, statistically not significant; mean ± SEM; n = 4).
Mentions: We next analysed the effects of the E682K mutation on APP processing in further detail. In primary neuronal cultures, this mutation increased C99 and sAPPβ levels two- to three-fold (Fig 2), which correlates well with the overall increases in Aβ levels as measured by ELISAs. Similar effects were observed in transiently transfected CHO cells, in which the E682K mutation caused a two- to three-fold increase in C99 and sAPPβ levels (Fig S1 of Supporting Information). These data show that the E682K mutation increased Aβ generation by favouring the β-site cleavage of APP. In contrast to the E682K mutation, the ‘Flemish’ A692G mutation did not significantly affect the β-secretase processing (as measured by C99 and sAPPβ generation), confirming that the increased Aβ generation with this mutant is caused by a different mechanism. It has indeed been shown that the ‘Flemish’ mutation affects an inhibitory domain in the APP sequence that modulates γ-secretase activity (Tian et al, 2010).

Bottom Line: Increasing exon- and exome-based sequencing efforts will identify many more putative pathogenic mutations without conclusive segregation-based evidence in a single family.Our study shows how functional analysis of such mutations allows to determine the potential pathogenic nature of these mutations.We propose to classify the E682K mutation as probable pathogenic awaiting further independent confirmation of its association with AD in other patients.

View Article: PubMed Central - PubMed

Affiliation: Department for Developmental and Molecular Genetics, VIB, Leuven, Belgium.

Show MeSH
Related in: MedlinePlus