Alzheimer's paired helical filaments share epitopes with neurofilament side arms.

Several cytoskeletal polypeptides as well as the protein ubiquitin have been implicated as components of the neurofibrillary tangles of Alzheimer disease. We have examined the relationship of ubiquitin staining with immunoreactivity for some of these proteins, both in frozen sections and in cytoskeletal fractions of Alzheimer brain material. We noted (a) antibodies specific solely for neurofflament and glial filament proteins failed to stain the fibrils stainable with ubiquitin. Tau-l antibody stained some but not all of the ubiquitin-stained profiles; fibers staining only for tau or only for ubiquitin were also seen. (il) The Tau-1-stained material was rather diffuse and granular, in contrast to the very sharply defined ubiquitin-positive profiles. (iiM) When Tau-l and ubiquitin stain the same fiber, Tau-l immunoreactivity is often visualized as a diffuse cortical layer of material surrounding a core of ubiquitin immunoreactivity. (iv) The tau immunoreactivity can be almost totally removed by boiling Alzheimer brain cytoskeletal material in 2% NaDodSO4 containing a sulfhydryl reducing agent, this procedure apparently having no effect on the ubiquitin immunoreactivity. If similar material is boiled in 2% NaDodSO4 in the absence of a sulfhydryl reducing agent, the tau immunoreactivity is removed less efficiently, suggesting that tau epitopes are bound to the ubiquitin reactive material in a manner partially dependent on covalent disulfide bridges. These results show that the tau and ubiquitin distributions, both characteristic of Alzheimer disease, are qualitatively different, and that the two markers define immunologically and biochemically distinct structures.

Section: Discussionmentioning

confidence: 99%

Ubiquitin and microtubule-associated protein tau immunoreactivity each define distinct structures with differing distributions and solubility properties in Alzheimer brain.

Shaw

Chau

1988

“…Monoclonal antibodies RT97, BF10, 8D8, and 1215 were produced and kindly provided (as ascites fluids) by B. Anderton and colleagues, who showed that they stain the Mr 200,000 and Mr 160,000 neurofilament proteins as well as neurofibrillary tangles (7,9). Monoclonal antibodies 147 and RS18, also provided as ascites fluids by Anderton and colleagues, stain neurofilaments but not tangles (9). Two monoclonal antibodies to the neurofilament Mr 200,000 and Mr 160,000 polypeptides (here designated BM200 and BM160, respectively) were obtained as purified IgG (20 ,g/ml) from Boehringer Mannheim.…”

Section: Methodsmentioning

confidence: 99%

“…(i) Tangles are stained by polyclonal antibodies to microtubule fractions (2, 3) and by polyclonal and monoclonal antibodies to microtubule-associated protein 2 (MAP2) (4,5). (ii) Antibodies to neurofilament proteins, particularly the Mr 200,000 and Mr 160,000 components, stain tangles (6)(7)(8)(9). (iii) Antibodies to partially purified PHF fractions extracted in NaDodSO4 react with no specific proteins in human brain homogenates, including neurofilament proteins, and produce a peculiar smear of immunoreactivity on electrophoretic transfer blots of Alzheimer disease brain homogenates (10).…”

mentioning

confidence: 99%

Recognition of Alzheimer paired helical filaments by monoclonal neurofilament antibodies is due to crossreaction with tau protein.

Nukina¹,

Kosik²,

Selkoe³

1987

151

Neurofibrillary tangles and senile plaques are the principal pathological features of Alzheimer disease. Neurofibrillary tangles and the neurites of senile plaques contain paired helical filaments (PHF) that consist of two 10-nm filaments twisted into a double helix. The precursor proteins of PHIF are not fully known. To identify these precursors, numerous immunochemical studies have been carried out during the past decade. Two apparently conflicting results have been reported. (I) Some, but not all, monoclonal antibodies to neuroframents stained neurofibrillary tangles. (is) Polyclonal antibodies prepared to PHF purified in NaDodSO4 because of their unusual insolubility did not recognize normal proteins, including neurofilaments, on electrophoretic transfer blots of human brain homogenates. These results have been confirmed in several laboratories, including by the use of electron microscopic labeling. Recently, we reported that polyconal PHF antibodies include antibodies to tau proteins, a family of heat-stable microtubule-associated phosphoproteins, and that antibodies to tau stain Alzheimer neurofibrillary tangles. Those monoclonal neuroframent antibodies that recognize tangles are reported to be directed against phosphorylated epitopes. These facts prompted us to reexamine certain neurofilament monoclonal antibodies that stain neurofibrillary tangles. All monoclonal neurofflament antibodies that stain tangles that we examined, including those initially reported, reacted with tau proteins. Our results suggest that these antibodies react with phosphorylated tau proteins in PH1F, not neurofilament proteins, highlighting the problem of using antibodies to phosphorylated protein epitopes in immunochemical studies. Independent evidence for the presence of neurofflament proteins in human paired helical filaments is now required.

“…Epitopes in neurofilament (NF) proteins and two microtubule-associated proteins (MAPs), r protein and MAP2, have been detected with monoclonal antibodies (mAbs) in Alzheimer disease (AD) neurofibrillary tangles (NFTs) (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Aberrant phosphorylation (3,9), transport (12), proteolysis (13,14), or crosslinking (15) may account for the incorporation of normal proteins or fragments thereof into NFTs.…”

mentioning

confidence: 99%

Alzheimer disease tangles share immunological similarities with multiphosphorylation repeats in the two large neurofilament proteins.

Lee

Ötvös

Schmidt

et al. 1988

104

Immunological and structural analyses of neurofilament (NF) proteins with >500 anti-NF monoclonal antibodies (mAbs) enumerated epitopes shared by NF proteins and Alzheimer neurofibrillary tangles. We identified the multiphosphorylation domain of the rat heaviest NF subunittandem repeats of Lys-Ser-Pro-Xaa (where Xaa is a small uncharged amino acid and serine is phosphorylated)-as the determinant recognized by 15 of the 16 mAbs from this collection of >500 mAbs that detected neurofibriflary tangles. Most (11) of these 16 mAbs also recognized the previously characterized multiphosphorylation repeat in the human middle sized NF subunit. However, although these mAbs shared the ability to recognize NFTs, the antigen-binding domains of these 16 mAbs represented 13 separate classes based on their differential recognition of 12 synthetic peptides derived from the rat heaviest NF subunit and the human middle-sized NF subunit multiphosphorylation sites, NF subunits of 10 diverse species, and normal human X protein. We conclude that NFTs share highly specific immunological and structural properties with specific rat heaviest NF subunit and human middle-sized NF subunit multiphosphorylation repeats.