TDP-43 is Recruited to Stress Granules in Conditions of Oxidative Insult

,,1 ,1

*Department of Neurology and Laboratory of Neuroscience, Dino Ferrari Center, Universita` degli Studi di Milano IRCCS Istituto

Auxologico Italiano, Milan, Italy

Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA

Neuromuscular Diseases Unit/ALS Clinic, Kantonsspital St. Gallen, St.Gallen, Switzerland

Institute of Pathology, Kantonsspital St. Gallen, St.Gallen, Switzerland

International Centre for Genetic Engineering and Biotechnology (ICGEB), AREA Science Park, Trieste, Italy

Transactive response DNA-binding protein 43 (TDP-43) isan ubiquitously expressed RNA-binding protein (RBP)belonging to the heterogeneous ribonucleoprotein (RNP)family and containing two RNA-recognition motif (RRM)domains for target RNA binding and a glycine-rich C-terminal tail for protein-protein interaction (Ayala et al.2005). The biological role of TDP-43 is associated both totranscriptional regulation and to post-transcriptional controlof RNA processing, ranging from splicing to mRNAstabilization and transport (Buratti and Baralle 2008).TDP-43 has recently emerged as the major neuropatholog-

ical hallmark of amyotrophic lateral sclerosis (ALS) andfrontotemporal lobar degeneration (FTLD) (Arai et al. 2006;Neumann et al. 2006). Although distinct neuronal populationsand brain areas selectively degenerate in ALS and in FTLD,

these two disorders represent a clinical continuum as FTLDpatients may also develop motor neuron disease and cognitivedecits have been observed in ALS. However, TDP-43

Received May 7, 2009; revised manuscript received July 30, 2009;accepted September 9, 2009.Address correspondence and reprint requests to Antonia Ratti,

Department of Neurology and Laboratory of Neuroscience, IRCCS Is-tituto Auxologico Italiano, Via Zucchi, 18 20095 Cusano Milanino,Milan, Italy. E-mail: [email protected] Silani and Antonia Ratti are Joint Senior Authors.

Abbreviations used: ALS, amyotrophic lateral sclerosis; FTLD,frontotemporal lobar degeneration; FUS/TLS, Fusion/Translocated inLipoSarcoma; HuR, Hu R antigen; P-bodies, processing bodies; RBP,RNA-binding protein; RNP, ribonucleoprotein; RRM, RNA-recognitionmotif; SG, stress granule; TDP-43, Transactive response DNA-bindingprotein 43; TIA-1, T cell-induced antigen 1; TIAR, TIA-related.

Abstract

Transactive response DNA-binding protein 43 (TDP-43) forms

abnormal ubiquitinated and phosphorylated inclusions in brain

tissues from patients with amyotrophic lateral sclerosis (ALS)

and frontotemporal lobar degeneration. TDP-43 is a DNA/

RNA-binding protein involved in RNA processing, such as

transcription, pre-mRNA splicing, mRNA stabilization and

transport to dendrites. We found that in response to oxidative

stress and to environmental insults of different types TDP-43

is capable to assemble into stress granules (SGs), ribonu-

cleoprotein complexes where protein synthesis is temporarily

arrested. We demonstrated that a specific aminoacidic interval

(216315) in the C-terminal region and the RNA-recognition

motif 1 domain are both implicated in TDP-43 participation in

SGs as their deletion prevented the recruitment of TDP-43

into SGs. Our data show that TDP-43 is a specific component

of SGs and not of processing bodies, although we proved that

TDP-43 is not necessary for SG formation, and its gene

silencing does not impair cell survival during stress. The

analysis of spinal cord tissue from ALS patients showed that

SG markers are not entrapped in TDP-43 pathological inclu-

sions. Although SGs were not evident in ALS brains, we

speculate that an altered control of mRNA translation in

stressful conditions may trigger motor neuron degeneration at

early stages of the disease.

Keywords: amyotrophic lateral sclerosis, RNA-binding pro-

tein, stress granules, Transactive response DNA-binding

protein 43.

J. Neurochem. (2009) 111, 10511061.

JOURNAL OF NEUROCHEMISTRY | 2009 | 111 | 10511061 doi: 10.1111/j.1471-4159.2009.06383.x

2009 The AuthorsJournal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 10511061 1051

inclusions have been identied also in Alzheimers, Parkin-sons and Huntingtons diseases, suggesting it may be acommon marker for many neurodegenerative disorders (Am-ador-Ortiz et al. 2007; Nakashima-Yasuda et al. 2007;Schwab et al. 2008). The fact that TDP-43 protein may beprimarily and specically involved in ALS onset has recentlyemerged from genetic studies showing TARDBP gene muta-tions in familial and sporadic ALS cases (Sreedharan et al.2008; Corrado et al. 2009). Whether TDP-43 protein aggre-gation is a pathogenic event that triggers neuronal degener-ation or whether TDP-43-positive inclusions are theconsequence of a neuroprotective mechanism still remainsan issue to be addressed.In condition of oxidative stress the regulation of gene

expression at post-transcriptional level, mediated by RBPs, isknown to be impaired. In particular, when a sub-lethaloxidative stress is induced in vitro, there is an immediateblock of the translation machinery with sequestration of theactively-translating mRNAs and distinct RBPs to cytoplas-mic foci, called stress granules (SGs) (Anderson andKedersha 2008). SGs represent a protective mechanism tobypass the cellular insult as the majority of mRNAs issilenced in these macromolecular structures in stalled 48Sribosomal complexes, while only specic and essentialtranscripts (i.e. Hsp70) are maintained in active translation(Anderson and Kedersha 2002; Kedersha and Anderson2002). During stress, SGs are in dynamic equilibriumbetween polysomes and processing bodies (P-bodies), thelatter being constitutive RNP complexes where both mRNAdegradation and microRNA-mediated translational arresttake place (Kedersha et al. 2005, 2008). It is experimentallyproven that once the insult is removed, these RNP complexessoon disaggregate in favour of a parallel polysome re-assembly and mRNA translation re-initiation. The molecularmechanisms and the signalling pathways triggering SGformation have been characterized, as well as the nature ofdistinct cellular insults which can induce these structures,including oxidative stress, proteasome inhibition, osmoticand heat shocks (Kedersha and Anderson 2007).In addition to mRNA, several RBPs have been described so

far to be components of these cytoplasmic foci in condition ofcellular insult. T cell-induced antigen 1 (TIA-1) and TIAR(TIA-related) are the essential RBPs which promote SGassembly because of their prion-like domains that favouraggregation of other RBPs/proteins in granules together withtheir target mRNAs. SGs also contain PolyA-Binding Protein1 (PABP-1), the Embryonic Lethal Abnormal Vision (ELAV)family member Hu R antigen (HuR), Survival Motor Neuron(SMN), Ras-GAP SH3 domain-binding protein (G3BP),Staufen, and Fragile X Mental Retardation Protein (FMRP)RBPs, together with the ribosomal 48S pre-initiation com-plex, early translation initiation factors, microRNA-associatedArgonaute proteins, p54/Rck helicase, XRN1 exonucleaseand cytoskeletal proteins (Anderson and Kedersha 2008).

The aim of our study was to investigate whether the RBPTDP-43 participates to the assembly of SGs in condition ofcellular stress in a motoneuronal cell line and whether suchcytoplasmic RNP complexes are also present in the spinalcord and/or in the TDP-43-positive pathological inclusions ofALS patients.

Experimental procedures

Cell culture and treatmentsThe motoneuronal cell line NSC34 (a kind gift of N.R. Cashman,

University of British Columbia, Vancouver, Canada) was cultured as

previously reported (Ratti et al. 2008). NSC34 cells were exposed to0.5 mM sodium arsenite for 30 min or pre-treated with emetine

(20 lg/mL for 2 h) or puromycin (20 lg/mL for 4 h) as described(Kedersha and Anderson 2007). MG132 (10 lM) was used for 4 has reported (Mazroui et al. 2007). All reagents were purchased fromSigma (Milan, Italy). For heat shock experiments, cells were

incubated at 44C for 30 min overlaid with mineral oil.

ImmunocytochemistryCells were xed with 4% paraformaldehyde in phosphate buffered

saline for 15 min, permeabilized with cold methanol and 0.2% Triton

X-100, and blocked with 10% normal goat serum solution (Vector

Laboratories, Burlingame, CA, USA). Incubation with primary

antibodies (TDP-43, 1 : 500, ProteinTech Group, Manchester, UK;

TIAR, 1 : 100, BD Transduction Laboratories, Milan, Italy; HuR,

1 : 500, Molecular Probes, Milan, Italy; Nova1, 1 : 200, Upstate

Biotechnology, Milan, Italy; FLAG, 1 : 1200, Sigma) was performed

in blocking solution for 1 h at 37C. The uorescent-taggedsecondary antibodies Alexa Fluor 488 and 555 (1 : 500, Invitrogen,

Milan, Italy) were used for detection and nuclei were visualized by

4-6-diamidino-2-phenylindole (DAPI) staining (Roche, Milan,Italy). As a negative control, primary antibodies were replaced by

normal goat serum. Slides were mounted with Fluorsave (Calbio-

chem, La Jolla, CA, USA) and acquired with a wideeld microscope

(DMIRE2/HCS, Leica Microsystems, Wetzlar, Germany).

Protein extraction, immunoprecipitation and western blottingNSC34 cells were homogenized in lysis buffer (150 mM NaCl,

20 mM Tris-HCl pH7.4, 1% Triton X-100, protease inhibitor

cocktail), centrifuged at 12 000 g for 15 min at 4C and superna-tants were collected. Proteins from cytoplasmic and nuclear

fractions were obtained with the ProteoJETTM kit (Fermentas,

Milan, Italy) following the manufacturers instructions. For immu-

noprecipitation experiments, 30 lL protein G Sepharose-beads pre-coated for 6 h with 1.5 lg of the selected antibody were incubatedwith 300 lg cytoplasmic protein lysate in NT2 buffer [50 mM Tris-HCl pH7.4, 15 mM NaCl, 1 mM MgCl2, 0.05% NP-40 (Sigma)],

containing 400 U RNase inhibitor, 1 mM dithiothreitol and 20 mM

EDTA. After overnight incubation and four washes in NT2 buffer,

recovered proteins were resolved on 10% sodium dodecyl sulfate

polyacrylamide gel electrophoresis and transferred to nitrocellulose

membrane. Western blot and immunoprecipitation assays were

performed with HuR, TIAR, TIA-1 and a-tubulin (all from SantaCruz Biotechnology, Santa Cruz, CA, USA), TDP-43 and p84

(Abcam, Cambridge, UK) antibodies.

Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 10511061 2009 The Authors

1052 | C. Colombrita et al.

Plasmid constructs, siRNA and transfectionsNSC34 cells were cultured on glass cover-slips and transiently

transfected with Lipofectamine 2000 (Invitrogen) following the

manufacturers instructions. After 48-h transfection cells were

exposed to 0.5 mM arsenite for 30 min prior to be processed for

immunocytochemistry. The FLAG-tagged full-length and deleted

(DRRM1, DC, 1315) TDP-43 plasmids used for transfection werepreviously described (Ayala et al. 2008). The Dcp1-enhanced GreenFluorescent Protein (EGFP) and TIA-1-enhanced Yellow Fluor-

escent Protein (EYFP) constructs were kindly provided by Dr. P.

Macchi (University of Trento, Italy). For gene silencing experiments

the following siRNA duplexes were used: 5-GCAAAGCCCA-GACGAGCCUdTdT-3 for mouse TDP-43; siGenome Non-Target-ing siRNA #2 (Dharmacon, Lafayette, CO, USA) against the rey

luciferase gene as a non-specic control.

ImmunohistochemistryParafn-embedded 8 lm-thick sections from cervical spinal cord ofthree sporadic ALS patients were processed and re-hydrated

following a standard protocol. After pre-treatment with 10 mM

sodium citrate buffer pH6 for 20 min at 80C, tissue sections werepermeabilized with 0.3% Triton X-100 and blocked with 10%

normal goat serum for 20 min. Double staining with TDP-43

(1 : 200) and TIAR (1 : 60, BD Transduction Laboratories) or HuR

(1 : 50, Santa Cruz Biotechnology) antibodies was performed

overnight at 4C in phosphate buffered saline and Alexa Fluor-conjugated secondary antibodies (1 : 500) were used for detection.

Nuclei were visualized by DAPI staining. As a negative control,

primary antibodies were replaced by normal goat serum. Slides were

acquired with a confocal microscope (LSM510 META, Zeiss, Jena,

Germany).

Results

TDP-43 forms stress granules after arsenite treatmentWe investigated whether TDP-43, like other RBPs, is able toassemble into SGs in condition of oxidative stress by treatingthe immortalized motoneuron-like NSC34 cells with 0.5 mMarsenite for 30 min as previously described (Kedersha andAnderson 2007). As SG markers in immunouorescenceassays we used antibodies against TIAR, which in physio-logical conditions shows both a nuclear and cytosolicdistribution (Fig. 1a), and HuR which is predominantlynuclear, like TDP-43 (Fig. 1b). After arsenite treatment weobserved the formation of cytoplasmic foci which stainedpositive for TIAR (80% of cells) and HuR (60% of cells). Asub-group of cells with TIAR or HuR-positive SGs (30% and50%, respectively) showed co-localization also with TDP-43protein in cytoplasmic granules (Fig. 1a and b). To furtherdemonstrate that TDP-43-positive foci were SGs, we incu-bated NSC34 cells with arsenite in the presence of twodistinct pharmacological inhibitors of translation, emetineand puromycin, known to prevent or favour SG formation,respectively (Kedersha and Anderson 2007). Induction ofoxidative stress in the presence of emetine was not able totrigger SG assembly (Fig. 1c), whilst with puromycin

pre-conditioning we observed the formation of SGs whichstained positive for HuR and TDP-43 proteins (Fig. 1d). Celltreatment with a milder dose of arsenite for a prolongedperiod of time (15 lM for 20 h) did not lead to the formationof SGs, but to a diffuse redistribution of TDP-43 protein inthe cytoplasm (data not shown).

TDP-43 distributes into the cytoplasm following oxidativestressWe conrmed the immunouorescence data by biochemicalanalyses of NSC34 protein extracts after arsenite insult.Western blot assays revealed no change in the total amount ofTDP-43 protein after induction of oxidative stress, as well asof the other two SG markers HuR and TIAR (Fig. 2a). Whatwe observed was a redistribution of the TDP-43 protein froma main nuclear localization to a cytoplasmic one (Fig. 2b).TIA-1 and TIAR-containing RNP complexes were recoveredby specic immunoprecipitation from the cytosolic fractionand probed for the presence of TDP-43 and HuR RBPs.Arsenite treatment signicantly increased the association ofthe two proteins with both TIA-1 and TIAR SG markers inthe cytoplasm of NSC34 cells (Fig. 2c).

TDP-43-positive SGs are induced by distinct cellular insultsWe studied whether other cellular insults, already reported tofavour SG assembly, were also able to change TDP-43 sub-cellular distribution. We observed that TDP-43 co-localizedwith the SG markers TIAR and HuR in granules after 30-minexposure of NSC34 cells to heat shock (Figs 3a,b and S1)and that pre-treatment with emetine abolished the formationof such cytoplasmic structures (Fig. 3c). Recently, also thepharmacological inhibition of the ubiquitin-proteasome sys-tem was shown to induce SG assembly (Mazroui et al.2007). We found that treatment of NSC34 cells with thespecic ubiquitin-proteasome system inhibitor MG132 pro-moted the recruitment of TDP-43 protein into cytoplasmicfoci which stained positive for the HuR marker (Fig. S1).Again, the use of the two translational inhibitors, puromycinand emetine, had opposite effects on SG formation and TDP-43 was present in granules only when cells were incubatedwith puromycin before treatment with MG132 (Fig. S1).

TDP-43 protein is specifically recruited to SGs and not toP-bodiesSeveral RBPs have been described to participate to SGassembly in condition of environmental stress (Anderson andKedersha 2006), although it is not clear if this recruitment isspecic or common to all RBPs. To address this issue weanalyzed the cell distribution of another RBP, Nova1, afterinducing oxidative stress by arsenite treatment. Nova1protein, which is an important alternative splicing factor inneurons (Jensen et al. 2000), neither changed its localizationnor co-localized with the SG marker TIAR in condition ofoxidative insult (Fig. 4a).

2009 The AuthorsJournal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 10511061

TDP-43 is a novel component of SGs | 1053

We also investigated whether TDP-43 was present in P-bodies, constitutive cytoplasmic RNP complexes whichcontrol mRNA fate and degradation. NSC34 cells weretransiently co-transfected with a Green Fluorescent Protein(GFP)-tagged plasmid encoding for the P-bodies markerDcp1 (Decapping enzyme 1) and a FLAG-tagged TDP-43construct. Recombinant TDP-43 protein did not show co-localization with Dcp1 both before and after arsenitetreatment, being TDP-43 mainly nuclear in unstressed con-dition and being present in distinct cytoplasmic punctategranules after induction of oxidative stress (Fig. 4b).

Both RRM1 domain and C-terminal region are necessaryfor TDP-43 aggregation in SGsWe used distinct deletion constructs to identify the amino-acidic regions responsible for TDP-43 assembly into SGs incondition of oxidative stress (Fig. 5a).The mutant DRRM1 protein, lacking the entire RRM1

domain responsible for target RNA binding, distributed in

discrete intra-nuclear bodies in physiological conditions asalready described (Ayala et al. 2008). After the arseniteinsult was applied, the mutant TDP-43 DRRM1 protein failedto shuttle into the cytoplasm where TIAR-positive SGs wereclearly evident (Fig. 5b).When we used two different FLAG-tagged constructs, 1

315 and DC (carrying residues 1216), with a partial and fulldeletion, respectively, of the TDP-43 C-terminal region, asimilar sub-cellular distribution of the two truncated proteinswas observed in untreated NSC34 cells. Both mutant proteinsshowed a variable localization, being present either exclu-sively in the nucleus or also in the cytoplasm (Fig. 5c and d),but their response to arsenite treatment was completelydifferent. In fact, the TDP-43 1315 protein formedcytoplasmic foci, which stained positive for the SG markerTIAR, in about 35% of the transfected cells (Fig. 5c), whilethe DC truncated protein was not able to assemble into SGsfollowing the cellular insult (Fig. 5d). These data indicatethat both TDP-43 RRM1 and C-terminal region, in particular

Fig. 1 Recruitment of TDP-43 protein into

SGs after arsenite treatment in vitro. (a)

Immunofluorescence images of TIAR

(green) and TDP-43 (red) proteins in un-

treated (Untr) and arsenite-treated (Ars)

NSC34 cells. Merged image shows co-

localization signals in cytoplasmic SGs after

induction of oxidative stress. (b) Immuno-

cytochemistry of HuR (green) and TDP-43

(red) proteins in untreated and Ars-treated

cells. The concurrent use of arsenite

and the translation inhibitors emetine (c)

and puromycin (d) prevented or favoured

SG formation, respectively. Nuclei were

counter-stained by DAPI (blue). Scale

bar, 10 lm.



the aminoacidic interval spanning from residue 216 to 315(Fig. 5a), are necessary for the recruitment of TDP-43 intoSGs in conditions of oxidative stress.

TDP-43 is neither an essential component of SGs nor aneuroprotective factor in stress conditionTo further assess the role of TDP-43 in SG assembly and instress response, we transfected the FLAG-tagged full-lengthTDP-43 construct in NSC34 cells and analyzed its capacityto induce SGs in the absence of any environmental insult.The over-expression of the RBPs TIA-1 and TIAR waspreviously described to promote SG assembly in physiolog-

ical conditions (Gilks et al. 2004) and this was alsoconrmed in our motoneuronal cell model (Fig. S2). Onthe contrary, we found that the over-expression of TDP-43protein was not sufcient per se to induce SG formation. Aswe have already observed for the endogenous TDP-43protein, FLAG- and TIAR-positive SGs only formedfollowing treatment with arsenite (Fig. S2).To address the issue of TDP-43 function in oxidative stress

response, we silenced its expression in NSC34 cells by usinga specic siRNA duplex. The efciency of TDP-43 knock-down was evaluated by western blot analysis at two differenttime points, obtaining a 70% and a 90% reduction at 72 and96 h post-transfection, respectively (Fig. S3). When cellswere treated with arsenite, we observed that TIAR-positiveSGs were able to form in TDP-43-depleted cells (Fig. 6a), aswell as in cells transfected with an irrelevant control siRNA(Fig. 6b). We also evaluated whether the lack of TDP-43would affect cell survival in response to oxidative stress in atime-course assay. The viability of TDP-43-knocked downcells, exposed to arsenite for 30, 60, 90 and 120 min, wassimilar to the control siRNA-transfected cells (Fig. S4).

TDP-43-positive inclusions in human ALS motor neuronsdo not contain SG markersAs ALS-affected motor neurons show abnormal cytoplasmicprotein aggregates, degenerating mitochondria and an in-creased level of reactive oxygen species, we investigatedwhether SGs were present in spinal cord tissue of ALS patientsand whether the described pathological TDP-43 inclusionsalso contained SG markers. We analyzed the sub-cellulardistribution of the RBPs TIAR andHuR in autoptic spinal cordfrom three patients affected by sporadic ALS. By uorescentimmunostaining assays we observed the presence of TDP-43-positive inclusions in many affected motor neurons in all theanalyzed patients, with either lamentous (Fig. 7) or compactround shapes (data not shown). Such large cytoplasmicinclusions did not stain positive for the two SG markers TIARand HuR (Fig. 7a and b). Additionally, we observed a greatintra-patient variability in the sub-cellular distribution of theTDP-43 protein. In some motor neurons we observed aphysiological localization of TDP-43 showing a main nuclearstaining (Fig. 7c), while in others TDP-43 appeared com-pletely mislocalized in the cytoplasm although no largelamentous or round inclusions were evident (Fig. 7d).Interestingly in these cases TDP-43 distributed in small anddiscrete cytoplasmic granules which did not overlap witheither HuR or TIARRBPs (see enlargements in Fig. 7c and d).

Discussion

In this paper, we proved that TDP-43 is capable to respond toan environmental insult by assembling into stress granules(SGs), cytoplasmic ribonucleoprotein foci which sequestermRNAs, several RBPs and stalled translation initiation

Fig. 2 Biochemical analyses of NSC34 protein extracts after arsenite

insult. (a) Representative western blot showing the total content of

TDP-43, HuR and TIAR proteins in NSC34 control cells (Untr) and

after 30-min treatment with arsenite (Ars). a-tubulin was used for

sample normalization. (b) Immunoblot of TDP-43 protein in nuclear

and cytoplasmic fractions in control (Untr) and Ars-treated NSC34

cells. p84 and a-tubulin were used to check for nuclear and cyto-

plasmic fractionation, respectively. (c) Western blot of RNP complexes

immunopurified from cytoplasmic fractions with anti-TIA-1 and anti-

TIAR antibodies in control (Untr) and Ars-treated cells. Antibodies

against TDP-43 and HuR revealed an enrichment of these two pro-

teins in the cytoplasm in association to TIA-1 and TIAR after inducing

oxidative stress. Western blots with TIAR and TIA-1 served as a po-

sitive control for immunoprecipitation. The arrowhead indicates the

antibody light chain.



complexes to temporarily arrest protein synthesis as aprotective response to cellular stress.The genetic ndings in ALS patients of causative muta-

tions in three proteins involved in RNA processing, namelySenataxin, TDP-43 and Fusion/Translocated in LipoSarcoma(FUS/TLS) (Chen et al. 2004; Sreedharan et al. 2008;Kwiatkowski et al. 2009; Vance et al. 2009), have focusedthe attention on the complex molecular mechanisms regulat-ing gene expression at post-transcriptional level as potentialpathogenic clues. Post-transcriptional control of mRNA fateis known to play an important role both during thedevelopment of the nervous system and for the maintenanceof neural activities in the adult brain. RBP-mediatedregulatory mechanisms allow a precise spatio-temporalcontrol of mRNA translation, associated to transport andsubcellular compartmentalization of mRNAs in dendrites andaxons (Besse and Ephrussi 2008), so that disruption of such

activities is supposed to severely impair neuronal cellmetabolism.So far, TDP-43 functions have been mainly associated to

alternative splicing processes and transcriptional activities(Bose et al. 2008; Buratti and Baralle 2008). However, TDP-43 protein has been recently demonstrated to have multipleroles in the regulation of mRNA fate in neuronal cells, suchas transcript stabilization and activity-dependent transport todendrites (Strong et al. 2007; Wang et al. 2008). In this view,the sequestration of TDP-43 in pathological aggregates issupposed to determine a loss of function of the protein withsevere consequences on mRNA metabolism and post-transcriptional regulation of gene expression.Our results show that in the motoneuron-like NSC34 cells

different types of environmental insults, ranging fromoxidative stress to proteasome inhibition and heat shock,are able to induce the assembly of TIAR- and HuR-positive

Fig. 3 TDP-43 forms SGs in response to

heat shock. Immunofluorescence images of

TIAR (green) and TDP-43 (red) protein

localization before (a) and after (b) expos-

ing NSC34 cells to a heat shock for 30 min

at 44C. (c) The presence of the transla-tional inhibitor emetine prevented SG

formation. Nuclei were counter-stained by

DAPI (blue). Scale bar, 10 lm.

Fig. 4 TDP-43 is a component of SGs, but

not of P-bodies. (a) Distribution of TIAR

(green) and Nova1 (red) proteins before

(Untr) and after exposing NSC34 cells to

arsenite (Ars) treatment. (b) Immunocyto-

chemistry of NSC34 cells after 48-h co-

transfection of the GFP-tagged plasmid

encoding for the P-bodies marker Dcp1

(green) and the FLAG-tagged TDP-43

construct (red) before and after inducing

oxidative stress. Nuclei were visualized by

DAPI (blue). Scale bar, 10 lm.



SGs, a subset of which also contains TDP-43 protein. Thesedata further indicate that TDP-43 has also a role in thecontrol of mRNA fate in the cytoplasmic compartment. It isinteresting that also FUS/TLS, another RBP causative of 5%of familial ALS cases (Lagier-Tourenne and Cleveland2009), forms SGs in conditions of oxidative stress (Anders-son et al. 2008). Indeed, several RBPs, enzymes andcytoskeletal elements have already been described to becomponents of SGs (Anderson and Kedersha 2008; Tsaiet al. 2009). However, we demonstrated that not every RBPis necessarily included in these structures after environmentalstress. In fact Nova1, a neuron-specic splicing factoressential for the development of the motor system and forthe survival of motor neurons (Jensen et al. 2000), did notchange its sub-cellular distribution in response to arseniteinsult. This observation is particularly interesting as bothNova1 and TDP-43 are mainly nuclear proteins regulatingalternative splicing of pre-mRNA, but also shuttle actively tothe cytoplasm (Ayala et al. 2008; Ratti et al. 2008; Wanget al. 2008). Therefore, TDP-43 RBP is specically recruitedto SGs but is not an essential component. This was shownboth by over-expression and gene silencing experiments andby the fact that TDP-43-positive SGs are present only in asubset of SG-forming NSC34 cells. Moreover, we demon-strated that TDP-43 is not able to inuence cell viability

Fig. 5 The RRM1 domain and the C-ter-

minal 216315 aminoacidic region are both

required for TDP-43 targeting to SGs. (a) A

schematic representation of TDP-43 con-

structs is shown. FLAG-tagged deleted

(DRRM1, 1315 and DC) plasmids wereused for transient transfection of NSC34

cells. (bd) Immunofluorescence images of

transfected NSC34 cells expressing the

different recombinant TDP-43 proteins in

physiological and oxidative (Ars) conditions.

TIAR (green) and FLAG (red) antibodies

were used for detecting SGs and trans-

fected cells, respectively. TIAR and FLAG

co-localization signals are shown (merge),

while nuclei are stained in blue (DAPI).

Scale bar, 10 lm.

Fig. 6 TDP-43 is not necessary for SG formation. Double immuno-

fluorescence staining of NSC34 cells knocked-down for TDP-43

(siTDP-43) and, as a control, for firefly luciferase (siCtrl) after treat-

ment with 0.5 mM arsenite for 30 min. TIAR (green) and TDP-43 (red)

antibodies were used. Arrows indicate TDP-43-depleted cells. Merged

images are shown and nuclei visualized by DAPI staining (blue). Scale

bar, 10 lm.



following stress conditions, supporting the idea that TDP-43participates to regulatory mechanisms of translational arrest,but it is not one of the promoting factors.Our data also show that TDP-43 is not involved in

targeting bound transcripts to the degradation machinery ofP-bodies, but rather in provisionally silencing them inpresence of a toxic insult. TDP-43 is known to specicallyrecognize and bind repeated (UG)n motifs and this may helpexplain why a low abundance of TDP-43-positive SGs isobserved, as it likeky reects the low frequency of (UG)n-containing mature mRNA. Few target mRNA have beenidentied and validated so far for TDP-43 (Buratti andBaralle 2008; Wang et al. 2008) and the future identicationof all its targets is expected to unravel the biological role ofthis RBP in neuronal cells and, at the same time, itspathomechanism in neurodegenerative diseases.In our attempt to dene the aminoacidic region responsible

for TDP-43 assembly into SGs, we found that the TDP-43protein lacking the RRM1 domain failed to shuttle from thenucleus to the cytoplasm after stress. The RRM1 domain isresponsible for target mRNA binding, in particular for therecognition of UG repeat motifs (Buratti and Baralle 2001)so that its disruption may determine a defective mRNA

binding and protein function. In fact, in physiologicalcondition TDP-43 DRRM1 mutant distributes in the nucleusin abnormal granular structures that may be associated withchanges in chromatin distribution of the DRRM1 protein(Fig. 5c; Ayala et al. 2008).An active and efcient shuttling from the nucleus to the

cytoplasm and vice-versa is necessary for the proper activityof many RBPs, including FUS/TLS and the EmbryonicLethal Abnormal Vision (ELAV) family member HuR. LikeTDP-43, FUS/TLS and HuR show a main nuclear distribu-tion in physiological conditions, but they also function asmRNA transport and stabilizing factors in the cytoplasm(Fujii and Takumi 2005; Hinman and Lou 2008). Interest-ingly, in familial ALS, mutant FUS/TLS protein shows anabnormal redistribution in the cytoplasm (Kwiatkowski et al.2009; Vance et al. 2009) similarly to what has been observedwith HuR in ALS animal and cellular models (Lu et al.2007), suggesting a potential perturbation of their originalfunction.A mislocalization of TDP-43 protein from the nucleus to

the cytoplasm already occurs in physiological conditionswith mutant forms lacking both the C-terminus and the N-terminal NLS (Nuclear Localization Signal) region (Ayala

(a)

(c)

(c)

(b)

Fig. 7 TDP-43 inclusions do not stain positive for SG markers in ALS

brain tissues. Human spinal cord tissue of patients with ALS were

stained with the SG markers TIAR (a, red) or HuR (b, red) together

with TDP-43 (green). Merged images (merge) show no distribution of

TIAR and HuR RBPs in TDP-43-positive filamentous inclusions.

Enlargements of the indicated areas are shown. Further magnification

on the z-plan of the granules pointed by the arrow is shown in the

inset. (c) Some motor neurons showed a normal distribution of TDP-43

(green) and HuR (red) proteins in the nucleus and a minor localization

in discrete granules in the cytoplasm with no evident co-localization

signals (merge and inset images). (d) Immunofluorescence image of

an affected motor neuron where TDP-43 (green) was completely

mislocalized in the cytoplasm in a granule-like distribution, without

forming inclusions. TIAR staining (red) does not overlap TDP-43 sig-

nals (merge and inset images). The asterisk (*) indicates lipofuscin

granules. Scale bar, 20 lm.



et al. 2008; Johnson et al. 2008; Winton et al. 2008; Nonakaet al. 2009). More importantly, we have found that, besidesthe RRM1 domain, also the selective lack of 100 aminoacids(216315) in the C-terminal region determines the failure ofTDP-43 to assemble into SGs. This glycine-rich C-terminalregion is highly conserved along phylogenesis and issupposed to be involved in protein-protein interactions.However, the ability of TDP-43 to aggregate into SGs seemsnot to be related to its interaction with heterogeneous RNPA/B proteins, essential for its splicing activity, as the bindingregion maps to residues 321366 (DAmbrogio et al. 2009).Interestingly, the C-terminal domain represents the mainlymutated region in ALS patients, although distinct mutantTDP-43 proteins (A382T, Q331K and M337V) distributenormally in the nucleus (Sreedharan et al. 2008), suggestingthat alteration in TDP-43 sub-cellular localization is deter-mined by multiple aminoacidic changes and/or additionalcontributing factors.Several pathogenic mechanisms have been demonstrated

to trigger motor neuron death in ALS, although theprimary or secondary role of these events in the patho-genesis of the disease is not clear yet, and the earlycausative processes still need to be claried. As thesemechanisms include oxidative stress, mithocondrial defects,protein aggregation, and proteasome impairment, weinvestigated if SGs may be implicated in ALS pathogen-esis. In human ALS motor neurons we failed to detectTIAR- and HuR-positive granules or inclusions co-local-izing with TDP-43 and, also in conditions of a mainphysiological TDP-43 distribution in the nucleus, we didnot observe overlapping signals with the SG markersTIAR and HuR in the cytoplasm. A previous paperreported the presence of TIA-1 and PolyA-Binding Protein1 (PABP-1), another RBP included in SGs, in the RNA-positive basophilic inclusions from patients presenting withadult-onset atypical motor neuron disease (Fujita et al.2008), conrming our hypothesis that translational arrestmechanisms are somehow involved in neuronal death invivo. The observation that SG structures are not associatedonly to environmental insults in vitro, but may form andbe detectable also in disease conditions, has already beenreported in animal models of brain ischemia and sciaticnerve axotomy (DeGracia et al. 2008; Moisse et al. 2009)as well as in tumors exposed to radiation-induced hypoxia(Moeller et al. 2004).One possible explanation to the fact that in ALS human

motor neurons SGs are not present is that such macromo-lecular complexes may assemble as a very early response toan environmental stress and that at the endstage of theneurodegenerative process other compensative mechanismsmay have occurred. On the other hand, we may alsohypothesize that in ALS affected tissues stressful processesare progressive and not acute enough to provoke SGformation, like in ischemia and axotomy conditions. We

have experimental evidence that TDP-43 does not formSGs, but distributes diffusely in the cytoplasm whentreating NSC34 cells with milder doses of arsenite forprolonged periods of time to mimic a chronic insult(unpublished results). Our ndings, together with theobserved cytoplasmic mislocalization of HuR in mutantSuperoxide dismutase 1 (SOD1) transgenic mice at veryearly stages of the disease (Lu et al. 2007), support the ideathat impairment of post-transcriptional regulatory mecha-nisms, including mRNA stabilization and translation, maybe actively involved in ALS pathogenesis and/or progres-sion. However, the reason of such a specic TDP-43, andnot HuR, pathological aggregation in human ALS braintissues needs further investigation. Although preliminarydata show the absence of FUS/TLS-positive cytoplasmicinclusions in sporadic ALS patients (Kwiatkowski et al.2009; Vance et al. 2009), a full comprehension of thepotential interplay of this protein with TDP-43 and HuRwill help elucidating the role of RBP-mediated regulation ofgene expression in neurodegeneration and motor neurondiseases.

Acknowledgements

We thank prof. F.E. Baralle for critically reading the manuscript, E.

Giovannini for her technical help and G. Bassell. This work was

nancially supported by the Italian Ministry of Health (Malattie

Neurodegenerative, ex Art.56, n.533F/N1), Fondazione Cariplo

(Grant n.2008.2307) and a donation of Peviani Family. EB is

supported by Telethon and Eurasnet.

Supporting information

Additional Supporting Information may be found in the online

version of this article:

Figure S1. TDP-43 is recruited to SGs in response to differentenvironmental insults.

Figure S2. TDP-43 does not promote SG formation.Figure S3. TDP-43 gene silencing in NSC34 cells.Figure S4. Depletion of TDP-43 does not impair cell viability

following oxidative stress.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such materials are

peer-reviewed and may be re-organized for online delivery, but are

not copy-edited or typeset. Technical support issues arising from

supporting information (other than missing les) should be

addressed to the authors.

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TDP-43 is Recruited to Stress Granules in Conditions of Oxidative Insult

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