IN VITRO MODEL FOR ALZHEIMER'S DISEASE DRUG DISCOVERY AND DEVELOPMENT FOCUSING ON OXIDATIVE-NITROSATIVE PATHWAYS

Purpose: The Alzheimer's Association estimates that in the absence of disease-modifying treatments the total costs of care for individuals with Alzheimer's disease by all payers will soar from about $170 billion today to more than $1 trillion in 2050. The goal of our project is to develop an in vitro model for testing drugs capable of preventing or slowing the progression of Alzheimer's disease (AD). Previous studies have demonstrated that oxidative-nitrosative stress is an important trigger for Alzheimer's disease and other neurodegenerative disorders. Different nitrosative mechanisms have been proposed for the pathology of this disease, including 3-nitrotyrosination of proteins. Nitric oxide synthase (NOS) is an enzyme that forms nitric oxide. Though important for normal brain function, nitric oxide can react with superoxide formed under oxidative stress conditions to create peroxynitrite, a damaging chemical species that reacts with tyrosine residues to produce aberrant 3-nitrotyrosinated proteins. Recent research has shown certain receptors can modulate NOS activity, but further experiments need to be done to confirm the clinical effectiveness of targeting such receptors with selective drugs as a new therapeutic approach to treating AD and other brain injury states. Our current research is aimed at establishing an in vitro model containing all the necessary elements of the oxidative-nitrosative stress signaling pathway for future drug testing in a cellular model. Methods: Towards this end we have begun to identify relevant pathway elements in a number of cell lines utilizing polymerase chain reaction (PCR) techniques to amplify RNA coding for these elements. This was achieved by culturing cells, extracting purified RNA and reverse transcribing it into DNA and then amplifying the DNA with oligonucleotide primers designed to target specific pathway components (e.g. NOS). Amplified PCR products were confirmed by size determination utilizing agarose gel DNA electrophoresis and DNA sequencing of the correctly-sized bands. Results: Three cell lines, including a neuronal and a glial cell line, have been identified so far that contain a number of the critical elements required for the in vitro model. Conclusions: The identification of a viable in vitro model will allow for further experimentation that can determine the effectiveness of new drugs acting on specific receptor targets to modulate NOS activity and thereby slow the progression of diseases with a nitrosative stress component.