Specific Aims: 1. Utilize the use of RNA interference technology to transiently silence gene expression of several candidate phosphoinositide kinase gene products that are directly inhibited by wortmannin and a phosphoinositide phosphate gene product that behaves like wortmannin when inhibited. 2. Identify inhibitory candidate sequences for stable expression in cell culture. 3. Preliminarily determine their usefulness in altering in vitro Aβ generation.

Background: Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder that destroys the brain and currently, there is no treatment or cure that addresses the mechanistic cause of the disease. After approximately two decades of intense research focus, physicians are still limited to temporarily preserving the benefits of the neurotransmitter, acetylcholine in affected brain regions by placing patients on a regimen of acetylcholinesterase inhibitors. This of course is only a temporary fix as the mechanism of neuronal and synaptic destruction is unknown and therefore cannot be addressed therapeutically. Currently, there are 4.5 million individuals in these United States that have AD and without a treatment or cure, the projections are that today’s estimate will triple by 2050 (Hebert et al 2003). Evidence gathered over the past two and half decades point convincingly to the amyloid ? (Aβ) peptide, the main component of the amyloid that deposits in the brains of individuals afflicted with AD, as playing a major role in its neuropathogenesis. This collection of evidence is known as the amyloid hypothesis and through subtle revisions over the years essentially states that Aβ of 42 residues, its soluble aggregates known as Aβ-derived diffusible ligands, or its deposits initiates the cascade of events that ultimately results in the neurodegeneration and the progressive dementia seen in AD (Golde 2005). Previous studies indicate that treatment with wortmannin, a known inhibitor of phosphoinositide kinases, significantly reduces soluble Aβ generation and plaque formation both in cell culture and in a transgenic mouse model of AD, respectively (Haugabook et al 2001, Petanceska & Gandy 1999). The mechanism by which wortmannin mediates this action is unknown and future studies are in the works to address this void by utilizing an approach that will mechanistically interfere with βAPP proteolysis and thus excessive Aβ generation, both in vivo and in vitro. The goal therefore of this study is to generate pilot data to enhance a future grant application to investigate the mechanism of action of wortmannin, as it relates to the processing of βAPP to reduce Aβ generation and plaque formation in a transgenic model of AD.