Alzheimer's disease: Immunoreactivity of neurofibrillary tangles with anti-neurofilament and anti-paired helical filament antibodies

521

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

521

337

“…Two monoclonal antibodies to the 200-and 150-kDa neurofilament proteins (9), respectively, as used by Rasool et al (15) failed to react with isolated Alzheimer tangles, although they did react in situ. Presumably, their reaction in situ may have been with a component of perikarya not actively associated with Alzheimer tangles.…”

Section: Discussionmentioning

confidence: 99%

“…Wisniewski et al (12) suggested that neurofilaments merely provide a passive "decoration" of Alzheimer tangles. Indeed, Igbal et al (13) (7) is essential for the formation of normal, compact axonal neurofilaments (3), the selected phosphorylation of a single site, as now observed in Alzheimer tangles, may result in irregular and incomplete assembly, morphologically identified as tangles, and immunocytochemically revealed by presentation of new epitopes (15) in addition to those shared with normal neurofilaments.…”

Section: Discussionmentioning

confidence: 99%

Aberrant neurofilament phosphorylation in Alzheimer disease.

Sternberger¹,

Sternberger²,

Ulrich³

1985

299

137

Alzheimer tangles, despite their location in neuronal perikarya, react immunocytochemically with monoclonal antibodies to phosphorylated epitopes of neurofilaments. Normal perikarya do not contain phosphorylated neurofilaments. The aberrant phosphorylation in both plaques and tangles seems to be largely restricted to individual phosphorylation sites among the many sites available in neurorilaments. It is suggested that the Alzheimer lesion involves an imbalance within specific kinases responsible for phosphorylation of different sites in neurofilaments. Subsequent studies have shown (3) that the macroheterogeneity was posttranslational and depended on phosphorylation. Thus, antibodies from group II reacted exclusively with phosphorylated neurofilaments; those ofgroup III, with nonphosphorylated epitopes in neurofilaments that are masked by phosphorylation; and antibodies from group IV, apparently with a more accessible, nonphosphorylated neurofilament epitope. In tissue sections, trypsin or phosphatase treatment alone had no effect on the immunocytochemical staining by antibodies from group II. However, trypsin followed by phosphatase reduced the staining. Trypsin treatment abolished the staining by antibodies from group III. However, the staining with these antibodies reappeared by subsequent phosphatase treatment but was converted to axonal staining-i.e., from a group III to a group II pattern. The data permitted the conclusions that neurofilaments in dendrites, perikarya, and proximal axons are nonphosphorylated and that phosphorylation occurs during transport along the axon. Furthermore, it was apparent that phosphorylated neurofilaments were more compact than nonphosphorylated forms.Alzheimer tangles are perikaryonal constituents. They have been shown by Selkoe et al. (4) to differ from normal neurofilaments in their resistance to solubilization by even extensive treatment with sodium dodecyl sulfate and, thus, can be considered highly compacted structures at least with regard to tertiary conformation. In contrast, normal perikaryonal neurofilaments, which are not phosphorylated, seem, according to our data, of noncompact configuration. It appeared, therefore, of interest to study phosphorylation of Alzheimer tangles and plaques and to examine the compactness of these structures with regard to susceptibility to dephosphorylation. MATERIALS AND METHODSThis study includes two cases of Alzheimer disease, one case of Down syndrome, and a case of cerebral infarct. The first three cases exhibited progressive dementia and revealed, on autopsy, severe changes of the Alzheimer type in hippocampus and neocortex. The last case had only few changes of the Alzheimer type.Paraffin sections were stained immunocytochemically (5) by using monoclonal first-layer antibodies, goat anti-mouse 4274 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

“…While the chemical composition of PHF has not been ascertained, it appears that some normal fibrous proteins or protein fragments are found within the NFT. Neurofilaments are likely to be among such proteins (13, [16][17][18][19][20]. We report here the raising of monoclonal antibodies (mAb) to microtubule-associated protein 2 (MAP-2) and the finding that certain MAP-2 epitopes are associated with Alzheimer NFT, both in situ and after isolation of the tangles under nondenaturing conditions.…”

mentioning

confidence: 99%

“…Clones that reacted positively by ELISA were analyzed by the immunoblot procedure of Towbin et al (41) using the microtubule preparation as the substrate. Tubulin and MAPs prepared either by the method of Shelanski et al (38) or by the taxol method of Vallee (42) were electrophoresed (43) (ii) Isolated NFT prepared under non-dena tions in a physiologic buffer (15,17) were imi Five to 10 g from NFT-rich areas of frozen A ease cerebral cortex were homogenized in 10 v line buffer (50 mM Tris HCl, pH 7.6/145 m1b homogenate was sieved through a 200-,m nyl centrifuged at 100,000 x g for 30 min at 4°C. T rehomogenized in Tris saline buffer and was k of 1 M sucrose in Tris saline and spun at 100,0 min.…”

mentioning

confidence: 99%

Microtubule-associated protein 2: monoclonal antibodies demonstrate the selective incorporation of certain epitopes into Alzheimer neurofibrillary tangles.

Kosik¹,

Duffy²,

Dowling³

et al. 1984

170

Neurofibrillary tangles (NFT) are the principal structural alteration of neuronal cell bodies in Alzheimer disease as well as in normal aging of the human brain. While the ultrastructure of these intraneuronal lesions has been extensively studied, the biochemical composition of the fibers comprising the NFT is unknown. We report the production of three monoclonal antibodies against the microtubule-associated protein 2 (MAP-2), one of which intensely labels Alzheimer NFT. All three antibodies specifically recognize MAP-2 on immunoblots and stain brain tissue in a characteristic dendritic pattern. The three antibodies are directed against at least two different antigenic sites on the MAP-2 molecule, and one appears to recognize a phosphorylation site on MAP-2. That only one of the three antibodies immunolabels NFT suggests that the formation of the tangle involves some modification of the MAP-2 molecule. Our findings suggest that one aspect of Alzheimer-type neurofibrillary pathology is an aggregation of MAP-2 or MAP-2 fragments with altered neurofilamentous elements present in NFT. Normal interactive function, which putatively occurs between neurofilaments and MAP-2, may thus be disrupted in Alzheimer disease.