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JOURNAL OF NEUROCHEMISTRY

| 2009 | 111 | 10511061

doi: 10.1111/j.1471-4159.2009.06383.x

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*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

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), ribonucleoprotein 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 inclusions. 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 protein, stress granules, Transactive response DNA-binding
protein 43.
J. Neurochem. (2009) 111, 10511061.

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


an 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 Cterminal tail for protein-protein interaction (Ayala et al.
2005). The biological role of TDP-43 is associated both to
transcriptional regulation and to post-transcriptional control
of RNA processing, ranging from splicing to mRNA
stabilization and transport (Buratti and Baralle 2008).
TDP-43 has recently emerged as the major neuropathological hallmark of amyotrophic lateral sclerosis (ALS) and
frontotemporal lobar degeneration (FTLD) (Arai et al. 2006;
Neumann et al. 2006). Although distinct neuronal populations
and brain areas selectively degenerate in ALS and in FTLD,

these two disorders represent a clinical continuum as FTLD


patients may also develop motor neuron disease and cognitive
decits 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 Istituto Auxologico Italiano, Via Zucchi, 18 20095 Cusano Milanino,
Milan, Italy. E-mail: antonia.ratti@unimi.it
1
Vincenzo Silani and Antonia Ratti are Joint Senior Authors.
Abbreviations used: ALS, amyotrophic lateral sclerosis; FTLD,
frontotemporal lobar degeneration; FUS/TLS, Fusion/Translocated in
LipoSarcoma; HuR, Hu R antigen; P-bodies, processing bodies; RBP,
RNA-binding protein; RNP, ribonucleoprotein; RRM, RNA-recognition
motif; SG, stress granule; TDP-43, Transactive response DNA-binding
protein 43; TIA-1, T cell-induced antigen 1; TIAR, TIA-related.

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1052 | C. Colombrita et al.

inclusions have been identied also in Alzheimers, Parkinsons and Huntingtons diseases, suggesting it may be a
common marker for many neurodegenerative disorders (Amador-Ortiz et al. 2007; Nakashima-Yasuda et al. 2007;
Schwab et al. 2008). The fact that TDP-43 protein may be
primarily and specically involved in ALS onset has recently
emerged from genetic studies showing TARDBP gene mutations in familial and sporadic ALS cases (Sreedharan et al.
2008; Corrado et al. 2009). Whether TDP-43 protein aggregation is a pathogenic event that triggers neuronal degeneration or whether TDP-43-positive inclusions are the
consequence of a neuroprotective mechanism still remains
an issue to be addressed.
In condition of oxidative stress the regulation of gene
expression at post-transcriptional level, mediated by RBPs, is
known to be impaired. In particular, when a sub-lethal
oxidative stress is induced in vitro, there is an immediate
block of the translation machinery with sequestration of the
actively-translating mRNAs and distinct RBPs to cytoplasmic foci, called stress granules (SGs) (Anderson and
Kedersha 2008). SGs represent a protective mechanism to
bypass the cellular insult as the majority of mRNAs is
silenced in these macromolecular structures in stalled 48S
ribosomal complexes, while only specic and essential
transcripts (i.e. Hsp70) are maintained in active translation
(Anderson and Kedersha 2002; Kedersha and Anderson
2002). During stress, SGs are in dynamic equilibrium
between polysomes and processing bodies (P-bodies), the
latter being constitutive RNP complexes where both mRNA
degradation and microRNA-mediated translational arrest
take place (Kedersha et al. 2005, 2008). It is experimentally
proven that once the insult is removed, these RNP complexes
soon disaggregate in favour of a parallel polysome reassembly and mRNA translation re-initiation. The molecular
mechanisms and the signalling pathways triggering SG
formation have been characterized, as well as the nature of
distinct cellular insults which can induce these structures,
including oxidative stress, proteasome inhibition, osmotic
and 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 of
cellular insult. T cell-induced antigen 1 (TIA-1) and TIAR
(TIA-related) are the essential RBPs which promote SG
assembly because of their prion-like domains that favour
aggregation of other RBPs/proteins in granules together with
their target mRNAs. SGs also contain PolyA-Binding Protein
1 (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 complex, early translation initiation factors, microRNA-associated
Argonaute proteins, p54/Rck helicase, XRN1 exonuclease
and cytoskeletal proteins (Anderson and Kedersha 2008).

The aim of our study was to investigate whether the RBP


TDP-43 participates to the assembly of SGs in condition of
cellular stress in a motoneuronal cell line and whether such
cytoplasmic RNP complexes are also present in the spinal
cord and/or in the TDP-43-positive pathological inclusions of
ALS patients.

Experimental procedures
Cell culture and treatments
The 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 to
0.5 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 h
as reported (Mazroui et al. 2007). All reagents were purchased from
Sigma (Milan, Italy). For heat shock experiments, cells were
incubated at 44C for 30 min overlaid with mineral oil.
Immunocytochemistry
Cells 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-tagged
secondary 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 (Calbiochem, La Jolla, CA, USA) and acquired with a wideeld microscope
(DMIRE2/HCS, Leica Microsystems, Wetzlar, Germany).
Protein extraction, immunoprecipitation and western blotting
NSC34 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 supernatants were collected. Proteins from cytoplasmic and nuclear
fractions were obtained with the ProteoJETTM kit (Fermentas,
Milan, Italy) following the manufacturers instructions. For immunoprecipitation experiments, 30 lL protein G Sepharose-beads precoated for 6 h with 1.5 lg of the selected antibody were incubated
with 300 lg cytoplasmic protein lysate in NT2 buffer [50 mM TrisHCl 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 Santa
Cruz Biotechnology, Santa Cruz, CA, USA), TDP-43 and p84
(Abcam, Cambridge, UK) antibodies.

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

TDP-43 is a novel component of SGs | 1053

Plasmid constructs, siRNA and transfections


NSC34 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 were
previously described (Ayala et al. 2008). The Dcp1-enhanced Green
Fluorescent Protein (EGFP) and TIA-1-enhanced Yellow Fluorescent 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-GCAAAGCCCAGACGAGCCUdTdT-3 for mouse TDP-43; siGenome Non-Targeting siRNA #2 (Dharmacon, Lafayette, CO, USA) against the rey
luciferase gene as a non-specic control.
Immunohistochemistry
Parafn-embedded 8 lm-thick sections from cervical spinal cord of
three 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 were
permeabilized 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 Fluorconjugated 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 treatment
We investigated whether TDP-43, like other RBPs, is able to
assemble into SGs in condition of oxidative stress by treating
the immortalized motoneuron-like NSC34 cells with 0.5 mM
arsenite for 30 min as previously described (Kedersha and
Anderson 2007). As SG markers in immunouorescence
assays we used antibodies against TIAR, which in physiological conditions shows both a nuclear and cytosolic
distribution (Fig. 1a), and HuR which is predominantly
nuclear, like TDP-43 (Fig. 1b). After arsenite treatment we
observed the formation of cytoplasmic foci which stained
positive for TIAR (80% of cells) and HuR (60% of cells). A
sub-group of cells with TIAR or HuR-positive SGs (30% and
50%, respectively) showed co-localization also with TDP-43
protein in cytoplasmic granules (Fig. 1a and b). To further
demonstrate that TDP-43-positive foci were SGs, we incubated NSC34 cells with arsenite in the presence of two
distinct pharmacological inhibitors of translation, emetine
and puromycin, known to prevent or favour SG formation,
respectively (Kedersha and Anderson 2007). Induction of
oxidative stress in the presence of emetine was not able to
trigger SG assembly (Fig. 1c), whilst with puromycin

pre-conditioning we observed the formation of SGs which


stained positive for HuR and TDP-43 proteins (Fig. 1d). Cell
treatment with a milder dose of arsenite for a prolonged
period of time (15 lM for 20 h) did not lead to the formation
of SGs, but to a diffuse redistribution of TDP-43 protein in
the cytoplasm (data not shown).
TDP-43 distributes into the cytoplasm following oxidative
stress
We conrmed the immunouorescence data by biochemical
analyses of NSC34 protein extracts after arsenite insult.
Western blot assays revealed no change in the total amount of
TDP-43 protein after induction of oxidative stress, as well as
of the other two SG markers HuR and TIAR (Fig. 2a). What
we observed was a redistribution of the TDP-43 protein from
a main nuclear localization to a cytoplasmic one (Fig. 2b).
TIA-1 and TIAR-containing RNP complexes were recovered
by specic immunoprecipitation from the cytosolic fraction
and probed for the presence of TDP-43 and HuR RBPs.
Arsenite treatment signicantly increased the association of
the two proteins with both TIA-1 and TIAR SG markers in
the cytoplasm of NSC34 cells (Fig. 2c).
TDP-43-positive SGs are induced by distinct cellular insults
We studied whether other cellular insults, already reported to
favour SG assembly, were also able to change TDP-43 subcellular distribution. We observed that TDP-43 co-localized
with the SG markers TIAR and HuR in granules after 30-min
exposure of NSC34 cells to heat shock (Figs 3a,b and S1)
and that pre-treatment with emetine abolished the formation
of such cytoplasmic structures (Fig. 3c). Recently, also the
pharmacological inhibition of the ubiquitin-proteasome system was shown to induce SG assembly (Mazroui et al.
2007). We found that treatment of NSC34 cells with the
specic ubiquitin-proteasome system inhibitor MG132 promoted the recruitment of TDP-43 protein into cytoplasmic
foci which stained positive for the HuR marker (Fig. S1).
Again, the use of the two translational inhibitors, puromycin
and emetine, had opposite effects on SG formation and TDP43 was present in granules only when cells were incubated
with puromycin before treatment with MG132 (Fig. S1).
TDP-43 protein is specifically recruited to SGs and not to
P-bodies
Several RBPs have been described to participate to SG
assembly in condition of environmental stress (Anderson and
Kedersha 2006), although it is not clear if this recruitment is
specic or common to all RBPs. To address this issue we
analyzed the cell distribution of another RBP, Nova1, after
inducing oxidative stress by arsenite treatment. Nova1
protein, which is an important alternative splicing factor in
neurons (Jensen et al. 2000), neither changed its localization
nor co-localized with the SG marker TIAR in condition of
oxidative insult (Fig. 4a).

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1054 | C. Colombrita et al.

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 untreated (Untr) and arsenite-treated (Ars)
NSC34 cells. Merged image shows colocalization signals in cytoplasmic SGs after
induction of oxidative stress. (b) Immunocytochemistry 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.

We also investigated whether TDP-43 was present in Pbodies, constitutive cytoplasmic RNP complexes which
control mRNA fate and degradation. NSC34 cells were
transiently co-transfected with a Green Fluorescent Protein
(GFP)-tagged plasmid encoding for the P-bodies marker
Dcp1 (Decapping enzyme 1) and a FLAG-tagged TDP-43
construct. Recombinant TDP-43 protein did not show colocalization with Dcp1 both before and after arsenite
treatment, being TDP-43 mainly nuclear in unstressed condition and being present in distinct cytoplasmic punctate
granules after induction of oxidative stress (Fig. 4b).
Both RRM1 domain and C-terminal region are necessary
for TDP-43 aggregation in SGs
We used distinct deletion constructs to identify the aminoacidic regions responsible for TDP-43 assembly into SGs in
condition 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 as


already described (Ayala et al. 2008). After the arsenite
insult was applied, the mutant TDP-43 DRRM1 protein failed
to shuttle into the cytoplasm where TIAR-positive SGs were
clearly evident (Fig. 5b).
When we used two different FLAG-tagged constructs, 1
315 and DC (carrying residues 1216), with a partial and full
deletion, respectively, of the TDP-43 C-terminal region, a
similar sub-cellular distribution of the two truncated proteins
was observed in untreated NSC34 cells. Both mutant proteins
showed a variable localization, being present either exclusively in the nucleus or also in the cytoplasm (Fig. 5c and d),
but their response to arsenite treatment was completely
different. In fact, the TDP-43 1315 protein formed
cytoplasmic foci, which stained positive for the SG marker
TIAR, in about 35% of the transfected cells (Fig. 5c), while
the DC truncated protein was not able to assemble into SGs
following the cellular insult (Fig. 5d). These data indicate
that both TDP-43 RRM1 and C-terminal region, in particular

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

TDP-43 is a novel component of SGs | 1055

ical conditions (Gilks et al. 2004) and this was also


conrmed in our motoneuronal cell model (Fig. S2). On
the contrary, we found that the over-expression of TDP-43
protein was not sufcient per se to induce SG formation. As
we have already observed for the endogenous TDP-43
protein, FLAG- and TIAR-positive SGs only formed
following 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 using
a specic siRNA duplex. The efciency of TDP-43 knockdown was evaluated by western blot analysis at two different
time points, obtaining a 70% and a 90% reduction at 72 and
96 h post-transfection, respectively (Fig. S3). When cells
were treated with arsenite, we observed that TIAR-positive
SGs were able to form in TDP-43-depleted cells (Fig. 6a), as
well as in cells transfected with an irrelevant control siRNA
(Fig. 6b). We also evaluated whether the lack of TDP-43
would affect cell survival in response to oxidative stress in a
time-course assay. The viability of TDP-43-knocked down
cells, exposed to arsenite for 30, 60, 90 and 120 min, was
similar to the control siRNA-transfected cells (Fig. S4).

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 cytoplasmic fractionation, respectively. (c) Western blot of RNP complexes
immunopurified from cytoplasmic fractions with anti-TIA-1 and antiTIAR antibodies in control (Untr) and Ars-treated cells. Antibodies
against TDP-43 and HuR revealed an enrichment of these two proteins in the cytoplasm in association to TIA-1 and TIAR after inducing
oxidative stress. Western blots with TIAR and TIA-1 served as a positive control for immunoprecipitation. The arrowhead indicates the
antibody light chain.

the aminoacidic interval spanning from residue 216 to 315


(Fig. 5a), are necessary for the recruitment of TDP-43 into
SGs in conditions of oxidative stress.
TDP-43 is neither an essential component of SGs nor a
neuroprotective factor in stress condition
To further assess the role of TDP-43 in SG assembly and in
stress response, we transfected the FLAG-tagged full-length
TDP-43 construct in NSC34 cells and analyzed its capacity
to induce SGs in the absence of any environmental insult.
The over-expression of the RBPs TIA-1 and TIAR was
previously described to promote SG assembly in physiolog-

TDP-43-positive inclusions in human ALS motor neurons


do not contain SG markers
As ALS-affected motor neurons show abnormal cytoplasmic
protein aggregates, degenerating mitochondria and an increased level of reactive oxygen species, we investigated
whether SGs were present in spinal cord tissue of ALS patients
and whether the described pathological TDP-43 inclusions
also contained SG markers. We analyzed the sub-cellular
distribution of the RBPs TIAR and HuR in autoptic spinal cord
from three patients affected by sporadic ALS. By uorescent
immunostaining assays we observed the presence of TDP-43positive inclusions in many affected motor neurons in all the
analyzed patients, with either lamentous (Fig. 7) or compact
round shapes (data not shown). Such large cytoplasmic
inclusions did not stain positive for the two SG markers TIAR
and HuR (Fig. 7a and b). Additionally, we observed a great
intra-patient variability in the sub-cellular distribution of the
TDP-43 protein. In some motor neurons we observed a
physiological localization of TDP-43 showing a main nuclear
staining (Fig. 7c), while in others TDP-43 appeared completely mislocalized in the cytoplasm although no large
lamentous or round inclusions were evident (Fig. 7d).
Interestingly in these cases TDP-43 distributed in small and
discrete cytoplasmic granules which did not overlap with
either HuR or TIAR RBPs (see enlargements in Fig. 7c and d).

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

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1056 | C. Colombrita et al.

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) exposing NSC34 cells to a heat shock for 30 min
at 44C. (c) The presence of the translational inhibitor emetine prevented SG
formation. Nuclei were counter-stained by
DAPI (blue). Scale bar, 10 lm.

complexes to temporarily arrest protein synthesis as a


protective response to cellular stress.
The genetic ndings in ALS patients of causative mutations in three proteins involved in RNA processing, namely
Senataxin, 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 focused
the attention on the complex molecular mechanisms regulating gene expression at post-transcriptional level as potential
pathogenic clues. Post-transcriptional control of mRNA fate
is known to play an important role both during the
development of the nervous system and for the maintenance
of neural activities in the adult brain. RBP-mediated
regulatory mechanisms allow a precise spatio-temporal
control of mRNA translation, associated to transport and
subcellular compartmentalization of mRNAs in dendrites and
axons (Besse and Ephrussi 2008), so that disruption of such

activities is supposed to severely impair neuronal cell


metabolism.
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, TDP43 protein has been recently demonstrated to have multiple
roles in the regulation of mRNA fate in neuronal cells, such
as transcript stabilization and activity-dependent transport to
dendrites (Strong et al. 2007; Wang et al. 2008). In this view,
the sequestration of TDP-43 in pathological aggregates is
supposed to determine a loss of function of the protein with
severe consequences on mRNA metabolism and posttranscriptional regulation of gene expression.
Our results show that in the motoneuron-like NSC34 cells
different types of environmental insults, ranging from
oxidative stress to proteasome inhibition and heat shock,
are able to induce the assembly of TIAR- and HuR-positive

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) Immunocytochemistry of NSC34 cells after 48-h cotransfection 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.

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

TDP-43 is a novel component of SGs | 1057

Fig. 5 The RRM1 domain and the C-terminal 216315 aminoacidic region are both
required for TDP-43 targeting to SGs. (a) A
schematic representation of TDP-43 constructs is shown. FLAG-tagged deleted
(DRRM1, 1315 and DC) plasmids were
used 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 transfected 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 immunofluorescence staining of NSC34 cells knocked-down for TDP-43
(siTDP-43) and, as a control, for firefly luciferase (siCtrl) after treatment 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.

SGs, a subset of which also contains TDP-43 protein. These


data further indicate that TDP-43 has also a role in the
control of mRNA fate in the cytoplasmic compartment. It is
interesting that also FUS/TLS, another RBP causative of 5%
of familial ALS cases (Lagier-Tourenne and Cleveland
2009), forms SGs in conditions of oxidative stress (Andersson et al. 2008). Indeed, several RBPs, enzymes and
cytoskeletal elements have already been described to be
components of SGs (Anderson and Kedersha 2008; Tsai
et al. 2009). However, we demonstrated that not every RBP
is necessarily included in these structures after environmental
stress. In fact Nova1, a neuron-specic splicing factor
essential for the development of the motor system and for
the survival of motor neurons (Jensen et al. 2000), did not
change its sub-cellular distribution in response to arsenite
insult. This observation is particularly interesting as both
Nova1 and TDP-43 are mainly nuclear proteins regulating
alternative splicing of pre-mRNA, but also shuttle actively to
the cytoplasm (Ayala et al. 2008; Ratti et al. 2008; Wang
et al. 2008). Therefore, TDP-43 RBP is specically recruited
to SGs but is not an essential component. This was shown
both by over-expression and gene silencing experiments and
by the fact that TDP-43-positive SGs are present only in a
subset of SG-forming NSC34 cells. Moreover, we demonstrated that TDP-43 is not able to inuence cell viability

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1058 | C. Colombrita et al.

(a)

(b)

(c)

(c)

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 signals (merge and inset images). The asterisk (*) indicates lipofuscin
granules. Scale bar, 20 lm.

following stress conditions, supporting the idea that TDP-43


participates 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 of
P-bodies, but rather in provisionally silencing them in
presence of a toxic insult. TDP-43 is known to specically
recognize and bind repeated (UG)n motifs and this may help
explain why a low abundance of TDP-43-positive SGs is
observed, as it likeky reects the low frequency of (UG)ncontaining mature mRNA. Few target mRNA have been
identied and validated so far for TDP-43 (Buratti and
Baralle 2008; Wang et al. 2008) and the future identication
of all its targets is expected to unravel the biological role of
this RBP in neuronal cells and, at the same time, its
pathomechanism in neurodegenerative diseases.
In our attempt to dene the aminoacidic region responsible
for TDP-43 assembly into SGs, we found that the TDP-43
protein lacking the RRM1 domain failed to shuttle from the
nucleus to the cytoplasm after stress. The RRM1 domain is
responsible for target mRNA binding, in particular for the
recognition of UG repeat motifs (Buratti and Baralle 2001)
so that its disruption may determine a defective mRNA

binding and protein function. In fact, in physiological


condition TDP-43 DRRM1 mutant distributes in the nucleus
in abnormal granular structures that may be associated with
changes 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 activity
of many RBPs, including FUS/TLS and the Embryonic
Lethal Abnormal Vision (ELAV) family member HuR. Like
TDP-43, FUS/TLS and HuR show a main nuclear distribution in physiological conditions, but they also function as
mRNA transport and stabilizing factors in the cytoplasm
(Fujii and Takumi 2005; Hinman and Lou 2008). Interestingly, in familial ALS, mutant FUS/TLS protein shows an
abnormal redistribution in the cytoplasm (Kwiatkowski et al.
2009; Vance et al. 2009) similarly to what has been observed
with HuR in ALS animal and cellular models (Lu et al.
2007), suggesting a potential perturbation of their original
function.
A mislocalization of TDP-43 protein from the nucleus to
the cytoplasm already occurs in physiological conditions
with mutant forms lacking both the C-terminus and the Nterminal NLS (Nuclear Localization Signal) region (Ayala

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

TDP-43 is a novel component of SGs | 1059

et al. 2008; Johnson et al. 2008; Winton et al. 2008; Nonaka


et al. 2009). More importantly, we have found that, besides
the RRM1 domain, also the selective lack of 100 aminoacids
(216315) in the C-terminal region determines the failure of
TDP-43 to assemble into SGs. This glycine-rich C-terminal
region is highly conserved along phylogenesis and is
supposed to be involved in protein-protein interactions.
However, the ability of TDP-43 to aggregate into SGs seems
not to be related to its interaction with heterogeneous RNP A/
B proteins, essential for its splicing activity, as the binding
region maps to residues 321366 (DAmbrogio et al. 2009).
Interestingly, the C-terminal domain represents the mainly
mutated region in ALS patients, although distinct mutant
TDP-43 proteins (A382T, Q331K and M337V) distribute
normally in the nucleus (Sreedharan et al. 2008), suggesting
that alteration in TDP-43 sub-cellular localization is determined by multiple aminoacidic changes and/or additional
contributing factors.
Several pathogenic mechanisms have been demonstrated
to trigger motor neuron death in ALS, although the
primary or secondary role of these events in the pathogenesis of the disease is not clear yet, and the early
causative processes still need to be claried. As these
mechanisms include oxidative stress, mithocondrial defects,
protein aggregation, and proteasome impairment, we
investigated if SGs may be implicated in ALS pathogenesis. In human ALS motor neurons we failed to detect
TIAR- and HuR-positive granules or inclusions co-localizing with TDP-43 and, also in conditions of a main
physiological TDP-43 distribution in the nucleus, we did
not observe overlapping signals with the SG markers
TIAR and HuR in the cytoplasm. A previous paper
reported the presence of TIA-1 and PolyA-Binding Protein
1 (PABP-1), another RBP included in SGs, in the RNApositive basophilic inclusions from patients presenting with
adult-onset atypical motor neuron disease (Fujita et al.
2008), conrming our hypothesis that translational arrest
mechanisms are somehow involved in neuronal death in
vivo. The observation that SG structures are not associated
only to environmental insults in vitro, but may form and
be detectable also in disease conditions, has already been
reported in animal models of brain ischemia and sciatic
nerve 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 macromolecular complexes may assemble as a very early response to
an environmental stress and that at the endstage of the
neurodegenerative process other compensative mechanisms
may have occurred. On the other hand, we may also
hypothesize that in ALS affected tissues stressful processes
are progressive and not acute enough to provoke SG
formation, like in ischemia and axotomy conditions. We

have experimental evidence that TDP-43 does not form


SGs, but distributes diffusely in the cytoplasm when
treating NSC34 cells with milder doses of arsenite for
prolonged periods of time to mimic a chronic insult
(unpublished results). Our ndings, together with the
observed cytoplasmic mislocalization of HuR in mutant
Superoxide dismutase 1 (SOD1) transgenic mice at very
early stages of the disease (Lu et al. 2007), support the idea
that impairment of post-transcriptional regulatory mechanisms, including mRNA stabilization and translation, may
be actively involved in ALS pathogenesis and/or progression. However, the reason of such a specic TDP-43, and
not HuR, pathological aggregation in human ALS brain
tissues needs further investigation. Although preliminary
data show the absence of FUS/TLS-positive cytoplasmic
inclusions in sporadic ALS patients (Kwiatkowski et al.
2009; Vance et al. 2009), a full comprehension of the
potential interplay of this protein with TDP-43 and HuR
will help elucidating the role of RBP-mediated regulation of
gene expression in neurodegeneration and motor neuron
diseases.

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 different
environmental 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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