Fused In Sarcoma/translated In Liposarcoma Research Articles

Multiple lines of evidence for disruption of nuclear lamina and nucleoporins in FUS amyotrophic lateral sclerosis.

Advanced pathological and genetic approaches have revealed that mutations in fused in sarcoma/translated in liposarcoma (FUS/TLS), which is pivotal for DNA repair, alternative splicing, translation and RNA transport, cause familial amyotrophic lateral sclerosis (ALS). The generation of suitable animal models for ALS is essential for understanding its pathogenesis and developing therapies. Therefore, we used CRISPR-Cas9 to generate FUS-ALS mutation in the non-classical nuclear localization signal (NLS), H517D (mouse position: H509D) and genome-edited mice. Fus WT/H509D mice showed progressive motor impairment (accelerating rotarod and DigiGait system) with age, which was associated with the loss of motor neurons and disruption of the nuclear lamina and nucleoporins and DNA damage in spinal cord motor neurons. We confirmed the validity of our model by showing that nuclear lamina and nucleoporin disruption were observed in lower motor neurons differentiated from patient-derived human induced pluripotent stem cells (hiPSC-LMNs) with FUS-H517D and in the post-mortem spinal cord of patients with ALS. RNA sequence analysis revealed that most nuclear lamina and nucleoporin-linking genes were significantly decreased in FUS-H517D hiPSC-LMNs. This evidence suggests that disruption of the nuclear lamina and nucleoporins is crucial for ALS pathomechanisms. Combined with patient-derived hiPSC-LMNs and autopsy samples, this mouse model might provide a more reliable understanding of ALS pathogenesis and might aid in the development of therapeutic strategies.

DNA damage stress-induced translocation of mutant FUS proteins into cytosolic granules and screening for translocation inhibitors.

Fused in sarcoma/translated in liposarcoma (FUS) is an RNA-binding protein, and its mutations are associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), through the DNA damage stress response, aberrant stress granule (SG) formation, etc. We previously reported that translocation of endogenous FUS into SGs was achieved by cotreatment with a DNA double-strand break inducer and an inhibitor of DNA-PK activity. In the present study, we investigated cytoplasmic SG formation using various fluorescent protein-tagged mutant FUS proteins in a human astrocytoma cell (U251) model. While the synergistic enhancement of the migration of fluorescent protein-tagged wild-type FUS to cytoplasmic SGs upon DNA damage induction was observed when DNA-PK activity was suppressed, the fluorescent protein-tagged FUSP525L mutant showed cytoplasmic localization. It migrated to cytoplasmic SGs upon DNA damage induction alone, and DNA-PK inhibition also showed a synergistic effect. Furthermore, analysis of 12 sites of DNA-PK-regulated phosphorylation in the N-terminal LC region of FUS revealed that hyperphosphorylation of FUS mitigated the mislocalization of FUS into cytoplasmic SGs. By using this cell model, we performed screening of a compound library to identify compounds that inhibit the migration of FUS to cytoplasmic SGs but do not affect the localization of the SG marker molecule G3BP1 to cytoplasmic SGs. Finally, we successfully identified 23 compounds that inhibit FUS-containing SG formation without changing normal SG formation. Highlights Characterization of DNA-PK-dependent FUS stress granule localization.A compound library was screened to identify compounds that inhibit the formation of FUS-containing stress granules.

Identification of hub molecules of FUS-ALS by Bayesian gene regulatory network analysis of iPSC model: iBRN

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.

Identification of Hub Molecules of FUS-ALA by Bayesian Gene Regulatory Network Analysis of iPSC Model: iBRN

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, etc., thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, non-biased Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, termed iBRN were applied to transcriptome of iPSC-derived MNs possessing wild-type form of FUS and FUSH517D (FUSH517D MNs). iBRN revealed “hub molecules”, which strongly influenced network, predicting miR-125b-5p-TIMELESS axis and PRKDC as key candidates for the molecular aetiology. Indeed, this axis was augmented in FUSH517D MNs and miR-125b-5p down-regulated DNA repair-related genes including TIMELESS. Importantly, both introduction of miR-125b-5p and knocking down Timeless caused DNA damage. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, iBRN provides the first compelling evidence to elucidate molecular aetiology.

Molecular determinants of cytochrome C oxidase IV mRNA axonal trafficking.

In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.

Genotype-phenotype relationships in familial amyotrophic lateral sclerosis with FUS/TLS mutations in Japan.

We investigated possible genotype-phenotype correlations in Japanese patients with familial amyotrophic lateral sclerosis (FALS) carrying fused in sarcoma/translated in liposarcoma (FUS/TLS) gene mutations. A consecutive series of 111 Japanese FALS pedigrees were screened for copper/zinc superoxide dismutase 1 (SOD1) and FUS/TLS gene mutations. Clinical data, including onset age, onset site, disease duration, and extramotor symptoms, were collected. Nine different FUS/TLS mutations were found in 12 pedigrees. Most of the patients with FUS/TLS-linked FALS demonstrated early onset in the brainstem/upper cervical region, and relatively short disease duration. A few mutations exhibited phenotypes that were distinct from typical cases. Frontotemporal dementia was present in 1 patient. This study revealed a characteristic phenotype in FUS/TLS-linked FALS patients in Japan. FUS/TLS screening is recommended in patients with FALS with this phenotype. Muscle Nerve 54: 398-404, 2016.

ALS-associated mutations in FUS disrupt the axonal distribution and function of SMN

Mutations in the RNA binding protein fused in sarcoma/translated in liposarcoma (FUS/TLS) cause amyotrophic lateral sclerosis (ALS). Although ALS-linked mutations in FUS often lead to a cytosolic mislocalization of the protein, the pathogenic mechanisms underlying these mutations remain poorly understood. To gain insight into these mechanisms, we examined the biochemical, cell biological and functional properties of mutant FUS in neurons. Expression of different FUS mutants (R521C, R521H, P525L) in neurons caused axonal defects. A protein interaction screen performed to explain these phenotypes identified numerous FUS interactors including the spinal muscular atrophy (SMA) causing protein survival motor neuron (SMN). Biochemical experiments showed that FUS and SMN interact directly and endogenously, and that this interaction can be regulated by FUS mutations. Immunostaining revealed co-localization of mutant FUS aggregates and SMN in primary neurons. This redistribution of SMN to cytosolic FUS accumulations led to a decrease in axonal SMN. Finally, cell biological experiments showed that overexpression of SMN rescued the axonal defects induced by mutant FUS, suggesting that FUS mutations cause axonal defects through SMN. This study shows that neuronal aggregates formed by mutant FUS protein may aberrantly sequester SMN and concomitantly cause a reduction of SMN levels in the axon, leading to axonal defects. These data provide a functional link between ALS-linked FUS mutations, SMN and neuronal connectivity and support the idea that different motor neuron disorders such as SMA and ALS may be caused, in part, by defects in shared molecular pathways.

Characterization of FUS Mutations in Amyotrophic Lateral Sclerosis Using RNA-Seq

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting in severe muscle weakness and eventual death by respiratory failure. Although little is known about its pathogenesis, mutations in fused in sarcoma/translated in liposarcoma (FUS) are causative for familial ALS. FUS is a multifunctional protein that is involved in many aspects of RNA processing. To elucidate the role of FUS in ALS, we overexpressed wild-type and two mutant forms of FUS in HEK-293T cells, as well as knocked-down FUS expression. This was followed by RNA-Seq to identify genes which displayed differential expression or altered splicing patterns. Pathway analysis revealed that overexpression of wild-type FUS regulates ribosomal genes, whereas knock-down of FUS additionally affects expression of spliceosome related genes. Furthermore, cells expressing mutant FUS displayed global transcription patterns more similar to cells overexpressing wild-type FUS than to the knock-down condition. This observation suggests that FUS mutants do not contribute to the pathogenesis of ALS through a loss-of-function. Finally, our results demonstrate that the R521G and R522G mutations display differences in their influence on transcription and splicing. Taken together, these results provide additional insights into the function of FUS and how mutations contribute to the development of ALS.

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.

Genetic Overlap between Apparently Sporadic Motor Neuron Diseases

Progressive muscular atrophy (PMA) and amyotrophic lateral sclerosis (ALS) are devastating motor neuron diseases (MNDs), which result in muscle weakness and/or spasticity. We compared mutation frequencies in genes known to be associated with MNDs between patients with apparently sporadic PMA and ALS. A total of 261 patients with adult-onset sporadic PMA, patients with sporadic ALS, and control subjects of Dutch descent were obtained at national referral centers for neuromuscular diseases in The Netherlands. Sanger sequencing was used to screen these subjects for mutations in the coding regions of superoxide dismutase-1 (SOD1), angiogenin (ANG), fused in sarcoma/translated in liposarcoma (FUS/TLS), TAR DNA-binding protein 43 (TARDBP), and multivesicular body protein 2B (CHMP2B). In our cohort of PMA patients we identified two SOD1 mutations (p.D90A, p.I113T), one ANG mutation (p.K17I), one FUS/TLS mutation (p.R521H), one TARDBP mutation (p.N352S), and one novel CHMP2B mutation (p.R69Q). The mutation frequency of these genes was similar in sporadic PMA (2.7%) and ALS (2.0%) patients, and therefore, our findings demonstrate a genetic overlap between apparently sporadic PMA and ALS.

Genetic analysis of the FUS/TLS gene in essential tremor

Although essential tremor (ET) has a genetic basis, specific genes have not been identified. Recently, in a large ET family (FET1) from Quebec, a non-sense mutation (p.Q290X) in the amyotrophic lateral sclerosis (ALS) gene fused in sarcoma/translated in liposarcoma (FUS/TLS) was identified by exome sequencing. No confirmatory studies have been published. Two-hundred and fifty-nine ET cases and 262 controls were enrolled in a study at Columbia University. We performed a comprehensive analysis of the FUS/TLS gene by sequencing all exons in a subsample of 116 ET cases with early-onset (≤40years) ET. We evaluated an association between ET and SNPs in the FUS/TLS gene by genotyping four haplotype tagging SNPs in all 259 ET cases and 262 controls. Additionally, seven variants associated with ALS, two variants of unknown pathogenicity detected in ALS cases, eight mis-sense variants predicted to be damaging, and six rare variants were genotyped in these 259 ET cases and 262 controls. FUS/TLS mutations previously reported in ALS, the FET1 family, or novel mutations were not found in any of the 116 early-onset ET cases. In the case-control analyses, although the power of the performed associations was limited, no significant association between tagging SNPs in FUS/TLS and ET was observed, and none of the analyzed SNPs showed evidence of association with ET. Our study suggests that pathogenic mutations in FUS/TLS are rare in a sample of early-onset ET cases in North America. We did not find evidence that the FUS/TLS gene is a risk factor for ET.

Evidence for an oligogenic basis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a substantial heritable component. In pedigrees affected by its familial form, incomplete penetrance is often observed. We hypothesized that this could be caused by a complex inheritance of risk variants in multiple genes. Therefore, we screened 111 familial ALS (FALS) patients from 97 families, and large cohorts of sporadic ALS (SALS) patients and control subjects for mutations in TAR DNA-binding protein (TARDBP), fused in sarcoma/translated in liposarcoma (FUS/TLS), superoxide dismutase-1 (SOD1), angiogenin (ANG) and chromosome 9 open reading frame 72 (C9orf72). Mutations were identified in 48% of FALS families, 8% of SALS patients and 0.5% of control subjects. In five of the FALS families, we identified multiple mutations in ALS-associated genes. We detected FUS/TLS and TARDBP mutations in combination with ANG mutations, and C9orf72 repeat expansions with TARDBP, SOD1 and FUS/TLS mutations. Statistical analysis demonstrated that the presence of multiple mutations in FALS is in excess of what is to be expected by chance (P = 1.57 × 10(-7)). The most compelling evidence for an oligogenic basis was found in individuals with a p.N352S mutation in TARDBP, detected in five FALS families and three apparently SALS patients. Genealogical and haplotype analyses revealed that these individuals shared a common ancestor. We obtained DNA of 14 patients with this TARDBP mutation, 50% of whom had an additional mutation (ANG, C9orf72 or homozygous TARDBP). Hereby, we provide evidence for an oligogenic aetiology of ALS. This may have important implications for the interpretation of whole exome/genome experiments designed to identify new ALS-associated genes and for genetic counselling, especially of unaffected family members.

Accumulation of insoluble forms of FUS protein correlates with toxicity in Drosophila

Recently, the fused in sarcoma/translated in liposarcoma (FUS) protein has been identified as a major constituent of nuclear and/or cytoplasmic ubiquitin-positive inclusions in patients with frontotemporal lobar degeneration or amyotrophic lateral sclerosis. The molecular mechanisms underlying FUS toxicity are currently not understood. To address aspects of FUS pathogenesis in vivo, we have generated new Drosophila transgenic models expressing a full-length wild-type isoform of human FUS protein. We found that when expressed in retinal cells, FUS proteins are mainly recovered as soluble forms, and their overexpression results in a mild eye phenotype, with malformed interommatidial bristles and the appearance of ectopic extensions. On the other hand, when FUS proteins are specifically targeted to adult differentiated neurons, they are mainly recovered as insoluble forms, and their overexpression drastically reduces fly life span. Importantly, FUS neurotoxicity occurs regardless of inclusion formation. Lastly, we showed that molecular chaperones reduce FUS toxicity by modulating protein solubility. Altogether, our data indicate that accumulation of insoluble non-aggregated FUS forms might represent the primary toxic species in human FUS proteinopathies.

RNA‐binding proteins in neurodegenerative disease: TDP‐43 and beyond

Neurodegenerative diseases are a diverse group of disorders that affect different neuron populations, differ in onset and severity, and can be either inherited or sporadic. One common pathological feature of most of these diseases is the presence of insoluble inclusions in and around neurons, which largely consist of misfolded and aggregated protein. For this reason, neurodegenerative diseases are typically thought to be disorders of aberrant protein processing, in which the cumulative effects of misfolded protein aggregates overwhelm the neuron's proteostatic capacity. However, a growing body of evidence suggests a role for abnormal RNA processing in neurodegenerative disease. The importance of RNA metabolism in disease was highlighted by the discovery of TDP-43 (TAR DNA-binding protein of 43 kDa), an RNA-binding protein (RBP), as a primary component of insoluble aggregates in patients with sporadic amyotrophic lateral sclerosis (ALS). Subsequently, inherited mutations in TDP-43 and the structurally related RBP, FUS/TLS (fused in sarcoma/translated in liposarcoma), were found to cause ALS. These exciting findings have ushered in a new era of ALS research in which the deregulation of RNA metabolism is viewed as a central cause of motor neuron deterioration. In addition, the fact that neuropathologically and anatomically distinct neurodegenerative diseases display altered RNA metabolism suggests that common pathologic mechanisms may underlie many of these disorders.

Arginine methylation by PRMT1 regulates nuclear-cytoplasmic localization and toxicity of FUS/TLS harbouring ALS-linked mutations

Mutations in FUS/TLS (fused in sarcoma/translated in liposarcoma) cause an inheritable form of amyotrophic lateral sclerosis (ALS6). In contrast to FUS(WT), which is concentrated in the nucleus, these mutants are abnormally distributed in the cytoplasm where they form inclusions and associate with stress granules. The data reported herein demonstrate the importance of protein arginine methylation in nuclear-cytoplasmic shuttling of FUS and abnormalities of ALS-causing mutants. Depletion of protein arginine methyltransferase 1 (PRMT1; the enzyme that methylates FUS) in mouse embryonic fibroblasts by gene knockout, or in human HEK293 cells by siRNA knockdown, diminished the ability of ALS-linked FUS mutants to localize to the cytoplasm and form inclusions. To examine properties of FUS mutants in the context of neurons vulnerable to the disease, FUS(WT) and ALS-linked FUS mutants were expressed in motor neurons of dissociated murine spinal cord cultures. In motor neurons, shRNA-mediated PRMT1 knockdown concomitant with the expression of FUS actually accentuated the shift in distribution of ALS-linked FUS mutants from the nucleus to the cytoplasm. However, when PRMT1 was inhibited prior to expression of ALS-linked FUS mutants, by pretreatment with a global methyltransferase inhibitor, ALS-linked FUS mutants were sequestered in the nucleus and cytoplasmic inclusions were reduced, as in the cell lines. Mitochondria were significantly shorter in neurons with cytoplasmic ALS-linked FUS mutants, a factor that could contribute to toxicity. We propose that arginine methylation by PRMT1 participates in the nuclear-cytoplasmic shuttling of FUS, particularly of ALS6-associated mutants, and thus contributes to the toxic gain of function conferred by these disease-causing mutations.

A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder characterized by the loss of motor neurons. Fused in sarcoma/translated in liposarcoma (FUS/TLS) and TAR DNA-binding protein (TDP)-43 are DNA/RNA-binding proteins found to be mutated in sporadic and familial forms of ALS. Ectopic expression of human ALS-causing FUS/TLS mutations in Drosophila caused an accumulation of ubiquitinated proteins, neurodegeneration, larval-crawling defect and early lethality. Mutant FUS/TLS localized to both the cytoplasm and nucleus, whereas wild-type FUS/TLS localized only to the nucleus, suggesting that the cytoplasmic localization of FUS/TLS is required for toxicity. Furthermore, we found that deletion of the nuclear export signal strongly suppressed toxicity, suggesting that cytoplasmic localization is necessary for neurodegeneration. Interestingly, we observed that FUS/TLS genetically interacts with TDP-43 in a mutation-dependent fashion to cause neurodegeneration in vivo. In summary, we demonstrate that ALS-associated mutations in FUS/TLS cause adult-onset neurodegeneration via a gain-of-toxicity mechanism that involves redistribution of the protein from the nucleus to the cytoplasm and is likely to involve an interaction with TDP-43.

Nuclear transport impairment of amyotrophic lateral sclerosis‐linked mutations in FUS/TLS

The fused in sarcoma/translated in liposarcoma (FUS/TLS) protein was recently identified as a cause of familial amyotrophic lateral sclerosis (ALS), as well as a major component of the inclusion bodies found in subtypes of frontotemporal lobar degeneration (FTLD). These diseases now are collectively known as the novel clinical spectrum, FUS proteinopathy. ALS-linked mutations of FUS are clustered in the C-terminal region; however, the molecular properties of mutant FUS remain unclear. To gain insight into the pathogenesis of FUS proteinopathy, we examined the biochemical and cellular characteristics of mutant FUS in expressing cells. Expression of ALS-linked FUS mutations resulted in their assembly into cytoplasmic stress granules (SGs), cellular structures that package mRNA and RNA-binding proteins during cell stress. A deletion mutant series revealed that the C-terminal region in FUS is critical for nuclear retention via Ran guanosine triphosphatase-dependent transport machinery. A parallel study of subcellular distribution revealed that ALS-linked mutants additively disturb the function of the C-terminus for nuclear traffic, resulting in cytoplasmic accumulation and the formation of SGs. This study demonstrates that mutant FUS, which is missing the nuclear traffic activity of the C-terminus, is dislocated to cytoplasm and assembled into SGs, indicating that disruption of translational regulation and metabolism of mRNA via inappropriate/excessive SGs may be crucial for FUS proteinopathies. Our findings provide new biological and pathological insights into the FUS protein that should help our understanding of the pathogenesis of ALS/FTLD.

Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA*

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that preferentially targets motor neurons. It was recently found that dominant mutations in two related RNA-binding proteins, TDP-43 (43-kDa TAR DNA-binding domain protein) and FUS/TLS (fused in sarcoma/translated in liposarcoma) cause a subset of ALS. The convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations are suggestive of a functional relationship; however, whether or not TDP-43 and FUS/TLS operate in common biochemical pathways is not known. Here we show that TDP-43 and FUS/TLS directly interact to form a complex at endogenous expression levels in mammalian cells. Binding was mediated by an unstructured TDP-43 C-terminal domain and occurred within the context of a 300-400-kDa complex that also contained C-terminal cleavage products of TDP-43 linked to neuropathology. TDP-43 C-terminal fragments were excluded from large molecular mass TDP-43 ribonucleoprotein complexes but retained FUS/TLS binding activity. The functional significance of TDP-43-FUS/TLS complexes was established by showing that RNAi silencing of either TDP-43 or FUS/TLS reduced the expression of histone deacetylase (HDAC) 6 mRNA. TDP-43 and FUS/TLS associated with HDAC6 mRNA in intact cells and in vitro, and competition experiments suggested that the proteins occupy overlapping binding sites. The combined findings demonstrate that TDP-43 and FUS/TLS form a functional complex in intact cells and suggest that convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations may reflect their participation in common biochemical processes.

La recherche sur la SLA en 2009 : compte rendu du groupe bibliographique de la coordination des centres SLA

This paper, written by French amyotrophic lateral sclerosis (ALS) center experts, presents an update of recent advances in fundamental, epidemiological and clinical research in ALS based on a review of the literature between September 2008 and November 2009. Among other pathophysiological mechanisms, the role of stress of the endoplasmic reticulum and the importance of energetic metabolic disturbances have been underscored. In the field of genetics, research has been advanced through the identification of mutations of the gene FUsed in Sarcoma/Translated in LipoSarcoma (FUS/TLS) in individuals with familial and sporadic ALS. This gene is involved in the regulation of transcription, splicing and RNA transport, and has functional homology to another ALS gene, TARDBP, which suggests that a common mechanism may underlie motor neuron degeneration. A report showed that mice expressing a mutant form of human TDP-43 develop a progressive and fatal neurodegenerative disease reminiscent of both ALS and frontotemporal lobar degeneration with ubiquitin aggregates (FTLD-U), providing a new animal model that may help to better understand the pathophysiology and test new therapeutics. Beside genetic studies, several epidemiologic studies have investigated the role of environmental factors. A recent study suggests that smoking is a risk factor for developing ALS and it is hypothesized that this could occur through lipid peroxidation via formaldehyde exposure. From a neuroprotective perspective, trials with IGF-1, sodium valproate, coenzyme Q or glatiramer acetate have failed to demonstrate any beneficial effect. A study published in 2008 argued that lithium may have a neuroprotective effect in ALS mice and also in patients. However, two preclinical studies failed to replicate the neuroprotective effect of lithium in ALS mice. Therapeutic trials have been performed or are currently ongoing in Europe and North America. Their results have not yet been published.

Two Italian kindreds with familial amyotrophic lateral sclerosis due to FUS mutation

Recently, fused in sarcoma/translated in liposarcoma (FUS/TLS) gene, located on chromosome 16p11.2, has been identified as a disease gene in familial amyotrophic lateral sclerosis (FALS). We have analyzed FUS/TLS in a cohort of 52 index cases from seven Italian regions with non- SOD1 and non -TARDBP FALS. We identified a heterozygous c.G1542C missense mutation in a family of northern Italian origin, and a heterozygous c.C1574T missense mutation in a family of Sicilian origin. Both variants are located in exon 15 encoding the RNA-recognition motif, and result in a substitution of an arginine with a serine in position 514 (p.R514S) and substitution of a proline with a leucine at position 525 (p.P525L), respectively. Overall, the two mutations accounted for 3.8% of 52 non-SOD1 and non-TDP43 index cases of FALS. The clinical phenotype was similar within each of the families, with a predominantly upper limb onset in the family carrying the p.R514S mutation and bulbar onset, with very young age and a rapid course in the family carrying the p.P525L mutation.