ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles - PubMed (original) (raw)

ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles

Haiyan An et al. Acta Neuropathol Commun. 2019.

Abstract

Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside paraspeckles, especially prominent under conditions of paraspeckle-inducing stress. Consistently, paraspeckle-dependent microRNA production, a readout for functionality of paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of paraspeckles accompanied by accumulation of non-paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS.

Keywords: Amyotrophic lateral sclerosis (ALS); Fused in sarcoma (FUS); NEAT1; Paraspeckle.

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Conflict of interest statement

Human samples were from clinically and histopathologically characterised ALS cases and neurologically healthy individuals. Samples were provided by the Sheffield Brain Tissue Bank and MRC London Neurodegenerative Diseases Brain Bank (Institute of Psychiatry, King’s College London). Consent was obtained from all subjects for autopsy and histopathological assessment, and research were performed in accordance with local and national Ethics Committee approved donation.

Not applicable.

Competing interests

Authors declare no competing interests.

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Figures

Fig. 1

Generation and characterisation of SH-SY5Y cell lines with targeted modifications of the endogenous FUS gene. a Structures of the FUS gene and FUS protein together with the positions of CRISPR/Cas9 target sites chosen to delete the NLS-encoding fragment. PAM sequences are in green, stop codon is highlighted in yellow and exons are in bold. b Subcellular distribution of FUS protein in FUS ΔNLS and FUS knockout (KO) clones detected with N-terminal FUS antibody. c PCR genotyping of FUS ΔNLS clones. PCR with primers flanking the fragment to be deleted (underlined in A) yields 595 and 265 bp fragments for WT and edited FUS alleles, respectively. d RNA-Seq reads for exons 14 and 15 of the FUS gene in WT cells as well as heterozygous and homozygous FUS ΔNLS lines. Dashed lines indicate the deletion. e Analysis of FUS mRNA levels by qRT-PCR in FUS ΔNLS lines. Diagram shows positions of primers for measuring total and WT mRNA (not drawn to scale, del denotes the deleted region). Average values for three heterozygous (“het pooled”) and three homozygous (“ho pooled”) lines are also shown. N = 4–6, *p < 0.05, ****p < 0.0001 (one-way ANOVA). f Western blot analysis of FUS in FUS ΔNLS and FUS KO lines using antibodies recognising its N-terminal (aa.1–50) or C-terminal (aa.500–526) segments. Note that mutant FUS possesses a FUS-unrelated C-terminal amino acid stretch both in ΔNLS1_ho and ΔNLS2_het lines causing slower migration of the mutant protein (for protein sequences see Additional file 1: Figure S1A). g FUS distribution in representative heterozygous and homozygous FUS ΔNLS lines. Nuclei border in homozygous cells is indicated with a dashed line. h FUS levels in total lysates and cytoplasmic fraction from WT and FUS ΔNLS lines. Ratio C/T, ratio cytoplasmic to total FUS levels. Note absence of histones (arrows) in the cytoplasmic fraction. Scale bars, 10 μm

Fig. 2

Accumulation of NEAT1 and augmented paraspeckle assembly in heterozygous FUS ΔNLS lines. a, b Cells heterozygous for the FUS NLS deletion (ΔNLS_het) have increased number of paraspeckles, whereas homozygous (ΔNLS_ho) and FUS knockout (KO) lines are almost devoid of paraspeckles. Arrows indicate clusters of paraspeckles in ΔNLS_het lines and arrowheads – residual paraspeckles in ΔNLS_ho lines (a). The number of NEAT1-positive foci and their area were quantified for ΔNLS_het lines (b). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Holm-Sidak test). c Paraspeckles in ΔNLS_het cells contain both NEAT1_2 and a core paraspeckle protein NONO. d NEAT1 isoforms are upregulated in FUS ΔNLS lines. Representative tracks for poly(A) capture RNA-Seq analysis of NEAT1 gene in a heterozygous (ΔNLS8_het) and a homozygous (ΔNLS4_ho) lines are shown. NEAT1_1 levels were measured by RNA-Seq and NEAT1_2 levels – by qRT-PCR. N = 4 per line. *p < 0.05, ***p < 0.001, ****p < 0.0001 (one-way ANOVA). e A NEAT1-repressed transcript ADARB2 is downregulated in FUS ΔNLS lines. ADARB2 mRNA levels were measured by RNA-Seq (left) and qRT-PCR (right). N = 3 per line. ****p < 0.0001 (one-way ANOVA with Dunnett’s test). f-h Overexpression of FUS or its mutants restores paraspeckles in FUS KO and ΔNLS_ho cells. Arrowheads indicate mature paraspeckles or their clusters (f, g). Inset in g shows paraspeckle primary units in a non-transfected FUS KO cell. Bar chart shows the fraction of transfected ΔNLS1_ho and FUS KO cells with one or more paraspeckle (large NEAT1-positive dot) (h). **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared to non-transfected (NT) cells (one-way ANOVA with Holm-Sidak test). All FUS variants were expressed as N-terminal GFP-fusions. Paraspeckles were visualised by NEAT1 RNA-FISH. Combined data for three heterozygous and three homozygous lines are referred as “het pooled” and “ho pooled”, respectively. In b and h, numbers of cells analysed are indicated within each bar. Scale bars, 10 μm

Fig. 3

Structural and functional deficiency of paraspeckles in FUS ΔNLS lines. a Interaction of FUS with SFPQ and NONO is reduced in FUS ΔNLS lines as revealed by proximity ligation assay (PLA). PLA was performed in a heterozygous (ΔNLS2_het) and a homozygous (ΔNLS1_ho) lines; FUS KO cells were used as a negative control. Representative images and quantification (number of single interactions (dots) per cell (foci per cell)) are shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Holm-Sidak test). b Extractability of NEAT1_2 is increased in FUS ΔNLS lines. NEAT1_2 extractability was analysed by determining its levels in QIAzol-lysed heated versus non-heated samples (“fold extraction”) by qRT-PCR. Note near-complete NEAT1_2 extractability in FUS KO cells (fold extraction ~ 1). See also Additional file 1: Figure S4B. N = 3 per line. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA). c NEAT1_1 accumulates in soluble nuclear extract (SNE) in FUS ΔNLS lines. Left, representative PCR (non-saturated conditions, 26 cycles); right, qRT-PCR analysis. A primer pair located immediately upstream NEAT1_1 polyA-tail (NEAT1 pA) was used to quantify NEAT1_1 in cDNA of polyadenylated RNA. Note that NEAT1_2 which is not polyadenylated is undetectable under these conditions. *p < 0.05, **p < 0.01 (one-way ANOVA with Holm-Sidak test). d NEAT1 displays diffuse distribution in poly(I:C)-stimulated ΔNLS_het lines. Cells were analysed 8 h after poly(I:C) transfection by NEAT1 RNA-FISH. Representative images and quantification of the fraction of cells with diffuse NEAT1 distribution are shown. e Paraspeckle-regulated miRNAs are decreased in FUS ΔNLS lines. Levels of six mature miRNAs produced from pri-miR17~92 were measured by qRT-PCR separately for heterozygous and homozygous FUS ΔNLS lines, and combined average values were plotted. *p < 0.05 (Mann-Whitney _U_-test). Combined data for three heterozygous and three homozygous lines are referred as “het pooled” and “ho pooled”, respectively. In a and d, numbers of cells analysed are indicated within each bar. Scale bars, 10 μm

Fig. 4

Localisation of NEAT1_1 outside paraspeckles in patient fibroblasts bearing FUS mutation. a FUS is predominantly nuclear in human patient fibroblasts bearing FUS P525L mutation. b Paraspeckle assembly is augmented in FUS P525L human fibroblasts. Paraspeckles were visualised by NEAT1_2 (3′ segment probe) RNA-FISH. *p < 0.05 (Mann-Whitney _U_-test). c Diffuse, non-paraspeckle distribution of NEAT1 in FUS P525L fibroblasts revealed using RNA-FISH with 5′ segment NEAT1 probe (total NEAT1). d NEAT1_1 is abnormally localised to nuclear speckles in FUS P525L fibroblasts. Representative images and quantification of the fraction of cells with speckle-localised NEAT1 are shown. Total NEAT1 (5′ segment probe) was used, and speckles were visualised by polyA+ RNA FISH. In b and d, numbers of cells analysed are indicated within bars. Scale bars, 10 μm

Fig. 5

Accumulation of paraspeckles in spinal neurons and glial cells in ALS-FUS. a Examples of paraspeckles in spinal neurons and glial cells of ALS-FUS and sALS patients visualised using RNA-FISH with fluorescently-labelled (Quasar 570) 5′ segment NEAT1 probe. Images were taken both in the orange and green channels to distinguish between specific NEAT1 signal and autofluorescence from lipofuscin. See also Additional file 1: Table S2. Arrowheads point to paraspeckles in a glial cell. Scale bars, 10 μm. b Examples of paraspeckles in spinal neurons (left panels) and glial cells (right panels) in an ALS-FUS patient visualised with RNAscope® ISH using NEAT1_2 probe. Neuronal nuclei are circled. Scale bars, 10 μm (left panels) and 50 μm (right panels)

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ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles - PubMed (original) (raw)