Studies from many laboratories have indicated that tau proteins and ubiquitin are important components of paired helical filaments (PHF).
The aim of this project is to .use quantitative immunochemical methods to examine the materials reactive to anti-PHF, anti-tau, and anti-ubiquitin antibodies, in soluble and insoluble fractions of Alzheimer and control tissues, including PHF-enriched fractions insoluble in hot SDS/2-mercaptoethanol (2-ME). Information on the relationship between the levels of these materials in soluble and insoluble fractions of Alzheimer and control tissues can be expected to help to elucidate the relationship between the metabolism of tau proteins and ubiquitin and their accumulation in insoluble aggregates in Alzheimer disease, and to indicate directions for future studies. Quantitative assays will be standardized for PHF, tau proteins, and ubiquitin, using ELISAs, Western blots, and dot blots. Part of this standardization will be a comparison, per mg protein, of the reactivity of preparations of purified PHF to that of purified tau proteins and pure ubiquitin with the antibodies used. These assays will then be utilized to compare the amount of immunoreactivity in soluble and insoluble fractions (including SDS/2-ME insoluble fractions) of Alzheimer brain and age and sex matched control brain. These comparisons will be made in areas affected and unaffected by the accumulation of neurofibrillary tangles. These assays will then be utilized to compare immunoreactivity to PHF and related proteins in fibroblasts from familial and sporadic Alzheimer subjects and control fibroblasts matched for age and sex of subject, and biological age in culture. Cells will be grown both under standard conditions and under conditions which lead to the accumulation of materials which stain with anti-PHF antibodies in the familial Alzheimer cells. In future studies, autopsy brain samples will be examined from patients with other dementing illnesses, including those associated with abnormalities of cytoskeletal proteins, such as Pick disease. If quantitative immunochemical techniques demonstrate significant differences between Alzheimer and control cells, these techniques will be incorporated into the comparison of the large groups of Alzheimer and control subjects, as part of Project 1.
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