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Accumulation of the DNA/RNA binding protein fused in sarcoma (FUS) as inclusions in neurons and glia is the pathological hallmark of amyotrophic lateral sclerosis patients with mutations in FUS (ALS-FUS) as well as in several subtypes of frontotemporal lobar degeneration (FTLD-FUS), which are not associated with FUS mutations. Despite some overlap in the phenotype and neuropathology of FTLD-FUS and ALS-FUS, significant differences of potential pathomechanistic relevance were recently identified in the protein composition of inclusions in these conditions. While ALS-FUS showed only accumulation of FUS, inclusions in FTLD-FUS revealed co-accumulation of all members of the FET protein family, that include FUS, Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 15 (TAF15) suggesting a more complex disturbance of transportin-mediated nuclear import of proteins in FTLD-FUS compared to ALS-FUS. To gain more insight into the mechanisms of inclusion body formation, we investigated the role of Transportin 1 (Trn1) as well as 13 additional cargo proteins of Transportin in the spectrum of FUS-opathies by immunohistochemistry and biochemically. FUS-positive inclusions in six ALS-FUS cases including four different mutations did not label for Trn1. In sharp contrast, the FET-positive pathology in all FTLD-FUS subtypes was also strongly labeled for Trn1 and often associated with a reduction in the normal nuclear staining of Trn1 in inclusion bearing cells, while no biochemical changes of Trn1 were detectable in FTLD-FUS. Notably, despite the dramatic changes in the subcellular distribution of Trn1 in FTLD-FUS, alterations of its cargo proteins were restricted to FET proteins and no changes in the normal physiological staining of 13 additional Trn1 targets, such as hnRNPA1, PAPBN1 and Sam68, were observed in FTLD-FUS. These data imply a specific dysfunction in the interaction between Trn1 and FET proteins in the inclusion body formation in FTLD-FUS. Moreover, the absence of Trn1 in ALS-FUS provides further evidence that ALS-FUS and FTLD-FUS have different underlying pathomechanisms.

We investigated a family manifesting amyotrophic lateral sclerosis (ALS) with a heterozygous E478G mutation in the optineurin (OPTN) gene. Clinically, slow deterioration of motor function, mood and personality changes, temporal lobe atrophy on neuroimaging, and bizarre finger deformity were noted. Neuropathologically, TAR DNA-binding protein 43 (TDP-43)-positive neuronal intracytoplasmic inclusions were observed in the spinal and medullary motor neurons. In these cells, the immunoreactivity of nuclear TDP-43 was reduced. Consecutive sections revealed that the inclusions were also reactive with anti-ubiquitin and anti-p62 antibodies, but noticeably negative for OPTN. In addition, TDP-43/p62-positive glial cytoplasmic inclusions (GCIs) were scattered throughout the spinal cord and the medullary motor nuclei. Furthermore, Golgi fragmentation was identified in 70% of the anterior horn cells (AHCs). The presence of AHCs with preserved nuclear TDP-43 and a fragmented Golgi apparatus, which are unrecognizable in sporadic ALS, indicates that patients with the E4787G OPTN mutation would manifest Golgi fragmentation before loss of nuclear TDP-43. In the neocortex, GCIs were sparsely scattered among the primary motor and temporal cortices, but no neuronal TDP-43-positive inclusions were detected. In the amygdala and the ambient gyrus, argyrophilic grains and ballooned neurons were seen. The thorough neuropathologic investigations performed in this work demonstrated that OPTN-positive inclusion bodies, if any, were not prominent. We postulate that optineurinopathy is closely linked with TDP-proteinopathy and speculate that this heterozygous E478G mutation would cause ALS by acting through a dominant-negative mechanism.

Most cases of frontotemporal lobar degeneration (FTLD) are characterized by abnormal intracellular accumulation of either tau or TDP-43 protein. However, in ~10% of cases, composed of a heterogenous collection of uncommon disorders, the molecular basis remains to be uncertain. We recently discovered that the pathological changes in several tau/TDP-43-negative FTLD subtypes are immunoreactive (ir) for the fused in sarcoma (FUS) protein. In this study, we directly compared the pattern of FUS-ir pathology in cases of atypical FTLD-U (aFTLD-U, N = 10), neuronal intermediate filament inclusion disease (NIFID, N = 5) and basophilic inclusion body disease (BIBD, N = 8), to determine whether these are discrete entities or represent a pathological continuum. All cases had FUS-ir pathology in the cerebral neocortex, hippocampus and a similar wide range of subcortical regions. Although there was significant overlap, each group showed specific differences that distinguished them from the others. Cases of aFTLD-U consistently had less pathology in subcortical regions. In addition, the neuronal inclusions in aFTLD-U usually had a uniform, round shape, whereas NIFID and BIBD were characterized by a variety of inclusion morphologies. In all cases of aFTLD-U and NIFID, vermiform neuronal intranuclear inclusions (NII) were readily identified in the hippocampus and neocortex. In contrast, only two cases of BIBD had very rare NII in a single subcortical region. These findings support aFTLD-U, NIFID and BIBD as representing closely related, but distinct entities that share a common molecular pathogenesis. Although cases with overlapping pathology may exist, we recommend retaining the terms aFTLD-U, NIFID and BIBD for specific FTLD-FUS subtypes.

Basophilic Inclusion Body Disease (BIBD) is a tau-negative form of frontotemporal lobar degeneration (FTLD), characterized by neuronal cytoplasmic inclusions (NCI) that are visible on hematoxylin and eosin stain (HE), contain RNA, and are inconsistently ubiquitin-immunoreactive (ir). The normal nuclear expression of TDP-43 is not altered. Here we investigate whether the distribution of the structurally and functionally related protein fused in sarcoma (FUS) is altered in BIBD. Mutations in the FUS gene have recently been identified as a cause of familial amyotrophic lateral sclerosis (ALS). In addition to these familial ALS cases, FUS protein has recently been demonstrated in NCI in a subset of FTLD with ubiquitinated inclusions (atypical FTLD-U) and in neuronal intermediate filament inclusion disease (NIFID). We examined seven BIBD brains of patients with average age at onset 46 (range 29-57) and average duration of disease 8 years (range 5-12). Three cases presented with the behavioural variant of fronto-temporal dementia (FTD-bv) and one with FTD-bv combined with severe dysarthria. All four developed motor neuron disease/ALS syndrome (MND/ALS) several years later. In the other three cases, presentation was predominantly with motor symptoms, construed as MND/ALS in two, and progressive supranuclear palsy (PSP) in one. Severity of cortical degeneration varied, but all cases shared severe nigrostriatal atrophy and lower motor neuron pathology. In spared areas of cortex, FUS antibodies showed intense labelling of neuronal nuclei and weak positivity of cytoplasm, whereas, in affected areas, intense labelling of NCI was accompanied by reduction or disappearance of the normal IR pattern. The number of FUS-ir NCI was much greater than the number detected by HE or with ubiquitin or P62 immunohistochemistry. FUS-ir glial cytoplasmic inclusions (GCI) were abundant in the grey and white matter in all cases, whereas neuronal intranuclear inclusions were rare and only seen in 2/7 cases. Thus, BIBD shares with atypical FTLD-U and NIFID the presence of FUS-ir NCI and GCI, and together comprise a new biochemical category of neurodegenerative disease (FUS proteinopathies). The consistent involvement of motorneurons in BIBD indicates that the association of FTLD and MND/ALS can occur on a FUS or TDP-43 pathological substrate.

This report concerns an immunohistochemical investigation on RNA-related proteins in the basophilic inclusions (BIs) from patients with adult-onset atypical motor neuron disease. Formalin-fixed, paraffin-embedded sections of the motor cortex and the lumbar spinal cord were examined. The BIs appeared blue in color with H&E and Nissl stain, and pink with methylgreen-pyronin stain. Ribonuclease pretreatment abolished the methylgreen-pyronin staining, suggesting that the BIs contained RNA. Immunohistochemically, the BIs were distinctly labeled with the antibodies against poly(A)-binding protein 1, T cell intracellular antigen 1, and ribosomal protein S6. These proteins are essential constituents of stress granules. In contrast, the BIs were not immunoreactive for ribosomal protein L28 and decapping enzyme 1, which are core components of transport ribonucleoprotein particles and processing bodies, respectively. Moreover, the BIs were not immunopositive for TDP-43. Our results imply that translation attenuation could be involved in the processes of BI formation in this disorder.

Phosphorylated Smad2/3 (pSmad2/3), the central mediators of transforming growth factor (TGF)-beta signaling, were recently identified in tau-positive inclusions in certain neurodegenerative disorders. To clarify whether the localization of pSmad2/3 is altered in amyotrophic lateral sclerosis (ALS), we immunohistochemically examined spinal cords from sporadic ALS (SALS), from familial ALS (FALS) patients with the A4V mutation in their Cu/Zn superoxide dismutase (SOD1) gene, and from G93A mutant SOD1 transgenic (mSOD1 Tg) mice. In control spinal cords, pSmad2/3 immunoreactivity was observed exclusively in neuronal and glial nuclei. In SALS and FALS patients the nuclei showed increased immunoreactivity for pSmad2/3. Noticeably, round hyaline inclusions (RHIs) and skein-like inclusions of SALS patients were immunoreactive for pSmad2/3. Double immunofluorescence staining for pSmad2/3 and transactive response-DNA-binding protein (TDP)-43 revealed co-localization of these proteins within RHIs. In contrast, Bunina bodies in SALS and Lewy body-like hyaline inclusions (LBHIs) in FALS were devoid of labeling for pSmad2/3. Similarly, in the mSOD1 Tg mice pSmad2/3 immunoreactivity was increased in the nuclei, while LBHIs were not labeled. These findings suggest increased TGF-beta-Smad signaling in SALS, FALS, and mSOD1 Tg mice, as well as impaired TGF-beta signal transduction in RHI-bearing neurons of SALS patients, presumably at the step of pSmad2/3 translocation into the nucleus. The pathomechanisms, including the process of inclusion development, appears to be different between SALS and mSOD1-related FALS or Tg mice.

Recent investigations have indicated that the nucleocytoplasmic transport system is essential for maintaining cell viability and cellular functions and that its dysfunction could lead to certain disorders. To investigate the involvement of this system in the pathomechanisms of amyotrophic lateral sclerosis (ALS), we examined the immunohistochemical localization of proteins associated with nucleocytoplasmic transport in the lumbar spinal cord in a mutant SOD1 (G93A) transgenic mouse model of ALS. This model is widely used for ALS research, and the mutant mice are known to exhibit neuronal loss and Lewy body-like hyaline inclusions (LBHIs) in the anterior horns, similar to the pathology seen in familial ALS patients associated with an SOD1 mutation and in several other transgenic rodent models. Using antibodies against the importin beta family of proteins, the major carrier proteins of nucleocytoplasmic transport, and those against their adapter protein, importin alpha, we found that the immunoreactivities were decreased within the nuclei and increased within the cytoplasm of a subset of the surviving anterior horn cells of the transgenic mice. In addition, LBHIs were invariably reactive toward these antibodies. Furthermore, the immunoreactivities for histone H1 and beta-catenin, representative cargo proteins transported by importin beta-dependent and beta-independent nucleocytoplasmic transport pathways, respectively, showed distributions similar to those for importin beta family and importin alpha proteins. The altered distributions of these proteins were not associated with caspase-3 expression, suggesting that the findings are unlikely to be a manifestation of apoptotic processes. Chronological quantitative analysis of importin beta-immunostained sections from the transgenic mice revealed a statistically significant progressive decrease in the proportion of the anterior horn cells exhibiting a more intense reactivity for these proteins in the nucleus than in the cytoplasm. To the contrary, we found that the anterior horn cells with the immunoreactivity in their cytoplasm, being more pronounced than that in their nucleus, were significantly increased in number along with the disease progression. This is the first report investigating nucleocytoplasmic transport in the ALS model mouse, and our present results imply that its dysfunction could be involved in the pathomechanisms underlying ALS.

The immunohistochemical localization of glucose-regulated protein 78/BiP (GRP78), a chaperone protein that primarily resides within the lumen of the endoplasmic reticulum, was investigated in the lumbar spinal cord of mutant copper/zinc superoxide dismutase (SOD1) transgenic mice. Re-staining techniques were used to determine the immunoreactivity with anti-GRP78 antibody of abnormal structures observed by hematoxylin and eosin staining. Besides its physiological localization in the neuronal and glial cytoplasm, GRP78 was expressed in Lewy body-like hyaline inclusions, in irregularly-shaped eosinophilic structures without an apparent halo, and in cord-like swollen neurites. These different sites were invariably also immunopositive for ubiquitin, suggesting them to be pathological structures. The topographic distribution of GRP78 expression closely resembled that of SOD1. Moreover, our chronological quantitative analysis demonstrated that virtually all the Lewy body-like hyaline inclusions were immunolabeled by the anti-GRP78 antibody, irrespective to the age of mice examined, even at the presymptomatic stages. These findings imply that GRP78 may bind to, or at least be closely associated with, SOD1, and may participate in the pathological processes leading to inclusion formation. Thus, the results suggest that dysfunction of GRP78 and subsequent derangement of the system responding to unfolded proteins may be involved in the pathogenesis of familial amyotrophic lateral sclerosis caused by a mutation of the human SOD1 gene.

Reducing body myopathy is a rare muscle disease defined by abnormal inclusions in affected muscle fibers that can be stained with menadione-nitroblue tetrazolium. The origin of these inclusions has not been determined. Here we show that reducing bodies bear characteristics of nucleoli. Ultrastructurally, muscle biopsy specimens of a patient with adult-onset reducing body myopathy showed granular structures of reducing bodies with features similar to the granules of nucleoli, which consisted of pre-ribosomes. In addition, reducing bodies were positive for histochemistry of argyrophilic nucleolar organizer regions (a method for detecting the areas where ribosomal RNA is actively transcribed in the nucleolus), and for antibodies against nucleoli and nuclear ribonucleoprotein. The current findings suggest that reducing bodies contain pre-ribosomes and their associated proteins of the nucleolus and that formation of reducing bodies may result from defects of processing and assembly of ribosomes.

The mechanisms of neuronal death in amyotrophic lateral sclerosis (ALS) are not known. A pathological aggregation of cytoplasmic constituents in the form of variety of inclusions may play a role in the pathogenesis of neuronal death. Cytoplasmic basophilic inclusions (BIs) in motor neurons are commonly found in sporadic juvenile ALS. The functional significance of these inclusions is not known, i.e., whether they represent a protective reaction for the isolation of abnormal products from the cytoplasm, or a sign of irreversible neuronal damage. To gain insights on the significance of BIs we asked whether neurons with BIs had an intact or fragmented Golgi apparatus (GA), a sign of neuronal degeneration reported not only in sporadic and familial ALS with mutations of the Cu/Zn superoxide dismutase gene (SOD1), but also in transgenic mice expressing the G93A mutation of SOD1. In these mice fragmentation of the GA of spinal cord motor neurons was found months before the onset of paralysis. We report here that all neurons bearing the inclusions showed fragmentation and reduced number of GA. These results suggest that common pathogenetic mechanisms are involved in the production of BIs and in the fragmentation of the GA.

We applied immunohistochemical techniques to study alterations of the Golgi apparatus, and to determine possible relationships between this complex and the intraneuronal inclusions of lower motor neurons of patients with the sporadic form of amyotrophic lateral sclerosis (ALS). Spinal cords from normal individuals served as controls. Monoclonal antibodies to the Golgi zone and to Golgi beta-COP protein were used. Immunoreactivity with these antibodies was seen in frozen sections of paraformaldehyde-fixed spinal cord tissue, but not in formalin-fixed, paraffin-embedded specimens. The immunoreaction products were seen as granular structures, diffusely distributed in neurons and glial cells. Although immunostaining of some ALS neurons was reduced, fragmentation of the Golgi apparatus was not evident with either antibody. Bunina bodies and skein-like inclusions, characteristically found in spinal anterior horn cells of ALS patients, were not stained by these antibodies to epitopes of the Golgi apparatus, nor were Lewy body-like hyaline inclusions, present in some cases of sporadic ALS and reported to be characteristic to familial ALS with posterior column degeneration. These results suggest that components of the Golgi apparatus are not directly incorporated into intraneuronal inclusions. However, the possibility that abnormal proteinaceous material of the Golgi apparatus may be involved in their genesis cannot be ruled out.

This report concerns the topographic immunohistochemical analysis of the putamen, globus pallidus (GP) and substantia nigra (SN) of two patients with adult-onset motor neuron disease with basophilic inclusions (MND/BIs), seven patients with sporadic classic amyotrophic lateral sclerosis (sporadic ALS) and five neurologically normal individuals. The striatal efferent terminals of the GP and SN were visualized immunohistochemically using antibodies to met-enkephalin (MEnk) and substance P (SP). In specimens from patients with sporadic ALS and normal subjects there was intense immunostaining for MEnk and SP throughout the external and internal segments of the GP, respectively. By contrast, a marked reduction of MEnk- and SP-positive striatal efferents was seen in the ventrocaudal portions of both GP segments from the MND/BIs patients. Moreover, while MEnk-positive striosomes was readily detected in the putamen of normals and sporadic ALS patients, there was significant reduction in MEnk immunoreactivity, and no evidence of striosomal organization in the putamen of MND/BIs patients. In addition, whereas the SN of patients with sporadic ALS expressed SP, the ventrolateral SN portion of the MND/BIs patient tested had reduced immunoreactivity. The present findings on patients with MND/BIs may represent a reflection of the topographic striatum degeneration in this disease and appear to provide additional evidence for the heterogeneity of MND.

This report concerns immunocytochemical and ultrastructural studies on the basophilic inclusions in two cases of sporadic juvenile amyotrophic lateral sclerosis (ALS). The inclusion had a globular, irregular-shaped, or sometimes fragmented appearance. Ultra-structurally, the inclusions consisted mainly of thick filamentous structures associated with granules. Focal neurofilamentous accumulations were occasionally observed among the granulofilamentous structures. The basophilic inclusions occasionally showed granular reaction product deposits with an antibody to ubiquitin. The inclusions did not react with antibodies to phosphorylated neurofilament and to tau protein.

A Schwann cell can form only one internode of myelin around an axon. However, we observed the formation by a single Schwann cell of myelin around two axons of different diameters in the sural nerve of a 45-year-old man with mononeuritis multiplex. Schwann cell processes spiraled in the same direction around each axon, forming mesaxons. The findings in this case appear to be an undescribed type of aberrant myelination.

Biopsy of the sural nerve in a 74-year-old man with chronic neuropathy demonstrated Schwann cells that possessed multiple cisterns of rough endoplasmic reticulum filled with 7- to 8-nm filaments and perinuclear cisterns that were markedly distended with fine granular substances and electron-dense globules. To our knowledge, this is the third case exhibiting filamentous inclusions in Schwann cells. Unlike the previous cases, however, this case showed inclusions in the distended perinuclear cisterns and axonal degeneration. The significance of these alterations remains to be elucidated.

A 36-year-old man developed motor neuron signs consisting of weakness and atrophy of the right upper limb, which progressed to involve the other limbs along with development of upper motor neuron signs including pseudobulbar palsy. He died 8.5 years after onset. Bilateral precentral gyri and putamina were grossly atrophic. In addition to severe degeneration of bilateral pyramidal tracts and marked neuronal cell loss of the precentral gyri and putamina, basophilic inclusions were widely distributed in the motor cortex, putamina, general somatic motor neurons such as the hypoglossal nucleus and spinal anterior horns, and other areas like the red nucleus and inferior olive. The inclusions were clearly shown with Nissl stain to be anilinophilic irregular masses with distinct rims. Ultrastructurally the inclusions appeared to consist of thick filamentous structures of 12-25 nm in diameter studded with electron-dense ribosome-like granules. Thick filamentous profiles were relatively short or occasionally fragmentary, haphazardly mingled with various amounts of granules and other organelles. No prominent accumulation of 10-nm neurofilaments or eosinophilic inclusions like Bunina bodies were found. The inclusions were indistinguishable from those reported in so-called "juvenile" amyotrophic lateral sclerosis.

A case of herpes simplex encephalitis was investigated at the ultrastructural level. Herpes simplex virions were observed, both in nuclei and perikarya of degenerated cells in the cerebrum. Needle-shaped mineralized precipitates were also present in the disintegrated cells. These mineralized depositions were confined to mitochondria. This finding suggests that mitochondria serve as initial nidus of calcification in herpes simplex encephalitis.

Ultrastructural features of chromatolytic neurons observed in a sporadic case with amyotrophic lateral sclerosis (ALS) are reported. A 70-year-old woman died of weakness and atrophy of the four limbs, bulbar and facial muscles, and hyperreflexia, of 3 1/2 years' duration. Neuronal loss was marked in the anterior horn of the spinal cord, with degeneration of the pyramidal tracts. Most of the remaining neurons showed chromatolysis. Some of the chromatolytic neurons contained faintly eosinophilic inclusions with a halo. Few spheroids were observed. Hypoglossal nuclei, nucleus ambiguus, motor nuclei of N.VII and N.V were well populated, but contained several chromatolytic neurons. Ultrastructurally, the chromatolytic neurons contained aggregates of fibrils thicker than the 10-nm neurofilaments. These fibrils were arranged randomly, and were closely associated with granular materials as well as rough endoplasmic reticulum. Neurofilamentous accumulations reported to be common in sporadic ALS were rare in this case. No Bunina body was observed.