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Reciprocal relationship between APP positioning relative to the membrane and PS1 conformation.

Uemura K, Farner KC, Nasser-Ghodsi N, Jones P, Berezovska O - Mol Neurodegener (2011)

Bottom Line: Several familial Alzheimer disease (FAD) mutations within the transmembrane region of the amyloid precursor protein (APP) increase the Aβ42/40 ratio without increasing total Aβ production.In the present study, we analyzed the impact of FAD mutations and γ-secretase modulators (GSMs) that alter the Aβ42/40 ratio on APP C-terminus (CT) positioning relative to the membrane, reasoning that changes in the alignment of the APP intramembranous domain and presenilin 1 (PS1) may impact the PS1/γ-secretase cleavage site on APP.Thus, we propose that there is a reciprocal relationship between APP-CT positioning relative to the membrane and PS1 conformation, suggesting that factors that modulate either APP positioning in the membrane or PS1 conformation could be exploited therapeutically.

View Article: PubMed Central - HTML - PubMed

Affiliation: Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Charlestown, MA 02129, USA. oberezovska@partners.org.

ABSTRACT

Background: Several familial Alzheimer disease (FAD) mutations within the transmembrane region of the amyloid precursor protein (APP) increase the Aβ42/40 ratio without increasing total Aβ production. In the present study, we analyzed the impact of FAD mutations and γ-secretase modulators (GSMs) that alter the Aβ42/40 ratio on APP C-terminus (CT) positioning relative to the membrane, reasoning that changes in the alignment of the APP intramembranous domain and presenilin 1 (PS1) may impact the PS1/γ-secretase cleavage site on APP.

Results: By using a Förster resonance energy transfer (FRET)-based technique, fluorescent lifetime imaging microscopy (FLIM), we show that Aβ42/40 ratio-modulating factors which target either APP substrate or PS1/γ-secretase affect proximity of the APP-CT to the membrane and change PS1 conformation.

Conclusions: Thus, we propose that there is a reciprocal relationship between APP-CT positioning relative to the membrane and PS1 conformation, suggesting that factors that modulate either APP positioning in the membrane or PS1 conformation could be exploited therapeutically.

No MeSH data available.


Related in: MedlinePlus

The Ab42/40 ratio-modulating APP mutations induce changes in APP positioning relative to the membrane . A) ELISA detection of the human Aβ40 and Aβ42 in conditioned media of the cells transiently transfected with human APP-RFP constructs with designated mutations. The amount of each Aβ species was normalized to that obtained from the cells expressing wild-type APP-RFP (Aβ40 ≈70 pMol/L, Aβ42 ≈1 pMol/L). Three independent experiments were performed. (mean ± SD; *p < 0.001 vs. wild-type APP-RFP, ANOVA, n: number of wells in the representative experiment shown). B) FLIM analysis of the proximity between APP-CT RFP and myrGFP labeled membrane. The pseudo-colour images show distribution of the EGFP and myrGFP donor fluorophore lifetimes in the presence (bottom) or absence (top) of the RFP acceptor fluorophore fused to the wild-type APP-CT. Only cells transfected with myrGFP as a donor fluorophore showed lifetime shortening (red and yellow pixels) in the presence of APP-RFP, with the shortest lifetime at the cell periphery. Scale bar: 10 μm. Colorimetric scale shows GFP fluorophore lifetime in picoseconds. C) Quantitative FLIM analysis of the GFP lifetimes in APP/APLP2 dKO cells expressing wild-type and mutant APP-RFP constructs. In cells transfected with the EGFP as a donor fluorophore (grey bars), the donor lifetime did not change significantly in the presence of either wild-type or V717I APP-RFP. In cells transfected with myrGFP as a donor (black bars), the donor lifetime was significantly shortened in the presence of RFP acceptor at the APP CT. FAD-linked APP mutations (V717I and I716F) significantly shortened, whereas V717K APP mutation significantly increased the lifetime of myrGFP donor, compared to that of the wild-type APP (mean ± SD; *p < 0.001, **p < 0.01, ANOVA). Data from one of the three independent experiments is shown; n = cell number. D) CHO cells were transfected with APP-GFP to serve as a donor fluorophore (green) in the FLIM assay. Plasma membrane was stained with CM-DiI to serve as an acceptor fluorophore (red). Merged image shows that APP signal is outlined by red membrane. Scale bar; 10 μm. E) The graph shows average lifetime of GFP donor fluorophore in CHO cells from (D). The lifetime of GFP donor was shortened in cells with CM-DiI membrane staining. In the presence of I716F and V717I mutations, the GFP lifetime was significantly shorter than that in wild-type APP-GFP transfected cells (mean ± SD; *p < 0.01, ANOVA). Three independent experiments were performed (n: total cell number).
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Figure 1: The Ab42/40 ratio-modulating APP mutations induce changes in APP positioning relative to the membrane . A) ELISA detection of the human Aβ40 and Aβ42 in conditioned media of the cells transiently transfected with human APP-RFP constructs with designated mutations. The amount of each Aβ species was normalized to that obtained from the cells expressing wild-type APP-RFP (Aβ40 ≈70 pMol/L, Aβ42 ≈1 pMol/L). Three independent experiments were performed. (mean ± SD; *p < 0.001 vs. wild-type APP-RFP, ANOVA, n: number of wells in the representative experiment shown). B) FLIM analysis of the proximity between APP-CT RFP and myrGFP labeled membrane. The pseudo-colour images show distribution of the EGFP and myrGFP donor fluorophore lifetimes in the presence (bottom) or absence (top) of the RFP acceptor fluorophore fused to the wild-type APP-CT. Only cells transfected with myrGFP as a donor fluorophore showed lifetime shortening (red and yellow pixels) in the presence of APP-RFP, with the shortest lifetime at the cell periphery. Scale bar: 10 μm. Colorimetric scale shows GFP fluorophore lifetime in picoseconds. C) Quantitative FLIM analysis of the GFP lifetimes in APP/APLP2 dKO cells expressing wild-type and mutant APP-RFP constructs. In cells transfected with the EGFP as a donor fluorophore (grey bars), the donor lifetime did not change significantly in the presence of either wild-type or V717I APP-RFP. In cells transfected with myrGFP as a donor (black bars), the donor lifetime was significantly shortened in the presence of RFP acceptor at the APP CT. FAD-linked APP mutations (V717I and I716F) significantly shortened, whereas V717K APP mutation significantly increased the lifetime of myrGFP donor, compared to that of the wild-type APP (mean ± SD; *p < 0.001, **p < 0.01, ANOVA). Data from one of the three independent experiments is shown; n = cell number. D) CHO cells were transfected with APP-GFP to serve as a donor fluorophore (green) in the FLIM assay. Plasma membrane was stained with CM-DiI to serve as an acceptor fluorophore (red). Merged image shows that APP signal is outlined by red membrane. Scale bar; 10 μm. E) The graph shows average lifetime of GFP donor fluorophore in CHO cells from (D). The lifetime of GFP donor was shortened in cells with CM-DiI membrane staining. In the presence of I716F and V717I mutations, the GFP lifetime was significantly shorter than that in wild-type APP-GFP transfected cells (mean ± SD; *p < 0.01, ANOVA). Three independent experiments were performed (n: total cell number).

Mentions: First, we measured the effect of V717I, V717K or I716F mutations located in the transmembrane domain of APP on the Aβ42/40 ratio in CHO cells transiently transfected with the mutant APP constructs. As reported previously [8,9], the amount of Aβ42 was increased while the amount of the Aβ40 was decreased in the conditioned medium of V717I and I716F APP expressing cells, leading to a significantly elevated Aβ42/40 ratio (Figure 1A). By contrast, artificial V717K APP mutation [32] significantly lowered the Aβ42/40 ratio.


Reciprocal relationship between APP positioning relative to the membrane and PS1 conformation.

Uemura K, Farner KC, Nasser-Ghodsi N, Jones P, Berezovska O - Mol Neurodegener (2011)

The Ab42/40 ratio-modulating APP mutations induce changes in APP positioning relative to the membrane . A) ELISA detection of the human Aβ40 and Aβ42 in conditioned media of the cells transiently transfected with human APP-RFP constructs with designated mutations. The amount of each Aβ species was normalized to that obtained from the cells expressing wild-type APP-RFP (Aβ40 ≈70 pMol/L, Aβ42 ≈1 pMol/L). Three independent experiments were performed. (mean ± SD; *p < 0.001 vs. wild-type APP-RFP, ANOVA, n: number of wells in the representative experiment shown). B) FLIM analysis of the proximity between APP-CT RFP and myrGFP labeled membrane. The pseudo-colour images show distribution of the EGFP and myrGFP donor fluorophore lifetimes in the presence (bottom) or absence (top) of the RFP acceptor fluorophore fused to the wild-type APP-CT. Only cells transfected with myrGFP as a donor fluorophore showed lifetime shortening (red and yellow pixels) in the presence of APP-RFP, with the shortest lifetime at the cell periphery. Scale bar: 10 μm. Colorimetric scale shows GFP fluorophore lifetime in picoseconds. C) Quantitative FLIM analysis of the GFP lifetimes in APP/APLP2 dKO cells expressing wild-type and mutant APP-RFP constructs. In cells transfected with the EGFP as a donor fluorophore (grey bars), the donor lifetime did not change significantly in the presence of either wild-type or V717I APP-RFP. In cells transfected with myrGFP as a donor (black bars), the donor lifetime was significantly shortened in the presence of RFP acceptor at the APP CT. FAD-linked APP mutations (V717I and I716F) significantly shortened, whereas V717K APP mutation significantly increased the lifetime of myrGFP donor, compared to that of the wild-type APP (mean ± SD; *p < 0.001, **p < 0.01, ANOVA). Data from one of the three independent experiments is shown; n = cell number. D) CHO cells were transfected with APP-GFP to serve as a donor fluorophore (green) in the FLIM assay. Plasma membrane was stained with CM-DiI to serve as an acceptor fluorophore (red). Merged image shows that APP signal is outlined by red membrane. Scale bar; 10 μm. E) The graph shows average lifetime of GFP donor fluorophore in CHO cells from (D). The lifetime of GFP donor was shortened in cells with CM-DiI membrane staining. In the presence of I716F and V717I mutations, the GFP lifetime was significantly shorter than that in wild-type APP-GFP transfected cells (mean ± SD; *p < 0.01, ANOVA). Three independent experiments were performed (n: total cell number).
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Figure 1: The Ab42/40 ratio-modulating APP mutations induce changes in APP positioning relative to the membrane . A) ELISA detection of the human Aβ40 and Aβ42 in conditioned media of the cells transiently transfected with human APP-RFP constructs with designated mutations. The amount of each Aβ species was normalized to that obtained from the cells expressing wild-type APP-RFP (Aβ40 ≈70 pMol/L, Aβ42 ≈1 pMol/L). Three independent experiments were performed. (mean ± SD; *p < 0.001 vs. wild-type APP-RFP, ANOVA, n: number of wells in the representative experiment shown). B) FLIM analysis of the proximity between APP-CT RFP and myrGFP labeled membrane. The pseudo-colour images show distribution of the EGFP and myrGFP donor fluorophore lifetimes in the presence (bottom) or absence (top) of the RFP acceptor fluorophore fused to the wild-type APP-CT. Only cells transfected with myrGFP as a donor fluorophore showed lifetime shortening (red and yellow pixels) in the presence of APP-RFP, with the shortest lifetime at the cell periphery. Scale bar: 10 μm. Colorimetric scale shows GFP fluorophore lifetime in picoseconds. C) Quantitative FLIM analysis of the GFP lifetimes in APP/APLP2 dKO cells expressing wild-type and mutant APP-RFP constructs. In cells transfected with the EGFP as a donor fluorophore (grey bars), the donor lifetime did not change significantly in the presence of either wild-type or V717I APP-RFP. In cells transfected with myrGFP as a donor (black bars), the donor lifetime was significantly shortened in the presence of RFP acceptor at the APP CT. FAD-linked APP mutations (V717I and I716F) significantly shortened, whereas V717K APP mutation significantly increased the lifetime of myrGFP donor, compared to that of the wild-type APP (mean ± SD; *p < 0.001, **p < 0.01, ANOVA). Data from one of the three independent experiments is shown; n = cell number. D) CHO cells were transfected with APP-GFP to serve as a donor fluorophore (green) in the FLIM assay. Plasma membrane was stained with CM-DiI to serve as an acceptor fluorophore (red). Merged image shows that APP signal is outlined by red membrane. Scale bar; 10 μm. E) The graph shows average lifetime of GFP donor fluorophore in CHO cells from (D). The lifetime of GFP donor was shortened in cells with CM-DiI membrane staining. In the presence of I716F and V717I mutations, the GFP lifetime was significantly shorter than that in wild-type APP-GFP transfected cells (mean ± SD; *p < 0.01, ANOVA). Three independent experiments were performed (n: total cell number).
Mentions: First, we measured the effect of V717I, V717K or I716F mutations located in the transmembrane domain of APP on the Aβ42/40 ratio in CHO cells transiently transfected with the mutant APP constructs. As reported previously [8,9], the amount of Aβ42 was increased while the amount of the Aβ40 was decreased in the conditioned medium of V717I and I716F APP expressing cells, leading to a significantly elevated Aβ42/40 ratio (Figure 1A). By contrast, artificial V717K APP mutation [32] significantly lowered the Aβ42/40 ratio.

Bottom Line: Several familial Alzheimer disease (FAD) mutations within the transmembrane region of the amyloid precursor protein (APP) increase the Aβ42/40 ratio without increasing total Aβ production.In the present study, we analyzed the impact of FAD mutations and γ-secretase modulators (GSMs) that alter the Aβ42/40 ratio on APP C-terminus (CT) positioning relative to the membrane, reasoning that changes in the alignment of the APP intramembranous domain and presenilin 1 (PS1) may impact the PS1/γ-secretase cleavage site on APP.Thus, we propose that there is a reciprocal relationship between APP-CT positioning relative to the membrane and PS1 conformation, suggesting that factors that modulate either APP positioning in the membrane or PS1 conformation could be exploited therapeutically.

View Article: PubMed Central - HTML - PubMed

Affiliation: Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Charlestown, MA 02129, USA. oberezovska@partners.org.

ABSTRACT

Background: Several familial Alzheimer disease (FAD) mutations within the transmembrane region of the amyloid precursor protein (APP) increase the Aβ42/40 ratio without increasing total Aβ production. In the present study, we analyzed the impact of FAD mutations and γ-secretase modulators (GSMs) that alter the Aβ42/40 ratio on APP C-terminus (CT) positioning relative to the membrane, reasoning that changes in the alignment of the APP intramembranous domain and presenilin 1 (PS1) may impact the PS1/γ-secretase cleavage site on APP.

Results: By using a Förster resonance energy transfer (FRET)-based technique, fluorescent lifetime imaging microscopy (FLIM), we show that Aβ42/40 ratio-modulating factors which target either APP substrate or PS1/γ-secretase affect proximity of the APP-CT to the membrane and change PS1 conformation.

Conclusions: Thus, we propose that there is a reciprocal relationship between APP-CT positioning relative to the membrane and PS1 conformation, suggesting that factors that modulate either APP positioning in the membrane or PS1 conformation could be exploited therapeutically.

No MeSH data available.


Related in: MedlinePlus