ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects - PubMed (original) (raw)

. 2014 Mar;124(3):981-99.

doi: 10.1172/JCI72723. Epub 2014 Feb 10.

Sebum Lee, Yulei Shang, Wen-Yuan Wang, Kin Fai Au, Sherry Kamiya, Sami J Barmada, Steven Finkbeiner, Hansen Lui, Caitlin E Carlton, Amy A Tang, Michael C Oldham, Hejia Wang, James Shorter, Anthony J Filiano, Erik D Roberson, Warren G Tourtellotte, Bin Chen, Li-Huei Tsai, Eric J Huang

ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects

Haiyan Qiu et al. J Clin Invest. 2014 Mar.

Erratum in

Abstract

Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C-associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions.

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Figures

Figure 1

Figure 1. Early onset motor behavioral deficits and postnatal lethality in FUS-R521C transgenic mice.

(A) A schematic diagram showing our strategy for generating transgenic mice that express human FUS-R521C mutant proteins using the Syrian hamster prion promoter. (B and C) The majority of N1F1 FUS-R521C mice show growth retardation, spastic paraplegia, and severe muscle wasting. (D and E) In addition, the FUS-R521C mice also exhibit prolonged hind limb clasping on tail-suspension test. (F) In a 2-minute period, FUS-R521C mice spend approximately 100 seconds with hind limbs clasped together, whereas nontransgenic control mice exhibit similar clasping phenotype for less than 10 seconds. 2-tailed Student’s t test, P < 0.001. (G and H) Due to the spastic paraplegia, FUS-R521C mice show reduced distance between hind paws during tandem walk tests. 2-tailed Student’s t test, P < 0.001. (I) The gait and motor coordination defects in FUS-R521C mice are further verified using computerized catwalk measurements. (J) In addition to the gait abnormalities, FUS-R521C N1F1 mice show poor performance and motor learning on rotarod tests.

Figure 2

Figure 2. FUS-R521C interacts with the WT FUS and perturbs its distribution.

(A and B) Western blots showing the level of FUS-R521C proteins is similar to that of the endogenous FUS proteins in 2 independent transgenic mice (P37). However, immunoprecipitation shows that FUS-R521C can be detected in a complex with the endogenous WT FUS. The relative levels of endogenous FUS and FUS-R521C in control (NTG) and FUS-R521C brain and spinal cord are quantified in B. Student’s t test, n = 3 for each group. *P < 0.017; ***P < 0.008. (C and D) Coimmunoprecipitation assays using lysates from transfected HEK293T cells demonstrate that FUS-R521C has a higher propensity to form protein complexes with FUS-R521C proteins than with WT FUS proteins. (E) Pulse-chase experiments in HEK293T cells showing FUS-R521C proteins are twice as stable as WT FUS proteins. (F and G) IHC using FLAG antibody shows FUS-R521C proteins are predominantly found in the nuclei of spinal motor neurons, but can also be detected in the cytoplasm. (H and I) FUS proteins are found predominantly in the nuclei of spinal motor neurons and in punctate synaptic structures in the spinal cord. The punctate FUS staining in synapses is diminished in FUS-R521C mice (P37). Scale bar: 25 μm.

Figure 3

Figure 3. FUS-R521C transgenic mice exhibit an impairment of FUS/HDAC1 interaction and an increase in DNA damage.

(A) Immunoprecipitation and Western blotting with the indicated antibodies of protein lysates from the control cortex and spinal cord show WT FUS in complex with HDAC1. However, protein lysates from FUS-R521C mutants show no evidence of protein complexes between FUS and HDAC1 or between FUS-R521C and HDAC1. Asterisk indicates nonspecific proteins that crossreact with FUS antibody. (B) Western blot analysis shows increased γH2AX, ATF3, and phosphorylated p53 levels in protein lysates from cortex and spinal cord of FUS-R521C transgenic mice. NTG, control nontransgenic mice; TG: transgenic mice. (C) DAB staining and immunofluorescent images of γH2AX (arrows) in cortex and spinal cord in control and FUS-R521C mice. The confocal images (bottom) show that most γH2AX+ staining is detected in spinal motor neurons (arrows), but smaller foci of γH2AX can also be detected in dying neurons or glial nuclei (arrowhead). Scale bars: 50 μm (γH2AX IHC panel); 5 μm (ChAT/γH2AX IF). (D) The number of γH2AX-positive cells per high-power field in FUS-R521C transgenic mice is increased compared with NTG mice. n = 3 mice, 7–9 sections per mouse (mean ± SEM, ***P < 0.001, Student’s t test). (E) Representative images of comet assays of cortical neurons isolated from NTG and FUS-R521C mice. Quantification shows the percentage of cells with a comet tail (mean ± SEM, unpaired Student’s t test). Scale bar: 50 μm.

Figure 4

Figure 4. Dendritic and synaptic defects in FUS-R521C spinal motor neurons.

(A and B) ChAT immunostaining shows a modest reduction of motor neurons in cervical spinal cord of FUS-R521C mice (P37–P44). Scale bar: 100 μm. (C and D) Golgi staining shows reduced dendritic arborization in FUS-R521C motor neurons. Scale bar: 200 μm. (E and F) Neurolucida tracing of dendrites in control and FUS-R521C motor neurons. Scale bar: 100 μm. (G and H) Sholl analyses show reduced dendritic intersections (from 50 to 250 μm) and reduced cumulative area of dendrites in FUS-R521C neurons. P < 0.0001, 2-way repeated measures ANOVA. (I–M) Extensive colocalization of FUS with ChAT+ boutons and SIPT is reduced in FUS-R521C spinal cord. Student’s t test. Scale bar: 10 μm. (N–Q) EM images of control cervical spinal cord show synapses adjacent to 2 motor neurons (pink and green), many showing rosette-like pattern (arrows in P). In contrast, synapse density and size (arrows in Q) are reduced in areas adjacent to FUS-R521C motor neuron (pink). Scale bars: 5 μm (O); 2 μm (Q). N, nucleus of spinal motor neurons. (R) Reduced PSD length and the density of synapse in FUS-R521C cervical spinal cord. P < 0.0001, 2-way repeated measures ANOVA. (S) Reduced cumulative frequency and PSD size distribution in FUS-R521C neurons.

Figure 5

Figure 5. Dendritic and synaptic defects in neurons of the sensorimotor cortex in FUS-R521C mice.

(A and B) Golgi staining shows reduced dendritic arborization in layers IV–V neurons in FUS-R521C sensorimotor cortex. Scale bar: 100 μm. (C and D) Neurolucida tracing of the apical and basal dendrites in control and FUS-R521C cortical neurons. Scale bar: 50 μm. (E and F) Reduced dendritic intersections in apical dendrite and cumulative areas of dendrites in FUS-R521C cortical neurons. P < 0.0001, 2-way repeated measures ANOVA. (G–K) Reduced mature dendritic spine density in dendrites of FUS-R521C cortical neurons. Scale bar: 10 μm. Statistical analyses use 2-tailed Student’s t test, P = 0.001 for total spine and P = 0.008 for mature spine. (L–M) EM shows reduced synapse in the sensorimotor cortex of FUS-R521C mice. 2-tailed Student’s t test, P < 0.0001. Scale bar: 1 μm. (N and O) FUS-R521C cortex shows reduction in the length of the PSD and the number of synapse. P < 0.0001, 2-way repeated measures ANOVA. (P) Reduced cumulative frequency and size of PSD in FUS-R521C cortex. Statistics for PSD length use 2-tailed Student’s t test, P < 0.0001, and for cumulative frequency use 2-tailed Mann-Whitney test, P = 0.0042.

Figure 6

Figure 6. FUS-R521C mice show increased DNA damage and splicing defects in 5′ noncoding exons in the Bdnf gene.

(A) Schematic diagrams of the qPCR-based FPG assay to identify oxidized (or “damaged”) nucleotides in genomic DNA. (B) FPG assays reveal DNA damage in the 5′ noncoding exons of the Bdnf gene in cortex and spinal cord in FUS-R521C mice. Statistics use Student’s t test, *P < 0.05; **P < 0.01. (C) A schematic diagram of the mouse Bdnf gene, which includes seven 5′ noncoding exons and exon 8, which contains the entire coding sequence and 3′ UTR. PCR primers to detect the retention of 5′ splice junction are highlighted as black arrows (forward) and blue arrows (reverse). Red bars indicate the synthetic oligoribonucleotides that contain the 5′ splice junctions in noncoding exons. (D) qRT-PCR assays detect the marked retention of 5′ splice junction sequences in the Bdnf mRNA in FUS-R521C brain. (E) Crosslinking immunoprecipitation (CLIP) qRT-PCR assays show that more FUS proteins are bound to the 5′ splice junction sequences in exons 2, 4, and 6 in the Bdnf mRNA in the brain of FUS-R521C mice. Statistics in D and E use Student’s t test.

Figure 7

Figure 7. FUS-R521C proteins form more stable complexes with Bdnf RNA and reduce splicing efficiency.

(A) EMSAs show both recombinant GST-WT FUS (GST-FUS-WT) and GST-FUS-R521C proteins form complexes with radioactive RNA oligos that contain sequences from the 5′ splice junction of Bdnf exons 2, 4, and 6. Compared with GST-FUS-WT, the RNA-protein complexes formed by GST-FUS-R521C tend to show slower migration, suggesting a higher molecular weight complex or more stable complex formation. (B) Both GST-FUS-WT and GST-FUS-R521C proteins also interact with RNA oligo-_Bdnf_-3′ UTR#2, but not _Bdnf_-3′ UTR#4. (C and D) Competition assays show that nonradioactive (“cold”) RNA oligos can displace WT FUS from the RNA-protein complex at the IC50 of 0.73 μM. In contrast, the GST-FUS-R521C-RNA complex was more stable and required higher concentration of cold probe to dissociate RNA-protein complexes (IC50 = 2.19 μM).

Figure 8

Figure 8. BDNF restores TrkB activation and partially ameliorates dendrite phenotype in cortical neurons expressing FUS-R521C.

(A) In situ hybridization shows reduced Bdnf mRNA in cortical and spinal motor neurons in FUS-R521C mice. Bdnf mRNA can be detected in the cell body and dendrites of control neurons (arrowheads), but is significantly reduced in FUS-R521C neurons. Scale bar: 20 μm. (B–D) Western blots and quantification showing reduced pro-BDNF, mature BDNF, and TrkB phosphorylation in FUS-R521C brain and spinal cord, without affecting total TrkB protein levels. (E and F) BDNF treatment (10 ng/ml) restores TrkB activation in cortical neurons from FUS-R521C embryos. *P < 0.01. (G) Cultured neurons expressing WT FUS or FUS-R521C show reduced dendritic growth from DIV1 to DIV7, which can be partially ameliorated by exogenous BDNF (10 ng/ml). Scale bar: 20 μm. (H) Quantification of total dendrite length in control neurons and neurons expressing WT FUS or FUS-R521C from DIV1 to DIV7. *P < 0.05; **P < 0.001. (I) BDNF treatment partially restores dendritic growth in neurons expressing FUS or FUS-R521C. Statistics in C, D, F, H, and I use 2-tailed Student’s t test, n = 3.

Figure 9

Figure 9. RNA-seq analyses reveal transcription and splicing defects in FUS-R521C spinal cord.

(A) DESeq analyses of RNA-seq data reveal 766 differentially expressed genes in FUS-R521C spinal cord. All mice were euthanized at P38, with the FUS-R521C mice reaching disease end-stage. (B) Fold change and enrichment scores of the GO terms from DAVID Bioinformatics analyses of the differentially expressed genes in FUS-R521C spinal cord. (C) Scattered plot generated by SpliceMap demonstrates intron retention events and the log ratio of intron retention index (RII) of FUS-R521C/WT (y axis) plotted against the log of the intron length (bp) (_x_-axis). (D) Significantly retained introns in the spinal cord of FUS-R521C mice and RefSeq introns are grouped by length and plotted as a percentage of the total introns. (E and F) The mouse Col7a1 gene encompasses 54 kb on chromosome 9 and contains 119 exons. In FUS-R521C spinal cord, Col7a1 mRNA shows evidence of excessive inclusion of cassette exons (bracketed) and intron retention (arrows). qRT-PCR using primers that detect the presence of 5′ and 3′ splice junctions in Col7a1 further confirmed the RNA-seq data. Quantification of the qRT-PCR data is shown in F. Statistics uses 2-tailed Student’s t test, n = 3.

Figure 10

Figure 10. Proposed working models for the dominant inhibitory effects of FUS-R521C on DNA damage response/repair machinery and RNA splicing.

(A) WT FUS is rapidly recruited to DNA damage foci caused by double-stranded breaks, where it interacts with chromatin remodeling factor HDAC1. Although FUS-R521C can still be recruited to DNA damage foci, it fails to interact with HDAC1. As a consequence, neurons in FUS-R521C transgenic mice show increased DNA damage (indicated by blue asterisks and the presence of double-stranded breaks). (B) Results from CLIP–RT-PCR and protein-RNA interactions in EMSA assays show that both WT FUS and FUS-R521C can interact with selective oligoribonucleotides from Bdnf exon-intron boundaries. Whereas the equilibrium of WT FUS-RNA interactions appears to be more dynamic, FUS-R521C tends to form more stable protein-RNA complexes that are more difficult to dissociate.

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