Alzheimer paired helical filaments: Bulk isolation,solubility, and protein composition

Summary A method has been developed for the bulk isolation of Alzheimer neurofibrillary tangles (ANT) of paired helical filaments (PHF) from histopathologically confirmed cases of Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). The fresh or frozen autopsied cerebral cortex affected with Alzheimer neurofibrillary changes is dissociated by homogenization and sieving through nylon bolting cloth and the ANT are separated by a combination of sucrose discontinuous density gradient centrifugation, glass bead column chromatography, and sodium dodecyl sulfate (SDS) treatment. The isolated ANT produce red-green birefringence when viewed through polarized light after staining with Congo red. Ultrastructurally, the isolated PHF are well preserved and have the dimensions of the PHF seen in situ. Two major Populations of ANT which exist in different proportions in AD/SDAT brains are identified on the basis of their solubility in SDS. The ANT I and the ANT II are soluble and insoluble respectively on treatment with 2% SDS at room temperature for 5 min. Solubilization of the ANT II requires several repeated extractions with a solution containing 10% each of SDS and -mercaptoethanol (BME) at 100°C for 10 min. Sonication of the ANT II greatly facilitates their solubilization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated ANT reveals the presence of two major polypeptides with molecular weights (MW) of 62,000 and 57,000, several minor polypeptides with MW below 57,000, and a significant amount of material not entering the stacking and the resolving gels. Re-electrophoresis of polypeptides extracted from various areas of the resolving gel or of the material which does not enter the gel generates the same polypeptide profile as on the first gel, suggesting that the PHF material which does not enter the gel may result from the reaggregation of the polypeptides that enter the resolving gel. None of the polpeptides observed in the isolated PHF comigrate in the SDS-PAGE with any of the neurofilament polypeptides, tubulin, actin, or myosin.Supported in part by NIH grants NS 17487 and NS 18105     

Young adult-form of dementia with neurofibrillary changes and lewy bodies

Summary Alzheimer's neurofibrillary tangles, Lewy bodies and chromatolytic neurons were found in the brain at autopsy of a 28-year-old male with pyramidal and extrapyramidal signs, and severe dementia of 7-year duration prior to his death. Review of histological material showed generalized changes involving both cortical and subcortical structures. These changes were characterized by the presence of neurofibrillary tangles, Lewy bodies and chromatolytic neurons. Neuritic plaques were not found. There was also loss of neurons and gliosis in the prefrontal cortex, hippocampus, amygdaloid nucleus, basal ganglia, midbrain and pons. There were spongiform changes due to loss of neurons. Myelin stain showed pallor of myelin in long tracts and in subcortical regions. The neurofibrillary tangles were mostly composed of Alzheimer's paired helical filaments (PHF). PHF were immunostained with both polyclonal and monoclonal antibodies to PHF and the microtubule-associated protein tau. Some Lewy bodies were immunolabelled with monoclonal antibodies to PHF. To the best of our knowledge it is the first reported case of a young adult-form of dementia with extensive formation of neurofibrillary changes and Lewy bodies.Supported in part by grants from the NIH NS18105 and POINS04220     

A progressive deposition of paired helical filaments (PHF) in the brain characterizes the evolution of dementia in Alzheimer's disease. An immunocytochemical study with a monoclonal antibody against the PHF core

Using the monoclonal antibody (mAb) 6.423 which recognizes epitopes of the pronase-resistant core of paired helical filaments (PHF), we studied postmortem frontal cortex from Alzheimer's disease (AD) patients with short (Group II) and long (Group III) histories of clinical dementia. Four cases with clinically unconfirmed dementia and a postmortem diagnosis of AD (Group I) were also studied. In Group I, the 6,423 mAb was negative whereas in Group II, the antibody recognized primarily neurofibrillary tangles (NFT). In contrast, brains in Group III contained a dense network of 6,423-immunoreactive (IR) thread-like structures ("ghost" neurites) and plaque-like structures with granular appearance, in addition to NFT. The number of 6,423-IR structures appeared to be related to the duration of clinical dementia and the age of onset. Furthermore, "ghost" neurites were more abundant in young AD cases. The possible significance of the 6,423-IR pattern in the pathogenesis of AD is discussed.     

Tau immunoreactivity and SDS solubility of two populations of paired helical filaments that differ in morphology

To further understand the processes that lead to the formation of neurofibrillary tangles from paired helical filaments (PHF) in Alzheimer brains, we studied two morphologically distinct fractions of PHF separated on sucrose density gradient. In a fraction with mostly short and non-aggregated PHF, the majority of filaments could be solubilized in SDS. In a fraction containing primarily PHF aggregated into clusters or bundles, sometimes resembling neurofibrillary tangles, filaments were less soluble in SDS. Immunogold labelling with a panel of tau-immunoreactive antibodies demonstrated that N-terminal epitopes of tau were preserved in the short filaments, but were reduced or absent in aggregated filaments. In contrast, C-terminal epitopes were present in both fractions. Furthermore, the accessibility of the microtubule-binding domain to immunolabelling was markedly impaired in short and non-aggregated filaments compared to aggregated filaments. These results are consistent with proteolytic degradation of the N-terminal epitopes and preservation of the C-terminal epitopes and the microtubule-binding domain of tau in the aggregated filaments. Partial proteolysis may be involved in the generation of aggregated PHF in neurofibrillary tangles.     

Quantitative solubilization and analysis of insoluble paired helical filaments from alzheimer disease

In this study, we evaluate the ability of several solvents to solubilize insoluble paired helical filaments (PHF) of Alzheimer disease. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents of PHF. Using sequential categories of protein solvent to analyze insoluble PHF, only alkali or exhaustive proteolysis are effective in completely solubilizing PHF, while a variety of denaturants are ineffective. Alkali does not affect the phosphorylation state of PHF and complete dephosphorylation of PHF with hydrofluoric acid does not affect PHF solubility. These findings suggest that the ‘hyperphos-sphorylation’ of PHF proteins is not responsible for PHF insolubility. However the in vitro glycation of τ generates PHF that are insoluble in SDS and soluble in alkali. These findings suggest that protein crosslinks, including advanced glycation endproduct-derived crosslinks which were recently described in Alzheimer disease, play a major role in effecting PHF insolubility in vivo.     

Alzheimer''s disease: exposure of neurofilament immunoreactivity in SDS-insoluble paired helical filaments

In Alzheimer's disease, neurofibrillary tangles (NFT) react with both antibodies to paired helical filaments (PHF) and certain antibodies to neurofilaments, for example, monoclonal RT97 against the 200,000 mol. wt. neurofilament protein. After isolating NFT by extraction in sodium dodecyl sulfate (SDS), PHF antibodies remain reactive while RT97 weakly stains only a minority of the extracted tangles. We now show that isolated NFT initially unreactive with RT97 apparently develop exposed antigenic sites following incubation under denaturing conditions. Our results suggest that at least one neurofilament epitope is buried in the SDS-insoluble PHF fiber and that conformational changes in PHF proteins allow its exposures.     

Senile dementia of Alzheimer type: Astroglial reaction to extracellular neurofibrillary tangles in the hippocampus

Summary Two types of Alzheimer neurofibrillary tangles may be found in the hippocampus in senile dementia of the Alzheimer type. Besides classical flameshaped intraneuronal tangles, there are less compact tangles representing extracellular remnants of destroyed neurons with neurofibrillary change. Strong immunoreactivity for glial fibrillary acidic protein (GFA) was found in the second type of tangles, which was due to penetration of fine processes of fibrous astrocytes into bundles of paired helical filaments (PHF). PHF appear to be a strong stimulus for astrocytic reaction when they are not segregated from the neuropil by the neuronal cell membrane.     

Three-dimensional structure of Alzheimer's neurofibrillary tangles of the aged human brain revealed by the quick-freeze,deep-etch and replica method

Summary The three-dimensional structure of Alzheimer's neurofibrillary tangles in the pyramidal cells of the hippocampus and in the nerve cells of the parahippocampal gyrus was examined by the quick-freeze, deep-etch and replica method. The tangles consisted of either parallel bundles of or randomly arranged paired helical filaments (PHF), occupying the perikaryotic cytoplasm and extending to the dendritic processes. On the stereophotographs the PHF, measuring 28 to 36 nm in width, had two component filaments of 14 to 18 nm in diameter which were coiled anti-clockwise (left-handed) around each other with periodicity of 70 to 90 nm. The PHF in compact parallel bundles were cross-linked to each other with thin filaments, of about 6 nm in diameter, at relatively regular intervals. Randomly arranged PHF had no cross-bridges or side arms. Straight-type tangles of about 24 nm in diameter were rarely found in the dendritic processes. There were no discernible differences between the PHF of the patients with senile dementia of Alzheimer type and those of nondemented brains.     

Glycosylation of microtubule-associated protein tau in Alzheimer’s disease brain

In the neurofibrillary pathology of Alzheimer’s disease (AD), neurofibrillary tangles (NFTs) contain paired helical filaments (PHFs) as their major fibrous component. Abnormally hyperphosphorylated, microtubule-associated protein tau is the major protein subunit of PHFs. A recent in vitro study showed that PHF tangles from AD brains are highly glycosylated, whereas no glycan is detected in normal tau. Deglycosylation of PHF tangles converts them into bundles of straight filaments and restores their accessibility to microtubules. We showed that PHF tangles from AD brain tissue were associated with specific glycan molecules by double immunostaining with peroxidase and alkaline phosphatase labeling. Intracellular tangles and dystrophic neurites in a neuritic plaque with abnormally hyperphosphorylated tau, detected with the monoclonal antibodies AT-8 and anti-tau-2, were also positive with lectin Galanthus nivalis agglutinin (GNA) which recognizes both the N- and O-glycosidically linked saccharides. Colocalization was not seen in the extracellular tangles and amyloid deposition, suggesting that the glycosylation of tau might be associated with the early phase of insoluble NFT formation. Thus, although abnormal phosphorylation might promote aggregation of tau and inhibition of the assembly of microtubules, glycosylation mediated by a GNA-positive glycan appears to be responsible for the formation of the PHF structures in vivo. Received: 25 June 1998 / Revised: 3 November 1998 / Accepted: 20 November 1998     

Alzheimer paired helical filaments: Identification of polypeptides with monoclonal antibodies

Summary Paired helical filaments (PHF) were isolated from autopsied brain of cases of Alzheimer dementia, and their polypeptides were identified with monoclonal antibodies to PHF by Western blots. The PHF polypeptide profile consisted of several bands with a size difference of less than 5 kilodalton (kDa) between adjacent bands; the most prominent bands were in the 45–62 kDa region. These PHF polypeptides were also labeled with tangles reactive antisera to microtubules from normal brain but had electrophoretic patterns different from those of microtubules and neurofilaments. These studies define the molecular weight profile of PHF polypeptides and suggest that they might originate from normal brain proteins.Supported in part by NIH grants NS 18105, NS 17487 and P01 AG/NS 04220     

New patterns of intraneuronal accumulation of the microtubular binding domain of tau in granulovacuolar degeneration.

Sixteen brains from Alzheimer's disease (AD) patients with varying duration of dementia were studied using the monoclonal antibody (mAb) 6.423 raised against the three repeated domains of the tau protein, and named the paired helical filament (PHF) core. In Ammon's horns of the AD cases 6.423 mAb, in addition to immunoreacting with neurofibrillary tangles (NFTs), dystrophic neurites, and plaquelike structures, also recognized a subpopulation of granulovacuolar degeneration elements (GVD). A new immunoreactive structure, a spherical inclusion, was also stained by 6.423. The immunoreactive GVD elements and the spherical inclusion were found in the aged controls (greater than 65 years of age) and in non-AD dementia cases, as well. The staining of the GVD was markedly decreased when the tissue was preincubated with alkaline phosphatase. In contrast, NFTs and the spherical inclusions resisted dephosphorylation. Neurons containing the spherical inclusion frequently lacked immunoreactive intracellular NFTs. Due to the similar immunohistochemical properties between the spherical bodies and immunoreactive NFTs, we named this new inclusion PHF core body. Our results suggest that the PHF core body may represent a successful attempt by hippocampal neurons to restrict the PHF core expression. Thus, the failure of this mechanism may lead to the NFT formation in a range of dementing processes. Alternatively, the PHF core body may be an early stage in the NFT formation.     

Alzheimer paired helical filaments: Cross-reacting polypeptide/s normally present in brain

Summary Antisera to microtubule-enriched fraction fron normal human brain (anti-MT sera) label neurofibrillary tangles and neurites of neuritic (senile) plaques in brain sections of cases with Alzheimer disease/senile demintia of the Alzheimer type (AD/SDAT); the plaque core amyloid is not labeled. These anti-MT sera label both tangles in tissue sections and smears of isolated tangles which had been extracted with sodium dodecyl sulfate (SDS) to remove impurities trapped in between the paired helical filaments (PHF). The tangle labeling of anti-MT sera is eliminated on their absorption both with microtubule-enriched fractions from human and animal brain and with the isolated PHF. Neurofilament triplet, actin, myosin, keratin, or fibroblasts do not absorb the tangles staining antibodies. Furthermore, antisera containing antibodies to tubulin, microtubule-associated high mol. wt. polypeptides (MAPS), neurofilament triplet, and the 50,000 mol. wt. contaminant of CNS neurofilament preparations do not label tangles. On immunoblots of SDS-polyacrylamide gels of isolated PHF anti-MT sera label some of the same polypeptides identified with antisera to PHF; affinity-purified antibodies to tubulin used as a control do not label any PHF polypeptide on the immunoblots. The anti-MT sera, when preabsorbed with the PHF polypeptides eluted from SDS-polyacrylamide gels, do not label tangles. These studies demonstrate that a polypeptide/s cross-reactive with Alzheimer PHF is indeed normally present in brain and that it is different from tubulin, neurofilament triplet, actin, myosin, vimentin, and keratin.Parts of this paper were reported at the XIIth International Congress of Gerontology, Satellite Symposium, Heidelberg, Federal Republic of Germany, 1981; at the IXth International Congress of Neuropathology, Vienna, Austria, 1982; and at the 60th Annual Meeting of the American Association of Neuropathologists, San Diego, CA, USA, 1984Supported by NIH grants NS 18105 and NS 17487     

Tau,paired helical filaments and amyloid in the neocortex: a morphometric study of 15 cases with graded intellectual status in aging and senile dementia of Alzheimer type

Summary Tau immunoreactivity was studied in temporal neocortex, area 22, in 15 cases with graded intellectual status and compared with the immunoreactivity observed with an antiserum against paired helical filaments (PHF) and with the density of amyloid revealed by thioflavin S. Samples came from women over 75 years either intellectually normal or affected by senile dementia of the alzheimer type at various degrees of severity. Mental status had been prospectively assessed by the Blessed's test score. Antitau labelled a neuropil meshwork, the density of which increased with the severity of the disease. This meshwork was denser in layers II, III and V in the most affected cases. The number and the size of the taupositive fibers within the senile plaques increased with the intellectual deficit. Senile plaques were more numerous in layers II and III and neurofibrillary tangles in layers III and V whatever the staining technique: tau or PHF immunocytochemistry, and thioflavin S. The densities of senile plaques and of neurofibrillary tangles (NFT) were correlated with the severity of the disease whatever the staining method. The three methods revealed a systematically different number of changes. This systematic difference could greatly influence the neuropathological diagnosis. It could be the consequence of various factors: different sensitivities of the staining methods or changes in the antigenic and amyloid composition of the lesion according to the stage of the disease. In line with the last hypothesis, a higher proportion of amyloid-rich plaques was noted in the less affected cases, suggesting that tau and PHF epitopes appeared secondarily. Tau epitopes seemed to be present at least as early as PHF epitopes in the NFT. The pathological changes best linked to dementia were NFT revealed by tau antiserum.Supported by a fellowship from the European Community Commission (PD)     

Alzheimer paired helical filaments: Immunochemical identification of polypeptides

Summary Antisera to isolated Alzheimer neurofibrillary tangles (ANT) of paired helical filaments (PHF) were raised in rabbits. These anti-PHF sera immunolabeled both ANT in sections of Alzheimer hippocampus and ANT which were isolated and extracted with sodium dodecyl sulfate (SDS). The immunostaining of ANT in tissue sections was removed by absorption of the anti-PHF serum with small amounts of PHF and also with 40-fold the amount of a fraction prepared identically from normal brain; neurofilament and brain microtubule preparations used at the same concentration as the normal brain control fraction did not eliminate the tangle staining. Furthermore, the tangle staining was also not removed with glial filaments or actin and myosin filaments. No labeling of the neurofilaments of axons and cerebellar basket fibers by anti-PHF sera was observed in tissue sections from non-neurologic brain. On paper blots of SDS-polyacrylamide gels anti-PHF serum reacted with neither polypeptides of the normal brain control fraction nor major microtubule and neurofilament polypeptides. However, the immunoblots of PHF preparations with the anti-PHF serum revealed staining of several polypeptide bands in the 45,000–70,000 molecular weight (MW) region, material on top of the gel and diffuse staining of the high MW region. The tangles staining in tissue sections by the anti-PHF serum was abolished by its absorption with PHF polypeptides extracted from high and low molecular weight areas of SDS polyacrylamide gels but not with identically prepared neurofilament polypeptides. These results indicate that (1) the antigen(s) recognized by the anti-PHF serum is inherent to the PHF, (2) this PHF polypeptide(s) is at least partly soluble in SDS, and (3) this polypeptide(s) occurs in normal brain but is not associated with microtubules, neurofilaments, actin, or myosin.Parts of this paper have been reported at the 57th Annual Meeting of the American Association of Neuropathologists, 1981, Vancouver, Canada (Grundke-Iqbal et al. 1981) and at the IXth International Congress of Neuropathology, Vienna, 1982Supported by NIH grants NS 18105 and NS 17487     

A neuropathologic study of long-term, Economo-type postencephalitic parkinsonism with a prolonged clinical course

Abstract In this report, the neuropathologic features of five autopsied cases of postencephalitic parkinsonism of the Economo-type (PEPE) with a mean age of 66.6 years and a mean duration of the illness of 53.6 years are described. All five patients had presented with personality changes and severe parkinsonism. In addition, four patients had also had ocular symptoms. A pronounced chronic progression of the symptoms characterized all five cases. Active degenerating lesions were found in the substantia nigra (patients 3, 4 and 5) and the oculomotor nucleus (patient 5) which might explain the clinical observation of chronic active disease in these patients. We found that the intraneuronal neurofibrillary tangles (NFT) were immunoreactive to paired helical filaments (PHF), tau and ubiquitin; but ghost tangles demonstrated immunoreactivity only to glial fibrillary acid protein (GFAP). The ghost tangles consisted of dispersed bundles of abnormal tubules, and electron-dense glial filaments would surround and occasionally invade the ghost tangles. The present study suggests that NFT in PEPE are similar in their immunohistochemistry and ultrastructure to those observed in the case of Alzheimer-type dementia.     

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.     

Expression of ubiquitin-binding protein p62 in ubiquitin-immunoreactive intraneuronal inclusions in amyotrophic lateral sclerosis with dementia: analysis of five autopsy cases with broad clinicopathological spectrum

Amyotrophic lateral sclerosis with dementia (ALSD), corresponding to the motor neuron disease type of frontotemporal dementia, is neuropathologically characterized by depletion of the motor neurons, degeneration of the extra-motor cerebral cortices and formation of ubiquitin-immunoreactive (not argyrophilic, tau-negative, -synuclein-negative) intraneuronal inclusions. Recently, immunoreactivity for ubiquitin-binding protein p62 has been reported in several ubiquitin-containing intraneuronal or intraglial inclusions (e.g. neurofibrillary tangles, Pick bodies, Lewy bodies, glial cytoplasmic inclusions) in various neurodegenerative diseases. We examined p62 immunoreactivity in ubiquitin-immunoreactive intraneuronal inclusions in five ALSD cases with a broad clinicopathological spectrum. p62 immunoreactivity in ubiquitin-immunoreactive intraneuronal inclusions was seen in all cases. The mean proportion of p62-immunoreactive inclusions to the total number of ubiquitin-immunoreactive inclusions (p62/Ub ratio) in the dentate gyrus was 27.5±16.6% (range 6.3–47.3%). There was no correlation between p62/Ub ratio and the severity of dementia, duration of illness or neuropathological severity. Although the main constituent of these inclusions is unknown, our study suggests that p62 contributes to the formation of the inclusions via the same mechanism as in other previously reported neurodegenerative diseases. Since p62 is believed to have a neuroprotective role, the formation of these inclusions may represent a non-harmful, rather protective effect against the neuronal degeneration in ALSD.     

Neuritic pathology and dementia in Alzheimer's disease

Previous studies of Alzheimer's disease (AD) have correlated the severity of dementia with either the number of senile plaques or neurofibrillary tangles. We used antibodies raised against amyloid beta/A4 protein of senile plaque cores and tau protein as well as thioflavine S and the Campbell-Switzer modification of the Hicks silver method to examine the hippocampal formation and five neocortical regions from 22 nondemented elderly control subjects and 34 demented patients with cerebral senile plaques and neurofibrillary tangles, without complicating disease processes. Ten control subjects (46%) had no beta/A4 protein deposition. Twelve control subjects (54%) had widespread beta/A4 protein deposition but no neocortical neuritic pathology. Of the 34 patients with AD-type changes, 27 (79%) had widespread senile plaques and neurofibrillary tangles, while 7 (21%) had neocortical senile plaques with few neurofibrillary tangles. All demented patients had widespread beta/A4 protein deposition and neocortical tau-immunoreactive, Hicks silver-positive dystrophic neurites. The neurites were found both free in the neuropil as well as surrounding senile plaques. Quantitative analysis showed that dystrophic neurites were significantly increased in patients with AD compared with control subjects and the number of dystrophic neurites and neurofibrillary tangles correlated with the clinical severity of dementia. Widespread cerebral beta/A4 protein deposition may be necessary but by itself is insufficient for the development of dementia in AD.     

About the presence of paired helical filaments in dystrophic neurites participating in the plaque formation

Summary Immunocytochemistry with monoclonal antibodies to the -protein and to antigens associated with paired helical filaments (PHF) allows us to selectively stain two major components of neuritic (senile) plaques (NP): PHF and amyloid deposits. Using this method, the structure of NP in the brains of Alzheimer disease victims was compared to their structure in the brains of non-demented aged individuals selected for high numbers of NP. It is demonstrated that the dystrophic neurites participating in the plaque formation contain PHF only when cortical nerve cells in the same brain area form neurofibrillary tangles (NFT). People with many NP and many NFT were always demented, whereas people with many NP but few, if any NFT were not. It is speculated that there is individual susceptibility to the formation of PHF and that their appearance may represent a nonspecific response of the neuronal network to different kinds of injuries, like the deposition of amyloid in Alzheimer disease, or other pathogenic factors associated with various dementive neurodegenerative diseases. It is hypothesized that the deposition of brain amyloid in people resistant to neurofibrillary pathology may induced too little dysfunction for the development of dementia.Supported in part by grants AGO-4220 and HD-22634 from the National Institutes of Health     

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

The origin of the paired helical filaments (PHP) that accumulate in human neurons during aging and in Alzheimer's disease and their relationship to normal neurofilaments remain unclear. The observation that a rabbit antiserum to highly enriched PHF fractions specifically labeled PHF in Alzheimer neurofibrillary tangles but showed no reaction with neurofilaments or other normal cytoskeletal proteins led us to compare this antiserum to two monoclonal antibodies, RT97 and BF10, previously found to cross-react with tangles and with the 210, 000 and 155, 000 mol. wt. neurofilament proteins, respectively. Both α-PHF serum and the neurofilament monoclonals strongly immunolabel almost all neurofibrillary tangles in Alzheimer cortical sections. Double-immunolabeling studies show that both reagents recognize the same tangles and usually show identical patterns of staining of intraneuronal fibrous material. Following prolonged extraction of cortex in sodium dodecyl sulfate, a step which removes normal neurofilaments but leaves PHF intact, almost all isolated tangles retain strong immunoreactivity with α-PHF serum at an intensity which is slightly reduced from that in cortical sections. In contrast, only a small number of isolated tangles are stained strongly by RT97 and BF10; most show much decreased or no reactivity with these monoclonal neurofilament antibodies. This differential immunoreactivity was confirmed by double-labeling studies. Tangles prepared under gentle extraction conditions show strong reactivity with α-PHF antibodies but again only a small number are strongly labeled by RT97 and BF10. We conclude that neurofibrillary tangles in Alzheimer's disease are heterogeneous as regards their filamentous content and contain both antigens cross-reacting with neurofilaments and antigens which are apparently unique to PHF and not shared with normal neurofilaments.