Valosin‐containing protein immunoreactivity in tauopathies,synucleinopathies, polyglutamine diseases and intranuclear inclusion body disease

Valosin‐containing protein (VCP) is associated with multiple cellular functions, including ubiquitin‐dependent protein degradation. Mutations in VCP are known to cause inclusion body myopathy with Paget's disease and frontotemporal dementia and familial amyotrophic lateral sclerosis (fALS; ALS14), both of which are characterized by trans‐activation response DNA protein 43 (TDP‐43)‐positive neuronal cytoplasmic and nuclear inclusions. Recently, immunoreactivity for fALS‐associated proteins (TDP‐43, fused in sarcoma (FUS), optineurin and ubiquilin‐2) were reported to be present in cytoplasmic and nuclear inclusions in various neurodegenerative diseases. However, the extent and frequency of VCP‐immunoreactive structures in these neurodegenerative diseases are uncertain. We immunohistochemically examined the brains of 72 cases with neurodegenerative diseases and five control cases. VCP immunoreactivity was present in Lewy bodies in Parkinson's disease and dementia with Lewy bodies, and neuronal nuclear inclusions in five polyglutamine diseases and intranuclear inclusion body disease, as well as in Marinesco bodies in aged control subjects. However, other neuronal and glial cytoplasmic inclusions in tauopathies and TDP‐43 proteinopathies were unstained. These findings suggest that VCP may have common mechanisms in the formation or degradation of cytoplasmic and nuclear inclusions of neurons, but not of glial cells, in several neurodegenerative conditions.     

Unusual clinical presentation and neuropathology in two subjects with fused‐in sarcoma (FUS) positive inclusions

We report two unusual autopsy cases with frontotemporal lobar degeneration (FTLD) that were hyperphosphorylated‐tau‐ and TAR DNA binding protein 43 (TDP‐43)‐ negative. The behavioral symptoms in both cases were compatible with frontotemporal dementia, but they exhibited more prominent speech and language related symptoms than previously reported. Moreover, they displayed a short duration of the disease; the male case had a disease onset age of 45 years, and duration of 5 years, and the female case suffered even shorter disease duration and a later onset of the symptoms, at the age of 67 years. Moreover, the motor functions had deteriorated in different ways in these cases. The male patient showed progressive motor symptoms, weakness of extremities and bulbar muscles suggesting motor neuron disease with a muscle biopsy supporting neurogenic deficits, whereas the female patient exhibited dyskinesias and tremor with progressive swallowing disorders. The father of the male case displayed dementia of similar type at the age of 68 years. In both cases, neuropathological examination showed fused‐in sarcoma (FUS)‐positive pathology. The male patient had intensely FUS‐positive cytoplasmic and intranuclear inclusions that resembled the characteristics previously reported in FTLD FUS, whereas the female patient did not exhibit any cytoplasmic inclusions but had roundish, dense FUS‐positive intranuclear inclusions. She also displayed a plethora of other pathologies including α‐synuclein, hyperphosphorylated‐tau, β‐amyloid aggregation and some neuronal polyglutamine aggregation (1C2) but no well‐demarcated inclusions were observed. We conclude that clinical phenotypes of FUS pathologies also include elderly patients and are more variable with motor and speech disorders than previously reported.     

Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell‐type‐specific

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions could be present in all brain cells. The effects of nuclear inclusion formation have been mainly studied in neurons, while the effect on glia has been comparatively disregarded. Astrocytes, microglia, and oligodendrocytes are glial cells that are essential for normal brain function and are implicated in several neurological diseases. Here we examined the number of nuclear mHTT inclusions in both neurons and various types of glia in the two brain areas that are the most affected in HD, frontal cortex, and striatum. We compared nuclear mHTT inclusion body formation in three HD mouse models that express either full‐length HTT or an N‐terminal exon1 fragment of mHTT, and we observed nuclear inclusions in neurons, astrocytes, oligodendrocytes, and microglia. When studying the frequency of cells with nuclear inclusions in mice, we found that half of the population of neurons contained nuclear inclusions at the disease end stage, whereas the proportion of GFAP‐positive astrocytes and oligodendrocytes having a nuclear inclusion was much lower, while microglia hardly showed any nuclear inclusions. Nuclear inclusions were also present in neurons and all studied glial cell types in human patient material. This is the first report to compare nuclear mHTT inclusions in glia and neurons in different HD mouse models and HD patient brains. GLIA 2016;65:50–61     

Numerous FUS‐positive inclusions in an elderly woman with motor neuron disease

We report an autopsy case of a 75‐year‐old Japanese woman with motor neuron disease (MND) showing numerous neuronal and glial inclusions immunostained with anti‐fused in sarcoma (FUS) antibody. At 73 years, she received a diagnosis of MND and died of respiratory insufficiency 2 years later. No mutation was found in all exons of the FUS gene. Neuropathological examination revealed a reduced number of anterior horn cells and degeneration of the pyramidal tracts. Neither Bunina bodies nor inclusions positive for ubiquitin/phosphorylated TAR DNA binding protein of 43 kD (pTDP‐43), such as skein‐like or round inclusions, were observed. However, basophilic inclusions (BIs) were frequently observed in the remaining neurons of the anterior horns, facial nuclei, hypoglossal nuclei, vestibular nuclei, dentate nuclei and inferior olivary nuclei. In an immunohistochemical analysis, the BIs showed strong immunoreactivity with anti‐FUS and anti‐ubiquitin‐binding protein p62 (p62) antibodies. The nuclear staining of FUS was preserved in some neurons with FUS‐positive inclusions, and a few FUS‐positive glial inclusions were found. FUS‐positive inclusions were more common than p62‐positive inclusions in some anatomical regions, and in some neurons, p62 immunoreactivity was observed in only parts of the BIs. These results suggest that BI formation and TDP‐43 aggregation have different pathogenic mechanisms, and FUS may play an important role in the pathogenesis of MND with BIs. This patient has the oldest reported age of onset for MND with BIs, and clinical features observed in this patient were indistinguishable from those of classic sporadic MND. Therefore, we consider that the age of onset and clinical features of FUS‐related disorders may be variable.     

Transportin 1 colocalization with Fused in Sarcoma (FUS) inclusions is not characteristic for amyotrophic lateral sclerosis‐FUS confirming disrupted nuclear import of mutant FUS and distinguishing it from frontotemporal lobar degeneration with FUS inclusions

C. Troakes, T. Hortobágyi, C. Vance, S. Al‐Sarraj, B. Rogelj and C. E. Shaw (2013) Neuropathology and Applied Neurobiology 39, 553–561 Transportin 1 colocalization with Fused in Sarcoma (FUS) inclusions is not characteristic for amyotrophic lateral sclerosis‐FUS confirming disrupted nuclear import of mutant FUS and distinguishing it from frontotemporal lobar degeneration with FUS inclusions Aims: Transportin 1 (TNPO 1) is an abundant component of the Fused in Sarcoma (FUS)‐immunopositive inclusions seen in a subgroup of frontotemporal lobar degeneration (FTLD‐FUS). TNPO 1 has been shown to bind to the C‐terminal nuclear localizing signal (NLS) of FUS and mediate its nuclear import. Amyotrophic lateral sclerosis (ALS)‐linked C‐terminal mutants disrupt TNPO 1 binding to the NLS and impair nuclear import in cell culture. If this held true for human ALS then we predicted that FUS inclusions in patients with C‐terminal FUS mutations would not colocalize with TNPO 1. Methods: Expression of TNPO 1 and colocalization with FUS was studied in the frontal cortex of FTLD‐FUS (n = 3) and brain and spinal cord of ALS‐FUS (n = 3), ALS‐C9orf72 (n = 3), sporadic ALS (n = 7) and controls (n = 7). Expression levels and detergent solubility of TNPO 1 was measured by Western blot. Results: Aggregates of TNPO 1 were abundant and colocalized with FUS inclusions in the cortex of all FTLD‐FUS cases. In contrast, no TNPO 1‐positive aggregates or FUS colocalization was evident in two‐thirds, ALS‐FUS cases and was rare in one ALS‐FUS case. Nor were they present in C9orf72 or sporadic ALS. No increase in the levels of TNPO 1 was seen in Western blots of spinal cord tissues from all ALS cases compared with controls. Conclusions: These findings confirm that C‐terminal FUS mutations prevent TNPO 1 binding to the NLS, inhibiting nuclear import and promoting cytoplasmic aggregation. The presence of TNPO 1 in wild‐type FUS aggregates in FTLD‐FUS distinguishes the two pathologies and implicates different disease mechanisms.     

Intranuclear Neuronal Inclusions in Huntington''s Disease and Dentatorubral and Pallidoluysian Atrophy: Correlation between the Density of Inclusions andIT15CAG Triplet Repeat Length

Huntington's disease (HD) is caused by CAG triplet repeat expansion inIT15which leads to polyglutamine stretches in the HD protein product, huntingtin. The pathological hallmark of HD is the degeneration of subsets of neurons, primarily those in the striatum and neocortex. Specific morphological markers of affected cells have not been identified in patients with HD, although a unique intranuclear inclusion was recently reported in neurons of transgenic animals expressing a construct encoding the N-terminal part (including the glutamine repeat) of huntingtin (Davieset al., 1997). In order to understand the importance of this finding, we sought for comparable nuclear abnormalities in autopsy material from patients with HD. In all 20 HD cases examined, anti-ubiquitin and N-terminal huntingtin antibodies identified intranuclear inclusions in neurons and the frequency of these lesions correlated with the length of the CAG repeat inIT15. In addition, examination of material from the related HD-like triplet repeat disorder, dentatorubral and pallidoluysian atrophy, also revealed intranuclear neuronal inclusions. These findings suggest that intranuclear inclusions containing protein aggregates may be a common feature of the pathogenesis of glutamine repeat neurodegenerative disorders.     

G protein‐coupled receptor 26 immunoreactivity in intranuclear inclusions associated with polyglutamine and intranuclear inclusion body diseases

G protein‐coupled receptor 26 (GPR26) is one of the G‐protein‐coupled receptors (GPCRs), which comprise the largest family of membrane proteins and mediate most of the physiological responses to hormones, neurotransmitters and environmental stimulants. Although GPCRs are considered to play an important role in the pathophysiology of neurodegenerative disorders, it is uncertain whether GPR26 is involved in the pathogenesis of polyglutamine and intranuclear inclusion body diseases. We immunohistochemically examined the brain tissues of patients with four polyglutamine diseases (Huntington's disease, dentatorubral‐pallidoluysian atrophy, and spinocerebellar ataxia types 1 and 3) and intranuclear inclusion body disease, and normal control subjects. In controls, anti‐GPR26 antibody immunolabeled the neuronal cytoplasm in a diffuse granular pattern. Neuronal nuclear inclusions in polyglutamine diseases were immunopositive for GPR26. In intranuclear inclusion body disease, GPR26‐positive nuclear inclusions were found in both neurons and glial cells. Marinesco bodies in aged control subjects were also positive for GPR26. Double immunofluorescence analysis revealed co‐localization of GPR26 with polyglutamine or ubiquitin in these nuclear inclusions. These findings suggest that GPR26 may have a common role in the formation or degradation of intranuclear inclusions in several neurodegenerative diseases.     

Overexpression of heat shock protein 70 in R6/2 Huntington's disease mice has only modest effects on disease progression

Huntington’s disease (HD) is a neurodegenerative disorder caused by expansion of a polyglutamine tract in a protein called huntingtin. The inducible form of heat shock protein 70 (Hsp70) has been shown to reduce polyglutamine-induced toxicity. To investigate if overexpression of Hsp70 can affect disease progression in a mouse model of HD, we crossed R6/2 mice, expressing exon 1 of the HD gene with an expanded CAG repeat, with mice overexpressing Hsp70 (both types of transgenic mice were of the CBAxC57BL/6 strain). The resulting R6/2-Hsp70 transgenics exhibited 5- to 15-fold increases in Hsp70 expression in neocortical, hippocampal and basal ganglia regions. This correlated with a delayed loss of body weight compared to R6/2 mice. However, the number or size of nuclear inclusions, the loss of brain weight, reduction of striatal volume, reduction in size of striatal projection neurons, downregulation of DARPP-32, development of paw clasping phenotype and early death of the mice were not affected by Hsp70 overexpression. Interestingly, the polyglutamine protein affected the potential rescuing agent, because in older R6/2-Hsp70 mice a large proportion of the Hsp70 protein was sequestrated in nuclear inclusions.     

Nuclear carrier and RNA‐binding proteins in frontotemporal lobar degeneration associated with fused in sarcoma (FUS) pathological changes

Y. S. Davidson, A. C. Robinson, Q. Hu, M. Mishra, A. Baborie, E. Jaros, R. H. Perry, N. J. Cairns, A. Richardson, A. Gerhard, D. Neary, J. S. Snowden, E. H. Bigio and D. M. A. Mann (2013) Neuropathology and Applied Neurobiology 39, 157–165 Nuclear carrier and RNA‐binding proteins in frontotemporal lobar degeneration associated with fused in sarcoma (FUS) pathological changes Aims: We aimed to investigate the role of the nuclear carrier and binding proteins, transportin 1 (TRN1) and transportin 2 (TRN2), TATA‐binding protein‐associated factor 15 (TAF15) and Ewing's sarcoma protein (EWS) in inclusion body formation in cases of frontotemporal lobar degeneration (FTLD) associated with fused in sarcoma protein (FTLD‐FUS). Methods: Eight cases of FTLD‐FUS (five cases of atypical FTLD‐U, two of neuronal intermediate filament inclusion body disease and one of basophilic inclusion body disease) were immunostained for FUS, TRN1, TRN2, TAF15 and EWS. Ten cases of FTLD associated with TDP‐43 inclusions served as reference cases. Results: The inclusion bodies in FTLD‐FUS contained TRN1 and TAF15 and, to a lesser extent, EWS, but not TRN2. The patterns of immunostaining for TRN1 and TAF15 were very similar to that of FUS. None of these proteins was associated with tau or TDP‐43 aggregations in FTLD. Conclusions: Data suggest that FUS, TRN1 and TAF15 may participate in a functional pathway in an interdependent way, and imply that the function of TDP‐43 may not necessarily be in parallel with, or complementary to, that of FUS, despite each protein sharing many similar structural elements.     

Huntington aggregates may not predict neuronal death in Huntington's disease

The mechanism by which polyglutamine expansion in Huntington's disease (HD) results in selective neuronal degeneration remains unclear. We previously reported that the immunohistochemical distribution of N-terminal huntingtin in HD does not correspond to the severity of neuropathology, such that significantly greater numbers of huntingtin aggregates are present within the cortex than in the striatum. We now show a dissociation between huntingtin aggregation and the selective pattern of striatal neuron loss observed in HD. Aggregate formation was predominantly observed in spared interneurons, with few or no aggregates found within vulnerable spiny striatal neurons. Multiple perikaryal aggregates were present in almost all cortical NADPH-diaphorase neurons and in approximately 50% of the spared NADPH-diaphorase striatal neurons from early grade HD cases. In severe grade HD patients, aggregates were more prominent as nuclear inclusions in NADPH-diaphorase neurons, with less perikaryal and neuropil aggregation. In contrast, nuclear or perikaryal huntingtin aggregates were present in less than 4% of the vulnerable calbindin striatal neurons in all HD cases. These findings support the hypothesis that polyglutamine aggregation may not be a predictor of cell loss. Rather than a harbinger of neuronal death, mutant huntingtin aggregation may be a cytoprotective mechanism against polyglutamine-induced neurotoxicity.     

Huntington's disease intranuclear inclusions contain truncated, ubiquitinated huntingtin protein

Intranuclear inclusion bodies are a shared pathological feature of Huntington's disease (HD) and its transgenic mouse model. Using a panel of antibodies spanning the entire huntingtin molecule, we have investigated the pattern of immunoreactivity within the intranuclear inclusions in the frontal cortex and striatum of patients with HD. The intranuclear inclusions reacted with anti-ubiquitin and antibodies against the N-terminal portion of huntingtin (CAG53b, HP1), but not with HD1 and the 1C2 antibodies that detect the expanded polyglutamine tract nor the more C-terminal antibodies. However, the 1C2, HP1, CAG53b, and HD1 antibodies detected granular cytoplasmic deposits in cortical and striatal neurons that also contained intranuclear N-terminal huntingtin immunoreactivity. These data show a differential intracellular location of truncated huntingtin in the HD brain. Both the cytoplasmic and the nuclear aggregates of the protein fragments could be neurotoxic. The frequency of the cortical intranuclear inclusions correlated with the size of CAG expansion and was inversely related to the age at onset and death. No such correlations were detected for the striatum, which most likely reflects a more advanced neuronal loss accrued by the time of death.     

B2 attenuates polyglutamine‐expanded androgen receptor toxicity in cell and fly models of spinal and bulbar muscular atrophy

Expanded polyglutamine tracts cause neurodegeneration through a toxic gain‐of‐function mechanism. Generation of inclusions is a common feature of polyglutamine diseases and other protein misfolding disorders. Inclusion formation is likely to be a defensive response of the cell to the presence of unfolded protein. Recently, the compound B2 has been shown to increase inclusion formation and decrease toxicity of polyglutamine‐expanded huntingtin in cultured cells. We explored the effect of B2 on spinal and bulbar muscular atrophy (SBMA). SBMA is caused by expansion of polyglutamine in the androgen receptor (AR) and is characterized by the loss of motor neurons in the brainstem and spinal cord. We found that B2 increases the deposition of mutant AR into nuclear inclusions, without altering the ligand‐induced aggregation, expression, or subcellular distribution of the mutant protein. The effect of B2 on inclusions was associated with a decrease in AR transactivation function. We show that B2 reduces mutant AR toxicity in cell and fly models of SBMA, further supporting the idea that accumulation of polyglutamine‐expanded protein into inclusions is protective. Our findings suggest B2 as a novel approach to therapy for SBMA. © 2010 Wiley‐Liss, Inc.     

ALS‐associated protein FIG4 is localized in Pick and Lewy bodies,and also neuronal nuclear inclusions,in polyglutamine and intranuclear inclusion body diseases

FIG4 is a phosphatase that regulates intracellular vesicle trafficking along the endosomal‐lysosomal pathway. Mutations of FIG4 lead to the development of Charcot‐Marie‐Tooth disease type 4J and amyotrophic lateral sclerosis (ALS). Moreover, ALS‐associated proteins (transactivation response DNA protein 43 (TDP‐43), fused in sarcoma (FUS), optineurin, ubiquilin‐2, charged mutivesicular body protein 2b (CHMP2B) and valosin‐containing protein) are involved in inclusion body formation in several neurodegenerative diseases. Using immunohistochemistry, we examined the brains and spinal cords of patients with various neurodegenerative diseases, including sporadic TDP‐43 proteinopathy (ALS and frontotemporal lobar degeneration). TDP‐43 proteinopathy demonstrated no FIG4 immunoreactivity in neuronal inclusions. However, FIG4 immunoreactivity was present in Pick bodies in Pick's disease, Lewy bodies in Parkinson's disease and dementia with Lewy bodies, neuronal nuclear inclusions in polyglutamine and intranuclear inclusion body diseases, and Marinesco and Hirano bodies in aged control subjects. These findings suggest that FIG4 is not incorporated in TDP‐43 inclusions and that it may have a common role in the formation or degradation of neuronal cytoplasmic and nuclear inclusions in several neurodegenerative diseases.     

Coexistence of Huntington’s disease and amyotrophic lateral sclerosis: a clinicopathologic study

We report a retrospective case series of four patients with genetically confirmed Huntington’s disease (HD) and sporadic amyotrophic lateral sclerosis (ALS), examining the brain and spinal cord in two cases. Neuropathological assessment included a polyglutamine recruitment method to detect sites of active polyglutamine aggregation, and biochemical and immunohistochemical assessment of TDP-43 pathology. The clinical sequence of HD and ALS varied, with the onset of ALS occurring after the mid-50’s in all cases. Neuropathologic features of HD and ALS coexisted in both cases examined pathologically: neuronal loss and gliosis in the neostriatum and upper and lower motor neurons, with Bunina bodies and ubiquitin-immunoreactive skein-like inclusions in remaining lower motor neurons. One case showed relatively early HD pathology while the other was advanced. Expanded polyglutamine-immunoreactive inclusions and TDP-43-immunoreactive inclusions were widespread in many regions of the CNS, including the motor cortex and spinal anterior horn. Although these two different proteinaceous inclusions coexisted in a small number of neurons, the two proteins did not co-localize within inclusions. The regional distribution of TDP-43-immunoreactive inclusions in the cerebral cortex partly overlapped with that of expanded polyglutamine-immunoreactive inclusions. In the one case examined by TDP-43 immunoblotting, similar TDP-43 isoforms were observed as in ALS. Our findings suggest the possibility that a rare subset of older HD patients is prone to develop features of ALS with an atypical TDP-43 distribution that resembles that of aggregated mutant huntingtin. Age-dependent neuronal dysfunction induced by mutant polyglutamine protein expression may contribute to later-life development of TDP-43 associated motor neuron disease in a small subset of patients with HD.     

Methylene Blue Modulates Huntingtin Aggregation Intermediates and Is Protective in Huntington's Disease Models

Huntington's disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB), has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD.     

PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.     

Overexpression of human wild-type FUS causes progressive motor neuron degeneration in an age- and dose-dependent fashion

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are relentlessly progressive neurodegenerative disorders with overlapping clinical, genetic and pathological features. Cytoplasmic inclusions of fused in sarcoma (FUS) are the hallmark of several forms of FTLD and ALS patients with mutations in the FUS gene. FUS is a multifunctional, predominantly nuclear, DNA and RNA binding protein. Here, we report that transgenic mice overexpressing wild-type human FUS develop an aggressive phenotype with an early onset tremor followed by progressive hind limb paralysis and death by 12 weeks in homozygous animals. Large motor neurons were lost from the spinal cord accompanied by neurophysiological evidence of denervation and focal muscle atrophy. Surviving motor neurons in the spinal cord had greatly increased cytoplasmic expression of FUS, with globular and skein-like FUS-positive and ubiquitin-negative inclusions associated with astroglial and microglial reactivity. Cytoplasmic FUS inclusions were also detected in the brain of transgenic mice without apparent neuronal loss and little astroglial or microglial activation. Hemizygous FUS overexpressing mice showed no evidence of a motor phenotype or pathology. These findings recapitulate several pathological features seen in human ALS and FTLD patients, and suggest that overexpression of wild-type FUS in vulnerable neurons may be one of the root causes of disease. Furthermore, these mice will provide a new model to study disease mechanism, and test therapies.     

An autopsy case of neuronal intermediate filament inclusion disease with regard to immunophenotypic and topographical analysis of the neuronal inclusions

We report an autopsy case of neuronal intermediate filament inclusion disease (NIFID), in which pyramidal motor dysfunction preceded cognitive disturbance for 3 years from the onset. A 41‐year‐old Japanese man presented progressive spastic tetraparesis followed by cognitive impairment. His neurological symptoms gradually deteriorated and he died of pneumonia 16 years from the onset. His brain showed severe generalized atrophy with enlargement of ventricles. The microscopic examination revealed severe neuronal loss with gliosis and sponginess predominantly in the fronto‐temporal cortices, caudate and putamen. Many hyaline conglomerate inclusions (HC) without immunoreactivity for ‘fused in sarcoma’ protein (FUS) and some granular and small round FUS‐immunoreactive (FUS‐ir) neuronal cytoplasmic inclusions (NCI) were observed in the remaining neurons. Some neurons with HC had small basophilic inclusions which showed positive FUS‐ir, attached to HC in the cytoplasm. Otherwise, FUS‐ir large compact inclusions (so‐called Pick‐like) were also observed but were scarce. In the cerebral cortex and the neostriatum, frequency of the inclusions was well correlated with neuronal loss. In the brainstem, neuronal loss was mild and FUS‐ir inclusions dominated. In the subthalamic nucleus and red nucleus, there was no HC but there were many FUS‐ir inclusions without neuronal loss. The above findings suggest that cytoplasmic mislocalization and aggregation of FUS appear at the early stage of the disease, and the FUS aggregate process may not be a direct precedent structure of HC.