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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

Copatching of PLAP and TfR del 5–41 with YFP-wtAPP and BACE1A-CFP. 10 h after transient transfection, the cells were incubated for 45 min at 10°C with the primary antibodies. Patching of YFP-wtAPP and BACE1A-CFP was achieved with polyclonal antibodies KG77 and 7523, respectively. PLAP and TfR del 5–41 were patched with mouse monoclonal antibodies from Dako and Roche, respectively. Thereafter, the cells were washed and incubated for 45 min with mixed Cy5- and Cy3-labeled secondary antibodies. (A) YFP-wtAPP and BACE1A-CFP segregate from TfR del 5–41. (B) Colocalization of cross-linked YFP-wtAPP and BACE1A-CFP with PLAP. Bar, 10 μm.
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fig2: Copatching of PLAP and TfR del 5–41 with YFP-wtAPP and BACE1A-CFP. 10 h after transient transfection, the cells were incubated for 45 min at 10°C with the primary antibodies. Patching of YFP-wtAPP and BACE1A-CFP was achieved with polyclonal antibodies KG77 and 7523, respectively. PLAP and TfR del 5–41 were patched with mouse monoclonal antibodies from Dako and Roche, respectively. Thereafter, the cells were washed and incubated for 45 min with mixed Cy5- and Cy3-labeled secondary antibodies. (A) YFP-wtAPP and BACE1A-CFP segregate from TfR del 5–41. (B) Colocalization of cross-linked YFP-wtAPP and BACE1A-CFP with PLAP. Bar, 10 μm.

Mentions: Both BACE1 and wtAPP colocalized with PLAP at the plasma membrane in the majority of cells, but they clearly segregated from TfR del 5–41 (Fig. 2). BACE1 and wtAPP could also be localized to the same patches upon cross-linking (Fig. 3). For quantitative analyses of the extent of copatching, images of 10 randomly selected cells on one coverslip were taken and assigned into four categories: (1) coclustering (>80% overlap); (2) partial coclustering (clearly overlapping spots 30–80%); (3) random distribution, and (4) segregation. The data from five independent experiments (Fig. 4) indicate that cross-linked wtAPP and BACE1 copatched with the raft protein PLAP and segregated from the nonraft protein TfR del 5–41.


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)

Copatching of PLAP and TfR del 5–41 with YFP-wtAPP and BACE1A-CFP. 10 h after transient transfection, the cells were incubated for 45 min at 10°C with the primary antibodies. Patching of YFP-wtAPP and BACE1A-CFP was achieved with polyclonal antibodies KG77 and 7523, respectively. PLAP and TfR del 5–41 were patched with mouse monoclonal antibodies from Dako and Roche, respectively. Thereafter, the cells were washed and incubated for 45 min with mixed Cy5- and Cy3-labeled secondary antibodies. (A) YFP-wtAPP and BACE1A-CFP segregate from TfR del 5–41. (B) Colocalization of cross-linked YFP-wtAPP and BACE1A-CFP with PLAP. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Copatching of PLAP and TfR del 5–41 with YFP-wtAPP and BACE1A-CFP. 10 h after transient transfection, the cells were incubated for 45 min at 10°C with the primary antibodies. Patching of YFP-wtAPP and BACE1A-CFP was achieved with polyclonal antibodies KG77 and 7523, respectively. PLAP and TfR del 5–41 were patched with mouse monoclonal antibodies from Dako and Roche, respectively. Thereafter, the cells were washed and incubated for 45 min with mixed Cy5- and Cy3-labeled secondary antibodies. (A) YFP-wtAPP and BACE1A-CFP segregate from TfR del 5–41. (B) Colocalization of cross-linked YFP-wtAPP and BACE1A-CFP with PLAP. Bar, 10 μm.
Mentions: Both BACE1 and wtAPP colocalized with PLAP at the plasma membrane in the majority of cells, but they clearly segregated from TfR del 5–41 (Fig. 2). BACE1 and wtAPP could also be localized to the same patches upon cross-linking (Fig. 3). For quantitative analyses of the extent of copatching, images of 10 randomly selected cells on one coverslip were taken and assigned into four categories: (1) coclustering (>80% overlap); (2) partial coclustering (clearly overlapping spots 30–80%); (3) random distribution, and (4) segregation. The data from five independent experiments (Fig. 4) indicate that cross-linked wtAPP and BACE1 copatched with the raft protein PLAP and segregated from the nonraft protein TfR del 5–41.

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