Autoantibodies to neurofibrillary tangles and brain tissue in Alzheimer's disease. Establishment of Epstein-Barr virus-transformed antibody-producing cell lines.

Multiple EBV-transformed B cell lines were established from five patients with a clinical diagnosis of Alzheimer's disease (AD) and six age-matched controls. The supernatants were screened for antibody activity against SDS-treated isolated neurofibrillary tangles (NFT). Reactive supernatants were identified from both the AD and control group. The frequencies of anti-NFT antibody-secreting lines were 6.3 and 1.6% for the AD and the control groups, respectively. A proportion of these supernatants also stained NFT in situ and neurons and/or glia in sections of the frontal and the temporal cortexes of autopsied AD and normal brains, as well as cells from three cell lines (HeLa, fibroblast, and neuroblastoma). Several patterns of staining were revealed by these supernatants, indicating different reactive antigens. One supernatant stained NFT and astrocytes in sections from AD brains. It did not stain sections from two normal brains. This cell line is the result of the immortalization of a circulating B cell making antibody specific for an antigen in AD. The present approach may provide new insights in the pathogenesis of AD.

A fibril-specific, conformation-dependent antibody recognizes a subset of Abeta plaques in Alzheimer disease, Down syndrome and Tg2576 transgenic mouse brain.

Beta-amyloid (Abeta) is thought to be a key contributor to the pathogenesis of Alzheimer disease (AD) in the general population and in adults with Down syndrome (DS). Different assembly states of Abeta have been identified that may be neurotoxic. Abeta oligomers can assemble into soluble prefibrillar oligomers, soluble fibrillar oligomers and insoluble fibrils. Using a novel antibody, OC, recognizing fibrils and soluble fibrillar oligomers, we characterized fibrillar Abeta deposits in AD and DS cases. We further compared human specimens to those obtained from the Tg2576 mouse model of AD. Our results show that accumulation of fibrillar immunoreactivity is significantly increased in AD relative to nondemented aged subjects and those with select cognitive impairments (p < 0.0001). Further, there was a significant correlation between the extent of frontal cortex fibrillar deposit accumulation and dementia severity (MMSE r = -0.72). In DS, we observe an early age of onset and age-dependent accumulation of fibrillar OC immunoreactivity with little pathology in similarly aged non-DS individuals. Tg2576 mice show fibrillar accumulation that can be detected as young as 6 months. Interestingly, fibril-specific immunoreactivity was observed in diffuse, thioflavine S-negative Abeta deposits in addition to more mature neuritic plaques. These results suggest that fibrillar deposits are associated with disease in both AD and in adults with DS and their distribution within early Abeta pathology associated with diffuse plaques and correlation with MMSE suggest that these deposits may not be as benign as previously thought.

The carboxyl third of tau is tightly bound to paired helical filaments.

To obtain definitive evidence that tau is a component of paired helical filaments (PHF) in Alzheimer's disease, we fractionated and sequenced PHF-derived peptides according to a previously described procedure. In the PHF digest, we found four independent tau peptides that were located in the carboxyl third of tau. Subsequent extensive analysis of the PHF digest did not provide any other tau peptides. The conventional PHF antiserum and a new antiserum directed toward formic acid-denatured PHF reacted with the distinct CNBr fragments of tau localized on the carboxy-terminal portion of tau by protein sequencing. From these observations, we conclude that the carboxyl third of tau is tightly bound to PHF.

Epitopes that span the tau molecule are shared with paired helical filaments.

Tau protein has been shown to be an integral component of Alzheimer paired helical filaments (PHF). However, the extent to which tau is incorporated into PHF has not been clear because the antibodies used to label PHF generally do not have precisely defined epitopes. Here we define the antigenic sites for five monoclonal antibodies that react with tau and cross-react with SDS-extracted neurofibrillary tangles. The reactive sites were determined by screening a lambda gt11 sublibrary expressing small fragments of the tau sequence. The mapped epitopes were found to span almost the entire length of tau, suggesting that PHF contains tau in its entirety or nearly in its entirety. One antibody was found to cross-react with microtubule-associated protein 2, implying some degree of homology between the two proteins.

Antigenic changes similar to those seen in neurofibrillary tangles are elicited by glutamate and Ca2+ influx in cultured hippocampal neurons.

In several neurological disorders including Alzheimer's disease, abnormal accumulations of cytoskeleton-associated proteins manifest as neurofibrillary tangles (NFTs) in vulnerable brain regions. Antibodies recognizing tau (5E2 and Alz-50) and ubiquitin epitopes in NFTs were used to examine the influence of glutamate and Ca2+ influx on antigen expression in cultured rat hippocampal neurons. Glutamate caused the degeneration of a subpopulation of pyramidal neurons, which exhibited increased immunostaining with all three antibodies. Subtoxic levels of glutamate also increased 5E2 and Alz-50 antigen levels in a subpopulation of neurons, particularly in the distal regions of the axons. Both glutamate-induced degeneration and increases in tau and ubiquitin immunostaining were prevented by removal of extracellular Ca2+. Increased immunostaining was also induced by Ca2+ ionophore A23187 or elevated levels of extracellular K+. The antigenic changes occurred within 1 hr of exposure to glutamate or A23187 and were not prevented by the protein synthesis inhibitor cycloheximide. These data indicate that Ca2+ influx caused by glutamate can lead to modifications of extant proteins similar to those seen in NFTs.

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 epitopes are incorporated into a range of lesions in Alzheimer's disease.

The neuronal microtubule-associated phosphoprotein, tau, has been identified as a major antigenic component of paired helical filaments in Alzheimer's disease (AD). The extent and distribution of altered tau antigens in AD brain, other than those found in neurofibrillary tangles (NFT) and senile plaque (SP) neurites, has not been widely discussed. We have examined tau immunoreactivity in AD using the monoclonal antibody (MAb), 5E2, raised against human fetal tau. Four types of abnormalities were recognized by MAb 5E2, each having some counterpart in Bielschowsky silver impregnations: 1) NFT; 2) thickened neurites in SP; 3) diffuse perikaryal staining seen in some neurons apparently lacking NFT; and 4) a dispersed network of randomly oriented thickened neurites not clustered into discrete plaques but found in NFT- and SP-rich cerebral cortex. These four alterations could also be recognized using three different polyclonal antibodies which had strong tau immunoreactivity but were optimally shown by MAb 5E2. Our findings demonstrate the complexity of altered tau-immunoreactive neuronal elements and emphasize the widespread abnormality of microtubule-associated proteins in AD cortex.

Alz-50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies.

Immunocytochemical and quantitative immunochemical techniques were used to study the expression of Alz-50 antigen, ubiquitin and Tau in neurologic disorders characterized by the formation of filamentous neuronal inclusions. Alz-50, anti-ubiquitin and Tau-1 immunostained the intraneuronal neurofibrillary tangles and the neuritic component of plaques, both in Alzheimer's disease and in the brains of patients without dementia, but extraneuronal tangles were largely unstained. These antibodies also reacted with Pick bodies, and with the neurofibrillary tangles of Kufs' disease and Guam Parkinsonism-dementia. In sections from the brain of a patient with progressive supranuclear palsy, virtually all of the tangles were immunostained with Tau-1 but only a few with Alz-50 or anti-ubiquitin. Anti-ubiquitin also labelled Lewy bodies and the inclusions of granulovacuolar degeneration. Quantitative analysis of immunoblots of homogenized frontal cortex showed significantly more Alz-50 antigen in the brains of patients with Alzheimer's and Pick's disease than in controls. The level of this antigen was increased both in the crude homogenates and in the cytosolic fraction. Ubiquitin immunoreactivity was increased only in the brains of patients with Alzheimer's disease and then only in the crude homogenates. The finding that antigenic determinants for Alz-50, anti-ubiquitin and Tau-1 are shared by several filamentous neuronal inclusions occurring in diverse neurologic disorders may reflect common metabolic defects underlying the formation of these inclusions, or common metabolic responses to their presence.

An antigenic profile of Lewy bodies: immunocytochemical indication for protein phosphorylation and ubiquitination.

An antigenic profile of subcortical and cortical Lewy bodies was determined in the presence or absence of neurofibrillary tangles in the same brain using antisera and monoclonal antibodies to various cytoskeletal elements as well as to determinants not present in the normal cytoskeleton. The cores of many Lewy bodies were strongly reactive with a monoclonal antibody to paired helical filaments which has been shown to recognize ubiquitin. This antibody also stained Marinesco bodies in the same tissue sections. Two monoclonal antibodies to phosphorylated epitopes of neurofilament proteins (SM I 31, SM I 34) stained the peripheries of about 40% of all discernable Lewy bodies on untreated paraffin sections. Reactivity with a monoclonal antibody to neurofilaments (SM I 33) appeared only after pretreatment of the sections with phosphatase. Lewy bodies did not bind antibodies to tau protein. Our results show that, as previously shown for neurofibrillary tangles, Lewy bodies also contain ubiquitin. The uncovering of neurofilament epitopes by treatment with phosphatase indicates that abnormal phosphorylation of cytoskeletal elements may play a role in the pathogenesis of the Lewy body.

Expression of immune system-associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer's disease.

HLA-DR is a class II major histocompatibility complex antigen which in the periphery confers antigen presenting capability. We have previously shown that this marker is profusely expressed in cortex of elderly and Alzheimer's disease (AD) patients, as is the receptor for the lymphokine interleukin-2. We now report presence of additional immune-related antigens in AD, and distributional differences from normal elderly controls. In gray matter, HLA-DR immunoreactivity is normally sparse, except in AD where it co-localizes with virtually all neuritic plaques. HLA-DR positive T cells can be demonstrated in Alzheimer's disease brain tissue, as can instances of apposition between putative brain microglia and T cells. In addition, cells with the morphologic characteristics of astrocytes label for natural killer cell antigen (Leu-11), and apparent lymphocytes bearing T helper and T cytotoxic/suppressor cell antigens are observed. These and other data suggest that the glial proliferation and scavenger activity characteristic of Alzheimer's disease may occur in an immune context and may play an important role in the pathogenesis of the disorder.

Antibodies to normal and Alzheimer human brain structures from non-immunised mice of various ages.

Supernatants from mouse spleen hybridoma lines established without previous immunisation were screened immunohistochemically against cryostat sections of human temporal cortex and found to stain a variety of brain structures, including Alzheimer plaques and tangles. The age of the mice had no effect on antibody production.

Preparation of antisera to neurofilament protein from chicken brain and human sciatic nerve.

Antigens isolated by hydroxyapatite chromatography from human sciatic nerve (SN1 protein) and from 8 M urea extracts of chicken brain were selectively localized by immunofluorescence to neurofibrils in rat and chicken CNS. Absorption of the antisera with SN1 protein, chicken antigen or GFA protein abolished the staining. Antisera raised against antigen isolated with the same procedure from buffer extracts of chicken brain stained both neurofibrils and glial fibrils by immunofluorescence. Neurofibrillary staining was selectively abolished by absorption of the antisera with SN1 protein. Antisera prepared against axonal preparations isolated from bovine white matter only stained astroglia and were thus undistinguishable from anti-GFA sera in this respect. The data suggested that the protein subunits of neurofilament and glial filaments, although difficult to separate in brain extracts by standard biochemical procedures and by subcellular fractionation in bovine white matter, still retain immunological specificity. In addition, the immunological cross reactivity between human and chicken antigens suggested that neurofilaments, as other constituents of the cytoskeleton such as microtubules and actin microfilaments, show a high degree of evolutionary stability.

Ganglioside monoclonal antibody (A2B5) labels Alzheimer's neurofibrillary tangles.

Ganglioside monoclonal antibody (A2B5) labels Alzheimer's neurofibrillary tangles both in isolated neurofibrillary tangle-bearing nerve cells and in partially purified preparations of tangle fibers. Antibody staining was preabsorbed by preincubation of antibody with neuronal ganglioside preparations. These results suggest that Alzheimer's neurofibrillary tangles have a ganglioside associated with them.

Serum antibodies to neurofilament antigens in patients with neurological and other diseases and in healthy controls.

A total of 529 sera from patients with a wide variety of neurological and non-neurological diseases, and 101 sera from healthy control subjects, were examined by indirect immunofluorescence for the presence of autoantibodies to neurofilament antigens. Antibodies were found in approximately 50% of sera from patients with spongiform encephalopathies, 15-30% of sera from patients with other neurological and non-neurological diseases, and 7% of sera from healthy controls. The antigenic stimulus to these autoantibodies in very diverse disease processes is unknown, but as presently assayed, they are not of sufficient specificity to be useful as an aid to clinical diagnosis.

Developmental expression of neurotypy revealed by immunocytochemistry with monoclonal antibodies.

Adult and developing rat cerebella were stained immunocytochemically with six neuron-specific monoclonal antibodies obtained from spleen cells of BALB/c mice immunized with hypothalamus. Monoclonal peroxidase-antiperoxidase complex was used in the staining procedure. In the adult, three different staining patterns were seen with these antibodies. The general patterns have been designated as (1) neurofibrillar, (2) perikaryal-neurofribillar, and (3) synapse-associated. In addition, each antibody within a designated group showed a different immunocytochemical distribution. Some antibodies reacted widely, for example, with many neuronal perikarya and fibers, and their distribution increased with the development of the brain. Other antibodies had a more restricted distribution and stained only some structures within an area or pathway. To account for this type of restriction of distribution, we propose that there may be microheterogeneity of some of the antigens visualized and have called this microheterogeneity 'neurotypy'. A second type of restriction was also observed. With several antibodies a loss of staining occurred in the adult cerebellum in structures that had reacted during early development. These differences in staining probably reflect developmental regulation of the antigens (neurotypes).

Immunocytologic analyses of 10 nm intermediate filaments in the nervous system of Myxicola.

Antibodies prepared in rabbits against Myxicola infundibulum neurofilaments have been employed to stain neurofilaments immunohistochemically in intact Myxicola infundibulum nervous tissue. Paraffin-embedded and frozen sections (5--6 mu) were examined at the light microscopic level with Sternberger's peroxidase-antiperoxidase method, and Vibratome (20--40 mu) sections were studied at the ultrastructural level with Nakane's conjugated peroxidase method. The neurofilament antibody stained only neurons and axons at the light microscopic level. The staining pattern at the electron microscopic level corresponded to the neurofilaments within axons and neurons. Glial cells, which surround the axons, contain large bundles of filaments that resemble astrocytic filaments in mammalian astrocytes. These filaments do not stain with the anti-neurofilament antibody. Neurons, neurofilaments, glial cells, glial filaments, and nonnervous tissue showed no peroxidase staining when specific antiserum absorbed with neurofilaments was used. These structures were also unstained when antiserum to the glial fibrillary acidic protein of mammalian central nervous system astrocytes was substituted for the neurofilament antiserum. Therefore, in Myxicola infundibulum, the antigenic determinants of the neurofilament protein, as recognized immunohistochemically by anti-neurofilament protein antibodies, are not shared with those of glial filaments.

Production and characterization of a monospecific antiserum (A128) to disaggregated Alzheimer paired helical filaments.

Paired helical filaments (PHF), which constitute neurofibrillary tangles (NFT) and neuritic plaque (NP) neurites, serve as a useful marker for Alzheimer disease (AD). We have isolated AD PHF in a highly purified and disaggregated form for use as an immunogen to produce a heterologous polyclonal antiserum in rabbits. One rabbit was maintained long-term for the high quality of the antiserum it produced. Through absorptions with normal brain tissue, we were able to produce a monospecific antiserum which reacts only with NFT and NP neurites in AD brain tissue sections. We further demonstrated the specificity of this antiserum by electron microscopic immunohistochemistry, gel diffusion analysis, and immunoblotting. This antiserum also showed immunoreactivity to NFT of Down syndrome and progressive supranuclear palsy, and to the Pick bodies of Pick disease, but not to the Lewy bodies of idiopathic Parkinson disease. This well-characterized antiserum, all from one rabbit, offers several unique advantages to the study of the nature, origin, and interrelationships of filamentous protein abnormalities in AD and other neurodegenerative disorders.

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells.

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.

Proteolytic fragments of Alzheimer's paired helical filaments.

We found that at least a part of Alzheimer's paired helical filaments (PHF) was cleaved by proteases to release three major polypeptides, the apparent molecular weights of which were 10K, 26K, and 36K daltons. These proteolytic fragments were strongly labeled with the antibodies specific to PHF. Absorption with highly purified PHF eliminated this labeling. The monospecific antibodies bound to the 10K daltons protein, the most intensely immunolabeled one, stained isolated neurofibrillary tangles composed of PHF. From these observations, we conclude that these polypeptides released by proteases, especially the 10K daltons protein, are derived from PHF themselves.

Monoclonal antibody binding to congophilic elements in human Alzheimer brain.

Immunohistochemical studies, using a monoclonal antibody, SMP, raised in a mouse against aged human peripheral blood white cells, are described. This antibody reacts with dendritic reticulum cells in lymph node and tonsil, and, in Alzheimer brain, with argyrophilic plaques, neurofibrillary tangles, and cerebral vascular amyloid.