FUS-positive Inclusions Research Articles

A postzygotic de novo NCDN mutation identified in a sporadic FTLD patient results in neurochondrin haploinsufficiency and altered FUS granule dynamics

Frontotemporal dementia (FTD) is a heterogeneous clinical disorder characterized by progressive abnormalities in behavior, executive functions, personality, language and/or motricity. A neuropathological subtype of FTD, frontotemporal lobar degeneration (FTLD)-FET, is characterized by protein aggregates consisting of the RNA-binding protein fused in sarcoma (FUS). The cause of FTLD-FET is not well understood and there is a lack of genetic evidence to aid in the investigation of mechanisms of the disease. The goal of this study was to identify genetic variants contributing to FTLD-FET and to investigate their effects on FUS pathology. We performed whole-exome sequencing on a 50-year-old FTLD patient with ubiquitin and FUS-positive neuronal inclusions and unaffected parents, and identified a de novo postzygotic nonsense variant in the NCDN gene encoding Neurochondrin (NCDN), NM_014284.3:c.1206G > A, p.(Trp402*). The variant was associated with a ~ 31% reduction in full-length protein levels in the patient’s brain, suggesting that this mutation leads to NCDN haploinsufficiency. We examined the effects of NCDN haploinsufficiency on FUS and found that depleting primary cortical neurons of NCDN causes a reduction in the total number of FUS-positive cytoplasmic granules. Moreover, we found that these granules were significantly larger and more highly enriched with FUS. We then examined the effects of a loss of FUS function on NCDN in neurons and found that depleting cells of FUS leads to a decrease in NCDN protein and mRNA levels. Our study identifies the NCDN protein as a likely contributor of FTLD-FET pathophysiology. Moreover, we provide evidence for a negative feedback loop of toxicity between NCDN and FUS, where loss of NCDN alters FUS cytoplasmic dynamics, which in turn has an impact on NCDN expression.

Characteristic Features of FUS Inclusions in Spinal Motor Neurons of Sporadic Amyotrophic Lateral Sclerosis.

Alterations of RNA metabolism caused by mutations in RNA-binding protein genes, such as transactivating DNA-binding protein-43 (TDP-43) and fused in sarcoma (FUS), have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Unlike the accumulation of TDP43, which is accepted as a pathological hall mark of sporadic ALS (sALS), FUS pathology in sALS is still under debate. Although immunoreactive inclusions of FUS have been detected in sALS patients previously, the technical limitation of signal detection, including the necessity of specific antigen retrieval, restricts our understanding of FUS-associated ALS pathology. In this study, we applied a novel detection method using a conventional antigen retrieval technique with Sudan Black B treatment to identify FUS-positive inclusions in sALS patients. We classified pathological motor neurons into 5 different categories according to the different aggregation characteristics of FUS and TDP-43. Although the granular type was more dominant for inclusions with TDP-43, the skein-like type was more often observed in FUS-positive inclusions, suggesting that these 2 proteins undergo independent aggregation processes. Moreover, neurons harboring FUS-positive inclusions demonstrated substantially reduced expression levels of dynactin-1, a retrograde motor protein, indicating that perturbation of nucleocytoplasmic transport is associated with the formation of cytoplasmic inclusions of FUS in sALS.

Neuronal intermediate filament inclusion disease may be incorrectly classified as a subtype of FTLD-FUS.

The majority of cases of frontotemporal lobar degeneration (FTLD) are characterized by focal cortical atrophy with an underlying tau or TDP-43 proteinopathy. A subset of FTLD cases, however, lack tau and TDP-43 immuno-reactivity, but have neuronal inclusions positive for ubiquitin, referred to as atypical FTLD (aFTLD-U). Studies have demonstrated that ubiquitin-positive inclusions in aFTLD-U are immuno-reactive for fused in sarcoma (FUS). As such, the current nosology for this entity is FTLD-FUS, which is thought to include not only aFTLD-U, but also neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease. To compare pathological features of cases of aFTLD-U and NIFID. We reviewed the neuropathology of 15 patients (10 males and 5 females; average age at death 54 years (range 41-69 years)) with an antemortem clinical diagnosis of a frontotemporal dementia and pathological diagnosis of aFTLD-U (n=8) or NIFID (n=7). Sections were processed for immunohistochemistry and immunoelectron microscopy with FUS, TDP-43, and α-internexin (αINX) antibodies. Eight cases had pathologic features consistent with FTLD-FUS, with severe striatal atrophy (7/8 cases), as well as FUS-positive neuronal cytoplasmic and vermiform intranuclear inclusions, but no αINX immuno-reactivity. Five cases had features consistent with NIFID, with neuronal inclusions positive for both FUS and αINX. Striatal atrophy was present in only 2 of the NIFID cases. Two cases had αINX-positive neuronal inclusions consistent with NIFID, but both lacked striatal atrophy and FUS immunoreactivity. Surprisingly, one of these two NIFID cases had lesions immunoreactive for TDP-43. While FUS pathology remains a prominent feature of aFTLD-U, there is pathologic heterogeneity, including rare cases of NIFID with TDP-43- rather than FUS-positive inclusions.

Antiviral Immune Response as a Trigger of FUS Proteinopathy in Amyotrophic Lateral Sclerosis

SummaryMutations in the FUS gene cause familial amyotrophic lateral sclerosis (ALS-FUS). In ALS-FUS, FUS-positive inclusions are detected in the cytoplasm of neurons and glia, a condition known as FUS proteinopathy. Mutant FUS incorporates into stress granules (SGs) and can spontaneously form cytoplasmic RNA granules in cultured cells. However, it is unclear what can trigger the persistence of mutant FUS assemblies and lead to inclusion formation. Using CRISPR/Cas9 cell lines and patient fibroblasts, we find that the viral mimic dsRNA poly(I:C) or a SG-inducing virus causes the sustained presence of mutant FUS assemblies. These assemblies sequester the autophagy receptor optineurin and nucleocytoplasmic transport factors. Furthermore, an integral component of the antiviral immune response, type I interferon, promotes FUS protein accumulation by increasing FUS mRNA stability. Finally, mutant FUS-expressing cells are hypersensitive to dsRNA toxicity. Our data suggest that the antiviral immune response is a plausible second hit for FUS proteinopathy.

FUS(1-359) transgenic mice as a model of ALS: pathophysiological and molecular aspects of the proteinopathy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the eventual death of motor neurons. Described cases of familial ALS have emphasized the significance of protein misfolding and aggregation of two functionally related proteins, FUS (fused in sarcoma) and TDP-43, implicated in RNA metabolism. Herein, we performed a comprehensive analysis of the in vivo model of FUS-mediated proteinopathy (ΔFUS(1-359) mice). First, we used the Noldus CatWalk system and confocal microscopy to determine the time of onset of the first clinical symptoms and the appearance of FUS-positive inclusions in the cytoplasm of neuronal cells. Second, we applied RNA-seq to evaluate changes in the gene expression profile encompassing the pre-symptomatic and the symptomatic stages of disease progression in motor neurons and the surrounding microglia of the spinal cord. The resulting data show that FUS-mediated proteinopathy is virtually asymptomatic in terms of both the clinical symptoms and the molecular aspects of neurodegeneration until it reaches the terminal stage of disease progression (120days from birth). After this time, the pathological process develops very rapidly, resulting in the formation of massive FUS-positive inclusions accompanied by a transcriptional "burst" in the spinal cord cells. Specifically, it manifests in activation of a pro-inflammatory phenotype of microglial cells and malfunction of acetylcholine synapse transmission in motor neurons. Overall, we assume that the highly reproducible course of the pathological process, as well as the described accompanying features, makes ΔFUS(1-359) mice a convenient model for testing potential therapeutics against proteinopathy-induced decay of motor neurons.

Neuron-to-Neuron Transfer of FUS in Drosophila Primary Neuronal Culture Is Enhanced by ALS-Associated Mutations.

The DNA- and RNA-binding protein fused in sarcoma (FUS) has been pathologically and genetically linked to amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Cytoplasmic FUS-positive inclusions were identified in the brain and spinal cord of a subset of patients suffering with ALS/FTLD. An increasing number of reports suggest that FUS protein can behave in a prion-like manner. However, no neuropathological studies or experimental data were available regarding cell-to-cell spread of these pathological protein assemblies. In the present report, we investigated the ability of wild-type and mutant forms of FUS to transfer between neuronal cells. We combined the use of Drosophila models for FUS proteinopathies with that of the primary neuronal cultures to address neuron-to-neuron transfer of FUS proteins. Using conditional co-culture models and an optimized flow cytometry-based methodology, we demonstrated that ALS-mutant forms of FUS proteins can transfer between well-differentiated mature Drosophila neurons. These new observations support that a propagating mechanism could be applicable to FUS, leading to the sequential dissemination of pathological proteins over years.

The presence of heterogeneous nuclear ribonucleoproteins in frontotemporal lobar degeneration with FUS-positive inclusions

Frontotemporal lobar degeneration with fused in sarcoma–positive inclusions (FTLD-FUS) is a disease with unknown cause. Transportin 1 is abundantly found in FUS-positive inclusions and responsible for the nuclear import of the FET proteins of which FUS is a member. The presence of all FET proteins in pathological inclusions suggests a disturbance of transportin 1–mediated nuclear import. FUS also belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) protein family. We investigated whether hnRNP proteins are associated with FUS pathology implicating dysfunctional nuclear export in the pathogenesis of FTLD-FUS. hnRNP proteins were investigated in affected brain regions in FTLD-FUS using immunohistochemistry, biochemical analysis, and the expression analysis. We demonstrated the presence of several hnRNP proteins in pathological inclusions including neuronal cytoplasmic inclusions and dystrophic neurites. The biochemical analysis revealed a shift in the location of hnRNP A1 from the nucleus to the cytoplasm. The expression analysis revealed an increase in several hnRNP proteins in FTLD-FUS. These results implicate a wider dysregulation of movement between intracellular compartments, than mechanisms only affecting the nuclear import of FUS proteins.

Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation

Mutations in the fused in sarcoma (FUS) gene can cause amyotrophic lateral sclerosis (ALS), and FUS gene mutations have been reported in sporadic ALS patients with basophilic cytoplasmic inclusions. Deficiency of adenosine deaminase acting on RNA 2 (ADAR2), an enzyme that specifically catalyzes GluA2 Q/R site-editing, has been reported in considerable proportions of spinal motor neurons of the majority of sporadic ALS patients. We describe the relationship between GluA2 Q/R site-editing efficiency and FUS-positive inclusions in a patient with FUSP525L. A 24-year-old woman with ALS presented with basophilic cytoplasmic inclusions, significantly reduced GluA2 Q/R site-editing efficiency in the spinal motor neurons, and markedly decreased ADAR2 mRNA levels. Neuropathologic examination showed that not all spinal motor neurons expressed ADAR2 and revealed FUS-positive cytoplasmic inclusions in motor neurons irrespective of ADAR2 immunoreactivity. There were no phosphorylated transactive response (TAR) DNA-binding protein 43 kDa (TDP-43)-positive inclusions, indicating that there was no tight correlation between ADAR2 deficiency and TDP-43 deposition. ADAR2 deficiency can occur in ALS patients with a FUSP525L mutation and is unrelated to the presence of FUS-positive inclusions. FUS-associated ALS may share neurodegenerative characteristics with classical sporadic ALS.

ALS-associated mutant FUS inhibits macroautophagy which is restored by overexpression of Rab1.

Amyotrophic lateral sclerosis (ALS) is characterised by the formation of intracellular misfolded protein inclusions that form in motor neurons. Autophagy is the major degradation pathway for aggregate-prone proteins within lysosomes. Autophagy begins by the production of the omegasome, forming the autophagosome membrane, which then fuses with the lysosome. Mutations in fused in sarcoma (FUS) cause 5% of familial ALS cases and FUS-positive inclusions are also formed in sporadic ALS tissues. In this study, we demonstrate that the expression of ALS-associated mutant FUS impairs autophagy in neuronal cells. In mutant FUS-expressing neuronal cells, accumulation of ubiquitinated proteins and autophagy substrates p62 and NBR1 was detected, and formation of both the omegasome and autophagosome was inhibited in these cells. However, overexpression of Rab1 rescued these defects, suggesting that Rab1 is protective in ALS. The number of LC3-positive vesicles was also increased in motor neurons from the spinal cord of an ALS patient carrying a FUS (R521C) mutation compared with a control patient, providing additional evidence that autophagy is dysregulated in mutant FUS-associated ALS. This study provides further understanding of the intricate autophagy system and neurodegeneration in ALS.

Accumulation of transportin 1 in the fused in sarcoma-positive neuronal inclusions in sporadic amyotrophic lateral sclerosis withoutFUSmutation

We describe a female patient with sporadic amyotrophic lateral sclerosis (ALS) with fused in sarcoma (FUS)-immunoreactive neuronal inclusions. She presented with weakness mainly in the upper extremities that had started at the age of 53 years. She died at the age of 61 years. Neuronal loss and gliosis accompanying FUS-positive neuronal cytoplasmic inclusions involved the upper and lower motor neurons, brainstem motor neurons, basal ganglia, substantia nigra, cerebellar dentate nuclei, thalamus, hypothalamic nuclei, and red nuclei. Most of these inclusions were positive for transportin 1, in a pattern similar to frontotemporal lobar degeneration with FUS and different from familial ALS-FUS. Mutational analysis showed no mutations in the FUS gene. Our findings show that transportin 1 accumulates in the FUS-positive inclusions in sporadic ALS-FUS.

An unusual autopsy case of progressive muscular atrophy with widespread fus positive inclusions

Progressive muscular atrophy (PMA) is the lower motor neuron-predominant form of motor neuron disease (MND). MND is a relatively common, progressive fatal neurodegenerative disease, the pathogenetic basis of which is not fully understood. Recent genetic studies have identified mutations in the Fused in sarcoma/ translated in liposarcoma (FUS/TLS) gene at chromosome 16 in approximately 3% of familial MND and less than 1% of sporadic patients. The mutation usually leads to an earlier onset and more aggressive phenotype of MND. In this case study we describe the autopsy neuropathology of a late-onset and seemingly sporadic case of PMA. The pathology was dominated by loss of lower motor neurons and widespread FUS positive cytoplasmic inclusions. These inclusions were negative for TDP-43, distinguishing them from typical sporadic MND cases. FUS positive cytoplasmic inclusions were also found in other regions, in both neurons and glia. They were present in brainstem nuclei, cerebellum and deep grey structures but were absent from hippocampus and neocortical areas. This unique case broadens the phenotypic and neuropatho-logical spectrum of FUS-related neurodegenerative conditions and may provide additional insights into the role of FUS in both sporadic and familial MND.

Phenotypic Variability Within the Inclusion Body Spectrum of Basophilic Inclusion Body Disease and Neuronal Intermediate Filament Inclusion Disease in Frontotemporal Lobar Degenerations With FUS-Positive Inclusions

Basophilic inclusion body disease and neuronal intermediate filament inclusion disease (NIFID) are rare diseases included among frontotemporal lobar degenerations with FUS-positive inclusions (FTLD-FUS). We report clinical and pathologic features of 2 new patients and reevaluate neuropathologic characteristics of 2 previously described cases, including an early-onset case of basophilic inclusion body disease (aged 38 years) with a 5-year disease course and abundant FUS-positive inclusion bodies and 3 NIFID cases. One NIFID case (aged 37 years) presented with early-onset psychiatric disturbances and rapidly progressive cognitive decline. Two NIFID cases had later onset (aged 64 years and 70 years) and complex neurologic deficits. Postmortem neuropathologic studies in late-onset NIFID cases disclosed α-internexin-positive "hyaline conglomerate"-type inclusions that were positive with 1 commercial anti-FUS antibody directed to residues 200 and 250, but these were negative to amino acids 90 and 220 of human FUS. Early-onset NIFID had similar inclusions that were positive with both commercial anti-FUS antibodies. Genetic testing performed on all cases revealed no FUS gene mutations. These findings indicate that phenotypic variability in NIFID, including clinical manifestations and particular neuropathologic findings, may be related to the age at onset and individual differences in the evolution of lesions.

Transportin 1 accumulates specifically with FET proteins but no other transportin cargos in FTLD-FUS and is absent in FUS inclusions in ALS with FUS mutations

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.

Requirements for Stress Granule Recruitment of Fused in Sarcoma (FUS) and TAR DNA-binding Protein of 43 kDa (TDP-43)

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.

FET proteins in frontotemporal dementia and amyotrophic lateral sclerosis

Mutations in the fused in sarcoma gene (FUS) cause amyotrophic lateral sclerosis (ALS) with TDP-43-negative, FUS-positive pathology. FUS is also the pathological protein in most tau/TDP-43-negative subtypes of frontotemporal lobar degeneration (FTLD-FUS). FUS, together with Ewing's sarcoma protein (EWS) and TATA-binding protein associated factor 15 (TAF15), make up the FET family of DNA/RNA binding proteins that share functional homology and have the potential to interact. We recently investigated the role of the other FET proteins in the clinicopathological spectrum of FUS-opathies. In all FTLD-FUS subtypes, FUS-positive pathology was also labeled for TAF15 and EWS and cells with inclusions showed a reduction in the normal nuclear staining of all FET proteins. In contrast, in cases of ALS-FUS, TAF15 and EWS remained localized to the nucleus and did not label FUS-positive inclusions. Cell culture models replicated the human diseases. These findings indicate that ALS-FUS and FTLD-FUS have different pathomechanisms and add TAF15 and EWS to the growing list of RNA-binding proteins involved in neurodegeneration.This article is part of a Special Issue entitled: RNA-Binding Proteins.

Transportin1: a marker of FTLD-FUS

The term frontotemporal lobar degeneration (FTLD) describes a group of disorders that are subdivided by the presence of one of a number of pathological proteins identified in the inclusion bodies observed post-mortem. The FUS variant is defined by the presence of the fused in sarcoma protein (FUS) in the pathological inclusions. However, similar to other FTLDs, the disease pathogenesis of FTLD-FUS remains largely poorly understood. Here we present data that the protein transportin1 (TRN1) is abundant in the FUS-positive inclusions. TRN1, the protein product of the TNP01 gene, is responsible for shuttling proteins containing an M9 nuclear localisation signal between the nuclear and cytoplasmic compartments. RNA interacting proteins, including FUS, have been implicated as targets of TRN1. Using TRN1 immunohistochemistry and Western blotting in this study, we investigated 13 cases of FTLD-FUS including 6 cases with neuronal intermediate filament inclusion disease (NIFID) and 7 atypical frontotemporal lobar degeneration with ubiquitinated inclusion (aFTLD-U) cases. The data from our immunohistochemical studies show that FUS-immunoreactive inclusions are also strongly labelled with the anti-TRN1 antibody and double-label immunofluorescence studies indicate good co-localisation between the FUS and TRN1 pathologies. Our biochemical investigations demonstrate that urea-soluble TRN1 is present in aFTLD-U and NIFID, but not in normal control brains. These findings implicate abnormalities of FUS transport in the pathogenesis of FTLD-FUS.

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.

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.

ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import

Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD-FUS). We show that a non-classical PY nuclear localization signal (NLS) in the C-terminus of FUS is necessary for nuclear import. The majority of fALS-associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease-causing mutations within a PY-NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co-deposit with inclusions in fALS and FTLD-FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS-opathies.

Glucose metabolism in progressive non-fluent aphasia patients with versus without parkinsonism

Conclusions: FUS protein accumulation is characteristic in a group of FTLDs with ubiquitin-positive pathology. The clinical features are heterogeneous although the most common association is with bvFTD. Clinical and pathological evidence of MND may be found in cases previously diagnosed as NIFID. FUS-positive inclusions in both cerebral cortex and motor neurons in NIFID and aFTLD-U extend the spectrum of diseases with frontotemporal degeneration and involvement of the motor system.