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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

Effect of antibody cross-linking on association of BACE1A-CFP and YFP-swAPP to DRMs. 10 h after adenovirus infection to express BACE1A-CFP or YFP-swAPP, the cells were labeled for 2 h with [35S]methionine and chased for 2 h in the presence of antibody KG77 (anti-FP) or antibody 7523 (anti-BACE1). The cells were subsequently lysed in 20 mM CHAPS/TNE at 4°C. (A) After flotation in an OptiPrep step gradient, BACE1A-CFP and YFP-swAPP were immunoprecipitated with antibody KG77 from the collected fractions. (B) Quantification; antibody-induced patching significantly increased the amount of APP (n = 3) and of BACE1 (n = 4) in the top two fractions (DRM associated). The amount in the top two fractions was correlated to the total amount of protein in all fractions.
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fig5: Effect of antibody cross-linking on association of BACE1A-CFP and YFP-swAPP to DRMs. 10 h after adenovirus infection to express BACE1A-CFP or YFP-swAPP, the cells were labeled for 2 h with [35S]methionine and chased for 2 h in the presence of antibody KG77 (anti-FP) or antibody 7523 (anti-BACE1). The cells were subsequently lysed in 20 mM CHAPS/TNE at 4°C. (A) After flotation in an OptiPrep step gradient, BACE1A-CFP and YFP-swAPP were immunoprecipitated with antibody KG77 from the collected fractions. (B) Quantification; antibody-induced patching significantly increased the amount of APP (n = 3) and of BACE1 (n = 4) in the top two fractions (DRM associated). The amount in the top two fractions was correlated to the total amount of protein in all fractions.

Mentions: Initial experiments revealed that in N2a cells only a minor amount (<5%) of both APP and BACE1 were resistant to extraction with 1% Triton X-100. However, when the cells were extracted with 20 mM CHAPS a significantly higher amount of BACE1 and APP floated to the low density membrane fraction in a cholesterol-dependent manner (unpublished data). Therefore, we used CHAPS-extracted membranes to examine the effect of antibody-induced patching on DRM association. N2a cells were infected with adenoviruses to express YFP-swAPP or BACE1A-CFP, metabolically labeled for 2 h with [35S]methionine, and chased for 2 h in the absence of antibody or in the presence of anti-FP (KG77) or anti-BACE1 (7523) antibodies, respectively. Cells were then extracted with 20 mM CHAPS, and the detergent extracts were subjected to OptiPrep step gradient centrifugation. A significantly higher fraction of APP and BACE1 floated with DRMs after antibody-induced patching (Fig. 5). Quantification revealed that without cross-linking ∼18.0 ± 2.6% of APP (n = 3) and 24.6 ± 2.3% of BACE1 (n = 4) were found in the upper two fractions (DRMs). Antibody cross-linking increased the DRM-associated fraction to 25.1 ± 1.2% (n = 3) and 32.3 ± 0.7% (n = 4) of APP and BACE1, respectively. Thus, both APP and BACE1 increased their detergent resistance upon cross-linking, probably reflecting increased raft affinity caused by oligomerization. Similar results have been obtained for other raft proteins, which increase their raft association by forming oligomers (Simons and Toomre, 2000; Cheng et al., 2001).


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 on association of BACE1A-CFP and YFP-swAPP to DRMs. 10 h after adenovirus infection to express BACE1A-CFP or YFP-swAPP, the cells were labeled for 2 h with [35S]methionine and chased for 2 h in the presence of antibody KG77 (anti-FP) or antibody 7523 (anti-BACE1). The cells were subsequently lysed in 20 mM CHAPS/TNE at 4°C. (A) After flotation in an OptiPrep step gradient, BACE1A-CFP and YFP-swAPP were immunoprecipitated with antibody KG77 from the collected fractions. (B) Quantification; antibody-induced patching significantly increased the amount of APP (n = 3) and of BACE1 (n = 4) in the top two fractions (DRM associated). The amount in the top two fractions was correlated to the total amount of protein in all fractions.
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Related In: Results  -  Collection

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

fig5: Effect of antibody cross-linking on association of BACE1A-CFP and YFP-swAPP to DRMs. 10 h after adenovirus infection to express BACE1A-CFP or YFP-swAPP, the cells were labeled for 2 h with [35S]methionine and chased for 2 h in the presence of antibody KG77 (anti-FP) or antibody 7523 (anti-BACE1). The cells were subsequently lysed in 20 mM CHAPS/TNE at 4°C. (A) After flotation in an OptiPrep step gradient, BACE1A-CFP and YFP-swAPP were immunoprecipitated with antibody KG77 from the collected fractions. (B) Quantification; antibody-induced patching significantly increased the amount of APP (n = 3) and of BACE1 (n = 4) in the top two fractions (DRM associated). The amount in the top two fractions was correlated to the total amount of protein in all fractions.
Mentions: Initial experiments revealed that in N2a cells only a minor amount (<5%) of both APP and BACE1 were resistant to extraction with 1% Triton X-100. However, when the cells were extracted with 20 mM CHAPS a significantly higher amount of BACE1 and APP floated to the low density membrane fraction in a cholesterol-dependent manner (unpublished data). Therefore, we used CHAPS-extracted membranes to examine the effect of antibody-induced patching on DRM association. N2a cells were infected with adenoviruses to express YFP-swAPP or BACE1A-CFP, metabolically labeled for 2 h with [35S]methionine, and chased for 2 h in the absence of antibody or in the presence of anti-FP (KG77) or anti-BACE1 (7523) antibodies, respectively. Cells were then extracted with 20 mM CHAPS, and the detergent extracts were subjected to OptiPrep step gradient centrifugation. A significantly higher fraction of APP and BACE1 floated with DRMs after antibody-induced patching (Fig. 5). Quantification revealed that without cross-linking ∼18.0 ± 2.6% of APP (n = 3) and 24.6 ± 2.3% of BACE1 (n = 4) were found in the upper two fractions (DRMs). Antibody cross-linking increased the DRM-associated fraction to 25.1 ± 1.2% (n = 3) and 32.3 ± 0.7% (n = 4) of APP and BACE1, respectively. Thus, both APP and BACE1 increased their detergent resistance upon cross-linking, probably reflecting increased raft affinity caused by oligomerization. Similar results have been obtained for other raft proteins, which increase their raft association by forming oligomers (Simons and Toomre, 2000; Cheng et al., 2001).

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