Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila

Neurobiology of Disease 74 (2015) 76–88

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Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila Sandra Jäckel a,b,⁎, Anna K. Summerer b, Catherine M. Thömmes b, Xia Pan c, Aaron Voigt c, Jörg B. Schulz c, Tobias M. Rasse d, Dorothee Dormann e,1, Christian Haass e,f, Philipp J. Kahle a,b,⁎ a

German Center for Neurodegenerative Diseases (DZNE) Tübingen, Germany Hertie Institute for Clinical Brain Research, Laboratory of Functional Neurogenetics, Tübingen, Germany Department of Neurology, University Medical Centre, RWTH Aachen, Aachen, Germany d Hertie Institute for Clinical Brain Research, Synaptic Plasticity Group, Tübingen Germany e Adolf Butenandt Institute, Metabolic Biochemistry, Ludwig Maximilians University, Munich, Germany f German Center for Neurodegenerative Diseases (DZNE) Munich, Germany b c

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Article history: Received 17 June 2014 Revised 14 October 2014 Accepted 3 November 2014 Available online 8 November 2014 Keywords: FUS Caz Transportin PRMT ALS FTLD Drosophila

a b s t r a c t Inclusions containing Fused in Sarcoma (FUS) are found in familial and sporadic cases of the incurable progressive motor neuron disease amyotrophic lateral sclerosis and in a common form of dementia, frontotemporal dementia. Most disease-associated mutations are located in the C-terminal proline–tyrosine nuclear localization sequence (PY-NLS) of FUS and impair its nuclear import. It has been shown in cell culture that the nuclear import of FUS is mediated by transportin, which binds the PY-NLS and the last arginine/glycine/glycine-rich (RGG) domain of FUS. Methylation of this last RGG domain by protein arginine methyltransferases (PRMTs) weakens transportin binding and therefore impairs nuclear translocation of FUS. To investigate the requirements for the nuclear import of FUS in an in vivo model, we generated different transgenic Drosophila lines expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. To rule out effects caused by heterologous hFUS expression, we analysed the corresponding variants for the Drosophila FUS orthologue Cabeza (Caz wt, P398L, Q349X). Expression of these variants in eyes and motor neurons confirmed the PY-NLS-dependent nuclear localization of FUS/Caz and caused neurodegenerative effects. Surprisingly, FUS/Caz toxicity was correlated to the degree of its nuclear localization in this overexpression model. High levels of nuclear FUS/Caz became insoluble and reduced the endogenous Caz levels, confirming FUS autoregulation in Drosophila. RNAi-mediated knockdown of the two transportin orthologues interfered with the nuclear import of FUS/Caz and also enhanced the eye phenotype. Finally, we screened the Drosophila PRMT proteins (DART1-9) and found that knockdown of Dart1 led to a reduction in methylation of hFUS P525L and aggravated its phenotype. These findings show that the molecular mechanisms controlling the nuclear import of FUS/Caz and FUS autoregulation are conserved between humans and Drosophila. In addition to the well-known neurodegenerative effects of FUS loss-of function, our data suggest toxic potential of overexpressed FUS in the nucleus and of insoluble FUS. © 2014 Elsevier Inc. All rights reserved.

Introduction

Abbreviations: FUS, Fused in Sarcoma; ALS, amyotrophic lateral sclerosis; FTLD, frontotemporal lobar degeneration; VNC, ventral nerve cord; NLS, nuclear localization signal; Caz, Cabeza; PRMT, protein arginine methyltransferase; DART, Drosophila arginine methyltransferase. ⁎ Corresponding authors at: German Center for Neurodegenerative Diseases (DZNE) and Hertie Institute for Clinical Brain Research, Laboratory of Functional Neurogenetics, University of Tübingen, Otfried Müller Str. 27, 72076 Tübingen, Germany. Fax: + 49 7071 29 4620. E-mail addresses: [email protected] (S. Jäckel), [email protected] (P.J. Kahle). Available online on ScienceDirect (www.sciencedirect.com). 1 Present address: Institute of Cell Biology, Ludwig Maximilians University, Schillerstr. 42, 80336 Munich, Germany.

http://dx.doi.org/10.1016/j.nbd.2014.11.003 0969-9961/© 2014 Elsevier Inc. All rights reserved.

The occurrence of ubiquitin-positive, cytoplasmic and nuclear inclusions of Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) in neurons and glia is the characteristic feature in subtypes of the two neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), termed ALS-FUS and FTLDFUS, respectively (Hewitt et al., 2010; Kwiatkowski et al., 2009; Munoz et al., 2009; Neumann et al., 2009; Vance et al., 2009; Woulfe et al., 2010). ALS causes the progressive loss of upper and lower motor neurons which leads to muscle weakening, usually resulting in the death of the patients within 1–5 years after disease onset, mostly due to respiratory paralysis (Boillée et al., 2006). FTLD is a common form of dementia with selective loss of neurons in the frontal and temporal

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lobes, which leads to changes in social behaviour and personality, semantic dementia and progressive non-fluent aphasia (Mackenzie et al., 2010). Because several genes like C9ORF72, TARDBP and FUS are linked to both diseases and some patients with ALS show symptoms of FTD and vice versa (Ringholz et al., 2005), these two diseases seem to share similar pathological mechanisms. FUS is a ubiquitously expressed DNA/RNA binding protein of 526 amino acids and belongs to the FET (FUS, EWS, TAF15) family of proteins together with Ewing sarcoma (EWS) protein and the TATAbinding protein-associated factor TAF15 (Bertolotti et al., 1996; Law et al., 2006). The N-terminal half of FUS contains a glutamine–glycine– serine–tyrosine rich region (QGSY), which functions as a potent transcriptional activation domain (Kwon et al., 2013; Law et al., 2006; Prasad et al., 1994; Schwartz et al., 2012; Tan et al., 2012). Additionally, FUS harbours a glycine-rich region involved in protein–protein interactions and three different RNA-binding regions: an RNA recognition motif (RRM), a zinc finger (ZnF) and three arginine/glycine/glycine (RGG)-rich regions (Fig. 1A) (Aman et al., 1996; Iko et al., 2004). Although FUS is found predominantly in the nucleus, FUS contains sequences for nuclear import and export, which enables it to shuttle between the nucleus and the cytoplasm (Zinszner et al., 1997). Accordingly, FUS is involved in processes taking place in both compartments like transcriptional activation (Kwon et al., 2013; Law et al., 2006; Prasad et al., 1994; Schwartz et al., 2012; Tan et al., 2012), DNA repair (Kuroda et al., 2000; Mastrocola et al., 2013), RNA splicing (Lagier-Tourenne et al., 2012; Meissner et al., 2003; Nakaya et al., 2013; Orozco et al., 2012; Rogelj et al., 2012; Yang et al., 1998), miRNA processing (Morlando et al., 2012), RNA transport and stability (Fujii and Takumi, 2005; Zinszner et al., 1997) and translational regulation (Andersson et al., 2008; Liu-Yesucevitz et al., 2011; Yasuda et al., 2013). Mutations in FUS, which are mostly dominant, are often the cause of familial ALS-FUS, but are only rarely observed in familial FTLD-FUS (Broustal et al., 2010; Kwiatkowski et al., 2009; Vance et al., 2009). The mutations cluster in the glycine-rich region and in the C-terminal nuclear localization signal, which is a non-classical R/H/KX2–5PY-NLS (Lee et al., 2006). This PY-NLS is required for the binding of FUS to its nuclear import receptor transportin (karyopherin ß2) (Dormann et al., 2010; Ito et al., 2011; Kino et al., 2011; Niu et al., 2012). In cell culture hFUS mutants with defective PY-NLS are found in the cytoplasm (Bosco et al., 2010; Dormann et al., 2010; Ito et al., 2011; Kino et al., 2011; Kwiatkowski et al., 2009; Vance et al., 2013). Moreover, the last RGG domain of FUS participates in transportin binding and methylation of this domain through protein arginine methyltransferases (PRMTs) weakens the interaction. Furthermore, it was shown that lowering the RGG methylation status through chemical or genetic inhibition of PRMTs partially restores the nuclear import of FUS mutants in cell culture (Dormann et al., 2012; Scaramuzzino et al., 2013; Tradewell et al., 2012). These new findings led to studies showing for the first time that ALS-FUS and FTLD-FUS may have different pathogenic mechanisms. It turned out that the FUS-positive cytoplasmic inclusions in ALS patients contain only the methylated FUS protein, whereas the inclusion in FTLD-patients contain hypomethylated FUS and in addition, the two other family members, EWS and TAF-15 as well as transportin (Brelstaff et al., 2011; Davidson et al., 2012; Dormann et al., 2012; Neumann et al., 2011; Neumann et al., 2012; Troakes et al., 2013). Thus, ALS-FUS seems to involve specific FUS nuclear import defects while FTLD-FUS could be due to a broader nuclear import defect of the FET family proteins. Drosophila has only one FET homolog, Cabeza (Caz)/SARFH (Sarcoma-associated RNA-binding fly homolog) (Immanuel et al., 1995; Stolow and Haynes, 1995) with 44,9% identity and 62,3% similarity to the human FUS protein. Loss of the endogenous Caz protein leads to neurodegenerative defects in eyes, reduced life span, defects at the neuromuscular junction and reduced locomotion (Azuma et al., 2014; Machamer et al., 2014; Sasayama et al., 2012; Shahidullah et al., 2013; Wang et al., 2011). These phenotypes can be rescued by the expression

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of human FUS wildtype (wt) (Wang et al., 2011), showing functional conservation between FUS and Caz at least regarding the assessed phenotypes. Interestingly, the overexpression of FUS wt or ALS-associated FUS mutants in Drosophila resembles the loss of Caz phenotypes pointing to a loss-of-function and a toxic gain-of-function mechanism (Chen et al., 2011; Daigle et al., 2013; Lanson et al., 2011; Wang et al., 2011; Xia et al., 2012). However, overexpression data need to be carefully interpreted, because overexpression of hFUS wt at a level of ~ 2fold compared to endogenous Caz levels did not cause any phenotypes (Wang et al., 2011), whereas stronger overexpression induced the above mentioned phenotypes (Chen et al., 2011; Daigle et al., 2013; Lanson et al., 2011; Wang et al., 2011; Xia et al., 2012). Although many studies showed that the cytoplasmic mislocalization of hFUS after disruption of the PY-NLS is conserved in Drosophila, it is unknown if transportin and PRMTs play a role in the nuclear import of hFUS in Drosophila. In this study we elucidated the factors involved in nuclear transport of hFUS in Drosophila. We generated flies expressing human FUS wt and the two mutant variants hFUS P525L and hFUS R495X, which show cytoplasmic mislocalization and are associated with aggressive forms of ALS-FUS causing juvenile disease onset with rapid progression (Bosco et al., 2010; Chiò et al., 2009; Kwiatkowski et al., 2009; Waibel et al., 2013). We show that the nuclear localization of hFUS in vivo is dependent on the integrity of its PY-NLS. The expression of hFUS wt and FUS P525L in eyes or motor neurons leads to strong eye phenotypes and impairs locomotion, respectively. In contrast, the more cytoplasmic hFUS R495X variant induced almost no phenotypes. We observed that predominantly nuclear localized FUS variants tended to become insoluble in strong transgenic lines and under such conditions reduced the endogenous Caz level, which could reflect an autoregulatory mechanism (Machamer et al., 2014; Zhou et al., 2013). Consistently, the hFUS R495X variant with the strongest cytoplasmic distribution, did not deplete endogenous Caz. Knockdown of Drosophila transportin caused some cytoplasmic retention of hFUS wt and enhanced eye phenotypes. Furthermore, we found that hFUS is being methylated in Drosophila and this methylation can be inhibited by reduction of the Drosophila PRMT1 homolog, DART1. Knockdown of Dart1 enhanced the eye phenotype of the PY-NLS impaired hFUS P525L. The mechanisms of FUS neurotoxicity are complex and involve insolubility, autoregulation and nuclear import. Material and methods Drosophila stocks All Drosophila melanogaster stocks were maintained on standard cornmeal-yeast agar based fly food. Experiments were performed at 25 °C unless otherwise noted. UAS-RNAi-lines used were UAS-dTNPORNAi (VDRC #6543), UAS-CG8219-RNAi (VDRC #30066), UAS-vasaRNAi (VDRC #46464), UAS-Dart1-RNAi #1 (VDRC #40388), UASDart1-RNAi #2 (VDRC #110391), UAS-Dart1-RNAi #3 (Bloomington #31348). GAL4 and other lines used were OK371-GAL4 (Bloomington #26160), GMR-GAL4 (Bloomington #1104), actin-GAL4 (BL #4414), hs-GAL4, tubGAL80ts and UAS-nlsGFP (Bloomington #4776). Generation of constructs and vectors For generation of transgenic flies, HA-FUS and HA-Caz variants were amplified using specific primers introducing 5′-KpnI and 3′-XbaI restriction sites and the specific mutations. FUS constructs were cloned from a pcDNA5-FRT-vector containing human FUS wt cDNA (kind gift from Manuela Neumann). Caz constructs were generated from cDNA prepared from control white larvae (see Fiesel et al., 2010). Subsequent digestion with respective enzymes allowed subcloning into the pUAST or pUASTattB vector (using the same restriction sites). The randominserted (w1118) and site-directed (insertion in 76A2, Bloomington

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stock #9732) transgenic lines were generated by germline transmission (BestGene). Immunofluorescence Brains or eye imaginal discs were fixed for 30 min with 4% PFA/PBS, permeabilized with 3 × 10 min 0.3% Triton-X-100/PBS (PBT) and blocked for 30 min with 5% normal goat serum (NGS)/PBT. Primary antibody incubation was performed in 5% NGS/PBT over night at 4 °C or in case of the Caz antibody for 4 h at room temperature. Tissues were incubated with the secondary antibodies for 1 h at RT in the dark. Antibodies used were anti-HA (rat, clone 3F10, Roche Cat. No. 11867423001, dilution 1:250), anti-HA (rabbit, Sigma H-6908, dilution 1:250), anti-Caz (equal mixture of clone 1G5 and 5G2, dilution 1:25, Immanuel et al., 1995, provided by E. Storkebaum) and anti-meFUS (9G6, dilution 1:10, Dormann et al., 2012). Secondary AlexaFluor-488 or − 568 conjugated antibodies from Invitrogen were used at a 1:500 dilution. Nuclei were stained with Hoechst 33258 dye (1 μg/ml). Whole brains or eye imaginal discs were mounted in fluorescent mounting medium (Dako) onto microscope slides. Images were obtained with a Zeiss AxioImager Z1 microscope equipped with a Zeiss ApoTome and processed with AxioVision software (Zeiss). Adult eye microscopy After freezing for 1 h at −20 °C adult flies were mounted onto black microscope slides. Using a Zeiss AxioImager Z1 microscope and the AxioVision software z-stacks of 5 μm slices were obtained. The planes were then stacked using Helicon focus software. Western blots For soluble protein extraction, adult heads were homogenized in Drosophila lysis buffer (10% glycerol, 50 mM HEPES pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA) containing 1× complete protease inhibitors with EDTA (Roche) using a hand pestle. After 15 min on ice, cell debris was pelleted at 14,000 ×g for 15 min at 4 °C and supernatants were collected. For total protein extraction, third instar larvae were directly homogenized in 3× Laemmli buffer. Denatured proteins were separated on 7.5 –10% polyacrylamide gels and transferred onto nitrocellulose membranes (Amersham Hybond ECL) or onto Hybond-P polyvinylidene fluoride (PVDF) membranes (Millipore) for detection of meFUS. After blocking in 5% skim-milk/ TBST membranes were incubated with primary antibody in Western Blocking Reagent (Roche) or in 5% skim-milk/TBST for horseradish peroxidase (HRP)-conjugated primary antibodies at 4 °C overnight. Detection of proteins was performed using the Immobilon Western Chemiluminescent HRP substrate (Millipore) on Amersham HyperfilmTM ECL (GE Healthcare). Primary antibodies used were antiHA-HRP (rat, clone 3F10, Roche Cat. No. 12013819001, dilution 1:10,000), anti-FUS (mouse, clone 4H11, Santa Cruz, sc-47711, dilution 1:500), anti-meFUS (9G6, Dormann et al., 2012, dilution 1:50), anti-Caz (clone 1G5, dilution 1:100, Immanuel et al., 1995, provided by E. Storkebaum), anti-α-tubulin (mouse, Sigma T-5168, dilution 1:100,000) and anti-syntaxin (mouse, clone 8C3, DSHB, dilution 1:2,500). Secondary HRP-conjugated antibodies from Jackson ImmunoResearch Laboratories were used at a 1:10,000 dilution. Protein solubility 20 fly heads were homogenized in 100 μl RIPA buffer (50 mM Tris pH 8.0, 0.15 M NaCl, 0.1% (v/v) SDS, 1% Triton, 0.5% sodium deoxycholate, 1 mM EDTA) with 1× complete protease inhibitors without EDTA (Roche). After centrifugation at 13,000 ×g for 20 min at 4 °C the supernatants were collected. This step was repeated two times to isolate the RIPA-soluble fraction, which was subsequently analysed via

Western blot. For the slot blot assay of the remaining RIPA-insoluble fraction, the pellet was washed five times with RIPA buffer and then dissolved in 100 μl UREA buffer (30 mM Tris pH 8.5, 7 M UREA, 2 M ThioUREA, 4% CHAPS). After centrifugation 20 μg of the supernatant containing the urea-soluble fraction was mixed with 6× DOT buffer (0.5 M Tris, 0.4% SDS pH 6.8, 30% Glycerol, 0.4 M SDS, 0.3 M DTT). Using a slot blot filtration unit, lysates were loaded on a nitrocellulose membrane and the membrane was washed three times with TBST. Afterwards blocking and detection with anti-HA were performed as described in the Western blots section. RNA isolation and RT-PCR RNA extraction of 20–25 third instar larvae was performed using the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. For sufficient homogenisation, whole larvae were ground in a 1.5 ml microcentrifuge tube using a hand pestle in 200 μl RLT buffer. After addition of 200 μl of RLT buffer the lysate was passed ten times through a blunt 20-gauge needle fitted to an RNase-free syringe. 600 ng total RNA was reverse transcribed with anchored oligo-dT primer using the Transcriptor High Fidelity cDNA Synthesis kit (Roche). cDNA was used as template for transcription amplification in a 25 μl reaction with 5 μl 5× GoTaq buffer, 0.1 μl GoTaq Polymerase (Promega) and 2 mM primer (sequences available on request). Amplified PCR products were subjected to electrophoresis using a 2% agarose gel stained with ethidium bromide. Behavioural assays For all larval locomotion assay, 4 day old larvae were collected and washed in PBS to remove any residual food before they were placed on apple juice agar plates. For the larval righting assay, larvae were turned ventral side up and the time taken to return back was measured. This assay was performed three times for each larva and terminated at 120 s. For the larval crawling assay, the distance the larvae crawled in 30 s was measured. For adult climbing assays, adult male flies were collected in groups of 10 flies on the day of eclosion (day 0) and tested on days 1 and 2. Flies were transferred into an empty fresh vial and given 10 s to climb 7 cm. The number of flies that crossed the 7 cm line was counted and the procedure was repeated three times for each group. In all assays significance was determined by one-way ANOVA analysis followed by Dunnett's multiple comparison test. Results Overexpression of hFUS wt or ALS-associated hFUS mutants in Drosophila eyes leads to age-dependent neurodegeneration In human and murine cells mutations in the PY-NLS of hFUS lead to its cytoplasmic mislocalization, which is thought to be directly correlated to disease strength in patients (Dormann et al., 2010). To analyse hFUS mutations affecting the PY-NLS in Drosophila, we used random P-element transformation to establish fly lines expressing N-terminal HA-tagged hFUS wt, P525L and R495X (Fig. 1A). In addition, we examined the corresponding HA-tagged Caz variants (Caz wt, P398L and Q349X) (Fig. 1A). To characterize the randomly inserted hFUS variants, we first expressed them in the eye using a GMR-GAL4 driver. For each hFUS variant a phenotypically weak, intermediate and strong line was selected by their relative degenerative eye phenotype (see Fig. 1B) for further analysis. Expression of hFUS wt or P525L resulted in a rough eye phenotype with pigment loss (Fig. 1B, left side, upper/middle panels). In contrast, the R495X variant caused only a mild ommatidial phenotype with very slight pigmentation defects (Fig. 1B, left side, lower panels). Please note that the relatively “strong” phenotype classification of hFUS R495X line #52 corresponds to very mild eye degeneration compared to hFUS

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Fig. 1. Overexpression of hFUS wt or ALS-associated hFUS mutants in Drosophila eyes leads to age-dependent neurodegeneration. A) Schematic representation of hFUS and Caz wt variants. The protein domain structures of human FUS wt and Drosophila Caz are presented including the positions of the introduced mutations (bold). ALS-associated mutations in the PY-NLS of hFUS are shown in the enlargement. For a summary of other ALS-related mutations in hFUS (see Mackenzie et al., 2010). B) Eye phenotypes of different hFUS/Caz variants. Randomly inserted hFUS wt, P525L and R495X (three different insertion lines for each variant, line numbers are indicated) and the corresponding Caz wt, P398L and Q349X variants were expressed in the eye using GMR-GAL4. For Caz also the eyes of the site-directed insertion lines – for equal expression levels of the different variants – are shown (right panels). Pictures were taken 2 days after eclosion. Arrows and arrowheads point to dents or black lesions in the eye, respectively. Scale bar = 100 μm. C) Comparison of hFUS variant expression levels. hFUS variants were expressed with hs-GAL4 and total protein lysates of third instar larvae were subjected to WB analysis with anti-FUS. α-tubulin serves as a loading control. Lines used: hFUS wt (#59), P525L (#56), R495X (#52). control = hsGAL4/+. D) Subcellular localization of hFUS variants in eye imaginal discs. Eye imaginal discs of wandering third instar larvae were stained with anti-HA. Nuclei (DNA) were counterstained with Hoechst 33258. Insets show magnifications of representative areas. Scale bar = 20 μm.

wt and P525L lines. The mild phenotype of hFUS R495X was unexpected because this C-terminal deletion mutation, which lacks the NLS, is associated with severe phenotypes in humans. To assess whether our observed phenotypes are artificially resulting from heterologous hFUS expression, we analysed the randomly inserted Caz variants (Caz wt, P398L and Q349X). As for hFUS, expression of Caz wt protein caused a more dramatic phenotype compared to the C-terminal deletion mutant, Caz Q349X (Fig. 1B). Of note, the eye phenotype of Caz wt expressing flies was more severe than that of hFUS wt expressing flies: dents (arrow) or black lesions (arrowhead) were never observed in hFUS wt flies. To demonstrate that the different eye phenotypes were not caused by random insertion effects of the hFUS and Caz UAS-cDNAs in the Drosophila genome, we inserted all Caz variants at the same genomic site via the attB/attP-system. Again the expression of Caz wt caused the strongest phenotype, whereas Caz Q349X had almost no phenotype (Fig. 1B, right panels). Expression levels of transgenic FUS proteins were examined by Western blot analysis. To first assess general protein expression and steady-state stability, we took advantage of the ubiquitous weak (non-toxic level) leaky expression from the hs-GAL4 driver without heat shock. Total protein extracts from third instar larvae demonstrated expression of all hFUS transgenic variants (Fig. 1C). Thus, the very weak hFUS R495X phenotypes are not simply a reflection of reduced protein expression.

Another possible reason for the unexpected mild phenotype of the hFUS R495X/Caz Q349X variants could arise from a different subcellular localization of the proteins in Drosophila compared to mammalian cells. Therefore, we analysed the cellular distribution of all HA-tagged hFUS variants in third instar eye-antennal imaginal discs. The immunofluorescence stainings showed that, as in human/mouse cells, hFUS wt was localized to the nucleus, whereas hFUS P525L showed an additional mild cytoplasmic staining and hFUS R495X was found at equal levels in the nucleus and cytoplasm (Fig. 1D). Although transgenic overexpressed hFUS occasionally appeared in a punctate, granular manner, we did not have the impression to observe visibly striking FUS inclusions typical for human neuropathology. The same staining pattern was observed for the respective Caz variants (data not shown). Thus, mutation or deletion of the PY-NLS leads to cytoplasmic mislocalization of hFUS/Caz in Drosophila. Interestingly, severe neurodegeneration is associated with the more nuclear localizing hFUS/Caz wt and hFUS P525L/Caz P398L and not by the more cytoplasmic localized hFUS R495X/Caz Q349X. Overexpression of hFUS wt or ALS-associated hFUS mutants in motor neurons affects viability and causes impaired locomotor behaviour We next expressed all hFUS variants in more ALS-relevant motor neurons using the OK371-GAL4 driver (Mahr and Aberle, 2006). This

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dramatically affected the viability of the flies. Consistent with their phenotypic strength in the eyes, hFUS wt and hFUS P525L-expressing flies developed only until the pharate adult stage or died as nonwandering third instar larvae/early pupae in the food (Fig. 2A). Only the phenotypically weak hFUS P525L (#55) flies, but in contrast all hFUS R495X-expressing flies developed to adulthood. These flies were then tested in adult climbing assays. Flies expressing only the OK371GAL4 driver served as controls. One day post-eclosion the hFUS P525L flies showed a significant reduction to 32.8% in their climbing ability in comparison to controls, which was further reduced the next day to 3.5% (Fig. 2B). Three days post-eclosion the FUS P525L flies were totally unable to walk and died after five days. In contrast, even the phenotypically strongest hFUS R495X line (#52) showed no significant reduction in climbing ability or viability in comparison to controls during the analysed time frame. As the expression of hFUS wt with OK371-GAL4 was lethal in the pupal stage for all lines examined, larval righting assays were performed, allowing a comparison of all different hFUS variants. Expression of hFUS wt or P525L led to a significant delay in righting

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time in a dose-dependent manner (Fig. 2C). In contrast, all R495Xexpressing larvae showed the same righting ability as control larvae. As OK371-GAL4 drives expression not only in motor neurons but in all glutamatergic neurons in Drosophila, we repeated the viability experiment with the pure motor neuronal driver D42-GAL4. Also with this driver the hFUS wt flies did not eclose, whereas hFUS R495X flies survived to adulthood (Fig. 2A). To confirm the localization pattern of the different hFUS variants stainings of motor neurons of the ventral nerve cord (VNC) were performed. All hFUS variants driven by OK371-GAL4 showed the same nuclear/cytosolic distribution pattern as in eye-imaginal discs (Figs. 2D and 1D). Again, despite some occasional patchy distribution, striking FUS inclusion body formation was not a prominent feature in the motor neurons, not even in the larvae with the weakest performance in the larval righting assays. Taken together, our data show that overexpression of hFUS variants in motor neurons affects viability and locomotor behaviour in larvae and adults. Consistent with our results from the eye analysis, the nuclear

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Fig. 2. Overexpression of hFUS wt or ALS-associated hFUS mutants in motor neurons affects viability and causes impaired locomotor behaviour. A) Viability of hFUS expressing flies. Flies were crossed to OK371-GAL4 (drives expression in all glutamatergic neurons) and D42-GAL4 (drives expression only in motor neurons) and the maximal developmental stage reached was determined. +* = third instar larvae unable to crawl up the vial wall, sometimes forming early pupae in the food. B) Adult climbing assay of hFUS flies. Surviving adults expressing hFUS P525L (#55) or R495X (#52) were tested for their ability to climb 7 cm in 10 s (control = OK371/+). Error bars indicate standard deviation. dpe = day post eclosion. 4–7 animal groups for each genotype were tested on two consecutive days. C) Larval righting assay of hFUS flies. hFUS variants were expressed with OK371-GAL4 and after 4 days larvae were subjected to the larval righting assay (control = OK371/+). Error bars indicate standard deviation. control: n = 97; hFUS wt #61 n = 14, #59 n = 25, #64 n = 58; hFUS P525L #56 n = 7, #57 n = 29, #55 = n = 30; hFUS R495X #52 n = 30, #50. n = 30, #51 n = 27. D) Subcellular localization of hFUS variants in motor neurons. Motor neurons in the ventral nerve cord of third instar larvae were stained with anti-HA. Nuclei (DNA) were counterstained with Hoechst 33258. Insets show magnifications of representive areas. Scale bar = 20 μm. p b0.01 = **; p b0.001 = ***.

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and not the cytoplasmic localized hFUS variants induce the neurodegenerative phenotypes. Overexpression of hFUS variants reduces endogenous Caz levels FUS can autoregulate its expression levels by binding to its own premRNA and promoting the splicing of exon 7, which results in a premature stop codon (Zhou et al., 2013), or via a miRNA-mediated regulatory loop targeting the 3′-UTR (Dini Modigliani et al., 2014). Moreover, a recent study in Drosophila shows that expression of hFUS wt affects endogenous Caz levels (Machamer et al., 2014). The hFUS variants investigated here may have different influences on the endogenous Caz levels — if this autoregulatory mechanism is conserved in Drosophila. Therefore, we expressed hFUS wt and hFUS R495X in motor neurons using OK371-GAL4 and compared the endogenous Caz levels in FUS-positive (Fig. 3A, arrowheads) and adjacent FUS-negative cells. To control for non-specific effects of high nuclear protein expression, we expressed a strong version of nlsGFP. As shown in Fig. 3A, the

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endogenous Caz levels were greatly reduced in motor neurons expressing hFUS wt, whereas less reduction was observed in hFUS R495Xexpressing motor neurons. The expression of high levels of a nuclear protein per se did not influence Caz levels as shown by strong Caz immunostaining in nlsGFP-expressing motor neurons (Fig. 3A, lower panel). To verify the influence of hFUS expression on endogenous Caz levels in another tissue, we expressed all hFUS variants in the eye. On account of the strong eye phenotypes of the hFUS wt and P525L variants using the developmental GMR-GAL4 driver (see Fig. 1B), we expected large differences in the relation of degenerating eye tissue versus overall head tissue among the different hFUS variants. As this could falsify our analysis of the expression levels of different hFUS variants, we decided to use the GAL4-tubGAL80ts-System. In this system the expression of the hFUS variants is repressed due to GAL80 binding to the GAL4 transcription factor at 18 °C. Shifting the temperature to 29 °C inhibits the temperature-sensitive GAL80ts and allows expression. Accordingly, flies were raised at 18 °C and switched to 29 °C after eclosion. After

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Fig. 3. Overexpression of hFUS reduces endogenous Caz levels. A) Effect of hFUS variants on endogenous Caz levels in motor neurons. Motor neurons of third instar larvae expressing HA-tagged hFUS constructs with OK371-GAL4 were stained with anti-HA and anti-Caz. hFUS-expressing (HA-positive) are marked with arrowheads to facilitate comparison to adjacent non-expressing cells. nlsGFP was expressed as a control for non-specific effects of high nuclear expression. For better comparison nlsGFP is shown in the red channel. Insets show magnifications of representative areas. Scale bar = 20 μm. B) Effect of hFUS variants on endogenous Caz levels in adult eyes. After eclosion hFUS variants were expressed using the GMR-GAL4, tubGAL80ts-System. After 4d soluble protein fractions of adult heads were isolated and subjected to Western blot analysis. Blots were probed with anti-FUS, anti-Caz and anti-α-tubulin (as loading control). control = GMR-GAL4, tubGAL80ts/+. C) Insolubility of hFUS wt. The pan-neuronal driver elav-GAL4 (with tubGAL80ts) was used to express hFUS wt (#59) and hFUS R495X (#51) in adult flies after eclosion for the indicated time points. Soluble proteins were analysed on Western blot, whereas the insoluble protein fraction were used for the slot blot assay and both blots were probed with anti-HA. Syntaxin served as a loading control. control = elav-GAL4, tubGAL80ts/+.

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4 days of expression we isolated the soluble protein fraction of adult heads. Of note, no degenerative eye phenotypes were visible at this time point (not shown). Western blot analysis revealed that hFUS wt (#61) and P525L (#56), which showed a strong phenotype in eyes and locomotor behaviour (Figs. 1B and 2C), indeed reduced endogenous Caz levels, whereas the R495X variants did not (Fig. 3B). We noticed that in contrast to the R495X variant the bands for the hFUS wt (#61) and P525L (#56) were weaker. To explore if this could be due to a shift from the soluble to the insoluble fraction in case of hFUS wt and P525L, we compared the solubility of hFUS wt and R495X driven with pan-neuronal elav-GAL4, tubGal80ts expression system. After eclosion, expression of both variants was induced at 29 °C and adult heads were analysed at the indicated time points (Fig. 3C). hFUS wt showed a mild insolubility already on day 1 post-induction, which strongly increased after 10 days of expression. In contrast, the R495X variant remained soluble even at 10 days post-induction. These experiments suggest that although hFUS wt and P525L variants become insoluble over time, they still have the capacity to reduce Caz mRNA levels (see discussion for details on insolubility and aggregation). In summary, our data suggest that autoregulation of FUS occurs in Drosophila and thus is a highly conserved mechanism. Nuclear import of hFUS is mediated via transportin in Drosophila We have shown that the nuclear localization of hFUS is dependent on the integrity of the PY-NLS. Next, we wanted to investigate if the nuclear import of hFUS is in Drosophila-like in human cell culture — also dependent on the nuclear import receptor transportin. For this purpose, we analysed the effects of RNAi-mediated transportin knockdown on hFUS localization. The only annotated transportin protein in Drosophila, dTNPO (CG7398), shows a high identity to the human proteins, hTNPO1 and hTNPO2 (74/72%, respectively). A second Drosophila transportin protein, CG8219 (Quan et al., 2008) shows a lower identity to both proteins (57/56%, respectively) (Fig. 4A; detailed protein sequence alignment, see Supp. Fig. 1). To test the efficiency of the RNAi-mediated knockdown, we separately expressed the RNAis in the whole animal using actin-GAL4 and performed semi-quantitative RT-PCR with RNA from third instar larvae. Larvae expressing vasa-RNAi served as controls. Both RNAis downregulated dTNPO very efficiently and led to a mild reduction of CG8219 expression (Fig. 4B). This was expected as the used RNAis are long (dTNPO-RNAi: 212 bp; CG8219-RNAi: 350 bp) and the targeted mRNA sequences are very well conserved among each other. To analyse the effect of transportin knockdown on hFUS localization, we stained motor neurons of late third instar larvae expressing hFUS wt, dTNPO-RNAi (stronger effect than CG8219-RNAi) and Dicer2 to enhance RNAi efficiency. Indeed, these motor neurons showed – in addition to the normal nuclear staining – a mild cytoplasmic localization of hFUS wt (Fig. 4C, upper panels). The effect of hFUS mislocalization was even more pronounced in salivary glands, where the cytoplasmic staining showed a strong increase at the expense of nuclear staining (Fig. 4C, middle panels). We expected that the localization of the endogenous Caz protein would also be affected by dTNPO silencing. Therefore, we analysed endogenous Caz distribution in larvae expressing only dTNPO-RNAi and Dicer2 with OK371-GAL4. To label the OK371expressing motor neurons in the VNC, we additionally expressed EGFP. In motor neurons with transportin knockdown (Fig. 4C, arrowheads), endogenous Caz wt becomes mislocalized to the cytoplasm, whereas it is nuclear in cells without dTNPO silencing (Fig. 4C, arrows). Therefore, hFUS and endogenous Caz are imported into the nucleus via transportin in Drosophila. Next, we wanted to examine the effects of transportin knockdown on the eye phenotypes in adult flies expressing different hFUS variants. Eye-specific expression (GMR-GAL4) of dTNPO- or CG8219-RNAi alone caused almost no phenotype (only some fused ommatidia mostly in

the dorsal anterior region) (Fig. 4D, left panels). We expected that the effect of dTNPO-RNAi on hFUS wt would be a phenocopy of the PY-NLS mutant variants, as the toxic nuclear hFUS wt levels should also be reduced. In contrast, we observed a strong enhancement of the eye phenotype of hFUS wt. Nevertheless, the effects of dTNPO- and CG8219-RNAi on the eye phenotypes of the transportin-binding impaired hFUS mutants P525L and R495X were much weaker (Fig. 4D). To verify that the phenotype was caused by the knockdown of transportin and not due to RNAi off-target effects, we performed rescue experiments with human TNPO1 and TNPO2. Unfortunately, the expression of hTNPO2 alone caused dramatic eye phenotypes, whereas hTNPO1-expression had no phenotype by itself (data not shown). Therefore, we used only hTNPO1 for our rescue experiments. Coexpression of hFUS wt, dTNPO-RNAi and hTNPO1 resulted in a strong amelioration (no black spots) of the phenotype (Fig. 4E). To rule out GAL4-titration effects we tried to rescue the phenotype by expressing a nlsGFP construct. This resulted only in a mild suppression (Fig. 4E), confirming that our phenotype is transportin-dependent. These data indicate that the nuclear import of exogenous hFUS and endogenous Caz in Drosophila requires transportin. Although the knockdown of transportin leads to cytoplasmic mislocalization of overexpressed hFUS wt, it does not phenocopy the PY-NLS mutant variants. Instead of amelioration of the phenotype transportin knockdown leads to a strong aggravation of the hFUS wt phenotype (see discussion).

Drosophila PRMT1 modifies hFUS via arginine methylation Having shown that the function of transportin for the nuclear import of hFUS is conserved in Drosophila, we wanted to analyse the effects of arginine methylation of hFUS. First we wanted to prove that hFUS wt can be methylated in Drosophila. Using an antibody specific for methylated FUS (Dormann et al., 2012) we could confirm the existence of methylated hFUS in immunostainings of eye imaginal disc expressing hFUS wt in the eye primordium with GMR-GAL4 (Fig. 5A). Of note, GMR-GAL4 is only expressed in differentiating cells posterior to the morphogenetic furrow. This can be seen in the overexposed picture, where meFUS-staining is absent in cells anterior to the morphogenetic furrow in the non-GMR-expressing cells, thereby proving the specificity of the antibody. After the confirmation that hFUS wt is being methylated in Drosophila, we aimed to identify the responsible arginine methyltransferases. It has recently been shown in cell culture that methylation of the last RGG domain of hFUS by protein arginine methyltransferase 1 (PRMT1) reduces its ability to bind to transportin and therefore interferes with its nuclear import. In line with this, lowering the methylation status of hFUS P525L counteracted its mutation-dependent reduced binding to transportin and partially restores its nuclear import (Dormann et al., 2012). Therefore, we used this hFUS P525L mutant to screen RNAi-lines for the nine Drosophila arginine methyltransferases (DARTs), the PRMT orthologues, for modification of the hFUS P525L eye phenotype (data not shown). Our analysis revealed that only the RNAi for Dart1 (#1, see Material and Methods section), the PRMT1 homolog, showed a clear enhancement of the hFUS P525L phenotype. This could be confirmed with two other Dart1-RNAis (Fig. 5B). In comparison to hFUS P525L, the hFUS wt phenotype was evidently too strong to be further enhanced by Dart1-RNAi (Fig. 5B). The C-terminal truncation mutant R495X lacks the meFUS epitope and therefore appears to be less subject to DART1 modifier effects (Fig. 5B). Dart1-RNAi efficiency was checked via RT-PCR of third instar larvae, which ubiquitously expressed the RNAis using an actin-GAL4 driver (Fig. 5C). In order to prove that knockdown of Dart1 leads to reduced methylation of hFUS P525L, we performed Western blots of adult head cell lysates. Comparison of the anti-meFUS with anti-HA blot clearly shows that the amount of methylated hFUS P525L is reduced when Dart1 is downregulated (Fig. 5D).

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Fig. 4. Nuclear import of hFUS is mediated via transportin in Drosophila. A) Protein comparison of human and Drosophila transportins. The percentage of identity/similarity respectively for pairwise alignments of the human and Drosophila transportin protein sequences are shown. B) Silencing of Drosophila transportins by RNAi. RT-PCR for both transportin genes (dTNPO and CG8219) was performed with lysates of third instar larvae with ubiquitous expression (act-GAL4) of dTNPO-RNAi, CG8219-RNAi or control (vasa)-RNAi. GAPDH served as control. C) Effect of dTNPO silencing on hFUS wt/Caz localization in motor neurons and salivary glands. dTNPO-RNAi and HA-hFUS wt (#64) were coexpressed with OK371-GAL4 and Dcr2. VNC/salivary glands of third instar larvae were stained with anti-HA. In the motor neurons, some nuclei had to be overexposed to visualize the cytoplasmic mislocalization of hFUS wt. To analyse the transportin silencing effect on endogenous Caz dTNPO-RNAi and Dcr2 were coexpressed in motor neurons of third instar wt larvae (OK371-GAL4) and staining with an antibody against Caz was performed. To identify the dTNPO-RNAi/Dcr2-expressing cells (arrowheads) in the brain, 2xEGFP was coexpressed. Arrows mark the non-expressing cells, which serve as internal controls. Insets show magnifications of representative areas. Scale bar = 20 μm. D) Effect of dTNPO/CG8219 silencing on hFUS eye phenotypes. Using the GMR-GAL4 driver different hFUS variants and both transportin-RNAis were coexpressed. Arrows point to the observed black lesions in adult eyes. Scale bar = 100 μm. E) Rescue of the Drosophila dTNPO silencing effect by overexpression of human TNPO1. For the rescue HA-FUS wt, dTNPO-RNAi and hTNPO1 were expressed with GMR-GAL4. To control for possible GAL4 titration effects nlsGFP instead of hTNPO1 was coexpressed. Scale bar = 100 μm.

These results show that hFUS is being arginine methylated by DART1 in Drosophila and that impairment of this methylation enhances the eye phenotype of hFUS P525L. Discussion In human forms of ALS and FTLD with cytoplasmic inclusions of the predominantly nuclear FUS protein, the cytoplasmic mislocalization of FUS is considered to be an important step in disease pathogenesis. Therefore, the elucidation of the mechanisms required for its nuclear

ex- or import is of great interest. Concerning its nuclear import, it was previously shown in mammalian cell cultures that hFUS is bound to its nuclear import receptor transportin via the PY-NLS, and that the methylation of the last RGG domain of hFUS reduces this interaction (Dormann et al., 2012; Dormann et al., 2010). In this study, we could show that these key aspects of FUS nuclear import are conserved in Drosophila. We demonstrate in two different Drosophila tissues, eyes and motor neurons, that the localization of hFUS and Drosophila Caz is dependent on the integrity of the C-terminal PY-NLS. Additionally, we find in immunostainings and on

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Fig. 5. Drosophila PRMT1 modifies hFUS via arginine methylation. A) hFUS can be methylated in Drosophila. Eye imaginal disc of third instar larvae expressing hFUS wt under control of GMR-GAL4 were stained with anti-meFUS. Of note, GMR-GAL4 drives expression only posterior to the morphogenetic furrow (MF). The overexposed image shows the absence of meFUS-staining in non-GMR-GAL4-expressing cells anterior to the MF. A = anterior, P = posterior. Scale bar = 20 μm. B) Effects of different Dart1-RNAis on hFUS eye phenotypes. Three different Dart1-RNAis were expressed with GMR-GAL4 alone (lower panels) or together with hFUS variants. Pictures were taken 2 days after eclosion. Scale bar = 100 μm. control = vasa-RNAi. C) Silencing of Drosophila Dart1 by RNAi. For efficiency testing, RT-PCR for Dart1 was performed with lysates of third instar larvae with ubiquitous expression (act-GAL4) of Dart1 #1–3-RNAi or vasa-RNAi (control). Expression levels were normalized to rp49. For mRNA quantification, three technical replicates were performed on two biological replicates (one biological replicate is shown as an example). Error bars indicate standard deviation. D) Effect of Dart1-RNAi on methylation of the hFUS P525L protein. For Western blots, Dart1 #2-RNAi (or vasa-RNAi as control) and HA-hFUS P525L were coexpressed in adult eyes using GMR-GAL4. Protein lysates of adult heads were prepared from 2 day-old flies and subjected to Western Blot, which was probed with meFUS- and HA-antibody. α-tubulin serves as a loading control.

Western blots that the overexpression of hFUS variants leads to a reduction of endogenous Caz levels dependent on the degree of hFUS nuclear localization. This autoregulation was previously shown in cell culture (Zhou et al., 2013), recently also in Drosophila (Machamer et al., 2014) and demonstrates the functionality of the expressed hFUS variants. The observation of this autoregulatory side effect of hFUS overexpression imposes a critical review of previous studies using hFUS overexpression in Drosophila and other model organisms, because the analysis of specific hFUS variant effects may be hampered by the additional effects of loss of endogenous FUS. Therefore, future studies will be required to generate and analyse knock-in animal models, where one endogenous FUS allele is replaced with a mutant variant, thereby achieving endogenous expression levels. In addition, such models would resemble more to the human genetic situation. Concerning the phenotypes caused by our different hFUS/Caz variants, we observed that the predominantly nuclear hFUS/Caz wt and hFUS P525L/Caz P398L caused strong phenotypes in eyes and motor neurons, whereas the expression of the more cytoplasmic localized hFUS

R495X/Caz Q349X results in almost no neurodegenerative phenotypes. This genotype/phenotype correlation is inverse to the human disease situation as the hFUS R495X mutation leads to one of the most aggressive forms of ALS (Bosco et al., 2010; Waibel et al., 2013). Of note, in humans heterozygous mutation of one FUS copy into hFUS R495X could lead to some cytoplasmic mislocalization and haploinsufficiency. Unfortunately, this loss-of-function aspect is not represented in our Drosophila model, which could be one reason for the different effects. In our system the hFUS R495X is overexpressed in addition to the two endogenous Caz alleles, thereby preventing loss of functional nuclear Caz. Although we observed an autoregulatory mechanism of overexpressed hFUS on endogenous Caz, we could show in immunostainings and Western blots that the nuclear levels of the hFUS R495X variant are too small to cause a strong reduction of endogenous Caz. Therefore, the analysis of this mutation in Drosophila has to be repeated in the appropriate knock-in animals, which have to be generated in the future. The discovered autoregulatory mechanism and therefore the loss of endogenous Caz cannot be responsible for the strong phenotypes of the

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mostly nuclear localized hFUS wt and hFUS P525L variants. As it was shown in Drosophila and zebrafish that hFUS wt can rescue the loss of the endogenous protein (Armstrong and Drapeau, 2013; Kabashi et al., 2011; Wang et al., 2011) – demonstrating the functional conservation of FUS protein function – hFUS wt should compensate for the loss of endogenous Caz in our system. Even if hFUS wt would not be able to fully compensate the loss of all Caz functions, Drosophila Caz wt should rescue. In contrast, we observed that also overexpression of Caz wt caused strong eye phenotypes. As overexpression of the C-terminal deletion mutants hFUS R495X/Caz Q349X caused only very mild effects, our data point to a nuclear toxic function of overexpressed hFUS/Caz. Comparable results for hFUS wt, Caz wt and a hFUSΔ32 variant have been described previously (Xia et al., 2012). Additionally, this group could show that the addition of a nuclear export signal to hFUS wt dramatically reduces its toxicity. The understanding of hFUS overexpression effects is also important for ALS patients as a recent study observed mutations in the 3′UTR of FUS leading to enhanced FUS levels (Sabatelli et al., 2013). The hypothesis that FUS could have toxic gain of function effects in the nucleus is underlined by the observation that besides the FUSpositive cytoplasmic inclusions, intranuclear inclusions are also found in ALS and FTLD patients (Bäumer et al., 2010; Neumann et al., 2009). Additionally, intranuclear FUS inclusions have been found in postmortem tissue of patients with neuronal intranuclear inclusion body disease (NIIBD), certain forms of FTLD and in polyglutamine diseases like Huntington's disease and spinocerebellar ataxia 1 and 3 (Doi et al., 2010; Mori et al., 2012; Mori et al., 2014; Woulfe et al., 2010). Moreover, it was shown for some disease-associated FUS mutations that they are not associated with cytoplasmic mislocalization and cytoplasmic inclusion formation. For example, the expression of mutant hFUS G156E (Ticozzi et al., 2009) in rat hippocampal neurons leads to the formation of intranuclear inclusions, which also sequester hFUS wt (Nomura et al., 2014). It is reasonable that FUS overexpression or loss-of function could have a profound influence on general protein expression as FUS regulates the transcription and the splicing of hundreds of target genes (Lagier-Tourenne et al., 2012; Schwartz et al., 2012). Moreover, recent studies demonstrate that FUS is required for the integrity of different nuclear structures such as nuclear gems, sites of SMN (survival of motor neurons) protein accumulation in the nucleus (Yamazaki et al., 2012) and paraspeckles (Naganuma et al., 2012; Page et al., 2011; Shelkovnikova et al., 2014). Interestingly, paraspeckles are involved in storage and rapid release of certain RNAs under stress conditions (Prasanth et al., 2005). Under basal conditions paraspeckles are absent in neurons, but recent studies suggest, that they form during early stages of ALS, triggered by increased synthesis of the core component long non-coding RNA NEAT1, suggesting participation of paraspeckles in response to neuronal stress (Nishimoto et al., 2013). In consequence, disruption of paraspeckle formation by mutant or overexpressed FUS could interfere with these protective mechanisms. Therefore, nuclear loss- or gain of toxic function of FUS is likely to affect a variety of different nuclear processes. In addition, the nuclear toxicity of overexpressed FUS observed in our study combined with the known neurodegenerative effects caused by FUS loss-of-function strongly suggests that too much or too little FUS is toxic, which is also supported by the evolutionary conservation of the autoregulatory mechanism. Although we did not get the impression that occasionally detectable patchy appearances particularly of cytosolically mislocalized FUS converged to human neuropathologically typical inclusions, we detected biochemically insoluble hFUS wt and P525L in the phenotypically strongest lines. Concomitantly, these lines showed massive reduction of endogenous Caz protein levels. It appears that functional FUS levels need to be kept in a tight balance. Reduction of FUS/Caz activity by forced expression of FUS might be a contributing factor to disease. Under severe circumstances the aggregation-prone nature of FUS might eventually deplete it into insoluble fractions that can no longer compensate for the down-regulated Caz. The discrepancy between the

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observed insolubility and the absence of visible aggregates could be due to the different time points analysed, as the immunostainings were performed on larval tissue, whereas adult fly heads were used for the insolubility assay. Therefore, we cannot exclude that neuropathological FUS inclusions are present in adult tissue. Nevertheless, aggregate formation does not seem to be a prerequisite for hFUS toxicity in Drosophila as we did not observe any aggregates in larvae with weak performance in the larval locomotion assay. Therefore, we think that the formation of FUS aggregates is a long-term process in humans, which may not occur to the same extent in short-lived animals. FUS neurodegeneration might involve a complex combination of perturbations by too much or too little “normal” FUS activity and autoregulation in the nucleus, adverse effects by biochemically insoluble FUS proteins, micro-aggregates or oligomers, both in the nucleus and cytoplasm, and eventually the neuropathologically authentic FUS inclusions, which might be modelled better in long-lived animal models. Our analysis of the dependency of hFUS localization on its PY-NLS suggested that the nuclear import of hFUS via transportin is conserved from fly to humans. To clearly verify the involvement of transportin in FUS nuclear import in Drosophila, we analysed the effects of transportin knockdown on hFUS localization via RNAi. As expected, we observed that in motor neurons and salivary glands transportin knockdown leads to cytoplasmic retention of overexpressed hFUS wt and of endogenous Caz. Concerning the phenotypic effects of transportin silencing in adult eyes we found that expression of dTNPO-RNAi resulted only in very mild effects in control eyes (GMR-GAL4). This shows that although a cytoplasmic retention of Caz or other transportin cargos occurs in this situation it is not causing severe defects. Similarly, we observed almost no effects of transportin knockdown on the hFUS P525L and R495X eye phenotypes. This is most likely due to the fact that these variants cannot bind to transportin and are therefore not affected by its downregulation. In contrast, silencing of transportin strongly enhanced the eye phenotype of hFUS wt. We could exclude that this is caused by off-target effects, as we could rescue this strong eye phenotype via the expression of human TNPO1, thereby also showing the evolutionary conservation of transportin function. The aggravation of the hFUS wt phenotype by transportin knockdown is puzzling at first glance. We expected that the effects of cytoplasmic mislocalization of hFUS wt after dTNPO silencing should be a phenocopy of the PY-NLS mutant variants as the nuclear import of FUS is impaired in both situations. As the eye phenotype of hFUS wt seems to be caused by high nuclear FUS levels, interfering with its nuclear import should reduce nuclear levels and therefore ameliorate the phenotype. Instead, it appears that differential Caz autoregulation and/ or FUS insolubility that differ dramatically between hFUS wt and R495X play a role. Particularly Caz autoregulation would be exacerbated by dTNPO knockdown, as Caz nuclear import depends on transportin. Finally, we examined whether the nuclear import of hFUS is also regulated by its methylation status. We could demonstrate in immunostainings and Western blots that a portion of hFUS wt and P525L is methylated in Drosophila. Moreover, this methylation can be reduced by knockdown of Dart1, the homolog of the arginine methyltransferase PRMT1. Additionally, we observed that the eye phenotype of hFUS P525L is enhanced after Dart1-silencing. This could be explained by increased nuclear import of hFUS P525L as it was reported, that reduced methylation of the RGG3 domain of hFUS P525L can rescue its weak binding to transportin (Dormann et al., 2012). However, silencing of PRMT1 only led to a partial rescue of the cytoplasmic mislocalization of hFUS P525L (Dormann et al., 2012). As our immunostainings already show a strong nuclear localization of hFUS P525L before Dart1silencing, we doubt that reduced methylation of hFUS P525L would led to a sufficient increase in nuclear hFUS and to be the sole cause of the eye phenotype modifier effect. Additionally, we suggest that the silencing of Dart1 affects hFUS function via other mechanisms. Note that in addition to the arginine residues of the nuclear import-associated RGG3 domain, also other arginine residues in RGG1 and RGG2 of FUS can be

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methylated (Ong et al., 2004; Rappsilber et al., 2003). Thus, methylation of FUS may not only influence its nuclear import but also affect other FUS functions. For example, a recent study in HeLa cells suggests that FUS methylation could effect its role as a coactivator in transcriptional regulation as the activation of the survivin promoter by FUS wt is dependent on its methylation by PRMT1 (Du et al., 2011). In an indirect way, silencing of Dart1 could also affect the function of FUS as a transcriptional regulator. As mentioned previously, FUS regulates the transcriptional down- and upregulation of over hundred target genes through direct interaction with the C-terminal domain of RNA polymerase II (Schwartz et al., 2012). In this scenario, Dart1-silencing could change the methylation status of histones, thereby changing the chromatin structure of FUS target genes and by that interfering with normal FUS function. In this context, it is interesting to note that the histone substrates of human PRMT1 and PRMT4 (CARM1) seem to be conserved in Drosophila. DART1 methylates arginine 3 on histone H4 like PRMT1 (Boulanger et al., 2004; Kimura et al., 2008; Strahl et al., 2001) and DART4 methylates histone H3 like CARM1 (Boulanger et al., 2004; Ma et al., 2001; Schurter et al., 2001). In summary, we could demonstrate that the localization of hFUS/Caz is dependent on the integrity of the PY-NLS. Expression of hFUS / Caz leads to neurodegenerative phenotypes in eyes and motor neurons, dependent on its nuclear level. High nuclear levels of hFUS lead to reduced levels of endogenous Caz most likely due to a conserved autoregulatory mechanism. Additionally, we show that the key mechanisms of hFUS nuclear import – transportin dependency and modulation via PRMTs – are conserved in Drosophila. These findings demonstrate that Drosophila is a suitable model system to analyse the mechanisms involved in the nuclear import of FUS and to identify other FUS interaction partners. Finally, our data suggest that FUS levels need to be tightly regulated and further point to a nuclear toxic potential of FUS, which has to be elucidated in future studies. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG grant KA1675/3-1 and -2), the German Competence Network “Degenerative Dementias” grant 01GI1005B, the Virtual Institute “RNA Dysfunctions” of the German Center for Neurodegenerative Diseases (DZNE), DZNE, and the Hertie Foundation. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.nbd.2014.11.003. References Aman, P., Panagopoulos, I., Lassen, C., Fioretos, T., Mencinger, M., Toresson, H., Höglund, M., Forster, A., Rabbitts, T.H., Ron, D., Mandahl, N., Mitelman, F., 1996. Expression patterns of the human sarcoma-associated genes FUS and EWS and the genomic structure of FUS. Genomics 37, 1–8. Andersson, M.K., Ståhlberg, A., Arvidsson, Y., Olofsson, A., Semb, H., Stenman, Gö, Nilsson, O., Aman, P., 2008. The multifunctional FUS, EWS and TAF15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response. BMC Cell Biol. 9, 37. Armstrong, G.A.B., Drapeau, P., 2013. Loss and gain of FUS function impair neuromuscular synaptic transmission in a genetic model of ALS. Hum. Mol. Genet. 22, 4282–4292. Azuma, Y., Tokuda, T., Shimamura, M., Kyotani, A., Sasayama, H., Yoshida, T., Mizuta, I., Mizuno, T., Nakagawa, M., Fujikake, N., Ueyama, M., Nagai, Y., Yamaguchi, M., 2014. Identification of ter94, Drosophila VCP, as a strong modulator of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS. Hum. Mol. Genet. 23, 3467–3480. Bäumer, D., Hilton, D., Paine, S.M., Turner, M.R., Lowe, J., Talbot, K., Ansorge, O., 2010. Juvenile ALS with basophilic inclusions is a FUS proteinopathy with FUS mutations. Neurology 75, 611–618. Bertolotti, A., Lutz, Y., Heard, D.J., Chambon, P., Tora, L., 1996. hTAF(II)68, a novel RNA/ ssDNA-binding protein with homology to the pro-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II. EMBO J. 15, 5022–5031. Boillée, Sé, Vande Velde, C., Cleveland, D.W., 2006. ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron 52, 39–59.

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