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β- but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia.

Tamayev R, Matsuda S, Arancio O, D'Adamio L - EMBO Mol Med (2012)

Bottom Line: Processing of β-CTF by γ-secretase releases amyloid-β (Aβ), which is assumed to cause AD.These results suggest that sAPPβ and/or β-CTF, rather than Aβ, are the toxic species causing dementia, and indicate that reducing β-cleavage of APP is an appropriate therapeutic approach to treating human dementias.Our data and the failures of anti-Aβ therapies in humans advise against targeting γ-secretase cleavage of APP and/or Aβ.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA.

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A BRI2-derived peptide binds APP and inhibits β-cleavage of APPA-B. HEK293-APP cells were incubated with the indicated peptides. β- and α-cleavage of APP were quantified by measuring sAPPβ and sAPPα in media by WB. WB of cell lysates detected APP and α-Tubulin.C. WB analysis of cell lysates and conditioned media from HEK293-APP cells treated either with the indicated concentrations of either N3-2A or N1. In a duplicate experiment, cells were treated with compound-E (+) while incubated with the indicated peptides. Lysates were probed for APP, APP-CTFs and α-Tubulin, culture media was probed for sAPPα and sAPPβ. In the right panel, the results of a similar experiment in HeLa-APP cells are shown.D-F. WB analysis of lysates (L) or α-Flag IP (IP) from HeLa/APP cells incubated for 2 h with Flag-tagged peptides.D. Cells were incubated at either 37 or 4 °C with or without 40 µM N3-2A-F.E. The indicated concentrations of N3-2A were added to the media containing 40 µM N3-2A-F.F. Cells were incubated with 40 µM N3-2A-F, N4-F or N3-4A-F.G. Brain cells were cultured as in (D).H. Biotinylated cells were cultured as in (D). The reduced and not reduced samples are indicated (+red and −red, respectively). Lysates (L), α-Flag IP eluted with Flag-peptide (E), eluted sample precipitated with streptavidin-beads [both the fraction unbound (U) and bound (B) to streptavidin-beads], were probed for APP in WB.I. Purified β-secretase was incubated with fluorescent β-secretase substrate for 30 min, resulting in β-cleavage that could be detected by fluorescence increase. In separate samples, the indicated concentrations of N3-2A or β-secretase-inhibitor IV were added to the reaction. The data are shown as % of inhibition of β-secretase activity in samples without inhibitors.J. Model depicting the mechanism of action of N3-2A/MoBA. The peptide interferes with processing of APP by β-secretase but, unlike full-length BRI2, does not modulate γ-cleavage of β-CTF.
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fig02: A BRI2-derived peptide binds APP and inhibits β-cleavage of APPA-B. HEK293-APP cells were incubated with the indicated peptides. β- and α-cleavage of APP were quantified by measuring sAPPβ and sAPPα in media by WB. WB of cell lysates detected APP and α-Tubulin.C. WB analysis of cell lysates and conditioned media from HEK293-APP cells treated either with the indicated concentrations of either N3-2A or N1. In a duplicate experiment, cells were treated with compound-E (+) while incubated with the indicated peptides. Lysates were probed for APP, APP-CTFs and α-Tubulin, culture media was probed for sAPPα and sAPPβ. In the right panel, the results of a similar experiment in HeLa-APP cells are shown.D-F. WB analysis of lysates (L) or α-Flag IP (IP) from HeLa/APP cells incubated for 2 h with Flag-tagged peptides.D. Cells were incubated at either 37 or 4 °C with or without 40 µM N3-2A-F.E. The indicated concentrations of N3-2A were added to the media containing 40 µM N3-2A-F.F. Cells were incubated with 40 µM N3-2A-F, N4-F or N3-4A-F.G. Brain cells were cultured as in (D).H. Biotinylated cells were cultured as in (D). The reduced and not reduced samples are indicated (+red and −red, respectively). Lysates (L), α-Flag IP eluted with Flag-peptide (E), eluted sample precipitated with streptavidin-beads [both the fraction unbound (U) and bound (B) to streptavidin-beads], were probed for APP in WB.I. Purified β-secretase was incubated with fluorescent β-secretase substrate for 30 min, resulting in β-cleavage that could be detected by fluorescence increase. In separate samples, the indicated concentrations of N3-2A or β-secretase-inhibitor IV were added to the reaction. The data are shown as % of inhibition of β-secretase activity in samples without inhibitors.J. Model depicting the mechanism of action of N3-2A/MoBA. The peptide interferes with processing of APP by β-secretase but, unlike full-length BRI2, does not modulate γ-cleavage of β-CTF.

Mentions: We tested if peptides spanning this domain duplicated BRI2's function. Two overlapping peptides N3 and N8 strongly reduced β-cleavage and moderately decreased α-processing of APP (Fig 2A). Mutagenesis of N3 showed that replacing any of amino acids 3–10 with Alanine reduced the inhibitory activity of N3 on β-cleavage of APP, showing the functional importance of these residues. However, replacing either the first or second residue (N3-1A/N3-2A) actually resulted in a stronger inhibitor of APP processing by β-secretase (Fig 2B). Notably, α-cleavage of APP is inhibited by N3, unaffected by N3-2A and, probably, increased by N3-1A. It is possible that these three peptides bind APP differently thereby reducing (N3), unaffecting (N3-2A) or increasing (N3-1A) access of α-secretase to the APP-docking/cleavage site. However, the mechanism underlying this potentially useful difference remains to be investigated.


β- but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia.

Tamayev R, Matsuda S, Arancio O, D'Adamio L - EMBO Mol Med (2012)

A BRI2-derived peptide binds APP and inhibits β-cleavage of APPA-B. HEK293-APP cells were incubated with the indicated peptides. β- and α-cleavage of APP were quantified by measuring sAPPβ and sAPPα in media by WB. WB of cell lysates detected APP and α-Tubulin.C. WB analysis of cell lysates and conditioned media from HEK293-APP cells treated either with the indicated concentrations of either N3-2A or N1. In a duplicate experiment, cells were treated with compound-E (+) while incubated with the indicated peptides. Lysates were probed for APP, APP-CTFs and α-Tubulin, culture media was probed for sAPPα and sAPPβ. In the right panel, the results of a similar experiment in HeLa-APP cells are shown.D-F. WB analysis of lysates (L) or α-Flag IP (IP) from HeLa/APP cells incubated for 2 h with Flag-tagged peptides.D. Cells were incubated at either 37 or 4 °C with or without 40 µM N3-2A-F.E. The indicated concentrations of N3-2A were added to the media containing 40 µM N3-2A-F.F. Cells were incubated with 40 µM N3-2A-F, N4-F or N3-4A-F.G. Brain cells were cultured as in (D).H. Biotinylated cells were cultured as in (D). The reduced and not reduced samples are indicated (+red and −red, respectively). Lysates (L), α-Flag IP eluted with Flag-peptide (E), eluted sample precipitated with streptavidin-beads [both the fraction unbound (U) and bound (B) to streptavidin-beads], were probed for APP in WB.I. Purified β-secretase was incubated with fluorescent β-secretase substrate for 30 min, resulting in β-cleavage that could be detected by fluorescence increase. In separate samples, the indicated concentrations of N3-2A or β-secretase-inhibitor IV were added to the reaction. The data are shown as % of inhibition of β-secretase activity in samples without inhibitors.J. Model depicting the mechanism of action of N3-2A/MoBA. The peptide interferes with processing of APP by β-secretase but, unlike full-length BRI2, does not modulate γ-cleavage of β-CTF.
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fig02: A BRI2-derived peptide binds APP and inhibits β-cleavage of APPA-B. HEK293-APP cells were incubated with the indicated peptides. β- and α-cleavage of APP were quantified by measuring sAPPβ and sAPPα in media by WB. WB of cell lysates detected APP and α-Tubulin.C. WB analysis of cell lysates and conditioned media from HEK293-APP cells treated either with the indicated concentrations of either N3-2A or N1. In a duplicate experiment, cells were treated with compound-E (+) while incubated with the indicated peptides. Lysates were probed for APP, APP-CTFs and α-Tubulin, culture media was probed for sAPPα and sAPPβ. In the right panel, the results of a similar experiment in HeLa-APP cells are shown.D-F. WB analysis of lysates (L) or α-Flag IP (IP) from HeLa/APP cells incubated for 2 h with Flag-tagged peptides.D. Cells were incubated at either 37 or 4 °C with or without 40 µM N3-2A-F.E. The indicated concentrations of N3-2A were added to the media containing 40 µM N3-2A-F.F. Cells were incubated with 40 µM N3-2A-F, N4-F or N3-4A-F.G. Brain cells were cultured as in (D).H. Biotinylated cells were cultured as in (D). The reduced and not reduced samples are indicated (+red and −red, respectively). Lysates (L), α-Flag IP eluted with Flag-peptide (E), eluted sample precipitated with streptavidin-beads [both the fraction unbound (U) and bound (B) to streptavidin-beads], were probed for APP in WB.I. Purified β-secretase was incubated with fluorescent β-secretase substrate for 30 min, resulting in β-cleavage that could be detected by fluorescence increase. In separate samples, the indicated concentrations of N3-2A or β-secretase-inhibitor IV were added to the reaction. The data are shown as % of inhibition of β-secretase activity in samples without inhibitors.J. Model depicting the mechanism of action of N3-2A/MoBA. The peptide interferes with processing of APP by β-secretase but, unlike full-length BRI2, does not modulate γ-cleavage of β-CTF.
Mentions: We tested if peptides spanning this domain duplicated BRI2's function. Two overlapping peptides N3 and N8 strongly reduced β-cleavage and moderately decreased α-processing of APP (Fig 2A). Mutagenesis of N3 showed that replacing any of amino acids 3–10 with Alanine reduced the inhibitory activity of N3 on β-cleavage of APP, showing the functional importance of these residues. However, replacing either the first or second residue (N3-1A/N3-2A) actually resulted in a stronger inhibitor of APP processing by β-secretase (Fig 2B). Notably, α-cleavage of APP is inhibited by N3, unaffected by N3-2A and, probably, increased by N3-1A. It is possible that these three peptides bind APP differently thereby reducing (N3), unaffecting (N3-2A) or increasing (N3-1A) access of α-secretase to the APP-docking/cleavage site. However, the mechanism underlying this potentially useful difference remains to be investigated.

Bottom Line: Processing of β-CTF by γ-secretase releases amyloid-β (Aβ), which is assumed to cause AD.These results suggest that sAPPβ and/or β-CTF, rather than Aβ, are the toxic species causing dementia, and indicate that reducing β-cleavage of APP is an appropriate therapeutic approach to treating human dementias.Our data and the failures of anti-Aβ therapies in humans advise against targeting γ-secretase cleavage of APP and/or Aβ.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA.

Show MeSH
Related in: MedlinePlus