Molecular profiling of paired helical filaments.

Fibrous deposition of neurofibrillary tangles (NFT) is one of the neuropathological hallmarks of Alzheimer’s disease (AD). The extent of the NFT lesions correlates well with the severity of dementia in AD. Therefore, it is of ultimate importance to understand the molecular basis of NFT. This would entails a formidable challenge that requires the isolation of a defined macromolecular assembly and characterization of individual constitutes by conventional biochemical analysis. Fortunately, the first step has been largely aided by the ultrastructural studies using electron microscopy that led to the identification of the major fibrous units of NFT as paired helical filaments (PHF) [1,2]. In the recent decade, major efforts and significant progress have been made in unraveling the molecular composition of PHF. However, as the article by Goux et al. (this issue) and other studies suggest, such an endeavor is far from over. Goux et al. argue that the current view of PHF as made mainly of tau protein may only represent a partial picture of molecules that are present in PHFs. Based on their analysis; they maintained that other non-protein components such as glycolipids might be more abundant than tau on a weight percent basis. While the view of Goux et al. is likely to be controversial, the involvement of molecules other than tau protein in the composition of or the assembly of PHF has been suggested by other investigators. In addition to glycolipid, ubiquitin [3], glycosaminoglycans [4,5], products of glycation and lipid oxidation [6], and metals [7,8] have been found to be associated with PHF. Like any other biological assembly, the key problem in differentiating authentic PHF components from “contaminants” is how PHF structures should be defined, analyzed and quantitated. Electron microscopy (EM) continues to be the standard method for verifying the presence of PHF through the observation of the characteristic, twisted fibrous structures. While it is possible to obtain estimates on mass per unit length of the PHF using scanning transmission EM (STEM) [2], other methods are necessary for analyzing the molecular nature and composition of PHF. The first insight into the major protein components came as antibodies against PHF cross-react with tau. After extensive analysis of isolated PHF, it is commonly accepted that hyperphosphorylated tau is a major protein component of PHF [9]. However, few studies have actually quantitated tau on a weight percent basis in their preparations. It turns out that quantitation of tau and other components in PHF is not as simple as it appears. Tau exists as six isoforms and is extensively modified by phosphorylation, and possibly by crosslinking. The endogenous proteolysis at different stages of AD pathology also results in tau molecules of various lengths. The insolubility of PHF also adds to the complexity in molecular analysis by conventional methods. PHF can be isolated as pronase-resistant tangle fragments (prcPHF) with truncated tau containing only the repeat region of 90 residues at the protein core. Since prcPHF is extremely insoluble and resists extraction by denaturants and detergents, there may be uncertainty in the purity analysis for the prcPHF preparations other than gross morphology examination by EM. It is in this type of preparations that Goux and co-workers initially find the evidence of glycolipids being abundant nonprotein components [10]. Using H and C NMR and GC/MS, they found evidence of carbohydrates (mostly glucose) and fatty acids with a molar ratio of about 6:1. Their data was then fit to a proposed structure of