Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis - PubMed (original) (raw)

Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis

Christophe Verbeeck et al. Mol Neurodegener. 2012.

Abstract

Background: Mutations in the gene encoding the RNA-binding protein fused in sarcoma (FUS) can cause familial and sporadic amyotrophic lateral sclerosis (ALS) and rarely frontotemproal dementia (FTD). FUS accumulates in neuronal cytoplasmic inclusions (NCIs) in ALS patients with FUS mutations. FUS is also a major pathologic marker for a group of less common forms of frontotemporal lobar degeneration (FTLD), which includes atypical FTLD with ubiquitinated inclusions (aFTLD-U), neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease (BIBD). These diseases are now called FUS proteinopathies, because they share this disease marker. It is unknown how FUS mutations cause disease and the role of FUS in FTD-FUS cases, which do not have FUS mutations. In this paper we report the development of somatic brain transgenic (SBT) mice using recombinant adeno-associated virus (rAAV) to investigate how FUS mutations lead to neurodegeneration.

Results: We compared SBT mice expressing wild-type human FUS (FUSWT), and two ALS-linked mutations: FUSR521C and FUSΔ14, which lacks the nuclear localization signal. Both FUS mutants accumulated in the cytoplasm relative to FUSWT. The degree of this shift correlated with the severity of the FUS mutation as reflected by disease onset in humans. Mice expressing the most aggressive mutation, FUSΔ14, recapitulated many aspects of FUS proteinopathies, including insoluble FUS, basophilic and eosiniphilic NCIs, and other pathologic markers, including ubiquitin, p62/SQSTM1, α-internexin, and the poly-adenylate(A)-binding protein 1 (PABP-1). However, TDP-43 did not localize to inclusions.

Conclusions: Our data supports the hypothesis that ALS or FTD-linked FUS mutations cause neurodegeneration by increasing cyotplasmic FUS. Accumulation of FUS in the cytoplasm may retain RNA targets and recruit additional RNA-binding proteins, such as PABP-1, into stress-granule like aggregates that coalesce into permanent inclusions that could negatively affect RNA metabolism. Identification of mutations in other genes that cause ALS/FTD, such as C9ORF72, sentaxin, and angiogenin, lends support to the idea that defective RNA metabolism is a critical pathogenic pathway. The SBT FUS mice described here will provide a valuable platform for dissecting the pathogenic mechanism of FUS mutations, define the relationship between FTD and ALS-FUS, and help identify therapeutic targets that are desperately needed for these devastating neurodegenerative disorders.

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Figures

Figure 1

Generation of Human Fused in Sarcoma (FUS) mouse models using rAAV1 and SBT. (A). Diagram of FUSWT, FUSR521C, and FUSΔ14 expression constructs. All FUS constructs were cloned with a V5 epitope tag on the amino-terminus to aid immunodetection. The major protein domains of FUS are highlighted. QGSY=Gln-Gly-Ser-Tyr rich region. Glycine rich region. NES=nuclear export signal. RRM= RNA recognition motif. RGG= Arg-Gly-Gly-rich motif. ZNF=zinc finger motif. PY-NLS=Pro-Tyr nuclear localisation signal. (B) to (G). Immunohistochemistry (anti-V5 antibody) detects widespread expression of FUSWT, FUSR521C, and FUSΔ14 in cerebral cortex and dentate gyrus (DG). Wild type FUS is mainly localized in the nuclei (B insert). Intense nuclear V5 staining in cerebral cortex (B) and weak cytoplasmic v5 staining in DG (E). In the FUSR521C model, FUS is no longer predominantly located in the nucleus, but also found in the soma and dendrites (C and F). FUSΔ14 mice have a dramatic translocation of FUS from the nucleus to the cytoplasm, and formation of neuronal cytoplasmic inclusions (NCIs) (D and G). Scale bar: 100 μm (H). Histogram showing the percent of nuclear and cytoplasmic v5 staining,cytoplasmic inclusion in cerebral cortex. (n=4; S.E.M.) * P<0.05, **P<0.01 and ***P<0.001, one way ANOVA.

Figure 2

FUS mutations cause an aberrant subcellular redistribution in mouse neurons. A representative immunoblot (A) of the V5 tagged FUS proteins extracted from AAV injected mouse brains. Tissue extracts from FUSWT, FUSR521C, and FUSΔ14 brain were separated into soluble fractions from the cytoplasm and nucleus. Histone 3 staining was used as a nuclear marker to verify extraction fidelity. (B) The ratio of cytoplasmic FUS to nuclear FUS was calculated based on quantification of immunoblots for different FUS constructs (n=4; S.E.M.). A higher ratio of FUSR521C and FUSΔ14 are found in the cytoplasm. ** P<0.01 and ***P<0.001. (C) FUSR521C and FUSΔ14 protein are more insoluble than FUSWT.

Figure 3

Neuropathology of SBT FUS Δ14 mice is similar to human FUS proteinopathies (A-P). Adjacent sections from the cerebral cortex of eGFP (A-D), FUSWT (E-H), FUSR521C (I-L), and FUSΔ14 (M-P) mice stained with protein NCI markers found in aFTLD-U, NIFID, BIBD and hematoxylin-eosin (H&E). (M) Only FUSΔ14 brains contain ubiquitinated NCIs. (N) Hematoxylin-eosin (H&E) staining of FUSΔ14 cerebral cortex shows cytoplasmic basophilic inclusions (arrows). (O) NCIs are infrequently positive for α-internexin, similar to the pathological NCIs found in NIFID cases. (P) Some NCI in the cerebral cortex of FUSΔ14 mice contain the stress granule marker protein PABP-1. Scale bar: 100 μm. Slides are orientated to a common blood vessel (arrow head) to serve as a landmark (M-P).

Figure 4

Ubiquitin co-localizes to FUS-positive inclusions in hFUS Δ14 mice. Confocal image showing FUS located in nucleus in FUSWT mice (A), increased cytoplasmic distribution of FUS in FUSR521C mice (B), and accumulation of FUS into NCIs in FUSΔ14 mice (C) that co-labels with ubiquitin (F and L). Nuclei were counterstained with DAPI (G, H and I). FUS detected with anti-V5 (A, B, C). Scale bar: 10 μm.

Figure 5

Multiple neuropathologic markers co-accumulate in FUS Δ14 mice NCIs. Double labelling for FUS (anti-V5; A, E and I) and P62 (B), PABP1 (F) and α-internexin (J). Colocalization shown in merged image (D, H and L). Nuclei were counterstained with DAPI (C, G, and K). Scale bar: 20 μm.

Figure 6

Endogenous TDP-43 is not redistributed in FUS WT, FUS R521C and FUS Δ14 mouse brain. Mouse TDP-43 is predominantly in the nucleus (DAB immunohistochemistry; A, B, and C). Double-label immunofluorescence of TDP-43 and FUSwt, FUSR521C or FUSΔ14 mice (anti-V5; D-O). FUS is distributed to the nucleus in FUSWT mice (D), increased in the neuronal cytoplasm in FUSR521C mice (H), and accumulates as inclusions in the neuronal cytoplasm in FUSΔ14 mice (L). TDP-43 staining is nuclear in FUSWT, FUSR521C and FUSΔ14 mice (E, I and M). Nuclei were counterstained with DAPI. Scale bar: 20 μm.

References

1. Vance C, Rogelj B, Hortobagyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P. et al.Mutations in FUS, an RNA Processing Protein, Cause Familial Amyotrophic Lateral Sclerosis Type 6. Science. 2009;323:1208–1211. - PMC - PubMed
1. Kwiatkowski TJ, Bosco DA, LeClerc AL, Tamrazian E, Vanderburg CR, Russ C, Davis A, Gilchrist J, Kasarskis EJ, Munsat T. et al.Mutations in the FUS/TLS Gene on Chromosome 16 Cause Familial Amyotrophic Lateral Sclerosis. Science. 2009;323:1205–1208. - PubMed
1. Ticozzi N, Tiloca C, Morelli C, Colombrita C, Poletti B, Doretti A, Maderna L, Messina S, Ratti A, Silani V. Genetics of Familial Amyotrophic Lateral Sclerosis. 2011. - PubMed
1. Kovar H, Dr J, Mr H. The Two Faces of the FUS/EWS/TAF15 Protein Family. Sarcoma. 2011;2011:837474. - PMC - PubMed
1. Law WJ, Cann KL, Hicks GG. TLS, EWS and TAF15: a model for transcriptional integration of gene expression. Brief Funct Genomic Proteomic. 2006;5:8–14. - PubMed

Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis - PubMed (original) (raw)