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Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts.

Ehehalt R, Keller P, Haass C, Thiele C, Simons K - J. Cell Biol. (2003)

Bottom Line: A beta generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre.Although APP inside raft clusters seems to be cleaved by beta-secretase, APP outside rafts undergoes cleavage by alpha-secretase.Thus, access of alpha- and beta-secretase to APP, and therefore A beta generation, may be determined by dynamic interactions of APP with lipid rafts.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, D-01307 Dresden, Germany.

ABSTRACT
Formation of senile plaques containing the beta-amyloid peptide (A beta) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by beta-secretase or by alpha-secretase to initiate amyloidogenic (release of A beta) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating A beta generation. Reducing cholesterol levels in N2a cells decreased A beta production. APP and the beta-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of A beta in a cholesterol-dependent manner. A beta generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by beta-secretase, APP outside rafts undergoes cleavage by alpha-secretase. Thus, access of alpha- and beta-secretase to APP, and therefore A beta generation, may be determined by dynamic interactions of APP with lipid rafts.

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Related in: MedlinePlus

Endocytosis is essential for the generation of Aβ, and antibody cross-linking overcomes the block caused by inhibition of endocytosis. (A) Inhibition of endocytosis and APP processing. After transient transfection with YFP-swAPP and RN-tre or dynamin K44A, N2a cells were metabolically labeled for 1 h with [35S]methionine. Immunoprecipitations from cell lysate (swAPP, αCTF, βCTF; antibody IP60) and medium (Aβ; antibody 70JE) show a decrease of fragments generated by β-cleavage (βCTF and Aβ) upon expression of endocytosis inhibitors. (B) The effect of copatching of APP and BACE1 on Aβ secretion under conditions where endocytosis is inhibited. Cells were transfected with YFP-swAPP/BACE1A-VSVG/empty vector (−inhibition) or YFP-swAPP/BACE1A-VSVG/dynamin K44A (+inhibition), labeled with [35S]methionine for 40 min, and chased for 2 h in the presence (+Ab) or absence (−Ab) of antibodies KG77 (anti-FP) and 7523 (anti-BACE1). Quantification of three independent experiments showed a significant increase in Aβ generation upon copatching under both conditions. The values are normalized to the total amount of APP present in the cell lysate. The ratio has been arbitrarily set to 100% in nonpatched cells transfected with YFP-swAPP/BACE1A VSVG/empty vector. (C) Effect of antibody cross-linking on internalization of biotin transferrin under conditions where endocytosis is inhibited by expression of dynamin K44A. Cells were transfected with human transferrin receptor (TfR) and dynK44A-GFP, incubated on ice with biotin transferrin, and chased for 10 min at 37°C in the presence (+Ab) or absence (−Ab) of anti–human TfR antibodies. Biotin transferrin was quantified on an Origen M8 analyzer in cells where remaining surface transferrin was removed by acid wash (black columns) or controls (no acid wash; grey columns). Cross-linking did not increase internalization of transferrin.
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fig7: Endocytosis is essential for the generation of Aβ, and antibody cross-linking overcomes the block caused by inhibition of endocytosis. (A) Inhibition of endocytosis and APP processing. After transient transfection with YFP-swAPP and RN-tre or dynamin K44A, N2a cells were metabolically labeled for 1 h with [35S]methionine. Immunoprecipitations from cell lysate (swAPP, αCTF, βCTF; antibody IP60) and medium (Aβ; antibody 70JE) show a decrease of fragments generated by β-cleavage (βCTF and Aβ) upon expression of endocytosis inhibitors. (B) The effect of copatching of APP and BACE1 on Aβ secretion under conditions where endocytosis is inhibited. Cells were transfected with YFP-swAPP/BACE1A-VSVG/empty vector (−inhibition) or YFP-swAPP/BACE1A-VSVG/dynamin K44A (+inhibition), labeled with [35S]methionine for 40 min, and chased for 2 h in the presence (+Ab) or absence (−Ab) of antibodies KG77 (anti-FP) and 7523 (anti-BACE1). Quantification of three independent experiments showed a significant increase in Aβ generation upon copatching under both conditions. The values are normalized to the total amount of APP present in the cell lysate. The ratio has been arbitrarily set to 100% in nonpatched cells transfected with YFP-swAPP/BACE1A VSVG/empty vector. (C) Effect of antibody cross-linking on internalization of biotin transferrin under conditions where endocytosis is inhibited by expression of dynamin K44A. Cells were transfected with human transferrin receptor (TfR) and dynK44A-GFP, incubated on ice with biotin transferrin, and chased for 10 min at 37°C in the presence (+Ab) or absence (−Ab) of anti–human TfR antibodies. Biotin transferrin was quantified on an Origen M8 analyzer in cells where remaining surface transferrin was removed by acid wash (black columns) or controls (no acid wash; grey columns). Cross-linking did not increase internalization of transferrin.

Mentions: N2a cells were transiently transfected with equal amounts of plasmids encoding for swAPP, RN-tre, or dynamin K44A and labeled for 1 h with [35S]methionine. Immunoprecipitations from cell lysates with antibody IP60 (raised against the COOH terminus of APP) and from media with antibody 70JE (Aβ) were performed (Fig. 7 A). APP biosynthesis was unchanged after expression of RN-tre or dynamin K44A; however, the COOH-terminal fragment generated by β-cleavage (βCTF) and secretion of Aβ were significantly reduced. Expression of dynamin K44A inhibited Aβ secretion by 80–90% (Fig. 7 A). Remarkably, the membrane-bound fragment generated by α-cleavage (αCTF) was only slightly increased (correlated to total APP). Thus, under our experimental conditions endocytosis was essential for β-cleavage to occur, whereas α-cleavage was not appreciably stimulated by inhibiting endocytosis.


Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts.

Ehehalt R, Keller P, Haass C, Thiele C, Simons K - J. Cell Biol. (2003)

Endocytosis is essential for the generation of Aβ, and antibody cross-linking overcomes the block caused by inhibition of endocytosis. (A) Inhibition of endocytosis and APP processing. After transient transfection with YFP-swAPP and RN-tre or dynamin K44A, N2a cells were metabolically labeled for 1 h with [35S]methionine. Immunoprecipitations from cell lysate (swAPP, αCTF, βCTF; antibody IP60) and medium (Aβ; antibody 70JE) show a decrease of fragments generated by β-cleavage (βCTF and Aβ) upon expression of endocytosis inhibitors. (B) The effect of copatching of APP and BACE1 on Aβ secretion under conditions where endocytosis is inhibited. Cells were transfected with YFP-swAPP/BACE1A-VSVG/empty vector (−inhibition) or YFP-swAPP/BACE1A-VSVG/dynamin K44A (+inhibition), labeled with [35S]methionine for 40 min, and chased for 2 h in the presence (+Ab) or absence (−Ab) of antibodies KG77 (anti-FP) and 7523 (anti-BACE1). Quantification of three independent experiments showed a significant increase in Aβ generation upon copatching under both conditions. The values are normalized to the total amount of APP present in the cell lysate. The ratio has been arbitrarily set to 100% in nonpatched cells transfected with YFP-swAPP/BACE1A VSVG/empty vector. (C) Effect of antibody cross-linking on internalization of biotin transferrin under conditions where endocytosis is inhibited by expression of dynamin K44A. Cells were transfected with human transferrin receptor (TfR) and dynK44A-GFP, incubated on ice with biotin transferrin, and chased for 10 min at 37°C in the presence (+Ab) or absence (−Ab) of anti–human TfR antibodies. Biotin transferrin was quantified on an Origen M8 analyzer in cells where remaining surface transferrin was removed by acid wash (black columns) or controls (no acid wash; grey columns). Cross-linking did not increase internalization of transferrin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172747&req=5

fig7: Endocytosis is essential for the generation of Aβ, and antibody cross-linking overcomes the block caused by inhibition of endocytosis. (A) Inhibition of endocytosis and APP processing. After transient transfection with YFP-swAPP and RN-tre or dynamin K44A, N2a cells were metabolically labeled for 1 h with [35S]methionine. Immunoprecipitations from cell lysate (swAPP, αCTF, βCTF; antibody IP60) and medium (Aβ; antibody 70JE) show a decrease of fragments generated by β-cleavage (βCTF and Aβ) upon expression of endocytosis inhibitors. (B) The effect of copatching of APP and BACE1 on Aβ secretion under conditions where endocytosis is inhibited. Cells were transfected with YFP-swAPP/BACE1A-VSVG/empty vector (−inhibition) or YFP-swAPP/BACE1A-VSVG/dynamin K44A (+inhibition), labeled with [35S]methionine for 40 min, and chased for 2 h in the presence (+Ab) or absence (−Ab) of antibodies KG77 (anti-FP) and 7523 (anti-BACE1). Quantification of three independent experiments showed a significant increase in Aβ generation upon copatching under both conditions. The values are normalized to the total amount of APP present in the cell lysate. The ratio has been arbitrarily set to 100% in nonpatched cells transfected with YFP-swAPP/BACE1A VSVG/empty vector. (C) Effect of antibody cross-linking on internalization of biotin transferrin under conditions where endocytosis is inhibited by expression of dynamin K44A. Cells were transfected with human transferrin receptor (TfR) and dynK44A-GFP, incubated on ice with biotin transferrin, and chased for 10 min at 37°C in the presence (+Ab) or absence (−Ab) of anti–human TfR antibodies. Biotin transferrin was quantified on an Origen M8 analyzer in cells where remaining surface transferrin was removed by acid wash (black columns) or controls (no acid wash; grey columns). Cross-linking did not increase internalization of transferrin.
Mentions: N2a cells were transiently transfected with equal amounts of plasmids encoding for swAPP, RN-tre, or dynamin K44A and labeled for 1 h with [35S]methionine. Immunoprecipitations from cell lysates with antibody IP60 (raised against the COOH terminus of APP) and from media with antibody 70JE (Aβ) were performed (Fig. 7 A). APP biosynthesis was unchanged after expression of RN-tre or dynamin K44A; however, the COOH-terminal fragment generated by β-cleavage (βCTF) and secretion of Aβ were significantly reduced. Expression of dynamin K44A inhibited Aβ secretion by 80–90% (Fig. 7 A). Remarkably, the membrane-bound fragment generated by α-cleavage (αCTF) was only slightly increased (correlated to total APP). Thus, under our experimental conditions endocytosis was essential for β-cleavage to occur, whereas α-cleavage was not appreciably stimulated by inhibiting endocytosis.

Bottom Line: A beta generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre.Although APP inside raft clusters seems to be cleaved by beta-secretase, APP outside rafts undergoes cleavage by alpha-secretase.Thus, access of alpha- and beta-secretase to APP, and therefore A beta generation, may be determined by dynamic interactions of APP with lipid rafts.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, D-01307 Dresden, Germany.

ABSTRACT
Formation of senile plaques containing the beta-amyloid peptide (A beta) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by beta-secretase or by alpha-secretase to initiate amyloidogenic (release of A beta) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating A beta generation. Reducing cholesterol levels in N2a cells decreased A beta production. APP and the beta-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of A beta in a cholesterol-dependent manner. A beta generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by beta-secretase, APP outside rafts undergoes cleavage by alpha-secretase. Thus, access of alpha- and beta-secretase to APP, and therefore A beta generation, may be determined by dynamic interactions of APP with lipid rafts.

Show MeSH
Related in: MedlinePlus