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

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.

Figure 1

Plasma membrane and cytosolic IDE have different turn-over. A-Immunofluorescence of PM-associated endogenous IDE in living N2a wild type (N2WT) (panel 1) and Na2WT-EGFP (panel 2) cells using 1C1 monoclonal antibody and Alexa 488 (green) or Cy3 (red) anti-mouse IgG, respectively. Expression of EGFP was visualized based on the EGFP fluorescence (green) and labeled the cytosolic compartment. Scale bars: 5 μm. B-Immunogold electron microscopy on cryosections of N2aSW cells showed a cluster of IDE molecules at the PM (panel 1) which were more evident at higher magnification of the framed region (panel 2). Scale bar, 50 nm. C- Representative phosphorimaging of the remaining IDE during the chase period in cytosol (upper panel) and PM (lower panel). N2aWT cells were pulse-labeled with [³⁵S]-methionine for 30 minutes and chased for the indicated times (0 to 96 hours). Cytosolic and PM fractions were immunoprecipitated as described in Material and Methods. The intensity of the bands was quantified and the maximal value was obtained at time cero and after 6 hours of chase for cytosolic and PM-associated IDE pools, respectively.

Figure 2

Endogenous IDE is localized in DRMs of living cells. A- Living N2aWT cells (panel 1–3) and primary hippocampal neurons (panel 4–6) were stained to label DRMs (panel 1 and 4) and IDE (panel 2 and 5). Merged images showed partial co-localization (arrows) on the PM (panel 3 and 6). Scale bar, 5 μm (panel 1–3) and 20 μm (panel 4–6).B- Immunogold electron microscopy on cryosections of N2aSW cells showed clusters of gold particles at the PM (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 flotillin-1 (black arrows, 6 nm size) and IDE (white arrows, 15 nm size). Scale bar, 50 nm.

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.

Figure 4

DRM- and DSM-associated IDE pools are proteolytically active in vitro. A- Western blotting with BC2 anti-IDE polyclonal antibody of immunoprecipated IDE from isolated DRM and DSM fractions of rat brain using 1C1/3A2 anti-IDE monoclonal antibodies. B- Representative phosphorimage scan showed degradation of [¹²⁵I]-insulin and [¹²⁵I]-Aβ after incubation with anti-IDE immunoprecipitates from DRM and DSM in the presence of a protease inhibitor cocktail (as defined in Antibodies and Chemicals) with or without metalloprotease inhibitors (EDTA/1,10 phenantroline). IgG, unrelated immunoglobulin used as a negative control for the immunoprecipitation. The intensity of the band in the presence of unrelated IgG and after incubation in degradation buffer was referred as intact substrate (0% degradation). C- Bars represent the semi-quantitative analysis of the percentage of [¹²⁵I]-insulin and [¹²⁵I]-Aβ degradation by IDE from DRMs and DSMs in the presence and absence of 1,10-Phe/EDTA (n = 3; *p < 0.001).

Figure 5

PM-IDE activity in vitro is impaired after manipulation of membrane lipid composition. A- Representative western blotting of IDE and flotillin-1 showed no differences in the protein levels in membranes isolated from control (CTL) and Mβ CD-treated cells.B- Representative PhosphorImage scan showed degradation of [¹²⁵I]-insulin after incubation with membranes isolated from cells treated or not with Mβ CD in the presence of a protease inhibitor cocktail (as described in Antibodies and Chemicals) with or without metalloprotease inhibitors (1,10 phenantroline/EDTA). Bars showed the semi-quantitative analysis of the percentage of [¹²⁵I]-insulin degradation by endogenous IDE from control and MβCD-treated membranes (n = 3; *p < 0.05).

Figure 6

Association of endogenous IDE with DRMs is sensitive to membrane-lipid composition. A-Sucrose gradient fractions from N2aWT cells control (CTL), treated with MβCD alone (MβCD) or replenished with soluble Chol (MβCD + Chol) analysed by western blotting with anti-IDE and anti-flotillin-1 antibodies, respectively. Framed region, DRMs (fractions 3 and 4). B- Bars showed the percentage of total endogenous IDE and flotillin-1 in sucrose gradient fractions 3 and 4 (DRMs) from CTL (filled bars), MβCD (open bars) and MβCD + Chol (dashed bars). IDE and flotillin-1 were dramatically affected by membrane-lipid manipulation (n = 3; *p < 0.05 as compared to CTL values in each fraction). Fraction 2: top of the gradient. Fraction 9: bottom of the gradient.

Figure 7

Effect of disorganized DRM on Aβ degradation mediated by IDE. A- Bars showed percentage of degraded [¹²⁵I]-Aβ by IDE immunoprecipitated from DRM and DSM fractions from membranes treated or not with MβCD (n = 2; *p < 0.05). B- Bars showed the semi-quantitative analysis of the percentage of degraded extracellular [¹²⁵I]-Aβ in the conditioned media of control, MβCD treated and Chol replenished N2aWT cells (n = 6; *p < 0.05). C- Upper panel, representative western blotting of C-terminal APP and tubulin levels from N2aSW cellular homogenates showed no differences between control (CTL) and MβCD-treated cells. Lower panel, bars represented Aβ 40 levels in the supernatants of CTL and MβCD-treated cells after 12 and 24 hours of incubation (n = 2; *p < 0.05).

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).