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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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Effect of antibody cross-linking and cholesterol depletion on Aβ secretion. (A) Cells were infected with adenoviruses to express YFP-wtAPP and BACE1-VSVG, metabolically labeled for 40 min with [35S]methionine, and chased for 2 h in the presence of the indicated antibodies. YFP-wtAPP was cross-linked with anti-FP antibody KG77, and BACE1 was cross-linked with antibody 7523. (B) Quantification of the two independent experiments of A. The ratio was arbitrarily set to 100% in cells not cross-linked with antibodies. Secreted Aβ was normalized to the total amount of APP found in the cell lysate. (C) Aβ secreted from cross-linked/cholesterol-depleted cells. N2a cells were grown in the presence (+depletion) or absence (−depletion) of lovastatin/mevalonate/lipid-deficient FCS. 10 h after infection with adenoviruses to express YFP-wtAPP, the cells were treated for 5 min with 10 mM MβCD, labeled for 40 min with [35S]methionine, and chased for 2 h in the presence (+Ab) or absence (−Ab) of anti-FP antibody KG77. (D) Quantification of five independent experiments. The amount of secreted Aβ was normalized to the total amount of APP present in the cell lysate. The ratio was arbitrarily set to 100% in cells neither cross-linked nor cholesterol depleted.
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fig6: Effect of antibody cross-linking and cholesterol depletion on Aβ secretion. (A) Cells were infected with adenoviruses to express YFP-wtAPP and BACE1-VSVG, metabolically labeled for 40 min with [35S]methionine, and chased for 2 h in the presence of the indicated antibodies. YFP-wtAPP was cross-linked with anti-FP antibody KG77, and BACE1 was cross-linked with antibody 7523. (B) Quantification of the two independent experiments of A. The ratio was arbitrarily set to 100% in cells not cross-linked with antibodies. Secreted Aβ was normalized to the total amount of APP found in the cell lysate. (C) Aβ secreted from cross-linked/cholesterol-depleted cells. N2a cells were grown in the presence (+depletion) or absence (−depletion) of lovastatin/mevalonate/lipid-deficient FCS. 10 h after infection with adenoviruses to express YFP-wtAPP, the cells were treated for 5 min with 10 mM MβCD, labeled for 40 min with [35S]methionine, and chased for 2 h in the presence (+Ab) or absence (−Ab) of anti-FP antibody KG77. (D) Quantification of five independent experiments. The amount of secreted Aβ was normalized to the total amount of APP present in the cell lysate. The ratio was arbitrarily set to 100% in cells neither cross-linked nor cholesterol depleted.

Mentions: If β-cleavage were to take place in cholesterol/sphingolipid-enriched microdomains, then antibody cross-linking should not only increase the association of APP and BACE1 with DRMs and induce their copatching at the surface of living cells, but cross-linking should also increase Aβ production. To find out whether this is the case, we analyzed the effect of antibody cross-linking on Aβ secretion. Cells were infected with adenoviruses to express YFP-wtAPP and BACE-VSVG. They were metabolically labeled for 40 min with [35S]-methionine and chased for 2 h in the presence of antibodies KG77 (anti-FP), 7523 (anti-BACE1), or both. Antibody cross-linking increased Aβ secretion significantly (Fig. 6, A and B).


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)

Effect of antibody cross-linking and cholesterol depletion on Aβ secretion. (A) Cells were infected with adenoviruses to express YFP-wtAPP and BACE1-VSVG, metabolically labeled for 40 min with [35S]methionine, and chased for 2 h in the presence of the indicated antibodies. YFP-wtAPP was cross-linked with anti-FP antibody KG77, and BACE1 was cross-linked with antibody 7523. (B) Quantification of the two independent experiments of A. The ratio was arbitrarily set to 100% in cells not cross-linked with antibodies. Secreted Aβ was normalized to the total amount of APP found in the cell lysate. (C) Aβ secreted from cross-linked/cholesterol-depleted cells. N2a cells were grown in the presence (+depletion) or absence (−depletion) of lovastatin/mevalonate/lipid-deficient FCS. 10 h after infection with adenoviruses to express YFP-wtAPP, the cells were treated for 5 min with 10 mM MβCD, labeled for 40 min with [35S]methionine, and chased for 2 h in the presence (+Ab) or absence (−Ab) of anti-FP antibody KG77. (D) Quantification of five independent experiments. The amount of secreted Aβ was normalized to the total amount of APP present in the cell lysate. The ratio was arbitrarily set to 100% in cells neither cross-linked nor cholesterol depleted.
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Related In: Results  -  Collection

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fig6: Effect of antibody cross-linking and cholesterol depletion on Aβ secretion. (A) Cells were infected with adenoviruses to express YFP-wtAPP and BACE1-VSVG, metabolically labeled for 40 min with [35S]methionine, and chased for 2 h in the presence of the indicated antibodies. YFP-wtAPP was cross-linked with anti-FP antibody KG77, and BACE1 was cross-linked with antibody 7523. (B) Quantification of the two independent experiments of A. The ratio was arbitrarily set to 100% in cells not cross-linked with antibodies. Secreted Aβ was normalized to the total amount of APP found in the cell lysate. (C) Aβ secreted from cross-linked/cholesterol-depleted cells. N2a cells were grown in the presence (+depletion) or absence (−depletion) of lovastatin/mevalonate/lipid-deficient FCS. 10 h after infection with adenoviruses to express YFP-wtAPP, the cells were treated for 5 min with 10 mM MβCD, labeled for 40 min with [35S]methionine, and chased for 2 h in the presence (+Ab) or absence (−Ab) of anti-FP antibody KG77. (D) Quantification of five independent experiments. The amount of secreted Aβ was normalized to the total amount of APP present in the cell lysate. The ratio was arbitrarily set to 100% in cells neither cross-linked nor cholesterol depleted.
Mentions: If β-cleavage were to take place in cholesterol/sphingolipid-enriched microdomains, then antibody cross-linking should not only increase the association of APP and BACE1 with DRMs and induce their copatching at the surface of living cells, but cross-linking should also increase Aβ production. To find out whether this is the case, we analyzed the effect of antibody cross-linking on Aβ secretion. Cells were infected with adenoviruses to express YFP-wtAPP and BACE-VSVG. They were metabolically labeled for 40 min with [35S]-methionine and chased for 2 h in the presence of antibodies KG77 (anti-FP), 7523 (anti-BACE1), or both. Antibody cross-linking increased Aβ secretion significantly (Fig. 6, A and B).

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