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Neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration are rare diseases characterized by ubiquitin-positive inclusions lacking transactive response DNA-binding protein-43 and tau. Recently, mutations in the fused in sarcoma gene have been shown to cause familial amyotrophic lateral sclerosis and fused in sarcoma-positive neuronal inclusions have subsequently been demonstrated in neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration with ubiquitinated inclusions. Here we provide clinical, imaging, morphological findings, as well as genetic and biochemical data in 14 fused in sarcoma proteinopathy cases. In this cohort, the age of onset was variable but included cases of young-onset disease. Patients with atypical frontotemporal lobar degeneration with ubiquitinated inclusions all presented with behavioural variant frontotemporal dementia, while the clinical presentation in neuronal intermediate filament inclusion disease was more heterogeneous, including cases with motor neuron disease and extrapyramidal syndromes. Neuroimaging revealed atrophy of the frontal and anterior temporal lobes as well as the caudate in the cases with atypical frontotemporal lobar degeneration with ubiquitinated inclusions, but was more heterogeneous in the cases with neuronal intermediate filament inclusion disease, often being normal to visual inspection early on in the disease. The distribution and severity of fused in sarcoma-positive neuronal cytoplasmic inclusions, neuronal intranuclear inclusions and neurites were recorded and fused in sarcoma was biochemically analysed in both subgroups. Fused in sarcoma-positive neuronal cytoplasmic and intranuclear inclusions were found in the hippocampal granule cell layer in variable numbers. Cortical fused in sarcoma-positive neuronal cytoplasmic inclusions were often ‘Pick body-like’ in neuronal intermediate filament inclusion disease, and annular and crescent-shaped inclusions were seen in both conditions. Motor neurons contained variable numbers of compact, granular or skein-like cytoplasmic inclusions in all fused in sarcoma-positive cases in which brainstem and spinal cord motor neurons were available for study (five and four cases, respectively). No fused in sarcoma mutations were found in any cases. Biochemically, two major fused in sarcoma species were found and shown to be more insoluble in the atypical frontotemporal lobar degeneration with ubiquitinated inclusions subgroup compared with neuronal intermediate filament inclusion disease. There is considerable overlap and also significant differences in fused in sarcoma-positive pathology between the two subgroups, suggesting they may represent a spectrum of the same disease. The co-existence of fused in sarcoma-positive inclusions in both motor neurons and extramotor cerebral structures is a characteristic finding in sporadic fused in sarcoma proteinopathies, indicating a multisystem disorder.

Background

Mutations in the gene encoding the RNA-binding protein fused in sarcoma (FUS) can cause familial and sporadic amyotrophic lateral sclerosis (ALS) and rarely frontotemproal dementia (FTD). FUS accumulates in neuronal cytoplasmic inclusions (NCIs) in ALS patients with FUS mutations. FUS is also a major pathologic marker for a group of less common forms of frontotemporal lobar degeneration (FTLD), which includes atypical FTLD with ubiquitinated inclusions (aFTLD-U), neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease (BIBD). These diseases are now called FUS proteinopathies, because they share this disease marker. It is unknown how FUS mutations cause disease and the role of FUS in FTD-FUS cases, which do not have FUS mutations. In this paper we report the development of somatic brain transgenic (SBT) mice using recombinant adeno-associated virus (rAAV) to investigate how FUS mutations lead to neurodegeneration.

Results

We compared SBT mice expressing wild-type human FUS (FUSWT), and two ALS-linked mutations: FUSR521C and FUSΔ14, which lacks the nuclear localization signal. Both FUS mutants accumulated in the cytoplasm relative to FUSWT. The degree of this shift correlated with the severity of the FUS mutation as reflected by disease onset in humans. Mice expressing the most aggressive mutation, FUSΔ14, recapitulated many aspects of FUS proteinopathies, including insoluble FUS, basophilic and eosiniphilic NCIs, and other pathologic markers, including ubiquitin, p62/SQSTM1, α-internexin, and the poly-adenylate(A)-binding protein 1 (PABP-1). However, TDP-43 did not localize to inclusions.

Conclusions

Our data supports the hypothesis that ALS or FTD-linked FUS mutations cause neurodegeneration by increasing cyotplasmic FUS. Accumulation of FUS in the cytoplasm may retain RNA targets and recruit additional RNA-binding proteins, such as PABP-1, into stress-granule like aggregates that coalesce into permanent inclusions that could negatively affect RNA metabolism. Identification of mutations in other genes that cause ALS/FTD, such as C9ORF72, sentaxin, and angiogenin, lends support to the idea that defective RNA metabolism is a critical pathogenic pathway. The SBT FUS mice described here will provide a valuable platform for dissecting the pathogenic mechanism of FUS mutations, define the relationship between FTD and ALS-FUS, and help identify therapeutic targets that are desperately needed for these devastating neurodegenerative disorders.

The FET family of proteins is composed of FUS/TLS, EWS/EWSR1, and TAF15 and possesses RNA- and DNA-binding capacities. The FET-proteins are involved in transcriptional regulation and RNA processing, and FET-gene deregulation is associated with development of cancer and protein granule formations in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and trinucleotide repeat expansion diseases. We here describe a comparative characterization of FET-protein localization and gene regulatory functions. We show that FUS and TAF15 locate to cellular stress granules to a larger extend than EWS. FET-proteins have no major importance for stress granule formation and cellular stress responses, indicating that FET-protein stress granule association most likely is a downstream response to cellular stress. Gene expression analyses showed that the cellular response towards FUS and TAF15 reduction is relatively similar whereas EWS reduction resulted in a more unique response. The presented data support that FUS and TAF15 are more functionally related to each other, and that the FET-proteins have distinct functions in cellular signaling pathways which could have implications for the neurological disease pathogenesis.

Neuronal intermediate filament inclusion disease (NIFID), a rare form of frontotemporal lobar degeneration (FTLD), is characterized neuropathologically by focal atrophy of the frontal and temporal lobes, neuronal loss, gliosis, and neuronal cytoplasmic inclusions (NCI) containing epitopes of ubiquitin and neuronal intermediate filament proteins. Recently, the ‘fused in sarcoma’ (FUS) protein (encoded by the FUS gene) has been shown to be a component of the inclusions of familial amyotrophic lateral sclerosis with FUS mutation, NIFID, basophilic inclusion body disease, and atypical FTLD with ubiquitin-immunoreactive inclusions (aFTLD-U). To further characterize FUS proteinopathy in NIFID, and to determine whether the pathology revealed by FUS immunohistochemistry (IHC) is more extensive than α-internexin, we have undertaken a quantitative assessment of ten clinically and neuropathologically well-characterized cases using FUS IHC. The densities of NCI were greatest in the dentate gyrus (DG) and in sectors CA1/2 of the hippocampus. Anti-FUS antibodies also labeled glial inclusions (GI), neuronal intranuclear inclusions (NII), and dystrophic neurites (DN). Vacuolation was extensive across upper and lower cortical layers. Significantly greater densities of abnormally enlarged neurons and glial cell nuclei were present in the lower compared with the upper cortical laminae. FUS IHC revealed significantly greater numbers of NCI in all brain regions especially the DG. Our data suggest: (1) significant densities of FUS-immunoreactive NCI in NIFID especially in the DG and CA1/2; (2) infrequent FUS-immunoreactive GI, NII, and DN; (3) widely distributed vacuolation across the cortex, and (4) significantly more NCI revealed by FUS than α-internexin IHC.

The RNA-binding proteins TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) play central roles in neurodegeneration associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Normally localized in the nucleus, in sites affected by ALS and FTLD-U they are mislocalized to the cytoplasm and form cytoplasmic inclusions. TDP-43 and FUS are transported to the nucleus in a Ran-GTPase-dependent manner via nuclear import receptors, but they also contribute to the formation of stress granules (SGs), which are intracytoplasmic structures incorporating RNA. C-terminal truncations of TDP-43 eliminate the nuclear transport signal and cause mislocalization of the protein to the cytoplasm, where it accumulates and forms SGs. ALS-associated FUS mutations impair nuclear transport and cause mislocalization of FUS to the cytoplasm, where it also contributes to assembly of SGs. Furthermore, the ALS susceptibility factor ataxin-2, recently identified as a potent modifier of TDP-43 toxicity, is also a predicted cytoplasmic RNA-binding protein and a constituent protein of SGs, suggesting that it is a part of the common pathologic cascade formed by TDP-43 and FUS. Thus, we propose that excessive mislocalization of the RNA-binding proteins TDP-43, FUS, and ataxin-2 into the cytoplasm leads to impairment of the RNA quality control system, forming the core of the ALS/FTLD-U degenerative cascade. In this review, we discuss the molecular basis of the novel disease spectrum of ALS/FTLD-U, including the neurodegenerative mechanism of the cytoplasmic RNA-binding proteins TDP-43 and FUS and the possibility of a novel therapeutic strategy.

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.

Electronic supplementary material

The online version of this article (doi:10.1007/s00401-012-1043-z) contains supplementary material, which is available to authorized users.

Frontotemporal dementia (FTD) is a clinical syndrome with a heterogeneous molecular basis. The neuropathology associated with most FTD is characterized by abnormal cellular aggregates of either transactive response DNA-binding protein with Mr 43 kDa (TDP-43) or tau protein. However, we recently described a subgroup of FTD patients, representing around 10%, with an unusual clinical phenotype and pathology characterized by frontotemporal lobar degeneration with neuronal inclusions composed of an unidentified ubiquitinated protein (atypical FTLD-U; aFTLD-U). All cases were sporadic and had early-onset FTD with severe progressive behavioural and personality changes in the absence of aphasia or significant motor features. Mutations in the fused in sarcoma (FUS) gene have recently been identified as a cause of familial amyotrophic lateral sclerosis, with these cases reported to have abnormal cellular accumulations of FUS protein. Because of the recognized clinical, genetic and pathological overlap between FTD and amyotrophic lateral sclerosis, we investigated whether FUS might also be the pathological protein in aFTLD-U. In all our aFTLD-U cases (n = 15), FUS immunohistochemistry labelled all the neuronal inclusions and also demonstrated previously unrecognized glial pathology. Immunoblot analysis of protein extracted from post-mortem aFTLD-U brain tissue demonstrated increased levels of insoluble FUS. No mutations in the FUS gene were identified in any of our patients. These findings suggest that FUS is the pathological protein in a significant subgroup of sporadic FTD and reinforce the concept that FTD and amyotrophic lateral sclerosis are closely related conditions.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are neurodegenerative diseases with clinical and pathological overlap. Landmark discoveries of mutations in the transactive response DNA-binding protein (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS) as causative of ALS and FTLD, combined with the abnormal aggregation of these proteins, have initiated a shifting paradigm for the underlying pathogenesis of multiple neurodegenerative diseases. TDP-43 and FUS/TLS are both RNA/DNA-binding proteins with striking structural and functional similarities. Their association with ALS and other neurodegenerative diseases is redirecting research efforts toward understanding the role of RNA processing regulation in neurodegeneration.

Fused in sarcoma (FUS)-immunoreactive neuronal and glial inclusions define a novel molecular pathology called FUS proteinopathy. FUS has been shown to be a component of inclusions of familial amyotrophic lateral sclerosis with FUS mutation and three FTLD entities, including neuronal intermediate filament inclusion disease (NIFID). The pathogenic role of FUS is unknown. In addition to FUS, many neuronal cytoplasmic inclusions (NCI) of NIFID contain aggregates of α-internexin and neurofilament proteins. Herein, we have: (1) shown that FUS becomes relatively insoluble in NIFID and there are no post-translational modifications; (2) shown there are no pathogenic abnormalities in the FUS gene in NIFID; (3) performed an immunoelectron microscopy analysis of the precise localizations of FUS in NIFID, as this has not previously been described. FUS localized to euchromatin, and strongly with paraspeckles, in nuclei, consistent with its RNA/DNA-binding functions. NCI of varying morphologies were observed. Most frequent were the ‘loosely aggregated cytoplasmic inclusions’ (LACI), 81% of which had moderate or high levels of FUS-immunoreactivity. Much rarer ‘compact cytoplasmic inclusions’ (CCI) and ‘Tangled twine ball inclusions’ (TTBI) were FUS-immunoreactive at their granular peripheries, or heavily FUS-positive throughout, respectively. Thus FUS may aggregate in the cytoplasm and then admix with neuronal intermediate filament accumulations.

RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer’s disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.

Electronic supplementary material

The online version of this article (doi:10.1007/s00401-012-1045-x) contains supplementary material, which is available to authorized users.

Fused in Sarcoma (FUS) proteinopathy is a feature of frontotemporal lobar dementia (FTLD), and mutation of the fus gene segregates with FTLD and amyotrophic lateral sclerosis (ALS). To study the consequences of mutation in the fus gene, we created transgenic rats expressing the human fus gene with or without mutation. Overexpression of a mutant (R521C substitution), but not normal, human FUS induced progressive paralysis resembling ALS. Mutant FUS transgenic rats developed progressive paralysis secondary to degeneration of motor axons and displayed a substantial loss of neurons in the cortex and hippocampus. This neuronal loss was accompanied by ubiquitin aggregation and glial reaction. While transgenic rats that overexpressed the wild-type human FUS were asymptomatic at young ages, they showed a deficit in spatial learning and memory and a significant loss of cortical and hippocampal neurons at advanced ages. These results suggest that mutant FUS is more toxic to neurons than normal FUS and that increased expression of normal FUS is sufficient to induce neuron death. Our FUS transgenic rats reproduced some phenotypes of ALS and FTLD and will provide a useful model for mechanistic studies of FUS–related diseases.

Author Summary

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are two related diseases characterized by degeneration of selected groups of neuronal cells. Neither of these diseases has a clear cause, and both are incurable at present. Mutation of the fus gene has recently been linked to these two diseases. Here, we describe a novel rat model that expresses a mutated form of the human fus gene and manifests the phenotypes and pathological features of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Establishment of this FUS transgenic rat model will allow not only for mechanistic study of FUS–related diseases, but also for quick development of therapies for these devastating diseases.

Neuronal intermediate filament inclusion disease (NIFID) is an uncommon neurodegenerative condition that typically presents as early-onset, sporadic frontotemporal dementia (FTD), associated with a pyramidal and/or extrapyramidal movement disorder. The neuropathology is characterized by frontotemporal lobar degeneration with neuronal inclusions that are immunoreactive for all class IV intermediate filaments (IF), light, medium and heavy neurofilament subunits and α-internexin. However, not all the inclusions in NIFID are IF-positive and the primary molecular defect remains uncertain. Mutations in the gene encoding the fused in sarcoma (FUS) protein have recently been identified as a cause of familial amyotrophic lateral sclerosis (ALS). Because of the recognized clinical, genetic and pathological overlap between FTD and ALS, we investigated the possible role of FUS in NIFID. We found abnormal intracellular accumulation of FUS to be a consistent feature of our NIFID cases (n = 5). More neuronal inclusions were labeled using FUS immunohistochemistry than for IF. Several types of inclusions were consistently FUS-positive but IF-negative, including neuronal intranuclear inclusions and glial cytoplasmic inclusions. Double-label immunofluorescence confirmed that many cells had only FUS-positive inclusions and that all cells with IF-positive inclusions also contained pathological FUS. No mutations in the FUS gene were identified in a single case with DNA available. These findings suggest that FUS may play an important role in the pathogenesis of NIFID.

A combination of yeast genetics and protein biochemistry define how the fused in sarcoma (FUS) protein might contribute to Lou Gehrig's disease.

TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.

Author Summary

Many human neurodegenerative diseases are associated with the abnormal accumulation of protein aggregates in the neurons of affected individuals. Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a fatal human neurodegenerative disease caused primarily by a loss of motor neurons. Recently, mutations in a gene called fused in sarcoma (FUS) were identified in some ALS patients. The basic mechanisms by which FUS contributes to ALS are unknown. We have addressed this question using protein biochemistry and the genetically tractable yeast Saccharomyces cerevisiae. We defined the regions of biochemically pure FUS protein that contribute to its aggregation and toxic properties. We then used genome-wide screens in yeast to identify genes and cellular pathways involved in the toxicity of FUS. Many of the FUS toxicity modifier genes that we identified in yeast have clear homologs in humans, suggesting that these might also be relevant for the human disease. Together, our studies provide novel insight into the basic mechanisms associated with FUS aggregation and toxicity. Moreover, our findings open new avenues that could be explored for therapeutic intervention.

TLS (translocated in liposarcoma), also known as FUS (fused in sarcoma), is an RNA/DNA-binding protein that plays regulatory roles in transcription, pre-mRNA splicing and mRNA transport. Mutations in TLS are responsible for familial amyotrophic lateral sclerosis (ALS) type 6. Furthermore, TLS-containing intracellular inclusions are found in polyglutamine diseases, sporadic ALS, non-SOD1 familial ALS and a subset of frontotemporal lobar degeneration, indicating a pathological significance of TLS in a wide variety of neurodegenerative diseases. Here, we identified TLS domains that determine intracellular localization of the murine TLS. Among them, PY-NLS located in the C-terminus is a strong determinant of intracellular localization as well as splicing regulation of an E1A-derived minigene. Disruption of PY-NLS promoted the formation of cytoplasmic granules that were partially overlapped with stress granules and P-bodies. Some of the ALS-linked mutations altered both intracellular localization and splicing regulation of TLS, while most mutations alone did not affect splicing regulation. However, phospho-mimetic substitution of Ser505 (or Ser513 in human) could enhance the effects of ALS mutations, highlighting interplay between post-translational modification and ALS-linked mutations. These results demonstrate that ALS-linked mutations can variably cause loss of nuclear functions of TLS depending on the degree of impairment in nuclear localization.

Frontotemporal lobar degeneration (FTLD) is a heterogeneous group of disorders characterized by disturbances of behavior and personality and different types of language impairment with or without concomitant features of motor neuron disease or parkinsonism. FTLD is characterized by atrophy of the frontal and anterior temporal brain lobes. Detailed neuropathological studies have elicited proteinopathies defined by inclusions of hyperphosphorylated microtubule-associated protein tau, TAR DNA-binding protein TDP-43, fused-in-sarcoma or yet unidentified proteins in affected brain regions. Rather than the type of proteinopathy, the site of neurodegeneration correlates relatively well with the clinical presentation of FTLD. Molecular genetic studies identified five disease genes, of which the gene encoding the tau protein (MAPT), the growth factor precursor gene granulin (GRN), and C9orf72 with unknown function are most frequently mutated. Rare mutations were also identified in the genes encoding valosin-containing protein (VCP) and charged multivesicular body protein 2B (CHMP2B). These genes are good markers to distinguish underlying neuropathological phenotypes. Due to the complex landscape of FTLD diseases, combined characterization of clinical, imaging, biological and genetic biomarkers is essential to establish a detailed diagnosis. Although major progress has been made in FTLD research in recent years, further studies are needed to completely map out and correlate the clinical, pathological and genetic entities, and to understand the underlying disease mechanisms. In this review, we summarize the current state of the rapidly progressing field of genetic, neuropathological and clinical research of this intriguing condition.

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43–positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the ∼25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.

Author Summary

The abnormal accumulation of disease proteins in neuronal cells of the brain is a characteristic feature of many neurodegenerative diseases. Rare mutations in the genes that encode the accumulating proteins have been identified in these disorders and are crucial for the development of cell and animal models used to study neurodegeneration. Recently, the TAR DNA-binding protein 43 (TDP-43) was identified as the disease accumulating protein in patients with frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) and in amyotrophic lateral sclerosis (ALS). TDP-43 was also found in the brains of 20–30% of patients with Alzheimer's disease (AD). Here, we evaluated whether mutations in TDP-43 cause disease in a cohort of 296 patients presenting with FTLD, ALS or AD. We identified three missense mutations in three out of 92 familial ALS patients (3.3%), and no mutations in AD or FTLD patients. All the identified mutations clustered in exon 6, which codes for a highly conserved region in the C-terminal part of the TDP-43 protein, which is known to be involved in the interaction of TDP-43 with other proteins. We conclude that mutations in TDP-43 are a rare cause of familial ALS, but so far are not found in other neurodegenerative diseases.

Neurodegenerative disorders characterized by neuronal and glial lesions containing aggregated pathological TDP-43 protein in the cytoplasm, nucleus, or neurites are collectively referred to as TDP-43 proteinopathies. Lesions containing aggregated TDP-43 protein are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). In addition, mutations in human TDP-43 cause ALS. We have developed a C. elegans model of TDP-43 proteinopathies to study the cellular, molecular, and genetic underpinnings of TDP-43 mediated neurotoxicity. Expression of normal human TDP-43 in all C. elegans neurons causes moderate motor defects, while ALS-mutant G290A, A315T, or M337V TDP-43 transgenes cause severe motor dysfunction. The model recapitulates some characteristic features of ALS and FTLD-U including age-induced decline in motor function, decreased lifespan, and degeneration of motor neurons accompanied by hyperphosphorylation, truncation, and ubiquitination of TDP-43 protein that accumulates in detergent insoluble protein deposits. In C. elegans, TDP-43 neurotoxicity is independent of activity of the cell death caspase CED-3. Furthermore, phosphorylation of TDP-43 at serine residues 409/410 drives mutant TDP-43 toxicity. This model provides a tractable system for further dissection of the cellular and molecular mechanisms underlying TDP-43 neuropathology.

Major discoveries have been made in the recent past in the genetics, biochemistry and neuropathology of frontotemporal lobar degeneration (FTLD). TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, has been identified as the major pathological protein of FTLD with ubiquitin-immunoreactive (ub-ir) inclusions (FTLD-U) with or without amyotrophic lateral sclerosis (ALS) and sporadic ALS. Recently, mutations in the TARDBP gene in familial and sporadic ALS have been reported which demonstrate that abnormal TDP-43 alone is sufficient to cause neurodegeneration. Several familial cases of FTLD-U, however, are now known to have mutations in the progranulin (GRN) gene, but granulin is not a component of the TDP-43- and ub-ir inclusions. Further, TDP-43 is found to be a component of the inclusions of an increasing number of neurodegenerative diseases. Other FTLD-U entities with TDP-43 proteinopathy include: FTLD-U with valosin-containing protein (VCP) gene mutation and FTLD with ALS linked to chromosome 9p. In contrast, chromosome 3-linked dementia, FTLD-U with chromatin modifying protein 2B (CHMP2B) mutation, has ub-ir, TDP-43-negative inclusions. In summary, recent discoveries have generated new insights into the pathogenesis of a spectrum of disorders called TDP-43 proteinopathies including: FTLD-U, FTLD-U with ALS, ALS, and a broadening spectrum of other disorders. It is anticipated that these discoveries and a revised nosology of FTLD will contribute toward an accurate diagnosis, and facilitate the development of new diagnostic tests and therapeutics.

Background:

Juvenile amyotrophic lateral sclerosis (ALS) with basophilic inclusions is a form of ALS characterized by protein deposits in motor neurons that are morphologically and tinctorially distinct from those of classic sporadic ALS. The nosologic position of this type of ALS in the molecular pathologic and genetic classification of ALS is unknown.

Methods:

We identified neuropathologically 4 patients with juvenile ALS with basophilic inclusions and tested the hypothesis that specific RNA binding protein pathology may define this type of ALS. Immunohistochemical findings prompted us to sequence the fused in sarcoma (FUS) gene.

Results:

Motor symptoms began between ages 17 and 22. Disease progression was rapid without dementia. No family history was identified. Basophilic inclusions were strongly positive for FUS protein but negative for TAR DNA binding protein 43 (TDP-43). Granular and compact FUS deposits were identified in glia and neuronal cytoplasm and nuclei. Ultrastructure of aggregates was in keeping with origin from fragmented rough endoplasmic reticulum. Sequencing of all 15 exons of the FUS gene in 3 patients revealed a novel deletion mutation (c.1554_1557delACAG) in 1 individual and the c.1574C>T (P525L) mutation in 2 others.

Conclusion:

Juvenile ALS with basophilic inclusions is a FUS proteinopathy and should be classified as ALS-FUS. The FUS c.1574C>T (P525L) and c.1554_1557delACAG mutations are associated with this distinct phenotype. The molecular genetic relationship with frontotemporal lobar degeneration with FUS pathology remains to be clarified.

GLOSSARY

= amyotrophic lateral sclerosis;

= frontotemporal lobar degeneration;

= frontotemporal lobar degeneration with FUS pathology;

= neuronal intermediate filament inclusion disease.

The clinical and pathological overlap between amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) suggests these diseases share common underlying mechanisms, a suggestion underscored by the discovery that TDP-43 inclusions are a key pathologic feature in both ALS and FTLD. This finding, combined with the identification of TDP-43 mutations in ALS, directly implicates this DNA/RNA binding protein in disease pathogenesis in ALS and FTLD. However, many key questions remain, including what is the normal function of TDP-43, and whether disease-associated mutations produce toxicity in the nucleus, cytoplasm or both. Furthermore, although pathologic TDP-43 inclusions are clearly associated with many forms of neurodegeneration, whether TDP-43 aggregation is a key step in the pathogenesis in ALS, FTLD and other disorders remains to be proven. This review will compare the features of numerous recently developed animal models of TDP-43-related neurodegeneration, and discuss how they contribute to our understanding of the pathogenesis of human ALS and FTLD.

Mutations in the gene encoding TDP-43 — the major protein component of neuronal aggregates characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusion bodies (FTLDu) — have been linked to familial forms of both disorders. Aggregates of TDP-43 in cortical and spinal motoneurons in ALS, or in neurons of the frontal and temporal cortices in FTLD, are closely linked to neuron loss and atrophy in these areas. However, the mechanism by which TDP-43 mutations lead to neurodegeneration is unclear. To investigate the pathogenic role of TDP-43 mutations, we established a model of TDP-43 proteinopathies by expressing fluorescently tagged wildtype and mutant TDP-43 in primary rat cortical neurons. Expression of mutant TDP-43 was toxic to neurons, and mutant-specific toxicity was associated with increased cytoplasmic mislocalization of TDP-43. Inclusion bodies were not necessary for the toxicity and did not affect the risk of cell death. Cellular survival was unaffected by the total amount of exogenous TDP-43 in the nucleus, but the amount of cytoplasmic TDP-43 was a strong and independent predictor of neuronal death. These results suggest that mutant TDP-43 is mislocalized to the cytoplasm, where it exhibits a toxic gain-of-function and induces cell death.

The identification of TAR DNA-binding protein 43 (TDP-43) as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) has defined a new class of neurodegenerative conditions: the TDP-43 proteinopathies. This breakthrough was quickly followed by mutation analysis of TARDBP, the gene encoding TDP-43. Herein, we provide a review of our previously published efforts that led to the identification of 3 TARDBP mutations (p.M337V, p.N345K, and p.I383V) in familial ALS patients, 2 of which were novel. With over 40 TARDBP mutations now discovered, there exists conclusive evidence that TDP-43 plays a direct role in neurodegeneration. The onus is now on researchers to elucidate the mechanisms by which mutant TDP-43 confers toxicity, and to exploit these findings to gain a better understanding of how TDP-43 contributes to the pathogenesis of disease. Our biochemical analysis of TDP-43 in ALS patient lymphoblastoid cell lines revealed a substantial increase in TDP-43 truncation products, including a ~25 kDa fragment, compared to control lymphoblastoid cell lines. We discuss the putative harmful consequence of abnormal TDP-43 fragmentation, as well as highlight additional mechanisms of toxicity associated with mutant TDP-43.

Ever since the significance of pathological 43-kDa transactivating responsive sequence DNA-binding protein (TDP-43) for human disease has been recognized in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U), a number of publications have emerged reporting on this pathology in a variety of neurodegenerative diseases. Given the heterogeneous and, in part, conflicting nature of the recent findings, we here review pathological TDP-43 and its relationship to human disease with a special focus on ALS and FTLD-U. To this end, we propose a classification scheme in which pathological TDP-43 is the major disease defining pathology in one group, or is present in addition to other neurodegenerative hallmark pathologies in a second category. We conclude that the TDP-43 proteinopathies represent a novel class of neurodegenerative disorders akin to α-synucleinopathies and tauopathies, with the concept of ALS and FTLD-U to be widened to a broad clinico-pathological multisystem disease, i.e., TDP-43 proteinopathy.

It is now established that pathological transactive response DNA-binding protein with a Mr of 43 kD (TDP-43) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitin-positive inclusions (now known as FTLD-TDP). In fact, the discovery of pathological TDP-43 solidified the idea that these disorders are multi-system diseases and this led to the concept of a TDP-43 proteinopathy as a spectrum of disorders comprised of different clinical and pathological entities extending from ALS to ALS with cognitive impairment/dementia and FTLD-TDP without or with motor neuron disease (FTLD-MND). These align along a broad disease continuum sharing similar pathogenetic mechanisms linked to pathological TDP-43. We here review salient findings in the development of a concept of TDP-43 proteinopathy as a novel group of neurodegenerative diseases similar in concept to α-synucleinopathies and tauopathies.

Frontotemporal lobar degeneration (FTLD) is clinically, pathologically and genetically heterogeneous. Three major proteins are implicated in its pathogenesis. About half of cases are characterized by depositions of the microtubule associated protein, tau (FTLD-tau). In most of the remaining cases, deposits of the transactive response (TAR) DNA-binding protein with Mw of 43 kDa, known as TDP-43 (FTLD-TDP), are seen. Lastly, about 5–10 % of cases are characterized by abnormal accumulations of a third protein, fused in sarcoma (FTLD-FUS). Depending on the protein concerned, the signature accumulations can take the form of inclusion bodies (neuronal cytoplasmic inclusions and neuronal intranuclear inclusions) or dystrophic neurites, in the cerebral cortex, hippocampus and subcortex. In some instances, glial cells are also affected by inclusion body formation. In motor neurone disease (MND), TDP-43 or FUS inclusions can present within motor neurons of the brain stem and spinal cord. This present paper attempts to critically examine the role of such proteins in the pathogenesis of FTLD and MND as to whether they might exert a direct pathogenetic effect (gain of function), or simply act as relatively innocent witnesses to a more fundamental loss of function effect. We conclude that although there is strong evidence for both gain and loss of function effects in respect of each of the proteins concerned, in reality, it is likely that each is a single face of either side of the coin, and that both will play separate, though complementary, roles in driving the damage which ultimately leads to the downfall of neurons and clinical expression of disease.