Beta-amyloid is the major proteinaceous component of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease (AD). Beta-protein is derived from a larger amyloid protein precursor (APP) which is encoded by a gene on human chromosome 21. The APP gene encodes multiple transcripts for a family of secreted proteins that are generated by alternate splicing. The APP gene is differentially expressed in all major tissues. The hypothesis is that the upregulation of the promoter activity of the APP gene resulting in its enhanced transcription can set in motion the series of events culminating in amyloid deposits. The long term goal is to study the transcriptional control of APP gene.
The specific aims are: 1) to examine the transcription at the APP promoter in different cell types and study the effect of different factors on the promoter; 2) to characterize the regulatory regions (cis-acting) of the APP promoter and identify the cell type-specific (trans-acting) factor(s) that might modulate the transcription of APP; 3) to study the effect of homeobox gene products on the promoter and 4) to determine whether there is a mutation in the APP gene promoter that is isolated from persons with familial Alzheimer's disease (FAD). Expression studies of the APP promoter will be done by constructing chloramphenicol acetyltransferase (CAT) based promoter plasmids with a 0.6-3.2kb fragment of the APP promoter. The strength of the promoter is measured by assaying CAT activity and determining the size of transcripts by ribonuclease protection assay in different cell extracts. Cell lines are of neuronal, glial, and chromaffin origin. The effect of interleukins, phorbol esters and heat shock treatment on the promoter will be studied in different stably transfected cells. To characterize the regulatory domains on the APP promoter, a progressive deletion from the 5' -end is made by limited nuclease digestion and using polymerase chain reaction (PCR). These deleted fragments are ligated to the reporter gene and the promoter activity is measured in different cell-types. Such experiments could detect the presence of an enhancer/silencer sequence in the promoter. The site-directed mutagenesis will be employed to determine the specificity of DNA regulatory elements of the APP promoter. The identification of any cell-specific factor(s) that might affect the promoter activity will be performed by gel shift assay using the appropriate promoter fragment and proteins from cell extracts. The promoter region of the APP gene contains 5 potential binding sites for a homeobox gene (Hox-1.3). Homeobox gene products act as transcriptional factors. We will examine the role of homeobox gene products with a cotransfection model using plasmid constructs with activator gene (Hox-3.l) and target gene (APP). To determine a mutation within the promoter region isolated from persons with presenile cases of FAD, two flanking oligonucleotide primers that span the O.8kb promoter will be synthesized. The flanked promoter from different sources will be amplified by PCR technique and then sequenced to locate a mutation.
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