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

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 PHF 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 neurofilaments stained neurofibrillary tangles. (ii) 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 polyclonal 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 neurofilament 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 neurofilament 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 PHF, not neurofilament proteins, highlighting the problem of using antibodies to phosphorylated protein epitopes in immunochemical studies. Independent evidence for the presence of neurofilament proteins in human paired helical filaments is now required.     

Measurement of distinct immunochemical presentations of tau protein in Alzheimer disease.

The tau protein is a microtubule-associated protein that is normally located in nerve axons. In Alzheimer disease, it is a constituent of paired helical filaments (PHFs), which are the principal fibrous component of the characteristic neurofibrillary tangles. The tau protein, therefore, is abnormally sequestered in an insoluble form in PHFs in the cell body and dendrites in Alzheimer disease. We have used two monoclonal antibodies (mAbs) to selectively measure the levels of normal, soluble tau protein and of PHF-associated tau protein in the brain. mAb 423 binds to PHFs and recognizes a 12-kDa fragment of tau protein released by formic acid treatment of PHFs, but it does not recognize normal tau protein. In contrast, mAb 7.51 recognizes normal tau protein as well as the PHF core-derived tau fragment, but its epitope is concealed in the PHF-bound form. The differential binding properties for these two mAbs have enabled us in this study to quantify insoluble PHF-associated tau protein in the somatodendritic compartment as well as normal soluble tau protein in its predominantly axonal location. Our findings demonstrate that a distinct immunochemical presentation of tau protein recognized by mAb 423, a PHF-specific marker, can be used to quantify neurofibrillary pathology in Alzheimer disease independently of the presence of normal tau proteins.     

Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology.

A monoclonal antibody to the microtubule-associated protein tau (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical filaments (PHF), in Alzheimer disease brain. The antibody also labeled isolated PHF that had been repeatedly washed with NaDodSO4. Dephosphorylation of the tissue sections with alkaline phosphatase prior to immunolabeling dramatically increased the number of tangles and plaques recognized by the antibody. The plaque core amyloid was not stained in either dephosphorylated or nondephosphorylated tissue sections. On immunoblots PHF polypeptides were labeled readily only when dephosphorylated. In contrast, a commercially available monoclonal antibody to a phosphorylated epitope of neurofilaments that labeled the tangles and the plaque neurites in tissue did not label any PHF polypeptides on immunoblots. The PHF polypeptides, labeled with the monoclonal antibody to tau, electrophoresed with those polypeptides recognized by antibodies to isolated PHF. The antibody to tau-labeled microtubules from normal human brains assembled in vitro but identically treated Alzheimer brain preparations had to be dephosphorylated to be completely recognized by this antibody. These findings suggest that tau in Alzheimer brain is an abnormally phosphorylated protein component of PHF.     

Senile plaque neurites in Alzheimer disease accumulate amyloid precursor protein.

Senile plaques are polymorphous beta-amyloid protein deposits found in the brain in Alzheimer disease and normal aging. This beta-amyloid protein is derived from a larger precursor molecule of which neurons are the principal producers in brain. We found that amyloid precursor protein (APP)-immunoreactive neurites were involved in senile plaques and that only a subset of these neurites showed markers for the abnormal filaments characteristic of neurofibrillary pathology. In the neocortex of nondemented individuals with senile plaques but spared of neurofibrillary pathology, dystrophic neurites in senile plaques showed only APP accumulation. In contrast, in the brains of Alzheimer patients, virtually all APP-immunoreactive neurites also showed immunoreactivity with ubiquitin, tau, and phosphorylated neurofilaments. The presence of tau and neurofilament epitopes in dystrophic neurites in senile plaques was correlated with the extent of neurofibrillary pathology in the surrounding brain tissue. Accumulation of APP and the formation of neurofibrillary pathology in senile plaque neurites are therefore distinct phenomena. Our findings suggest that APP accumulation in senile plaque neurites occurs prior to tau accumulation and is therefore more closely related to appearance of neuritic dystrophy.     

Altered phosphorylation of tau protein in heat-shocked rats and patients with Alzheimer disease.

Six hours after heat shocking 2- to 3-month-old male and female Sprague-Dawley rats at 42 degrees C for 15 min, we analyzed tau protein immunoreactivity in SDS extracts of cerebrums and peripheral nerves by using immunoblot analysis and immunohistochemistry with the anti-tau monoclonal antibody Tau-1, which recognizes a phosphate-dependent non-phosphorylated epitope, and with 125I-labeled protein A. In the cerebral extracts, we found altered phosphorylation of tau in heat-shocked females, characterized by a marked reduction in the amount of nonphosphorylated tau, a doubling of the ratio of total (phosphorylated plus nonphosphorylated) tau to nonphosphorylated tau, and the appearance of the slowest moving phosphorylated tau polypeptide (68 kDa). Similar, but milder, changes were observed in male rats. These changes progressively increased in females from 3 to 6 h after heat shocking. In contrast, both phosphorylated tau and nonphosphorylated tau were reduced in peripheral nerves after heat shocking. In immunoblots of SDS extracts from Alzheimer disease-affected brain, the two slowest moving phosphorylated tau polypeptides (62 kDa and 66 kDa, respectively) were detected by Tau-1 after dephosphorylation and by Tau-2 (an anti-tau-monoclonal antibody that recognizes a phosphate-independent epitope) without prior dephosphorylation only in regions that contained tau immunoreactivity in histologic preparations. In addition, quantitative immunoblot analysis of cortex and the underlying white matter with Tau-1 and 125I-labeled protein A showed that the amount of phosphorylated tau progressively increased in the Alzheimer disease-affected cerebral cortex, while concurrently a proportionally lesser amount of tau entered the white matter axons. The similar findings for the rat heat-shock model and Alzheimer disease suggest that life stressors may play a role in the etiopathogenesis of Alzheimer disease.     

Aberrant neurofilament phosphorylation in Alzheimer disease.

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 neurofilaments. It is suggested that the Alzheimer lesion involves an imbalance within specific kinases responsible for phosphorylation of different sites in neurofilaments.     

Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease.

The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death.     

Identification of the major multiphosphorylation site in mammalian neurofilaments.

The sequence Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly repeats six times serially in the human midsized neurofilament (NF) protein (NF-M). To establish whether Lys-Ser-Pro-Val(Ala) is the major site for in vivo NF phosphorylation, peptides based on the human NF-M repeat were synthesized and chemically phosphorylated. These synthetic peptides were probed with 515 monoclonal antibodies (mAbs) that were raised to, and distinguished, several differentially phosphorylated forms of NF proteins. Studies with 95 of those mAbs that recognized the peptides before and after chemical phosphorylation demonstrated that a highly immunogenic epitope shared by the peptides is present in NFs from all species tested, including invertebrates. This suggests the phylogenetic conservation of a major NF phosphorylation site. Lastly, a cross-reactive antigenic determinant shared by the peptides and the major NF phosphorylation site was shown to exist in neurofibrillary tangles of patients with Alzheimer disease as well as in two neuron-specific microtubule-associated proteins (MAPs)--i.e., MAP2 and tau.     

Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains

Neurofibrillary tangles (NFT) and neurites associated with senile plaques (SP) in Alzheimer disease-affected brain tissues were specifically immunostained with affinity-purified antibody preparations directed against ubiquitin. In addition, a class of neurites seen in brain regions containing NFT and SP were also specifically stained. Cross-reactivity of the ubiquitin antisera for tau protein, neurofilament proteins, and high molecular weight microtubule-associated proteins (MAPs) were ruled out by (i) the inability of the ubiquitin antisera to stain these proteins in immunoblotting experiments and (ii) the inability of tau, neurofilament, and MAP preparations, when preincubated with the ubiquitin antisera, to inhibit the selective neurofibrillar staining observed. Our results are consistent with the suggestion that ubiquitin is covalently associated with the insoluble neurofibrillary material of NFT and SP. We propose that the ubiquitin-mediated degradative pathway may be ineffective in removing these fibrillar structures in Alzheimer disease brain.     

Monoclonal antibodies to human thyroglobulin as probes for thyroglobulin structure

A panel of monoclonal antibodies (mAbs) against human thyroglobulin (hTg) was obtained by somatic fusion of the nonsecreting myeloma cell line P3X66 Ag8/0 and spleen cells of Balb/c mice immunized with purified hTg. Antibody secreting clones were selected by solid phase enzyme immunoassay and analyzed for cross-reaction with Tg from several animal species. Twelve out of 15 mAbs cross-reacted with both rat and mouse Tg and 11 Mabs cross-reacted with bovine Tg. The cross-reaction with mouse Tg paralleled that of rat Tg, whereas discrete differences between the cross-reactivity patterns with bovine Tg were observed. Two clones secreted mAbs specific for hTg. We further characterized the mAbs and found that three mAbs recognized T4-containing determinants and one mAb reacted with both T4 and T3-containing determinants on the Tg molecule. The binding of the mAbs to hormonogenic determinants depended upon the thyroid hormone content of the molecule and the integrity of the three dimensional structure of Tg. One other mAb reacted with four peptides of CNBr-cleaved hTg, indicating the recognition of a repetitive determinant in the hTg molecule distinct from the hormonogenic regions.     

Apolipoprotein E fragments present in Alzheimer''s disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons

Human apolipoprotein (apo) E4, a major risk factor for Alzheimer's disease (AD), occurs in amyloid plaques and neurofibrillary tangles (NFTs) in AD brains; however, its role in the pathogenesis of these lesions is unclear. Here we demonstrate that carboxyl-terminal-truncated forms of apoE, which occur in AD brains and cultured neurons, induce intracellular NFT-like inclusions in neurons. These cytosolic inclusions were composed of phosphorylated tau, phosphorylated neurofilaments of high molecular weight, and truncated apoE. Truncated apoE4, especially apoE4(Delta 272--299), induced inclusions in up to 75% of transfected neuronal cells, but not in transfected nonneuronal cells. ApoE4 was more susceptible to truncation than apoE3 and resulted in much greater intracellular inclusion formation. These results suggest that apoE4 preferentially undergoes intracellular processing, creating a bioactive fragment that interacts with cytoskeletal components and induces NFT-like inclusions containing phosphorylated tau and phosphorylated neurofilaments of high molecular weight in neurons.     

Paired helical filaments from Alzheimer disease patients contain cytoskeletal components.

Neurofibrillary tangles from Alzheimer disease patients share antigenic determinants with neurofilaments and microtubule-associated proteins, as shown by light microscopy immunocytology. The present study addresses the issue of whether these determinants are located on the paired helical filaments or on other components of the neurofibrillary tangle. Sections from postmortem brains from Alzheimer disease patients were stained by using Bodian's silver method or immunostained by using poly- and monoclonal antibodies to neurofilaments and polyclonal antibodies to microtubules. Bodian's silver stain has an intense affinity for neurofibrillary tangles and has been shown to bind to specific domains of neurofilament subunits. The antibodies to neurofilaments used here immunostain most or all of the neurofibrillary tangles present in the sections whereas the antiserum to microtubule protein immunoreacted with about half of the neurofibrillary tangles. All of the antibodies as well as Bodian's silver stain reacted with the paired helical filaments. The epitopes that we have shown to be present in the paired helical filament, in contrast to the corresponding epitopes present in normal neuronal cytoskeleton, are insoluble in ionic detergent. It is concluded that these epitopes are integral components of the paired helical filaments and that, at least in part, paired helical filaments are derived from altered elements of the normal neuronal cytoskeleton.     

Protease inhibitors and indoleamines selectively inhibit cholinesterases in the histopathologic structures of Alzheimer disease.

Neurofibrillary tangles and amyloid plaques express acetylcholinesterase and butyrylcholinesterase activity in Alzheimer disease. We previously reported that traditional acetylcholinesterase inhibitors such as BW284C51, tacrine, and physostigmine were more potent inhibitors of the acetylcholinesterase in normal axons and cell bodies than of the acetylcholinesterase in plaques and tangles. We now report that the reverse pattern is seen with indoleamines (such as serotonin and its precursor 5-hydroxytryptophan), carboxypeptidase inhibitor, and the nonspecific protease inhibitor bacitracin. These substances are more potent inhibitors of the cholinesterases in plaques and tangles than of those in normal axons and cell bodies. These results show that the enzymatic properties of plaque and tangle-associated cholinesterases diverge from those of normal axons and cell bodies. The selective susceptibility to bacitracin and carboxypeptidase inhibitor indicates that the catalytic sites of plaque and tangle-bound cholinesterases are more closely associated with peptidase or protease-like properties than the catalytic sites of cholinesterases in normal axons and cell bodies. This shift in enzymatic affinity may lead to the abnormal protein processing that is thought to play a major role in the pathogenesis of Alzheimer disease. The availability of pharmacological and dietary means for altering brain indoleamines raises therapeutic possibilities for inhibiting the abnormal cholinesterase activity associated with Alzheimer disease.     

Abeta and tau form soluble complexes that may promote self aggregation of both into the insoluble forms observed in Alzheimer's disease

To date, there is no reasonable explanation as to why plaques and tangles simultaneously accumulate in Alzheimer's disease (AD). We demonstrate here by Western blotting and ELISA that a stable complex can form between tau and amyloid-beta protein (Abeta). This complex enhances tau phosphorylation by GSK3beta, but the phosphorylation then promotes dissociation of the complex. We have localized the sites of this interaction by using peptide membrane arrays. Abeta binds to multiple tau peptides, especially those in exons 7 and 9. This binding is sharply reduced or abolished by phosphorylation of specific serine and threonine residues. Conversely, tau binds to multiple Abeta peptides in the mid to C-terminal regions of Abeta. This binding is also significantly decreased by GSK3beta phosphorylation of tau. We used surface plasmon resonance to determine the binding affinity of Abeta for tau and found it to be in the low nanomolar range and almost 1,000-fold higher than tau for itself. In soluble extracts from AD and control brain tissue, we detected Abeta bound to tau in ELISAs. We also found by double immunostaining of AD brain tissue that phosphorylated tau and Abeta form separate insoluble complexes within the same neurons and their processes. We hypothesize that in AD, an initial step in the pathogenesis may be the intracellular binding of soluble Abeta to soluble nonphosphorylated tau, thus promoting tau phosphorylation and Abeta nucleation. Blocking the sites where Abeta initially binds to tau might arrest the simultaneous formation of plaques and tangles in AD.     

Tau protein and beta protein

The main pathological features of Alzheimer's disease are Alzheimer neurofibrillary tangles and senile plaques. Recent biochemical research revealed that tangles are composed of tau protein and ubiquitin and amyloid in senile plaques is composed of beta protein which is a fragment of the membrane receptor protein. Although fraction, other data suggest that whole molecules become abnormal and aggregate into PHF. On the other hand, beta protein is a small cleavage product of the precursor protein. It is not concluded yet whether abnormal precursor proteins exist or not. Recent research in this field is reviewed.     

Neurofibrillary tangles of Alzheimer disease share antigenic determinants with the axonal microtubule-associated protein tau (tau)

The relationship of the neurofibrillary tangle, found in Alzheimer disease and aged brains, to normal or abnormal cytoskeletal proteins remains elusive. Although immunohistochemical studies have yielded disparate results, most antigenic determinants localized to neurofibrillary tangles are cytoskeletal constituents normally present in neuronal perikarya or dendrites. We report light and electron microscopic immunolabeling of neurofibrillary tangles by a monoclonal antibody to the microtubule-associated protein tau (tau). Dephosphorylation of tissue slices not only increased the number of tau-positive tangles but also produced marked positive immunoreactivity of neuritic plaques. The localization of tau, an axonal protein, to neurofibrillary tangles in the perikaryon in particular suggests that abnormal synthesis, modification, or aggregation of tau may induce aberrant cytoskeletal--cell organelle interactions, subsequent interference with axonal flow, and resultant tangle formation.     

Activation of a neurofilament kinase, a tau kinase, and a tau phosphatase by decreased ATP levels in nerve growth factor-differentiated PC-12 cells.

Brain pathology in Alzheimer disease and in aged controls shows hyperphosphorylation of tau and of neurofilament proteins. Roder and Ingram [Roder, H.M. & Ingram, V.M. (1991) J. Neurosci. 11, 3325-3343 and Roder, H.M., Eden, P.A. & Ingram, V.M. (1993) Biochem. Biophys. Res. Commun. 193, 639-647] previously reported that the brain protein kinase PK40erk can hyperphosphorylate both tau and neurofilaments and interestingly, is strongly inhibited by ATP uncomplexed with Mg2+. We now report that the mitochondrial uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone decreases ATP levels in rat pheochromacytoma (PC-12) cells differentiated with nerve growth factor and activates a neurofilament kinase, a tau kinase, and, unexpectedly, a tau phosphatase--either PP1 or PP2A. Such aberrant modulation of protein phosphorylation patterns could be the common biochemical basis for senile dementia and for Alzheimer disease and could explain the late-onset etiology of both conditions.     

Isoform-specific interactions of apolipoprotein E with microtubule-associated protein tau: implications for Alzheimer disease.

The apolipoprotein E (apoE) type 4 allele (APOE4) is a susceptibility gene for late-onset familial and sporadic Alzheimer disease. ApoE is found in some neurofibrillary tangle-bearing neurons, one of the major pathologic hallmarks of the disease. Neurofibrillary tangles contain paired helical filaments formed from hyperphosphorylated microtubule-associated protein tau. In vitro, tau binds avidly to apoE3, but not to apoE4, forming a bimolecular complex. Tau phosphorylated with a brain extract does not bind either isoform. ApoE3 binds to the microtubule-binding repeat region of tau, which is also the region that is thought to cause self-assembly into the paired helical filament. Binding studies with fragments of ApoE demonstrate that the tau-binding region of apoE3 corresponds to its receptor-binding domain and is distinct from the region that binds lipoprotein particles or beta/A4 peptide. Isoform-specific interactions of apoE with tau may regulate intraneuronal tau metabolism in Alzheimer disease and alter the rate of formation of paired helical filaments and neurofibrillary tangles.     

Immunohistochemical investigation of the brain of aged dogs. I. Detection of neurofibrillary tangles and of 4-hydroxynonenal protein, an oxidative damage product, in senile plaques.

In the aging dog brain lesions develop spontaneously. They share some morphological characteristics with those of Alzheimer 's disease in man. Diffuse and primitive plaques are well known, whereas neuritic plaques rarely develop. Neurofibrillary tangles have not been seen in the canine. The aim of the present investigation was to study major age-related changes of the dog's brain using paraffin sections with respect to cross-immunoreactivity of tau, A beta protein and other immunoreactive components including hydroxynonenal protein, which is a marker for oxidative damage. The occurrence of neurofibrillary tangles and of the protein tau therein was studied in serial brain sections of two dogs with the Gallyas stain and by immunohistochemistry with three different antibodies against tau. Senile plaques were stained with a monoclonal anti-A beta (residues 8-17), polyclonal anti-apolipoprotein E and a monoclonal antibody against 4-hydroxynonenal (HNE). Amyloid deposits and controls were screened by Congo red staining viewed in fluorescent light, followed by polarized light for green birefringence. With the Gallyas stain and one of the antisera against tau, neurofibrillary tangles were revealed in a similar dispersed pattern, whereas the other antitau antisera gave negative results. With the anti-HNE a positive reaction was found in cerebral amyloid deposits and in vascular wall areas where amyloid deposition was confirmed by Congo-red staining, and in perivascular cells and in some neurons. These results indicate that the canine with his tangles and plaques which show oxidative changes, forms a spontaneous modelfor understanding the early changes and their interrelationships in Alzheimer's disease.     

Recognition of tau epitopes by anti-neurofilament antibodies that bind to Alzheimer neurofibrillary tangles.

Eleven anti-neurofilament (anti-NF) monoclonal antibodies were studied for their reactivity with heat-stable, microtubule-associated proteins and Alzheimer neurofibrillary tangles (ANT). On immunoblots of NF proteins, the antibodies recognized epitopes that were variably sensitive to Escherichia coli alkaline phosphatase. Eight of the antibodies showed reactivity with ANT and decreased binding to electroblotted NF after phosphatase treatment. The same eight antibodies reacted with tau proteins from bovine and rat brain, binding to tau proteins was also substantially reduced by phosphatase. Of the eight antibodies that bound to animal tau proteins, five also bound to tau proteins from normal human brain. All of the antibodies that bound to animal tau proteins stained ANT in frozen tissue sections. Brief treatment of tissue sections with trypsin in most cases enhanced antibody binding to ANT. All antibodies that lacked reactivity with tau proteins failed to bind ANT. Phosphatase treatment of Alzheimer tissue sections did not change the immunoreactivity of ANT and neurites in senile plaques with ANT-reactive, anti-NF antibodies, except for two antibodies that showed decreased binding to ANT. In contrast, axonal staining was decreased or eliminated by phosphatase treatment, similar to the response of electroblotted NF and tau proteins. These results suggest that staining of ANT by anti-NF antibodies may be due to cross-reaction of anti-NF with epitopes in tau proteins, the epitopes in axons, NF, and tau are sensitive to the effect of phosphatase, whereas the majority of those in ANT are not, and some of the epitopes in ANT that are shared with NF and tau proteins are not readily accessible to antibody binding.