Alzheimer's Disease (AD) is a progressive neurodegenerative disorder which culminates in senile dementia. The etiology of AD is unknown although a variety of data suggests that excessive extracellular deposition of amyloid beta/A4 protein preceded by intracellular accumulation of its coding mRNA may be important. Nucleotide sequence analysis of amyloid protein precursor (APP) -695, 714, 751 and 770 mRNAs reveals the presence of 4 reiterations of the destabilizing pentanucleotide motif adenosine-uridine- uridine-uridine-adenosine (AUUUA) which targets cytokine and oncogene mRNAs for rapid, cytoplasmic degradation. We have recently identified a novel, cytoplasmic protein which specifically binds to the AUUUA elements of cytokine and oncogene mRNAs. This protein (which we have denoted as the adenosine-uridine binding factor or AUBF) is upregulated through phosphorylation by phorbol ester, ionophore or cytokines and likely participates in the stabilization of cytokine mRNAs by binding to the AUUUA motifs. Cytoplasmic lysates from rodent and human brain and a human neuroblastoma cell line constitutively express AUBF activity as well as three additional, novel, brain-specific AUUUA binding activities (denoted AUBF-B). These activities can be modulated by cytokines and specifically bind to APP mRNAs through the AUUUA motifs. Therefore, we hypothesize that the AUUUA containing APP mRNAs are normally unstable. The binding of AUBF or AUBF-B to APP mRNAs through the AUUUA motifs stabilizes the APP message leading to a relative accumulation of APP mRNA and subsequent APP production in the brain of AD patients. Thus the specific aims of this proposal are to: 1). Establish the turnover rates of the four wild type APP mRNAs (695, 714, 751, 770) as well as mutated APP transcripts with reduced numbers of AUUUA motifs after the stable transfection of their coding cDNAs into a human neuroblastoma cell line (SH-SY-5Y). The effects of IL-1, tumor necrosis factor, nerve growth factor and calcium ionophore on the turnover of wild type and mutant APP mRNAs and the level of AUBF and AUBF-B activities will be determined; 2). Purify AUBF-B activities from human brain by RNA affinity chromatography and generate polyclonal and monoclonal antibodies against these proteins. 3). Employ a cell-free RNA degradation system derived from rabbit reticulocyte lysate to assess the turnover rates of wild-type and mutant APP mRNAs; 4). Establish the function of AUBF-B by adding purified protein(s) the cell-free degradation system followed by measurement of the turnover rate of wild-type and mutant APP mRNAs. Cumulatively these studies will clarify the roles of posttranscriptional gene regulation and AUBF-B in the accumulation of APP mRNAs.
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