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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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Related in: MedlinePlus

Mature forms of APP and BACE1 show association with Lubrol-resistant membranes. (a) Mature forms of APP were efficiently recovered in DRM fractions derived from mouse brains solubilized with Lubrol WX but poorly with Triton X-100 or Brij 97. All three detergents showed recovery of PrP (a DRM marker) in DRM fractions well separated from calnexin (a detergent-soluble membrane marker). Arrows, different forms of FL-APP; m, mature forms; im, immature form of APP. (b) Quantitative comparison of distributions of mature and immature forms of APP across the gradients after Lubrol WX or Brij 97 solubilization. Each point represents a percentage of the total. Results are mean ± SD based on three to four independent experiments. (c) Mature forms and CTFs of APP and BACE1 associate with DRM fractions derived from primary cultured cortical neurons solubilized with Lubrol WX, whereas ADAM17 mainly resides in Lubrol-soluble membranes. GPI-anchored proteins Thy-1 and PrP were used as DRM markers, and calnexin and GM130 were used as detergent-soluble membrane markers. We used longer exposure to detect APP-CTFs compared with FL-APP. (d) Lubrol WX causes only negligible levels of artifactual intermingling of Thy-1. Rat and mouse brain tissues were cohomogenized in 1% Lubrol WX or 1% Brij 97, and DRMs were prepared. Thy-1 on DRMs was immunoaffinity purified and probed with species-specific antibodies. Lubrol caused only negligible levels of mixing of Thy-1 on DRMs during solubilization, centrifugation, and immunoisolation.
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fig1: Mature forms of APP and BACE1 show association with Lubrol-resistant membranes. (a) Mature forms of APP were efficiently recovered in DRM fractions derived from mouse brains solubilized with Lubrol WX but poorly with Triton X-100 or Brij 97. All three detergents showed recovery of PrP (a DRM marker) in DRM fractions well separated from calnexin (a detergent-soluble membrane marker). Arrows, different forms of FL-APP; m, mature forms; im, immature form of APP. (b) Quantitative comparison of distributions of mature and immature forms of APP across the gradients after Lubrol WX or Brij 97 solubilization. Each point represents a percentage of the total. Results are mean ± SD based on three to four independent experiments. (c) Mature forms and CTFs of APP and BACE1 associate with DRM fractions derived from primary cultured cortical neurons solubilized with Lubrol WX, whereas ADAM17 mainly resides in Lubrol-soluble membranes. GPI-anchored proteins Thy-1 and PrP were used as DRM markers, and calnexin and GM130 were used as detergent-soluble membrane markers. We used longer exposure to detect APP-CTFs compared with FL-APP. (d) Lubrol WX causes only negligible levels of artifactual intermingling of Thy-1. Rat and mouse brain tissues were cohomogenized in 1% Lubrol WX or 1% Brij 97, and DRMs were prepared. Thy-1 on DRMs was immunoaffinity purified and probed with species-specific antibodies. Lubrol caused only negligible levels of mixing of Thy-1 on DRMs during solubilization, centrifugation, and immunoisolation.

Mentions: Biochemically, membrane microdomains are characterized as DRMs by insolubility in nonionic detergents at 4°C and flotation on density gradients. Triton X-100 is the standard detergent for this purpose, but other detergents are also useful for showing microdomain heterogeneity or other types of microdomains (Madore et al., 1999; Roper et al., 2000; Pike, 2004). We prepared DRMs for two main purposes: (1) the efficient and reproducible recovery of APP in DRMs from brains and (2) to maintain the separate identity of membranes without substantial intermingling of protein components to validate immunoaffinity purification. We tried several nonionic detergents, including Brij 97 and Lubrol WX. All detergents tested showed a clear enrichment of a DRM marker, prion protein (PrP), in DRM fractions and separation from a detergent-soluble membrane marker, calnexin (Fig. 1 a). DRM fractions were also rich in cholesterol and GM1-ganglioside (unpublished data).


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)

Mature forms of APP and BACE1 show association with Lubrol-resistant membranes. (a) Mature forms of APP were efficiently recovered in DRM fractions derived from mouse brains solubilized with Lubrol WX but poorly with Triton X-100 or Brij 97. All three detergents showed recovery of PrP (a DRM marker) in DRM fractions well separated from calnexin (a detergent-soluble membrane marker). Arrows, different forms of FL-APP; m, mature forms; im, immature form of APP. (b) Quantitative comparison of distributions of mature and immature forms of APP across the gradients after Lubrol WX or Brij 97 solubilization. Each point represents a percentage of the total. Results are mean ± SD based on three to four independent experiments. (c) Mature forms and CTFs of APP and BACE1 associate with DRM fractions derived from primary cultured cortical neurons solubilized with Lubrol WX, whereas ADAM17 mainly resides in Lubrol-soluble membranes. GPI-anchored proteins Thy-1 and PrP were used as DRM markers, and calnexin and GM130 were used as detergent-soluble membrane markers. We used longer exposure to detect APP-CTFs compared with FL-APP. (d) Lubrol WX causes only negligible levels of artifactual intermingling of Thy-1. Rat and mouse brain tissues were cohomogenized in 1% Lubrol WX or 1% Brij 97, and DRMs were prepared. Thy-1 on DRMs was immunoaffinity purified and probed with species-specific antibodies. Lubrol caused only negligible levels of mixing of Thy-1 on DRMs during solubilization, centrifugation, and immunoisolation.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Mature forms of APP and BACE1 show association with Lubrol-resistant membranes. (a) Mature forms of APP were efficiently recovered in DRM fractions derived from mouse brains solubilized with Lubrol WX but poorly with Triton X-100 or Brij 97. All three detergents showed recovery of PrP (a DRM marker) in DRM fractions well separated from calnexin (a detergent-soluble membrane marker). Arrows, different forms of FL-APP; m, mature forms; im, immature form of APP. (b) Quantitative comparison of distributions of mature and immature forms of APP across the gradients after Lubrol WX or Brij 97 solubilization. Each point represents a percentage of the total. Results are mean ± SD based on three to four independent experiments. (c) Mature forms and CTFs of APP and BACE1 associate with DRM fractions derived from primary cultured cortical neurons solubilized with Lubrol WX, whereas ADAM17 mainly resides in Lubrol-soluble membranes. GPI-anchored proteins Thy-1 and PrP were used as DRM markers, and calnexin and GM130 were used as detergent-soluble membrane markers. We used longer exposure to detect APP-CTFs compared with FL-APP. (d) Lubrol WX causes only negligible levels of artifactual intermingling of Thy-1. Rat and mouse brain tissues were cohomogenized in 1% Lubrol WX or 1% Brij 97, and DRMs were prepared. Thy-1 on DRMs was immunoaffinity purified and probed with species-specific antibodies. Lubrol caused only negligible levels of mixing of Thy-1 on DRMs during solubilization, centrifugation, and immunoisolation.
Mentions: Biochemically, membrane microdomains are characterized as DRMs by insolubility in nonionic detergents at 4°C and flotation on density gradients. Triton X-100 is the standard detergent for this purpose, but other detergents are also useful for showing microdomain heterogeneity or other types of microdomains (Madore et al., 1999; Roper et al., 2000; Pike, 2004). We prepared DRMs for two main purposes: (1) the efficient and reproducible recovery of APP in DRMs from brains and (2) to maintain the separate identity of membranes without substantial intermingling of protein components to validate immunoaffinity purification. We tried several nonionic detergents, including Brij 97 and Lubrol WX. All detergents tested showed a clear enrichment of a DRM marker, prion protein (PrP), in DRM fractions and separation from a detergent-soluble membrane marker, calnexin (Fig. 1 a). DRM fractions were also rich in cholesterol and GM1-ganglioside (unpublished data).

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