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Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing.

Sakurai T, Kaneko K, Okuno M, Wada K, Kashiyama T, Shimizu H, Akagi T, Hashikawa T, Nukina N - J. Cell Biol. (2008)

Bottom Line: However, the molecular mechanisms underlying this effect remain to be elucidated.We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains.We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.

ABSTRACT
Neuronal activity has an impact on beta cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-beta peptide (Abeta). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of beta cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1-containing microdomains through X11-Munc18, which inhibits the APP-BACE1 interaction and beta cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Abeta overproduction, promotes the switching of APP microdomain association as well as beta cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

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Successful immunoisolation of DRM subfractions rich in APP or BACE1. Protein compositions of immunoisolated DRM subfractions were compared by silver staining (a) and WB (b). Brain-derived Lubrol DRMs were immunoaffinity purified with anti-APPc or anti-BACE1c. Protein band patterns of the DRM subfractions were distinct from that of the input (a). WB analysis showed minimal overlap between APP and BACE1 (b). X11, CASK, Munc18, and syntaxin 1 were enriched in APP DRMs. Proteins in the bound fraction derived from the same amount of DRM were loaded. The total amount of bound proteins in BACE1 DRMs estimated by SYPRO Ruby staining was roughly 50% of that in APP DRMs. PrP showed preferential association with APP DRMs over BACE1 DRMs.
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fig2: Successful immunoisolation of DRM subfractions rich in APP or BACE1. Protein compositions of immunoisolated DRM subfractions were compared by silver staining (a) and WB (b). Brain-derived Lubrol DRMs were immunoaffinity purified with anti-APPc or anti-BACE1c. Protein band patterns of the DRM subfractions were distinct from that of the input (a). WB analysis showed minimal overlap between APP and BACE1 (b). X11, CASK, Munc18, and syntaxin 1 were enriched in APP DRMs. Proteins in the bound fraction derived from the same amount of DRM were loaded. The total amount of bound proteins in BACE1 DRMs estimated by SYPRO Ruby staining was roughly 50% of that in APP DRMs. PrP showed preferential association with APP DRMs over BACE1 DRMs.

Mentions: To analyze APP-rich Lubrol-DRM subfractions derived from brain tissue or primary neurons, we performed immunomagnetic isolation using an affinity-purified antibody against the APP cytoplasmic domain. Successful isolation was shown by WB analysis, demonstrating almost complete precipitation of APP in DRMs (Fig. 2 b) and blocking by antigen peptide preabsorption (not depicted). The anti-APP antibody precipitated a discrete set of proteins from the input DRMs (Fig. 2 a) corresponding to 2% of the total input (estimation by SYPRO Ruby staining), indicating an association of APP with specialized subpopulations of DRMs. Identification of protein components by mass spectrometry (MS) and WB analysis revealed syntaxin 1 as a probable link between APP and the cholesterol-dependent microdomains distinct from rafts (Lang et al., 2001) and a possible connection with synaptic activity. As candidate links between APP and syntaxin 1, X11/X11L and Munc18 were detected in APP-containing DRMs by WB (Fig. 2 b) but not by MS analysis. Because APP–X11–Munc18 and X11–Munc18–syntaxin 1 interactions have been reported, we hypothesized that X11/X11L–Munc18 provides a link between APP and syntaxin 1.


Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing.

Sakurai T, Kaneko K, Okuno M, Wada K, Kashiyama T, Shimizu H, Akagi T, Hashikawa T, Nukina N - J. Cell Biol. (2008)

Successful immunoisolation of DRM subfractions rich in APP or BACE1. Protein compositions of immunoisolated DRM subfractions were compared by silver staining (a) and WB (b). Brain-derived Lubrol DRMs were immunoaffinity purified with anti-APPc or anti-BACE1c. Protein band patterns of the DRM subfractions were distinct from that of the input (a). WB analysis showed minimal overlap between APP and BACE1 (b). X11, CASK, Munc18, and syntaxin 1 were enriched in APP DRMs. Proteins in the bound fraction derived from the same amount of DRM were loaded. The total amount of bound proteins in BACE1 DRMs estimated by SYPRO Ruby staining was roughly 50% of that in APP DRMs. PrP showed preferential association with APP DRMs over BACE1 DRMs.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2568028&req=5

fig2: Successful immunoisolation of DRM subfractions rich in APP or BACE1. Protein compositions of immunoisolated DRM subfractions were compared by silver staining (a) and WB (b). Brain-derived Lubrol DRMs were immunoaffinity purified with anti-APPc or anti-BACE1c. Protein band patterns of the DRM subfractions were distinct from that of the input (a). WB analysis showed minimal overlap between APP and BACE1 (b). X11, CASK, Munc18, and syntaxin 1 were enriched in APP DRMs. Proteins in the bound fraction derived from the same amount of DRM were loaded. The total amount of bound proteins in BACE1 DRMs estimated by SYPRO Ruby staining was roughly 50% of that in APP DRMs. PrP showed preferential association with APP DRMs over BACE1 DRMs.
Mentions: To analyze APP-rich Lubrol-DRM subfractions derived from brain tissue or primary neurons, we performed immunomagnetic isolation using an affinity-purified antibody against the APP cytoplasmic domain. Successful isolation was shown by WB analysis, demonstrating almost complete precipitation of APP in DRMs (Fig. 2 b) and blocking by antigen peptide preabsorption (not depicted). The anti-APP antibody precipitated a discrete set of proteins from the input DRMs (Fig. 2 a) corresponding to 2% of the total input (estimation by SYPRO Ruby staining), indicating an association of APP with specialized subpopulations of DRMs. Identification of protein components by mass spectrometry (MS) and WB analysis revealed syntaxin 1 as a probable link between APP and the cholesterol-dependent microdomains distinct from rafts (Lang et al., 2001) and a possible connection with synaptic activity. As candidate links between APP and syntaxin 1, X11/X11L and Munc18 were detected in APP-containing DRMs by WB (Fig. 2 b) but not by MS analysis. Because APP–X11–Munc18 and X11–Munc18–syntaxin 1 interactions have been reported, we hypothesized that X11/X11L–Munc18 provides a link between APP and syntaxin 1.

Bottom Line: However, the molecular mechanisms underlying this effect remain to be elucidated.We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains.We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.

ABSTRACT
Neuronal activity has an impact on beta cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-beta peptide (Abeta). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of beta cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1-containing microdomains through X11-Munc18, which inhibits the APP-BACE1 interaction and beta cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Abeta overproduction, promotes the switching of APP microdomain association as well as beta cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

Show MeSH
Related in: MedlinePlus