Pamela G. Galloway

Hirano Body Filaments Contain Actin and Actin-Associated Proteins

Hirano bodies are eosinophilic, rod-shaped intraneuronal inclusions whose frequency increases with age and with Alzheimers 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, α-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.

Lewy Bodies Contain Epitopes Both Shared and Distinct from Alzheimer Neurofibrillary Tangles

Most of the identified constituents of the filamentous inclusions characteristic of the neurodegenerative diseases of aging are derived from the cytoskeleton. This study was undertaken to define immunocytochemically the cytoskeletal constituents of the filamentous cytopathologic marker of idiopathic Parkinson disease, the Lewy body (LB). An array of antibodies specific to neurofilaments, tubulin, microtubule associated proteins (tau, MAP1 and MAP2) and Alzheimer neurofibrillary tangles (NFT) were used to immunostain sections containing LB. All the antibodies to tubulin, MAP1 and MAP2 and the majority of the antibodies to neurofilaments and NFT recognized LB. The two monoclonal antibodies to NFT that recognize LB also react with ubiquitin, which has been identified in NFT. The prominent NFT component, tau, is apparently not incorporated into LB. These findings suggest that the presence of tau might not be a prerequisite to the formation of abnormal filaments. Therefore, although LB contain elements of neurofilaments, microtubules and ubiquitin, as do other abnormal neuronal filaments, they are distinct in composition. These distinctive and shared features may provide useful insights regarding the mechanisms underlying the formation of filaments in LB as well as those of other neuronal inclusions.

Hirano bodies contain tau protein

Hirano bodies are intraneuronal inclusions whose frequency increases with age and Alzheimers 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 Alzheimers disease. In this study, we show that Hirano bodies bind antibodies to the microtubule-associated protein tau, a component of Alzheimer neurofibrillary tangles.

Widespread serum amyloid P immunoreactivity in cortical amyloid deposits and the neurofibrillary pathology of Alzheimer's disease and other degenerative disorders

Amyloid P (AP) component is present in all types of systemic amyloid deposits. Recently, it has been shown to be also present in cerebral amyloid lesions of Alzheimers disease (AD). In this study, we used immunocytochemical methods to extend these findings at the electron microscope level and characterize the spectrum of AP immunoreactivity in neurofibrillary pathology (NFP) of AD and other neurodegenerative disorders including Downs syndrome (DS), Creutzfeldt‐Jakob, Parkinsons, Picks and diffuse Lewy body diseases and progressive supranuclear palsy. In AD and DS, AP immunoreaction product was evident in all the classical amyloid lesions and NFP in a large sample of all cortical areas examined. The distribution and relative intensity of immunostaining was similar to that of thioflavin S staining in serial sections. In many cases, however, plaques and vessels stained by anti‐AP serum were not apparent with thioflavin S. Serial sections immunostained with antiserum to amyloid A, C‐reactive protein or to other proteins involved in systemic amyloidoses and the acute phase response showed no evidence of staining in any of the cerebral lesions. Electron microscopy confirmed that AP immunoreactivity was associated with the abnormal filaments characteristic of NFP as well as amyloid fibrils found in plaques and vessels showing congophilic amyloid angiopathy. Plaques of Creutzfeldt‐Jakob disease, Pick bodies of Picks disease, tangles and Lewy bodies in Parkinsons disease and a subpopulation of Lewy bodies in the diffuse Lewy body disease coexistent with AD were also stained. With the exception of vessels in two of the five cases, AP was not detected in age‐matched controls. Our observations indicate AP to be a consistent feature of cerebral NFP and amyloid deposits.

The presence of tau distinguishes Lewy bodies of diffuse Lewy body disease from those of idiopathic Parkinson disease

The antigenic components of Lewy bodies in the cerebral cortex and substantia nigra in 5 cases of diffuse Lewy body disease were examined by immunocytochemistry, using antibodies to neurofilaments (in the phosphorylated or non-phosphorylated forms); to ubiquitin; to the microtubule-associated proteins MAP1, MAP2 and tau; to isolated Alzheimer paired helical filaments, and to tubulin, in the tyrosinated and non-tyrosinated forms. Immunoreactivity with antibodies to cytoskeletal components was identical to that previously described for Lewy bodies of idiopathic Parkinson disease, with the exception that the inclusions of diffuse Lewy body disease (in both cortex and substantia nigra) were stained by an antibody to tau protein. Our findings indicate that although the inclusions found in diffuse Lewy body disease share structural and epitopic features with the inclusions of idiopathic Parkinson disease, they also have distinguishing characteristics (in addition to the differing neuronal populations involved). Also, they suggest that although the inclusions in both conditions appear similar, they probably have different pathogenetic origins.

Basic fibroblast growth factor binds to filamentous inclusions of neurodegenerative diseases

The extracellular matrix protein heparin sulfate proteoglycans (HSPG) has been found in the neurofibrillary pathology of Alzheimer disease. This study was performed to determine if similar proteoglycans might be present in the fibrillary inclusions of other neurodegenerative diseases. Basic fibroblast growth factor (bFGF) binding to heparinase sensitive sites was used as an assay for HSPGs. We found that the inclusions of Pick and Parkinson diseases as well as progressive supranuclear palsy contained heparinase sensitive bFGF binding sites while the inclusions of diffuse Lewy body disease lacked bFGF binding sites. These findings indicate that HSPGs interactions and possible role in the formation of intraneuronal inclusions are not limited to Alzheimer disease.

Tropomyosin distinguishes lewy bodies of parkinson disease from other neurofibrillary pathology

Research into the cellular changes in the degenerative diseases of the central nervous system has focused on the alterations in the constituent proteins of the neuronal cytoskeleton. Although both microtubule and neurofilament proteins have been implicated in the formation of neurofibrillary pathology in Alzheimer, Pick, diffuse Lewy body and Parkinson diseases and progressive supranuclear palsy (PSP), until recently there has been little consideration of whether other cytoskeletal systems are involved. With the findings that epitopes of the microfilament associated protein tropomyosin are present in the neurofibrillary pathology of Alzheimer disease, we decided to investigate the presence of this protein in these related diseases. To address whether the inclusion bodies of other degenerative diseases share this property, sections of brain were immunostained with antibodies to smooth and skeletal muscle tropomyosin. Although neurofibrillary tangles in PSP, Pick bodies and some diffuse Lewy bodies stained, Lewy bodies of idiopathic Parkinson disease did not. This property further distinguishes the Lewy body of Parkinson disease from other neurofibrillary pathologies.

Endoscopic and radiographic evaluation of the murine colon

Endoscopic and radiographic techniques have not been widely applicable in the evaluation of chemically induced murine colon cancer. the authors investigated methods of cleansing the rat colon and refined endoscopic and radiographic techniques. They compared total colonoscopy (TC) and air‐contrast (ACBE) and single‐contrast barium enema (SCBE) findings with those obtained at necropsy in rats with 1,2‐dimethylhydrazine (DMH)‐induced colon cancer. Gastrograffin enemas with bisacodyl suppositories showed complete evacuation of solid feces. Sprague‐Dawley rats treated with DMH had their colons cleansed and then underwent TC (5.0‐mm Olympus bronchoscope) and either SCBE or ACBE. Colonoscopy and ACBE were equally sensitive (81.5% and 76.3%, respectively), although SCBE was significantly insensitive in identifying lesions (P < 0.001). This study demonstrates that: (1) mechanical cleansing of the rat colon is feasible, (2) TC and barium radiology can be done routinely after mechanical cleansing, and (3) TC and double‐contrast BE are sensitive in identifying colon lesions. These techniques will provide a means for manipulation of murine tumors and in vivo surveillance.

Antigenic characteristics of neurofibrillary tangles in progressive supranuclear palsy

The antigenic components of neurofibrillary tangles in the basal forebrain and brainstem were studied in 4 cases of progressive supranuclear palsy (PSP) at the light and electron microscopic levels, using antibodies to neurofilaments (in the phosphorylated and non-phosphorylated forms); the high, middle and low molecular weight neurofilament subunits; ubiquitin; the microtubule associated proteins MAP1, MAP2 and tau; isolated Alzheimer paired helical filaments and to tubulin, in the tyrosinated and detyrosinated forms. Although PSP neurofibrillary tangles appear to have most antigenic sites in common with those of Alzheimer disease, PSP tangles share epitopes with tyrosinated and detyrosinated tubulin, which has not been demonstrated in Alzheimer neurofibrillary tangles.

Ubiquitin in normal, reactive and neoplastic human astrocytes

Ubiquitin, a protein important in regulating non-lysosomal proteolysis, has previously been shown to be present in cytoskeletal inclusions of the neurodegenerative diseases. Its role in other pathological processes of the central nervous system, such as neoplastic transformation of cells, is not known. The astrocytoma, a tumor of complex biology derived from the astrocyte, is the most common primary parenchymal human brain tumor in both children and adults. Until recently, ubiquitin was not known to form stable conjugates in cells. We have shown using immunocytochemistry on sections of astrocytomas that both glial fibrillary acidic protein (GFAP) (the major intermediate filament protein present in normal, reactive and neoplastic astrocytes) and ubiquitin are simultaneously present in the cytoplasm and cell processes of tumor cells. The presence of ubiquitin and GFAP was also found in astrocytoma cells in short- and long-term culture, and confirmed by immunostaining of blots of tumor homogenates subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis.