Insulin degrading enzyme: the secretory pathway implicated in the clearance of amyloid ß.

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

Pinamar, Argentina

Congreso; PABMB 10th Congress, SAIB 41th Annual Meeting and SAN 20th Annual Meeting.; 2005

Newly generated soluble amyloid b (Ab) peptide is rapidly cleared from the brain and this process may be defective in Alzheimer`s disease. 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 recently proposed that Ab generation and oligomerization takes place in cholesterol and glycosphingolipids-rich membrane regions defined as lipid-rafts. We hypothesize that membrane associated and secreted IDE isoforms are critical in the process that sustains the levels of soluble brain Ab peptide within a physiological range. Aims: 1- Characterization of membrane-associated IDE. 2- Identification of the mechanisms involved in IDE secretion. Methods: Detergent resistant microdomains (DRMs) were isolated from rat brain and N2a cells by Triton X-100 treatment and sucrose gradient flotation. To visualize IDE in lipid rafts of living cells immunofluorescence was made using Alexa fluor 594-cholera toxin subunit B and 1C1 anti-IDE monoclonal antibody. IDE/lipid rafts-cholesterol association was study by Methyl â-cyclodextrin (MâCD) treatment followed by DRMs isolation. IDE release was tested by pulse chase and immunoprecipitation in the presence of Monensin, Brefeldin A, MâCD and low temperature. IDE post-translational (PT) modification were assessed by metabolic labeling using 35S-methionine, 3H-etanolamine and 3H-palmitic acid. Exosomes were purified from culture media of N2a cells by sequential ultra-centrifugation. Characterization of the pellets was performed by western blot using BC2 (anti-IDE polyclonal antibody), anti-Bip, and anti-flotillin. An aliquot was fixed with 4 % paraformaldehide, negatively stained with uranyl acetate and visualized under electron microscopy. Results: 1-A pool of membrane IDE resists alkaline treatment and is active in DRMs. 2- IDE lacks GPI and palmitoylation PT modifications 3- Cholesterol depletion disrupts IDE-DRMs association, 4- IDE is associated with lipid rafts in living cell, 5- IDE release is not mediated by the ER-Golgi pathway and exosomes may be involved in this process. Conclusions: our experimental evidences indicate that specific pools of IDE may associate with lipid raft or translocate from the cytosol to extracellular compartment to allow IDE-mediated proteolysis of soluble Ab.