Quantitative solubilization and analysis of insoluble paired helical filaments from Alzheimer disease

Brain Res. 1996 Apr 22;717(1-2):99-108. doi: 10.1016/0006-8993(95)01473-x.

Abstract

In this study, we evaluate the ability of several solvents to solubilize insoluble paired helical filaments (PHF) of Alzheimer disease. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents of PHF. Using sequential categories of protein solvent to analyze insoluble PHF, only alkali or exhaustive proteolysis are effective in completely solubilizing PHF, while a variety of denaturants are ineffective. Alkali does not affect the phosphorylation state of PHF and complete dephosphorylation of PHF with hydrofluoric acid does not affect PHF solubility. These findings suggest that the 'hyperphosphorylation' of PHF proteins is not responsible for PHF insolubility. However the in vitro glycation of tau generates PHF that are insoluble in SDS and soluble in alkali. These findings suggest that protein crosslinks, including advanced glycation endproduct-derived crosslinks which were recently described in Alzheimer disease, play a major role in effecting PHF insolubility in vivo.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Aged, 80 and over
  • Alkalies
  • Alzheimer Disease / metabolism*
  • Cell Fractionation
  • Cross-Linking Reagents / analysis
  • Cross-Linking Reagents / metabolism
  • Densitometry
  • Endopeptidases
  • Formates
  • Glycation End Products, Advanced / analysis
  • Guanidine
  • Guanidines
  • Hemostatics
  • Humans
  • Immunoblotting
  • Microscopy, Electron
  • Middle Aged
  • Neurofilament Proteins / analysis*
  • Neurofilament Proteins / metabolism
  • Neurofilament Proteins / ultrastructure
  • Phosphorylation
  • Protein Denaturation
  • Sodium Dodecyl Sulfate
  • Solubility
  • Urea

Substances

  • Alkalies
  • Cross-Linking Reagents
  • Formates
  • Glycation End Products, Advanced
  • Guanidines
  • Hemostatics
  • Neurofilament Proteins
  • formic acid
  • Sodium Dodecyl Sulfate
  • Urea
  • Endopeptidases
  • Guanidine