Our long term objective is to understand the pathogenesis of Alzheimer disease (AD). Our short term objective is to understand the mechanism of neurofibrillary degeneration in AD brain. We propose to definitively identify by gold-immuno-labeling the approximate 2.1nm diameter filaments that have been found in Alzheimer neurofibrillary tangles (NFT) isolated in the absence of detergent. These approximate 2.1 nm filaments have the same structure as the approximate 2.1.nm bovine tau polymer filaments which are triple-stranded and left-hand helical. Isolated whole NFT will be labeled in separate preparations with gold-conjugated-antibodies to tau, ubiquitin, and amyloid-Beta-protein. The structure of the NFT and the localization of the immuno-gold labels will be investigated in whole NFT that have been freeze-dried on a millipore filter and vertically platinum-carbon (Pt-C) replicated. We also propose to investigate how the tau monomers (about 1.0nm diam. by about 63 nm long) are aligned with one another within the three stranded tau polymer. We will study the tau polymers structure by binding antibody fragments, Fab' or F(ab)'2, to the N or C-terminal region and to the microtubule binding domain first in separate spreading experiments and then in a double immuno-labelling experiment. We propose to investigate whether the tau polymer or the monomer configuration is active in stabilizing long microtubules by direct visualization of the microtubule surfaces in vertically Pt-C replicated freeze-dried preparations. Finally we propose to correlate tau's inverse temperature transition at 197 nm in the circular dichroism (CD) spectrum with tau's length at the inverse temperature transition (about 30 degrees C), as well as above (about 37 degrees C) and below this temperature(4-10 degrees C. Since the tau polymers are of different lengths, immuno-labelled periodic intervals will be measured as a function of temperature. Since the inverse temperature transition at 197 nm in the CD spectrum appears to correlate with tau's ability to stimulate tubulin polymerization above 30 degrees C and not below 20 degrees C, its important that tau's length be correlated with this temperature effect. The work on tau polymer, is very relevant to Alzheimers disease, since normal tau's structure and function have to first be understood to assess how pathological abnormally phosphorylated tau in NFT differs from it.
