FUS Mutations Research Articles

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition.

SummaryNeuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

A novel splice site FUS mutation in a familial ALS case: effects on protein expression

Objective: To investigate the impact of a novel heterozygous FUS mutation in the acceptor splice site of intron 14 (c.1542 − 1 g > t) on protein expression in Peripheral Blood Mononuclear Cells (PBMC) from a familial ALS patient. Methods: PBMC were isolated for mRNA analysis (cDNA synthesis, sequencing and one-step RT-PCR), Western Immunoblot (WI), and Immunofluorescence (IF). Results: cDNA analysis revealed the skipping of exon 15 and a premature stop codon at c.228. RT-PCR showed reduced FUS mRNA by more than half compared to a healthy control (HC) and an ALS patient without genetic mutations (wtALS). In WI FUS band intensity in the proband was 30–50% compared to HC and wtALS. An antibody expected to detect only the wild-type protein did not reveal any reduction of FUS band intensity compared to the other antibodies. IF showed no difference among HC, wtALS, and the proband. Discussion: The reduction of FUS mRNA and protein in PBMC suggests the absence of the truncated protein, probably due to nonsense-mediated decay, leading to loss of function.

A serum microRNA sequence reveals fragile X protein pathology in amyotrophic lateral sclerosis.

Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS.

Targeted sequencing panels in Italian ALS patients support different etiologies in the ALS/FTD continuum

Background5–10% of amyotrophic lateral sclerosis (ALS) patients presented a positive family history (fALS). More than 30 genes have been identified in association with ALS/frontotemporal dementia (FTD) spectrum, with four major genes accounting for 60–70% of fALS. In this paper, we aimed to assess the contribution to the pathogenesis of major and rare ALS/FTD genes in ALS patients.MethodsWe analyzed ALS and ALS/FTD associated genes by direct sequencing or next-generation sequencing multigene panels in ALS patients.ResultsGenetic abnormalities in ALS major genes included repeated expansions of hexanucleotide in C9orf72 gene (7.3%), mutations in SOD1 (4.9%), FUS (2.1%), and TARDBP (2.4%), whereas variants in rare ALS/FTD genes affected 15.5% of subjects overall, most frequently involving SQSTM1 (3.4%), and CHMP2B (1.9%). We found clustering of variants in ALS major genes in patients with a family history for “pure” ALS, while ALS/FTD related genes mainly occurred in patients with a family history for other neurodegenerative diseases (dementia and/or parkinsonism).ConclusionsOur data support the presence of two different genetic components underlying ALS pathogenesis, related to the presence of a family history for ALS or other neurodegenerative diseases. Thus, family history may help in optimizing the genetic screening protocol to be applied.

Concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis.

Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis, a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOFs) through DPRs versus loss of C9ORF72 functions (LOFs). Through multiparametric high-content (HC) live profiling in spinal MNs from induced pluripotent stem cells and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA double-strand breaks (DSBs), RNA foci, DPRs, and apoptosis. We show that both GOFs and LOFs were necessary to yield the overall C9ORF72 pathology. Increased RNA foci and DPRs concurred with onset of axon trafficking defects, DSBs, and cell death, although DSB induction itself did not phenocopy C9ORF72 mutants. Interestingly, the majority of LOF-specific DEGs were shared with HRE-mediated GOF DEGs. Finally, C9ORF72 LOF was sufficient-albeit to a smaller extent-to induce premature distal axonal trafficking deficits and increased DSBs.

Emerging role of microRNAs in the pathogenesis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a deadly motor neuron disease (MND) and the most frequent MND in adults. ALS is recognized by degenerative alterations in both upper and lower motor neurons. This disorder is classified to familial and sporadic classes. Disease-causing mutations in SOD1, C9ORF72, FUS, and TARDBP have been recognized in familial ALS cases. However, in spite of conduction of several genetic association studies, heritable genetic risk elements in sporadic have not been identified completely. Several miRNAs have been dysregulated in the serum samples or brain tissues of ALS patients. Moreover, a number of miRNAs have been suggested as putative biomarkers for sporadic ALS. In the current manuscript, we review of miRNAs in the development of ALS.

Real-time observation of structure and dynamics during the liquid-to-solid transition of FUS LC

A subset of the proteins found in pathological protein fibrils also exhibit tendencies for liquid-liquid phase separation (LLPS) both in vitro and in cells. The mechanisms underlying the connection between these phase transitions have been challenging to study due to the heterogeneous and dynamic nature of the states formed during the maturation of LLPS protein droplets into gels and solid aggregates. Here, we interrogate the liquid-to-solid transition of the low-complexity domain of the RNA-binding protein FUS (FUS LC), which has been shown to adopt LLPS, gel-like, and amyloid states. We employ magic-angle-spinning NMR spectroscopy, which has allowed us to follow these transitions in real time and with residue-specific resolution. We observe the development of β-sheet structure through the maturation process and show that the final state of FUS LC fibrils produced after LLPS is distinct from that grown from fibrillar seeds. We also apply our methodology to FUS LC G156E, a clinically relevant FUS mutant that exhibits accelerated fibrillization rates. We observe significant changes in dynamics during the transformation of the FUS LC G156E construct and begin to unravel the sequence specific contributions to this phenomenon with computational studies of the phase-separated state of FUS LC and FUS LC G156E.

Mechanism of karyopherin-\u03b22 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X)

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid–liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-β2 (Kapβ2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapβ2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapβ2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapβ2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapβ2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapβ2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Impairment of mitochondrial oxidative phosphorylation in skin fibroblasts of SALS and FALS patients is rescued by in vitro treatment with ROS scavengers

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive, neurodegenerative disorder affecting upper and lower motor neurons. Approximately 10% of patients suffer from familial ALS (FALS) with mutations in different ubiquitously expressed genes including SOD1, C9ORF72, TARDBP, and FUS. There is compelling evidence for mitochondrial involvement in the pathogenic mechanisms of FALS and sporadic ALS (SALS), which is believed to be relevant for disease. Owing to the ubiquitous expression of relevant disease-associated genes, mitochondrial dysfunction is also detectable in peripheral patient tissue. We here report results of a detailed investigation of the functional impairment of mitochondrial oxidative phosphorylation (OXPHOS) in cultured skin fibroblasts from 23 SALS and 17 FALS patients, harboring pathogenic mutations in SOD1, C9ORF72, TARDBP and FUS. A considerable functional and structural mitochondrial impairment was detectable in fibroblasts from patients with SALS. Similarly, fibroblasts from patients with FALS, harboring pathogenic mutations in TARDBP, FUS and SOD1, showed mitochondrial defects, while fibroblasts from C9ORF72 associated FALS showed a very mild impairment detectable in mitochondrial ATP production rates only. While we could not detect alterations in the mtDNA copy number in the SALS or FALS fibroblast cultures, the impairment of OXPHOS in SALS fibroblasts and SOD1 or TARDBP FALS could be rescued by in vitro treatments with CoQ10 (5 μM for 3 weeks) or Trolox (300 μM for 5 days). This underlines the role of elevated oxidative stress as a potential cause for the observed functional effects on mitochondria, which might be relevant disease modifying factors.

Effect of familial clustering in the genetic screening of 235 French ALS families

ObjectivesTo determine whether the familial clustering of amyotrophic lateral sclerosis (ALS) cases and the phenotype of the disease may help identify the pathogenic genes involved.MethodsWe conducted a targeted next-generation sequencing...

A single copy transgenic mutant FUS strain reproduces age-dependent ALS phenotypes in C. elegans.

Mutations in the human DNA/RNA binding protein FUS are associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration, including some aggressive and juvenile onset forms. Cytoplasmic inclusions of human FUS proteins are observed in various neurodegenerative disorders, such as Huntington’s disease or spinocerebellar ataxia, suggesting that FUS proteinopathy may be a key player in neurodegeneration. To better understand the pathogenic mechanisms of FUS, we created single copy transgenic Caenorhabditis elegans strains expressing full-length, untagged human FUS in the worm’s GABAergic neurons. These transgenic worms expressing human mutant FUS (mFUS) display the same ALS-associated phenotypes than our previous multiple copy transgenic model, including adult-onset age-dependent loss of motility, progressive paralysis and GABAergic neurodegeneration. These phenotypes are distinct from the transgenic worms expressing human wild-type FUS (wtFUS). We introduce here our C. elegans single copy transgenic for human mutant FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening.

First person – Sebastian Markert

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Sebastian Markert is first author on ‘Overexpression of an ALS-associated FUS mutation in C. elegans disrupts NMJ morphology and leads to defective neuromuscular transmission’, published in BiO. Sebastian conducted the research described in this article while a PhD student in Christian Stigloher's lab at Imaging Core Facility, Biocenter, University of Wuerzburg, Germany. He is now a Postdoc in the lab of Shigeki Watanabe in Baltimore, USA, investigating how neurons communicate with each other on the molecular level.

The FUS gene is dual-coding with both proteins contributing to FUS-mediated toxicity

Novel functional coding sequences (altORFs) are camouflaged within annotated ones (CDS) in a different reading frame. We show here that an altORF is nested in the FUS CDS, encoding a conserved 170 amino acid protein, altFUS. AltFUS is endogenously expressed in human tissues, notably in the motor cortex and motor neurons. Over-expression of wild-type FUS and/or amyotrophic lateral sclerosis-linked FUS mutants is known to trigger toxic mechanisms in different models. These include inhibition of autophagy, loss of mitochondrial potential and accumulation of cytoplasmic aggregates. We find that altFUS, not FUS, is responsible for the inhibition of autophagy, and pivotal in mitochondrial potential loss and accumulation of cytoplasmic aggregates. Suppression of altFUS expression in a Drosophila model of FUS-related toxicity protects against neurodegeneration. Some mutations found in ALS patients are overlooked because of their synonymous effect on the FUS protein. Yet, we show they exert a deleterious effect causing missense mutations in the overlapping altFUS protein. These findings demonstrate that FUS is a bicistronic gene and suggests that both proteins, FUS and altFUS, cooperate in toxic mechanisms.

A Comparison of Low Read Depth QuantSeq 3' Sequencing to Total RNA-Seq in FUS Mutant Mice.

Transcriptomics is a developing field with new methods of analysis being produced which may hold advantages in price, accuracy, or information output. QuantSeq is a form of 3′ sequencing produced by Lexogen which aims to obtain similar gene-expression information to RNA-seq with significantly fewer reads, and therefore at a lower cost. QuantSeq is also able to provide information on differential polyadenylation. We applied both QuantSeq at low read depth and total RNA-seq to the same two sets of mouse spinal cord RNAs, each comprised by four controls and four mutants related to the neurodegenerative disease amyotrophic lateral sclerosis. We found substantial differences in which genes were found to be significantly differentially expressed by the two methods. Some of this difference likely due to the difference in number of reads between our QuantSeq and RNA-seq data. Other sources of difference can be explained by the differences in the way the two methods handle genes with different primary transcript lengths and how likely each method is to find a gene to be differentially expressed at different levels of overall gene expression. This work highlights how different methods aiming to assess expression difference can lead to different results.

Identification of novel FUS and TARDBP gene mutations in Chinese amyotrophic lateral sclerosis patients with HRM analysis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons. More than 30 genes have been linked to ALS to date, including FUS and TARDBP, which exhibit similar roles in RNA metabolism. This study explored the use of high-resolution melting (HRM) analysis to screen for FUS and TARDBP mutation hotspot regions in 146 Chinese ALS patients, which achieved 100% detection. Two FUS mutations were observed in two different familial ALS probands, a missense mutation (p.R521H) and a novel splicing mutation (c.1541+1G>A). Five TARDBP mutations were identified in six ALS patients, including a novel 3’UTR mutation (c.*731A>G) and four missense mutations (p.G294V, p.M337V, p.G348V, and p.I383V). We found that FUS mutations were present in 1.4% of Chinese ALS patients, whereas TARDBP mutations were responsible for 4.1% of Chinese ALS cases. Here, we describe the accuracy of using highly sensitive HRM analysis to identify two novel FUS and TARDBP mutations in Chinese sporadic and familial ALS cases. Our study contributes to the further understanding of the genetic and phenotypic diversity of ALS.

ALS/FTLD-Linked Mutations in FUS Glycine Residues Cause Accelerated Gelation and Reduced Interactions with Wild-Type FUS

The RNA-binding protein fused in sarcoma (FUS) can form pathogenic inclusions in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Over 70 mutations in Fus are linked to ALS/FTLD. In patients, all Fus mutations are heterozygous, indicating that the mutant drives disease progression despite the presence of wild-type (WT) FUS. Here, we demonstrate that ALS/FTLD-linked FUS mutations in glycine (G) strikingly drive formation of droplets that do not readily interact with WT FUS, whereas arginine (R) mutants form mixed condensates with WT FUS. Remarkably, interactions between WT and G mutants are disfavored at the earliest stages of FUS nucleation. In contrast, R mutants physically interact with the WT FUS such that WT FUS recovers the mutant defects by reducing droplet size and increasing dynamic interactions with RNA. This result suggests disparate molecular mechanisms underlying ALS/FTLD pathogenesis and differing recovery potential depending on the type of mutation.

Ubiquitin Homeostasis Is Disrupted in TDP-43 and FUS Cell Models of ALS.

SummaryA major feature of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitin (Ub) into intracellular inclusions. This sequestration of Ub may reduce the availability of free Ub, disrupting Ub homeostasis and ultimately compromising cellular function and survival. We previously reported significant disturbance of Ub homeostasis in neuronal-like cells expressing mutant SOD1. Here, we show that Ub homeostasis is also perturbed in neuronal-like cells expressing either TDP-43 or FUS. The expression of mutant TDP-43 and mutant FUS led to UPS dysfunction, which was associated with a redistribution of Ub and depletion of the free Ub pool. Redistribution of Ub is also a feature of sporadic ALS, with an increase in Ub signal associated with inclusions and no compensatory increase in Ub expression. Together, these findings suggest that alterations to Ub homeostasis caused by the misfolding and aggregation of ALS-associated proteins play an important role in the pathogenesis of ALS.

A Human-Based Functional NMJ System for Personalized ALS Modeling and Drug Testing.

Loss of the neuromuscular junction (NMJ) is an early and critical hallmark in all forms of ALS. The study design was to develop a functional NMJ disease model by integrating motoneurons (MNs) differentiated from multiple ALS-patients' induced pluripotent stem cells (iPSCs) and primary human muscle into a chambered system. NMJ functionality was tested by recording myotube contractions while stimulating MNs by field electrodes and a set of clinically relevant parameters were defined to characterize the NMJ function. Three ALS lines were analyzed, 2 with SOD1 mutations and 1 with a FUS mutation. The ALS-MNs reproduced pathological phenotypes, including increased axonal varicosities, reduced axonal branching and elongation and increased excitability. These MNs formed functional NMJs with wild type muscle, but with significant deficits in NMJ quantity, fidelity and fatigue index. Furthermore, treatment with the Deana protocol was found to correct the NMJ deficits in all the ALS mutant lines tested. Quantitative analysis also revealed the variations inherent in each mutant lines. This functional NMJ system provides a platform for the study of both fALS and sALS and has the capability of being adapted into subtype-specific or patient-specific models for ALS etiological investigation and patient stratification for drug testing.

Novel FUS mutation Y526F causing rapidly progressive familial amyotrophic lateral sclerosis

FUS gene is one of the most common mutated genes in amyotrophic lateral sclerosis (ALS). We sequenced for FUS mutations in a cohort of 15 familial ALS and 275 sporadic ALS of Chinese origin. All 15 exons of the FUS gene were sequenced by targeted next-generation sequencing in a cohort of 15 familial ALS indexes and 275 sporadic ALS patients of Chinese origin. One novel p.Y526F mutation in FUS was detected in one familial ALS proband. Another novel FUS p.Q140R variant and two known FUS mutations (p.R495Efs*33 and p.R521C) were identified in four sporadic ALS cases. The frequency of FUS mutation in our cohort is 6.7% in familial ALS and 1.5% in sporadic ALS. The familial ALS proband carrying the FUS p.Y526F mutation presented with juvenile-onset lower limbs weakness and demonstrated an aggressive course, with respiratory muscles involvement 6 months after onset. The other patients in the family all had limbs weakness and died 1–2 years after disease onset. Our results strengthen that FUS mutations are the most frequent genetic causes of young-onset aggressive ALS. Genetic testing of the FUS gene should be performed in early-onset ALS patients especially those with a rapid progression.

Proteomics analysis of FUS mutant human motoneurons reveals altered regulation of cytoskeleton and other ALS-linked proteins via 3\u2032UTR binding

Increasing evidence suggests that in Amyotrophic Lateral Sclerosis (ALS) mutated RNA binding proteins acquire aberrant functions, leading to altered RNA metabolism with significant impact on encoded protein levels. Here, by taking advantage of a human induced pluripotent stem cell-based model, we aimed to gain insights on the impact of ALS mutant FUS on the motoneuron proteome. Label-free proteomics analysis by mass-spectrometry revealed upregulation of proteins involved in catabolic processes and oxidation–reduction, and downregulation of cytoskeletal proteins and factors directing neuron projection. Mechanistically, proteome alteration does not correlate with transcriptome changes. Rather, we observed a strong correlation with selective binding of mutant FUS to target mRNAs in their 3′UTR. Novel validated targets, selectively bound by mutant FUS, include genes previously involved in familial or sporadic ALS, such as VCP, and regulators of membrane trafficking and cytoskeleton remodeling, such as ASAP1. These findings unveil a novel mechanism by which mutant FUS might intersect other pathogenic pathways in ALS patients’ motoneurons.