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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

Cholesterol depletion inhibits β-cleavage. (A) N2a cells were grown in the presence (+) or absence (−) of lovastatin/ mevalonate/delipidated FCS and 10 h after infection with adenoviruses to express wtAPP treated with 10 mM MβCD for the indicated times. Cells were then labeled with [35S]methionine for 40 min and chased for 2 h. Immunoprecipitations of extracellular medium (Aβ; antibody 70JE) and cell lysate (total wtAPP; antibody IP60) revealed that Aβ secretion decreased after cholesterol depletion. The extent of cholesterol depletion was determined as described in Materials and methods. (B) 10 h after infection with adenoviruses to express swAPP, N2a cells were labeled for 30 min and chased for 30 min in the presence of 10 mM MβCD (leading to an ∼40–50% decrease in total cellular cholesterol). Immunoprecipitations from extracellular medium (antibody 6E10) and cell lysate (antibody IP60) showed a decreased production of Aβ and the COOH-terminal fragment generated by β-cleavage (βCTF). At the same time, the COOH-terminal fragment generated by α-cleavage (αCTF) and the soluble ectodomain generated by α-cleavage (αAPPs) were increased.
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fig1: Cholesterol depletion inhibits β-cleavage. (A) N2a cells were grown in the presence (+) or absence (−) of lovastatin/ mevalonate/delipidated FCS and 10 h after infection with adenoviruses to express wtAPP treated with 10 mM MβCD for the indicated times. Cells were then labeled with [35S]methionine for 40 min and chased for 2 h. Immunoprecipitations of extracellular medium (Aβ; antibody 70JE) and cell lysate (total wtAPP; antibody IP60) revealed that Aβ secretion decreased after cholesterol depletion. The extent of cholesterol depletion was determined as described in Materials and methods. (B) 10 h after infection with adenoviruses to express swAPP, N2a cells were labeled for 30 min and chased for 30 min in the presence of 10 mM MβCD (leading to an ∼40–50% decrease in total cellular cholesterol). Immunoprecipitations from extracellular medium (antibody 6E10) and cell lysate (antibody IP60) showed a decreased production of Aβ and the COOH-terminal fragment generated by β-cleavage (βCTF). At the same time, the COOH-terminal fragment generated by α-cleavage (αCTF) and the soluble ectodomain generated by α-cleavage (αAPPs) were increased.

Mentions: To easily monitor the influence of cholesterol depletion on APP processing, N2a cells were infected with adenoviruses to express either human wild-type APP (wtAPP) or the Swedish mutant of APP (swAPP). swAPP is dominantly β cleaved, resulting in a several-fold higher production of Aβ than for wtAPP. After MβCD extraction, the cells were metabolically labeled with [35S]methionine and chased for up to 2 h. Immunoprecipitations from conditioned medium revealed that Aβ production was dependent on cellular cholesterol levels (Fig. 1 A). Decreasing cellular cholesterol by 85% totally abolished Aβ secretion. Remarkably, already relatively small changes in total cellular cholesterol levels were found to have strong effects. A 20–30% decrease showed a 50–60% reduction in Aβ secretion. βCTF, which is generated by β-cleavage, was also clearly reduced. On the other hand, the production of αCTF by α-cleavage was increased (Fig. 1 B). As expected from results of Kojro et al. (2001), the soluble ectodomain generated by α-cleavage was also strongly increased in cholesterol-depleted cells. In general, processing of wtAPP and swAPP were similarly affected by cholesterol depletion.


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)

Cholesterol depletion inhibits β-cleavage. (A) N2a cells were grown in the presence (+) or absence (−) of lovastatin/ mevalonate/delipidated FCS and 10 h after infection with adenoviruses to express wtAPP treated with 10 mM MβCD for the indicated times. Cells were then labeled with [35S]methionine for 40 min and chased for 2 h. Immunoprecipitations of extracellular medium (Aβ; antibody 70JE) and cell lysate (total wtAPP; antibody IP60) revealed that Aβ secretion decreased after cholesterol depletion. The extent of cholesterol depletion was determined as described in Materials and methods. (B) 10 h after infection with adenoviruses to express swAPP, N2a cells were labeled for 30 min and chased for 30 min in the presence of 10 mM MβCD (leading to an ∼40–50% decrease in total cellular cholesterol). Immunoprecipitations from extracellular medium (antibody 6E10) and cell lysate (antibody IP60) showed a decreased production of Aβ and the COOH-terminal fragment generated by β-cleavage (βCTF). At the same time, the COOH-terminal fragment generated by α-cleavage (αCTF) and the soluble ectodomain generated by α-cleavage (αAPPs) were increased.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172747&req=5

fig1: Cholesterol depletion inhibits β-cleavage. (A) N2a cells were grown in the presence (+) or absence (−) of lovastatin/ mevalonate/delipidated FCS and 10 h after infection with adenoviruses to express wtAPP treated with 10 mM MβCD for the indicated times. Cells were then labeled with [35S]methionine for 40 min and chased for 2 h. Immunoprecipitations of extracellular medium (Aβ; antibody 70JE) and cell lysate (total wtAPP; antibody IP60) revealed that Aβ secretion decreased after cholesterol depletion. The extent of cholesterol depletion was determined as described in Materials and methods. (B) 10 h after infection with adenoviruses to express swAPP, N2a cells were labeled for 30 min and chased for 30 min in the presence of 10 mM MβCD (leading to an ∼40–50% decrease in total cellular cholesterol). Immunoprecipitations from extracellular medium (antibody 6E10) and cell lysate (antibody IP60) showed a decreased production of Aβ and the COOH-terminal fragment generated by β-cleavage (βCTF). At the same time, the COOH-terminal fragment generated by α-cleavage (αCTF) and the soluble ectodomain generated by α-cleavage (αAPPs) were increased.
Mentions: To easily monitor the influence of cholesterol depletion on APP processing, N2a cells were infected with adenoviruses to express either human wild-type APP (wtAPP) or the Swedish mutant of APP (swAPP). swAPP is dominantly β cleaved, resulting in a several-fold higher production of Aβ than for wtAPP. After MβCD extraction, the cells were metabolically labeled with [35S]methionine and chased for up to 2 h. Immunoprecipitations from conditioned medium revealed that Aβ production was dependent on cellular cholesterol levels (Fig. 1 A). Decreasing cellular cholesterol by 85% totally abolished Aβ secretion. Remarkably, already relatively small changes in total cellular cholesterol levels were found to have strong effects. A 20–30% decrease showed a 50–60% reduction in Aβ secretion. βCTF, which is generated by β-cleavage, was also clearly reduced. On the other hand, the production of αCTF by α-cleavage was increased (Fig. 1 B). As expected from results of Kojro et al. (2001), the soluble ectodomain generated by α-cleavage was also strongly increased in cholesterol-depleted cells. In general, processing of wtAPP and swAPP were similarly affected by cholesterol depletion.

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