Insulin degrading enzyme from human brain microvessels degrades amyloid β and its Flemish, Dutch and Italian vasculotropic variants. Laura Morelli, Ramiro E. Llovera, Blas Frangione, Jorge Ghiso, Eduardo M. Castaño.

MORELLI, L; LLOVERA, RE; FRANGINE, B; GHISO, J; CASTAÑO, EM

Philadelphia, USA

Congreso; Annual Meeting Alzheimer Disease Association.; 2004

Background: Amyloid ß (Aß) deposition in the walls of cerebral arteries is associated with diseases characterized by dementia or stroke. A higher tendency of Aß to aggregate, a defective transport to the systemic circulation and insufficient proteolytic removal of Aß have been proposed as mechanisms leading to its accumulation in the brain. Insulin degrading enzyme (IDE) is believed to play a significant role as a physiologic Aâ protease in the mammalian brain. Despite the recent advances on Aß proteolysis by IDE, very little is known about the cellular and regional expression and activity in the human brain Objective(s): 1- Investigate the presence and activity of IDE in microvessels from human cerebral cortex. 2- Examine its activity against synthetic Aß1-40 wt and the major genetic variants associated with hereditary stroke: Flemish (A21G), Dutch (E22Q) and Italian (E22K). Methods: Brain microvessels were analysed by immunofluorescence and western blot using anti-IDE, anti-á actin and anti-neuronal specific enolase antibodies. IDE activity was examined in brain microvessels by immunoprecipitation with anti-IDE or anti-á actin and protein G-sepharose, respectively. Immunocomplexes were incubated with 125I-insulin in the presence or absence of inhibitors and degradation estimated by SDS-PAGE. To study specificity of degradation upon Aß variants, peptides were incubated with anti-IDE immunocomplexes and samples analyzed by MALDI-TOF mass spectrometry. Results: Immunofluorescence suggested that IDE was localized mainly within the cytoplasm of endothelial cells, pericytes and smooth muscle cells. Immunoprecipitation and western blots showed the presence of a single, specific 115 kDa component in a soluble fraction obtained from microvessels compatible with one isoform of IDE from human brain. IDE was immunoprecipitated in an active form, as assessed by 125I-insulin degradation. Moreover, we determined that endogenous IDE cleaved Aâwt, A21G and E22K primarily at positions 13-14, 14-15, 19-20 and 20-21 while a more restricted pattern was found with E22Q. Conclusions: Our results suggest that IDE is a component of the Aâ clearance system in the human brain microvasculature and support the upregulation of endogenous IDE as a potential therapeutic strategy in sporadic and inherited Aß cerebrovascular amyloidoses