QUANTITATIVE SOLUBILIZATION AND ANALYSIS OF INSOLUBLE PAIRED HELICAL FILAMENTS FROM ALZHEIMER-DISEASE

In this study, we evaluate the ability of several solvents to solubili ze insoluble paired helical filaments (PHF) of Alzheimer disease. Spec ifically, we use protein extraction and reduction in the volume of ins oluble material as quantitative assays to establish solvents of PHF. U sing sequential categories of protein solvent to analyze insoluble PHF , only alkali or exhaustive proteolysis are effective in completely so lubilizing PHF, while a variety of denaturants are ineffective. Alkali does not affect the phosphorylation state of PHF and complete dephosp horylation of PHF with hydrofluoric acid does not affect PHF solubilit y. These findings suggest that the 'hyperphosphorylation' of PHF prote ins is not responsible for PHF insolubility. However the in vitro glyc ation of tau generates PHF that are insoluble in SDS and soluble in al kali. These findings suggest that protein crosslinks, including advanc ed glycation endproduct-derived crosslinks which were recently describ ed in Alzheimer disease, play a major role in effecting PHF insolubili ty in vivo.