. 2016 May 17;35(10):1077-97.

doi: 10.15252/embj.201592559. Epub 2016 Mar 7.

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

Jelena Scekic-Zahirovic¹, Oliver Sendscheid², Hajer El Oussini¹, Mélanie Jambeau³, Ying Sun³, Sina Mersmann², Marina Wagner², Stéphane Dieterlé¹, Jérome Sinniger¹, Sylvie Dirrig-Grosch¹, Kevin Drenner³, Marie-Christine Birling⁴, Jinsong Qiu⁵, Yu Zhou⁵, Hairi Li⁵, Xiang-Dong Fu⁵, Caroline Rouaux¹, Tatyana Shelkovnikova⁶, Anke Witting⁷, Albert C Ludolph⁷, Friedemann Kiefer⁸, Erik Storkebaum⁹, Clotilde Lagier-Tourenne¹⁰, Luc Dupuis¹¹

Affiliations

¹ Faculté de Médecine, INSERM U1118, Strasbourg, France Université de Strasbourg UMR_S1118, Strasbourg, France.
² Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Muenster, Germany Faculty of Medicine, University of Muenster, Muenster, Germany.
³ Department of Neurosciences, University of California, San Diego La Jolla, CA, USA Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA, USA.
⁴ Institut Clinique de la souris, Illkirch-Graffenstaden, France.
⁵ Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA.
⁶ University of Cardiff, Cardiff, UK.
⁷ Department of Neurology University of Ulm, Ulm, Germany.
⁸ Mammalian Cell Signaling Laboratory, Department of Vascular Cell Biology, Max Planck Institute for Molecular Biomedicine, Muenster, Germany.
⁹ Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Muenster, Germany Faculty of Medicine, University of Muenster, Muenster, Germany erik.storkebaum@mpi-muenster.mpg.de clagier-tourenne@mgh.harvard.edu ldupuis@unistra.fr.
¹⁰ Department of Neurosciences, University of California, San Diego La Jolla, CA, USA Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA, USA erik.storkebaum@mpi-muenster.mpg.de clagier-tourenne@mgh.harvard.edu ldupuis@unistra.fr.
¹¹ Faculté de Médecine, INSERM U1118, Strasbourg, France Université de Strasbourg UMR_S1118, Strasbourg, France erik.storkebaum@mpi-muenster.mpg.de clagier-tourenne@mgh.harvard.edu ldupuis@unistra.fr.

PMID: 26951610
PMCID: PMC4868956
DOI: 10.15252/embj.201592559

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

Jelena Scekic-Zahirovic et al. EMBO J. 2016.

. 2016 May 17;35(10):1077-97.

doi: 10.15252/embj.201592559. Epub 2016 Mar 7.

Authors

Affiliations

¹ Faculté de Médecine, INSERM U1118, Strasbourg, France Université de Strasbourg UMR_S1118, Strasbourg, France.
² Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Muenster, Germany Faculty of Medicine, University of Muenster, Muenster, Germany.
³ Department of Neurosciences, University of California, San Diego La Jolla, CA, USA Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA, USA.
⁴ Institut Clinique de la souris, Illkirch-Graffenstaden, France.
⁵ Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA.
⁶ University of Cardiff, Cardiff, UK.
⁷ Department of Neurology University of Ulm, Ulm, Germany.
⁸ Mammalian Cell Signaling Laboratory, Department of Vascular Cell Biology, Max Planck Institute for Molecular Biomedicine, Muenster, Germany.
⁹ Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Muenster, Germany Faculty of Medicine, University of Muenster, Muenster, Germany erik.storkebaum@mpi-muenster.mpg.de clagier-tourenne@mgh.harvard.edu ldupuis@unistra.fr.
¹⁰ Department of Neurosciences, University of California, San Diego La Jolla, CA, USA Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA, USA erik.storkebaum@mpi-muenster.mpg.de clagier-tourenne@mgh.harvard.edu ldupuis@unistra.fr.
¹¹ Faculté de Médecine, INSERM U1118, Strasbourg, France Université de Strasbourg UMR_S1118, Strasbourg, France erik.storkebaum@mpi-muenster.mpg.de clagier-tourenne@mgh.harvard.edu ldupuis@unistra.fr.

PMID: 26951610
PMCID: PMC4868956
DOI: 10.15252/embj.201592559

Abstract

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.

Keywords: FUS; PY‐NLS; amyotrophic lateral sclerosis; frontotemporal dementia; motor neuron degeneration.

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Figures

**Figure EV1. Relevance of *Fus* ^Δ ^NLS mice to human ALS**
Upper panel: Scheme of the wild‐type FUS protein. The NLS, encoded by exon 15, includes the C‐terminal amino acids (aa 507–526, boundaries shown by the two dashed lines). Middle panels: 11 frameshift mutations (upper middle panel) and 2 truncating mutations (lower middle panel) in the *FUS* gene have been identified in ALS families. The corresponding mutant FUS proteins are shown. Insertions of abnormal polypeptide sequences induced by frameshift mutations are shown as red boxes. Lower panel: structure of FUS ^∆ ^NLS protein in *Fus* ^Δ ^NLS mice.

**Figure 1. FUS mislocalization in *Fus* ^Δ ^NLS ^/Δ ^NLS mice**
Schematic representation of the *Fus* gene locus (upper panel). Lower panels depict exons 11–15 in the wild‐type allele (left) and ∆NLS allele (right) with localization of PCR primers used for genotyping (gDNA, used in B) and for RT–PCR (Total and ∆NLS, used in C). Arrow: translational start site. STOP cassettes are indicated in red; loxP sites as black triangles; coding regions are in dark blue and UTRs in light blue. Location of the region encoding the nuclear localization signal (NLS) is indicated in exon 15.
Representative PCR genotyping results from 2 *Fus* ^+/+, 2 *Fus* ^Δ ^NLS ^/+, and 2 *Fus* ^Δ ^NLS ^/Δ ^NLS knockin mice using primers designed around the distal loxP site of the *Fus* ^Δ ^NLS allele and shown as gDNA in (A). The expected size of the PCR product of the ∆NLS allele is 240 bp; the size of wild‐type allele is 160 bp.
RT–PCR analysis of brain from 2 *Fus* ^+/+, 2 *Fus* ^Δ ^NLS ^/+, and 2 *Fus* ^Δ ^NLS ^/Δ ^NLS knockin P0 mice using primers located in the STOP cassette, and thus specific to the ∆NLS mRNA (∆NLS, upper panel), or primers located in exon 11, that is, upstream of the floxed cDNA insertion, and thus amplifying total *Fus*‐derived mRNA (Total, middle panel). PCR amplification of 18S rRNA is shown as a standard gene (lower panel).
Immunoblot analysis of FUS protein in cerebral cortex of 2 *Fus* ^+/+, 2 *Fus* ^Δ ^NLS ^/+, and 2 *Fus* ^Δ ^NLS ^/Δ ^NLS knockin mice using a combination of two different antibodies targeting either the C‐terminal (C‐ter. 1 and C‐ter. 2) NLS, the N‐terminal part (N‐ter. 1), or an internal part (N‐ter. 2) of FUS. Molecular weight markers are shown on the left, and apparent MW is indicated.
Double immunostaining for the motor neuronal marker ChAT and Fus (N‐terminal part) in the ventral horn of spinal cord.
Double immunostaining for nuclei (DAPI, blue) and Fus (N‐terminal part) in the cerebral cortex.
Source data are available online for this figure.

**Figure EV2. Expression of the *Fus* gene in various tissues of *Fus* ^Δ ^NLS mice**
RT–PCR analysis of spinal cord and gastrocnemius muscle from 2 *Fus* ^+/+, 2 *Fus* ^Δ ^NLS ^/+, and 2 *Fus* ^Δ ^NLS ^/Δ ^NLS P0 mice using primers located in the STOP cassette, and thus specific to the *Fus* ∆NLS mRNA (∆NLS, upper panel), or primers located in exon 11 and 12, that is, upstream of the floxed cDNA insertion, and thus amplifying total *Fus* mRNA (Total, lower panel).
Immunoblot analysis of FUS protein in spinal cord and gastrocnemius of 2 *Fus* ^+/+, 2 *Fus* ^Δ ^NLS ^/+, and 2 *Fus* ^Δ ^NLS ^/Δ ^NLS mice using two different antibodies targeting the C‐terminal (C‐ter. 1 and C‐ter. 2) NLS or antibodies targeting the N‐terminal (N‐ter. 1) and internal parts (N‐ter. 2) of FUS.
Representative confocal images for fluorescence immunocytochemical localization of FUS protein in mouse embryonic fibroblasts (MEFs).

**Figure 2. Perinatal lethality in *Fus* ^∆ ^NLS ^/∆ ^NLS mice**
A
Photographs of *Fus* ^+/+ and *Fus* ^Δ ^NLS ^/Δ ^NLS pups immediately after birth (P0 animals).
B, C
*Fus* ^Δ ^NLS ^/Δ ^NLS mice showed significantly reduced body weight (B) and length (C). Weight and length values normalized to wild type (*Fus* ^+/+) are presented (mean ± SEM). N = 11 *Fus* ^+/+, N = 26 *Fus* ^Δ ^NLS ^/+ and N = 14 *Fus* ^Δ ^NLS ^/Δ ^NLS; *P < 0.05, **P < 0.01 versus *Fus* ^+/+, ^# P < 0.05 versus *Fus* ^∆ ^NLS ^/+; one‐way ANOVA followed by Tukey's *post hoc* test.
D
Representative hematoxylin and eosin stainings of lungs of *Fus* ^+/+ and *Fus* ^Δ ^NLS ^/Δ ^NLS at birth.

**Figure 3. Generation of a complete *Fus* ^−/− loss‐of‐function mouse model**
A
Schematic representation of the *Fus* gene locus (upper panel). Lower panels depict exons 1–3 in the wild‐type allele (left) and loss‐of‐function allele (right). Arrow: translational start site; SA: splice acceptor; βgeo: β‐galactosidase/neomycin phosphotransferase fusion gene; pA: polyA.
B
Representative immunoblot for FUS on protein extracts of E18.5 brain. Histone 3 is used as a loading control.
C
Quantification of FUS protein levels from immunoblots.
D
Quantitative real‐time PCR for *Fus* transcript in *Fus* ^+/+ *and Fus* ^−/− mice. ND: not detected.
E
Immunostaining for the neuronal marker NeuN and FUS on the spinal cord ventral horn of E18.5 *Fus* ^+/+ and *Fus* ^−/− mice.
F, G
Body weight (F) and length (G) of *Fus* ^+/+, *Fus* ^+/−, and *Fus* ^−/− pups at birth; N = 14 *Fus* ^+/+, N = 36 *Fus* ^+/−, and N = 13 *Fus* ^−/− for body weight; N = 6 per genotype for body length.
Data information: Data represent mean ± SEM. **P < 0.01 versus *Fus* ^+/+, ^## P < 0.01 versus *Fus* ^+/−; one‐way ANOVA followed by Tukey's *post hoc* test.Source data are available online for this figure.

**Figure EV3. Genomewide expression changes identified by RNA‐seq in *Fus^Δ^NLS^/Δ^NLS* and *Fus* ^−/− brains**
Quantification of *Fus* RNA levels by strand‐specific RNA sequencing in brains from *Fus* ^Δ ^NLS ^/Δ ^NLS (blue bars), *Fus* ^−/− (red bars), and control littermates (*Fus* ^+/+, black bars). RNA levels were determined by fragments per kilobase of transcript per million mapped reads (FPKM) value.
Unsupervised hierarchical cluster analysis using all RNAs expressed in brains of *Fus* ^Δ ^NLS ^/Δ ^NLS mice (KI‐1 to KI‐5) and their control littermates (Ctrl‐1 to Ctrl‐4).
Unsupervised hierarchical cluster analysis using all RNAs expressed in brains of *Fus* ^−/− mice (KO‐1 to KO‐5) and their control littermates (Ctrl‐1 to Ctrl‐5).

**Figure 4. FUS‐dependent expression changes in mouse brain**
RNA‐seq reads from brain of homozygous knockin (*Fus* ^Δ ^NLS ^/Δ ^NLS, upper panel), homozygous knockout (*Fus* ^−/−, middle panel), and control (*Fus* ^+/+, lower panel) mice showing the absence of exon 15 (red arrow) in *Fus* mRNA in *Fus* ^Δ ^NLS ^/Δ ^NLS mice, while the entire *Fus* transcript is absent in *Fus* ^−/− mice (green arrows).
Heat map with hierarchical clustering of RNA‐seq data from biological replicates of *Fus* ^Δ ^NLS ^/Δ ^NLS (N = 5) and control littermates (N = 4), showing genes differentially regulated between both genotypes among which 237 are upregulated and 549 are downregulated in *Fus* ^Δ ^NLS ^/Δ ^NLS animals as defined by P < 0.05 adjusted for multiple testing.
Heat map with hierarchical clustering of RNA‐seq data from biological replicates of *Fus* ^−/− (N = 5) and control littermates (N = 5), showing genes differentially regulated between both genotypes, among which 669 are upregulated and 889 are downregulated in *Fus* ^−/− animals as defined by P < 0.05 adjusted for multiple testing.
Venn diagram showing the number of overlapping genes misregulated in *Fus* ^Δ ^NLS ^/Δ ^NLS (blue circle) and *Fus* ^−/− (red circle) brains with 353 genes similarly downregulated or upregulated upon cytoplasmic mislocalization or complete loss of FUS.
Normalized expression (based on FPKM from RNA‐seq) of genes identified by RNA‐seq to be significantly downregulated (*Ahi1, Kcnip1, Nefm, Nefl, Tuba4a, Dmpk, Rad9b, Stac3, Hist1h2bc, Hist1h1c*) or upregulated (*Fam193b, Pmm2, Bphl, Taf15*) in both *Fus* ^Δ ^NLS ^/Δ ^NLS and *Fus* ^−/− compared to their control. Error bars represent SEM in 4‐5 biological replicates. **P < 0.01, two‐tailed student's t‐test.
Normalized expression (based on FPKM from RNA‐seq) of genes identified by RNA‐seq to be uniquely changed in *Fus* ^Δ ^NLS ^/Δ ^NLS mice (*Trove2, Uhmk1, Ssh3, Vtn, Snrpb, Ephb3*). Error bars represent SEM in 4–5 biological replicates. *P < 0.05, **P < 0.01, two‐tailed Student's t‐test.

**Figure EV4. FUS‐dependent splicing alterations identified by RASL‐seq**
Schematic representation of the RASL‐seq strategy to measure ratios of alternative splicing isoforms from thousands of selected splicing events by high‐throughput sequencing.
Unsupervised hierarchical cluster analysis using all splicing events sequenced in brains of *Fus* ^Δ ^NLS ^/Δ ^NLS mice (KI‐1 to KI‐4), *Fus* ^Δ ^NLS ^/+ (HET‐1 to HET‐4), and their control littermates (Ctrl‐1 to Ctrl‐4).
Unsupervised hierarchical cluster analysis using all splicing events sequenced in brains of *Fus* ^−/− mice (KO‐1 to KO‐5) and their control littermates (Ctrl‐1 to Ctrl‐5).
Venn diagram showing the number of overlapping splicing events that are misregulated in *Fus* ^Δ ^NLS ^/Δ ^NLS (blue circle) and *Fus* ^−/− (red circle) brains with 75 exons similarly altered upon cytoplasmic mislocalization or complete loss of FUS.
Heat map using the fold changes of the 75 splicing events commonly regulated in *Fus* ^Δ ^NLS ^/Δ ^NLS and *Fus* ^−/− mice showing that 100% of the events were differentially included or excluded in the same direction.
Semi‐quantitative RT–PCR analyses of selected targets shown in Fig 5C with alternatively spliced exons depicted in orange boxes with their flanking constitutive exons in blue boxes.

**Figure 5. FUS‐dependent alternative splicing alterations in mouse brain**
Heat map with hierarchical clustering of RASL‐seq data from biological replicates of *Fus* ^Δ ^NLS ^/Δ ^NLS (N = 4) and control littermates (N = 4), showing 173 alternative splicing alterations associated with expression of cytoplasmic FUS in knockin animals.
Heat map with hierarchical clustering of RASL‐seq data from biological replicates of *Fus* ^−/− (N = 5) and control littermates (N = 5), showing 252 alternative splicing alterations associated with loss of FUS in knockout animals.
Semi‐quantitative RT–PCR analyses of selected targets. Left panels show representative acrylamide gel pictures of RT–PCR products. Quantification of splicing changes from at least three biological replicates of *Fus* ^Δ ^NLS ^/Δ ^NLS (blue bars) and *Fus* ^−/− (red bars) compared to their control littermates (*Fus* ^+/+, black bars) by semi‐quantitative RT–PCR (middle panel) and RASL‐seq (right panel) are shown. Error bars represent SEM. *P < 0.05, **P < 0.01, two‐tailed Student's t‐test.

**Figure 6. Motor neuron loss in *Fus* ^Δ ^NLS ^/Δ ^NLS mice**
A
Representative light microscopy images of spinal cord sections of *Fus* ^+/+, *Fus* ^Δ ^NLS ^/+, *Fus* ^Δ ^NLS ^/Δ ^NLS, and *Fus* ^−/− mice at birth stained with cresyl violet (Nissl, A¹, A³ A⁵, A⁷), or anti‐choline acetyltransferase (ChAT, A², A⁴ A⁶, A⁸).
B, C
Quantification of ChAT ⁺ motor neurons (B) and Nissl⁺ motor neurons (defined as Nissl‐positive cells with a soma area > 80 µm²) (C) per spinal cord ventral horn in *Fus* ^Δ ^NLS ^/Δ ^NLS mice (mean ± SEM). For Nissl⁺ N = 8 *Fus* ^+/+, N = 5 *Fus* ^Δ ^NLS ^/+, N = 7 *Fus* ^Δ ^NLS ^/Δ ^NLS, and for ChAT ⁺ N = 7 per genotype, **P < 0.01 versus *Fus* ^+/+, ^## P < 0.01 versus *Fus* ^∆ ^NLS ^/+; one‐way ANOVA followed by Tukey's *post hoc* test.
D, E
Quantification of ChAT ⁺ (D) and Nissl⁺ (E) motor neurons per spinal cord ventral horn in *Fus* ^−/− mice (mean ± SEM). N = 6 per genotype, no significant differences were found, by Student's unpaired t‐test.

**Figure 7. Motor neuron apoptosis in *Fus* ^Δ ^NLS ^/Δ ^NLS mice**
A
Representative images of TUNEL assay in spinal cord of *Fus* ^Δ ^NLS ^/Δ ^NLS mice (A⁴‐A⁶) and *Fus* ^+/+ mice (A¹‐A³).
B
Quantification of the total number of TUNEL and DRAQ5 (blue) double‐positive cells in *Fus* ^Δ ^NLS ^/Δ ^NLS and *Fus* ^+/+ per spinal cord section. Mean ± SEM, N = 3 per genotype, *P < 0.05, by Student's unpaired t‐test.
C, D
Immunofluorescence microscopy of spinal cord of *Fus* ^+/+ (C) and *Fus* ^Δ ^NLS ^/Δ ^NLS (D) mice showing active caspase‐3 (green), ChAT (red), and DNA (cyan, DRAQ5).
E
Quantification of caspase‐3 (Cas3)/ChAT/DRAQ5 triple‐positive cells in *Fus* ^Δ ^NLS ^/Δ ^NLS mice. Mean ± SEM, N = 7 per genotype, **P < 0.01, by Student's unpaired t‐test.

**Figure 8. Alterations of SMN, HDAC1, and eIF2α in *Fus* ^Δ ^NLS ^/Δ ^NLS mice**
Representative images of SMN (green) immunofluorescence in spinal cord. Nuclear gems, corresponding to SMN‐immunoreactive foci in nuclei, are marked by arrows.
HDAC1 immunoreactivity in spinal cord sections of *Fus* ^+/+ and *Fus* ^Δ ^NLS ^/Δ ^NLS mice. Arrows point to HDAC1‐immunoreactive nuclear foci.
Representative images of immunofluorescence staining of motor neurons, labeled with ChAT (red) and HDAC1 (green). Examples of HDAC1 immunoreactive foci in motor neurons are indicated by arrows.
Representative images of immunofluorescence staining of motor neurons labeled with ChAT (red) and phosphorylated eIF2α (green), a general translational stress response marker. DRAQ5 (cyan) was used to label nuclei.

**Figure EV5. The absence of protein aggregates and stress granules in *Fus* ^Δ ^NLS ^/Δ ^NLS mice**
Representative images of ubiquitin staining (green) in the ventral spinal cord.
Representative images of immunofluorescence staining of neurofilament heavy chain (green) and poly‐ubiquitin (lysine 48; red). The neurofilament immunostaining shows normal filamentous staining, and the poly‐ubiquitin staining is very weak and shows no positive aggregates.
Representative images of immunofluorescence staining of neurons labeled with NeuN (green) and TIAR (red), a stress granule marker.

**Figure 9. Selective restoration of FUS nuclear import in motor neurons rescues motor neuron loss**
A
Double immunolabeling of spinal cord neurons with ChAT (red) and N‐terminal FUS antibody (green). Nuclei were visualized with DRAQ5 (blue). Cellular localization of FUS was analyzed in the ventral spinal cord of *Fus* ^+/+/*ChAT*‐CRE (A¹‐A⁴), *Fus* ^Δ ^NLS ^/Δ ^NLS/− (A⁵‐A⁸), and *Fus* ^Δ ^NLS ^/Δ ^NLS/*ChAT*‐CRE (A⁹‐A¹²). FUS was completely nuclear in ChAT ⁺ neurons of *Fus* ^+/+/*ChAT*‐CRE, while cytoplasmic in *Fus* ^Δ ^NLS ^/Δ ^NLS/−. In the ventral horn of *Fus* ^Δ ^NLS ^/Δ ^NLS/*ChAT*‐CRE mice, ChAT ⁺ neurons (motor neurons, e.g., within the dashed square) displayed nuclear FUS immunoreactivity, while ChAT‐negative cells retained cytoplasmic FUS immunoreactivity (arrows).
B
Representative light microscopy images of spinal cord sections of *Fus* ^+/+/*ChAT*‐CRE (B¹, B⁴), *Fus* ^Δ ^NLS ^/Δ ^NLS/− (B², B⁵), and *Fus* ^Δ ^NLS ^/Δ ^NLS/*ChAT*‐CRE (B³, B⁶) mice at birth stained with cresyl violet (Nissl, B¹‐B³) or anti‐choline acetyltransferase (ChAT, B⁴‐B⁶).
C, D
Quantification of Nissl⁺ (C) and ChAT ⁺ (D) motor neurons per spinal cord ventral horn. Mean ± SEM, N = 9 *Fus* ^+/+/*ChAT*‐CRE, N = 8 *Fus* ^Δ ^NLS ^/Δ ^NLS/−, and N = 4 *Fus* ^Δ ^NLS ^/Δ ^NLS/*ChAT*‐CRE for Nissl⁺, and N = 11 *Fus* ^+/+/*ChAT*‐CRE, N = 7 *Fus* ^Δ ^NLS ^/Δ ^NLS/−, and N = 8 *Fus* ^Δ ^NLS ^/Δ ^NLS/*ChAT*‐CRE for ChAT ⁺; (**) P < 0.01 versus *Fus* ^+/+, ^## P < 0.01 versus *Fus* ^∆ ^NLS ^/+; (ns) non‐significant; one‐way ANOVA followed by Tukey's *post hoc* test.
E
Total numbers of caspase‐3 (Cas3)/ChAT/DAPI triple‐positive cells in *Fus* ^+/+/*ChAT*‐CRE, *Fus* ^Δ ^NLS ^/Δ ^NLS/−, and *Fus* ^Δ ^NLS ^/Δ ^NLS/*ChAT*‐CRE mice. N = 9 *Fus* ^+/+/*ChAT*‐CRE, N = 7 *Fus* ^Δ ^NLS ^/Δ ^NLS/−, and N = 8 *Fus* ^Δ ^NLS ^/Δ ^NLS/*ChAT*‐CRE; **P < 0.01 versus *Fus* ^+/+, ^# P < 0.05 versus *Fus* ^∆ ^NLS ^/+; (ns) non‐significant; one‐way ANOVA followed by Tukey's *post hoc* test.

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Comment in

FUScinating insights into motor neuron degeneration.
Dormann D. Dormann D. EMBO J. 2016 May 17;35(10):1015-7. doi: 10.15252/embj.201694397. Epub 2016 Apr 6. EMBO J. 2016. PMID: 27053723 Free PMC article.

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

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

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