Alzheimer's disease (AD), the most common type of progressive dementia in the elderly, is characterized by the deposition of beta-amyloid peptides (Abeta) in the brain parenchyma and cerebral vessels. A subset of AD, classified as familial early-onset AD (FAD), is inherited as an autosomal dominant disorder. Mutations in genes encoding polytopic membrane proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2), account for the majority of early-onset cases of AD. Presenilins (PS) play an important role in the generation of Abeta peptides. Abeta production is abrogated in PS1-deficient (PS-1-) cells. Moreover, FAD-linked mutant PS1 increases the production of highly fibrillogenic Abeta42 peptides. The precise role of PS1 in Abeta production, and the molecular mechanisms by which FAD-linked PS1 mutations lead to elevations in Abeta42 production have not been defined. Understanding these issues is of central importance to AD research. It is our view that molecular and structural domain analysis of PS1 will provide information critical for a clear understanding of how genetic mutations in PS1 might influence the normal function(s) of PS1, and confer pathogenic properties to mutant PS1 polypeptides. At present, very little is known regarding the molecular and structural domains of PS1. To address this issue, we will generate a series of PS1 polypeptides harboring experimental deletions and assess the influence on: PS1 endoproteolysis, """"""""gamma-secretase"""""""" processing of amyloid precursor protein, the intramembranous cleavage of Notch1, as well as evaluate the potential of the deletion polypeptides harboring FAD-linked missense mutations to elevate the levels of Abeta42. It is known that PS1 expression is tightly regulated at the post-translational level by complex formation with other proteins; however the mechanism(s) responsible for this regulation have not been defined. To gain insights regarding the regulation of PS1 protein accumulation, we will perform a functional screen based on a novel retroviral expression cloning strategy to identify proteins that participate in regulating PS1 accumulation. Because little or no Abeta is produced in the absence of PS1, identity(ies) of proteins that regulate PS levels is critical for the design of rational therapeutic strategies aimed at reducing Abeta burden. Finally, we have outlined transgenic strategies to examine the in vivo role of the hydrophilic domain of PS1, which is the domain least conserved between PS1 and PS homologues. Recent studies have predicted important function(s) for this domain based on phosphorylation, caspase cleavage, and protein interactions. Our efforts will focus on the role played by PS1 hydrophilic loop domain during mammalian embryonic development, and in the process of amyloid production/deposition in the brains of transgenic mice.
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