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Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice*

  • Author Footnotes
    1 Supported by the Short-Term European Molecular Biology Organization (EMBO) Fellowship (Grant ASTF 351-2011).
    Tatyana A. Shelkovnikova
    Footnotes
    1 Supported by the Short-Term European Molecular Biology Organization (EMBO) Fellowship (Grant ASTF 351-2011).
    Affiliations
    From the School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, Wales, United Kingdom,

    the Institute of Physiologically Active Compounds, Russian Academy of Sciences, 1 Severniy proezd, Chernogolovka 142432, Moscow Region, Russian Federation,
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  • Owen M. Peters
    Affiliations
    From the School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, Wales, United Kingdom,
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  • Alexey V. Deykin
    Affiliations
    the Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, Moscow 119334, Russian Federation,
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  • Natalie Connor-Robson
    Affiliations
    From the School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, Wales, United Kingdom,
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  • Author Footnotes
    2 Supported by the Cardiff Neuroscience and Mental Health Research Institute (NMHRI) 4-year Ph. D. Studentship Programme.
    Hannah Robinson
    Footnotes
    2 Supported by the Cardiff Neuroscience and Mental Health Research Institute (NMHRI) 4-year Ph. D. Studentship Programme.
    Affiliations
    From the School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, Wales, United Kingdom,
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  • Alexey A. Ustyugov
    Affiliations
    From the School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, Wales, United Kingdom,

    the Institute of Physiologically Active Compounds, Russian Academy of Sciences, 1 Severniy proezd, Chernogolovka 142432, Moscow Region, Russian Federation,
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  • Sergey O. Bachurin
    Affiliations
    the Institute of Physiologically Active Compounds, Russian Academy of Sciences, 1 Severniy proezd, Chernogolovka 142432, Moscow Region, Russian Federation,
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  • Tatyana G. Ermolkevich
    Affiliations
    the Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, Moscow 119334, Russian Federation,
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  • Igor L. Goldman
    Affiliations
    the Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, Moscow 119334, Russian Federation,
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  • Elena R. Sadchikova
    Affiliations
    the Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, Moscow 119334, Russian Federation,
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  • Elena A. Kovrazhkina
    Affiliations
    the Pirogov Russian National Research Medical University, Ostrovitianov Str. 1, Moscow 117997, Russian Federation, and
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  • Veronica I. Skvortsova
    Affiliations
    the Pirogov Russian National Research Medical University, Ostrovitianov Str. 1, Moscow 117997, Russian Federation, and
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  • Shuo-Chien Ling
    Affiliations
    the Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093
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  • Sandrine Da Cruz
    Affiliations
    the Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093
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  • Philippe A. Parone
    Affiliations
    the Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093
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  • Author Footnotes
    3 Both authors contributed equally to this work.
    Vladimir L. Buchman
    Correspondence
    To whom correspondence may be addressed. Tel.: 44-2920-879068;
    Footnotes
    3 Both authors contributed equally to this work.
    Affiliations
    From the School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, Wales, United Kingdom,

    the Institute of Physiologically Active Compounds, Russian Academy of Sciences, 1 Severniy proezd, Chernogolovka 142432, Moscow Region, Russian Federation,
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  • Author Footnotes
    3 Both authors contributed equally to this work.
    Natalia N. Ninkina
    Correspondence
    To whom correspondence may be addressed. Tel.: 44-2920-879068;
    Footnotes
    3 Both authors contributed equally to this work.
    Affiliations
    From the School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, Wales, United Kingdom,

    the Institute of Physiologically Active Compounds, Russian Academy of Sciences, 1 Severniy proezd, Chernogolovka 142432, Moscow Region, Russian Federation,
    Search for articles by this author
  • Author Footnotes
    * This work was supported by research grants from the Wellcome Trust (Grant 075615/Z/04/z) and Russian Federation Program (Agreement 8829) (to V. L. B.), the Russian Foundation for Basic Research (Grant 12-04-01610) and Russian Federation State (Contract 14.518.11.7040) (to E. R. S.), and the Program of Russian Academy of Sciences “Fundamental Sciences for Medicine” (to N. N. N.).
    This article contains supplemental Video S1.
    ♦ This article was selected as a Paper of the Week.
    1 Supported by the Short-Term European Molecular Biology Organization (EMBO) Fellowship (Grant ASTF 351-2011).
    2 Supported by the Cardiff Neuroscience and Mental Health Research Institute (NMHRI) 4-year Ph. D. Studentship Programme.
    3 Both authors contributed equally to this work.
Open AccessPublished:July 18, 2013DOI:https://doi.org/10.1074/jbc.M113.492017
      Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.
      Background: FUS inclusions are hallmarks of certain neurodegenerative diseases.
      Results: Expression of a highly aggregate prone FUS variant in transgenic mice causes proteinopathy and severe motor phenotype.
      Conclusion: Aggregation of FUS is sufficient to recapitulate motor pathology typical for amyotrophic lateral sclerosis.
      Significance: Understanding the role of protein aggregation in the development of human neurodegenerative diseases is crucial for designing efficient therapeutic approaches.

      Introduction

      Multiple missense and nonsense mutations in genes encoding DNA/RNA-binding proteins FUS
      The abbreviations used are: FUS, fused in sarcoma; TDP-43, TAR DNA-binding protein of 43 kDa; ALS, amyotrophic lateral sclerosis; NLS, nuclear localization signal; GFAP, glial fibrillary acidic protein; TG, transgenic.
      and TDP-43 were strongly linked with the development of ALS and related diseases, although it is still unclear how changes in the structure and/or metabolism of these proteins mediate pathology. In motor neurons of patients with FUS gene mutations, the encoded protein loses its normal nuclear localization and forms characteristic cytoplasmic inclusions (
      • Kwiatkowski Jr., T.J.
      • Bosco D.A.
      • Leclerc A.L.
      • Tamrazian E.
      • Vanderburg C.R.
      • Russ C.
      • Davis A.
      • Gilchrist J.
      • Kasarskis E.J.
      • Munsat T.
      • Valdmanis P.
      • Rouleau G.A.
      • Hosler B.A.
      • Cortelli P.
      • de Jong P.J.
      • Yoshinaga Y.
      • Haines J.L.
      • Pericak-Vance M.A.
      • Yan J.
      • Ticozzi N.
      • Siddique T.
      • McKenna-Yasek D.
      • Sapp P.C.
      • Horvitz H.R.
      • Landers J.E.
      • Brown Jr., R.H.
      Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis.
      ,
      • Vance C.
      • Rogelj B.
      • Hortobágyi T.
      • De Vos K.J.
      • Nishimura A.L.
      • Sreedharan J.
      • Hu X.
      • Smith B.
      • Ruddy D.
      • Wright P.
      • Ganesalingam J.
      • Williams K.L.
      • Tripathi V.
      • Al-Saraj S.
      • Al-Chalabi A.
      • Leigh P.N.
      • Blair I.P.
      • Nicholson G.
      • de Belleroche J.
      • Gallo J.M.
      • Miller C.C.
      • Shaw C.E.
      Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6.
      ). Moreover, FUS-positive inclusions have been observed in neurons of some patients with sporadic ALS (
      • Deng H.X.
      • Zhai H.
      • Bigio E.H.
      • Yan J.
      • Fecto F.
      • Ajroud K.
      • Mishra M.
      • Ajroud-Driss S.
      • Heller S.
      • Sufit R.
      • Siddique N.
      • Mugnaini E.
      • Siddique T.
      FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis.
      ), frontotemporal lobar degeneration (
      • Neumann M.
      • Rademakers R.
      • Roeber S.
      • Baker M.
      • Kretzschmar H.A.
      • Mackenzie I.R.
      A new subtype of frontotemporal lobar degeneration with FUS pathology.
      ), atypical neuronal intermediate filament inclusion disease (
      • Neumann M.
      • Roeber S.
      • Kretzschmar H.A.
      • Rademakers R.
      • Baker M.
      • Mackenzie I.R.
      Abundant FUS-immunoreactive pathology in neuronal intermediate filament inclusion disease.
      ), basophilic inclusion body disease (
      • Munoz D.G.
      • Neumann M.
      • Kusaka H.
      • Yokota O.
      • Ishihara K.
      • Terada S.
      • Kuroda S.
      • Mackenzie I.R.
      FUS pathology in basophilic inclusion body disease.
      ), and Unverricht-Lundborg disease (
      • Cohen N.R.
      • Hammans S.R.
      • Macpherson J.
      • Nicoll J.A.
      New neuropathological findings in Unverricht-Lundborg disease: neuronal intranuclear and cytoplasmic inclusions.
      ), signifying a role for non-genetic protein modifications in the development of FUS-induced neuropathology.
      However, the question of whether FUS aggregation is sufficient to cause pathological changes typical for FUSopathies or whether its altered function in RNA metabolism plays a primary role in the pathology development is still to be answered. Findings supporting the latter mechanism were reported (
      • Lagier-Tourenne C.
      • Polymenidou M.
      • Hutt K.R.
      • Vu A.Q.
      • Baughn M.
      • Huelga S.C.
      • Clutario K.M.
      • Ling S.C.
      • Liang T.Y.
      • Mazur C.
      • Wancewicz E.
      • Kim A.S.
      • Watt A.
      • Freier S.
      • Hicks G.G.
      • Donohue J.P.
      • Shiue L.
      • Bennett C.F.
      • Ravits J.
      • Cleveland D.W.
      • Yeo G.W.
      Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs.
      ), but the importance of FUS aggregation with formation of FUS positive inclusions in the affected neurons as triggers of pathological changes has never been directly addressed. This is largely caused by the apparent difficulty of separating the effects of deregulation of FUS RNA targets by overexpressed and mislocalized protein from the immediate and RNA target-independent consequences of FUS aggregation and formation of insoluble inclusions in available in vivo models. Furthermore, it appeared extremely hard to achieve aggregation and respective proteinopathy in models with expression of full-length FUS or FUS lacking functional NLS (
      • Chen Y.
      • Yang M.
      • Deng J.
      • Chen X.
      • Ye Y.
      • Zhu L.
      • Liu J.
      • Ye H.
      • Shen Y.
      • Li Y.
      • Rao E.J.
      • Fushimi K.
      • Zhou X.
      • Bigio E.H.
      • Mesulam M.
      • Xu Q.
      • Wu J.Y.
      Expression of human FUS protein in Drosophila leads to progressive neurodegeneration.
      ,
      • Xia R.
      • Liu Y.
      • Yang L.
      • Gal J.
      • Zhu H.
      • Jia J.
      Motor neuron apoptosis and neuromuscular junction perturbation are prominent features in a Drosophila model of Fus-mediated ALS.
      ,
      • Murakami T.
      • Yang S.P.
      • Xie L.
      • Kawano T.
      • Fu D.
      • Mukai A.
      • Bohm C.
      • Chen F.
      • Robertson J.
      • Suzuki H.
      • Tartaglia G.G.
      • Vendruscolo M.
      • Kaminski Schierle G.S.
      • Chan F.T.
      • Moloney A.
      • Crowther D.
      • Kaminski C.F.
      • Zhen M.
      • St George-Hyslop P.
      ALS mutations in FUS cause neuronal dysfunction and death in Caenorhabditis elegans by a dominant gain-of-function mechanism.
      ,
      • Huang C.
      • Zhou H.
      • Tong J.
      • Chen H.
      • Liu Y.J.
      • Wang D.
      • Wei X.
      • Xia X.G.
      FUS transgenic rats develop the phenotypes of amyotrophic lateral sclerosis and frontotemporal lobar degeneration.
      ), indicating that an additional event(s) is probably required to trigger aggregation of these proteins. To overcome these limitations, we have designed a FUS variant that would be predominantly cytoplasmic due to the lack of NLS and would not be able to interact with RNA and thus, directly affect RNA metabolism due to the deletion of major RNA binding domains (two C-terminal RGG boxes and a zinc finger). On the other hand, this truncated FUS 1–359 protein retained an N-terminal prion-like domain (
      • Cushman M.
      • Johnson B.S.
      • King O.D.
      • Gitler A.D.
      • Shorter J.
      Prion-like disorders: blurring the divide between transmissibility and infectivity.
      ), allowing its efficient aggregation. Moreover, because in FUS protein similar functional domains follow an inverse C- to N-terminal order to that of TDP-43, this C-terminally truncated FUS protein structurally resembled an N-terminally truncated 25-kDa product of caspase cleavage of TDP-43 that has been previously implicated in the development of neuronal pathology (
      • Neumann M.
      • Sampathu D.M.
      • Kwong L.K.
      • Truax A.C.
      • Micsenyi M.C.
      • Chou T.T.
      • Bruce J.
      • Schuck T.
      • Grossman M.
      • Clark C.M.
      • McCluskey L.F.
      • Miller B.L.
      • Masliah E.
      • Mackenzie I.R.
      • Feldman H.
      • Feiden W.
      • Kretzschmar H.A.
      • Trojanowski J.Q.
      • Lee V.M.
      Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
      ).
      Here we demonstrate that expression of a relatively low level of FUS 1–359 protein in neurons of transgenic mice triggers FUSopathy and severe motor neuron pathology, recapitulating certain key features of human diseases associated with FUS aggregation and dysfunction.

      DISCUSSION

      We produced a transgenic mouse model that has enabled us to address the effects of in vivo FUS aggregation independently of its roles in cellular RNA metabolism. Possible direct effects of exogenous FUS at the RNA-related pathways were eliminated by employing a protein with deletion of essential RNA binding domains along with the NLS. The intact N-terminal region that includes the presumptive prion-like domain (
      • Cushman M.
      • Johnson B.S.
      • King O.D.
      • Gitler A.D.
      • Shorter J.
      Prion-like disorders: blurring the divide between transmissibility and infectivity.
      ) enabled rapid aggregation of exogenous truncated protein and even engagement of endogenous mouse FUS into inclusions, suggesting that pathologically modified forms of FUS are able to seed aggregation of other, including structurally normal, FUS proteins. It is feasible that a similar mechanism might be involved in the formation of inclusions and even spreading of pathology in human FUSopathies. Moreover, sequestering of endogenous FUS in inclusions might cause depletion of its functional soluble forms in affected neurons and consequent malfunction of RNA metabolism. Such secondary loss of function along with direct toxicity of products of FUS aggregation might play a role in the development of neurodegenerative changes in the nervous system of FUS transgenic mice and patients with FUSopathy.
      An abrupt development of prominent FUSopathy in hemizygous line 19 transgenic mice expressing FUS 1–359 at a level lower than the level of endogenous protein led to severe damage of myelinated axons in the peripheral nerves, loss of spinal motor neurons, and consequently, muscle paralysis and atrophy. This resulted in the death of young animals several days after the first signs of pathology, which resembles the clinical pattern of typical ALS if an average mouse lifespan is extrapolated to the human one.
      A selective loss of brainstem motor neurons in line 19 mice also emulates the pattern of brainstem motor nuclei damage typical for ALS where motor neuron populations involved in eye movements remain spared. Therefore, FUS aggregation mediated by its N terminus is sufficient to cause degeneration and death of motor neurons in a very selective fashion. Surprisingly, we did not observe neuroinflammatory reaction in brainstem motor nuclei affected by the neurodegenerative process, whereas both microgliosis and astrogliosis were evident in anterior horns of the spinal cord of line 19 transgenic mice. Further studies are required to explain this phenomenon.
      We also produced a second line of transgenic mice expressing FUS 1–359 in their nervous system. Consistent with the significantly lower level of transgene expression, when compared with the first line, these mice start to develop neuronal FUS-positive inclusions (Fig. 2H) and compromised performance in motor tests (data not shown) only at the age of 9 months. Further studies of older line 6 animals will require us to establish whether their phenotype recapitulates certain features of late onset and slow developing ALS or other FUS-related neurodegenerative diseases.
      Animal models available so far could not be used to discriminate between the effects exerted by RNA binding-competent FUS proteins directly at the cellular RNA metabolism and effects caused by their aggregation and deposition in the cytoplasm. Moreover, in these models, either efficient FUS aggregation and inclusion formation have not been achieved or/and only certain aspects of ALS FUS were recapitulated. Overexpression of FUS in Drosophila melanogaster (
      • Chen Y.
      • Yang M.
      • Deng J.
      • Chen X.
      • Ye Y.
      • Zhu L.
      • Liu J.
      • Ye H.
      • Shen Y.
      • Li Y.
      • Rao E.J.
      • Fushimi K.
      • Zhou X.
      • Bigio E.H.
      • Mesulam M.
      • Xu Q.
      • Wu J.Y.
      Expression of human FUS protein in Drosophila leads to progressive neurodegeneration.
      ,
      • Miguel L.
      • Avequin T.
      • Delarue M.
      • Feuillette S.
      • Frébourg T.
      • Campion D.
      • Lecourtois M.
      Accumulation of insoluble forms of FUS protein correlates with toxicity in Drosophila.
      ) and Caenorhabditis elegans (
      • Murakami T.
      • Yang S.P.
      • Xie L.
      • Kawano T.
      • Fu D.
      • Mukai A.
      • Bohm C.
      • Chen F.
      • Robertson J.
      • Suzuki H.
      • Tartaglia G.G.
      • Vendruscolo M.
      • Kaminski Schierle G.S.
      • Chan F.T.
      • Moloney A.
      • Crowther D.
      • Kaminski C.F.
      • Zhen M.
      • St George-Hyslop P.
      ALS mutations in FUS cause neuronal dysfunction and death in Caenorhabditis elegans by a dominant gain-of-function mechanism.
      ), although confirming toxic gain of function for mutant variants, did not result in the formation of FUS-positive inclusions. In a previously described line of FUS transgenic mice, the increase of human wild type FUS expression by breeding animals to transgene homozygosity was required for the development of neurological phenotype (
      • Mitchell J.C.
      • McGoldrick P.
      • Vance C.
      • Hortobagyi T.
      • Sreedharan J.
      • Rogelj B.
      • Tudor E.L.
      • Smith B.N.
      • Klasen C.
      • Miller C.C.
      • Cooper J.D.
      • Greensmith L.
      • Shaw C.E.
      Overexpression of human wild-type FUS causes progressive motor neuron degeneration in an age- and dose-dependent fashion.
      ). In a rat model, transgenic expression of an ALS-associated variant, FUS R521C, although leading to paralysis due to muscle denervation, was not sufficient to cause proteinopathy and motor neuron loss in the spinal cord (
      • Huang C.
      • Zhou H.
      • Tong J.
      • Chen H.
      • Liu Y.J.
      • Wang D.
      • Wei X.
      • Xia X.G.
      FUS transgenic rats develop the phenotypes of amyotrophic lateral sclerosis and frontotemporal lobar degeneration.
      ). Similarly, expression of FUS R521C or wild type FUS in the nervous system of mice via recombinant AAV1-driven somatic brain transfer technique did not cause formation of inclusions (
      • Verbeeck C.
      • Deng Q.
      • Dejesus-Hernandez M.
      • Taylor G.
      • Ceballos-Diaz C.
      • Kocerha J.
      • Golde T.
      • Das P.
      • Rademakers R.
      • Dickson D.W.
      • Kukar T.
      Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis.
      ). However, in the same experimental system, a truncated FUS variant lacking both NLS and a portion of RGG3 aggregated and formed inclusions in the brain neurons, although these mice did not develop a motor phenotype or neurodegeneration at the age of 3 months (
      • Verbeeck C.
      • Deng Q.
      • Dejesus-Hernandez M.
      • Taylor G.
      • Ceballos-Diaz C.
      • Kocerha J.
      • Golde T.
      • Das P.
      • Rademakers R.
      • Dickson D.W.
      • Kukar T.
      Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis.
      ). It is not clear whether the latter was due to the nature of aggregates formed by the FUS variant used or less efficient expression of the ectopic protein in the most sensitive neuronal populations, i.e. motor neurons. Unexpectedly, nuclear localization and aggregation of FUS variants completely lacking C-terminal NLS were observed in a significant number of mouse neurons in the aforementioned study (
      • Verbeeck C.
      • Deng Q.
      • Dejesus-Hernandez M.
      • Taylor G.
      • Ceballos-Diaz C.
      • Kocerha J.
      • Golde T.
      • Das P.
      • Rademakers R.
      • Dickson D.W.
      • Kukar T.
      Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis.
      ) and in our study, and the reason for this is not clear. However, it should be noticed that the majority of data about the importance of certain motifs within the FUS molecule for nuclear import and export have been obtained in cultured non-neuronal cells. These processes might be regulated differently in various cell populations in vivo.
      The requirement of additional factors for triggering irreversible aggregation of FUS protein in vivo, for example compromised binding to target RNA coupled with cell stress, might explain difficulties with establishing adequate models of FUSopathy in short-lived laboratory animals. In our model, we managed to bypass this requirement by using a highly aggregate-prone protein lacking RNA binding capacity. The phenotype in our transgenic mice reproduces key features of human ALS FUS: i.e. (i) the presence of FUS-positive inclusions; (ii) abrupt disease onset and fast progression; (iii) motor deficits including asymmetrical limb paralysis; (iv) muscle wasting and denervation; and (v) loss of spinal motor neurons and selective involvement of brainstem nuclei. Importantly, and unlike other available models, our FUS transgenic mice allowed us to distinguish between direct effects on the cellular RNA metabolism exerted by RNA binding-competent proteins and effects triggered solely by FUS aggregation.
      In conclusion, our studies produced the first direct in vivo evidence that aggregation of FUS protein can per se trigger FUSopathy with severe damage to susceptible neurons. This suggests that protein aggregation might be considered as a promising target for therapeutic intervention in FUSopathies.

      Acknowledgments

      We are grateful to Justin Rochford and Don Cleveland for critical reading of the manuscript.

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