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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

Endogenous IDE co-localizes with flotillin-1 and Aβ on the plasma membrane. A- Brain rat membranes were processed as described in Materials and Methods and fractions analyzed by refractive index (□), distribution of total protein (○) and GPI-anchored alkaline phosphatase activity (◆). DRMs, fraction 3–4; DSMs, fractions 8–9. Fraction 1, top of the gradient; fraction 9, bottom of the gradient.B- Representative western blotting of the same amount of total protein from DRMs and DSMs isolated from cortical tissue of a FAD brain showed co-localization of IDE and flotilin-1 in DRMs. IF, intermediate fraction. C- Significant increased amount of Aβ 42 was detected in DRM compared to DSM by ELISA. Bars represent means ± S.E.M of 2 independent experiments. *p < 0.001. D- Immunogold electron microscopy on cryosections of N2aSW cells showed clusters of gold particles at the plasma membrane (panel 1). A higher magnification of the section framed in panel 1 (panel 2) clearly indicated co-localization of gold particles of different size corresponding to Aβ (white arrow, 15 nm) and IDE (black arrows, 6 nm). Scale bar, 50 nm.
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Figure 3: Endogenous IDE co-localizes with flotillin-1 and Aβ on the plasma membrane. A- Brain rat membranes were processed as described in Materials and Methods and fractions analyzed by refractive index (□), distribution of total protein (○) and GPI-anchored alkaline phosphatase activity (◆). DRMs, fraction 3–4; DSMs, fractions 8–9. Fraction 1, top of the gradient; fraction 9, bottom of the gradient.B- Representative western blotting of the same amount of total protein from DRMs and DSMs isolated from cortical tissue of a FAD brain showed co-localization of IDE and flotilin-1 in DRMs. IF, intermediate fraction. C- Significant increased amount of Aβ 42 was detected in DRM compared to DSM by ELISA. Bars represent means ± S.E.M of 2 independent experiments. *p < 0.001. D- Immunogold electron microscopy on cryosections of N2aSW cells showed clusters of gold particles at the plasma membrane (panel 1). A higher magnification of the section framed in panel 1 (panel 2) clearly indicated co-localization of gold particles of different size corresponding to Aβ (white arrow, 15 nm) and IDE (black arrows, 6 nm). Scale bar, 50 nm.

Mentions: We next used a biochemical approach to characterize the association of IDE with DRMs. DRMs are sub-cellular compartments that although do not correspond to lipid rafts as they exist in vivo, their biochemical separation are the only approach for assessing protein interactions with lipid rafts [27,28]. N2a cell membranes were isolated, treated with detergent at 4°C and subjected to sucrose gradient centrifugation. All fractions from the sucrose density gradient were analyzed by total protein distribution, refractive index and GPI-alkaline phosphatase activity (Fig. 3A). In agreement with a previous report [25], most of the alkaline phosphatase activity was located at the 5–30% sucrose interface, representing the DRMs (fraction 3 and 4) while detergent-soluble membranes (DSMs) were limited to fractions 8–9. Interestingly, as shown in Fig. 3B IDE immunoreactivity was detected in DRM fraction together with flotillin-1 which is over-represented in DRMs as compared to DSM indicating that the biochemical fractionation of brain DRMs was properly performed. To further investigate the co-residence of IDE with Aβ in DRMs we isolated DRMs and DSMs from the cerebral cortex of a familial AD case (carrying the PS1 mutation M146L), known to present extensive Aβ accumulation [19]. The presence of Aβ42 in DRM and DSM was corroborated by sandwich ELISA assay (Fig. 3C). Substantial peptide concentration was detected in DRM as compared to DSM in agreement with previous reports [29]. Taking into account the technical impossibility to perform a triple-immuno gold electron microscopy to detect IDE, Aβ and flotillin we performed IEM on N2aSW cells to localize IDE and Aβ (Fig. 3D1). A higher magnification of the portion framed in Fig. 3D1 (Fig. 3D2) showed clusters of colloidal gold particles of different sizes corresponding to IDE (black arrows, 6 nm) and Aβ (white arrows, 15 nm) detected on the PM. The experimental evidences that Aβ levels are significantly increased in DRM as compared to DSM and the highly specificity of the immunogold strongly suggest that DRMs may be one of the sub-cellular compartments where IDE and Aβ interact.


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)

Endogenous IDE co-localizes with flotillin-1 and Aβ on the plasma membrane. A- Brain rat membranes were processed as described in Materials and Methods and fractions analyzed by refractive index (□), distribution of total protein (○) and GPI-anchored alkaline phosphatase activity (◆). DRMs, fraction 3–4; DSMs, fractions 8–9. Fraction 1, top of the gradient; fraction 9, bottom of the gradient.B- Representative western blotting of the same amount of total protein from DRMs and DSMs isolated from cortical tissue of a FAD brain showed co-localization of IDE and flotilin-1 in DRMs. IF, intermediate fraction. C- Significant increased amount of Aβ 42 was detected in DRM compared to DSM by ELISA. Bars represent means ± S.E.M of 2 independent experiments. *p < 0.001. D- Immunogold electron microscopy on cryosections of N2aSW cells showed clusters of gold particles at the plasma membrane (panel 1). A higher magnification of the section framed in panel 1 (panel 2) clearly indicated co-localization of gold particles of different size corresponding to Aβ (white arrow, 15 nm) and IDE (black arrows, 6 nm). Scale bar, 50 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2648957&req=5

Figure 3: Endogenous IDE co-localizes with flotillin-1 and Aβ on the plasma membrane. A- Brain rat membranes were processed as described in Materials and Methods and fractions analyzed by refractive index (□), distribution of total protein (○) and GPI-anchored alkaline phosphatase activity (◆). DRMs, fraction 3–4; DSMs, fractions 8–9. Fraction 1, top of the gradient; fraction 9, bottom of the gradient.B- Representative western blotting of the same amount of total protein from DRMs and DSMs isolated from cortical tissue of a FAD brain showed co-localization of IDE and flotilin-1 in DRMs. IF, intermediate fraction. C- Significant increased amount of Aβ 42 was detected in DRM compared to DSM by ELISA. Bars represent means ± S.E.M of 2 independent experiments. *p < 0.001. D- Immunogold electron microscopy on cryosections of N2aSW cells showed clusters of gold particles at the plasma membrane (panel 1). A higher magnification of the section framed in panel 1 (panel 2) clearly indicated co-localization of gold particles of different size corresponding to Aβ (white arrow, 15 nm) and IDE (black arrows, 6 nm). Scale bar, 50 nm.
Mentions: We next used a biochemical approach to characterize the association of IDE with DRMs. DRMs are sub-cellular compartments that although do not correspond to lipid rafts as they exist in vivo, their biochemical separation are the only approach for assessing protein interactions with lipid rafts [27,28]. N2a cell membranes were isolated, treated with detergent at 4°C and subjected to sucrose gradient centrifugation. All fractions from the sucrose density gradient were analyzed by total protein distribution, refractive index and GPI-alkaline phosphatase activity (Fig. 3A). In agreement with a previous report [25], most of the alkaline phosphatase activity was located at the 5–30% sucrose interface, representing the DRMs (fraction 3 and 4) while detergent-soluble membranes (DSMs) were limited to fractions 8–9. Interestingly, as shown in Fig. 3B IDE immunoreactivity was detected in DRM fraction together with flotillin-1 which is over-represented in DRMs as compared to DSM indicating that the biochemical fractionation of brain DRMs was properly performed. To further investigate the co-residence of IDE with Aβ in DRMs we isolated DRMs and DSMs from the cerebral cortex of a familial AD case (carrying the PS1 mutation M146L), known to present extensive Aβ accumulation [19]. The presence of Aβ42 in DRM and DSM was corroborated by sandwich ELISA assay (Fig. 3C). Substantial peptide concentration was detected in DRM as compared to DSM in agreement with previous reports [29]. Taking into account the technical impossibility to perform a triple-immuno gold electron microscopy to detect IDE, Aβ and flotillin we performed IEM on N2aSW cells to localize IDE and Aβ (Fig. 3D1). A higher magnification of the portion framed in Fig. 3D1 (Fig. 3D2) showed clusters of colloidal gold particles of different sizes corresponding to IDE (black arrows, 6 nm) and Aβ (white arrows, 15 nm) detected on the PM. The experimental evidences that Aβ levels are significantly increased in DRM as compared to DSM and the highly specificity of the immunogold strongly suggest that DRMs may be one of the sub-cellular compartments where IDE and Aβ interact.

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