Rabbit IgG cross-reacts with Alzheimer neurofibrillary tangles

Biochemical studies have demonstrated that the paired helical filaments (PHF) of Alzheimer neurofibrillary tangles are mostly made up of tau and to a lesser degree of ubiquitin and other proteins. In addition, immunocytochemical labeling of tangles with antibodies to various other neuronal proteins has been shown previously. We report here the labeling of the locations of PHF, i.e., Alzheimer neurofibrillary tangles, neuropil threads and plaque neurites in tissue sections with a goat antiserum to rabbit IgG (GAR-T). The labeling is comparable in strength and distribution to that of tau and ubiquitin antibodies. The PHF-staining antibodies could be removed by absorption with native rabbit IgG but not with human IgG, IgG-depleted rabbit serum, rabbit IgG heavy chains or light chains eluted from nitrocellulose membranes. Furthermore, the PHF reactivity was obliterated by absorption with brain homogenate and a fraction enriched in soluble abnormally phosphorylated tau, but not with purified bovine tau or SDS-washed preparations of the relatively insoluble population of PHF. On immunoblots of both normal human tau and Alzheimer abnormally phosphorylated tau-enriched preparations, GAR-T labeled a set of three to five polypeptides in the tau region. Some of these polypeptides co-migrated with the tau bands. These results indicate (i) that PHF in Alzheimer's disease brain cross-react with a structural epitope/s present on native rabbit IgG, and (ii) that the cross-reactivity with PHF is probably due to tau.Supported in part by the New York State Office of Mental Retardation and Developmental Disabilities and grants NS 18105, AG04220, AG05892 and AG08076 from the National Institutes of Health     

Amyloid-like (Congophilic) neurofibrillary tangles do not react with neurofilament antisera in Alzheimer's cerebral cortex

Summary The immunohistological properties of Alzheimer's neurofibrillary tangles (NFT) were studied by immunofluorescence with neurofilament (NF) antisera and with antiserum raised to paired helical filaments (PHF) in NFT preparations, brain smears, and cryostat sections. NFT decorated by NF antisera were Congo red-negative. Conversely, PHF antisera stained Congo red-positive NFT but failed to decorate NF-positive NFT. It is concluded that NF do not cross react with typical NFT, i.e., NFT displaying amyloid-like birefringence, under the conditions reported in this study.Supported by the Veterans Administration, by NIH grants NS 13034 and AG 01307 (DJS)     

Induction of epitopes associated with neurofibrillary tangles in clonal mouse neuroblastoma (S20Y) cells

Summary Accumulation of paired helical filaments (PHF) in neurofibrillary tangles is a key neuropathological hallmark in Alzheimer's disease (AD). To date, PHF have been found primarily in humans. Cultured murine cholinergic neuroblastoma (S20Y) cells, following exposure to a serum-free medium or a differentiation medium, developed immunoreactivity to anti-PHF antibodies, and to the Alz-50 by immunocytochemical and immunoblot analyses. Electron microscopic examination revealed abundant fascicles of 10-nm filaments coursing tortuously amongst organelles, such as mitochondria, endoplasmic reticulum and dense-core vesicles, in perikarya and in neuritic extensions. However, subcellular structures identical or similar to PHF could not be found in these non-human cells. This convenient cell culture model may prove to be useful for studying certain aspects of the mechanisms underlying the abnormal cytoskeletal alterations which are characteristic of AD and related neurodegenerative disorders.Supported by grants from the Overbrook Foundation, the Will Rogers Institute, the Dr. I. Fund Foundation, the Winifred Masterson Burke Relief Foundation, the Alzheimer's Disease Research Program of the American Health Assistance Foundation and the National Institute of Aging (AG03853)     

Immunocytochemistry of neurofibrillary tangles with antibodies to subregions of tau protein: identification of hidden and cleaved tau epitopes and a new phosphorylation site

Summary Antibodies to multiple epitopes spanning the length of the tau molecule were used to study Alzheimer neurofibrillary tangles (NFT) using immunocytochemical methods and several differnt methods of fixation and tissue processing, including staining of vibratome sections, hydrated autoclaving of paraffin sections and immunofluorescence of NFT isolated from fresh brain tissue. Smears and sections were pretreated with trypsin and/or phosphatase to further characterize antibody binding. In tissue fixed briefly in periodate-lysine-paraformaldehyde, tau immunoreactivity was detected in astrocytes, but only a few tau epitopes were detected in NFT with this fixation method. In contrast, all tau epitopes were detected in NFT in tissue fixed in formaldehyde for prolonged periods of time. In the hippocampus, the number of NFT detected in the dentate fascia was in proportion to the duration of dementia, as we previously noted. Dentate fascia NFT were intracellular (i-NFT) and were reactive with antibodies recognizing epitopes in both the carboxy- and amino-terminal regions of tau, but not the microtubule-binding domain of tau, suggesting that microtubule-binding domain epitopes are hidden in i-NFT. In contrast, NFT in the subiculum and layer II of the parahippocampal cortex were mostly extracellular (e-NFT), especially in severe cases of long duration, e-NFT were immunoreactive with antibodies to the microtubule-binding domain, but only weakly reactive with antibodies to carboxy- or amino-terminal epitopes, suggesting that e-NFT may contain fragments of tau. In both isolated NFT and NFT in sections, amino-terminal epitopes, including the Alz-50 epitope, were sensitive to trypsin proteolysis, which suggests that the lack of staining of e-NFT by antibodies to the amino-terminal regions of tau is due to proteolysis. Antibodies reactive with amino-terminal epitopes also stained fewer NFT following hydrated autoclaving, while those reacting with the carboxy half of tau stained more NFT after hydrated autoclaving. Thus, although carboxy-terminal regions are not detected in e-NFT, they are probably masked, rather than proteolytically cleaved, since they can be revealed by hydrated autoclaving. Finally, phosphatase treatment of isolated NFT revealed enhanced immunostaining not only with Tau-1, as in previous studies demonstrating abnormal phosphorylation of tau proteins in NFT, but also with an antibody to exon 2, which reveals yet another phosphorylation site in tau of NFT.Supported by NIA AG06803, AG01136 and AG04145     

Neurofibrillary tangles in human upper cervical ganglia

Summary Neurofibrillary tangles (NFTs) are one of the main pathological hallmarks of Alzheimer's disease or senile dementia, and are seen in the cerebral cortex and some other nuclei in the central nervous system (CNS). No NFTs have been reported in the human peripheral nervous system, although NFTs were recognized in the dorsal root ganglion of the aged rodents. We report here the presence of NFTs in the upper cervical ganglia (UCGs), but not in the stellate nor in the celiac ganglia, of an elderly patient, who was not demented and had only minimal senile changes in the CNS. Immunohistochemically the antibodies to microtubule-associated protein 2, paired helical filaments and ubiquitin stained positively the NFTs in the UCGs. On electron microscopic examination a periodical twisted pattern of the filaments was identified; these findings suggest that the NFTs of the UCGs have just the same properties as those of the cerebral cortex. This is the first report of the demonstration of NFTs in the peripheral ganglia and might contribute to the study of mechanism of NFT production.     

Paired helical filaments are not the major binding sites for wheat germ and Dolichos biflorus agglutinins in the neurofibrillary tangles of Alzheimer's disease

Summary The isolated paired helical filaments (PHF) that occur in the neurofibrillary tangles of Alzheimer's disease were assayed to determine if they contained N-acetyl-glucosamine and N-acetyl-galactosamine residues. The enzyme-linked lectin assay was used to detect their total content in the PHF preparation. The assay employed biotinylated Dolichos biflorus and wheat germ agglutinins and was developed with avidin-horseradish peroxidase. The total PHF preparation was shown to contain very little of these glycosylated control proteins. Colloidal gold-labeled lectins were used to study the PHF by electron microscopy to assess whether the minor amount of lectin binding in the total preparation was directly associated with the PHF. These studies showed no significant association of the colloidal gold-labeled lectins with the isolated filaments. We conclude that the PHF themselves contain few or no N-acetyl-glucosamine or N-acetyl-galactosamine residues.Supported by an Advanced Technology Grant (J08166) from the Texas Higher Education Coordinating Board, a National Institutes of Health Alzheimer's Disease Research Center Grant (AG08013), and a grant from the French Foundation for Alzheimer Research     

Solubility of neurofibrillary tangles and ultrastructure of paired helical filaments in sodium dodecylsulphate

Summary Temporal cortex from 14 cases of Alzheimer-type dementia and 6 cases of Down's syndrome, all selected for severe Alzheimer pathology, was homogenised in distilled water, NaOH, or sodium dodecylsulphate (SDS) containing 0.1% -mercaptoethanol. The homogenates were stained with Congo red, and the neurofibrillary tangles and plaque cores were counted under crossed-polarisation microscopy. The number of tangles and plaque cores in the water-treated extracts was not related to age, sex, postmortem interval or duration of dementia. The number of tangles after extraction in SDS or NaOH, as a percentage of tangles in water-treated extracts, was 57±25 (mean±SD) for 1% SDS, 43±17 for 5% SDS and 37±22 for 0.2 M NaOH. Plaque cores were essentially insoluble in all three agents. The percentage of tangles insoluble in 1% SDS did not correlated with age or post-mortem interval but decreased with increasing duration of dementia. Enhanced tangle solubility with increasing duration of dementia suggests that the nature of tangles changes with time; one possibility is that this reflects transformation of intracellular to extracellular tangles. Paired helical filament (PHF) length and the number of repeats per PHF were measured in electron micrographs of PHF prepared with and without treatment by 1% SDS. There was no significant multimodality of PHF length to suggest that PHF broke at regular intervals. The mean repeat length (PHF length/number of repeats) was greater for PHF isolated in the presence of 1% SDS than in its absence, showing that SDS affects ultrastructure by untwisting PHF. An untwisting process may also occur in vivo producing the straight filaments found, together with PHF, in tangles and neurites.Supported by Miss E. Buchan (to the MRC Brain Metabolism Unit) and the British Foundation for Age Research and the Wellcome Trust (to P. A. M. Eagles). S. Hussey was in receipt of an MRC Partnership Award.     

Alzheimer neurofibrillary tangles: Monoclonal antibodies to inherent antigen(s)

Summary Using isolated Alzheimer neurofibrillary tangles as the immunogen, nine mouse hybridomas were generated which produced antibodies to the tangles as tested in both tissue sections and isolated neurons from Alzheimer brain. Extraction of isolated neurofibrillary tangles with 2% SDS could not remove the antigen(s) with which these monoclonal antibodies reacted. Immunocytochemical study revealed that each of the monoclonal antibodies reacted with one or more of other tissue antigens in addition to the Alzheimer tangles. However, no reaction with either neurofilaments or microtubules was observed with any one of these antibodies. This is the first demonstration of monoclonal antibodies which have been generated against isolated Alzheimer neurofibrillary tangles; these antibodies react with antigen(s) inherent to the tangles.Supported in part by NIH grants NS 18105 and NS 17487     

Neurofibrillary tangles of the Indiana kindred of Gerstmann-Stra¨ussler-Scheinker disease share antigenic determinants with those of Alzheimer disease

In the Indiana kindred of Gerstmann-Stra¨ussler-Scheinker disease, neurofibrillary tangles (NFT) with paired helical filaments (PHF) are numerous, widespread and consistently present in the cerebral cortex and several subcortical nuclei. Such tangles share antigenic determinants with those of Alzheimer disease; in fact, they are recognized by Alz50, anti-PHF and anti-ubiquitin antibodies. Thus, NFT with structural and immunocytochemical similarities are present in two distinct forms of amyloidosis of the central nervous system, i.e. the Indiana kindred of Gerstmann-Stra¨ussler-Scheinker disease and Alzheimer disease.     

Long survival in Fukuyama congenital muscular dystrophy: occurrence of neurofibrillary tangles in the nucleus basalis of meynert and locus ceruleus

Summary Three patients with Fukuyama congenital muscular dystrophy (FCMD), who died aged 23, 29 and 34 years, are reported. There was extensive brain malformation, but the most severe pattern of cortical dysplasia was absent in one case and in the other two localized bilaterally to small areas near the occipital poles. In two cases, numerous neurofibrillary tangles were observed in the locus ceruleus and nucleus basalis of Meynert. Electron microscopy revealed paired helical filaments, with a maximal width of about 25 nm and regular constrictions at approximately 80 nm intervals. The occurrence of neurofibrillary tangles, which is unknown in younger patients, suggests the presence of degenerative processes in the brains of the older patients with FCMD.Partly supported by the Grant Nos. 85-04, 85-07, 85-16 from National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare of Japan     

Mapping of neurofibrillary degeneration in Alzheimer's disease: evaluation of heterogeneity using the quantification of abnormal tau proteins

Summary A biochemical mapping of neurofibrillary degeneration was performed in Brodmann areas of the brains of five patients with senile dementia of the Alzheimer type (AD). To quantify the degenerating process, we used an immunoblot method with antibodies directed against the abnormally phosphorylated tau proteins named Tau 55, 64 and 69, known to be early and reliable markers of the degenerating process in AD. The detection intensities were assessed using a numerical rating scale for each area and scores were grouped by lobe. In all cases, the detection of Tau 55, 64 and 69 was positive in all areas except in primary visual cortex (area 17) for two patients. The detections were especially strong in temporal neocortical and limbic areas and were higher in associative cortex than in primary sensory cortex. Scores from the occipital and frontal lobes differed strongly between patients as compared to the uniform degree of detection in the limbic, temporal and parietal lobes. Despite a relatively identical duration of the disease and an apparently global involvement of the cerebral cortex, the distribution of neurofibrillary degeneration in AD varies significantly across cortical areas and displays striking heterogeneity patterns along the rostrocaudal axis. The immunodetection of abnormal tau proteins using the Western blot method may provide complete and rapid quantitative data of the degenerating process in AD.Supported by Conseil Regional Nord-Pas, Schering SA, ADERMA and INSERM clinical network CAR 489016     

Ultrastructural analysis of neurofibrillary tangles of Alzheimer's disease using computerized digital processing

Summary The ultrastructure of neurofibrillary tangles of Zlzheimer's disease was analyzed by computerized digital processing of electron micrographs. Processing of the electron micrographs consists of four steps: digitizing the electron micrograph, Fourier transformation, noise filtering and inverse Fourier transformation and Laplacian operation. In the present study, we have confirmed that neurofibrillary tangles are composed of a pair of helical filaments (PHF), which appear characteristically as an unbranched rigid structure. The periodicity of PHF is 78nm on the diffractogram. The dimensions of PHF obtained by our analysis, although basically similar to those described earlier by other investigators using conventional techniques, more precisely defines its structural conformation. We have also demonstrated that the spatial relationship of two filaments appears symmetrical after two-way tilting of the specimen about the axis of rotation. Our observations emphasize the importance of digital image processing as an effective tool for structural analytical research in biology and medicine.Supported in part by the Japanese Ministry of Culture (61570527) and the Research Committee on Senile Dementia of the Ministry of Welfare     

Substructures of paired helical filaments from alzheimer's disease neurofibrillary tangles

Summary Presented studies reveal that each of the approximately 10 nm filaments forming the paired helical filaments (PHF) is made up of four protofilaments. Each of the protofilaments is a beaded structure, consisting of globules connected by longitudinal bars. A cross-view of PHF shows eight globules linked by transverse bars. The transverse bars are shorter than the longitudinal bars. Comparison between PHF and neurofilament protofilaments indicates structural differences between these profiles, i.e., the globules making the PHF protofilaments are larger and the longitudinal bars are longer than those in the normal neurofilaments. A three-dimensional diagram of PHF structure is presented.Supported in part by grants from the NIA (1P01 AG/NS 04220) and from the Aluminium Association (914-1065A)     

Senile plaque neurites fail to demonstrate anti-paired helical filament and anti-microtubule-associated protein-tau immunoreactive proteins in the absence of neurofibrillary tangles in the neocortex

Summary Although much work has been directed recently towards unravelling the protein chemistry of neurofibrillary tangle (NFT) and senile plaque (SP) components in Alzheimer's disease, the pathogeneses of these lesions remains largely unknown and the problem of their relationship is unresolved. In particular, although paired helical filaments (PHF) have long been documented in SP neurites, we do not know if they are of pathogenetic relevance for the formation of the SP. To investigate the relationship between NFT and SP, we examined antigenic properties of proteins in SP neurites in neocortical tissues of patients with senile dementia of Alzheimer type, in the presence or absence of NFT in the same cortical area. We used two polyclonal antibodies directed against PHF and microtubule-associated protein (MAP)-tau and three monoclonal antibodies (MAbs) (RT97, BF10, 147) to phosphorylated epitopes of human neurofilament polypeptides, as well as the Gallyas silver impregnation method which specifically stains PHF in NFT and neurites. The main finding of our investigations consists in a differential pattern of immunoreactivity of SP neurites depending on the presence or absence of NFT in the neocortex. In the presence of NFT, there were numerous neuropil threads and SP neurites containing Gallyas-positive, as well as anti-PHF- and anti-tau-labelled material. In the absence of NFT in the neocortex there was a striking absence of any Gallyas-positive or PHF- and tau-immunoreactive structure in the cortical neuropil and in SP neurites, irrespective of the maturation stage of the SP. In contrast with these results, the number of neurites labelled by MAbs RT97, BF10 and 147 in SP and in the neuropil was apparently unaffected by the presence or absence of NFT. Amyloid in SP, remained consistently unstained by all antibodies of the panel as well as by the Gallyas stain. Our findings indicate that PHF and tau polypeptides are facultative components of SP neurites and suggest that the development of SP may occur independently of PHF pathology in neocortical neurons.Supported by the Wellcome Trust and the Medical Research Council     

Scanning electron microscopical study of the neurofibrillary tangles of Alzheimer’s disease

Neurofibrillary tangles (NFTs) have been ultrastructually studied by various methods, leading to several three-dimensional models of paired helical filaments (PHFs). In this study, we present the scanning electron microscopic findings of NFTs in an autopsy case of Alzheimer’s disease and clarify the three-dimensional structures of NFTs. NFTs were clearly defined in freeze-cracked nerve cells and consisted of two types of filamentous structures, straight and helical filaments. Straight filaments measured from 20 to 25 nm in diameter and had a smooth surface. They were slightly bent but mostly straight with no constrictions. One type of straight filaments ran in a bundle in the same direction, another was intertwined to each other. Most of the helical profiles of filaments usually measured about 28 nm in diameter, with a distance of 100 nm between periodic constrictions. They seemed to consist of a pair of isodiametric filaments of 10 nm in diameter. In addition, two unusual types of helical filaments were occasionally observed. One comprised thick filaments of about 38 nm in diameter, with a distance of 100 nm between constrictions; these helical filaments appeared to consist of two or more strands. The other comprised thin helical filaments of about 20 nm in diameter and regularly constricted at an interval of 50 nm. All types of the helical filaments examined in this case were leotropic. This result supports a protofilament model of PHFs. Scanning electron microscopy using the freeze-cracked and maceration method is a useful and simple method for three-dimensional observation of the filamentous structures in NFTs. Received: 10 October 1996 / Revised, accepted: 15 January 1997     

Abnormal phosphorylation of tau precedes ubiquitination in neurofibrillary pathology of Alzheimer disease

On tissue sections of Alzheimer brain, 4 antibodies to tau immunolabel not only neurofibrillary tangles, neuritic plaques and neuropil threads but also the tangle-free cytoplasm of a subset of hippocampal and cortical neurons we believe to be at a stage of alteration preceding the formation of paired helical filaments (PHF). Pretreatment of tissue sections with alkaline phosphatase leads to an increase in staining intensity and in number of immunoreactive lesions with antibodies directed to an amino terminal and to a mid-region of the tau molecule. The diffuse neuronal staining could not be observed with any of 7 monoclonal antibodies recognizing ubiquitin. We conclude (1) that abnormal phosphorylation of tau occurs prior to its incorporation into PHF and leads to its accumulation in the nerve cell body and (2) that ubiquitin is seen associated only when a neurofibrillary tangle is already formed.     

Alzheimer's neurofibrillary tangles and paired helical filaments in the pheochromocytoma cells of the adrenal medulla —Electron microscopic and immunoelectron microscopic observations

Summary Alzheimer's neurofibrillary tangles (NFT) and paired helical filaments (PHF) were found in the pheochromocytoma cells of the adrenal gland removed from a 54-year-old female. By electron microscopy they were identical to those found in the brains affected by dementia of Alzheimer type. In the tumor cells, most of the PHF were found dispersed loosely in the cytoplasm, while typical NFT were infrequent. By immunoelectron microscopy using peroxidase-antiperoxidase method, both NFT and dispersed PHF were stained positively with a polyclonal antiserum to human tau protein. This is the first observation of NFT and PHF in paraneuronal tumor cells. The patient has no obvious Alzheimer's disease.     

阿尔茨海默病患者脑中双螺旋丝的体外去磷酸化作用

目的探讨阿尔茨海默病（ＡＤ）脑损伤逆转的可能性及其途径。方法用去磷酸化和免疫印迹法研究ＡＤ脑损伤可逆性。结果蛋白磷酸酯酶（ＰＰ）２Ａ和ＰＰ２Ｂ可使ＡＤ神经原纤维缠结中的Ｉ型双螺旋丝（ＰＨＦＩｔａｕ）在Ｓｅｒ１９９／Ｓｅｒ２０２去磷酸化，Ｓｅｒ３９６／Ｓｅｒ４０４部分去磷酸化；此外，ＰＰ２Ａ和ＰＰ２Ｂ可分别使ＰＨＦＩＩｔａｕ的Ｓｅｒ４６和Ｓｅｒ２３５去磷酸化；去磷酸化后ＰＨＦＩＩｔａｕ的相对电泳迁移率加快。Ｍｎ２＋和Ｍｇ２＋可增加上述酶对ＰＨＦＩＩｔａｕ的去磷酸化作用。酶的去磷酸化作用具有浓度依赖性，随着酶浓度的增加，去磷酸化作用增强。结论因为ｔａｕ蛋白异常过度磷酸化并形成ＰＨＦ被认为是ＡＤ神经原纤维退化的基础，ＰＨＦ可在体外被蛋白磷酸酯酶去磷酸化的结果提示，ＡＤ脑损伤可能是可逆的。     

Scanning electron microscopical study of neurofibrillary tangles in a presenile patient with Down’s syndrome

An autopsy case of a 64-year old woman with Down’s syndrome (DS) is reported with a special reference to the ultrastructure of neurofibrillary tangles (NFTs). NFTs and senile plaques were diffusely observed throughout the brain. The most severe changes were seen in the amygdaloid nuclear complex and hippocampus. Immunohistochemistry of the NFTs and senile plaques indicated the features identical to those in Alzheimer’s disease (AD). Ultrastructurally, NFTs were composed of straight filaments and two profiles of paired helical filaments (PHFs). By transmission electron microscopy, straight filaments measured 25–28 nm in diameter. As to the PHFs, one type was 33 nm in maximum diameter and constricted at a 75– to 80-nm interval. The other was 16–18 nm in maximum diameter and constricted at a 35– to 40-nm interval. By scanning electron microscopy, the diameter of the straight filaments measured up to 28–30 nm. Two profiles of PHFs were observed. One type of PHF showed thick filaments about 34 nm in maximum diameter and constrictions at an 80-nm interval. The other was about 17 nm in diameter and constricted at a 40-nm interval. The helical directions of both PHFs were left-handed. The frequency of PHFs with short interval was much higher in DS than AD. Furthermore, the length of the periodicity of this type of PHF was somewhat less than that of AD. Thus, these findings suggest that the neuropathological changes in DS and AD share a common etiopathology, but that some differences in the PHFs between DS and AD may reflect on molecular difference in the proteins or peptides associated with PHF formation. Received: 17 November 1997 / Revised, accepted: 29 January 1998     

Alzheimer's paired helical filaments: Amyloid precursor protein epitope mapping

Paired helical filaments (PHF) were electro-phoretically purified and solubilized from Alzheimer's neurofibrillary tangles and consisted of a primary 66 kDa protein on SDS-PAGE analysis. A panel of antibodies raised against restricted regions of the beta-amyloid precursor protein (APP) were employed for epitope mapping studies of this 66 kDa PHF protein. Western blot studies revealed that C-terminal APP antibodies were immunoreactive with the 66 kDa PHF protein. Further analysis revealed that only antisera raised against peptides that include the beta/A4-amyloid region within the C-terminal portion of APP were immunoreactive with PHF proteins. These data complement previous immunocytochemical studies which indicated that C-terminal APP antibodies preferentially label PHF-containing neurofibrillary tangles in Alzheimer's brain. The present data suggest a similarity of secondary or tertiary structure between beta/A4-amyloid and PHF which accounts for the cross-reactivity of beta/A4-amyloid antibodies with PHF proteins.