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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.

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Related in: MedlinePlus

Effects of E682K mutant C99 on γ-secretase activity in cell-based assayA-C. CHO cells were transfected with WT or E682K mutant C99, conditioned media were analysed by Aβ ELISAs (**p < 0.01; *p < 0.05; ns, statistically not significant; mean ± SEM; n = 3).D. Western blotting analysis of C99 expression levels. Cell lysates containing either C99WT or C99E682K were loaded in a series of two times dilution.
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fig04: Effects of E682K mutant C99 on γ-secretase activity in cell-based assayA-C. CHO cells were transfected with WT or E682K mutant C99, conditioned media were analysed by Aβ ELISAs (**p < 0.01; *p < 0.05; ns, statistically not significant; mean ± SEM; n = 3).D. Western blotting analysis of C99 expression levels. Cell lysates containing either C99WT or C99E682K were loaded in a series of two times dilution.

Mentions: One interesting observation is that the E682K mutation increased the Aβ1–42/Aβ1–40 ratio in neuronal and CHO cell cultures. It is known that pathogenic mutations near the C-terminus of the Aβ sequence in APP can lead to increased generation of Aβ42 by modulating the active sites of γ-secretase. A recent report has shown that the ‘Flemish’ A692G mutation, which is located in the middle of Aβ, has unexpectedly, in addition to its slight inhibitory effect on α-secretase processing (De Strooper et al, 1995; Haass et al, 1994), a quite pronounced effect on γ-secretase activity (Tian et al, 2010), suggesting that the interaction between γ-secretase and C99 may also occur at sites that are remote from the actual cleavage site in C99. Therefore, we wondered whether the increase in the Aβ42/Aβ40 ratio caused by the E682K mutation might be caused by an additional effect on the modulation of γ-secretase. We transiently transfected CHO cells with WT or E682K mutant C99. This APP fragment is the direct substrate of γ-secretase and its processing will, therefore, largely be determined by γ-secretase. The expression levels of both C99WT and C99E682K were investigated by Western blotting, no difference was observed. Conditioned media were analysed by Aβ ELISAs (Fig 4). The E682K mutation generated the same amounts of Aβ1–42 but significantly less Aβ1–40, leading to an increased Aβ1–42/Aβ1–40 ratio. These data suggested that the E682K mutation affects also to a certain extent the Aβ1–42/Aβ1–40 ratio via modulation of γ-secretase.


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 mutant C99 on γ-secretase activity in cell-based assayA-C. CHO cells were transfected with WT or E682K mutant C99, conditioned media were analysed by Aβ ELISAs (**p < 0.01; *p < 0.05; ns, statistically not significant; mean ± SEM; n = 3).D. Western blotting analysis of C99 expression levels. Cell lysates containing either C99WT or C99E682K were loaded in a series of two times dilution.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3377078&req=5

fig04: Effects of E682K mutant C99 on γ-secretase activity in cell-based assayA-C. CHO cells were transfected with WT or E682K mutant C99, conditioned media were analysed by Aβ ELISAs (**p < 0.01; *p < 0.05; ns, statistically not significant; mean ± SEM; n = 3).D. Western blotting analysis of C99 expression levels. Cell lysates containing either C99WT or C99E682K were loaded in a series of two times dilution.
Mentions: One interesting observation is that the E682K mutation increased the Aβ1–42/Aβ1–40 ratio in neuronal and CHO cell cultures. It is known that pathogenic mutations near the C-terminus of the Aβ sequence in APP can lead to increased generation of Aβ42 by modulating the active sites of γ-secretase. A recent report has shown that the ‘Flemish’ A692G mutation, which is located in the middle of Aβ, has unexpectedly, in addition to its slight inhibitory effect on α-secretase processing (De Strooper et al, 1995; Haass et al, 1994), a quite pronounced effect on γ-secretase activity (Tian et al, 2010), suggesting that the interaction between γ-secretase and C99 may also occur at sites that are remote from the actual cleavage site in C99. Therefore, we wondered whether the increase in the Aβ42/Aβ40 ratio caused by the E682K mutation might be caused by an additional effect on the modulation of γ-secretase. We transiently transfected CHO cells with WT or E682K mutant C99. This APP fragment is the direct substrate of γ-secretase and its processing will, therefore, largely be determined by γ-secretase. The expression levels of both C99WT and C99E682K were investigated by Western blotting, no difference was observed. Conditioned media were analysed by Aβ ELISAs (Fig 4). The E682K mutation generated the same amounts of Aβ1–42 but significantly less Aβ1–40, leading to an increased Aβ1–42/Aβ1–40 ratio. These data suggested that the E682K mutation affects also to a certain extent the Aβ1–42/Aβ1–40 ratio via modulation of γ-secretase.

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