This is a resubmission of a proposal to develop a small animal model of Alzheimer's disease (AD) using gene targeting in embryonic stem (ES) cells to introduce mutations to the mouse amyloid precursor protein (APP) gene. Despite much research the only animal model currently available is the aging non-human primate. The lack of a small animal model has slowed not only the progress in our understanding of the molecular pathogenesis of AD but also the development of rational therapeutic strategies for the treatment of the disease. The identification of several mutations within the APP gene in a number of families with an autosomal dominant form of AD has opened up the possibility of creating a genetic model of AD in mice. Three of these mutations are caused substitutions in the same codon, APP717. The primary aim of this research project is to test the hypothesis that these mutations are sufficient to cause AD. To test this hypothesis we plan to use gene targeting via homologous recombination in ES cells to introduce a point mutation into codon 717 of the mouse APP gene. Introduction of these ES cells into mouse blastocysts and the subsequent breeding of chimeras derived will produce animals heterozygous for the condon 717 mutation. It is these animals which will provide the test of the hypothesis that the codon 717 mutation cause AD. If indeed these mutations are sufficient to cause disease these animals will develop a spontaneous neurodegenerative disorder. Unlike aging humans, aging mice do not develop beta-amyloid plaques. Several hypotheses have been proposed which address this issue. These are: that mice do not live long enough to develop senile plaques; that APP is processed differently in mouse brain or that there are sequence differences between mouse and human beta-amyloid which confer different physical properties upon the two proteins making mouse beta-amyloid less prone to fibril formation. To control for this last possibility, we have made constructs in which three point mutations have been introduced into exon 16 in addition to the exon 17 mutation at codon 717. The result of these three point mutations is that the beta-amyloid sequence derived from these constructs is identical to the human beta-amyloid sequence. To minimize the effects of the first possibility we will engineer a construct containing both the Swedish and a 717 mutation, this should accelerate b-amyloid deposition. If any of the constructs produce AD neuropathology, these animals will be bred to establish a colony which will be used for studies to understand the pathogenic mechanisms of disease, to characterize the phenotype and ultimately to test therapeutic strategies.
