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Gallyas-positive argyrophilic and ubiquitinated filamentous inclusions in rapidly progressive motor neuron disease: immunohistochemical and electron microscopic studies.

In an autopsy case of sporadic rapidly progressive lower motor neuron disease (MND), Gallyas-positive argyrophilic and ubiquitinated filamentous intracytoplasmic inclusions were found in the neurons. Clinically, 7 months prior to death, a 68-year-old woman experienced a history of rapidly progressive muscle weakness of all four extremities and bulbar sign, without sensory and autonomic disturbance. Two months later, she became unable to stand or walk. Four months after onset, she needed respiratory support, and subsequently died due to cardiorespiratory arrest. Neuropathological examinations revealed neuronal loss and associated gliosis in the lower motor neurons, except for ocular motor nuclei, Clark's column, and accessory cuneate nucleus, and tract degeneration was observed in the middle root zone of the posterior column and spinocerebellar tract. No Bunina bodies or Lewy body-like hyaline inclusions were found in the anterior horns. Gallyaspositive argyrophilic filamentous inclusions were found in the lower motor neurons and in nerve cells of the Clark's column, intermediate zone, posterior horn and accessory cuneate nucleus. These were positive with anti-ubiquitin antibody but negative with anti-tau (tau-2 and AT8) and neurofilament antibodies. Electron microscopic examinations disclosed randomly arranged tubular-like filamentous profiles, with a diameter of 12-14 nm, sometimes with amorphous granules in the perikaryon. This is the first report on the Gallyas-positive argyrophilic and ubiquitinated filamentous inclusions in neurons in MND.

Aged↗

Rice stripe virus 23.9 K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells.

The genome of Rice stripe virus (RSV, genus Tenuivirus) contains seven open reading frames (ORFs). Little is known about the products of four of these ORFs, including the 23.9 K protein encoded by the virus-sense ORF of RNA3. Western blotting revealed that the 23.9 K protein was synthesized in the host plant and also in the planthopper vector of RSV. Using a baculovirus vector, the 23.9 K protein was expressed, both unfused and fused with red-shifted green fluorescent protein, in Spodoptera frugiperda cells. Inclusion bodies were observed by light microscope in cells expressing fused or unfused proteins. Inclusion bodies in cells expressing the fused protein fluoresced under blue light. By immunoelectron microscopy, electron-dense inclusion bodies in cells expressing the unfused protein were specifically labeled with 23.9 K protein antiserum. Moreover, electron-dense masses labeled with 23.9 K protein antiserum were observed in virus-infected wheat tissue by electron microscopy. This paper thus demonstrates that RSV 23.9 K protein can aggregate in vivo and form inclusion bodies in infected plant tissue.

Animals↗

Brome mosaic virus replicase proteins localize with the movement protein at infection-specific cytoplasmic inclusions in infected barley leaf cells.

Subcellular localization of the Brome mosaic virus replicase-related 1a and 2a proteins, and the 3a movement protein in infected barley leaves was examined by immunogold electron microscopy. The 1a and 2a proteins colocalized at infection-specific electron-dense cytoplasmic inclusions. The 3a protein was also detected in these inclusions. The inclusions were oval or amorphous, and contained electron-lucent regions. Electron microscopic autoradiography showed that the inclusions were sites of [3H]uridine incorporation, suggesting that they are sites of viral RNA synthesis.

Bromovirus↗

Presence of beta-arrestin in cellular inclusions in metamphetamine-treated PC12 cells.

Cellular inclusions containing ubiquitin and alpha-synuclein were observed in PC12 cells treated with metamphetamine (MA). To study the possible involvement of beta-arrestin in inclusion formation, we treated PC12 cells with MA for different times and analyzed the ubiquitin proteosome pathway (UPP). We found that beta-arrestin is ubiquitinated in the MA-treated PC12 cell line. The involvement of beta-arrestin in UPP was further supported by electron microscopy and by confocal microscopy, which documented the presence of beta-arrestin in these Lewy body-like inclusions. Our experiments reveal an interesting and previously unappreciated connection between beta-arrestin and ubiquitination and suggest that beta-arrestin could be involved in the development of the inclusion bodies.

Animals↗

Stress protein co-localization to autofluorescent astrocytic inclusions in situ and in cysteamine-treated glial cultures.

In the aging brain, a unique subpopulation of limbic and periventricular astrocytes accumulates red autofluorescent, peroxidase-positive cytoplasmic inclusions distinct from lipofuscin. Cysteamine (CSH) exposure rapidly induces identical inclusions in cultured, immature astroglia. CSH induces a cellular stress response prior to astrocyte granulation. To determine whether stress proteins are actual constituents of the autofluorescent granules, 12-week-old rat brain sections and CSH-treated astroglial cultures were immunostained with various anti-stress protein antibodies and evaluated by laser scanning confocal microscopy. We observed intense co-localization of heat shock protein (HSP) 27 and ubiquitin (Ub) to the autofluorescent astrocyte granules in situ and in CSH-treated glial cultures. In both preparations, glucose regulated protein (GRP) 94 consistently exhibited partial co-localization to the granule periphery and adjacent cytoplasm. In contrast, HSP72 co-localization to these inclusions was only occasionally seen and the granules appeared entirely devoid of HSP90 and alpha B-crystallin. Acute exposure of cultured astroglia to CSH induced intense cytoplasmic Ub staining, suggesting that activation of the Ub pathway may be an early event in the biogenesis of these astrocytic granules. Taken together, our results support the notion that the autofluorescent astrocyte inclusions are stress or heat shock granules which progressively accumulate in the aging periventricular brain. Moreover, CSH greatly accelerates the appearance of this senescent astrocyte phenotype in primary culture.

Animals↗

Acid fast inclusions in tissues of dogs dosed with lead.

Liver, kidney and bone from dogs with experimental lead toxicosis were examined to study the frequency of occurrence of acid fast inclusions and the results were correlated with the lead content of these tissues. Light and electron microscopic studies revealed that bone and kidney had characteristic acid fast lead inclusions in 95 and 68 per cent of cases, respectively. Liver, although it had a high lead content, did not often show inclusions. For histopathological diagnosis of lead poisoning in dogs, we suggest that Ziehl-Nielsen stained sections of the epiphyseal area of long bone be examined for the presence of acid fast inclusions in osteoclasts.

Animals↗

Cytoplasmic crystalline inclusions in hepatocytes of a steer with shipping fever.

Needle-shaped crystalline inclusion bodies were found in vacuolated liver cells of a steer affected with pneumonic pasteurellosis. The inclusions were eosinophilic and refractive, and ranged in size from 3 to 6 microns long and from 150 to 250 nm wide. Histochemical examination revealed that they contained lipid and protein. Electron microscopic examination showed finely fibrillar or granular material. Cross-striations with a periodicity of 45 nm were seen in longitudinal section of the inclusions. The possible origin and pathogenic significance of the inclusions are discussed.

Animals↗

A new myopathy with tubulomembranous inclusions.

A muscle biopsy performed on a 16-year-old boy with progressive myopathy revealed hitherto unrecognized peculiar inclusions which consisted of 3 types of structures. The first type consisted of laminated tubulomembranous structures and most of the inclusions belonged to this type. The lamellae were regularly spaced with a periodicity of 8.5--9 nm and curving a little, and were observed as concentric lamellae according to the plane of sectioning. The second type of inclusions consisted of curvifilamentous material. The third type had the appearance of moderately electron-dense granular material surrounded by a single unit membrane. The origin and nature of these inclusions is obscure, but the diagnosis of some kind of storage myopathy was suspected in this case.

Adolescent↗

Skein-like inclusions in the anterior horn cells in motor neuron disease.

Skein-like inclusions (SLIs) in the anterior horn cells of patients with motor neuron diseases, including familial amyotrophic lateral sclerosis with posterior column degeneration, sporadic lower motor neuron disease and classical amyotrophic lateral sclerosis, were investigated morphologically with hematoxylin and eosin preparations, immunostaining for ubiquitin and immunoelectron microscopy. The SLIs were thready linear or tubular structures which immunostained with antiubiquitin antibodies. They were detected on hematoxylin and eosin preparations as eosinophilic thread-like structures often surrounded by pale areas. SLIs were occasionally present as networks of threads or tubules. Sometimes, they were aggregated and formed larger pale inclusions. Ultrastructurally, the SLIs were bundles of filaments which appeared thicker than neurofilaments. The SLIs tended to have central hollow spaces which were devoid of filaments. When the SLIs were clustered, fuzzy thick filaments were randomly and loosely arranged among the individual SLIs. The SLIs were histologically and ultrastructurally distinct from other inclusions such as Bunina bodies and hyaline inclusions. This unique morphology of SLIs may provide a novel perspective on the degenerative processes of the anterior horn cells in MND.

Adult↗

Hereditary distal myopathy with granulo-filamentous cytoplasmic inclusions containing desmin, dystrophin and vimentin.

A 56-year-old female and her 34-year-old daughter presented with a predominantly distal myopathy affecting the peroneal and calf muscles, neck flexors and hand muscles. Both patients and two other daughters had cardiac arrhythmias, three requiring the insertion of cardiac pacemakers. Skeletal muscle biopsies revealed a complex myopathic process with granular degeneration, rimmed vacuoles and eosinophilic cytoplasmic inclusions. Ultrastructurally, the inclusions were composed of electron dense granular material and filaments forming linear masses beneath the sarcolemma and rounded masses within the cytoplasm of the fibres. Immunohistochemistry revealed labelling of the inclusions for desmin, dystrophin and vimentin, but not for alpha-actinin, spectrin, utrophin or myosin heavy chains. This family shows a hereditary distal myopathy with some features in common with previously-reported cases in which biopsies showed cytoplasmic inclusion bodies containing desmin. This group of diseases is clinically and pathologically heterogeneous. In the present cases, the accumulation of cytoplasmic filaments may reflect a generalised disturbance of filamentous protein metabolism rather than a specific disorder of desmin.

Adult↗

Immunohistochemical study of intracytoplasmic inclusion bodies of the thalamus in myotonic dystrophy.

Intracytoplasmic inclusion bodies of the thalamus in eight patients with myotonic dystrophy (MyD) were studied immunohistochemically. The intracytoplasmic inclusion bodies of the thalamus (thalamic inclusions, TIs) were strongly immunostained with anti-ubiquitin antibody (Ab) and some of them were mildly stained with anti-microtubule associated protein 1 (MAP 1) and anti-MAP 2 antibodies. However, TIs did not react with any of the following: anti-neurofilament protein Ab, anti-tau Ab, anti-paired helical filament Ab, anti-tubulin Abs (alpha and beta), anti-neuron-specific enolase Ab, anti-glial fibrillary acidic protein Ab, anti-synaptophysin Ab, anti-myelin basic protein Ab, anti-actin Ab and anti-phosphorylated epitope of neurofilaments Ab. Thus, our study demonstrates the unique immunohistochemistry of TIs in MyD which differentiates them from other intracytoplasmic inclusions in various neurodegenerative disorders.

Aged↗

X-ray powder pattern analysis of cytoplasmic polyhedrosis virus inclusion bodies.

Cytoplasmic polyhedrosis virus is an insect reovirus which is occluded in crystalline inclusion bodies that form in the mid-gut of certain insects. Inclusion bodies of cytoplasmic polyhedrosis virus from Bombyx mori were isolated and purified. These crystalline bodies, about 1-3 microns in linear size, were compacted in a capillary tube while immersed in buffer. X-ray diffraction photographs showed powder rings, extending to 8.2 A resolution, which could be indexed with a cell measuring a = b = 49.9 +/- 0.4 A, c = 41.5 +/- 0.4 A, alpha = beta = gamma = 90 degrees. The polyhedrin protein, which is the major component of the inclusion body, has a molecular weight of about 30,000 daltons and, hence, there are probably two molecules in the unit cell. Thus, the unit cell is monoclinic or possibly triclinic. A Patterson derived from the measured powder pattern intensities, assuming monoclinic symmetry, could be interpreted in terms of a molecule with two larger globes. Such a structure is roughly consistent with the breakdown of the polyhedrin into two larger fragments of molecular weight 17,000 and 14,000 when raising the pH to near 10. Under these conditions the inclusion bodies disintegrate, releasing virus and catalyzing the proteolysis of the polyhedrin.

Electron Probe Microanalysis↗

Cytoplasmic inclusions in neocortical astrocytes associated with arteriopathic encephalopathy and dementia.

We describe what we believe to be the fifth case of a degenerative condition of the brain characterized by unusual intracytoplasmic inclusions in neocortical astrocytes, and we review four previous reports of what appears to be the same condition. Whereas these previous cases were characterized by prolonged clinical mental and psychomotor retardation, our case describes a rapid onset in a previously fit and mentally able patient in whom the astrocytic inclusions showed a close association with fibrohyaline vascular degeneration and changes resembling those of Alzheimer's disease. The inclusions, which were most frequent in the second to fourth layers of the frontal, temporal, and occipital cortices but absent from subcortical regions, consisted of large, irregular hyaline bodies surrounding the nucleus and extending into the proximal parts of cell processes. Ultrastructurally they consisted of free ribosomes in a granular and filamentous matrix. They were not bound by a membrane. Lipofuscin granules were associated with them. It is suggested that the inclusions might result from a disturbance of protein metabolism in protoplasmic astrocytes, but their true significance is unknown.

Astrocytes↗

A filamentous inclusion body within anterior horn neurones in motor neurone disease defined by immunocytochemical localisation of ubiquitin.

Using an immunocytochemical method to localise antibodies to ubiquitin, filamentous inclusion bodies were seen in spinal anterior horn neurones in cases of motor neurone disease (MND) but not in any control cases. These inclusion bodies appeared to be closely associated with classical Bunina bodies and immuno-electron microscopy suggested that they were based on arrays of straight 10-15 nm filaments together with some granular material. These observations link the protein ubiquitin with a chronic neurodegenerative disease and extend previous observations of a close association between filamentous inclusion bodies and ubiquitin. Ubiquitin-filament inclusions should be regarded as a new hallmark in the histological diagnosis of MND.

Adult↗

Immunolocalization of ubiquitin in muscle biopsies of patients with inclusion body myositis and oculopharyngeal muscular dystrophy.

In 10/10 inclusion body myositis (IBM) patients and 2/2 oculopharyngeal muscular dystrophy (OPMD) patients, vacuolated muscle fibers contained darkly stained ubiquitin (Ub)-immunoreactive cytoplasmic inclusions. By electronmicroscopy, Ub-immunoreactive material was strictly localized to the 15-21 nm pathologic cytoplasmic tubulofilaments (CTFs). None of 18 control muscle biopsies contained the Ub-immunoreactive inclusions that are typical for IBM and OPMD. Thus, (a) finding that CTFs are ubiquitinated places their protein in the Ub-mediated turnover pathway and provides their first molecular marker; (b) easy accessibility, as compared to the central nervous system, of muscle tissue containing ubiquitinated inclusions should be advantageous for biochemical and molecular studies and may provide information important to both systems.

Adolescent↗

Abnormal accumulation of phospholipase C-delta in filamentous inclusions of human neurodegenerative diseases.

We have previously demonstrated that an antibody to phosphoinositide-specific phospholipase C (PLC) isozyme, PLC-delta, intensely stained neurofibrillary tangles (NFT) in the brain tissue of Alzheimer's disease (AD). This study was performed to determine if abnormal PLC-delta accumulation might be present in the filamentous inclusions of other neurodegenerative diseases. We found that the anti-PLC-delta antibody stained neuronal inclusions of Pick's disease, progressive supranuclear palsy and diffuse Lewy body disease while the inclusions of idiopathic Parkinson's disease lacked PLC-delta accumulation. These results suggest a possible role for PLC-delta interaction in the formation of intraneuronal filamentous inclusions in human neurodegenerative diseases.

Aged↗

Nondenaturing solubilization of beta2 microglobulin from inclusion bodies by L-arginine.

Expression of beta2 microglobulin (beta2m) in Escherichia coli resulted in formation of inclusion bodies. Attenuated total reflectance Fourier transform infrared analysis suggested a native-like secondary structure of beta2m in the inclusion bodies. Nondenaturing solubilization of the native-like beta2m from inclusion bodies was achieved using L-arginine solution, which enables an efficient recovery of beta2m with little aggregation. Greater beta2m solubilization from inclusion bodies was obtained at higher temperatures. Low-temperature solubilization yielded beta2m with fluorescence properties identical to those of native beta2m, but its secondary structure was slightly nonnative. Solubilization at moderate temperature gave beta2m with an apparently native structure. We propose an efficient nondenaturing solubilization method combining L-arginine and moderate temperature.

Arginine↗

A rat model of human FENIB (familial encephalopathy with neuroserpin inclusion bodies).

FENIB (familial encephalopathy with neuroserpin inclusion bodies) is caused by intracellular accumulation/polymerization of mutant neuroserpins in the endoplasmic reticulum (ER). Transgenic rats overexpressing megsin (Tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid Schiff (PAS)-positive inclusions distributed throughout deeper layers of cerebral cortex, CA1 of the hippocampus, and substantia nigra. Hippocampal extracts from Tg meg rats showed increased expression of ER stress proteins, and activation of caspases-12 and -3, associated with decreased neuronal density. Enhanced ER stress was also observed in dopaminergic neurons in the substantia nigra, in parallel with decreased neuronal viability and motor coordination. In each case, PAS-positive inclusions were also positive for megsin. These data suggest that overexpression of megsin results in ER stress, eventuating in the formation of PAS-positive inclusions. Tg meg rats provide a novel model of FENIB, where accumulation of serpins in the ER induces selective dysfunction/loss of specific neuronal populations.

Animals↗