Molecular Diversity Techniques in the Study of Alzheimer's Disease

In this thesis, I have applied a number of molecular diversity techniques, to the study of Alzheimer' s disease (AD). In particular, I have investigated the nature of the epitope of an antibody, MN423, which is highly specific for the intracelullar tangles found in AD, and have attempted to derive antibodies specific for the different isoforms of tau, a protein important in microtubule organization and AD. MN423 recognizes a truncated form of Tau protein that is structurally constrained in Paired Helical Filaments (PHFs) from Alzheimer brain. In this thesis I attempted to identify the epitopes recognized by MN423 using a number of different techniques. First, by screening two cDNA expression libraries derived from AD, clones with sequence similarity to the C terminus of truncated tau were identified. No other clones which may explain the exquisite sensitivity of this antibody for AD brain were found. On the basis of this, and previous work, which identified the truncated tau epitope as being C terminal, the attempt was made to create a C terminal peptide library based on filamentous phage. This was unsuccessful for reasons described in the thesis, and may explain why a commonly cited cDNA display system (pJuFo) has not been widely used. Shortly after this work was carried out, a novel phage display vector based on lambda phage which has a random peptide library displayed at the C terminus of pD, was described. This library was used to identify a motif, consistent with the truncated tau sequence, and also confirmed in small diversity expression libraries prepared in the context of truncated tau. Both methods come to similar conclusions, showing an absolute requirement for a glycine at position -3 and preference for an alanine at position -2. However, further analysis of some of the identified peptides showed that selected peptides may have very different affinities. Bearing in mind the importance of tau in AD, and the fact that it has six different isoforms created by alternative splicing, I also attempted to derive isoform specific phage antibodies, with a final goal of precisely determining the ratio between different isoforms of Tau in AD versus normal brain. This was attempted in two different ways. One was to design peptides specific for regions which were isoform specific. While the results of this approach were not conclusive, polyclonal phage antibodies recognizing some of these peptides and their corresponding tau proteins were derived. The other approach was to utilize schemes of subtractive selections or depletive selections using two or more Tau isoforms. Eventually, after many such attempts, one single chain was isolated that recognizes one of the exons, an amino terminal region of 29 aa, and so was specific for Tau (441) and Tau (410) but not the others. The AD brain immunoreactivity to the antibody MN423 is the basis for the idea that tau is proteolytically processed, perhaps as a result of early apoptotic processes, during the AD neurodegenerative process. This is consistent with studies which have shown that tau can be processed by apoptotic proteases. With the aim of clarifying this possibility, within the context of a broader pool of observations, a number of tau mutants were created which identified the exact point at which the molecule is effectively processed by a defined apoptotic protease.