Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss

FUS is an RNA‐binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS‐containing aggregates are often associated with concomitant loss of nuclear FUS. Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell‐specific CRE‐mediated expression of wild‐type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.     

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.     

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

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

Phosphorylation of the FUS low‐complexity domain disrupts phase separation,aggregation, and toxicity

Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation‐prone, yeast prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.     

FUS contributes to mTOR-dependent inhibition of translation

The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)–linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.     

FUS and TARDBP but not SOD1 interact in genetic models of amyotrophic lateral sclerosis

Mutations in the SOD1 and TARDBP genes have been commonly identified in Amyotrophic Lateral Sclerosis (ALS). Recently, mutations in the Fused in sarcoma gene (FUS) were identified in familial (FALS) ALS cases and sporadic (SALS) patients. Similarly to TDP-43 (coded by TARDBP gene), FUS is an RNA binding protein. Using the zebrafish (Danio rerio), we examined the consequences of expressing human wild-type (WT) FUS and three ALS-related mutations, as well as their interactions with TARDBP and SOD1. Knockdown of zebrafish Fus yielded a motor phenotype that could be rescued upon co-expression of wild-type human FUS. In contrast, the two most frequent ALS-related FUS mutations, R521H and R521C, unlike S57Δ, failed to rescue the knockdown phenotype, indicating loss of function. The R521H mutation caused a toxic gain of function when expressed alone, similar to the phenotype observed upon knockdown of zebrafish Fus. This phenotype was not aggravated by co-expression of both mutant human TARDBP (G348C) and FUS (R521H) or by knockdown of both zebrafish Tardbp and Fus, consistent with a common pathogenic mechanism. We also observed that WT FUS rescued the Tardbp knockdown phenotype, but not vice versa, suggesting that TARDBP acts upstream of FUS in this pathway. In addition we observed that WT SOD1 failed to rescue the phenotype observed upon overexpression of mutant TARDBP or FUS or upon knockdown of Tardbp or Fus; similarly, WT TARDBP or FUS also failed to rescue the phenotype induced by mutant SOD1 (G93A). Finally, overexpression of mutant SOD1 exacerbated the motor phenotype caused by overexpression of mutant FUS. Together our results indicate that TARDBP and FUS act in a pathogenic pathway that is independent of SOD1.     

Liquidizing FUS via prion‐like domain phosphorylation

FUS is an RNA‐binding protein (RBP) with a prion‐like domain (PrLD) that condenses into functional liquids, which can aberrantly phase transition into solid aggregates comprised of pathological fibrils connected to neurodegenerative disease. How cells prevent aberrant phase transitions of FUS and other disease‐linked RBPs with PrLDs is poorly understood. In this issue of The EMBO Journal, Monahan et al ( 2017 ) establish that phosphorylation of specific serine and threonine residues in the FUS PrLD inhibits aberrant phase separation and toxicity.     

Knockdown of the Drosophila fused in sarcoma (FUS) homologue causes deficient locomotive behavior and shortening of motoneuron terminal branches

Mutations in the fused in sarcoma/translated in liposarcoma gene (FUS/TLS, FUS) have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). FUS is an RNA-binding protein that is normally localized in the nucleus, but is mislocalized to the cytoplasm in ALS, and comprises cytoplasmic inclusions in ALS-affected areas. However, it is still unknown whether the neurodegeneration that occurs in ALS is caused by the loss of FUS nuclear function, or by the gain of toxic function due to cytoplasmic FUS aggregation. Cabeza (Caz) is a Drosophila orthologue of human FUS. Here, we generated Drosophila models with Caz knockdown, and investigated their phenotypes. In wild-type Drosophila, Caz was strongly expressed in the central nervous system of larvae and adults. Caz did not colocalize with a presynaptic marker, suggesting that Caz physiologically functions in neuronal cell bodies and/or their axons. Fly models with neuron-specific Caz knockdown exhibited reduced climbing ability in adulthood and anatomical defects in presynaptic terminals of motoneurons in third instar larvae. Our results demonstrated that decreased expression of Drosophila Caz is sufficient to cause degeneration of motoneurons and locomotive disability in the absence of abnormal cytoplasmic Caz aggregates, suggesting that the pathogenic mechanism underlying FUS-related ALS should be ascribed more to the loss of physiological FUS functions in the nucleus than to the toxicity of cytoplasmic FUS aggregates. Since the Caz-knockdown Drosophila model we presented recapitulates key features of human ALS, it would be a suitable animal model for the screening of genes and chemicals that might modify the pathogenic processes that lead to the degeneration of motoneurons in ALS.     

THE LIFE CYCLE AND TOXICITY OF PFIESTERIA PISCICIDA REVISITED1

Despite use of excellent molecular techniques, Litaker et al. (2002) cannot provide insights about the life history of toxic Pfiesteria piscicida because they showed no data in support of having used toxic strains; rather they presented evidence that they used non‐inducible strains. Litaker et al. did not find amoeboid stages or a chrysophyte‐like cyst stage in several cultures and unequivocally concluded that the stages do not exist in all P. piscicida strains. Thus, they did not consider the tenet that absence of evidence does not constitute proof of absence. Apparent discrepancies between the research by Litaker et al. and previous research on Pfiesteria can be resolved as follows: First, Litaker et al. did not use toxic strains. We have reported findings (similar to Litaker et al.) showing few amoeboid transformations in non‐inducible strains, which manifest some but not all of the forms that have been documented in some toxic strains. We, and others, have documented active toxicity to fish, transformations to amoebae, and chrysophyte‐like cysts in some clonal toxic strains. Second, the data from several recent publications, which were available but not mentioned by Litaker et al. or by Coats (2002) in accompanying commentary, have verified P. piscicida amoebae, chrysophyte‐like cysts, and other stages in some toxic strains through a combination of approaches including PCR data from clonal cultures.     

Engineered Yeasts as Reporter Systems for Odorant Detection

Abstract

The functional expression of olfactory receptors (ORs) is a primary requirement to utilize olfactory detection systems. We have taken advantage of the functional similarities between signal transduction cascades in the budding yeast Saccharomyces cerevisiae and mammalian cells. The yeast pheromone response pathway has been adapted to allow ligand‐dependent signaling of heterologous expressed G‐protein coupled receptors (GPCRs) via mammalian or chimeric yeast/mammalian Gα proteins. Two different strategies are reported here which offer a positive screen for functional pairs. The OR and Gα protein are introduced into the modified yeast cells such that they hijack the pheromone response pathway usually resulting in cell cycle arrest. The first strategy utilizes ligand‐induced expression of a FUS1‐HIS3 reporter gene to permit growth on a selective medium lacking histidine; the second to induce ligand‐dependent expression of a FUS1‐Hph reporter gene, conferring resistance to hygromycin. Validation of the systems was performed using the rat I7 receptor response to a range of aldehyde odorants previously characterized as functional ligands. Of these only heptanal produced a positive growth response in the concentration range 5 × 10^?8 to 5 × 10^?6 M. Induction conditions appear to be critical for functional expression, and the solvents of odorants have a toxic effect for the highest odorant concentrations. The preference of rat I7 receptor for the ligand heptanal in yeast has to be compared to concurrent results obtained with mammalian expression systems.     

ArsP: a methylarsenite efflux permease

Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3‐nitro‐4‐hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study, C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III) > Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus, ArsP is the first identified efflux system specific for trivalent organoarsenicals.     

Impact of fusaproliferin on primary roots of maize cultivars differing in their susceptibility to <Emphasis Type="Italic">Fusarium</Emphasis>

Effects of fusaproliferin (FUS) on membrane potential (E _M), electrolyte leakage, enzymes activity and respiration of roots, were studied in two maize cultivars (Zea mays L.), differing in their susceptibility to this toxin. In short-term experiments (≤ 6 h), E _M has been rapidly and significantly depolarized by FUS. The rapidity of E _M depolarization in tolerant cv. Lucia was more expressive in comparison with susceptible cv. Pavla, but the extent of E _M depolarization was higher in cv. Pavla. In both maize cultivars, higher depolarization of E _M was registered in cells of root zone I. In long-term experiments after the first E _M depolarization, which occurred during the first 6 h of FUS treatment, gradual depolarization continued up to 24 h and was represented not only by the active component (E _P) but also by the passive component (E _D) of E _M. The decrease in E _M and E _D was followed by a loss of K⁺ ions from FUS treated roots of both cultivars. The leak of K⁺ ions from the root cells of both root zones as well as both maize cultivars increased with the time of FUS treatment and was significantly higher in susceptible cv. Pavla than in tolerant cv. Lucia. FUS treatment of maize roots resulted in a significant decrease of root respiration which was higher in susceptible cv. Pavla than in tolerant cv. Lucia.     

FUS transgenic rats develop the phenotypes of amyotrophic lateral sclerosis and frontotemporal lobar degeneration

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.     

<Emphasis Type="Italic">LEAFY COTYLEDON1</Emphasis>, <Emphasis Type="Italic">FUSCA3</Emphasis> expression and auxin treatment in relation to somatic embryogenesis induction in Arabidopsis

The expression pattern of the LEC1 and FUS3 genes during somatic embryogenesis in Arabidopsis explants (immature zygotic embryos) induced in vitro was analysed, using Real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC1 but not FUS3 within a 30 day time course of somatic embryo development, and a significant auxin-dependent upregulation of LEC1 was found over the time course. In contrast to embryogenic culture, the level of LEC1 and FUS3 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. In addition, the expression profile of LEC1 and FUS3 was followed in the embryogenic culture derived from 35S::LEC2-GR explants. A significant increase of LEC1 but not FUS3 activity was observed under LEC2 overexpression induced in auxin-treated explants. The work provides further experimental evidence on LEC gene involvement in the embryogenic response in Arabidopsis somatic cells, and also implicates LEC1 function in more advanced stages of SE culture in relation to somatic embryo differentiation and development.     

Effects of prey quality on social wasps when given a choice of prey

The effect of prey quality on foraging behavior and colony demographics of the social wasp Polistes fuscatus was examined by providing a choice between non‐toxic prey (Manduca sexta caterpillars) and sublethally toxic prey (Junonia coenia caterpillars), and then comparing the performance of these colonies to others given only the non‐toxic prey. In the choice, one of two types of Manduca were used: those fed an artificial diet or those fed plantain (Plantago lanceolata), which contains iridoid glycosides (IGs) that Junonia coenia store but which Manduca does not. Despite the negative correlation between the number of Junonia prey used and number of adult offspring produced, when a surplus of non‐toxic prey was available, the wasps did not completely avoid the toxic prey. However, they were more discriminating when the choice was between Manduca fed an artificial diet and Junonia fed plantain vs. when both prey species had eaten the plantain. Because the wasps had a choice of prey types and had a surplus of prey on about one‐third of the days, the wasps were able to take enough non‐toxic prey to avoid some of the negative consequences of IGs. For example, the total number of wasp offspring per nest was not affected, but mean weight of female offspring per colony was less for colonies given both prey types eating plantain, compared to that for colonies fed only non‐toxic prey, or those given a choice of non‐toxic prey vs. toxic prey. In addition, compared to the control (only non‐toxic prey), the proportion of males produced was less in the treatment that provided a clear contrast between non‐toxic and toxic prey. Why these wasps did not avoid the toxic prey is discussed.