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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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Munc18 is required for APP–syntaxin 1 copatching and regulates β cleavage in N2a cells. (a) The effect of RNAi-mediated knockdown of endogenous Munc18 on APP–syntaxin 1 copatching. RNAi reduced APP–syntaxin 1 copatching, indicating a requirement of Munc18 for APP–syntaxin 1 copatching. Results are means + SD of 32–38 measurements. (b) The effect of Munc18 knockdown on β cleavage. N2a cells were transfected with pHVenus-APP and RNAi vector. Released sAPP in medium was captured with anti-GFP and quantified with anti-sAPP end-specific antibodies. Munc18 knockdown promoted β cleavage, which is consistent with an inhibitory role of X11–Munc18–syntaxin 1 on β cleavage. Data are means + SD based on four independent experiments (n = 8). (c) The effect of Munc18 knockdown on the interaction between APP and syntaxin 1. N2a cells were lyzed with Lubrol and used for IP with anti-APPex. In the lysate of N2a cells transfected with RNAi vector, syntaxin 1 association with APP was reduced. ***, P < 0.001.
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fig5: Munc18 is required for APP–syntaxin 1 copatching and regulates β cleavage in N2a cells. (a) The effect of RNAi-mediated knockdown of endogenous Munc18 on APP–syntaxin 1 copatching. RNAi reduced APP–syntaxin 1 copatching, indicating a requirement of Munc18 for APP–syntaxin 1 copatching. Results are means + SD of 32–38 measurements. (b) The effect of Munc18 knockdown on β cleavage. N2a cells were transfected with pHVenus-APP and RNAi vector. Released sAPP in medium was captured with anti-GFP and quantified with anti-sAPP end-specific antibodies. Munc18 knockdown promoted β cleavage, which is consistent with an inhibitory role of X11–Munc18–syntaxin 1 on β cleavage. Data are means + SD based on four independent experiments (n = 8). (c) The effect of Munc18 knockdown on the interaction between APP and syntaxin 1. N2a cells were lyzed with Lubrol and used for IP with anti-APPex. In the lysate of N2a cells transfected with RNAi vector, syntaxin 1 association with APP was reduced. ***, P < 0.001.

Mentions: To confirm the requirement of Munc18 for microdomain association of APP and β cleavage regulation, we analyzed the effects of RNAi-mediated knockdown of Munc18 (Fig. 5). Ablation of Munc18 reduced the association of syntaxin 1 with APP observed by co-IP (Fig. 5 c). Reduction of APP–syntaxin 1 copatching (Fig. 5 a) confirmed the requirement of Munc18 for APP's association with syntaxin 1 microdomains. An increase in β cleavage (Fig. 5 b) was consistent with the negative regulation of β cleavage via X11–Munc18.


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)

Munc18 is required for APP–syntaxin 1 copatching and regulates β cleavage in N2a cells. (a) The effect of RNAi-mediated knockdown of endogenous Munc18 on APP–syntaxin 1 copatching. RNAi reduced APP–syntaxin 1 copatching, indicating a requirement of Munc18 for APP–syntaxin 1 copatching. Results are means + SD of 32–38 measurements. (b) The effect of Munc18 knockdown on β cleavage. N2a cells were transfected with pHVenus-APP and RNAi vector. Released sAPP in medium was captured with anti-GFP and quantified with anti-sAPP end-specific antibodies. Munc18 knockdown promoted β cleavage, which is consistent with an inhibitory role of X11–Munc18–syntaxin 1 on β cleavage. Data are means + SD based on four independent experiments (n = 8). (c) The effect of Munc18 knockdown on the interaction between APP and syntaxin 1. N2a cells were lyzed with Lubrol and used for IP with anti-APPex. In the lysate of N2a cells transfected with RNAi vector, syntaxin 1 association with APP was reduced. ***, P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Munc18 is required for APP–syntaxin 1 copatching and regulates β cleavage in N2a cells. (a) The effect of RNAi-mediated knockdown of endogenous Munc18 on APP–syntaxin 1 copatching. RNAi reduced APP–syntaxin 1 copatching, indicating a requirement of Munc18 for APP–syntaxin 1 copatching. Results are means + SD of 32–38 measurements. (b) The effect of Munc18 knockdown on β cleavage. N2a cells were transfected with pHVenus-APP and RNAi vector. Released sAPP in medium was captured with anti-GFP and quantified with anti-sAPP end-specific antibodies. Munc18 knockdown promoted β cleavage, which is consistent with an inhibitory role of X11–Munc18–syntaxin 1 on β cleavage. Data are means + SD based on four independent experiments (n = 8). (c) The effect of Munc18 knockdown on the interaction between APP and syntaxin 1. N2a cells were lyzed with Lubrol and used for IP with anti-APPex. In the lysate of N2a cells transfected with RNAi vector, syntaxin 1 association with APP was reduced. ***, P < 0.001.
Mentions: To confirm the requirement of Munc18 for microdomain association of APP and β cleavage regulation, we analyzed the effects of RNAi-mediated knockdown of Munc18 (Fig. 5). Ablation of Munc18 reduced the association of syntaxin 1 with APP observed by co-IP (Fig. 5 c). Reduction of APP–syntaxin 1 copatching (Fig. 5 a) confirmed the requirement of Munc18 for APP's association with syntaxin 1 microdomains. An increase in β cleavage (Fig. 5 b) was consistent with the negative regulation of β cleavage via X11–Munc18.

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