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.     

Hirano bodies and related neuronal inclusions

Hirano bodies are bright eosinophilic intracytoplasmic inclusions which have a highly characteristic crystalloid fine structure. They occur preferentially in the neuronal processes of the CA1 area in Amnion's horn in a wide variety of conditions, especially amyotrophic lateral sclerosis and parkinsonism–dementia complex on Guam, Alzheimer's disease, Pick's disease and 'normal' elderly individuals. Hirano bodies contain epitopes of actin, actin–associated proteins, tau, middle molecular weight neurofilaments subunit and a C–terminal fragment of β-amyloid precursor protein. In addition to the CA1 area of Amnion's horn, they have also been identified in many other areas of the nervous system in humans and various experimental animals. Although usually observed in neurons. Hirano bodies may also be present in other cell types. It is the consensus that Hirano bodies in the pyramidal layer of CA1 originate largely from an age-related alteration of the microfilamentous system.     

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     

Accumulation of hippocampal cholinergic neurostimulating peptide (HCNP)-related components in Hirano bodies

We have previously isolated from the hippocampus of young rats a novel peptide termed 'hippocampal cholinergic neurostimulating peptide' (HCNP) which specifically enhances the cholinergic activity of the septohippocampal system in vitro . Cloning and base sequence analysis of HCNP-specific cDNA from rat and human cDNA libraries revealed that the 1.1 kDa peptide aligns at the N-terminal region of its 21 kDa precursor protein. An affinity-purified rabbit antibody to rat HCNP prepared by us recognizes the C-terminal domain of the peptide. while an antibody against human HCNP binds to a large portion of the peptide. In this report we show that both antibodies react with HCNP-related components present in the soluble cytosol fraction of human brain tissue.
Immunohistochemical examination of human nervous system tissues from elderly individuals revealed that Hirano bodies in Sommer's sector of the hippocampus were specifically stained by anti-HCNP antibodies. The number of HCNP-positive Hirano bodies was greater in patients with Alzheimer's disease than in normal, age-matched individuals. The immunohistochemical results were substantiated by immunoelectron microscopy. The present findings indicate that HCNP-related components accumulate in Hirano bodies, suggesting that patients who bear these inclusions may have a disturbance of the septohippocampal cholinergic system, considered to be of importance for learning and memory formation.     

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.     

Hirano bodies contain tau protein

Hirano bodies are intraneuronal inclusions whose frequency increases with age and Alzheimer's disease. These paracrystalline inclusions have been shown previously using immunocytochemistry to share epitopes with actin, tropomyosin, alpha-actinin and vinculin. Hirano bodies have not previously been demonstrated to share components with neurofibrillary tangles, another intraneuronal inclusion characteristic of Alzheimer's disease. In this study, we show that Hirano bodies bind antibodies to the microtubule-associated protein tau, a component of Alzheimer neurofibrillary tangles.     

New approaches to the study of human dystrophic muscle cells in culture

Tissue culture provides a system for studying the growth and differentiation of muscle cells in a controlled environment. Several studies have been carried out on diseased muscle cells in culture in attempts to elucidate the aetiology of Duchenne muscular dystrophy (DMD) but the results were equivocal. Work in our laboratory in recent years has yielded an improved method for preparing primary muscle cell cultures from dissociated biopsies which permits the morphological and biochemical evaluation of these cultures at all stages of growth and development. Our results have shown abnormalities in cell behaviour, ultrastructure and creatine kinase synthesis. The background to these studies is reviewed. Recently we have developed a cell cloning procedure that allows the accumulation of a large number of cells from a single selected cell. We can with this technique monitor quantitative and qualitative cellular and cytochemical differences between individual cell types without the ambiguities inherent in the use of mixed cell populations. The results obtained with 4 different clonal preparations derived from dystrophic muscle have shown that a number of specific features were expressed by each of the 4 clones with respect to their growth pattern, ultrastructure, synthesis of muscle specific protein and cell surface antigen. These findings clearly illustrate the potential of these cloning procedures for studying the genetic expression of homogeneous cell populations derived from normal adult human muscle and patients with X-linked muscle disease.     

The structure analysis of Hirano bodies by digital processing on electron micrographs

Summary To clarify the structure of Hirano bodies, electron micrographs of Hirano bodies taken at various tilting angles have been studied by digital image analysis. On the electron micrographs, the beaded filaments of Hirano bodies were turned into a pattern of lattice-like arrays by changing the tilting angles. Based on computer-procesed diffraction patterns and filtered images, it is proposed that the filaments of Hirano bodies are helical strands with a pitch of 185 Å. A model for the helical strand drawn by microcomputer at various angles of rotation is in accordance with the filtered images of the tilted filaments. Computer simulation also reveals that the helical strands appear to be lattice-like when they are arranged in parallel.Supported by a grant from Ministry of Education, Science and Culture, Japan (No. 59770568)     

Ultrastructural alterations of neuronal cells in a brain stem ganglioglioma

Summary A brain stem ganglioglioma in a 9-year-old female was examined ultrastructurally. The constituent neuronal (ganglion) cells displayed various ultrastructural features of neuronal degeneration including Hirano, Lafora and zebra bodies, inclusion-like aggregates of neurofilaments and large dilatations of rough endoplasmic reticulum. Although similar observations have been reported in peripheral neuronal tumors, this is the first reported occurrence in ganglioglioma, an uncommon tumor in the central nervous system. The coincidence of these alterations in the present tumor appeared to be of great interest, however, their exact etiology remained uncertain.     

Progressive multifocal leukoencephalopathy with paramyxovirus-like structures,Hirano bodies and neurofibrillary tangles

Summary We have identified three structures that have not previously been reported in electron microscopic examinations of brain from patients with PML: 1. coiled nuclear bodies that may possibly represent tubular paramyxovirus nucleocapsids, 2. Hirano bodies and 3. Neurofibrillary tangles.The paramyxovirus-like material is interesting in view of reports of a transition between nuclear bodies and tubular paramyxovirus nucleocapsids and because of a possible interaction between syncytiogenic paramyxoviruses and Papova viruses in the pathogenesis of SSPE and PML. A study of the Hirano bodies in this case, taken in conjunction with the findings of others leads us to believe that these structures are formed from an overgrowth of neurofilaments. The presence of tangled masses of neurofilaments in the processes of neurons also seen in the present case is consistent with this hypothesis.     

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.     

Perisomatic granules (non-plaque dystrophic dendrites) of hippocampal CA1 neurons in Alzheimer's disease and Pick's disease: a lesion distinct from granulovacuolar degeneration

A number of pathological changes have been reported in relation to CA1 pyramidal cells in Alzheimer's disease (AD), among them hyperphosphorylation of tau protein followed by the formation of filamentous tau lesions, granulovacuolar degeneration (GVD), Hirano bodies and spindle-shaped dilatations of distal apical dendrites. Juxtacellular clusters of glutamate receptor (GluR)-positive granules around pyramidal cells of the CA1 sector have been recently reported under the term "non-plaque dystrophic dendrites". We independently found that CA1 pyramidal cells in AD patients are regularly surrounded by ubiquitin-positive granules measuring 1-4 microns in diameter, which we have termed perisomatic granules (PSG). Using confocal microscopy, ubiquitin- and GluR-reactive granules were found to largely coincide and to correspond to the same structure. By immunoelectron microscopy PSG were found to consist of GluR1-2-reactive enlarged synaptic boutons containing tubulo-filamentous or floccular material. PSG were found to be consistently associated with pyramidal (principal) cells but not with interneurons of the CA1 sector. Dual-labeling experiments have shown that PSG are preferentially associated with tau-immunoreactive "pretangle" neurons but not with cells containing filamentous tau inclusions or with tau-negative nerve cell bodies. The number of PSG was found to increase with the severity of AD changes with almost no PSG found in Braak stages I and II and few in stage III. Furthermore, PSG were not AD specific, as shown by their presence around CA1 pyramidal cells in Pick's disease. The reasons for GluR reactivity and ubiquitin complex formation in enlarged perisomatic boutons are unclear. Marked changes in GluR subunits have been observed in association with even moderate AD pathology in hippocampal pyramidal cells in AD and our findings suggest a pathogenic link between PSG and early tau pathology in CA1 neurons. PSG might represent residual and abnormally clustered GluR subunits in degenerating perisomatic neurites. Our work confirms and extend previous study on perisomatic "non-plaque dystrophic dendrites" in AD and establish PSG as a pathological entity distinct from GVD. In addition PSG should be acknowledged among main histological changes associated with hippocampal neurons in AD and Pick's disease.     

Caspase-cleaved actin (fractin) immunolabelling of Hirano bodies

Hirano bodies are eosinophilic rod-like inclusions that are found predominantly in neuronal processes in the hippocampal CA1 sector with increasing age and are particularly numerous in Alzheimer's disease. They contain a variety of cytoskeletal epitopes, especially actin and actin-binding proteins. Actin cleavage by cysteinyl aspartate-specific proteases (caspases) is a feature of apoptosis. Cleavage at aspartate 244 generates N-terminal 32 kDa and C-terminal 15 kDa actin fragments. This has led to the development of a rabbit polyclonal antibody specific for caspase-cleaved actin, directed to the last five C-terminal amino acids of the 32 kDa fragment of actin ('fractin'). Fractin immunohistochemistry was performed on hippocampal sections from Alzheimer's disease and control cases containing numerous Hirano bodies, in addition to immunolabelling with CM1 antiserum which recognizes activated caspase-3. The Hirano bodies showed strong diffuse fractin immunoreactivity. They did not label with CM1 antiserum, perhaps reflecting too low a level of activated caspase-3 for immunodetection, or involvement of a different member of the caspase family. The finding of fractin immunoreactivity of Hirano bodies suggests that caspase-like cleavage of actin may play a role in their formation and further supports caspase-like activity in neuronal processes, distinct from that associated with acute perikaryal apoptosis.     

Differential effects of chemokines on oligodendrocyte precursor proliferation and myelin formation in vitro

Chemokines have recently been postulated to have important functions in the central nervous system (CNS) in addition to their principal role of directional migration of leukocytes. In particular, it has been shown that chemokines may play a role in the regulation of oligodendrocyte biology. Here, we have chosen to study the role of certain chemokines in regulating myelination. We have used the murine oligodendrocyte precursor-like cell line, Oli-neu, and primary mixed cortical cultures as experimental systems to assess their activities on oligodendrocyte precursor proliferation and developmental in vitro myelination. GRO-alpha, IL-8, SDF-1alpha and RANTES dose-dependently increased proliferation of this mouse A2B5 precursor-like cell line, while MCP-1 did not. Furthermore, the CXC chemokines GRO-alpha, IL-8 and SDF-1alpha stimulated myelin basic protein synthesis in a dose-dependent manner in primary myelinating cultures and enhanced myelin segment formation in this system, while the CC chemokines MCP-1 and RANTES did not. We also demonstrate that the receptor for SDF-1alpha, CXCR4, is expressed in mixed cortical cultures by PDGFalphaR positive oligodendrocyte precursors (OLPs) as well as by Oli-neu cells. SDF-1alpha induced proliferation in primary mixed cultures and the Oli-neu cell line was mediated through this receptor. We propose, therefore, that CXC chemokines and in particular SDF-1alpha regulates CNS myelination via their effects on cells of the oligodendrocyte lineage, specifically stimulation of OLP proliferation.     

Neuron-specific enolase levels in primary cultures of neurons

Primary neuronal cell cultures, derived by centrifugal elutriation of cells dissociated from embryonic rat cerebra, have been analyzed for their content of neuron-specific enolase (NSE) and non-neuronal enolase (NNE). Both immunocytochemical staining and radioimmunoassay establish that NSE is present in these cells. Furthermore, the observed increase in NSE and decrease in the NNE/NSE ratio with time in culture parallels that observed in vivo. It has recently been shown that during in vivo neurogenesis and maturation a “switch over” occurs from NNE to NSE which correlates with neuronal differentiation. The presence of a neuron-specific protein in these primary neuronal cultures and the changes observed during growth further support their use as a model system for the study of nervous tissue differentiation.     

Intracytoplasmic inclusion of Hirano type in Purkinje cells

Intracytoplasmic, rod-shaped and eosinophilic inclusions were recognized only in Purkinje cells in a case of Crow-Fukase syndrome. The ultrastructure of the inclusions was similar to that of the Hirano body and was suspected to have some relation to neurofilaments.     

Light and electron microscopic observations on the relationship between Hirano bodies,neuron and glial perikarya in the human hippocampus

Summary Hippocampi from two intellectually normal and four demented subjects were examined in autopsy material. Large Hirano bodies seen by light and electron microscopy were thought to be glial in origin and not to be produced by the perikarya of neurons as has been suggested in the literature. Myelination of two Hirano bodies found in the stratum lacunosum-granulosum where neuron perikarya are rare suggests that these bodies are produced by oligodendroglia. Hirano bodies were found to be associated with neurons showing granulovacuolar degneration. With electron microscope they were frequently seen to be divided by clefts filled with amorphous material which possibly consisted of free ribosomes.This work was supported in part by a grant from the Research Committee of the Newcastle Area Health Authority     

Isolation,identification, and culture of normal mouse colonic glia

We have developed a novel method of isolating and culturing murine colonic mucosal glial cells. Two morphologies are appreciated, a small flat bi or tri polar cell and a larger multipolar cell. The glial cultures have been freed of contaminating fibroblasts and epithelial cells and have been passaged by trypsinization. By intermediate filament (IF) typing, the glial cells have been further characterized as astrocyte-like. All cells expressed glial fibrillary acid protein but not neurofilament 160 protein. The glial cultures expressed the neuropeptides, substance P and substance K. Central nervous system astrocytes synthesize neuropeptides, prostaglandins and cytokines, and can express major histocompatibility class II antigens. It is likely that enteric mucosal glia will also prove to have varied functions. These cultures can now be used to define the role of enteric mucosal glia and to further study their complex interaction with other cells of the colonic mucosa.     

Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions.