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Detergent resistant membrane-associated IDE in brain tissue and cultured cells: Relevance to Abeta and insulin degradation.

Bulloj A, Leal MC, Surace EI, Zhang X, Xu H, Ledesma MD, Castaño EM, Morelli L - Mol Neurodegener (2008)

Bottom Line: DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo.We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele.Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fundación Instituto Leloir, IIBBA-CONICET, Ave, Patricias Argentinas 435, Ciudad de Buenos Aires C1405BWE, Argentina. xuh@burnham.org.

ABSTRACT

Background: Insulin degrading enzyme (IDE) is implicated in the regulation of amyloid beta (Abeta) steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD). Although IDE sub-cellular localization has been well studied, the compartments relevant to Abeta degradation remain to be determined.

Results: Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM)-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-beta-cyclodextrin (MbetaCD), endogenous Abeta accumulated in the extracellular space and exogenous Abeta proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Abeta degradation in vitro.

Conclusion: Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Abeta degrading proteases away from DRMs may impair the physiological turn-over of Abeta in vivo deserves further investigation in light of therapeutic strategies based on enhancing Abeta proteolysis in which DRM protease-targeting may need to be taken into account.

No MeSH data available.


Related in: MedlinePlus

Impact of brain cholesterol levels on endogenous IDE localization and activity. A-Sucrose gradient fractions from sel-1 (+/+) and (+/-) mouse brains analyzed by western blotting with anti-IDE and anti-flotillin, respectively. Framed region, DRMs (fractions 3 and 4). Fraction 2: top of the gradient. Fraction 9: bottom of the gradient. B- Upper panel, bars showed the semi-quantitative analysis of the percentage of total endogenous IDE and flotillin-1 immunoreactivity in sucrose gradient fractions 3 and 4 (DRMs) from control (open bars) and partially knocked-out mice (filled bars). IDE and flotillin-1 present in DRM fraction were significant affected in sel-1 (+/-) mice brain compared to (+/+) mice (n = 3; *p < 0.05). C- Bars showed the semi-quantitative analysis of the percentage of [125I]-insulin degradation by endogenous DRMs-bound IDE from sel (+/+) and sel (+/-) brain membranes in the presence of a protease inhibitor cocktail as described in Antibodies and Chemicals (n = 2; *p < 0.05).
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Figure 8: Impact of brain cholesterol levels on endogenous IDE localization and activity. A-Sucrose gradient fractions from sel-1 (+/+) and (+/-) mouse brains analyzed by western blotting with anti-IDE and anti-flotillin, respectively. Framed region, DRMs (fractions 3 and 4). Fraction 2: top of the gradient. Fraction 9: bottom of the gradient. B- Upper panel, bars showed the semi-quantitative analysis of the percentage of total endogenous IDE and flotillin-1 immunoreactivity in sucrose gradient fractions 3 and 4 (DRMs) from control (open bars) and partially knocked-out mice (filled bars). IDE and flotillin-1 present in DRM fraction were significant affected in sel-1 (+/-) mice brain compared to (+/+) mice (n = 3; *p < 0.05). C- Bars showed the semi-quantitative analysis of the percentage of [125I]-insulin degradation by endogenous DRMs-bound IDE from sel (+/+) and sel (+/-) brain membranes in the presence of a protease inhibitor cocktail as described in Antibodies and Chemicals (n = 2; *p < 0.05).

Mentions: To determine the impact of brain Chol levels on IDE localization in vivo, mice with a targeted deletion of one seladin-1 (sel-1) allele (+/-) were used. Membranes from sel-1 (+/-) mouse brains showed reduced levels of Chol as compared to wild type sel-1 (+/+) mice (2.57 μg/mg vs. 5.55 μg/mg) and disorganized DRMs [31]. The flotation profiles of IDE and flotillin from sel-1 (+/+) mouse brains (Fig. 8A, upper panel) showed no significant differences as compared to rat or human brain as described above. In agreement with a DRM-specific shortage, reduction of brain sel-1 levels resulted in the displacement of IDE and flotillin (Fig. 8A, lower panel) from DRM. Thus, while in sel-1 (+/+) mice 27.7 ± 1.36% of the total IDE and 41.2 ± 0.76% of total flotillin resides in fractions 3 and 4, respectively, only 11.9 ± 0.4% and 34.8 ± 0.85% (n = 3; p < 0.05) of IDE and flotillin, respectively, was observed in sel-1 (+/-) brains (Fig. 8B). Decreased Chol levels induced a rise of IDE immunoreactivity in "intermediate fractions" (fractions 5–7) of sel (+/-) as compared to sel (+/+) mice brains (62.0 ± 1.3% vs. 28.3 ± 1.5%, respectively). As expected, a drop in [125I]-insulin degradation by IDE in sel-1 (+/-) was observed as compared to sel-1 (+/+) brains (2.6 ± 1.7% vs. 21.9 ± 0.9%; n = 2; p <0.05) (Fig. 8C).


Detergent resistant membrane-associated IDE in brain tissue and cultured cells: Relevance to Abeta and insulin degradation.

Bulloj A, Leal MC, Surace EI, Zhang X, Xu H, Ledesma MD, Castaño EM, Morelli L - Mol Neurodegener (2008)

Impact of brain cholesterol levels on endogenous IDE localization and activity. A-Sucrose gradient fractions from sel-1 (+/+) and (+/-) mouse brains analyzed by western blotting with anti-IDE and anti-flotillin, respectively. Framed region, DRMs (fractions 3 and 4). Fraction 2: top of the gradient. Fraction 9: bottom of the gradient. B- Upper panel, bars showed the semi-quantitative analysis of the percentage of total endogenous IDE and flotillin-1 immunoreactivity in sucrose gradient fractions 3 and 4 (DRMs) from control (open bars) and partially knocked-out mice (filled bars). IDE and flotillin-1 present in DRM fraction were significant affected in sel-1 (+/-) mice brain compared to (+/+) mice (n = 3; *p < 0.05). C- Bars showed the semi-quantitative analysis of the percentage of [125I]-insulin degradation by endogenous DRMs-bound IDE from sel (+/+) and sel (+/-) brain membranes in the presence of a protease inhibitor cocktail as described in Antibodies and Chemicals (n = 2; *p < 0.05).
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Related In: Results  -  Collection

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Figure 8: Impact of brain cholesterol levels on endogenous IDE localization and activity. A-Sucrose gradient fractions from sel-1 (+/+) and (+/-) mouse brains analyzed by western blotting with anti-IDE and anti-flotillin, respectively. Framed region, DRMs (fractions 3 and 4). Fraction 2: top of the gradient. Fraction 9: bottom of the gradient. B- Upper panel, bars showed the semi-quantitative analysis of the percentage of total endogenous IDE and flotillin-1 immunoreactivity in sucrose gradient fractions 3 and 4 (DRMs) from control (open bars) and partially knocked-out mice (filled bars). IDE and flotillin-1 present in DRM fraction were significant affected in sel-1 (+/-) mice brain compared to (+/+) mice (n = 3; *p < 0.05). C- Bars showed the semi-quantitative analysis of the percentage of [125I]-insulin degradation by endogenous DRMs-bound IDE from sel (+/+) and sel (+/-) brain membranes in the presence of a protease inhibitor cocktail as described in Antibodies and Chemicals (n = 2; *p < 0.05).
Mentions: To determine the impact of brain Chol levels on IDE localization in vivo, mice with a targeted deletion of one seladin-1 (sel-1) allele (+/-) were used. Membranes from sel-1 (+/-) mouse brains showed reduced levels of Chol as compared to wild type sel-1 (+/+) mice (2.57 μg/mg vs. 5.55 μg/mg) and disorganized DRMs [31]. The flotation profiles of IDE and flotillin from sel-1 (+/+) mouse brains (Fig. 8A, upper panel) showed no significant differences as compared to rat or human brain as described above. In agreement with a DRM-specific shortage, reduction of brain sel-1 levels resulted in the displacement of IDE and flotillin (Fig. 8A, lower panel) from DRM. Thus, while in sel-1 (+/+) mice 27.7 ± 1.36% of the total IDE and 41.2 ± 0.76% of total flotillin resides in fractions 3 and 4, respectively, only 11.9 ± 0.4% and 34.8 ± 0.85% (n = 3; p < 0.05) of IDE and flotillin, respectively, was observed in sel-1 (+/-) brains (Fig. 8B). Decreased Chol levels induced a rise of IDE immunoreactivity in "intermediate fractions" (fractions 5–7) of sel (+/-) as compared to sel (+/+) mice brains (62.0 ± 1.3% vs. 28.3 ± 1.5%, respectively). As expected, a drop in [125I]-insulin degradation by IDE in sel-1 (+/-) was observed as compared to sel-1 (+/+) brains (2.6 ± 1.7% vs. 21.9 ± 0.9%; n = 2; p <0.05) (Fig. 8C).

Bottom Line: DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo.We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele.Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fundación Instituto Leloir, IIBBA-CONICET, Ave, Patricias Argentinas 435, Ciudad de Buenos Aires C1405BWE, Argentina. xuh@burnham.org.

ABSTRACT

Background: Insulin degrading enzyme (IDE) is implicated in the regulation of amyloid beta (Abeta) steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD). Although IDE sub-cellular localization has been well studied, the compartments relevant to Abeta degradation remain to be determined.

Results: Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM)-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-beta-cyclodextrin (MbetaCD), endogenous Abeta accumulated in the extracellular space and exogenous Abeta proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Abeta degradation in vitro.

Conclusion: Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Abeta degrading proteases away from DRMs may impair the physiological turn-over of Abeta in vivo deserves further investigation in light of therapeutic strategies based on enhancing Abeta proteolysis in which DRM protease-targeting may need to be taken into account.

No MeSH data available.


Related in: MedlinePlus