Monoclonal antibodies show that neurofibrillary tangles and neurofilaments share antigenic determinants

523

337

Information about the structure of the paired helical filaments (PHF) that accumulate within human neurons and the amyloid fibers that accumulate in the extracellular spaces between neurons in Alzheimer disease has so far depended on electron microscopy of thin-sectioned or negatively stained material. To determine the protein conformation of these abnormal fibers, we have obtained x-ray diffraction patterns from unfixed human brain fractions highly enriched in PHF and from purified amyloid cores isolated from senile plaques. The predominant x-ray scatter evident from both types of samples, either wet or dry, is a sharp reflection at 4.76-A spacing and a diffuse one at about 10.6-A spacing. These features are characteristic of a beta-pleated sheet type of protein conformation. In doubly oriented dried pellets of PHF fractions, the two reflections are accentuated at right angles to each other and the arc at 4.76-A spacing is in the fiber direction indicating a cross-beta conformation. From the integral widths of the reflections we estimate the cross-beta crystallite to be about 80 A long in the fiber direction and about 40 A thick. These dimensions correspond to approximately four pleated sheets, each of which consists of approximately 16 hydrogen-bonded polypeptide chains running normal to the fiber direction. The cross-beta conformation of PHF and amyloid fibers that we have found from x-ray diffraction is in contrast to the predominant alpha-helical coiled-coil conformation of the neurofilaments with which they share epitopes and from which they have been postulated to derive.

Section: Discussionmentioning

confidence: 99%

X-ray diffraction from intraneuronal paired helical filaments and extraneuronal amyloid fibers in Alzheimer disease indicates cross-beta conformation.

Kirschner¹,

Abraham²,

Selkoe³

1986

523

337

“…Multimeric peptides (i.e., 1-12nP, 1-12P, 1-24nP, 1-24P, 1-36nP, and 1-36P) comprised of the Lys-SerPro-Ala-Glu-Ala repeat in rat NF-H were used in ELISA and immunodot tests to classify the peptide specificities of over 500 anti-NF mAbs generated with rat, cow, and human NF-H and/or NF-M immunogens. Other mAbs raised to or crossreactive with NF proteins (SMI33, SM134, RT97, UCD7H11) were also tested because they bind NFTs (1,9,16,17).…”

Section: Resultsmentioning

confidence: 99%

“…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.

“…Many previous studies have suggested that various intermediate filament proteins may be constituents of NFT (3)(4)(5). Our immunological evidence shows that no antibody directed solely against any neuronal or glial intermediate filament protein showed any staining for the ubiquitin-or tau-positive material.…”

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

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.