Active Insulin Degrading Enzyme in lipid rafts: relevance for Aß degradation in the brain.

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

Madrid, España

Congreso; International Conference of Alzheimer`s Disease & Related Disorders; 2006

Alzheimr`s Disease Association

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 membrane regions enriched in cholesterol and glycosphingolipids defined as 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. Aims: 1. To investigate if proteolitically active IDE is associated with lipid rafts. 2. To study the interaction of this IDE isoform with Ab. Methods: 1. Preparation of membrane homogenates from cells and brain, alkaline treatment and IDE detection by western blot. 2. Isolation of lipid rafts from N2a cells, rat and human brain by Triton X-100 treatment and sucrose gradient flotation. 3. Detection of IDE in lipid rafts of living cells by immunofluorescence (IF) using cholera toxin subunit B (CTB) and 1C1 anti-IDE monoclonal antibody. 4. Depletion of cellular cholesterol with methyl â-cyclodextrin. 5. Activity of endogenous IDE by degradation of 125I-Insulin and 125I-Ab in IDE immunoprecipitates. 6. IDE/ã-secretase protein complex localization in lipid raft by western blot. 7. Detection of Ab42 levels in lipid rafts from AD human brain by sandwich ELISA. 8. IDE/Ab localization in cells by immunogold electron microscopy (IEM). Results: 1. IDE remains in membrane pellets after alkaline treatment. 2.  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. 3. Cholesterol depletion disrupts IDE-lipid raft association. 4. Lipid raft associated IDE is proteolytically active using Ab and insulin as substrates. 5. IDE colocalizes with flotillin-1 and Ab at the plasma membrane. 6.  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.