Neurofibrillary changes in human brain. An immunocytochemical study with a neurofilament antiserum

Brain samples from cases of Alzheimer's disease, postencephalitic Parkinson's disease, progressive supranuclear palsy, amyotrophic lateral sclerosis, and Pick's disease, as well as from a case of Alzheimer's disease with a large number of Hirano bodies, were stained with the peroxidase-anti-peroxidase method using an antiserum previously shown to immunoreact with normal neurofilaments and neurofilament polypeptides. The specificity of this serum was confirmed by absorption an purified neurofilament proteins. Neurofibrillary tangles of Alzheimer's disease, postencephalitic Parkinson's disease, and progressive supranuclear palsy, Pick's bodies, and the fibrillary inclusions of amyotrophic lateral sclerosis were all immunostained. Hirano bodies showed no immunostaining. Thus, with the exception of the Hirano bodies, all the neuronal fibrillary inclusions examined appeared to share common antigenic characteristics. The orgin of all these structures from normal neurofilaments is postulated.     

Hirano body filaments contain actin and actin-associated proteins

Hirano bodies are eosinophilic, rod-shaped intraneuronal inclusions whose frequency increases with age and with Alzheimer's disease. To investigate their composition and possible relationship to the neuronal cytoskeleton, we employed immunocytochemistry and immunoelectronmicroscopy by using antisera to cytoskeletal proteins. The presence of actin, alpha-actinin, vinculin and tropomyosin was demonstrated diffusely throughout the Hirano body. The presence of these proteins supports the contention that Hirano bodies are derived from an abnormal organization of the neuronal cytoskeleton. The staining of Hirano bodies with fluorescent labelled phalloidin, a probe with a unique affinity for F-actin, indicates that the actin in Hirano bodies is in the F-state. Results of high voltage electron microscopy on 1.0 and 0.5 micron sections confirm the purely filamentous nature of Hirano bodies. These findings suggest that the mechanism of formation of Hirano bodies is different from that of the neurofibrillary tangle, another characteristic intraneuronal inclusion seen in Alzheimer patients.     

Neuropathology of Alzheimer's disease

In 1907, Alois Alzheimer described the presence of plaques and neurofibrillary tangles in a demented patient. Currently, Alzheimer's disease is known to be the most common cause of dementia in elderly patients. In this article, we summarize the most important neuropathologic features of Alzheimer's disease, including amyloid plaques, neurofibrillary tangles, neuronal loss, synaptic depletion, cerebral amyloid angiopathy, Hirano bodies, and granulovacuolar degeneration. We also review the history and application of Alzheimer's disease diagnostic criteria.     

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.     

Sequestration of Tubulin in Neurons in Alzheimer's disease

In Alzheimer's disease (AD), a variety of populations of neurons exhibits cytoskeletal abnormalities, including neurofibrillary tangles (NFT) in perikarya, Hirano bodies in dendrites and filament-distended axons/terminals/dendrites (neurites) in senile plaques. Some nerve cells, particularly pyramidal neurons of the hippocampus, also develop Hirano bodies (paracrystalline arrays of actin) and granulovacuolar degeneration (GVD; granular inclusions in cytoplasmic vacuoles). Since abnormalities of cytoskeletal elements have been implicated in the formation of NFT, neurites and Hirano bodies, the present study was designed to determine whether GVD also may represent a type of cytoskeletal pathology. Sections of hippocampus from controls and from individuals with AD were stained by immunocytochemical methods using monoclonal and polyclonal antibodies directed against a variety of cytoskeletal antigens. Granules of GVD contained tubulin-like immunoreactivity and absorption with purified tubulin abolished staining. Other antigens were not demonstrated in granules when antibodies directed against other cytoskeletal antigens were used. The observation of sequestration of tubulin in granules is consistent with the concept that abnormalities of the neuronal cytoskeleton are an important part of the cellular pathology of AD.     

Extrahypophyseal distribution of alpha-melanocyte stimulating hormone (alpha-MSH)-like immunoreactivity in postmortem brains from normal subjects and Alzheimer-type dementia patients

In Alzheimer's disease (AD), a variety of populations of neurons exhibits cytoskeletal abnormalities, including neurofibrillary tangles (NFT) in perikarya, Hirano bodies in dendrites and filament-distended axons/terminals/dendrites (neurites) in senile plaques. Some nerve cells, particularly pyramidal neurons of the hippocampus, also develop Hirano bodies (paracrystalline arrays of actin) and granulovacuolar degeneration (GVD; granular inclusions in cytoplasmic vacuoles). Since abnormalities of cytoskeletal elements have been implicated in the formation of NFT, neurites and Hirano bodies, the present study was designed to determine whether GVD also may represent a type of cytoskeletal pathology. Sections of hippocampus from controls and from individuals with AD were stained by immunocytochemical methods using monoclonal and polyclonal antibodies directed against a variety of cytoskeletal antigens. Granules of GVD contained tubulin-like immunoreactivity and absorption with purified tubulin abolished staining. Other antigens were not demonstrated in granules when antibodies directed against other cytoskeletal antigens were used. The observation of sequestration of tubulin in granules is consistent with the concept that abnormalities of the neuronal cytoskeleton are an important part of the cellular pathology of AD.     

The new neuropathology of degenerative frontotemporal dementias

The clinical features and recent developments in the neuropathology of frontotemporal dementia are reviewed. The five main neurodegenerative disorders that underlie the clinical syndrome of frontotemporal dementia are distinguished using immunohistochemistry with antisera to ubiquitin and tau proteins. Motor neuron disease-type dementia is characterised by ubiquitin-immunoreactive intraneuronal inclusions in cortical layer II and the hippocampal dentate granule cells. A diagnosis of Alzheimer's disease changes is based upon the presence of neurofibrillary tangles, which immunostain with antibodies to tau and ubiquitin, and many associated neuritic plaques. Corticobasal degeneration is diagnosed by the presence of tau-immunoreactive, but ubiquitin-non-reactive intraneuronal inclusions in cortical layer II and the substantia nigra. Pick's disease is restricted to cases with tau- and ubiquitin-immunoreactive spherical cortical intraneuronal inclusions (Pick bodies), best seen in the hippocampal dentate gyrus and frontotemporal cortex. Dementia of frontal type is the preferred term for cases in which no intraneuronal inclusions are seen with antisera to tau and ubiquitin. A practical approach to the pathological diagnosis of frontotemporal dementia and the differential diagnosis of the five disorders using immunohistochemical studies is provided. Received: 7 August 1995 / Revised, accepted: 10 October 1995     

The presence of FAC1 protein in Hirano bodies

We have previously reported that the FAC1 protein is contained in hippocampal structures that resemble Hirano bodies. Hirano bodies are cytoplasmic inclusions containing actin filaments that are numerous in the hippocampus of many Alzheimer's disease patients. FAC1 is a developmentally regulated protein that is localized to the cytoplasm of neurons during development and is predominately a nuclear protein in adult brain. In hippocampal sections from non-demented adults, Alzheimer's disease, and dementia with Lewy bodies patients, Hirano bodies were immunolabelled with antibodies to the FAC1 protein. Confocal laser microscopy demonstrated the presence of actin in FAC1 labelled Hirano bodies, and ultrastructural analysis confirmed the presence of a lattice structure within FAC1 labelled Hirano bodies. Numerous FAC1 immunoreactive swollen dendrites were also present in the hippocampus of Alzheimer's disease and dementia with Lewy bodies patients. Within any one case the total number of FAC1 positive swollen dendrites correlated with the total number of Hirano bodies, suggesting an association between the two structures. Thus, FAC1 protein is contained in Hirano bodies and swollen dendrites in the hippocampus of patients with Alzheimer's disease and dementia with Lewy bodies.     

RNA sequestration to pathological lesions of neurodegenerative diseases

Cytoplasmic RNA species have been identified recently within neurofibrillary tangles and senile plaques of Alzheimer’s disease brain. To determine whether RNA sequestration is a common feature of other lesions found in progressive neurodegenerative disorders, acridine orange histofluorescence was employed, alone or in combination with immunohistochemistry and thioflavine-S staining to identify RNA species in paraffin-embedded brain tissue sections. Postmortem samples came from 39 subjects with the following diagnoses: Alzheimer’s disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, corticobasal degeneration, diffuse Lewy body disease, normal controls, multiple system atrophy, Parkinson’s disease, Pick’s disease, progressive supranuclear palsy, and Shy-Drager syndrome. RNAs were detected in neurofibrillary tangles and neuritic senile plaques as well as in Pick bodies. However, Lewy bodies, Hirano bodies, and cytoplasmic glial inclusions did not contain abundant cytoplasmic RNA species. These observations demonstrate the selective localization of RNA species to distinct pathological lesions of neurodegenerative disease brains. Received: 15 April 1998 / Revised, accepted: 25 June 1998     

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.     

Atypical senile dementia with widespread Lewy type inclusion in the cerebral cortex

Summary An atypical case of senile dementia is reported which shows senile plaques, neurofibrillary tangles, granulovacuolar degeneration, and Hirano bodies and numerous Lewy bodies in the pigmented neurons of the brain stem, and Lewy-body-like inclusions in the cerebral cortex. The inclusions in the cerebral cortex had a strong resemblance to the Lewy bodies in the substantia nigra, though a few histochemical and ultrastructural differences were observed.The pathological findings were unexpected since the parkinsonian syndrome was not recognized.     

Topography of Pick inclusion bodies in hippocampi of demented patients. A quantitative study.

Topographic analysis was performed on the distribution of argyrophilic inclusions in hippocampal neurones of patients with Pick's dementia. A semiautomated scanning stage microscope linked potentiometrically to an XY pen recorder permitted the plotting of cytoarchitectonic "scattergrams" from the sequentially screened hippocampal formations. The density of Pick body-bearing nerve cells per cubic mm. of (pyramidal) cortex was quantified by measuring the area of each of six "zones" with a digitizer and programmable calculator. The ranking orders of relative severity showed that neurones in Rose's H1 field, the adjacent subiculum, and the entorhinal cortex are severely involved; that H2 and the presubiculum are less afflicted; and that the endplate is the least affected of all. The similarity of these predilections to those already demonstrated for Hirano bodies, granulovacuoles and neurofibrillary tangles in Alzheimer's disease suggests that a specific neurotransmitter defect may also underlie the dementia of Pick's disease.     

Quantitative neuropathologic analysis of Pick's disease cases: cortical distribution of Pick bodies and coexistence with Alzheimer's disease

Pick's disease is characterized morphologically by severe atrophy of the frontal and temporal lobes and the presence in the cerebral cortex of degenerative neuronal lesions referred to as Pick bodies. In the present study, we analyzed the regional and laminar distribution of Pick bodies in a series of 16 Pick's disease cases. These distribution and density patterns were compared with those observed for neurofibrillary tangles in Alzheimer's disease. Very high densities of Pick bodies were observed Ammon's horn, subiculum, entorhinal cortex, and in the granule cell layer of the dentate gyrus. In the frontal and temporal neocortex, they were preferentially distributed in layers II and VI. All of the Pick's disease cases also exhibited neurofibrillary tangles in the frontal and temporal areas and in the hippocampal formation, with higher densities in layers II–III than in layers V–VI of the neocortical regions. Interestingly, this laminar distribution of neurofibrillary tangles was strikingly different from that observed in Alzheimer's disease cases, where they were more numerous in the infragranular layers than in the supragranular layers. In addition, a few Pick's disease cases also had cortical senile plaques. These results suggest that the presence of neurofibrillary tangles in Pick's disease may be more frequent than previously reported, and that Pick's disease and Alzheimer's disease may coexist in certain cases. The lesion distribution patterns suggest that different populations of cortical neurons are affected in Pick's and Alzheimer's diseases, and that alterations of select corticocortical and corticosubcortical projections may distinguish these forms of dementia. It is also possible that these two disorders share certain pathogenetic mechanisms, even though both display specific patterns of regional and neuronal vulner-ability to the degenerative processes.Supported by the Brookdale Foundation, and NIH grants AG06647, AG05138 and AG08802     

Presenile dementia with Alzheimer-, Pick- and Lewy-body changes

An autopsy case of unclassifiable presenil dementia is reported. The outstanding pathological findings were as follows; 1. presence of senile plaques, neurofibrillary changes, Pick bodies, Hirano bodies, granulovacuolar degeneration of neurons, etc. 2. numerous Lewy bodies in the brain stem and diencephalon, 3. peculiar swollen neurons with intracytoplasmic, eosinophilic and argentophilic inclusions ("Lewy-like-bodies") in the cerebral cortices. Detailed study of the last mentioned inclusions indicates that they are almost identical to Lewy bodies, though there are some minor differences, in histochemical and electronmicroscopic findings. Nosologically, this case may represent either a combination of Alzheimer's disease, Pick's disease and idiopathic Parkinsonism with "Lewy-like-bodies" in the cerebral cortices, or a single disease. As far as we know, no similar case been reported.     

Atypical protein kinase C in neurodegenerative disease II: PKCiota/lambda in tauopathies and alpha-synucleinopathies

To study the role of atypical protein kinase C (aPKC) in neurodegenerative disease, we investigated the distribution of PKCiota/lambda, an aPKC isoform, in a variety of tauopathies and alpha-synucleinopathies. Immunohistochemical study revealed PKCiota/lambda within tau-positive neurofibrillary inclusions in Alzheimer disease (AD), progressive supranuclear palsy, corticobasal degeneration (CBD), and Pick disease (PiD), within alpha-synuclein-positive Lewy bodies in idiopathic Parkinson disease and dementia with Lewy bodies, as well as within glial inclusions in multisystem atrophy. We also observed PKCiota/lambda label of actin-rich Hirano bodies in AD, PiD, and elderly individuals. Double immunolabeling and fluorescence resonance energy transfer demonstrated close physical association between PKCiota/lambda and phospho-tau or alpha-synuclein in some neurofibrillary tangles and Lewy bodies. Furthermore, PKCiota/lambda colocalized with p62, a chaperone protein that binds to both aPKC and ubiquitin, in most of these inclusions. PKCiota/lambda also closely associated with the inactivated form of glycogen synthase kinase-3beta, GSK-3beta[ser9]. Together, these findings suggest that PKCiota/lambda may play a role in common mechanisms involving the pathogenesis of neurodegenerative disease.     

Neuronal and glial tau-positive inclusions in diverse neurologic diseases share common phosphorylation characteristics

Tau accumulating as paired helical filaments (PHF) in Alzheimer's disease brain is considered to be abnormally phosphorylated on distinct sites. To compare the phosphorylation state of tau-positive neuronal inclusions among diverse neurologic diseases, we have probed these lesions with three well-defined PHF/tau monoclonals, C5, M4 and tau 1, that most likely recognize three proline-directed phosphorylation sites in PHF-tau. In Alzheimer's disease brain all three monoclonals intensely immunostained intracellular neurofibrillary tangles, neuropil threads, senile plaque neurites, and pretangle neurons in a phosphorylation-dependent manner. They also stained, in the same manner, Pick bodies in Pick's disease, and neurofibrillary tangles and neuropil threads in various tangle-forming neurologic diseases. In most of these diseases (including Pick's disease, progressive supranuclear palsy, subacute sclerosing panencephalitis, and Alzheimer's disease) astrocytes and oligodendrocytes were found to contain tau-positive inclusions which showed the same immunocytochemical characteristics. Thus, the widely occurring tau-positive inclusions share common phosphorylation characteristics irrespective of underlying diseases or cell types.Supported by Grants-in-Aid for Specially Promoted Reseach (no. 03102008) and for Scientific Research on Priority Areas (no. 05261203 and 05251205) from the Ministry of Education, Science and Culture, and a Grant-in-Aid for Scientific Research from the Ministry of Health and Welfare, Japan     

Immunohistochemical localization of neurofilament protein in neuronal degenerations

Summary The immunohistochemical localization of human neurofilament proteins was studied in a variety of neuronal changes by the peroxidase-antiperoxidase method using antisera raised against each of the subunit proteins of human neurofilament. Torpedoes of the cerebellum, axonal spheroids of amyotrophic lateral sclerosis as well as of infantile neuroaxonal dystrophy, and neurofibrillary changes in a case of Pick's disease were consistently immunostained. Occasionally, a positive immunoreactivity was also observed in Lewy bodies, in neurofibrillary tangles of progressive supranuclear palsy, and in neuritic processes of senile plaques. Neurofibrillary tangles of Alzheimer type and Pick's bodies, however, did not react with the antisera. These data indicate that the human neurofilament doesnot share major antigenic determinants of its subunit protein with either Alzheimer's neurofibrillary tangles or Pick's bodies.     

Alpha-synuclein-positive structures in cases with sporadic Alzheimer's disease: morphology and its relationship to tau aggregation

Alzheimer's disease (AD) and Parkinson's disease share common clinical and pathological features. In this study, we examined the relationship between AD pathology and alpha-synuclein aggregation. The frequency and distribution of alpha-synuclein-positive structures were systematically investigated in 27 cases with sporadic AD by alpha-synuclein immuno-histochemistry. Thirteen (48.2%) of 27 cases had various alpha-synuclein-positive structures as well as Lewy bodies. The frequency and density of senile plaques and neurofibrillary tangles were not significantly different between cases with alpha-synuclein structures and those without. alpha-Synuclein-positive structures were found most frequently in the amygdala. The alpha-synuclein-positive inclusions that are different from Lewy bodies were observed at the highest rate in the hippocampus. The discovery of alpha-synuclein as the constituent of Lewy bodies facilitated the detection of Lewy-related structures even in AD cases with widespread and numerous neurofibrillary tangles. alpha-Synuclein-positive inclusions except for Lewy bodies are exposed, and the distribution of them indicates that Lewy body formation may be influenced by the degree of tau aggregation. This study also supports the suggestion that cases with AD pathology can be classified into two groups according to the existence or absence of alpha-synuclein aggregation.     

Association of selenoprotein p with Alzheimer's pathology in human cortex

Selenium is known for its antioxidant properties, making selenoproteins candidate molecules for mitigation of neurological disorders in which oxidative stress has been implicated. The selenium transport protein, selenoprotein P, is essential for neuronal survival and function. We sought to determine whether selenoprotein P expression is associated with Alzheimer's disease pathology. We examined postmortem tissue from individuals with the hallmark lesions of Alzheimer's disease and individuals without these lesions. Selenoprotein P immunoreactivity was co-localized with amyloid-beta plaques and neurofibrillary tangles. Dense-core and other non-diffuse amyloid-beta plaques were nearly always associated with selenoprotein P immunopositive cells. Analysis of spatial distribution showed a significant association between amyloid-beta plaques and selenoprotein P. Numerous cells also exhibited immunoreactivity to selenoprotein P and intraneuronal neurofibrillary tangles. Confocal microscopy confirmed co-localization of amyloid-beta protein and selenoprotein P. These findings suggest an association of selenoprotein P with Alzheimer's pathology.     

High molecular weight microtubule-associated proteins bind to actin lattices (Hirano bodies)

Summary Hirano bodies are filamentous, paracrystalline inclusions that are found in dendrites and cell bodies of neurons in Alzheimer's and other neurodegenerative diseases. Actin appears to be a major component of these structures. We present evidence that tropomyosin and high molecular weight microtubule-associated proteins (MAPs) are also components of Hirano bodies. Although an association betwen actin and MAPs has been noted in vitro, interactions in vivo have not heretofore been demonstrated. Since microtubules are not present in Hirano bodies, and anti-tubulin and anti-neurofilament antibodies do not bind to Hirano bodies, the association between MAPs and these inclusions is likely a result of interactions between MAPs and actin, and not MAPs and microtubules or neurofilaments.Supported in part by grants AG05386, The Alzheimer's Disease and Related Disorders Association, The John Douglas French Foundation, and The Wolf Memorial Fund