Alzheimer's disease (AD) is the fourth leading cause of death over age 60 in the United States. A characteristic pathological change found in the brains of patients with AD is the deposition of abnormal protein aggregates, called beta-amyloid in neuritic plaques (NP) and cerebrovascular amyloidosis (CVA). beta-amyloid protein (Abeta), a main constituent of beta-amyloid, consists of 39-43 amino acids and is proteolytically produced from a much larger precursor protein (betaPP). Although crucial roles of Abeta and betaPP in the pathogenesis of AD are strongly supported by discoveries of missense mutations in the gene encoding betaPP in certain families with an autosomal dominant form of AD, little is understood about the molecular mechanisms of development of the pathology, such as NP and CVA. Although important and multiple functions of betaPP have been suggested by the high degree of evolutionary conservation, widespread tissue expression, and its characteristics of the betaPP promoter, such functions still remain to be clarified. The long term goals of this research are to elucidate the roles of Abeta and betaPP in the etiology of AD by a better understanding of physiological functions and normal metabolism of betaPP and to establish the logical basis for prevention and therapy of AD. Several forms of betaPP are produced by alternative splicing of original betaPP transcripts. betaPP with the Kunitz protease inhibitor (KPI) domain is expressed in almost all tissues other than neurons and betaPP without the KPI domain is exclusively expressed in neurons. The correlation between the disease status and specific forms of betaPP has been suggested. Therefore it is highly desirable for a better understanding of the etiology of AD to investigate specific functions and metabolism for each form of betaPP. Thus, the Specific Aims of this research proposal are to: (1) mutagenize the Kunitz protease inhibitor (KPI) domain of the betaPP gene in embryonic stem (ES)cells; (2) establish transgenic mouse lines (including heterozygous and homozygous mutants) using ES cells established in Specific Aim 1; (3) abolish all forms of betaPP via gene targeting of ES cells; and (4) establish a transgenic mouse line in which all forms of betaPP are abolished using ES cells established in Specific Aim 3. Biochemical, histopathological, and immunohistochemical techniques will be used to analyze heterozygous and homozygous mutant mice established in these aims for studying the physiological functions and metabolism of the specific forms of betaPP. These mice may be useful to establish better transgenic models for AD by expressing human Abeta or betaPP knockout background.
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