Proteomic study of proteins tightly associated with tau in neurofibrillary tangles of Alzheimer brains

Abstract

Alzheimer's disease (AD) is the commonest cause of dementia in the elderly. Histopathologically AD brains are characterized by the presence of neurofibrillary tangles (NFTs), intracellular and extracellular (I-NFT and E-NFT, respectively) and neuritics plaques. Both structures represent dense accumulations of abnormal insoluble filaments. NFTs are formed by paired helical filaments (PHFs), which are structurally composed by assembled tau protein. To date, the integral constituents of PHF remain unknown mainly due to the highly insolubility of such structures. The purpose of this study was to perform a proteomic analysis of NFTs in order to identify those potential proteins which bound closely to tau into the PHF and may be have a possible role in the pathological assembly of tau. We combined mass espectometry with the use of a laser microdissector. Sixteen-μm-thick paraformaldehyde-fixed-brain tissue from 3 AD cases were immunolabelled with the conformational phospho-dependent-monoclonal antibody (mAb) AT100 (Ser 199, Ser202, Thr205, Thr 212, Ser 214) . For each immunolabelled slice we obtained approximately 7,000 isolated NFTs. The pull of these structures were tried to several harsh conditions of solubility. For each condition, samples were purified and digested prior to their analysis with bidimentional HPLC, electro spray ionization and tandem mass spectrometry (MS/MS). Candidate proteins were analyzed in silico with different databases (Uniprot Knowledgebase and IPI). We were able to identify 57 proteins. Preliminary analysis have demonstrated that among these proteins are included some related to metabolism (24%), cytoskeleton (13%), anti-oxidants (6%), kinases (2%), and ubiquitin (2%). Some of these findings have been verified using immunohistochemistry and confocal microscopy. We expect to identity a sub-group of proteins as potential candidates to be playing a role in the PHF formation in AD. This study demonstrates the advantages to combine techniques of high quality isolation and proteomics in fixed brain tissue.