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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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Association of APP with the syntaxin 1 microdomain through X11–Munc18 is controlled by cdk5 in N2a cells. (a) The effect of cdk5 on APP–syntaxin 1 and APP–BACE1 copatching. Cdk5/p35 expression caused switching of APP association in copatching. Results are means + SD of 20–31 measurements. (b) Effects of Munc18-1 mutants at the cdk5 phosphorylation site on APP–BACE1 copatching. Phosphorylation-resistant Munc18-1 T574A abolished the effect of cdk5, suggesting that the effect of cdk5 is mainly mediated by Munc18-1. A phosphorylation-mimetic mutant with reduced affinity to syntaxin 1, Munc18-1 T574E, had an effect similar to cdk5, indicating an involvement of syntaxin 1 and Munc18 in the regulation of the APP–BACE1 interaction. Data are means + SD based on three independent experiments (n = 32–44). (c) Effects of cdk5/p35 on interaction between syntaxin 1 and APP. N2a cells expressing Venus–syntaxin 1a with cdk5/p35 were used for IP with anti-GFP after cell lysis. Cdk5 reduced the association of Munc18, X11, and APP with syntaxin 1. **, P < 0.01.
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fig6: Association of APP with the syntaxin 1 microdomain through X11–Munc18 is controlled by cdk5 in N2a cells. (a) The effect of cdk5 on APP–syntaxin 1 and APP–BACE1 copatching. Cdk5/p35 expression caused switching of APP association in copatching. Results are means + SD of 20–31 measurements. (b) Effects of Munc18-1 mutants at the cdk5 phosphorylation site on APP–BACE1 copatching. Phosphorylation-resistant Munc18-1 T574A abolished the effect of cdk5, suggesting that the effect of cdk5 is mainly mediated by Munc18-1. A phosphorylation-mimetic mutant with reduced affinity to syntaxin 1, Munc18-1 T574E, had an effect similar to cdk5, indicating an involvement of syntaxin 1 and Munc18 in the regulation of the APP–BACE1 interaction. Data are means + SD based on three independent experiments (n = 32–44). (c) Effects of cdk5/p35 on interaction between syntaxin 1 and APP. N2a cells expressing Venus–syntaxin 1a with cdk5/p35 were used for IP with anti-GFP after cell lysis. Cdk5 reduced the association of Munc18, X11, and APP with syntaxin 1. **, P < 0.01.

Mentions: Phosphorylation of Munc18 by cdk5 is reported to induce conformational changes in syntaxin 1 to facilitate SNARE complex formation, although its physiological role is not yet elucidated (Fletcher et al., 1999; Rizo and Sudhof, 2002). We tested the effect of cdk5 on APP's microdomain association as a possible modulator. Because co-IP experiments demonstrated more efficient dissociation of APP, X11, and Munc18 from syntaxin 1 by cdk5/p35 (Fig. 6 c) than by cdk5/p25 (not depicted), we used cdk5/p35 for our subsequent experiments. Expression of cdk5/p35 in N2a cells caused a reduction of APP–syntaxin 1 copatching and a simultaneous increase in APP–BACE1 copatching (Fig. 6 a). To address whether the switching is mediated through Munc18 phosphorylation by cdk5, we used phosphorylation site mutants of Munc18, phosphorylation-resistant T574A that maintains affinity to syntaxin 1 (Fletcher et al., 1999), and a phosphorylation-mimetic T574E with reduced affinity to syntaxin 1 (Fig. 6 b; Liu et al., 2004). T574A blocked the effect of cdk5 on APP copatching, whereas T574E facilitated APP–BACE1 copatching and reduced APP–syntaxin 1 copatching without cdk5 expression. These results demonstrate that the effect of cdk5 is largely mediated by Munc18. Importantly, these data also indicate a requirement of syntaxin 1 for preventing the APP–BACE1 interaction. Copatching experiments showed segregation between BACE1 and syntaxin 1 and association between PrP and syntaxin 1 regardless of cdk5 expression (unpublished data). Similarly, we tested the effect of a phosphorylation-resistant mutant of APP, T668A, which maintains affinity to X11 but reduces the association with Fe65 (Ando et al., 2001). This mutant did not affect APP–syntaxin 1 or APP–BACE1 copatching or cdk5-induced switching, suggesting a major role of X11–Munc18–syntaxin 1 in the cdk5-dependent regulation of APP–BACE1 interaction (unpublished data). Consistent with these results, there was no significant change in APP processing in APP T668A knock-in mice (Sano et al., 2006).


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)

Association of APP with the syntaxin 1 microdomain through X11–Munc18 is controlled by cdk5 in N2a cells. (a) The effect of cdk5 on APP–syntaxin 1 and APP–BACE1 copatching. Cdk5/p35 expression caused switching of APP association in copatching. Results are means + SD of 20–31 measurements. (b) Effects of Munc18-1 mutants at the cdk5 phosphorylation site on APP–BACE1 copatching. Phosphorylation-resistant Munc18-1 T574A abolished the effect of cdk5, suggesting that the effect of cdk5 is mainly mediated by Munc18-1. A phosphorylation-mimetic mutant with reduced affinity to syntaxin 1, Munc18-1 T574E, had an effect similar to cdk5, indicating an involvement of syntaxin 1 and Munc18 in the regulation of the APP–BACE1 interaction. Data are means + SD based on three independent experiments (n = 32–44). (c) Effects of cdk5/p35 on interaction between syntaxin 1 and APP. N2a cells expressing Venus–syntaxin 1a with cdk5/p35 were used for IP with anti-GFP after cell lysis. Cdk5 reduced the association of Munc18, X11, and APP with syntaxin 1. **, P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Association of APP with the syntaxin 1 microdomain through X11–Munc18 is controlled by cdk5 in N2a cells. (a) The effect of cdk5 on APP–syntaxin 1 and APP–BACE1 copatching. Cdk5/p35 expression caused switching of APP association in copatching. Results are means + SD of 20–31 measurements. (b) Effects of Munc18-1 mutants at the cdk5 phosphorylation site on APP–BACE1 copatching. Phosphorylation-resistant Munc18-1 T574A abolished the effect of cdk5, suggesting that the effect of cdk5 is mainly mediated by Munc18-1. A phosphorylation-mimetic mutant with reduced affinity to syntaxin 1, Munc18-1 T574E, had an effect similar to cdk5, indicating an involvement of syntaxin 1 and Munc18 in the regulation of the APP–BACE1 interaction. Data are means + SD based on three independent experiments (n = 32–44). (c) Effects of cdk5/p35 on interaction between syntaxin 1 and APP. N2a cells expressing Venus–syntaxin 1a with cdk5/p35 were used for IP with anti-GFP after cell lysis. Cdk5 reduced the association of Munc18, X11, and APP with syntaxin 1. **, P < 0.01.
Mentions: Phosphorylation of Munc18 by cdk5 is reported to induce conformational changes in syntaxin 1 to facilitate SNARE complex formation, although its physiological role is not yet elucidated (Fletcher et al., 1999; Rizo and Sudhof, 2002). We tested the effect of cdk5 on APP's microdomain association as a possible modulator. Because co-IP experiments demonstrated more efficient dissociation of APP, X11, and Munc18 from syntaxin 1 by cdk5/p35 (Fig. 6 c) than by cdk5/p25 (not depicted), we used cdk5/p35 for our subsequent experiments. Expression of cdk5/p35 in N2a cells caused a reduction of APP–syntaxin 1 copatching and a simultaneous increase in APP–BACE1 copatching (Fig. 6 a). To address whether the switching is mediated through Munc18 phosphorylation by cdk5, we used phosphorylation site mutants of Munc18, phosphorylation-resistant T574A that maintains affinity to syntaxin 1 (Fletcher et al., 1999), and a phosphorylation-mimetic T574E with reduced affinity to syntaxin 1 (Fig. 6 b; Liu et al., 2004). T574A blocked the effect of cdk5 on APP copatching, whereas T574E facilitated APP–BACE1 copatching and reduced APP–syntaxin 1 copatching without cdk5 expression. These results demonstrate that the effect of cdk5 is largely mediated by Munc18. Importantly, these data also indicate a requirement of syntaxin 1 for preventing the APP–BACE1 interaction. Copatching experiments showed segregation between BACE1 and syntaxin 1 and association between PrP and syntaxin 1 regardless of cdk5 expression (unpublished data). Similarly, we tested the effect of a phosphorylation-resistant mutant of APP, T668A, which maintains affinity to X11 but reduces the association with Fe65 (Ando et al., 2001). This mutant did not affect APP–syntaxin 1 or APP–BACE1 copatching or cdk5-induced switching, suggesting a major role of X11–Munc18–syntaxin 1 in the cdk5-dependent regulation of APP–BACE1 interaction (unpublished data). Consistent with these results, there was no significant change in APP processing in APP T668A knock-in mice (Sano et al., 2006).

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