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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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Copatching demonstrates a preferential association between APP and PrP and BACE1 and Thy-1 as well as the relative separation between these two groups. (a) Preferential copatching between endogenous APP and PrP on axons of primary neurons. Mouse hippocampal neurons were treated with rabbit anti-APPex and rat anti–Thy-1.2 or mouse anti-PrP followed by fluorescently labeled secondary antibodies to induce patches of endogenous proteins on neurons. Colocalized APP and PrP patches are indicated by arrowheads. Bar, 5 μm. (b) Quantitative measurements of copatching demonstrated a statistically significant difference between APP–PrP and APP–Thy-1. n = 60. (c and d) Copatching on N2a cells demonstrates a preferential association between APP and PrP or syntaxin 1–HA and BACE1 and Thy-1 as well as the relative separation between these two groups. (c) Copatching is indicated by arrowheads. Bar, 1 μm. Quantitative measurements are shown in panel d. n = 34–44. (b and d) Data are means + SD based on three independent experiments. **, P < 0.01.
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fig3: Copatching demonstrates a preferential association between APP and PrP and BACE1 and Thy-1 as well as the relative separation between these two groups. (a) Preferential copatching between endogenous APP and PrP on axons of primary neurons. Mouse hippocampal neurons were treated with rabbit anti-APPex and rat anti–Thy-1.2 or mouse anti-PrP followed by fluorescently labeled secondary antibodies to induce patches of endogenous proteins on neurons. Colocalized APP and PrP patches are indicated by arrowheads. Bar, 5 μm. (b) Quantitative measurements of copatching demonstrated a statistically significant difference between APP–PrP and APP–Thy-1. n = 60. (c and d) Copatching on N2a cells demonstrates a preferential association between APP and PrP or syntaxin 1–HA and BACE1 and Thy-1 as well as the relative separation between these two groups. (c) Copatching is indicated by arrowheads. Bar, 1 μm. Quantitative measurements are shown in panel d. n = 34–44. (b and d) Data are means + SD based on three independent experiments. **, P < 0.01.

Mentions: In living cells, Thy-1 segregates away from PrP (Madore et al., 1999) and syntaxin 1 clusters (Lang et al., 2001) but associates with BACE1 in microdomains (Abad-Rodriguez et al., 2004). Conversely, APP segregates away from BACE1 in copatching experiments on primary neurons (Abad-Rodriguez et al., 2004) and in our DRM analysis but shows a preferential association with syntaxin 1 and PrP in DRMs (Fig. 2 b). These results prompted us to study the relationship between APP and microdomains rich in Thy-1 or PrP. These two GPI-anchored proteins are useful markers to analyze distinct microdomains in living neurons (Madore et al., 1999). We performed copatching of endogenous cell surface proteins on living hippocampal neurons as an established method to observe coresidence in the same microdomains (Harder et al., 1998; Abad-Rodriguez et al., 2004). To visualize antibody-induced protein clusters (patches) on the cell surface, we incubated live cells with two different sets of primary and fluorescently labeled secondary antibodies. Overlap between clusters of different proteins would indicate coresidence in the same microdomain. On primary neurons, patching of endogenous proteins revealed relative segregation between Thy-1 and APP as well as a preferential association between PrP and APP (Fig. 3, a and b). Exogenously expressed BACE1 showed a preferential association with Thy-1 and segregation from APP, as described previously (Abad-Rodriguez et al., 2004), and PrP (unpublished data). These results consistently indicate an association between PrP and APP and between Thy-1 and BACE1 as well as segregation of these two groups.


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)

Copatching demonstrates a preferential association between APP and PrP and BACE1 and Thy-1 as well as the relative separation between these two groups. (a) Preferential copatching between endogenous APP and PrP on axons of primary neurons. Mouse hippocampal neurons were treated with rabbit anti-APPex and rat anti–Thy-1.2 or mouse anti-PrP followed by fluorescently labeled secondary antibodies to induce patches of endogenous proteins on neurons. Colocalized APP and PrP patches are indicated by arrowheads. Bar, 5 μm. (b) Quantitative measurements of copatching demonstrated a statistically significant difference between APP–PrP and APP–Thy-1. n = 60. (c and d) Copatching on N2a cells demonstrates a preferential association between APP and PrP or syntaxin 1–HA and BACE1 and Thy-1 as well as the relative separation between these two groups. (c) Copatching is indicated by arrowheads. Bar, 1 μm. Quantitative measurements are shown in panel d. n = 34–44. (b and d) Data are means + SD based on three independent experiments. **, P < 0.01.
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Related In: Results  -  Collection

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fig3: Copatching demonstrates a preferential association between APP and PrP and BACE1 and Thy-1 as well as the relative separation between these two groups. (a) Preferential copatching between endogenous APP and PrP on axons of primary neurons. Mouse hippocampal neurons were treated with rabbit anti-APPex and rat anti–Thy-1.2 or mouse anti-PrP followed by fluorescently labeled secondary antibodies to induce patches of endogenous proteins on neurons. Colocalized APP and PrP patches are indicated by arrowheads. Bar, 5 μm. (b) Quantitative measurements of copatching demonstrated a statistically significant difference between APP–PrP and APP–Thy-1. n = 60. (c and d) Copatching on N2a cells demonstrates a preferential association between APP and PrP or syntaxin 1–HA and BACE1 and Thy-1 as well as the relative separation between these two groups. (c) Copatching is indicated by arrowheads. Bar, 1 μm. Quantitative measurements are shown in panel d. n = 34–44. (b and d) Data are means + SD based on three independent experiments. **, P < 0.01.
Mentions: In living cells, Thy-1 segregates away from PrP (Madore et al., 1999) and syntaxin 1 clusters (Lang et al., 2001) but associates with BACE1 in microdomains (Abad-Rodriguez et al., 2004). Conversely, APP segregates away from BACE1 in copatching experiments on primary neurons (Abad-Rodriguez et al., 2004) and in our DRM analysis but shows a preferential association with syntaxin 1 and PrP in DRMs (Fig. 2 b). These results prompted us to study the relationship between APP and microdomains rich in Thy-1 or PrP. These two GPI-anchored proteins are useful markers to analyze distinct microdomains in living neurons (Madore et al., 1999). We performed copatching of endogenous cell surface proteins on living hippocampal neurons as an established method to observe coresidence in the same microdomains (Harder et al., 1998; Abad-Rodriguez et al., 2004). To visualize antibody-induced protein clusters (patches) on the cell surface, we incubated live cells with two different sets of primary and fluorescently labeled secondary antibodies. Overlap between clusters of different proteins would indicate coresidence in the same microdomain. On primary neurons, patching of endogenous proteins revealed relative segregation between Thy-1 and APP as well as a preferential association between PrP and APP (Fig. 3, a and b). Exogenously expressed BACE1 showed a preferential association with Thy-1 and segregation from APP, as described previously (Abad-Rodriguez et al., 2004), and PrP (unpublished data). These results consistently indicate an association between PrP and APP and between Thy-1 and BACE1 as well as segregation of these two groups.

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