The central hypothesis underlying our research is that Alzheimer's disease is a multi-gene syndrome in which numerous distinct gene defects (and perhaps certain environmental factors) result in a chronic imbalance between Abeta production and Abeta clearance that leads to the microglial, astrocytic, neuronal and synaptic pathology which produce the symptoms of dementia. Based on this hypothesis, many labs including ours have focused substantial attention on the cell biology of APP and the mechanisms of Abeta production, both normally and in AD. To date, molecular causes of Abeta overproduction have explained only a fraction of AD, in all cases of which Abeta accumulates excessively. Yet the nature of Abeta degradation and clearance has hardly been studied. We have chosen to direct our further experiments in this renewal to this largely unexplored topic because of compelling data generated in the current period that demonstrate a time-dependent loss of Abeta40 and 42 peptides in several cell types, particularly microglia, and implicate the thiol metalloendopeptidase, insulin degrading enzyme (IDE), as the principal mediator of this loss.
Our Specific Aims are: 1) to establish quantitatively (Km, kcat, etc.) the role of IDE (and other possible Abeta-degrading proteases) in the proteolysis of extra- and intracellular Abeta in neural and non-neural cultured cells and define where in the cell IDE contacts Abeta; 2) to assess the extent to which IDE (and other proteases identified in Aim l) is expressed and actually degrades Abeta in brain regions prone vs not prone to Abeta build-up, and whether this catabolism is altered with age or in AD in humans and APP tg mice; 3) to prove that IDE can regulate Abeta levels and deposition in vivo by creating PDGF-IDE tg mice, crossing them with PDGF-APP tg mice and assessing the progeny biochemically and pathologically; and 4) to extend our intriguing-data that IDE, besides cleaving Abeta, can mediate its conversion to stable oligomers at physiological levels. In short, we will systematically explore how Abeta is degraded extra- and intracellularly, whether IDE plays a key role in this, and how extracellular levels of Abeta monomer are regulated simultaneously by cleavage and oligomerization. Our plan utilizes the 3 complementary approaches of cell culture, in situ analyses in human and mouse brain tissues, and in vivo modelling in IDE/APP transgenic mice to decipher how Abeta proteolysis and aggregation occur under physiological conditions. The results should help open up a new area of AD pathobiology, with attendant therapeutic implications.
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