Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides in...

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Title:
Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis
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English
Creator:
Verbeeck, Christophe
Deng, Qiudong
DeJesus-Hernandez, Mariely
Taylor, Georgia
Ceballos-Diaz, Carolina
Kocerha, Jannet
Golde, Todd
Das, Pritam
Rademakers, Rosa
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BioMed Central (Molecular Neurodeneration)
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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. Keywords: Amyotrophic lateral sclerosis, Frontotemporal lobar degeneration, Fused in sarcoma proteinopathies, Transgenic mouse models, Adeno-associated virus, Neuronal cytoplasmic inclusions, Ubiquitin, p62/SQSTM1, α-internexin, PABP-1, Stress granules, RNA dysfunction
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Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/UFir) at the time of funding. The institutional Repository at the University of Florida community, with research, news, outreach, and educational materials.
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Verbeeck et al. Molecular Neurodegeneration 2012, 7:53 http://www.molecularneurodegeneration.com/content/7/1/53; Pages 1-13
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doi:10.1186/1750-1326-7-53 Cite this article as: Verbeeck et al.: Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis. Molecular Neurodegeneration 2012 7:53.

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dochead Research article
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title
p Expression of it Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis
aug
au id A1 ce yes snm Verbeeckfnm Christopheinsr iid I1 email verbeeck.christophe@mayo.edu
A2 DengQiudongI2 qiudong.deng@emory.edu
A3 DeJesus-HernandezMarielydejesushernandez.mariely@mayo.edu
A4 TaylorGeorgiageorgia.taylor@emory.edu
A5 Ceballos-DiazCarolinaI3 caro.ceb81@ufl.edu
A6 KocerhaJannetI4 jannet.kocerha@emory.edu
A7 GoldeToddtgolde@mbi.ufl.edu
A8 DasPritamdas.pritam@mayo.edu
A9 RademakersRosarademakers.rosa@mayo.edu
A10 Dicksonmi WDennisdickson.dennis@mayo.edu
A11 ca KukarThomasthomas.kukar@emory.edu
insg
ins Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
Department of Pharmacology and Neurology, Emory University School of Medicine, Atlanta, GA, USA
Center for Translational Research in Neurodegenerative Disease, College of Medicine, University of Florida, Gainesville, FL, USA
Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA
source Molecular Neurodegeneration
issn 1750-1326
pubdate 2012
volume 7
issue 1
fpage 53
url http://www.molecularneurodegeneration.com/content/7/1/53
xrefbib pubidlist pubid idtype doi 10.1186/1750-1326-7-53pmpid 23046583
history rec date day 7month 6year 2012acc 2792012pub 10102012
cpyrt 2012collab Verbeeck et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Amyotrophic lateral sclerosis
Frontotemporal lobar degeneration
Fused in sarcoma proteinopathies
Transgenic mouse models
Adeno-associated virus
Neuronal cytoplasmic inclusions
Ubiquitin
p62/SQSTM1
α-internexin
PABP-1
Stress granules
RNA dysfunction
abs
sec
st
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 (FUSsub WT), 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.
bdy
Background
Mutations in the Fused in Sarcoma (FUS) gene were recently discovered in some cases of familial and sporadic amyotrophic lateral sclerosis (ALS) and more rarely fronto-temproal dementia (FTD)
abbrgrp
abbr bid B1 1
B2 2
B3 3
. FUS is a 526 amino acid DNA/RNA binding protein member of the FET family (FUS/Ewing’s sarcoma/TATA-binding protein-associated factor)
B4 4
. The FUS gene, also known as TLS (translated in sarcoma), was first described as a N-terminal fusion that produced hybrid oncogenes
B5 5
. The full length FUS protein is now appreciated to play a role in a number of critical cellular functions, including gene expression, RNA processing, RNA transport, and genomic integrity
5
B6 6
. The highest levels of FUS are found in the nucleus, driven by a highly conserved carboxyl (C) terminal PY nuclear localization signal (PY-NLS)
B7 7
. FUS is found in the cytoplasm at lower levels and can shuttle rapidly between the nucleus and the cytoplasm
B8 8
B9 9
. The majority of disease-linked FUS mutations cluster in the C-terminus and disrupt nuclear import, but the precise pathogenic mechanism of FUS mutations is currently unknown.
The identification of FUS mutations and accumulation of FUS within ubiquitin-positive neuronal cytoplasmic inclusions (NCI) in a portion of ALS cases led to the re-examination of other neurological diseases with NCI of unknown origin. Subsequently, abnormal FUS was detected within NCI, as well as glial inclusions, in several uncommon forms of frontotemporal lobar degeneration (FTLD), which is the term for the pathology underlying the clinical syndrome FTD. These rare subsets of FTLD were previously referred to as atypical FTLD with ubiquitinated inclusions (aFTLD-U), neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease (BIBD)
B10 10
. These disorders have now been grouped together as the FUS proteinopathies, because they share a common pathology and a presumed underlying disease mechanism
10
. The two major clinical and pathological types are known as frontotemporal lobar degeneration with FUS pathology (FTLD-FUS) and ALS with FUS pathology (ALS-FUS). This nomenclature is analogous to the classification that has been developed for the TDP-43 proteinopathies (ALS-TDP and FTLD-TDP), which have inclusions that contain the RNA-binding protein TDP-43 protein
B11 11
.
In this report we have utilized a technique called somatic brain transgenesis (SBT) to investigate how FUS mutations lead to neurodegeneration. SBT uses recombinant adeno-associated virus (rAAV) to express a cDNA predominantly in neurons throughout much of the brain for the lifetime of the mouse, beginning a few weeks after birth
B12 12
B13 13
. We compared over expression of wild-type human FUS (FUSWT), and two mutations associated with ALS: FUSR521C, or FUSΔ14. Expression of both FUS mutants led to increased FUS protein in the neuronal cytoplasm, the degree of which correlated with the severity of the mutation as reflected by disease onset in humans. Mice expressing the most aggressive mutation, FUSΔ14, recapitulated many aspects of human FUS proteinopathies, including insoluble FUS protein, basophilic and eosiniphilic neuronal cytoplasmic inclusions (NCI), and presence of other pathologic markers, including ubiquitin, p62/SQSTM1, α-internexin, and the polyadenylate-binding protein 1 (PABP-1).
Results
Generation of mice overexpressing FUS using somatic brain transgenesis (SBT)
We utilized SBT to express wild type human FUS and two FUS mutations associated with ALS in the brains of mice to investigate the role of FUS in neurodegeneration using an in vivo model. In this experimental paradigm, newly born (P0) litters of mice were administered recombinant AAV1 encoding FUSWT, FUSR521C, or FUSΔ14, through bilateral intracerebroventricular injection. The FUS R521C mutation, which has been identified in 16 ALS families to date, occurs within the PY nuclear localization signal (PY-NLS) region, and results in an average age of onset of 40 years
1
B14 14
B15 15
. The third model, FUSΔ14,was based on a de novo mutation found in a patient with sporadic ALS that we reported previously
B16 16
. Briefly, a mutation in intron 13 of the FUS gene (g.10747A>G) causes skipping of exon 14, a frame shift, and premature termination in exon 15, leading to a truncated FUS protein of 478 amino acids that lacks the C-terminal PY-NLS (Figure 
figr fid F1 1A). This mutation (FUSΔ14) is associated with early disease onset (20 years) and a rapid disease progression (22 months).
fig Figure 1caption Generation of Human Fused in Sarcoma (FUS) mouse models using rAAV1 and SBTtext
b 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.
graphic file 1750-1326-7-53-1
Characterization of pathology in FUS mice
Three months after viral injection, mice were killed and one brain hemisphere was fixed for neuropathologic characterization, while the other hemisphere was flash frozen for biochemical fractionation. Mice appeared healthy at the time of death and did not display obvious motor impairment or an abnormal grasping phenotype (data not shown). FUS constructs contained a V5 epitope tag on the amino-terminus to aid visualization of protein expression and do not interfere with protein function or cellular localization
16
. Based on V5 immunohistochemistry FUSWT, FUSR521C, and FUSΔ14 mice had widespread FUS protein expression, throughout the brain, with the highest levels in the cerebral cortex and the hippocampus (Figure 
1B-G). Using the SBT paradigm transgene expression is neuronal with no detectable glial expression, as assessed by double immunofluorescence (Additional file
supplr sid S1 1: Figure S1). Neurons expressing FUSWT showed predominantly nuclear localization, with low, but detectable, levels of cytoplasmic protein based on immunohistochemistry and subcellular fractionation (Figure 
1 and
F2 2). FUSR521C mice had marked increases in FUS immunoreactivity in the neuronal cytoplasm. The presence of nuclear FUSR521C was a consistent feature; however, FUSR521C was also detected in the soma, dendrites, and axons of neurons in mice, especially in the hippocampus (Figure 
1C and F). Despite increased cytoplasmic levels of FUSR521C, no obvious inclusions or aggregates of FUS were observed in mice injected with FUSWT or FUSR521C. FUSΔ14 mice showed the greatest cytoplasmic redistribution, with some neurons showing no nuclear FUS reactivity but strong labelling of the cell body and processes in cortex. A portion of neurons in FUSΔ14 mice contained FUS-positive neuronal cytoplasmic inclusions (NCIs), which bared striking resemblance to the NCIs that are a characteristic pathologic feature of ALS and FTD-FUS (Figure 
1D and
1G). In cortex, the percentage of transduced neurons with cytoplasmic distribution of FUS significantly increased in FUSR521C and FUSΔ14 mice (Figure 
1H). The FUSΔ14 mice were the only group that had NCI, reaching ~20% of neurons expressing FUS (Figure 
1H). Mutation-dependent FUS redistribution also was confirmed by double labelling with V5 and a neuronal marker (Additional file
S2 2: Figure S2). Despite the presence of NCI, we did not observe any obvious neuronal loss or degeneration when examining haematoxylin and eosin (H&E) stained sections. Activated caspase-3 and TUNEL assays were also negative (data not shown), suggesting that apoptosis is not occurring in FUS mice at this age. Further, we did not observe marked astrocytosis or microglial activation at this age (Additional file
S3 3: Figure S3).
suppl
Additional file 1
Figure S1. Neuron-specific expression of FUS in mouse cortex and hippocampus. Confocal imaging shows that cells transduced with rAAV1 expressing FUSWT (anti-V5; A, E, I, and M) colocalize with the neuronal marker NeuN (B and F) but not an astrocyte marker (GFAP; J and N) in cortex and hippocampus. Nuclei were counterstained with DAPI (C, G, K and O). Scale bar: 50μm.
name 1750-1326-7-53-S1.pdf
Click here for file
Additional file 2
Figure S2. Mutation-dependent redistribution of FUS. Double-immunofluorescence staining of V5 and a neuronal marker NeuN in FUSWT, FUSR521C, and FUSΔ14 mice (A-L). V5 staining mainly located in nucleus in FUSWT mouse (A-D). Strong nuclear and some cytoplasmic V5 staining in FUSR521C mouse neurons (E-H). Cytoplasmic accumulation of V5 staining in FUSΔ14 mice (I-L). Scale bar:10μm.
1750-1326-7-53-S2.pdf
Click here for file
Additional file 3
Figure S3. No marked astrocytosis or microglial activation in FUSΔ14 mice. Double-label immunofluorescence of a microglia marker IBA-1and V5 in the cerebral cortex of FUSWT and FUSΔ14 mice (A-H). Double-label immunofluorescence of a astrocyte marker GFAP and V5 in cerebral cortex of FUSWT and FUSΔ14 mice (A-H). Nuclei were counterstained with DAPI (C, G, K, and O). Scale bar: 50 μm.
1750-1326-7-53-S3.pdf
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Figure 2FUS mutations cause an aberrant subcellular redistribution in mouse neurons
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.
1750-1326-7-53-2
We next examined the biochemical solubility of the different FUS proteins expressed in the SBT mice. The cytoplasm and intact nuclei were isolated from the frozen brain hemisphere of the FUS mice analysed above using immunohistochemistry (Figure 
1). Nuclei were lysed in detergent to isolate the soluble nuclear fraction. The cytoplasmic and nuclear extracts were centrifuged at 20,000 x g to pellet insoluble proteins. Samples were separated by SDS/PAGE, analysed on immunoblots, and quantified using densitometry (Figure 
2A). Comparison of the ratio of soluble cytoplasmic to nuclear FUS protein using densitometry confirmed that the steady state levels of the FUS mutants are higher in the cytoplasm, with FUSΔ14 showing the strongest shift (Figure. 
2A and B). Intriguingly, the levels of FUSΔ14 detected in the cytoplasmic and nuclear lysates did not appear to match the immunohistochemistry (Figure 
1B-H) that suggested robust expression of all FUS constructs. We then analysed the pellets resulting from the protocol used biochemical isolation of the cytoplasm and nucleus, and discovered that the majority of FUSΔ14 protein partitions into the insoluble fraction (Figure 
2C). No FUSWT, but a portion of FUSR521C protein was also detected in the insoluble fraction.
Formation of neuronal cytoplasmic inclusions in FUSΔ14 mice
We next examined FUS mice for the presence of neuropathologic markers found in ALS or FTD. Sections from eGFP, FUSWT and FUSR521C mice had diffuse ubiquitin staining with no detectable inclusions (Figure 
F3 3A, E and I). In contrast, FUSΔ14 mice had frequent ubiquitin-positive NCIs (Figure 
3M and Additional file
S4 4: Figure S4). NCIs varied in size from small puncta to large, round inclusions (Additional file
S5 5: Figure S5). Double-label immunofluorescence confirmed that FUSΔ14 and ubiquitin co-localize to the same inclusion (Figure 
F4 4). We did not observe an increase in high molecular weight smearing of FUS, an indicator of poly-ubiquitination, on immunoblots of brain tissue of FUSΔ14 compared to FUSWT (data not shown). Furthermore, immunoblots directly for poly-ubiquitin did not detect a difference between FUSWT, FUSR521C or FUSΔ14 mice, suggesting that FUS is not robustly ubiquitinated (data not shown).
Additional file 4
Figure S4. Quantification of ubiquitin levels in SBT FUS mice. The levels of ubiqutin immunoreactivity in the brain of FUSWT, FUSR521C, and FUSΔ14 mice were quantified using positive pixel counts (default strong positive DAB threshold) and analyzed relative to the total pixels in the analysis area using the ImageScope software (Aperio). Compared to FUSWT and FUSR521C mice, FUSΔ14 mice have significantly greater accumulation of ubiquitin (***p < 0.001, One-way analysis of variance; Graph Pad Prism 5). Values represent mean ± SEM (n=6 for each experimental group).
1750-1326-7-53-S4.pdf
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Additional file 5
Figure S5. Diversity of neuronal cytoplasmic inclusions in the brains of FUSΔ14 mice. The different sizes of inclusions may represent a spectrum of growth from small aggregates to large, insoluble NCIs. The first phase is characterized by nuclear and cytoplasmic location of FUS (A), followed by formation of small, round shaped aggregates (B). FUS accumulates in the cytoplasm (darker staining), associated with depletion of FUS from the nucleus (lighter staining), and the aggregates eventually merge into one or two amorphous inclusions (C). In the end stage, the NCI’s are round or oval shaped and FUS is no longer localized in the nucleus. (D).
1750-1326-7-53-S5.pdf
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Figure 3Neuropathology of SBT FUSΔ14 mice is similar to human FUS proteinopathies (A-P)
Neuropathology of SBT FUSΔ14mice 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).
1750-1326-7-53-3
Figure 4Ubiquitin co-localizes to FUS-positive inclusions in hFUSΔ14 mice
Ubiquitin co-localizes to FUS-positive inclusions in hFUSΔ14mice. 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.
1750-1326-7-53-4
Based on the robust formation of NCIs in the FUSΔ14 mice, we examined a panel of protein markers that were previously described in the NCIs of FUS proteinopathies. Many of the NCI in FUSΔ14 mice were basophilic by H&E staining, similar to the NCI found in BIBD, aFTLD-U, and NIFID cases (Figure 
3N)
B17 17
B18 18
B19 19
. Many of the basophilic inclusions were cytoplasmic, adjacent to the nucleus, and had a rounded, “Pick-body” like structure (Figure 
3N insert). Eosinophilic staining was also noted on the periphery of inclusions, distinct spots within basophilic NCIs, or as distinct inclusions. We then stained for the neuronal intermediate filament protein α-internexin, which is one of the most abundant markers for NIFID
B20 20
. A portion of the NCIs found in the FUSΔ14 model were positive for α-internexin (Figure 
3O), but they were less frequent than NCIs labelled with ubiquitin (Figure 
3M). NCI in FUSΔ14 were also immunoreactive for PABP-1, which was previously reported to stain NCI in BIBD and NIFID cases (Figure 
3P)
B21 21
. There was diffuse cytoplasmic staining of PABP-1 in eGFP, FUSWT or FUSR521C mice, but no obvious NCI (Figure 
3D, H and L). Double-label immunofluorescence confirmed that α-internexin and PABP-1 co-localized with FUS inclusions in the FUSΔ14 model (Figure 
F5 5E-L).
Figure 5Multiple neuropathologic markers co-accumulate in FUSΔ14 mice NCIs
Multiple neuropathologic markers co-accumulate in FUSΔ14mice 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.
1750-1326-7-53-5
We also examined the staining patterns of the p62/ sequestosome-1 (p62/SQSTM1) and optineurin (OPTN) proteins in FUS mice, which have been reported to be mutated in some familial and sporadic ALS cases
B22 22
B23 23
. P62 robustly co-localized with NCI in FUSΔ14 mice (Figure 
5A-D). OPTN immunostaining was diffuse and widespread in the neuronal cytoplasm of FUSWT, FUSR521C, as well as FUSΔ14 mice. We found occasional increased OPTN staining in the neuronal cytoplasm in brain regions of FUSΔ14 mice with ubiquitin positive NCI, but no definite labelling of NCIs (Additional file
S6 6: Figure S6).
Additional file 6
Figure S6. Optineurin is not a robust marker of NCI in FUSsΔ14 mice. Immunohistochemistry of cerebral cortex of FUSWT (A), FUSR521C (B) and FUSΔ14 (C) mice shows no optineurin (OPTN) positive inclusions. There are occasional neurons in the cortex of FUSΔ14 mice with small extranuclear protein aggregates that are positive for OPTN (arrows C). Detection of endogenous mouse optineurin with this antibody was weak, making it difficult to draw definitive conclusions about co-localization with FUS-positive NCI. Scale bar: 50 μm.
1750-1326-7-53-S6.pdf
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No redistribution or accumulation of TDP-43 in FUS mice
Because FUS and TDP-43 have such striking structural and functional similarities, the relationship between their pathology and disease mechanism is an important unanswered question in the field. We did not find any evidence of TDP-43 redistribution from the nucleus to the cytoplasm or presence of TDP-43 within NCI in FUSWT, FUSR521C and FUSΔ14 mice (Figure 
F6 6A, B and C). In contrast, over expression of TDP-43 with a mutated nuclear localization signal in mice using SBT leads to cytoplasmic accumulation of TDP-43 (Additional file
S7 7: Figure S7). Double-label immunofluorescence data also showed TDP-43 predominantly distributed in the nucleus in eGFP, FUSWT, FUSR521C and FUSΔ14 mice, even in neurons with well-defined NCIs (Additional file
S8 8: Figure S8 and Figure 
6C-J).
Additional file 7
Figure S7. Expression of human TDP-43 with a NLS mutation in mouse brain using SBT leads to increased cytoplasmic levels in mouse neurons. Mutant human TDP-43NLS accumulates in the soma, dendrites, and axons of neurons. Scale bar, 100 μm.
1750-1326-7-53-S7.pdf
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Additional file 8
Figure S8. Expression of eGFP in mouse brain using SBT did not lead to redistribution of TDP-43. Double labeling of EGFP (A) and TDP-43(B). Scale bar: 20μm.
1750-1326-7-53-S8.pdf
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Figure 6Endogenous TDP-43 is not redistributed in FUSWT, FUSR521C and FUSΔ14 mouse brain
Endogenous TDP-43 is not redistributed in FUSWT,FUSR521Cand FUSΔ14mouse 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.
1750-1326-7-53-6
Discussion
Our study is the first to use SBT to model FUS gene mutations in the mammalian central nervous system. The SBT paradigm was chosen because 1) mice can be generated quickly (a few months) compared to traditional transgenic techniques (a few years), 2) gene expression reaches a maximum ~3 weeks after birth, potentially avoiding toxicity during development, as has been recently observed for TDP-43
B24 24
, and 3) recombinant AAV vectors can be rapidly generated to test different constructs in vivo, such as alternative promoters or putative disease associated mutations.
A key question in the field is how mutations in FUS cause neurodegeneration in ALS or FTD. Different pathogenic mechanisms for FUS mutants including toxic gain-of-function, loss-of-function, or a combination of effects have been hypothesized
10
B25 25
B26 26
. The SBT FUS mice we have described provide additional insight into this issue. Over expression of either FUSWT, FUSR521C, or FUSΔ14 was not overtly toxic to mice on an organismal level after 3 months. Similarly, transgenic rats expressing wild-type human FUS do not have acute neuronal degeneration or behavioural impairment up to the first year of life; although transgenic lines expressing FUSR521C have rapid motor impairment and neuronal degeneration
B27 27
. Despite this ALS-like phenotype, FUS R521C rat lines did not have classic neuropathology associated with FUS proteinopathies. Intriguingly, both FUS WT and R521C rats accumulated ubiquitin; however FUS did not co-localize with ubiquitin and there was no formation of distinct NCI
27
. Similar to this result we did not detect NCI in our FUSR521C mice. In contrast, SBT generated FUSΔ14 mice have FUS and ubiquitin positive NCI, suggesting that we observed a much greater accumulation of neuropathology due to the use of this mutation, which causes a dramatic redistribution of FUS into the cytoplasm
16
. One deficiency of the SBT FUSR521C or FUSΔ14 mice we have described is the lack of a motor phenotype or neurodegeneration. A simple explanation is that neuronal death is not present at the three-month time point we have examined. Larger cohorts of SBT FUS mice are being generated and aged to answer this question.
To date 46 mutations in FUS that are associated with ALS or FTD have been discovered, but the mechanism of their toxicity is still being deciphered
26
B28 28
B29 29
. A majority of these mutations cluster in or near the C-terminal PY-NLS signal, and a number of groups have now reported that in cell culture these mutations inhibit nuclear import of FUS to varying levels and increase cytoplasmic levels of FUS
27
B30 30
B31 31
B32 32
. Our data provide the first in vivo evidence in mouse neurons that both ALS mutations studied, FUSR521C and FUSΔ14, translocate to the cytoplasm at higher levels compared to control. FUSΔ14, which lacks the entire PY-NLS domain, had the highest levels of FUS in the neuronal cytoplasm, lowest levels in the nucleus, and was the only mutation that developed robust inclusions and insoluble FUS. The degree of FUS re-localization caused by a mutation and age of disease onset has been interpreted to mean that cytoplasmic accumulation of FUS is a primary event that drives neurodegneration
23
. Experiments in yeast
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, Drosophila
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, and C. elegans
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support the concept that cytoplasmic accumulation of FUS is toxic. In contrast, Xia et al. have reported that FUS toxicity in Drosophila requires nuclear localization
B41 41
. Our observation that FUSΔ14, which produces the earliest disease onset in humans, accumulates at the highest levels in the cytoplasm and rapidly induces multiple pathological features of FUS proteinopathies, broadly supports the hypothesis that cytoplasmic FUS is toxic Further experiments will be necessary to dissect whether chronic cytoplasmic accumulation of FUSΔ14 in our mice leads to neurodegeneration and if so by what molecular mechanism.
Many neurodegenerative diseases have NCI or glial inclusions and the identity of the aggregated molecule(s) has proven to be a useful tool to characterize disease sub-types and help define disease pathogenesis
B42 42
. Despite the lack of an obvious motor or behavioural phenotype, the SBT FUSΔ14 mice recapitulate many key features of FUS proteinopathies
10
19
. The most striking feature in FUSΔ14 mice is the robust formation of NCIs, which are immunopositive for FUS, ubiquitin, PABP1and p62/SQSTM1. NCIs containing ubiquitin and p62 are common to all sub-types of FTD and ALS-FUS. More informative is the frequent presence of basophilic NCI in FUSΔ14 mice, which are numerous in BIBD cases, but also present in aFTLD-U and NIFID to a lesser extent
19
. Basophilic staining of NCIs has recently been linked to the presence of RNA and RNA-binding proteins, which is logical based on the function of FUS
21
. In contrast, we only detected infrequent α-internexin staining of NCI. This may indicate that FUSΔ14 pathology more closely resembles BIBID and aFTLD-U. Alternatively, NCI formation may start with FUS aggregation and accumulation of α-internexin is a downstream event. Further, when FUSΔ14 NCIs do stain with α-internexin, it is only a portion of the total inclusion (see Figure 
3 and
5). We also asked if OPTN occurred in FUS NCI based on recent reports that OPTN is a prominent marker of NCI in a subset of ALS and FTLD
B43 43
B44 44
. In our FUSΔ14 mouse model, only a small percentage of neurons had small extra-nuclear aggregates of OPTN and these did not robustly overlap with NCI detected by FUS and ubiquitin immunohistochemistry (Additional file
5: Figure S5). α-internexin inclusions were more frequent and distinct than OPTN, but still only labelled a fraction of the total FUS positive NCIs (Figure 
3 and Figure 
5). This observation is reminiscent of recent pathological studies of NIFID cases which found that many NCI were immunoreactive for FUS and in some cases FUS-immunoreactive NCI were more numerous than α-internexin immunoreactive NCI
17
B45 45
. The lack of α-internexin or OPTN positive NCI in FUSWT or FUSR521C mice implies that inclusion formation is a requirement for the development of this pathology. Based on these findings, and the referenced pathological findings in human cases, we suggest that α-internexin and OPTN pathology are downstream events and are not a major driver of pathology and neurodegeneration in most FUS proteinopathies. Electron microscopy of NIFID tissue supports the idea that neuronal intermediate filament accumulates following FUS aggregation in the cytoplasm
B46 46
. On-going experiments with FUSΔ14 mice will address whether aging increases the amount of α-internexin staining.
An interesting question raised by our data is the identity of the ubiquitinated protein(s) in FUSΔ14 inclusions. Ubiquitin is the most enriched marker, besides FUS, in the NCI of FUSΔ14, but we do not detect mono or poly-ubiquitination of FUS. This data is in agreement with multiple reports that aggregated FUS isolated from human brain is not modified by post-translational modifications, such as ubiquitin or phosphorylation
2
46
B47 47
B48 48
. Taken together, we hypothesize that accumulation of FUSΔ14 into NCI recruits other protein(s) that are ubiquitinated. The identity of these proteins remains to be determined and may reveal additional insights into FUS pathogenesis.
PABP-1 was another protein frequently detected in FUSΔ14 NCI. PABP-1 binds the poly(A) tail of mRNA and is involved in multiple steps of mRNA metabolism, including pre-mRNA splicing and regulation of translation. PABP-1 has recently gained attention in the neurodegeneration field due to its involvement in the formation of stress granules. Stress granules are dense cytoplasmic foci composed of non-translated messenger RNA, ribonucleoproteins, and other proteins that vary depending on the cell type and stress inducer
B49 49
. Stress granules are thought to protect mRNA from harmful conditions or serve as a mechanism to rapidly modulate the types and quantities of mRNA in response to changes in the environment
B50 50
. PABP-1 is one of the more common RNA-binding proteins that reliably associates with the various types of stress granules and is therefore commonly used as a specific marker
49
. PABP-1 labels NCI in ALS-FUS with a R521C mutation, as well as NCI in FTLD-FUS, BIBD and NIFID
31
. In cell culture, mutation of the PY-NLS can efficiently redistribute FUS into the cytoplasm, but an additional stressor appears necessary to induce localization to stress granules
31
. This finding lead the authors to speculate that two hits may be necessary to induce abnormal accumulation of FUS into stress granules and eventually end-stage NCIs
31
. This does not appear to be the case in the FUSΔ14 mice, because we observe numerous NCIs that co-localize with ubiquitin, p62, and PABP-1. However, milder mutations such as FUS R521C or sporadic cases may indeed require additional genetic or environmental factors to induce abnormal FUS pathology.
A major difference between the FUS positive NCI found in ALS-FUS or FTLD-FUS is that they are much larger and more insoluble than the stress granules observed in cell culture. More detailed examination of the spectrum of FUSΔ14 transduced neurons reveals a spectrum of aggregates ranging from multiple small foci in a neuron to a single large inclusion filling the cell body (Additional file
5: Figure S5). We hypothesize that FUS-immunoreactive inclusions evolve in stages, and may represent a transition from stress granules, which are reversible and can rapidly be dissolve, to the large, insoluble, basophilic inclusions found in end-stage FUS pathology.
Mutations in FUS were first identified in ALS cases because sequencing of the FUS gene was prioritized based on its functional similarity to TDP-43, another RNA-binding protein that had been discovered to harbour causative mutations in ALS patients. Abnormal function of FUS, TDP-43, and other RNA-binding proteins has been recently proposed to be part of a common pathway linking defects in RNA quality control to neurodegeneration in ALS and FTLD
B51 51
. Therefore it is imperative to determine if FUS and TDP-43 share pathogenic mechanisms or interact in some way. To date, most ALS cases with FUS mutations or FTLD cases with FUS pathologies do not show abnormal TDP-43 redistribution or pathology, although one group has reported co-deposition of both proteins in NCIs
18
B52 52
. Experiments in Drosophila imply that both proteins share a common pathway, with FUS acting downstream of TDP-43
25
. Other model systems suggest that FUS and TDP-43 act through distinct pathways and cause disease through independent mechanisms, but a consensus has not yet been reached in the field
28
B53 53
B54 54
. We find no evidence of TDP-43 redistribution into the cytoplasm or co-aggregation into NCI in any of the FUS mice examined, even in the presence of NCIs (Figure 
5). Thus in our mouse model, FUS and TDP-43 aggregation appear distinct, and lead us to speculate that despite their many similarities
6
, FUS and TDP-43 have unique biological functions and their dysfunction may cause neurodegeneration through RNA dysfunction, but the precise targets and pathways are distinct.
Conclusions
We find that SBT is a viable and rapid method to investigate the mechanism and disease relevance of genes in the nervous system of mice. The rAAV-1 vector we used in this study targets gene expression to neurons, but other rAAV vectors and promoter combinations are available to target expression to most cell types in the CNS
B55 55
. We find that expression of a disease-associated FUS mutation (FUSΔ14) validates it as a pathogenic mutation, because expression of this mutation produced a number of pathological features of FUS proteinopathies. The finding that FUSΔ14 expression can reproduce many pathologic features observed in subtypes of FTLD and ALS FUS proteinopathies was surprising, and provides additional evidence that these diseases may share a common disease mechanism.
Overexpression of human FUSWT did not induce neurodegeneration or abnormal neuropathology. Expression of the ALS mutation FUSR521C was also not obviously toxic to animals at 3 months. Although FUSR521C mice did not have distinct NCI, they did have a large increase in the amount of FUS present in the cell bodies and processes of neurons, as well as accumulation of biochemically insoluble FUS. The presence of aggregated FUS in FUSR521C, mice but no detectable NCI may indicate that we have captured an early stage of the disease process before inclusions form. Alternatively, the insoluble nature of a portion of FUSR521C may indicate that small NCIs or oligomers of FUS may already be present in these animals, but are not detectable using classic immunohistochemistry. Experiments are on-going to examine behavioural and neuropathic changes overtime in SBT FUS mice.
In summary, our data supports the hypothesis that many ALS/FTD-linked mutations cause disease by increasing the cyotplasmic levels of FUS, with unknown consequences. One possibility is that cytoplasmic FUS recruits other RNA-binding proteins, such as TAF15 and PABP-1, into stress-granule like aggregates that overtime coalesce into permanent, insoluble inclusions (Additional file
5: Figure S5). Sequestration of RNA-binding proteins could dramatically affect RNA metabolism and would have devastating effects on numerous cellular events. The recent identification of an expanded hexanucleotide repeat in C9ORF72 as a frequent cause of the ALS/FTD clinical spectrum in addition to causative mutations in RNA-binding proteins, including TDP-43, FUS, sentaxin, and angiogenin, strongly implicates defects in RNA metabolism as a critical pathogenic pathway in both ALS and FTD
29
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. The SBT FUS mice described in this manuscript will provide a valuable platform for further 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.
Methods
Cloning
The generation of the N-terminally V5 tagged FUS constructs, AAV1-wild type human FUS (FUSWT), AAV1-human pR521C mutant FUS (FUSR521C) and AAV1-human p.G466VfsX14 truncated FUS (FUSΔ14) was previously described
16
. Inserts were cloned into the AAV1-vector using BamHI and XhoI restriction sites. The V5 tag does not alter the normal location or function of FUS and was added to the N-terminus of all constructs to facilitate detection and analysis without interference from endogenous FUS protein
32
. The sequences of all AAV1-FUS expression constructs were confirmed by direct sequencing of the complete cDNA inserts and flanking vector sequences.
AAV1 generation and injection
All experiments with mice were approved by the Emory University and Mayo Clinic Institutional Animal Care and Use Committees and performed according to the guidelines for the care and use of laboratory animals. Animals were housed under circadian conditions and had free access to food and water. Recombinant adeno-associated virus serotype 1 (AAV1) generation and neonatal injection procedures were previously described
B60 60
. Briefly, through viral transduction of the neuron, the protein of interest is expressed under the control of the cytomegalovirus enhancer/chicken β-actin promoter. P0 mouse pups (0–12 hours old) were cryoanesthetized on ice and bilaterally injected with 2 μl of virus (10sup 13 particles/ml) per cerebral ventricle. After injection, the pups were wrapped in cage bedding, recovered on a heating pad then returned to their mother. Three groups of wild type B6C3F1 mice were injected with virus encoding FUSWT (n=9), FUSR521C (n=16) and FUSΔ14 (n=11).
Tissue preparation
Brains were harvested at 3 months of age, except the hTDP43M337V mice, which were sacrificed at 3 weeks of age. Half of the brain was immersion fixed in 4% paraformaldehyde for 24 hours and washed in Tris Buffered Saline (TBS). Sections were embedded in paraffin, sectioned in the sagittal plain (5 μm thick) and mounted on glass slides. The other half of the brain was frozen on dry ice for biochemistry.
Histology and immunohistochemistry
Sections were deparaffinized in xylene and rehydrated in a graded series of alcohol followed by dH2O. Antigen retrieval was performed in a dH2O steam bath for 30 minutes. Immunohistochemistry was performed on an automated stainer (DAKO Auto Machine Corporation) and the DAKO EnVision+ HRP system. All sections were briefly counterstained with hematoxylin. For Double labelling immunofluorescence.
After deparaffinised and rehydrated, sections were incubated in retrieval solution (DAKO) for 30 min at 95 degree. Tissues were immunostained with the following primary antibodies at the indicated concentrations: V5 (monoclonal, Invitrogen, 1:1,000 and polyclonal, Bethyl Labs, 1:1000), FUS (polyclonal, Sigma, 1:2,500), TDP-43 (monoclonal human specific, Novus Biologicals, 1:3,000 and polyclonal, Proteintech, 1:5,000), PABP1 (polyclonal, Cell signaling, 1:100), α-internexin (monoclonal IgG; from Gerry Shaw, University of Florida, 1:50), OPTN (polyclonal, Abcam, 1:100) and ubiquitin (monoclonal, Millipore,1:60K and polyconal, DAKO, 1:200), TAF15(polyclonal, Bethyl Lab, 1:250), P62(monoclonal, BD Biosciences, 1:100), NeuN(polyclonal, Millipore, 1:200), IBA1(polyclonal, WAKO, 1:1000), EWS(polyclonal, Epitomics, 1:250), GFAP(polyclonal, Millipore, 1:1000). For Double labelling immunofluorescence, the sections were incubated in the secondary antibodies conjugated to Cy3 and fluorescein(1:250, Invitrogen). Hematoxylin-eosin (H&E) staining was performed on paraffin sections. Stained sections were captured using the ScanScope XT image scanner (Aperio, Vista, CA, USA) and processed with ImageScope software. Ubiquitin accumulation was quantified using the ImageScope positive pixel count algorithm for DAB staining (Aperio). Other photomicrographs were captured on an Olympus BX50 microscope with DP12 digital camera (Olympus, PA, USA). Confocal images were collected with a Zeiss LSM 510 NLO META system.
Biochemistry
Nuclear and cytoplasmic enriched protein fractions were isolated from the tissue using the ProteoJET Cytoplasmic and Nuclear Extraction Kit (Fermentas, Ontario, Canada) following the manufacturer’s protocol. Protein separation and immunoblot were performed as previously described
B61 61
B62 62
. Briefly, proteins were separated on 4-12% Bis-Tris XT gels (Bio-Rad, CA, USA) with XT-MES running buffer and transferred to a 0.2μm nitrocellulose membrane. After overnight blocking at 4°C in a 0.5% casein block solution, blots were probed with anti-V5 (monoclonal, Invitrogen, 1:1K) and anti-histone 3 (polyclonal, Cell Signaling, 1:1,000) primary antibody, followed by horseradish peroxidase (HRP)-conjugated secondary antibody against mouse and rabbit IgG (Jackson Immuno Research, 1:5K). Relative band intensity was quantified using ImageJ software (NIH).
Abbreviations
ALS: Amyotrophic lateral sclerosis; FTD: Fronto-temproal dementia; FTLD: Frontotemporal lobar degeneration; aFTLD-U: Atypical FTLD with ubiquitinated inclusions; NIFID: Neuronal intermediate filament inclusion disease; BIBD: Basophilic inclusion body disease; FUS or TLS: Fused in sarcoma or translocated in sarcoma; NCIs: Neuronal cytoplasmic inclusions; NLS: Nuclear localization sequence; NES: Nuclear export signal; SBT: Somatic brain transgenesis; AAV: Adeno-associated virus; PABP-1: Polyadenylate-binding protein 1.
Competing interests
The authors declare that they have no conflict of interest.
Authors’ contributions
CV, QD, PD, and TK carried out animal experiments, western blots, and analysis of data. MDH, JK, and CCD cloned constructs and produced AAV. CV, QD, GT, DWD, and TK performed tissue immunostaining and analysis. TG, PD, RR, DWD, and TK conceived of the study, participated in design, experiment coordination, and data analysis. CV, QD, and TK drafted the manuscript. All authors read and approved the final manuscript.
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ack
Acknowledgements
The authors thank Monica Castanedes-Casey, Virginia Phillips and Linda Rousseau for their excellent histological and immunohistochemical support. Dr. Gerry Shaw at the University of Florida kindly provided the monoclonal antibody against α-internexin. TK is supported by the National Institutes of Health grants (P30NS069289 and P50AG016574-13). RR is supported by the National Institutes of Health grants (P50AG016574-13 and R01NS065782). QD is supported by National institutes of Health training grant T32 “Training and translational research in Neurology” (2T32 NS 007480). This research project was supported in part by the Microscopy Core Facility of the Emory Neuroscience NINDS Core Facilities grant (P30NS055077).
refgrp Mutations in FUS, an RNA Processing Protein, Cause Familial Amyotrophic Lateral Sclerosis Type 6VanceCRogeljBHortobagyiTDe VosKJNishimuraALSreedharanJHuXSmithBRuddyDWrightPetal Science20093231208lpage 1211Mutations in the FUS/TLS Gene on Chromosome 16 Cause Familial Amyotrophic Lateral SclerosisKwiatkowskiTJBoscoDALeClercALTamrazianEVanderburgCRRussCDavisAGilchristJKasarskisEJMunsatTScience200932312051208TicozziNTilocaCMorelliCColombritaCPolettiBDorettiAMadernaLMessinaSRattiASilaniVGenetics of Familial Amyotrophic Lateral Sclerosis2011The Two Faces of the FUS/EWS/TAF15 Protein FamilyKovarHDrJMrHSarcoma20112011837474TLS, EWS and TAF15: a model for transcriptional integration of gene expressionLawWJCannKLHicksGGBrief Funct Genomic Proteomic20065814Rethinking ALS: The FUS about TDP-43Lagier-TourenneCClevelandDWCell200913610011004Identification and Characterization of the Nuclear Localization/Retention Signal in the EWS Proto-oncoproteinZakaryanRPGehringHJ Mol Biol20063632738The multifunctional FUS, EWS and TAF15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress responseAnderssonMStahlbergAArvidssonYOlofssonASembHStenmanGNilssonOAmanPBMC Cell Biol2008937TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttlingZinsznerHSokJImmanuelDYinYRonDJ Cell Sci199711017411750TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementiaMackenzieIRARademakersRNeumannMLancet Neurol201099951007Nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration: an updateMackenzieINeumannMBigioECairnsNAlafuzoffIKrilJKovacsGGhettiBHallidayGHolmIActa Neuropathol201011914Intracranial adeno-associated virus-mediated delivery of anti-pan amyloid beta, amyloid beta40, and amyloid beta42 single-chain variable fragments attenuates plaque pathology in amyloid precursor protein miceLevitesYJansenKSmithsonLADakinRHollowayVMDasPGoldeTEJ Neurosci2006261192311928Intraventricular Brain Injection of Adeno-Associated Virus Type 1 (AAV1) in Neonatal Mice Results in Complementary Patterns of Neuronal Transduction to AAV2 and Total Long-Term Correction of Storage Lesions in the Brains of {beta}-Glucuronidase-Deficient MicePassiniMAWatsonDJViteCHLandsburgDJFeigenbaumALWolfeJHJ Virol20037770347040Multiple system degeneration with basophilic inclusions in Japanese ALS patients with bm
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RESEARCHARTICLEOpenAccessExpressionof Fusedinsarcoma mutationsinmice recapitulatestheneuropathologyofFUS proteinopathiesandprovidesinsightinto diseasepathogenesisChristopheVerbeeck1 †,QiudongDeng2 †,MarielyDeJesus-Hernandez1,GeorgiaTaylor2,CarolinaCeballos-Diaz3, JannetKocerha4,ToddGolde3,PritamDas1,RosaRademakers1,DennisWDickson1andThomasKukar2* †AbstractBackground: MutationsinthegeneencodingtheRNA-bindingproteinfusedinsarcoma(FUS)cancausefamilial andsporadicamyotrophiclateralsclerosis(ALS)andrarelyfrontotemproaldementia(FTD).FUSaccumulatesin neuronalcytoplasmicinclusions(NCIs)inALSpatientswithFUSmutations.FUSisalsoamajorpathologicmarker foragroupoflesscommonformsoffrontotemporallobardegeneration(FTLD),whichincludesatypicalFTLDwith ubiquitinatedinclusions(aFTLD-U),neuronalintermediatefilamentinclusiondisease(NIFID)andbasophilicinclusion bodydisease(BIBD).ThesediseasesarenowcalledFUSproteinopathies,becausetheysharethisdiseasemarker.It isunknownhowFUSmutationscausediseaseandtheroleofFUSinFTD-FUScases,whichdonothaveFUS mutations.Inthispaperwereportthedevelopmentofsomaticbraintransgenic(SBT)miceusingrecombinant adeno-associatedvirus(rAAV)toinvestigatehowFUSmutationsleadtoneurodegeneration. Results: WecomparedSBTmiceexpressingwild-typehumanFUS(FUSWT),andtwoALS-linkedmutations:FUSR521CandFUS 14,whichlacksthenuclearlocalizationsignal.BothFUSmutantsaccumulatedinthecytoplasmrelativeto FUSWT.ThedegreeofthisshiftcorrelatedwiththeseverityoftheFUSmutationasreflectedbydiseaseonsetin humans.Miceexpressingthemostaggressivemutation,FUS 14,recapitulatedmanyaspectsofFUSproteinopathies, includinginsolubleFUS,basophilicandeosiniphilicNCIs,andotherpathologicmarkers,includingubiquitin,p62/SQSTM1, -internexin,andthepoly-adenylate(A)-bindingprotein 1(PABP-1).However,TDP-43didnotlocalizetoinclusions. Conclusions: OurdatasupportsthehypothesisthatALSorFTD-l inkedFUSmutationscauseneurodegenerationby increasingcyotplasmicFUS.AccumulationofFUSinthecytoplasmmayretainRNAtargetsandrecruitadditional RNA-bindingproteins,suchasPABP-1,intostress-granulelikeaggregatesthatcoalesceintopermanentinclusions thatcouldnegativelyaffectRNAmetabolism.IdentificationofmutationsinothergenesthatcauseALS/FTD,suchas C9ORF72,sentaxin,andangiogenin,lendssupporttotheideathatdefectiveRNAmetabolismisacriticalpathogenic pathway.TheSBTFUSmicedescribedherewillprovideavaluableplatformfordissectingthepathogenicmechanism ofFUSmutations,definetherelationshipbetweenFTDandALS-FUS,andhelpidentifytherapeutictargetsthatare desperatelyneededforthesedevastatingneurodegenerativedisorders. Keywords: Amyotrophiclateralsclerosis,Frontotemporalloba rdegeneration,Fusedinsarcomaproteinopathies, Transgenicmousemodels,Adeno-associatedvirus,Neur onalcytoplasmicinclusions,Ubiquitin,p62/SQSTM1, -internexin, PABP-1,Stressgranul es,RNAdysfunction *Correspondence: thomas.kukar@emory.edu†Equalcontributors2DepartmentofPharmacologyandNeurology,EmoryUniversitySchoolof Medicine,Atlanta,GA,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Verbeecketal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Verbeeck etal.MolecularNeurodegeneration 2012, 7 :53 http://www.molecularneurodegeneration.com/content/7/1/53

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BackgroundMutationsinthe FusedinSarcoma ( FUS )genewere recentlydiscoveredinsomecasesoffamilialandsporadicamyotrophiclateralsclerosis(ALS)andmore rarelyfronto-temproaldementia(FTD)[1-3].FUSisa 526aminoacidDNA/RNAbindingproteinmemberof theFETfamily(FUS/Ewing ’ ssarcoma/TATA-binding protein-associatedfactor)[4].TheFUSgene,also knownasTLS( translatedinsarcoma ),wasfirst describedasaN-terminalfusionthatproducedhybrid oncogenes[5].ThefulllengthFUSproteinisnowappreciatedtoplayaroleinanumberofcriticalcellularfunctions,includinggeneexpression,RNAprocessing,RNA transport,andgenomicintegrity[5,6].Thehighestlevels ofFUSarefoundinthenucleus,drivenbyahighlyconservedcarboxyl(C)terminalPYnuclearlocalizationsignal(PY-NLS)[7].FUSisfoundinthecytoplasmatlower levelsandcanshuttlerapidlybetweenthenucleusand thecytoplasm[8,9].Themajorityofdisease-linkedFUS mutationsclusterintheC-terminusanddisruptnuclear import,buttheprecisepathogenicmechanismofFUS mutationsiscurrentlyunknown. TheidentificationofFUSmutationsandaccumulation ofFUSwithinubiquitin-positiveneuronalcytoplasmic inclusions(NCI)inaportionofALScasesledtothereexaminationofotherneurologicaldiseaseswithNCIof unknownorigin.Subsequently,abnormalFUSwas detectedwithinNCI,aswellasglialinclusions,inseveraluncommonformsoffrontotemporallobardegeneration(FTLD),whichisthetermforthepathology underlyingtheclinicalsyndromeFTD.TheseraresubsetsofFTLDwerepreviouslyreferredtoasatypical FTLDwithubiquitinatedinclusions(aFTLD-U),neuronalintermediatefilamentinclusiondisease(NIFID)and basophilicinclusionbodydisease(BIBD)[10].ThesedisordershavenowbeengroupedtogetherastheFUSproteinopathies,becausetheyshareacommonpathology andapresumedunderlyingdiseasemechanism[10].The twomajorclinicalandpathologicaltypesareknownas frontotemporallobardegenerationwithFUSpathology (FTLD-FUS)andALSwithFUSpathology(ALS-FUS). Thisnomenclatureisanalogoustotheclassificationthat hasbeendevelopedfortheTDP-43proteinopathies (ALS-TDPandFTLD-TDP),whichhaveinclusionsthat containtheRNA-bindingproteinTDP-43protein[11]. Inthisreportwehaveutilizedatechniquecalledsomaticbraintransgenesis(SBT)toinvestigatehowFUS mutationsleadtoneurodegeneration.SBTusesrecombinantadeno-associatedvirus(rAAV)toexpressa cDNApredominantlyinneuronsthroughoutmuchof thebrainforthelifetimeofthemouse,beginningafew weeksafterbirth[12,13].Wecomparedoverexpression ofwild-typehumanFUS(FUSWT),andtwomutations associatedwithALS:FUSR521C,orFUS 14.Expressionof bothFUSmutantsledtoincreasedFUSproteininthe neuronalcytoplasm,thedegreeofwhichcorrelatedwith theseverityofthemutationasreflectedbydiseaseonset inhumans.Miceexpressingthemostaggressivemutation,FUS 14,recapitulatedmanyaspectsofhumanFUS proteinopathies,includinginsolubleFUSprotein,basophilicandeosiniphilicneuronalcytoplasmicinclusions (NCI),andpresenceofotherpathologicmarkers,includingubiquitin,p62/SQSTM1, -internexin,andthe polyadenylate-bindingprotein1(PABP-1).ResultsGenerationofmiceoverexpressingFUSusingsomatic braintransgenesis(SBT)WeutilizedSBTtoexpresswildtypehumanFUSand twoFUSmutationsassociatedwithALSinthebrainsof micetoinvestigatetheroleofFUSinneurodegeneration usingan invivo model.Inthisexperimentalparadigm, newlyborn(P0)littersofmicewereadministeredrecombinantAAV1encodingFUSWT,FUSR521C,orFUS 14, throughbilateralintracerebroventricularinjection.The FUSR521Cmutation,whichhasbeenidentifiedin16 ALSfamiliestodate,occurswithinthePYnuclearlocalizationsignal(PY-NLS)region,andresultsinanaverageageofonsetof40years[1,14,15].Thethirdmodel, FUS 14,wasbasedona denovo mutationfoundina patientwithsporadicALSthatwereportedpreviously [16].Briefly,amutationinintron13ofthe FUS gene (g.10747A>G)causesskippingofexon14,aframeshift, andprematureterminationinexon15,leadingtoatruncatedFUSproteinof478aminoacidsthatlacksthe C-terminalPY-NLS(Figure1A).Thismutation(FUS 14) isassociatedwithearlydiseaseonset(20years)andarapid diseaseprogression(22months).CharacterizationofpathologyinFUSmiceThreemonthsafterviralinjection,micewerekilledand onebrainhemispherewasfixedforneuropathologic characterization,whiletheotherhemispherewasflash frozenforbiochemicalfractionation.Miceappeared healthyatthetimeofdeathanddidnotdisplayobvious motorimpairmentoranabnormalgraspingphenotype (datanotshown).FUSconstructscontainedaV5epitope tagontheamino-terminustoaidvisualizationofprotein expressionanddonotinterferewithproteinfunctionor cellularlocalization[16].BasedonV5immunohistochemistryFUSWT,FUSR521C,andFUS 14micehadwidespreadFUSproteinexpression,throughoutthebrain, withthehighestlevelsinthecerebralcortexandthe hippocampus(Figure1B-G).UsingtheSBTparadigm transgeneexpressionisneuronalwithnodetectableglial expression,asassessedbydoubleimmunofluorescence (Additionalfile1:FigureS1).NeuronsexpressingFUSWTshowedpredominantlynuclearlocalization,withlow,butVerbeeck etal.MolecularNeurodegeneration 2012, 7 :53Page2of13 http://www.molecularneurodegeneration.com/content/7/1/53

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Figure1 GenerationofHuman FusedinSarcoma (FUS)mousemodelsusingrAAV1andSBT. ( A ).DiagramofFUSWT,FUSR521C,andFUS 14expressionconstructs.AllFUSconstructswereclonedwithaV5epitopetagontheamino-terminustoaidimmunodetection.Themajorprotein domainsofFUSarehighlighted.QGSY=Gln-Gly-Ser-Tyrrichregion.Glycinerichregion.NES=nuclearexportsignal.RRM=RNArecognitionmotif. RGG=Arg-Gly-Gly-richmotif.ZNF=zincfingermotif.PY-NLS=Pro-Tyrnuclearlocalisationsignal.( B )to( G ).Immunohistochemistry(anti-V5 antibody)detectswidespreadexpressionofFUSWT,FUSR521C,andFUS 14incerebralcortexanddentategyrus(DG).WildtypeFUSismainly localizedinthenuclei( B insert).IntensenuclearV5stainingincerebralcortex( B )andweakcytoplasmicv5staininginDG( E ).IntheFUSR521Cmodel,FUSisnolongerpredominantlylocatedinthenucleus,butalsofoundinthesomaanddendrites( C and F ).FUS 14micehaveadramatic translocationofFUSfromthenucleustothecytoplasm,andformationofneuronalcytoplasmicinclusions(NCIs)( D and G ). Scalebar: 100 m( H ). Histogramshowingthepercentofnuclearandcytoplasmicv5staining,cytoplasmicinclusionincerebralcortex.(n=4;S.E.M.)*P<0.05,**P<0.01 and***P<0.001,onewayANOVA. Verbeeck etal.MolecularNeurodegeneration 2012, 7 :53Page3of13 http://www.molecularneurodegeneration.com/content/7/1/53

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detectable,levelsofcytoplasmicproteinbasedonimmunohistochemistryandsubcellularfractionation(Figure1 and2).FUSR521CmicehadmarkedincreasesinFUS immunoreactivityintheneuronalcytoplasm.ThepresenceofnuclearFUSR521Cwasaconsistentfeature;however,FUSR521Cwasalsodetectedinthesoma,dendrites, andaxonsofneuronsinmice,especiallyinthehippocampus(Figure1CandF).Despiteincreasedcytoplasmic levelsofFUSR521C,noobviousinclusionsoraggregatesof FUSwereobservedinmiceinjectedwithFUSWTor FUSR521C.FUS 14miceshowedthegreatestcytoplasmic redistribution,withsomeneuronsshowingnonuclear FUSreactivitybutstronglabellingofthecellbodyand processesincortex.AportionofneuronsinFUS 14mice containedFUS-positiveneuronalcytoplasmicinclusions (NCIs),whichbaredstrikingresemblancetotheNCIs thatareacharacteristicpathologicfeatureofALSand FTD-FUS(Figure1DandG).Incortex,thepercentageof transducedneuronswithcytoplasmicdistributionofFUS significantlyincreasedinFUSR521CandFUS 14mice (Figure1H).TheFUS 14miceweretheonlygroupthat hadNCI,reaching~20%ofneuronsexpressingFUS (Figure1H).Mutation-dependentFUSredistributionalso wasconfirmedbydoublelabellingwithV5andaneuronalmarker(Additionalfile2:FigureS2).Despitethe presenceofNCI,wedidnotobserveanyobviousneuronallossordegenerationwhenexamininghaematoxylin andeosin(H&E)stainedsections.Activatedcaspase-3 andTUNELassayswerealsonegative(datanotshown), suggestingthatapoptosisisnotoccurringinFUSmiceat thisage.Further,wedidnotobservemarkedastrocytosis ormicroglialactivationatthisage(Additionalfile3: FigureS3). Wenextexaminedthebiochemicalsolubilityofthe differentFUSproteinsexpressedintheSBTmice.The cytoplasmandintactnucleiwereisolatedfromthefrozen brainhemisphereoftheFUSmiceanalysedaboveusing immunohistochemistry(Figure1).Nucleiwerelysedin detergenttoisolatethesolublenuclearfraction.The cytoplasmicandnuclearextractswerecentrifugedat 20,000xgtopelletinsolubleproteins.Sampleswere separatedbySDS/PAGE,analysedonimmunoblots,and quantifiedusingdensitometry(Figure2A).Comparison oftheratioofsolublecytoplasmictonuclearFUSprotein usingdensitometryconfirmedthatthesteadystatelevels oftheFUSmutantsarehigherinthecytoplasm,with FUS 14showingthestrongestshift(Figure.2AandB). Intriguingly,thelevelsofFUS 14detectedinthecytoplasmicandnuclearlysatesdidnotappeartomatchthe immunohistochemistry(Figure1B-H)thatsuggested robustexpressionofallFUSconstructs.Wethenanalysedthepelletsresultingfromtheprotocolusedbiochemicalisolationofthecytoplasmandnucleus,and discoveredthatthemajorityofFUS 14proteinpartitions intotheinsolublefraction(Figure2C).NoFUSWT,buta portionofFUSR521Cproteinwasalsodetectedintheinsolublefraction.FormationofneuronalcytoplasmicinclusionsinFUS 14miceWenextexaminedFUSmiceforthepresenceofneuropathologicmarkersfoundinALSorFTD.Sectionsfrom eGFP,FUSWTandFUSR521Cmicehaddiffuseubiquitin stainingwithnodetectableinclusions(Figure3A,Eand I).Incontrast,FUS 14micehadfrequentubiquitinpositiveNCIs(Figure3MandAdditionalfile4:Figure S4).NCIsvariedinsizefromsmallpunctatolarge, roundinclusions(Additionalfile5:FigureS5).DoublelabelimmunofluorescenceconfirmedthatFUS 14and ubiquitinco-localizetothesameinclusion(Figure4). Figure2 FUSmutationscauseanaberrantsubcellular redistributioninmouseneurons. Arepresentativeimmunoblot ( A )oftheV5taggedFUSproteinsextractedfromAAVinjected mousebrains.TissueextractsfromFUSWT,FUSR521C,andFUS 14brainwereseparatedintosolublefractionsfromthecytoplasmand nucleus.Histone3stainingwasusedasanuclearmarkertoverify extractionfidelity.( B )TheratioofcytoplasmicFUStonuclearFUS wascalculatedbasedonquantificationofimmunoblotsfordifferent FUSconstructs(n=4;S.E.M.).AhigherratioofFUSR521CandFUS 14arefoundinthecytoplasm.**P<0.01and***P<0.001.( C )FUSR521CandFUS 14proteinaremoreinsolublethanFUSWT. Verbeeck etal.MolecularNeurodegeneration 2012, 7 :53Page4of13 http://www.molecularneurodegeneration.com/content/7/1/53

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Wedidnotobserveanincreaseinhighmolecular weightsmearingofFUS,anindicatorofpoly-ubiquitination,onimmunoblotsofbraintissueofFUS 14comparedtoFUSWT(datanotshown).Furthermore, immunoblotsdirectlyforpoly-ubiquitindidnotdetecta differencebetweenFUSWT,FUSR521CorFUS 14mice, suggestingthatFUSisnotrobustlyubiquitinated(data notshown). BasedontherobustformationofNCIsintheFUS 14mice,weexaminedapanelofproteinmarkersthatwere previouslydescribedintheNCIsofFUSproteinopathies. ManyoftheNCIinFUS 14micewerebasophilicby H&Estaining,similartotheNCIfoundinBIBD,aFTLDU,andNIFIDcases(Figure3N)[17-19].Manyofthe basophilicinclusionswerecytoplasmic,adjacenttothe nucleus,andhadarounded, “ Pick-body ” likestructure (Figure3Ninsert).Eosinophilicstainingwasalsonoted ontheperipheryofinclusions,distinctspotswithinbasophilicNCIs,orasdistinctinclusions.Wethenstainedfor theneuronalintermediatefilamentprotein -internexin, whichisoneofthemostabundantmarkersforNIFID [20].AportionoftheNCIsfoundintheFUS 14model werepositivefor -internexin(Figure3O),buttheywere lessfrequentthanNCIslabelledwithubiquitin (Figure3M).NCIinFUS 14werealsoimmunoreactive forPABP-1,whichwaspreviouslyreportedtostainNCI inBIBDandNIFIDcases(Figure3P)[21].Therewas diffusecytoplasmicstainingofPABP-1ineGFP,FUSWTorFUSR521Cmice,butnoobviousNCI(Figure3D,Hand L).Double-labelimmunofluorescenceconfirmedthat -internexinandPABP-1co-localizedwithFUSinclusions intheFUS 14model(Figure5E-L). Wealsoexaminedthestainingpatternsofthep62/ sequestosome-1(p62/SQSTM1)andoptineurin(OPTN) proteinsinFUSmice,whichhavebeenreportedtobe mutatedinsomefamilialandsporadicALScases [22,23].P62robustlyco-localizedwithNCIinFUS 14mice(Figure5A-D).OPTNimmunostainingwasdiffuse andwidespreadintheneuronalcytoplasmofFUSWT, FUSR521C,aswellasFUS 14mice.Wefoundoccasional increasedOPTNstainingintheneuronalcytoplasmin brainregionsofFUS 14micewithubiquitinpositive NCI,butnodefinitelabellingofNCIs(Additionalfile6: FigureS6). Figure3 NeuropathologyofSBTFUS 14miceissimilartohumanFUSproteinopathies(A-P). Adjacentsectionsfromthecerebralcortexof eGFP( A D ),FUSWT( E H ),FUSR521C( I L ),andFUS 14( M P )micestainedwithproteinNCImarkersfoundinaFTLD-U,NIFID,BIBDandhematoxylineosin( H & E ).( M )OnlyFUS 14brainscontainubiquitinatedNCIs.( N )Hematoxylin-eosin( H & E )stainingofFUS 14cerebralcortexshowscytoplasmic basophilicinclusions(arrows).( O )NCIsareinfrequentlypositivefor -internexin,similartothepathologicalNCIsfoundinNIFIDcases.( P )Some NCIinthecerebralcortexofFUS 14micecontainthestressgranulemarkerproteinPABP-1. Scalebar :100 m.Slidesareorientatedtoacommon bloodvessel(arrowhead)toserveasalandmark( M P ). Verbeeck etal.MolecularNeurodegeneration 2012, 7 :53Page5of13 http://www.molecularneurodegeneration.com/content/7/1/53

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NoredistributionoraccumulationofTDP-43inFUSmiceBecauseFUSandTDP-43havesuchstrikingstructural andfunctionalsimilarities,therelationshipbetweentheir pathologyanddiseasemechanismisanimportantunansweredquestioninthefield.WedidnotfindanyevidenceofTDP-43redistributionfromthenucleustothe cytoplasmorpresenceofTDP-43withinNCIinFUSWT,FUSR521CandFUS 14mice(Figure6A,BandC).In contrast,overexpressionofTDP-43withamutatednuclearlocalizationsignalinmiceusingSBTleadstocytoplasmicaccumulationofTDP-43(Additionalfile7: FigureS7).Double-labelimmunofluorescencedataalso showedTDP-43predominantlydistributedinthenucleusineGFP,FUSWT,FUSR521CandFUS 14mice,even inneuronswithwell-definedNCIs(Additionalfile8: FigureS8andFigure6C-J).DiscussionOurstudyisthefirsttouseSBTtomodelFUSgene mutationsinthemammaliancentralnervoussystem. TheSBTparadigmwaschosenbecause1)micecanbe generatedquickly(afewmonths)comparedtotraditionaltransgenictechniques(afewyears),2)geneexpressionreachesamaximum~3weeksafterbirth, potentiallyavoidingtoxicityduringdevelopment,ashas beenrecentlyobservedforTDP-43[24],and3)recombinantAAVvectorscanberapidlygeneratedtotestdifferentconstructs invivo ,suchasalternativepromoters orputativediseaseassociatedmutations. AkeyquestioninthefieldishowmutationsinFUS causeneurodegenerationinALSorFTD.Different pathogenicmechanismsforFUSmutantsincludingtoxic gain-of-function,loss-of-function,oracombinationof effectshavebeenhypothesized[10,25,26].TheSBTFUS micewehavedescribedprovideadditionalinsightinto thisissue.OverexpressionofeitherFUSWT,FUSR521C, orFUS 14wasnotovertlytoxictomiceonanorganismallevelafter3months.Similarly,transgenicrats expressingwild-typehumanFUSdonothaveacute neuronaldegenerationorbehaviouralimpairmentupto thefirstyearoflife;althoughtransgeniclinesexpressing FUSR521Chaverapidmotorimpairmentandneuronal degeneration[27].DespitethisALS-likephenotype,FUS R521Cratlinesdidnothaveclassicneuropathology associatedwithFUSproteinopathies.Intriguingly,both FUSWTandR521Cratsaccumulatedubiquitin;howeverFUSdidnotco-localizewithubiquitinandthere wasnoformationofdistinctNCI[27].SimilartothisresultwedidnotdetectNCIinourFUSR521Cmice.In contrast,SBTgeneratedFUS 14micehaveFUSandubiquitinpositiveNCI,suggestingthatweobservedamuch greateraccumulationofneuropathologyduetotheuse ofthismutation,whichcausesadramaticredistribution ofFUSintothecytoplasm[16].Onedeficiencyofthe SBTFUSR521CorFUS 14micewehavedescribedisthe lackofamotorphenotypeorneurodegeneration.Asimpleexplanationisthatneuronaldeathisnotpresentat thethree-monthtimepointwehaveexamined.Larger cohortsofSBTFUSmicearebeinggeneratedandaged toanswerthisquestion. Todate46mutationsinFUSthatareassociatedwith ALSorFTDhavebeendiscovered,butthemechanism oftheirtoxicityisstillbeingdeciphered[26,28,29].A majorityofthesemutationsclusterinorneartheCterminalPY-NLSsignal,andanumberofgroupshave nowreportedthatincellculturethesemutationsinhibit nuclearimportofFUStovaryinglevelsandincrease cytoplasmiclevelsofFUS[27,30-32].Ourdataprovide thefirst invivo evidenceinmouseneuronsthatboth ALSmutationsstudied,FUSR521CandFUS 14,translocatetothecytoplasmathigherlevelscomparedtocontrol.FUS 14,whichlackstheentirePY-NLSdomain,had thehighestlevelsofFUSintheneuronalcytoplasm, lowestlevelsinthenucleus,andwastheonlymutation thatdevelopedrobustinclusionsandinsolubleFUS.The degreeofFUSre-localizationcausedbyamutationand ageofdiseaseonsethasbeeninterpretedtomeanthat cytoplasmicaccumulationofFUSisaprimaryeventthat drivesneurodegneration[23].Experimentsinyeast Figure4 Ubiquitinco-localizestoFUS-positiveinclusionsin hFUS 14mice. ConfocalimageshowingFUSlocatedinnucleusin FUSWTmice( A ),increasedcytoplasmicdistributionofFUSin FUSR521Cmice( B ),andaccumulationofFUSintoNCIsinFUS 14mice( C )thatco-labelswithubiquitin( F and L ).Nucleiwere counterstainedwithDAPI( G H and I ).FUSdetectedwithanti-V5 ( A B C ). Scalebar :10 m. Verbeeck etal.MolecularNeurodegeneration 2012, 7 :53Page6of13 http://www.molecularneurodegeneration.com/content/7/1/53

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[33,34], Drosophila [35-38],and C.elegans [39,40]supporttheconceptthatcytoplasmicaccumulationofFUS istoxic.Incontrast,Xiaetal.havereportedthatFUS toxicityin Drosophila requiresnuclearlocalization[41]. OurobservationthatFUS 14,whichproducestheearliestdiseaseonsetinhumans,accumulatesatthehighest levelsinthecytoplasmandrapidlyinducesmultiple pathologicalfeaturesofFUSproteinopathies,broadly supportsthehypothesisthatcytoplasmicFUSistoxic. Furtherexperimentswillbenecessarytodissectwhether chroniccytoplasmicaccumulationofFUS 14inourmice leadstoneurodegenerationandifsobywhatmolecular mechanism. ManyneurodegenerativediseaseshaveNCIorglial inclusionsandtheidentityoftheaggregatedmolecule(s) hasproventobeausefultooltocharacterizedisease sub-typesandhelpdefinediseasepathogenesis[42]. Despitethelackofanobviousmotororbehavioural phenotype,theSBTFUS 14micerecapitulatemanykey featuresofFUSproteinopathies[10,19].ThemoststrikingfeatureinFUS 14miceistherobustformationof NCIs,whichareimmunopositiveforFUS,ubiquitin, PABP1andp62/SQSTM1.NCIscontainingubiquitinand p62arecommontoallsub-typesofFTDandALS-FUS. Moreinformativeisthefrequentpresenceofbasophilic NCIinFUS 14mice,whicharenumerousinBIBD cases,butalsopresentinaFTLD-UandNIFIDtoa lesserextent[19].BasophilicstainingofNCIshasrecentlybeenlinkedtothepresenceofRNAandRNAbindingproteins,whichislogicalbasedonthefunction ofFUS[21].Incontrast,weonlydetectedinfrequent internexinstainingofNCI.Thismayindicatethat FUS 14pathologymorecloselyresemblesBIBIDand aFTLD-U.Alternatively,NCIformationmaystartwith FUSaggregationandaccumulationof -internexinisa downstreamevent.Further,whenFUS 14NCIsdostain with -internexin,itisonlyaportionofthetotalinclusion(seeFigure3and5).WealsoaskedifOPTNoccurredinFUSNCIbasedonrecentreportsthatOPTN isaprominentmarkerofNCIinasubsetofALSand FTLD[43,44].InourFUS 14mousemodel,onlyasmall percentageofneuronshadsmallextra-nuclearaggregatesofOPTNandthesedidnotrobustlyoverlapwith NCIdetectedbyFUSandubiquitinimmunohistochemistry(Additionalfile5:FigureS5). -internexininclusionsweremorefrequentanddistinctthanOPTN,but stillonlylabelledafractionofthetotalFUSpositive NCIs(Figure3andFigure5).ThisobservationisreminiscentofrecentpathologicalstudiesofNIFIDcases whichfoundthatmanyNCIwereimmunoreactivefor FUSandinsomecasesFUS-immunoreactiveNCIwere morenumerousthan -internexinimmunoreactiveNCI Figure5 Multipleneuropathologicmarkersco-accumulateinFUS 14miceNCIs. DoublelabellingforFUS(anti-V5; A E and I )andP62( B ), PABP1( F )and -internexin( J ).Colocalizationshowninmergedimage( D H and L ).NucleiwerecounterstainedwithDAPI( C G ,and K ). Scalebar : 20 m. Verbeeck etal.MolecularNeurodegeneration 2012, 7 :53Page7of13 http://www.molecularneurodegeneration.com/content/7/1/53

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[17,45].Thelackof -internexinorOPTNpositiveNCI inFUSWTorFUSR521Cmiceimpliesthatinclusionformationisarequirementforthedevelopmentofthis pathology.Basedonthesefindings,andthereferenced pathologicalfindingsinhumancases,wesuggestthat internexinandOPTNpathologyaredownstreamevents andarenotamajordriverofpathologyandneurodegenerationinmostFUSproteinopathies.ElectronmicroscopyofNIFIDtissuesupportstheideathatneuronal intermediatefilamentaccumulatesfollowingFUSaggregationinthecytoplasm[46].On-goingexperimentswith FUS 14micewilladdresswhetheragingincreasesthe amountof -internexinstaining. Aninterestingquestionraisedbyourdataistheidentityoftheubiquitinatedprotein(s)inFUS 14inclusions. Ubiquitinisthemostenrichedmarker,besidesFUS,in theNCIofFUS 14,butwedonotdetectmonoorpolyubiquitinationofFUS.Thisdataisinagreementwith multiplereportsthataggregatedFUSisolatedfrom humanbrainisnotmodifiedbypost-translational modifications,suchasubiquitinorphosphorylation [2,46-48].Takentogether,wehypothesizethataccumulationofFUS 14intoNCIrecruitsotherprotein(s)that areubiquitinated.Theidentityoftheseproteinsremains tobedeterminedandmayrevealadditionalinsightsinto FUSpathogenesis. PABP-1wasanotherproteinfrequentlydetectedin FUS 14NCI.PABP-1bindsthepoly(A)tailofmRNA andisinvolvedinmultiplestepsofmRNAmetabolism, includingpre-mRNAsplicingandregulationoftranslation.PABP-1hasrecentlygainedattentionintheneurodegenerationfieldduetoitsinvolvementinthe formationofstressgranules.Stressgranulesaredense cytoplasmicfocicomposedofnon-translatedmessenger RNA,ribonucleoproteins,andotherproteinsthatvary dependingonthecelltypeandstressinducer[49].Stress granulesarethoughttoprotectmRNAfromharmful conditionsorserveasamechanismtorapidlymodulate thetypesandquantitiesofmRNAinresponseto changesintheenvironment[50].PABP-1isoneofthe Figure6 EndogenousTDP-43isnotredistributedinFUSWT,FUSR521CandFUS 14mousebrain. MouseTDP-43ispredominantlyinthe nucleus(DABimmunohistochemistry; A B ,and C ) Double-labelimmunofluorescenceofTDP-43andFUSwt,FUSR521CorFUS 14mice(anti-V5; D-O).FUSisdistributedtothenucleusinFUSWTmice( D ),increasedintheneuronalcytoplasminFUSR521Cmice( H ),andaccumulatesas inclusionsintheneuronalcytoplasminFUS 14mice( L ).TDP-43stainingisnuclearinFUSWT,FUSR521CandFUS 14mice( E I and M ).Nucleiwere counterstainedwithDAPI. Scalebar :20 m. Verbeeck etal.MolecularNeurodegeneration 2012, 7 :53Page8of13 http://www.molecularneurodegeneration.com/content/7/1/53

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morecommonRNA-bindingproteinsthatreliably associateswiththevarioustypesofstressgranulesandis thereforecommonlyusedasaspecificmarker[49]. PABP-1labelsNCIinALS-FUSwithaR521Cmutation, aswellasNCIinFTLD-FUS,BIBDandNIFID[31].In cellculture,mutationofthePY-NLScanefficientlyredistributeFUSintothecytoplasm,butanadditional stressorappearsnecessarytoinducelocalizationto stressgranules[31].Thisfindingleadtheauthorsto speculatethattwohitsmaybenecessarytoinduceabnormalaccumulationofFUSintostressgranulesand eventuallyend-stageNCIs[31].Thisdoesnotappearto bethecaseintheFUS 14mice,becauseweobservenumerousNCIsthatco-localizewithubiquitin,p62,and PABP-1.However,mildermutationssuchasFUSR521C orsporadiccasesmayindeedrequireadditionalgenetic orenvironmentalfactorstoinduceabnormalFUS pathology. AmajordifferencebetweentheFUSpositiveNCI foundinALS-FUSorFTLD-FUSisthattheyaremuch largerandmoreinsolublethanthestressgranules observedincellculture.Moredetailedexaminationof thespectrumofFUS 14transducedneuronsrevealsa spectrumofaggregatesrangingfrommultiplesmallfoci inaneurontoasinglelargeinclusionfillingthecell body(Additionalfile5:FigureS5).Wehypothesizethat FUS-immunoreactiveinclusionsevolveinstages,and mayrepresentatransitionfromstressgranules,which arereversibleandcanrapidlybedissolve,tothelarge, insoluble,basophilicinclusionsfoundinend-stageFUS pathology. MutationsinFUSwerefirstidentifiedinALScases becausesequencingoftheFUSgenewasprioritized basedonitsfunctionalsimilaritytoTDP-43,another RNA-bindingproteinthathadbeendiscoveredto harbourcausativemutationsinALSpatients.Abnormal functionofFUS,TDP-43,andotherRNA-bindingproteinshasbeenrecentlyproposedtobepartofacommonpathwaylinkingdefectsinRNAqualitycontrolto neurodegenerationinALSandFTLD[51].Thereforeit isimperativetodetermineifFUSandTDP-43share pathogenicmechanismsorinteractinsomeway.To date,mostALScaseswithFUSmutationsorFTLD caseswithFUSpathologiesdonotshowabnormalTDP43redistributionorpathology,althoughonegrouphas reportedco-depositionofbothproteinsinNCIs[18,52]. Experimentsin Drosophila implythatbothproteins shareacommonpathway,withFUSactingdownstream ofTDP-43[25].OthermodelsystemssuggestthatFUS andTDP-43actthroughdistinctpathwaysandcause diseasethroughindependentmechanisms,butaconsensushasnotyetbeenreachedinthefield[28,53,54].We findnoevidenceofTDP-43redistributionintothecytoplasmorco-aggregationintoNCIinanyoftheFUS miceexamined,eveninthepresenceofNCIs(Figure5). Thusinourmousemodel,FUSandTDP-43aggregation appeardistinct,andleadustospeculatethatdespite theirmanysimilarities[6],FUSandTDP-43haveunique biologicalfunctionsandtheirdysfunctionmaycause neurodegenerationthroughRNAdysfunction,butthe precisetargetsandpathwaysaredistinct.ConclusionsWefindthatSBTisaviableandrapidmethodtoinvestigatethemechanismanddiseaserelevanceof genesinthenervoussystemofmice.TherAAV-1 vectorweusedinthisstudytargetsgeneexpression toneurons,butotherrAAVvectorsandpromoter combinationsareavailabletotargetexpressionto mostcelltypesintheCNS[55].Wefindthatexpressionofadisease-associatedFUSmutation(FUS 14) validatesitasapathogenicmutation,becauseexpressionofthismutationproducedanumberofpathologicalfeaturesofFUSproteinopathies.Thefinding thatFUS 14expressioncanreproducemanypathologicfeaturesobservedinsubtypesofFTLDandALS FUSproteinopathieswassurprising,andprovidesadditionalevidencethatthesediseasesmayshareacommondiseasemechanism. OverexpressionofhumanFUSWTdidnotinduceneurodegenerationorabnormalneuropathology.Expression oftheALSmutationFUSR521Cwasalsonotobviously toxictoanimalsat3months.AlthoughFUSR521Cmice didnothavedistinctNCI,theydidhavealargeincrease intheamountofFUSpresentinthecellbodiesandprocessesofneurons,aswellasaccumulationofbiochemicallyinsolubleFUS.ThepresenceofaggregatedFUSin FUSR521C,micebutnodetectableNCImayindicatethat wehavecapturedanearlystageofthediseaseprocess beforeinclusionsform.Alternatively,theinsolublenatureofaportionofFUSR521Cmayindicatethatsmall NCIsoroligomersofFUSmayalreadybepresentin theseanimals,butarenotdetectableusingclassic immunohistochemistry.Experimentsareon-goingto examinebehaviouralandneuropathicchangesovertime inSBTFUSmice. Insummary,ourdatasupportsthehypothesisthat manyALS/FTD-linkedmutationscausediseasebyincreasingthecyotplasmiclevelsofFUS,withunknown consequences.OnepossibilityisthatcytoplasmicFUS recruitsotherRNA-bindingproteins,suchasTAF15and PABP-1,intostress-granulelikeaggregatesthatovertime coalesceintopermanent,insolubleinclusions(Additionalfile5:FigureS5).SequestrationofRNA-binding proteinscoulddramaticallyaffectRNAmetabolismand wouldhavedevastatingeffectsonnumerouscellular events.TherecentidentificationofanexpandedhexanucleotiderepeatinC9ORF72asafrequentcauseoftheVerbeeck etal.MolecularNeurodegeneration 2012, 7 :53Page9of13 http://www.molecularneurodegeneration.com/content/7/1/53

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ALS/FTDclinicalspectruminadditiontocausative mutationsinRNA-bindingproteins,includingTDP-43, FUS,sentaxin,andangiogenin,stronglyimplicates defectsinRNAmetabolismasacriticalpathogenicpathwayinbothALSandFTD[29,56-59].TheSBTFUS micedescribedinthismanuscriptwillprovideavaluable platformforfurtherdissectingthepathogenicmechanismofFUSmutations,definetherelationshipbetween FTDandALS-FUS,andhelpidentifytherapeutictargets thataredesperatelyneededforthesedevastatingneurodegenerativedisorders.MethodsCloningThegenerationoftheN-terminallyV5taggedFUS constructs,AAV1-wildtypehumanFUS(FUSWT), AAV1-humanpR521CmutantFUS(FUSR521C)and AAV1-humanp.G466VfsX14truncatedFUS(FUS 14) waspreviouslydescribed[16].Insertswerecloned intotheAAV1-vectorusing BamH Iand Xho Irestrictionsites.TheV5tagdoesnotalterthenormal locationorfunctionofFUSandwasaddedtothe N-terminusofallconstructstofacilitatedetection andanalysiswithoutinterferencefromendogenous FUSprotein[32].ThesequencesofallAAV1-FUS expressionconstructswereconfirmedbydirectsequencingofthecompletecDNAinsertsandflanking vectorsequences.AAV1generationandinjectionAllexperimentswithmicewereapprovedbytheEmory UniversityandMayoClinicInstitutionalAnimalCare andUseCommitteesandperformedaccordingtothe guidelinesforthecareanduseoflaboratoryanimals. Animalswerehousedundercircadianconditionsand hadfreeaccesstofoodandwater.Recombinantadenoassociatedvirusserotype1(AAV1)generationandneonatalinjectionprocedureswerepreviouslydescribed [60].Briefly,throughviraltransductionoftheneuron, theproteinofinterestisexpressedunderthecontrolof thecytomegalovirusenhancer/chicken -actinpromoter. P0mousepups(0 – 12hoursold)werecryoanesthetized oniceandbilaterallyinjectedwith2 lofvirus(1013particles/ml)percerebralventricle.Afterinjection,the pupswerewrappedincagebedding,recoveredona heatingpadthenreturnedtotheirmother.Threegroups ofwildtypeB6C3F1micewereinjectedwithvirusencodingFUSWT(n=9),FUSR521C(n=16)andFUS 14(n=11).TissuepreparationBrainswereharvestedat3monthsofage,exceptthe hTDP43M337Vmice,whichweresacrificedat3weeksof age.Halfofthebrainwasimmersionfixedin4% paraformaldehydefor24hoursandwashedinTrisBufferedSaline(TBS).Sectionswereembeddedinparaffin, sectionedinthesagittalplain(5 mthick)andmounted onglassslides.Theotherhalfofthebrainwasfrozenon dryiceforbiochemistry.HistologyandimmunohistochemistrySectionsweredeparaffinizedinxyleneandrehydratedin agradedseriesofalcoholfollowedbydH2O.AntigenretrievalwasperformedinadH2Osteambathfor30minutes.Immunohistochemistrywasperformedonan automatedstainer(DAKOAutoMachineCorporation) andtheDAKOEnVision+HRPsystem.Allsections werebrieflycounterstainedwithhematoxylin.For Doublelabellingimmunofluorescence. Afterdeparaffinisedandrehydrated,sectionswere incubatedinretrievalsolution(DAKO)for30minat95 degree.Tissueswereimmunostainedwiththefollowing primaryantibodiesattheindicatedconcentrations:V5 (monoclonal,Invitrogen,1:1,000andpolyclonal,Bethyl Labs,1:1000),FUS(polyclonal,Sigma,1:2,500),TDP-43 (monoclonalhumanspecific,NovusBiologicals,1:3,000 andpolyclonal,Proteintech,1:5,000),PABP1(polyclonal, Cellsignaling,1:100), -internexin(monoclonalIgG; fromGerryShaw,UniversityofFlorida,1:50),OPTN (polyclonal,Abcam,1:100)andubiquitin(monoclonal, Millipore,1:60Kandpolyconal,DAKO,1:200),TAF15 (polyclonal,BethylLab,1:250),P62(monoclonal,BD Biosciences,1:100),NeuN(polyclonal,Millipore,1:200), IBA1(polyclonal,WAKO,1:1000),EWS(polyclonal,Epitomics,1:250),GFAP(polyclonal,Millipore,1:1000).For Doublelabellingimmunofluorescence,thesectionswere incubatedinthesecondaryantibodiesconjugatedtoCy3 andfluorescein(1:250,Invitrogen).Hematoxylin-eosin (H&E)stainingwasperformedonparaffinsections. StainedsectionswerecapturedusingtheScanScopeXT imagescanner(Aperio,Vista,CA,USA)andprocessed withImageScopesoftware.Ubiquitinaccumulationwas quantifiedusingtheImageScopepositivepixelcountalgorithmforDABstaining(Aperio).OtherphotomicrographswerecapturedonanOlympusBX50microscope withDP12digitalcamera(Olympus,PA,USA).Confocal imageswerecollectedwithaZeissLSM510NLO METAsystem.BiochemistryNuclearandcytoplasmicenrichedproteinfractions wereisolatedfromthetissueusingtheProteoJET CytoplasmicandNuclearExtractionKit(Fermentas, Ontario,Canada)followingthemanufacturer ’ sprotocol. Proteinseparationandimmunoblotwereperformed aspreviouslydescribed[61,62].Briefly,proteinswere separatedon4-12%Bis-TrisXTgels(Bio-Rad,CA, USA)withXT-MESrunningbufferandtransferredtoVerbeeck etal.MolecularNeurodegeneration 2012, 7 :53Page10of13 http://www.molecularneurodegeneration.com/content/7/1/53

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a0.2 mnitrocellulosemembrane.Afterovernight blockingat4Cina0.5%caseinblocksolution,blots wereprobedwithanti-V5(monoclonal,Invitrogen,1:1K) andanti-histone3(polyclonal,CellSignaling,1:1,000) primaryantibody,followedbyhorseradishperoxidase (HRP)-conjugatedsecondaryantibodyagainstmouse andrabbitIgG(JacksonImmunoResearch,1:5K). RelativebandintensitywasquantifiedusingImageJ software(NIH).AdditionalfilesAdditionalfile1: FigureS1. Neuron-specificexpressionofFUSin mousecortexandhippocampus.Confocalimagingshowsthatcells transducedwithrAAV1expressingFUSWT(anti-V5;A,E,I,andM)colocalizewiththeneuronalmarkerNeuN(BandF)butnotanastrocyte marker(GFAP;JandN)incortexandhippocampus.Nucleiwere counterstainedwithDAPI(C,G,KandO).Scalebar:50 m. Additionalfile2: FigureS2. Mutation-dependentredistributionofFUS. Double-immunofluorescencestainingofV5andaneuronalmarkerNeuN inFUSWT,FUSR521C,andFUS 14mice(A-L).V5stainingmainlylocatedin nucleusinFUSWTmouse(A-D).StrongnuclearandsomecytoplasmicV5 staininginFUSR521Cmouseneurons(E-H).Cytoplasmicaccumulationof V5staininginFUS 14mice(I-L).Scalebar:10 m. Additionalfile3: FigureS3. Nomarkedastrocytosisormicroglial activationinFUS 14mice.Double-labelimmunofluorescenceofa microgliamarkerIBA-1andV5inthecerebralcortexofFUSWTandFUS 14mice(A-H).Double-labelimmunofluorescenceofaastrocytemarker GFAPandV5incerebralcortexofFUSWTandFUS 14mice(A-H).Nuclei werecounterstainedwithDAPI(C,G,K,andO). Scalebar :50 m. Additionalfile4: FigureS4. QuantificationofubiquitinlevelsinSBT FUSmice.ThelevelsofubiqutinimmunoreactivityinthebrainofFUSWT, FUSR521C,andFUS 14micewerequantifiedusingpositivepixelcounts (defaultstrongpositiveDABthreshold)andanalyzedrelativetothetotal pixelsintheanalysisareausingtheImageScopesoftware(Aperio). ComparedtoFUSWTandFUSR521Cmice,FUS 14micehavesignificantly greateraccumulationofubiquitin(***p<0.001,One-wayanalysisof variance;GraphPadPrism5).ValuesrepresentmeanSEM(n=6foreach experimentalgroup). Additionalfile5: FigureS5. Diversityofneuronalcytoplasmic inclusionsinthebrainsofFUS 14mice.Thedifferentsizesofinclusions mayrepresentaspectrumofgrowthfromsmallaggregatestolarge, insolubleNCIs.Thefirstphaseischaracterizedbynuclearand cytoplasmiclocationofFUS(A),followedbyformationofsmall,round shapedaggregates(B).FUSaccumulatesinthecytoplasm(darker staining),associatedwithdepletionofFUSfromthenucleus(lighter staining),andtheaggregateseventuallymergeintooneortwo amorphousinclusions(C).Intheendstage,theNCI ’ sareroundoroval shapedandFUSisnolongerlocalizedinthenucleus. Additionalfile6: FigureS6. OptineurinisnotarobustmarkerofNCIin FUSs 14mice.ImmunohistochemistryofcerebralcortexofFUSWT( A ), FUSR521C( B )andFUS 14( C )miceshowsnooptineurin(OPTN)positive inclusions.ThereareoccasionalneuronsinthecortexofFUS 14mice withsmallextranuclearproteinaggregatesthatarepositiveforOPTN ( arrows C ).Detectionofendogenousmouseoptineurinwiththis antibodywasweak,makingitdifficulttodrawdefinitiveconclusions aboutco-localizationwithFUS-positiveNCI.Scalebar:50 m. Additionalfile7: FigureS7. ExpressionofhumanTDP-43withaNLS mutationinmousebrainusingSBTleadstoincreasedcytoplasmiclevels inmouseneurons.MutanthumanTDP-43NLSaccumulatesinthesoma, dendrites,andaxonsofneurons. Scalebar ,100 m. Additionalfile8: FigureS8. ExpressionofeGFPinmousebrainusing SBTdidnotleadtoredistributionofTDP-43.DoublelabelingofEGFP(A) andTDP-43(B).Scalebar:20 m. Abbreviations ALS:Amyotrophiclateralsclerosis;FTD:Fronto-temproaldementia; FTLD:Frontotemporallobardegeneration;aFTLD-U:AtypicalFTLDwith ubiquitinatedinclusions;NIFID:Neuronalintermediatefilamentinclusion disease;BIBD:Basophilicinclusionbodydisease;FUSorTLS:Fusedin sarcomaortranslocatedinsarcoma;NCIs:Neuronalcytoplasmicinclusions; NLS:Nuclearlocalizationsequence;NES:Nuclearexportsignal;SBT:Somatic braintransgenesis;AAV:Adeno-associatedvirus;PABP-1:Polyadenylatebindingprotein1. Competinginterests Theauthorsdeclarethattheyhavenoconflictofinterest. Authors'contributions CV,QD,PD,andTKcarriedoutanimalexperiments,westernblots,and analysisofdata.MDH,JK,andCCDclonedconstructsandproducedAAV.CV, QD,GT,DWD,andTKperformedtissueimmunostainingandanalysis.TG,PD, RR,DWD,andTKconceivedofthestudy,participatedindesign,experiment coordination,anddataanalysis.CV,QD,andTKdraftedthemanuscript.All authorsreadandapprovedthefinalmanuscript. Acknowledgements TheauthorsthankMonicaCastanedes-Casey,VirginiaPhillipsandLinda Rousseaufortheirexcellenthistologicalandimmunohistochemicalsupport. Dr.GerryShawattheUniversityofFloridakindlyprovidedthemonoclonal antibodyagainst -internexin.TKissupportedbytheNationalInstitutesof Healthgrants(P30NS069289andP50AG016574-13).RRissupportedbythe NationalInstitutesofHealthgrants(P50AG016574-13andR01NS065782).QD issupportedbyNationalinstitutesofHealthtraininggrantT32 “ Trainingand translationalresearchinNeurology ” (2T32NS007480).Thisresearchproject wassupportedinpartbytheMicroscopyCoreFacilityoftheEmory NeuroscienceNINDSCoreFacilitiesgrant(P30NS055077). Authordetails1DepartmentofNeuroscience,MayoClinic,Jacksonville,FL,USA.2DepartmentofPharmacologyandNeurology,EmoryUniversitySchoolof Medicine,Atlanta,GA,USA.3CenterforTranslationalResearchin NeurodegenerativeDisease,CollegeofMedicine,UniversityofFlorida, Gainesville,FL,USA.4DepartmentofHumanGenetics,EmoryUniversity SchoolofMedicine,Atlanta,GA,USA. Received:7June2012Accepted:27September2012 Published:10October2012 References1.VanceC,RogeljB,HortobagyiT,DeVosKJ,NishimuraAL,SreedharanJ,Hu X,SmithB,RuddyD,WrightP, etal : MutationsinFUS,anRNAProcessing Protein,CauseFamilialAmyotrophicLateralSclerosisType6. Science 2009, 323: 1208 – 1211. 2.KwiatkowskiTJ,BoscoDA,LeClercAL,TamrazianE,VanderburgCR,RussC, DavisA,GilchristJ,KasarskisEJ,MunsatT, etal : MutationsintheFUS/TLS GeneonChromosome16CauseFamilialAmyotrophicLateralSclerosis. 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epdcx:valueString Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis
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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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Verbeeck, Christophe
Deng, Qiudong
DeJesus-Hernandez, Mariely
Taylor, Georgia
Ceballos-Diaz, Carolina
Kocerha, Jannet
Golde, Todd
Das, Pritam
Rademakers, Rosa
Dickson, Dennis W
Kukar, Thomas
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Christophe Verbeeck et al.; licensee BioMed Central Ltd.
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Molecular Neurodegeneration. 2012 Oct 10;7(1):53
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