Journal Pre-proof Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS

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Journal Pre-proof Functional characterization of a FUS

mutant zebrafish line as a novel genetic model for ALS

Annis-Rayan Bourefis, Maria-Letizia Campanari, Valérie Buée-Scherrer, Edor

Kabashi

To cite this version:

Annis-Rayan Bourefis, Maria-Letizia Campanari, Valérie Buée-Scherrer, Edor Kabashi. Journal Pre-proof Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS. Neurobiology of Disease, Elsevier, 2020, pp.104935. �10.1016/j.nbd.2020.104935�. �hal-02573425�

Journal Pre-proof

Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS

Annis-Rayan Bourefis, Maria-Letizia Campanari, Valerie Buee-Scherrer, Edor Kabashi

PII: S0969-9961(20)30210-2

DOI: https://doi.org/10.1016/j.nbd.2020.104935

Reference: YNBDI 104935

To appear in: Neurobiology of Disease

Received date: 10 February 2020 Revised date: 22 April 2020 Accepted date: 29 April 2020

Please cite this article as: A.-R. Bourefis, M.-L. Campanari, V. Buee-Scherrer, et al., Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS, Neurobiology of Disease (2019),https://doi.org/10.1016/j.nbd.2020.104935

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1

Functional characterization of a FUS mutant zebrafish line as a

novel genetic model for ALS

Annis-Rayan Bourefis1,2*, Maria-Letizia Campanari1,2*, Valerie Buee-Scherrer3 and Edor

Kabashi1,2,

* These authors contributed equally to this work

Affiliations: (1) Imagine Institute, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 1163, Paris Descartes Université, 75015, Paris, France; (2) Sorbonne

Université, Université Pierre et Marie Curie (UPMC), Université de Paris 06, INSERM Unité 1127, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 7225

Institut du Cerveau et de la Moelle Épinière (ICM), 75013, Paris, France; (3) Université de

Lille, Inserm, CHU-Lille, Alzheimer & Tauopathies, Lille, France.

Correspondence should be addressed to: maria-letizia.campanari@institutimagine.org or edor.kabashi@institutimagine.org

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2 Abstract

Mutations in Fused in sarcoma (FUS), an RNA-binding protein, are known to cause

Amyotrophic Lateral Sclerosis (ALS). However, molecular mechanisms due to loss of FUS

function remain unclear and controversial. Here, we report the characterization and phenotypic

analysis of a deletion mutant of the unique FUS orthologue in zebrafish where Fus protein levels

are depleted. The homozygous mutants displayed a reduced lifespan as well as impaired motor

abilities associated with specific cellular deficits, including decreased motor neurons length and

neuromuscular junctions (NMJ) fragmentation. Furthermore, we demonstrate that these cellular

impairments are linked to the misregulation of mRNA expression of acetylcholine receptor

(AChR) subunits and histone deacetylase 4, markers of denervation and reinnervation processes

observed in ALS patients. In addition, fus loss of function alters tau transcripts favoring the

expression of small tau isoforms. Overall, this new animal model extends our knowledge on FUS

and supports the relevance of FUS loss of function in ALS physiopathology.

Keywords: Amyotrophic Lateral Sclerosis (ALS); Motor neuron; Neuromuscular Junction; Zebrafish; FUS; tau; Frontotemporal Dementia; Genetics; Neurodegeneration.

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3 1 Introduction:

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder caused

by progressive degeneration of upper and lower motor neurons (MNs) with a very rapid clinical

course. It is one of the most common neuromuscular disease with an incidence in Europe of 2.16

per 100,000 each year (1). ALS leads to muscle weakness and atrophy progressing to paralysis,

with respiratory failure being the major cause of death within years following clinical diagnosis

(2). A major gene which is mutated in ALS patients is the RNA-binding proteins FUS (FUSed in

sarcoma; also known as TLS, translocated in liposarcoma) (3–6). FUS is implicated in multiple

aspects of RNA metabolism including RNA splicing, trafficking and translation (7–9). This

factor shuttles between the nucleus and the cytoplasm (8,10,11) where it controls more than

5,500 identified RNA targets in both human and mouse brain (9).

FUS mutations were initially identified in 2009 (6,12), with currently more than 50 missense mutations reported mostly located in the exon 15 which encodes for the NLS (nuclear localization signal) at the C-terminal region of the protein (13). These mutations are known to cause the redistribution of FUS into the cytoplasm with the consequent clearance from the

nucleus (7,13,14). Nonsense mutations have also been described, two in exon 14 (R495X and

G478LfsX23) (15,16) and one in exon 15 (Q519X) (17) which leads to truncated forms of FUS

without the essential domains for nucleic acids binding. Interestingly, the aforementioned

nonsense mutations are characterized by juvenile onset, rapid course (usually <24 months) and predominant bulbar phenotype, with early respiratory involvement and lower motor neuron

disease suggesting that FUS deletion mutants could exacerbate clinical features associated with

ALS (16).

Numerous animal models featuring depleted or reduced FUS have been generated and

analyzed including vertebrate and invertebrate models. However, the major phenotypic features

obtained in these models are complex and often controversial. Several Fus knockout mice have

been described in the literature. Fus-/- mice were reported to die within 16 hours of birth and

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4 affected cells were showing an increase in aneuploidy and chromosomal aberrations (18).

Furthermore, these mice were also analyzed and found to lack DNA pairing and recombination

activity in their reproductive system (19). More recently, another Fus knockout mouse model has

been described (20). Its progeny survives without displaying any ALS-like phenotypes.

Interestingly, the murine model developed by Kino and colleagues displayed certain behavioral

abnormalities, reminiscent of frontotemporal dementia (FTD) (20). Furthermore, high levels of

Taf15 and Ews, homologs of Fus, were reported in this model suggesting a functional compensatory regulation within the same protein family that could explain the absence of early

developmental and neuropathological signs (20). Other studies using mouse models demonstrated that the complete loss of Fus nuclear function does not trigger motor neuron

degeneration (21) but is sufficient to induce alterations at genes expression and splicing (11).

These observations are in concordance with a very recent work conducted on human induced

pluripotent stem cell-derived motor neurons where loss of FUS in nuclei could cause early DNA

damage response (DDR) before cytosolic aggregate formation (22).

In Drosophila, the decreased or loss of expression of Cabeza (Caz), orthologue of human

FUS, during development is necessary and sufficient to induce adult motor performance defects and shortened life span (23,24), but it is not responsible for adult neuronal function maintenance,

where additive contributions from FUS cytosolic aggregate and non-neuronal cell type

pathological changes are probably required (24). Very recently, Caz mediated-toxicity has been

related to the upregulation of the nuclear chromatin-binding protein Xrp1. Indeed, down

regulation of Xrp1 rescues the gene expression dysregulation in Caz-affected neurons (25), thus,

confirming the involvement of FUS in DNA defective repair mechanisms. Similarly, in

zebrafish, knockdown of fus causes motor neuron degeneration and swimming defects (26,27).

On the other hand, deletion mutants generated by CRISPR/Cas9 did not display motor deficits

(28). These conflicting results define the need for further animal models to better understand the

role of FUS in ALS pathogenesis.

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5 Among numerous FUS targets tau mRNA has been described (29). Tau proteins are

microtubule-associated proteins, and their main function relates to the stabilization of

microtubules and thus axonal transport. Tau proteins undergo several post-translational

modifications, among them phosphorylation is the most abundant one (30).

Hyperphosphorylation of tau leads to their aggregation and thus degeneration of the neurons,

giving rise to a number of neurodegenerative disorders called tauopathies (31). It has been shown

that FUS could regulate tau splicing, by skipping exons 3 and 10 (29). Moreover, loss of FUS

and SFQP has been shown to affect exon 10 splicing leading to an increase in 4R/3R ratio and

tau hyperphosphorylation and thus neurodegeneration (32).

Here, we describe for the first time a deletion mutant for the unique fus orthologue in

zebrafish generated by the Wellcome Sanger Institute (33). In this genetic line, we observed in

homozygous animals a reduced life span as well as a severe motor phenotype in both

stimulus-induced locomotion and in spontaneous locomotion tests. These behavioral deficits were

accompanied by anatomical defects, including reduced length of motor neurons and neuromuscular junctions (NMJ) fragmentation. Importantly, the major motor defects were

rescued by overexpression of human FUS mRNA but not by human TARDBP mRNA. Finally,

we measured a general misregulation of Tau transcripts associated with Fus loss of function. In

conclusion, this genetic model of ALS reinforces the knowledge of FUS and its role in

neurodegeneration and could provide essential insight in pathogenic mechanisms that lead to

motor neuron degeneration.

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6 2 Results

2.1 Generation and characterization of a stable fus deletion mutant zebrafish.

In order to study the consequences of fus loss of function in zebrafish, we obtained a deletion

mutant genetic line for fus from the Zebrafish Mutation Project (reference: sa15506, mutation

c.155T>A; p.Y52X, Sanger Institute) (33). The mutation carriers were crossed, and the

homozygous generation was generated. Sanger high-precision sequencing was used to confirm

the presence of the point mutation (Fig 1A). The mutation leads to a translation termination

(stop) codon at the beginning of exon 3, responsible for the prion-like domain generation at the

N-terminus of Fus protein (Fig 1B). To ascertain that this mutation leads to fus KO animals, we used Western blot analyses to determine the levels of Fus in this model. At 48 hours

post-fertilization (hpf), homozygous mutant embryos showed a drastic reduction of Fus protein level

compared to WT and heterozygotes (Fig 1C). Coherently, the decrease in the protein

corresponds to an increase of fus transcript, measured by RT-PCR (Fig 1D). Indeed, a feed-back

FUS autoregulation was proposed in human cells (9,34) and mouse models (35). Briefly, FUS is a repressor of its own exon 7, leading to alternative splicing and NMD (nonsense-mediated

decay). Since this mechanism was unexplored in zebrafish, we checked for levels of fus

transcript with primers for the exon 1 and observed a significant increase of this transcript

confirming the autoregulatory mechanisms in the homozygous fus deletion mutant, fus-/- (Fig

1D).

The morphological aspects and main phenotypic features of WT and fus-/- embryos were assessed

at 2 days post-fertilization (dpf). Compared with WT embryos, fus+/- and fus-/- embryos showed no major morphological deficits with absence of body and head malformations (Fig 1E). To

exclude developmental delays of the locomotor system, we assessed the spontaneous twitching

of the body axis, which comprise the first movements that zebrafish embryos begin to display

around 18 hpf (36). Both homozygous and heterozygous larvae did not show signs of impaired

motility at this stage compared to the wild-type condition (Supplementary Material, Fig S1).

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7 Despite the normal development, the viability in fus-/- embryos is severely reduced (Fig 1F) with

more than 80% of homozygous larvae dying starting from day 11 post-fertilization. Importantly,

no major difference of viability was observed between the WT and fus+/-.

2.2 fus deletion causes zebrafish mobility defects at the touch-evoked escape response.

Zebrafish is an excellent model to study motor function. In fact, by 48 hpf, zebrafish embryos

exhibit stereotyped swimming behavior that can be efficiently quantified under controlled

conditions. To analyze swimming features, we utilized the touch-evoked escape response

(TEER) assay. Similar to previous studies (26,37), we induced embryos locomotor activity at 2

dpf by gently touching the tail with a glass pipette (Fig 2A). In WT (light brown), this results in

a powerful swim away from the stimulus. Also, in heterozygous fish (orange), the stimulated

locomotion is similar to WT. On the contrary, homozygous fus-/- embryos (dark brown) presents

an aberrant locomotion characterized by early fatigue and, occasionally, paralysis. Using a tracking system to monitor swimming behavior, we measured the distance, the time and the

velocity for each animal (Fig 2B). We found that in fus-/-, the locomotor abilities are significantly

reduced, confirming an important role for fus in the proper development of the motor circuitry

(23,38).

Human FUS (NM_004960) and zebrafish fus (NM_201083) share functional similarities at

approximately 60% at the transcript level and 80% at the protein level. Thus, we tested the

ability of human FUS to rescue the motor defects in fus-/-. Injecting the human FUS RNA at one

cell stage embryos resulted in the restoration of embryos motility, increasing swimming distance

and speed (Fig 2A and B, light blue). These results are in concordance with functional rescue

previously observed in KD fus model in zebrafish (26) and Drosophila (38).

2.3 Depletion of the FUS orthologue leads to behavioral impairments.

Zebrafish are diurnal animals, meaning that they are particularly active during light exposition,

where predator, food finding and sexual behaviors are evident (39). It also means that their visual

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8 system is refined (40) and essential for their proper development and survival. For these reasons

light stimuli tests are widely used in zebrafish research to assess their motor performances

independently from vision defects (40,41). Since vision develops rapidly in zebrafish over the

course of 2 days (Supplementary Material, Fig S2A), we determined whether the motor

impairment in homozygous zebrafish exist and persist at 3, 4 and 5 dpf after light stimulation.

Using the ZebraLab platform, we designed a behavior tracking protocol in which single

zebrafish larvae, placed in each well of a 96-well plate for a period of 7 minutes, are

simultaneously stimulated by light after 5 minutes of adaptation in darkness (Fig 3A). With this

test, we have been able to track the larval displacement, which is severely compromised in fus -/-(Fig 3B) and the global activity over time -/-(Fig 3C, D, E). At day 3, homozygous larvae showed

a significant decrease in both spontaneous and induced (startle) motor activity with respect to the

WT and heterozygous fish (Fig 3C). Interestingly, at day 4, the fus+/- presented a reduced startle

response similarly to homozygous larvae (Fig 3D). At day 5, the spontaneous movements of WT

larvae are approximately 15-fold augmented with respect to previous stages (Fig 3E). On the contrary, fus+/- larvae displayed spontaneous locomotion diminution that is comparable to fus

-/-zebrafish larvae. Taken together, these locomotion impairments suggest that fus is necessary for

motor development in zebrafish. Also, the progressive motor impairment developed by

heterozygous zebrafish indicates a cumulative effect of fus partial deletion.

2.4 fus LoF (Loss of Function) causes defects at the zebrafish NMJs.

To better refine the phenotypic features in the homozygous line, we performed an

immunolabeling analysis for NMJ components both at the pre-synaptic and post-synaptic

terminals. We used an anti-SV2 antibody to mark the pre-synaptic vesicles and the

α-bungarotoxin to mark the acetylcholine receptor clusters at the post-synaptic level. By 48 hpf, in

WT embryos the caudal primary motor neuron had extended branches and formed synapses

along the length of individual muscle fibers (Supplementary Material, Fig S3A). To evaluate the correct conformation of the NMJ, we used the Imaris software’s “spot” detection in

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9 conjunction with the colocalization function in 3D reconstructions of confocal imaging

(Supplementary Material, Fig S3B). At 48hpf, pre-synaptic terminals are almost always

precisely opposed to AChR clusters (Fig 4Ai). A similar immunostaining pattern was observed

in fus+/- embryos as compared to WT controls (Fig 4A ii). In contrast, in fus-/- pre and

post-synaptic clustering was impaired (Fig 4Aiii). We measured an increasing number of both αBTX

and SV2 orphan clusters (Fig 4B, C), suggesting the presence of disconnected motor neurons

terminals. Moreover, the αBTX staining showed the appearance of smaller and diffused AChR

clusters (Fig 4Aa,b,c), reminiscent of those seen during the early stage of development, prior to

any motor innervation (42). We also observed shorter axons (Fig 4D) in fus-/- embryos compared with WT. Similar results were observed in a previous zebrafish model developed by knocking

down the fus orthologue confirming the role that FUS can play in the neuromuscular junction

formation (27,28).

Next, we analyzed whether the aberrant phenotype at the motor and NMJs levels was caused by

denervation or collateral reinnervation of previously denervated muscle fibers. Functional AChR

in adult muscle is an heterooligomeric membrane protein composed of α, β, ε and δ subunits in

which the α subunits bear the acetylcholine binding sites (43) (Fig 4E). In mammals and fish, the γ subunit is part of the pentamers during development, before being replaced by ε (44,45). In

ALS patients, where muscle denervation and abnormal reinnervation are pathological hallmarks

of the disease, both subtypes exist (46,47). For these reasons, abnormal levels of subunit α (chrn α 1), γ (chrn γ) and ε (chrn ε) are normally considered as markers of denervation/reinnervation together with muscle histone deacetylase 4 (HDAC4) level which is dramatically altered upon denervation in ALS mice (48) as well as in rapidly progressive patients with ALS (49). In fus-/- at

2dpf we observed an important transcript induction of AChR γ and AChR ε, with a slight but not significant increase of AChR α as compared to age-matched fus

and WT controls (Fig 4E,

bottom). Reversely, hdac4 level is decreased compared to controls. These results confirm the

denervation/reinnervation caused by the absence of a functional fus in the zebrafish model

described here.

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10 2.5 TDP-43 overexpression does not restore fus-/- phenotype.

Previous studies using animal models and primary cortical neurons have described that the gene

TARDBP, that encodes for the RNA-binding protein TDP-43, and FUS are part of a common pathogenic mechanism (26,38). Furthermore, these important factors for ALS pathogenesis,

share common transcriptome profiles (50), suggesting a collaboration of these two genes in

mRNA maturation and transportation (51). FUS share structural and functional features with a

set of RNA-binding proteins, such as TATA box-binding protein (TBP)-associated factor 15

(TAF15) (52).

Considering the known association between these genes and their functional and

structural analogy, we measured tardbp and taf15 expression, in order to rule out possible

compensation or additive effects that could influence phenotypic features in the homozygous

deletion mutants. Transcript levels of tardbp and taf15 did not show any significant difference between WT and mutant conditions (Fig 5A). Moreover, tdp-43 protein levels were not altered

in both fus-/- and fus+/- conditions compared to controls (Fig 5B). Finally, overexpression of

human TARDBP was not able to rescue the phenotypic features of the fus-/- genetic line (Fig 5C).

These results are consistent with previous studies presenting TDP-43 upstream of FUS in a

common pathway (27). Our data suggest no compensatory effect due to TAF15 or TDP-43

expression in the fus-related phenotypes observed in this novel zebrafish model of disease.

2.6 tau isoforms disequilibrium in fus-/- fish. To better explore the cause of the motor deficit seen in fus-/- embryos, we measured the transcript levels of the microtubule-associated protein tau

(MAPT) gene, which is implicated in the outgrowth of neural processes and in the development

of neuronal polarity (53). Interestingly, MAPT has been identified as a physiological splicing

target of FUS (29), with FUS Lof disrupting tau isoform equilibrium and inducing FTLD-like

behavioral impairments and tauopathy in rodents (32). In zebrafish two paralogues of MAPT

have been annotated mapta and maptb (54). At 48 hpf, maptb transcript is significantly increased

in fus-/- embryos (Fig 6A). We also evaluated the transcript levels of a set of genes that play a

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11 central role in neuronal development and synaptic plasticity: the Muscle blind like splicing

regulator 1 (MBNL1) gene (55–57), the fragile X mental retardation 1 (FMR1) gene (58–61), and the Ca2+/calmodulin-dependent protein kinase IIa (CaM-KIIa) gene (62). However, their

levels were found unchanged, confirming the specific implication of tau in the motor deficit seen

in fus-/- embryos. Therefore, we analyzed the complex pattern of alternative splicing of the mapta

and maptb transcripts using the primers previously described by Nik and colleagues (63)

designed to amplify all zebrafish Tau isoforms (Fig 6B). Normally, the expression level of small

and longer tau isoforms are similar at 48 hpf (54). Although the mapta 6R and maptb 4R

expression are not altered (Fig S4 A, B), the increase of the smallest transcripts causes an overall decrease of 6R/4R and 4R/3R ratios of tau transcripts in fus-/- embryos at this embryonic stage

(Fig 6C). Since small tau proteins are predominant in early stages (54), these results indicate

either a delay in development or a recurrence of fetal features in fus-/- embryos. This is consistent

with the increase of the transcript levels of tra2b (Fig 6D), a splicing regulator which putative

binding sites have been detected in exon 8 of mapta and exon 9 of maptb (63). Thus, tra2b may contribute to 3R and 4R isoforms levels by increasing the exon 8 and 9 exclusion, as previously

described for the corresponding human TRA2B and the Tau exon 10 (64). Finally, we performed

western blot analysis to check if mapt increase at 48 hpf was corresponding to an increase of the

tau protein level. As expected, the total amount of tau is higher in fus-/- zebrafish when compared

to WT control (Fig 6E). Our data demonstrate altered tau transcription and tau expression upon

fus inactivation.

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12 3 Discussion

In this paper we report the generation and the phenotypic characterization of a stable mutant

zebrafish line for the unique FUS orthologue, implicated in familial and sporadic forms of ALS. We demonstrated that loss of fus drastically reduces the life span in zebrafish and leads to

locomotor disabilities that persist over time, whether the movement is spontaneous or induced by

touch or light stimulation. The motor impairments were significantly restored upon expression of

the human FUS, but not TARDBP, and we did not observe any misregulation of tardbp and taf15

transcripts, indicating the specificity of the motor phenotype due to fus deletion, as well as the

functional conservation between human FUS and zebrafish fus. To extend this characterization,

we associated these phenotypic features with the observation of aberrant axonal projections from

motor neurons and disorganized NMJ morphology. Furthermore, we describe here a significant

misregulation of several AChR subunits associated with a decrease of Hdac4 highlighting

possible denervation and reinnervation processes in our model. At the molecular level, we

demonstrated transcriptomic and protein expression changes for the genes encoding for different

isoforms of the protein tau, suggesting a link between fus and altered tau function that could play

a role in the development of tauopathies.

These results indicate that the orthologue of FUS in zebrafish is necessary for the proper

development of motor circuits. These results are in contrast with the fus-CRISPR/Cas9 zebrafish

model where no major motor deficits were described (28). It is quite possible that the genetic background could have an important influence on gene expression and the resulting phenotypic

display. Indeed, in the fus mutant generated by CRISPR/Cas9, the authors used an outbred

zebrafish (AB x TU) line to generate the fus knockout mutants (28). In this report, we obtained a

pure, inbred AB zebrafish line in which the fus point mutation was generated and subsequently

maintained. Indeed, genetic compensations have been described in CRISPR-generated mutants

in zebrafish as compared to conditions when genes are knocked down by morpholinos (65).

Furthermore, we can speculate the existence of functional compensatory pathways that could

mask the phenotypic features due to reduced levels of Fus, as previously seen in a Fus knockout

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13 mouse model where increased levels of TAF15 and EWS were observed (20). FUS, TAF15 and

EWS belong to the FET family of RNA-binding proteins, with ubiquitous nuclear expression and

implicated in regulation of gene expression, maintenance of genomic integrity and

mRNA/microRNA processing (66–68). Interestingly, in frontotemporal dementia (FTD), all

types of neuronal and glial cytoplasmic and intranuclear FUS-positive inclusions are also labeled

for TAF15 and EWS. In contrast to ALS-FUS-positive inclusions, where no abnormal

immunostaining has been demonstrated for TAF15 or EWS (52). In our model, the expression of

taf15 transcript was not altered, underlining the specificity of the fus homozygous phenotype.

However, it is still plausible that upregulation of other transcripts and proteins could be necessary to compensate for the loss of fus. Transcriptomic and proteomic studies could unravel

important alterations in this novel Fus zebrafish model.

Furthermore, we describe that the locomotor impairments can be rescued by human FUS,

revealing a remarkable conservation of protein function during evolution. In contrast, excess

production of human TARDBP did not have the ability to rescue the homozygous phenotypes,

and fus depletion did not induce an increase of Tdp-43 expression. These data are consistent with

a genetic relationship in which fus is downstream of tardbp as previously established in zebrafish (26) and drosophila (38). fus protein is highly and predominantly expressed in the CNS of

zebrafish from developmental stages to adulthood (26) and its depletion induces locomotive

deficits as well as reduction of life span, both features of ALS pathology. Thus, our results

demonstrate that fus activity is necessary to regulate motor function and could reduce viability in

vivo. These results suggest that the disease onset in ALS patients carrying FUS mutations could be due to FUS loss of function complemented by formation of insoluble aggregates at later

stages of disease. These data are in line with the increasing evidence of an early involvement of

FUS in ALS development, where the progressive and additive defects at the mRNA and DNA

processing could play an important role in the onset and severity of the disease (11,19,25). This

hypothesis is supported by the decreasing of “spontaneous” locomotor behavior that was

observed in the fus heterozygous animals. However, some motor deficits are not maintained over

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14 time, as we observed for the “startle response”, probably because the partial depletion of FUS

induce a combination of minor defects that are covered or corrected by compensatory

mechanisms.

Zebrafish is particularly well suited for a detailed analysis of the NMJ due to its transparency, its

well-defined spinal network architecture and similarity to higher vertebrates. As previously

described (27), by double-labeling of a pre-synaptic marker and post-synaptic acetylcholine

receptors, we observed an increased number of misaligned NMJ structures suggesting the

presence of denervated endplates. However, based on the transcriptional results presented here

both denervation and reinnervation phenomena could coexist in the fus-/- model. In fact, both chrn γ and chrn ε are elevated whereas the expression of the hdac4 transcript was decreased.

Hdac4 is upregulated in aged SOD1G93A and SMARD1 mice, both examples of neuromuscular diseases as well as in WT denervated mice. On the other hand, HDAC levels are not significantly

elevated in myogenic muscular dystrophies (48). These results suggest that Hdac4 is specifically

induced in response to neuronal degeneration as opposed to specific muscle dysfunction (48).

Hdac4 is responsible for the coordinated induction of synaptic genes upon denervation, and its

upregulation is associated with lower muscle reinnervation ability, particularly visible in patients with rapidly progressive ALS (49,69). In the fus-/- zebrafish embryos, the expression of the hdac4

transcript is significantly reduced as compared to controls suggesting a reinnervation effect on

muscles, as previously seen in knockout mice for Hdac4, where mutant muscles were

reinnervated more rapidly than those from the controls after nerve injury (70,71). Therefore, fus/-

zebrafish may recapitulate certain hallmarks of ALS degeneration observed in human muscle biopsies (49,69) and thus, it could represent an optimal model where very early NMJ

dysfunctions can be detected and studied. FUS has been recently shown to be important for

muscle development in iPSCs derived from patients and animal models (72). Indeed, hFUS

overexpression in the fus-/- leads to significantly increased velocity parameters indicating

improved muscular force in this novel zebrafish model.

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15 The identification of expression changes of mapt genes in this model is particularly interesting as

it follows multiple evidence linking ALS and FTD. Indeed, it is estimated that up to 50% of ALS

patients present signs of altered behavior and cognitive impairment with approximately 15% of

them reaching the diagnostic criteria of FTD (73,74). The same has been reported for FTD

patients with up to 15% of them having ALS motor dysfunctions (75,76). Interestingly, it was

demonstrated that in FTD, FUS was involved in tau splicing by skipping exons 3 and 10 (29),

and that loss of FUS and its partner SFPQ in mice could cause an increase in 4R-tau expression

and tau hyper phosphorylation leading to neurodegeneration and FTD-like behaviors (32).

Although duplicated, the zebrafish MAPT genes have a complex alternative splicing regulation

which leads to higher concentration of small tau isoforms during early developmental stages to

maintain microtubule dynamics as observed in mammalians (77,78). The absence of fus in our model leads to a change in the ratio of expression of tau isoforms, with the prevalence of smaller

forms at 48 hpf when compared to the WT controls. This result could be explained by a delay in

development in the homozygous zebrafish or by an attempt to increase neuronal plasticity. Since

no morphological differences were evident among the different genetic conditions, we excluded

the first option in favor of the second one. In fact, the increase of small tau isoforms could represent a return to early developmental stages, where microtubules are dynamic to favor the

possible phenomena of reinnervation in the fus deletion mutant. These results coupled with the

altered expression of hdac4 and the AChRs suggest an altered denervation/reinnervation pattern

in this model that needs to be further studied.

This novel zebrafish deletion mutant could represent an excellent model to study FUS, tau and

their implication in neurodegeneration. In this context, the transcriptomic and proteomic

sequencing will be particularly important. Also, in the context of ALS-FTD molecular

mechanistic insight, it would be interesting to examine motor and cognitive features of fus

heterozygous and homozygous deletion mutant animals, as well as the fus-/- survival, at later

stages during juvenile and adult age. In particular, this model may provide a pre-clinical tool to

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16 test genetic and chemical modifiers that could rescue the phenotype due to FUS inactivation.

Overall, our results demonstrate that the fus-/- zebrafish model recapitulates some features of ALS

pathology and demonstrate the importance of fus loss of function in the development of this

disease. Therefore, it can provide additional information that could be translated rapidly into the

human ALS physiopathology in order to generate efficient therapeutic strategies for ALS and

related neurodegenerative disorders.

4 Materials and Methods

4.1 Animal Care: Adult and larval zebrafish (Danio rerio) were maintained at the ICM (Institut du Cerveau et de la Moelle épinière, Paris) zebrafish facility and bred according to the National

and European Guidelines for AnimalWelfare. All procedures were approved by the Institutional

Ethics Committees at the Research Centers of ICM and Imagine.

4.2 Screening of mutant lines: The fus mutant zebrafish line was obtained from the European Zebrafish Resource Center (EZRC) (reference: sa15506) and originates from the mutagenesis

screen performed at the Wellcome Trust Sanger Institute (Stemple Laboratory). The mutation (c.155T>A; p.Y52X) in the fus genetic line is a nonsense point mutation at the exon 3 of fus.

Heterozygous parents and homozygous offspring were screened through high-precision

sequencing (GATC Biotech, Eurofins Genomics). DNA extraction was performed by cutting a

part of the fin from adult zebrafish and used for genotyping. Prior to fin-clipping, the adult

zebrafish were anesthetized in tricaine (160 µg/mL). A part of their caudal fin was cut and put in

75 µL of extraction solution (25mM NaOH, 0,2 mM EDTA). The samples were then incubated

at 98°C for 1 hour and 75 µL of a Tris-HCl (40 mM, pH 5.5) solution was added to the tubes

before centrifugation at 4000 rpm for 3 minutes. 1 µL of supernatant was used for PCR using

Taq polymerase Master Mix (Thermo Scientific K1071) and the following primers:

CTGCCCAGAACTACAGTCAG (forward primer) and GCCACCAGAGCTATACGACT

(reverse primer). The PCR products were then sent to GATC Biotech for sequencing.

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17 4.3 Survival assay: Embryos were maintained according to the procedures of the ICM (Institut du Cerveau et de la Moelle épinière, Paris) and IMAGINE fish facility. Larvae were checked

daily and maintained in petri dish in a 28.5°C incubator for the first 5 days post-fertilization

(dpf). After 5 dpf, the fish were transferred in tanks with continuous water flux. They were then

fed regularly and the number of dead larvae was scored each day for 20 days by a blinded

observer.

4.4 Western Blot: 15 to 20 fish were deyolked and resuspended in ice-cold extraction buffer: 50 mM Tris-HCl, pH 7.4/ 500 mM NaCl/ 5 mM EDTA/ 1% (w/v) Nonidet P-40/ 0.5% (w/v) Triton

X-100 supplemented with a cocktail of protease inhibitors. Fish were then sonicated and

centrifuged at 14000×g at 4 ºC for 20 minutes. Samples were denatured at 98°C for 7 minutes.

30 ug of proteins from zebrafish lysates (equal amount of protein in each lane) were separated by sodium dodecylepolyacrylamide gel electrophoresis. The separated proteins were transferred to

nitrocellulose membranes (0.45µm, Life Sciences) and probed with the following primary

antibodies: c-terminal FUS antibody (sc-47711, Santacruz), C-terminal TDP-43 antibody

(12892-1-AP, Proteintech) and the C-terminal tau antibody (antibody raised against amino-acids

426-441 (79). An α-tubulin antibody (T5168, Sigma-Aldrich) was used as a loading control. The blots were incubated with the corresponding fluorescent secondary antibody and the signal was

detected using the ODYSSEY® CLx. The intensity of bands in each of the lanes from the

Western blots were measured by ImageJ.

4.5 RNA isolation and analysis of transcripts by q-PCR: Total RNA was isolated from fish using TRIzol Reagent (Sigma) according to the manufacturer’s protocol. First-strand cDNAs

were obtained by reverse transcription of 1 μg of total RNA using the High Capacity cDNA

Reverse Transcription Kit (Roche), according to the manufacturer’s instructions (primers are

listed in Supplementary Material, Table S1). Quantitative PCR amplification was performed

with 2X SYBR Green Master mix (Bimake). Data were analyzed transforming raw Cq values

into relative quantification data using the delta Cq method.

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18 4.6 Locomotion assessment: Touch-evoked escape response (TEER): 2 days post-fertilization (dpf) zebrafish embryos were tested for behavior under a stereomicroscope (Zeiss, Germany) by

inducing a light touch-stimulus at the level of the tail with a tip and were scored for the distance

travelled, the time spent swimming and speed in a 15-cm diameter petri dish. Their responses

were recorded with a Grasshopper digital camera (Point Grey) at a rate of 30 frame/s and

quantified using ImageJ manual tracking plug-in. Spontaneous locomotion: 3 dpf-zebrafish

larvae were maintained in a 96-well plate and assayed for total locomotor activity in ZebraBox

(Viewpoint Life Sciences, Lyon, France). The embryos were tested for 10 min in a 5 min dark, 2

min light (100%), 3 min dark paradigm, repeated 3 times. The experiment was done on the same fish at 4 and 5 dpf. Total swim activity was analysed using ZebraLab V3 software (Viewpoint

Life Sciences, Lyon, France).

4.7 Immunohistochemistry: Animals were fixed in 4% paraformaldehyde for 3 hours at RT. After fixation the larvae were rinsed several times with PBS and then incubated in PBS

containing 1 mg/ml collagenase (20 min). The collagenase was washed off with PBS Triton

X-100 (PBST; 1 h) and heads were cut away. After an incubation of 30 min in blocking solution

(1% BSA, 1% triton, PBS, 2% Goat serum), the embryos were incubated overnight at 4°C in synaptic vesicle 2 (SV2, 1:200; DSHB) antibody diluted in blocking solution. The embryos were then washed and incubated for 30 min in PBST containing α-bungarotoxin conjugated to Alexa

488 (αBTX, 1:1000; Abcam). The larvae were then rinsed several times with PBST and then

incubated in freshly prepared block solution containing a secondary antibody (Alexa Fluor 568,

1:1000; Life Technologies) for 3h at RT before mounting on glass slide in 50% glycerol. The NMJs were visualized using a Spinning disk system (Intelligent Imaging Innovations, USA), an

Examiner.Z1 upright stand (Carl Zeiss, Germany), a CSU-W1 head (Yokogawa, Japan), and an

ORCA-Flash 4.0 camera (Hamamatsu, Japan). All images were captured at 40x and stained

embryos were processed using Imaris Image Analysis software and ImageJ.

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19 4.8 TDP-43 and FUS overexpression studies: Human WT TARDBP (WT TDP-43) and FUS mRNAs were transcribed from NotI-linearized pCS2 using SP6 polymerase with the

mMESSAGE Machine Kit (Ambion). The RNA quality and concentration were assessed using

the Bioanalyzer (Agilent). Injections of 100 ng of RNA were then performed in 1–4 cell stage blastulae. Embryos were maintained at 28°C and manually dechorionated using fine forceps at

24 hpf. After behavioral test, 15 to 20 fish were deyolked and resuspended in ice-cold extraction

buffer (see section 4.4 Western Blot).

4.9 Statistical analysis: All data values for the zebrafish experiments are represented as average standard error of mean (SEM) with significance determined using one-way ANOVAs (values are

listed in Supplementary Material, Table S2). Differences between groups were identified via

post hoc comparisons, specified in the legend of each Fig. All analyses were performed using

Prism 5.0 (Graph Pad, CA). Significance level was set at p<0.05.

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