Active Insulin Degrading Enzyme in lipid rafts: Relevance for Aâ degradation in the brain.

BULLOJ A; LEAL MC; CASTAÑO EM; XU H; MORELLI L

Antigua

Simposio; The Second ISN Special Neurochemistry Conference on Neural Glycoproteins and Glycolipids.; 2006

International Society for Neurochemistry

Newly generated amyloid b peptide (Ab) is rapidly cleared from the brain and this process may be defective in Alzheimer`s disease (AD). Insulin degrading enzyme (IDE) is a major brain protease capable of degrading Ab but little is known about the sub-cellular compartment where IDE-Ab interaction occurs. It has been proposed that Ab generation and its oligomerization in AD takes place in lipid-rafts. We hypothesize that membrane -rather than cytosolic- IDE isoforms are critical in keeping the levels of soluble brain Ab within a physiological range. Methods: 1.Isolation of lipid rafts by Triton X-100 treatment and sucrose gradient flotation. 2.Detection of IDE in lipid rafts of living cells by immunofluorescence (IF) using cholera toxin subunit B (CTB). 3.Depletion of cellular cholesterol with methyl â-cyclodextrin. 4.Activity of endogenous IDE by degradation of 125I-Insulin and 125I-Ab.  5.Detection of Ab42 levels in lipid rafts from AD human brain by sandwich ELISA. 7.IDE/Ab localization in cells by immunogold electron microscopy (IEM). Results: 1.A pool of IDE is associated with lipid rafts in different cell lines and brain homogenates, as shown by IEM, CTB clustering and sucrose gradients. 2.Cholesterol depletion disrupts IDE-lipid raft association. 3. Lipid raft associated IDE is proteolytically active. 4.IDE colocalizes with flotillin-1 and Ab at the plasma membrane. 5.IDE, Ab and ã-secretase protein complex reside in lipid rafts from human brain. Conclusions: Our results support that Ab degradation by IDE may occur in lipid rafts and underscores the importance of these domains in Ab metabolism under normal and pathogenic conditions.