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Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer's disease mutants.

Gorman PM, Kim S, Guo M, Melnyk RA, McLaurin J, Fraser PE, Bowie JU, Chakrabartty A - BMC Neurosci (2008)

Bottom Line: We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.The dissociation constants are correlated to both the Abeta42/Abeta40 ratio and the mean age of disease onset in AD patients.We also show that these TM-peptides reduce Abeta production and Abeta42/Abeta40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and gamma-secretase components, potentially revealing a new class of gamma-secretase inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada. pgorman@westwindpartners.ca

ABSTRACT

Background: Amyloid precursor protein (APP) is enzymatically cleaved by gamma-secretase to form two peptide products, either Abeta40 or the more neurotoxic Abeta42. The Abeta42/40 ratio is increased in many cases of familial Alzheimer's disease (FAD). The transmembrane domain (TM) of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains.

Results: Using synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.

Conclusion: The dissociation constants are correlated to both the Abeta42/Abeta40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Abeta production and Abeta42/Abeta40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and gamma-secretase components, potentially revealing a new class of gamma-secretase inhibitors.

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Related in: MedlinePlus

APP-TM peptides are not toxic to HEK293 cells stably overexpressing APP and γ-secretase components. HEK293 cells stably expressing Swedish mutant APP and the γ-secretase components were incubated overnight with APP-TM peptides. The SRB cytotoxicity assay was used to determine whether wild-type (black bars) and V717G (clear bars) affected cell survival. Note that neither peptide caused a reduction in cell survival.
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Figure 8: APP-TM peptides are not toxic to HEK293 cells stably overexpressing APP and γ-secretase components. HEK293 cells stably expressing Swedish mutant APP and the γ-secretase components were incubated overnight with APP-TM peptides. The SRB cytotoxicity assay was used to determine whether wild-type (black bars) and V717G (clear bars) affected cell survival. Note that neither peptide caused a reduction in cell survival.

Mentions: The addition of wild-type and V717G peptides to the HEK293 cells resulted in a drastic reduction in the levels of Aβ relative to control samples and displayed a strong concentration dependence (Fig. 7A,B). The ability of the V717G peptide to suppress Aβ production was greater than that of the wild-type peptide. Both peptides had a greater inhibitory effect on Aβ42 production (Fig. 7B) than on Aβ40 production (Fig. 7A). Because of their greater inhibitory effect on Aβ42 production, both peptides caused a reduction in the Aβ42/40 ratio relative to control samples (Fig. 7C), which were comparable to in vivo Aβ42/Aβ40 ratios [15]. Importantly, the reduction in Aβ production is not caused by any cytotoxic activity of the peptides. Examination of the effect of APP-TM peptides on the viability of these HEK293 cells demonstrated that they do not reduce cell viability (Fig. 8). Analysis of the cell extracts revealed an accumulation of the α- and β-stub fragments of APP, which are γ-secretase substrates. Accumulation of γ-secretase substrates is evidence of γ-secretase inhibition (Fig. 9). While the mechanism by which these peptides reduce Aβ production requires further investigation, their action may be caused by interactions between the γ-secretase complex and monomeric/dimeric forms of the APP-TM peptide or heterodimeric complexes of the peptides with endogenous APP (see below).


Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer's disease mutants.

Gorman PM, Kim S, Guo M, Melnyk RA, McLaurin J, Fraser PE, Bowie JU, Chakrabartty A - BMC Neurosci (2008)

APP-TM peptides are not toxic to HEK293 cells stably overexpressing APP and γ-secretase components. HEK293 cells stably expressing Swedish mutant APP and the γ-secretase components were incubated overnight with APP-TM peptides. The SRB cytotoxicity assay was used to determine whether wild-type (black bars) and V717G (clear bars) affected cell survival. Note that neither peptide caused a reduction in cell survival.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266763&req=5

Figure 8: APP-TM peptides are not toxic to HEK293 cells stably overexpressing APP and γ-secretase components. HEK293 cells stably expressing Swedish mutant APP and the γ-secretase components were incubated overnight with APP-TM peptides. The SRB cytotoxicity assay was used to determine whether wild-type (black bars) and V717G (clear bars) affected cell survival. Note that neither peptide caused a reduction in cell survival.
Mentions: The addition of wild-type and V717G peptides to the HEK293 cells resulted in a drastic reduction in the levels of Aβ relative to control samples and displayed a strong concentration dependence (Fig. 7A,B). The ability of the V717G peptide to suppress Aβ production was greater than that of the wild-type peptide. Both peptides had a greater inhibitory effect on Aβ42 production (Fig. 7B) than on Aβ40 production (Fig. 7A). Because of their greater inhibitory effect on Aβ42 production, both peptides caused a reduction in the Aβ42/40 ratio relative to control samples (Fig. 7C), which were comparable to in vivo Aβ42/Aβ40 ratios [15]. Importantly, the reduction in Aβ production is not caused by any cytotoxic activity of the peptides. Examination of the effect of APP-TM peptides on the viability of these HEK293 cells demonstrated that they do not reduce cell viability (Fig. 8). Analysis of the cell extracts revealed an accumulation of the α- and β-stub fragments of APP, which are γ-secretase substrates. Accumulation of γ-secretase substrates is evidence of γ-secretase inhibition (Fig. 9). While the mechanism by which these peptides reduce Aβ production requires further investigation, their action may be caused by interactions between the γ-secretase complex and monomeric/dimeric forms of the APP-TM peptide or heterodimeric complexes of the peptides with endogenous APP (see below).

Bottom Line: We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.The dissociation constants are correlated to both the Abeta42/Abeta40 ratio and the mean age of disease onset in AD patients.We also show that these TM-peptides reduce Abeta production and Abeta42/Abeta40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and gamma-secretase components, potentially revealing a new class of gamma-secretase inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada. pgorman@westwindpartners.ca

ABSTRACT

Background: Amyloid precursor protein (APP) is enzymatically cleaved by gamma-secretase to form two peptide products, either Abeta40 or the more neurotoxic Abeta42. The Abeta42/40 ratio is increased in many cases of familial Alzheimer's disease (FAD). The transmembrane domain (TM) of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains.

Results: Using synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.

Conclusion: The dissociation constants are correlated to both the Abeta42/Abeta40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Abeta production and Abeta42/Abeta40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and gamma-secretase components, potentially revealing a new class of gamma-secretase inhibitors.

Show MeSH
Related in: MedlinePlus