Neurobiology of Disease 60 (2013) 5160

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Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi

Posttranscriptional regulation of SOD1 gene expression under oxidative

stress: Potential role of ELAV proteins in sporadic ALS
Pamela Milani a,b,,1, Marialaura Amadio c,,1, Umberto Laforenza d, Michela Dell'Orco a,b, Luca Diamanti b,e,
Valentina Sardone a,b, Stella Gagliardi a, Stefano Govoni c, Mauro Ceroni b,e, Alessia Pascale c, Cristina Cereda a
a
Laboratory of Experimental Neurobiology, C. Mondino National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy
b
Department of Public Health, Neuroscience, Experimental and Forensic Medicine, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy
c
Department of Drug Sciences, Pharmacology Section, University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy
d
Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy
e
Division of General Neurology, C. Mondino National Institute of Neurology Foundation, IRCCS, Via Mondino 2, 27100 Pavia, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Increased levels of SOD1 mRNA have been observed in sporadic ALS patients (SALS) compared to controls. Hence,
Received 4 March 2013 the understanding of the mechanisms by which SOD1 gene expression is modulated may shed new light on SOD1
Revised 5 July 2013 involvement in ALS. Of interest, some adenine/uracil-rich elements (AREs) in SOD1 3-untranslated region have
Accepted 7 August 2013
been identied. These sequences represent the docking sites for several RNA-binding proteins such as ELAV pro-
Available online 20 August 2013
teins (ELAVs), positive regulators of gene expression. We rst investigated in SH-SY5Y cells whether SOD1 mRNA
Keywords:
represents a target of ELAVs. Results from RNA Electrophoretic Mobility Shift and RNA-immunoprecipitation
Sporadic amyotrophic lateral sclerosis assays showed a molecular interaction between ELAVs and SOD1 mRNA. We also observed that the treatment
SOD1 with H2O2 induced a signicant increase of the amount of SOD1 mRNA bound by ELAVs and an up-regulation
mRNA of SOD1 protein levels. We found a specic increase in ELAV/HuR phosphorylation, suggesting activation of
Posttranscriptional regulation this protein, in peripheral blood mononuclear cells from SALS patients compared to controls. Finally, we found
ELAV proteins increased levels of ELAV proteins in the motor cortex and spinal cord from SALS patients compared to controls,
Peripheral blood mononuclear cells in parallel with SOD1 up-regulation in the same areas. This study suggests, for the rst time, that ELAVs are
Motor cortex
involved in the regulation of SOD1 gene expression at post-transcriptional level and that these proteins are
Spinal cord
more activated in ALS pathology. The link between ELAVs and SOD1 may open novel perspectives for ALS
research, paving the way for new therapeutic options.
2013 Elsevier Inc. All rights reserved.

Introduction
Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenera-
tive disease clinically characterized by progressive muscular paralysis
reecting the degeneration of both upper and lower motor neurons.
The majority (90%) of cases is sporadic (SALS), while the remainder
presents a family history (FALS). As recently reviewed by Cozzolino
et al. (2012), ALS is a complex and multifactorial disease characterized
by the involvement of several pathogenic conditions, including oxida-
tive stress. In particular, the occurrence of distinctive oxidation markers,
such as elevated protein carbonyl levels, increased lipid peroxidation
and DNA/RNA oxidative modications, has been reported in the ner-
vous and peripheral tissues in both sporadic and familial ALS patients
Corresponding author.
Corresponding author. Fax: +39 0382 987405.
E-mail addresses: pame.milani@gmail.com (P. Milani), amadio@unipv.it (M. Amadio).
Available online on ScienceDirect (www.sciencedirect.com).
1
These authors equally contributed to the work.
(Babu et al., 2008; Barber and Shaw, 2010; Bogdanov et al., 2000;
Bonnefont-Rousselot et al., 2000; Chang et al., 2008; Cova et al., 2010;
Robberecht, 2000; Shaw et al., 1995; Simpson et al., 2003). An important
contribution towards the understanding of ALS pathogenesis came from
the discovery of mutations within the gene encoding for Cu/Zn superox-
ide dismutase (SOD1) which accounts for the 1520% of FALS patients.
In addition, there is increasing evidence that SOD1 protein is also
involved in sporadic ALS, since the wild-type form of the protein un-
dergoes conformational changes due to either age- or environmental-
dependent posttranslational modications, such as oxidation, rendering
its behavior similar to mutSOD1 (Bosco et al., 2010; Ezzi et al., 2007;
Guareschi et al., 2012). Furthermore, we reported that also the increase
of SOD1 mRNA expression is a distinctive feature of sporadic ALS pathol-
ogy compared to other neurodegenerative diseases and healthy controls
(Gagliardi et al., 2010). Notably, SOD1 mRNA level is elevated in both
nervous areas typically affected by ALS disease (i.e., the brain stem and
spinal cord) and peripheral blood mononuclear cells (PBMCs) from
SALS patients compared to control population. The increased SOD1
mRNA expression in lymphocytes of SALS patients has been recently

0969-9961/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nbd.2013.08.005
52 P. Milani et al. / Neurobiology of Disease 60 (2013) 5160
conrmed by Mougeot et al. (2011). Considering that the genetic
analysis of the SOD1 promoter region has not revealed DNA sequence
variations with functional effects for SOD1 mRNA expression in ALS
(Milani et al., 2012), alterations in SOD1 mRNA content may be due to
the activation of specic intracellular molecular cascades as a response
to stressful stimuli. Indeed, increasing evidence is showing that SOD1
levels are ne-tuned by complex pathways which involve positive and
negative regulatory elements potentially acting in concert (Milani
et al., 2011). Specically, numerous cis-acting regulatory elements in
the SOD1 promoter region and the corresponding trans-acting factors
have been identied. These transcription factors, involved in the consti-
tutive and inducible SOD1 gene expression under specic extra- and
intracellular conditions, include Sp1, Egr1, AP1, Nrf2, NF-B and AHR.
Several papers reported SOD1 transcriptional induction mediated by
Nrf2 and NF-B under oxidative stress conditions (Park and Rho,
2002; Rojo et al., 2004); conversely, at the present, posttranscriptional
control mechanisms of SOD1 expression are still almost unknown.
Increasing evidence demonstrate that posttranscriptional control repre-
sents a specic and punctual regulatory mechanism for gene expression
which can rapidly modify protein levels in response to extracellular
stimuli (Abdelmohsen et al., 2008; Keene, 2007; Mata et al., 2005).
Stress-induced transcriptional and posttranscriptional mechanisms
often act together to collectively determine the overall variations in
expression proles of specic genes. Regarding posttranscriptional
events, very few authors have focused on SOD1 mRNA structure and
metabolism. Among them, in 1995 Kilk and co-workers (Kilk et al.,
1995) identied some adenine/uracil-rich stretches in the 3UTR of
SOD1 mRNA, and hypothesized that these sequences may affect the
expression of the correspondent protein potentially allowing a faster
cellular response to environmental stressors. In general, the adenine/
uracil-rich elements (AREs) represent the docking sites for many RNA-
binding proteins (RBPs), which can inuence one or more steps of the
metabolism of the target transcripts (Chen and Shyu, 1995). Among
ARE-binding RBPs, a relevant place is taken by ELAV proteins (Antic
and Keene, 1997). In vertebrates, the ELAV (or Hu) family comprises
the neuron-specic members HuB, HuC and HuD (Pascale et al., 2008),
and the ubiquitously expressed HuR (a.k.a. HuA). ELAV proteins act
mainly as positive regulators of gene expression, since they can increase
the stability and/or translation of target mRNAs whose corresponding
proteins are fundamental for key cellular functions. Of interest, ELAVs
are demonstrated to be involved in various physio-pathological pro-
cesses where inammation and oxidation play a primary role (Pascale
and Govoni, 2012; Srikantan and Gorospe, 2012).
Within this context, we rst investigated in a cellular model, the
human neuroblastoma SH-SY5Y cells, whether SOD1 mRNA is a target
of ELAV proteins, and whether the binding between ELAV proteins
and SOD1 mRNA is favored by oxidative stress, a condition observed
in ALS.
Moreover, we tested the potential implication of ELAV proteins in
ALS, studying the activation of their pathway in PBMCs, motor cortex
and spinal cord, two areas of the nervous system specically involved
in the disease.
Materials and methods
Patient selection criteria Ethics statement
Peripheral blood mononuclear cells (PBMCs) were obtained from 15
SALS subjects and 14 sex- and age-matched healthy volunteers free
from any pharmacological treatment. The patients were recruited at
the National Neurological Institute C. Mondino (Pavia, Italy) and
clinical diagnosis was made according to the El Escorial revised criteria
(Brooks et al., 2000). All ALS patients have been previously screened
for SOD1, TARDBP, FUS/TLS, ANG, UBQLN2, PFN1 and C9ORF72 gene muta-
tions: patients harboring mutations in these genes were excluded from
this study. Control donors were all unrelated and the normal phenotype
was identied on interviews based on personal health histories. The
study was approved by the Ethics Committee, and patients and controls
signed informed consent prior to the study. Samples of the motor cere-
bral cortex from 10 clinically diagnosed post-mortem SALS patients and
6 non-neurodegenerative disease control individuals were obtained
from the Human Brain and Spinal Fluid Resource Center (UCLA Los
Angeles, CA).
PBMC isolation
PBMCs were immediately isolated from peripheral venous blood
using Histopaque-1077 (Sigma-Aldrich, Italy), washed twice with
PBS and centrifuged at 1500 g for 5 min. Cell counts were performed
using trypan blue to stain non-viable cells, which were excluded from
the count. An aliquot of PBMCs corresponding to 5 105 cells was re-
covered and employed for immunouorescence assay. The remaining
cells were used for cytoplasmic protein extraction and Western blotting
analysis.
Cell culture and treatments
All reagents for cell culture were purchased from PAA Laboratories
(Pasching, Austria). Human neuroblastoma SH-SY5Y cells were grown
in Dulbecco's modied Eagle/F12 medium supplemented with 15%
fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 10 mg/ml
streptomycin, at 37 C in an atmosphere of 5% CO2 and 95% humidity.
Cell medium was changed every 2 days and the cells were subcultured
once they reached 8090% conuence. SH-SY5Y cells were exposed to
the solvent (PBS) or to 1 mM H2O2 (Sigma-Aldrich, Italy) for different
times as indicated in the results/gures. For translation inhibition exper-
iments SH-SY5Y cells were incubated with 1 mM puromycin (Sigma-
Aldrich, Italy).
Preparation of cytosolic and nuclear extracts
Nuclear and cytoplasmic extracts were prepared according to a pub-
lished protocol (Schreiber et al., 1989). Before freezing in aliquots at
80 C, cytoplasmic and nuclear protein contents were determined using
bicinchoninic acid (BCA) method (Sigma-Aldrich, Italy) using BSA as a
standard.
Preparation of mRNP (messenger-RiboNucleoProteic complexes)
Lysates from cultured cells were obtained as previously described
(Tenenbaum et al., 2002). Protein extracts were quantied using the
BCA method (Sigma-Aldrich, Italy) and stored at 20 C until use.
Western blotting
SDS-PAGE and membrane blotting were performed as described in
Gagliardi et al. (2010). The complete list of the antibodies and dilutions
is reported in the Supplementary material. Densitometric analysis of
the bands was performed using ImageJ software (http://rsb.info.nih.
gov/ij/).
SOD1 mRNA primary and secondary structure analysis
We aligned the primary sequence of the entire SOD1 mRNA with
published ELAV-target motifs (Lpez de Silanes et al., 2004), and we
performed RNA secondary structure analysis since ELAV target motifs
are predicted to form a stemloop conformation (Saunus et al., 2008).
Different RNA folding programs, such as the mfold and Vienna RNA
package (Hofacker, 2003; Zuker, 2003), were used to perform the
secondary structure predictions and folding calculations of SOD1
transcript. Overall these studies highlighted, within SOD1 3UTR, a can-
didate region, comprising the canonical class I ARE and two other uracil-
 P. Milani et al. / Neurobiology of Disease 60 (2013) 5160
rich sequences, which is predicted to assume a stemloop structure
(Supplementary Figs. S1AB). This region was consequently chosen to
design a probe for RNA electrophoretic mobility shift assay.
RNA electrophoretic mobility shift assay (REMSA)
We used the puried recombinant HuR (ELAVL1; OriGene Technolo-
gies, Rockville, MD, USA) protein because HuR is ubiquitously expressed
in comparison to the others which are neuron-specic. An ARE-bearing
probe of TNF was employed as positive control of the experiment
given that it contains, in tandem, multiple ARE motifs, which are well
characterized ELAV-target sequences (Schaljo et al., 2009). Both TNF
and SOD1 probes were predicted to assume a stemloop conformation
(see Supplementary Figs. S1BC). Cy5.5-labeled SOD1 and TNF probes
were purchased from Eurogentec (Seraing, Belgium). Probe sequences
were as follows: SOD1: 5-AAA CUG AUU UAU GAU CAC UUG GAA
GAU UUG UAU AGU UUU AUA AAA CUC AG-3 and TNF: 5-AUU AUU
UAU UAU UUA UUU AUU AUU UA-3. Both probes were dye-labeled at
5-terminus. 250 ng of recombinant HuR protein were incubated with
500 fmol of RNA probes in 1 REMSA binding buffer (10 mM TrisHCl,
pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM DTT, 4% glycerol and 10 U
RNasin Ribonuclease Inhibitor). To reduce non-specic binding, yeast
tRNA (Sigma-Aldrich, Italy; 0.1 g/l nal concentration) was added.
Reaction mixtures were incubated for 30 min at RT in the dark. Binding
reactions were then loaded on a non-denaturating 4% polyacrylamide
gel (37.5:1 ratio of acrylamide to bis-acrylamide) in 0.5 Tris/borate/
EDTA buffer. The gel had been pre-run for 30 min prior to loading the
samples. Samples were resolved at 100 V (constant voltage) at 4 C in
the dark. Probe signals were detected and analyzed using the Odyssey
Infrared Imaging System (LI-COR Bioscience).
Immunoprecipitation
Immunoprecipitation was performed according to a previously pub-
lished protocol with minor modications (Amadio et al., 2008). Immu-
noprecipitation was performed at room temperature for 2 h using 1 g
of mouse monoclonal antibody anti-ELAV/HuR (3A2, sc-5261; Santa
Cruz Biotechnology, Inc., USA) per 50 g of proteins diluted in the NT2
buffer [50 mM Tris, pH 7.4; 150 mM NaCl; 1 mM MgCl2; 0.05% Nonidet
P-40] plus RNase and protease inhibitors, 1 mM DTT, 20 mM EDTA and
in the presence of 50 l of protein A/G plus agarose (Santa Cruz Biotech,
CA, USA). The anti-ELAV/HuR antibody employed in this study recog-
nized both the ubiquitously expressed HuR protein and the neuronal-
specic ELAV proteins (HuB, HuC and HuD). The samples were nally
subjected to either Western blotting or RNA extraction. The negative
control was obtained in the same conditions, but in the presence of an
irrelevant antibody with the same isotype of the specic immuno-
precipitating antibody. For binding assay, 100 l of the immunoprecipi-
tation mixes were collected from each sample and used as input
signals to normalize the real time qPCR data.
Quantitative real-time reverse-transcription polymerase chain reaction
Total RNA from SH-SY5Y cells and from the motor cortex of 10 ALS
patients and 6 control subjects was extracted with Trizol reagent
(Invitrogen, Milan, Italy) according to the manufacturer's recommenda-
tions. RNA quality and quantity were determined using NanoDrop spec-
trophotometer (Celbio, Milan, Italy) and 1 g was reverse transcribed
using the iScript cDNA synthesis kit (BioRad, Milan, Italy) according to
the manufacturer's protocol. The quantitative (q) PCR was performed
by real time qPCR with TaqMan technology according to a previously
described protocol (Gagliardi et al., 2010). Primer sequences are
described in the Supplementary material.
53
Immunocytouorescence
SH-SY5Y cells plated on coverslips were exposed to the solvent
(PBS) or to 1 mM H2O2 for 30 min. After treatment, the medium was re-
moved and cells were rinsed twice with PBS. Freshly isolated PBMCs
from 4 ALS patients and 4 healthy donors were harvested, washed
with PBS and resuspended in RPMI-1640 medium. Approximately
5 105 cells were placed on pre-coated poly-L-lysine slide (Thermo
Fisher Scientic Inc., MA, USA), incubated at 37 C for 15 min to allow
them to attach to the slide and rinsed twice with PBS. Both SH-SY5Y
cells and PBMCs were then xed (15 min at RT) using a solution of 4%
paraformaldehyde in PBS. Fixed cells were washed three times with
PBS, treated with a blocking solution containing 5% goat serum in 0.1%
Tween-PBS for 1 h, and nally incubated overnight with the primary
antibody (mouse monoclonal anti-ELAV/HuR, 3A2, sc-5261, Santa
Cruz Biotechnology, Inc., USA, with a dilution 1:250 in blocking
solution) at 4 C. Cells were then washed three times with PBS, and
incubated with secondary antibody (Alexa 594 goat anti-mouse,
dilution 1:700 in blocking solution; Invitrogen, Carlsbad, CA, USA), for
1 h at RT. Samples were nally washed three times with PBS and incu-
bated for 30 s with DAPI solution (Sigma-Aldrich, Italy, dilution 1:2500
in deionized water). After twice washing, slides were mounted up-side-
down with 60% glycerol solution, dried for 2 h and nail-polished. Local-
ization of ELAV proteins was dened using a confocal laser microscope
(Leica DM IRBE, Leica Microsystems Srl, Italy).
Immunohistochemistry
Motor cortex specimens from 2 ALS patients and 2 non-neuro-
degenerative disease control subjects were obtained at autopsy and
immediately frozen at 80 C. Samples were processed as described in
Laforenza et al. (2009).
Data analysis
All the experiments were performed at least three times. Values
were expressed as means S.E.M. Statistical analysis was performed
using GraphPad Prism version 5 (San Diego, CA, USA). The data were an-
alyzed by Student's t-test and analysis of variance (ANOVA) followed,
when signicant, by an appropriate post hoc comparison test. Differ-
ences were considered statistically signicant when p values were
b0.05.
Results
Effect of H2O2 exposure on SOD1 gene expression
SH-SY5Y human neuroblastoma cells were treated with vehicle or
1 mM H2O2 for 30 and 60 min, and SOD1 mRNA expression was evalu-
ated by real time quantitative (q) PCR. As shown in Fig. 1, the oxidative
stimulus enhanced SOD1 mRNA levels in a time-dependent manner
(+51.7% and +96.7% at 30 and 60 min, respectively). Although a
positive trend was evident at 30 min, the increase of SOD1 mRNA was
statistically signicant only at 60 min (p b 0.05).
3UTR SOD1 mRNA as a novel target of ELAV proteins
We analyzed the SOD1 3UTR primary sequence to identify the
cis-acting elements and the corresponding trans-acting factors poten-
tially affecting SOD1 mRNA stability and/or translation. We found
within the SOD1 3UTR the presence of ARE sequences, one of which
representing the canonical class I ARE sequence (AUUUA), generally rec-
ognized as targets of the RNA-binding ELAV proteins (Supplementary
Fig. S1A). This region was consequently chosen to design a probe for
RNA electrophoretic mobility shift assay (REMSA), an in vitro cell-free
assay employed to evaluate the possible binding between ELAV proteins
54 P. Milani et al. / Neurobiology of Disease 60 (2013) 5160

H2O2-treated SH-SY5Y cells showed that the oxidative stimulus deter-

mines an increased intensity of the gel-shifted complex in comparison
to control (Supplementary Fig. S1D).

Oxidative stress induces early translocation/activation of ELAV proteins

To further investigate the effects of oxidative stress on ELAV protein

Fig. 1. Effect of H2O2 exposure on SOD1 gene expression. Determination of the steady state
levels of SOD1 mRNA by quantitative real-time RT-PCR in human neuroblastoma SH-SY5Y
cells following treatments with 1 mM H2O2 for 30 and 60 min. SOD1 mRNA expression in
untreated cells was taken as 100%. The values obtained from total cellular mRNA have
been normalized to the level of GAPDH mRNA and expressed as mean S.E.M. The
data were analyzed by ANOVA, followed by NewmanKeuls Multiple Comparison Test;
*p b 0.05, n = 3.
and SOD1 mRNA. As shown in Fig. 2A, HuR protein physically interacts
with the SOD1 probe, causing the formation of a stable proteinRNA
complex that migrates more slowly than the free probe.
We then performed RNA-immunoprecipitation (RNA-IP) assay on
SH-SY5Y cells, which express all the four ELAV proteins, to corroborate
this molecular interaction and to test whether ELAV proteins bind SOD1
mRNA also in our biological model. In particular, we carried out RNA-IP
on messenger-RiboNucleoProteic (mRNP) complexes from control and
H2O2-treated cells because mRNPs have been shown to be enriched in
transcripts directed towards the translational machinery. We thus
extracted the bound mRNA from the immunoprecipitated ELAV pro-
teins in mRNP complexes and we measured SOD1 mRNA levels by real
time qPCR. We found that SOD1 mRNA content in immunoprecipitated
ELAV proteins was signicantly higher in comparison to the amount
obtained in the same conditions with an irrelevant antibody (not
shown). Furthermore, RNA-IP experiments were carried out also in
mRNP from 30 min H2O2-treated cells to assess whether the interaction
between ELAV proteins and SOD1 mRNA could be modulated by oxida-
tive stress. Indeed, as shown in Fig. 2B, after 30 min H2O2 treatment the
amount of SOD1 mRNA in the immunoprecipitated ELAV proteins was
signicantly higher (about 5-fold) in comparison to control, suggesting
that following a stress stimulus ELAV proteins early bind to SOD1 mRNA
in mRNP complexes. Accordingly, REMSA assay performed using
SOD1 probe and cytoplasmic extracts from either control or 30 min
activation, we evaluated their subcellular localization in 30 and 60 min
H2O2-treated cells by both Western blotting (WB) and immunouores-
cence analysis. Although the changes of ELAV protein level in the nucle-
ar compartment were undetectable by WB, we found a signicant
increase of ELAV protein amounts in the cytoplasm after H2O2 treatment
(+49.1% and +36.1% at 30 and 60 min, respectively; Fig. 3A). Confocal
images and straight line-scan analysis conrmed that oxidative stress
induced a marked translocation of ELAV proteins from the nucleus to
the cytoplasm at 30 min (Fig. 3B). This stress-induced translocation of
ELAV proteins from the nucleus to the cytoplasm was likely accompa-
nied by their activation; to this regard, indeed, in the same in vitro
model, previous data demonstrated that H2O2 treatment induced
ELAV protein activation through a Protein Kinase C-mediated phosphor-
ylation in their serine- and threonine residues (Amadio et al., 2008).
Oxidative stress enhances SOD1 translation
Given that ELAV proteins can form mRNP complexes that associate
to the translational machinery, and have been demonstrated to enhance
translation of some target mRNAs (Abdelmohsen et al., 2008), we tested
whether the treatment with H2O2 also inuenced SOD1 protein levels.
To this purpose, we rst evaluated by WB SOD1 protein level in mRNP
complexes after 30 and 60 min of H2O2 exposure in SH-SY5Y cells. As
shown in Fig. 4A, we found increased (+30%) SOD1 protein levels in
60 min H2O2-treated cells with respect to control. The up-regulation
of SOD1 protein level following 60 min H2O2 was due to the new syn-
thesis of the protein itself, since it was prevented by the treatment
with the translation inhibitor puromycin (Fig. 4B).
HuR protein phosphorylation is affected in PBMCs of sporadic ALS patients
SOD1 mRNA levels have been shown to be up-regulated in PBMCs
from SALS patients compared to healthy controls (Gagliardi et al.,
2010); furthermore, we also observed an increased ROS expression in
patients' cells vs control group (Supplementary Fig. S2). On the basis
of our in vitro ndings on the binding between ELAV proteins and
SOD1 mRNA, we consequently investigated whether also ELAV protein
expression and/or activation are affected by SALS disease. To this aim,

Fig. 2. SOD1 mRNA as a novel target of ELAV proteins. (A) Puried HuR protein binds to SOD1 RNA probe. RNA electrophoretic mobility shift assay was performed with 500 fmol
Cy5.5-labeled RNA probes and 250 ng of puried HuR protein, as described in Materials and methods. The TNF probe represents the positive control of the experiment. The arrow
indicates the RNA:protein complexes. The image is representative of three independent experiments. (B) The amount of SOD1 transcript bound by ELAV proteins in mRNP complexes
is increased following H2O2 stress stimulus. Fold enrichment detected by quantitative real-time RT-PCR of SOD1 mRNA in control (CTR) and 30 min H2O2-treated (H2O2) SH-SY5Y
cells following immunoprecipitation with anti-ELAV antibody in mRNP complexes. **p = 0.0077, Student's t-test, n = 3. The data of SOD1 were normalized with respect to the data
obtained from immunoprecipitation with an irrelevant antibody.
 P. Milani et al. / Neurobiology of Disease 60 (2013) 5160 55

Fig. 3. H2O2 causes ELAV protein accumulation in the cytoplasm of SH-SY5Y cells. (A) Increased levels of ELAV proteins in the cytoplasmic compartment of SH-SY5Y cells following 30 and
60 min of H2O2. Representative Western blotting (left panel) and densitometric analysis (right panel) of ELAV proteins in the nuclear and cytoplasmic compartments of control (0) and
H2O2-treated cells (30, 60 min). Markers for nuclear (Sp1) and cytoplasmic (-tubulin) compartment were used to exclude a cross-contamination between the two fractions and to verify
equal loading and transfer. Results are expressed as means S.E.M.; ELAV protein expression in untreated cells was taken as 100%. The data were analyzed by ANOVA, followed by
NewmanKeuls Multiple Comparison Test; *p b 0.05, **p b 0.01, n = 7. (B) Redistribution of ELAV proteins after 30 min oxidative stimulation. Representative confocal uorescence
microscopy images (left panel) of ELAV proteins (red) intracellular localization in control (CTR) and 30 min H2O2-treated SH-SY5Y cells (H2O2 30 min). Nuclei were visualized using
the uorescent nuclear dye DAPI (blue). Scale bar: 15 m. Nuclear localization of ELAV proteins was also evaluated by the straight line-scan image analysis method (right panel). Pixels
located under the straight line were scanned and the average channel-specic intensity of the pixels calculated. Red and blue lines, representing proles for the respective color channels,
were plotted together. Comparison of the two proles clearly conrms ELAV protein accumulation in the cytoplasm after oxidative stress induction.

Fig. 4. Oxidative stress up-regulates SOD1 protein expression. (A) H2O2 treatment induces an increase of SOD1 protein level in messenger-RiboNucleoProteic complexes (mRNPs) of
SH-SY5Y cells. Representative Western blotting (left) of SOD1 protein from control (0) and H2O2-treated cells (30, 60 min). The samples were normalized according to -tubulin values.
The histogram (right) shows the densitometric analysis of the bands. Results are expressed as mean S.E.M.; SOD1 protein expression in untreated cells was taken as 100%. A signicant
increase in SOD1 level is found in 60 min H2O2-treated cells compared to untreated cells (paired Student's t-test, *p b 0.05, n = 5). (B) Translation inhibition prevents stress-induced
SOD1 protein increase in mRNPs. SH-SY5Y cells were exposed to puromycin (Puro) and H2O2 for 60 min as indicated in Materials and methods. Representative Western blotting (left)
and densitometric analysis (right panel) of SOD1 protein in mRNPs from control (0) and treated cells (60). The samples were normalized according to -tubulin values. Results are
expressed as mean S.E.M.; SOD1 protein expression in untreated cells was taken as 100%.
56 P. Milani et al. / Neurobiology of Disease 60 (2013) 5160
we rst evaluated both the expression and cellular localization of the
ubiquitous ELAV/HuR protein in PBMCs from SALS patients and healthy
controls. Characteristics of SALS patients and control subjects are
reported in Table 1. By performing immunocytochemistry and WB
experiments, we found no difference between control and SALS groups,
neither in the levels nor in the cellular localization of the ELAV/HuR pro-
tein, which in PBMCs seems to be mainly located in the nuclear com-
partment (Figs. 5A and B). We then evaluated possible changes in the
activation of the ELAV/HuR protein in PBMCs from the two groups. In
particular, as already mentioned, considering that ELAV proteins can
be activated by phosphorylation (Amadio et al., 2008; Latorre et al.,
2012), and that the cytoplasm is the site where activated ELAV proteins
localize to affect mRNA stability/translation, we isolated the ELAV/HuR
protein by performing an immunoprecipitation on the cytoplasmic
fraction of PBMCs from control and SALS subjects, and analyzed its
possible phosphorylation. Notably, we found an increase in both threo-
nine (+38.6%, p b 0.05; Fig. 5C) and serine (+46.5% not statistically
signicant; not shown) phosphorylations of the ELAV/HuR protein in
SALS samples with respect to controls, suggesting that this protein is
activated in PBMCs from SALS patients.
The expression of ELAV proteins and SOD1 mRNA is increased in the
cerebral motor cortex of SALS patients
Considering that in Alzheimer's disease and other neurodegenera-
tive pathology, ELAV protein levels are altered in the brain tissues
primarily affected by this pathology (Amadio et al., 2009); we wanted
to investigate possible changes of ELAV protein expression in the
motor cortex, a cerebral area specically involved by SALS. We thus
evaluated ELAV protein expression and intracellular localization by
performing immunohistochemistry (IHC) analysis in the motor cortical
tissues from SALS patients and healthy controls. As shown in Fig. 6A, the
ELAV-positive signal was much more intense in the cerebral sections
from SALS patients than in the ones from control subjects. Moreover,
in the control tissues ELAV proteins appeared mainly conned to the
nucleus, while in the sections from SALS subjects they were strongly lo-
calized also in the perinuclear region and in the cytoplasm. SOD1 mRNA
and protein levels are increased in the cerebral motor cortex from SALS
patients in comparison to controls, as resulted by real time qPCR
and IHC reported in Figs. 6B and C, respectively. IHC experiments on
spinal cord specimens revealed that ELAV protein expression is up-
regulated in SALS patients also in this area, as shown in Supplementary
Fig. S3.
Discussion
The discovery that mutations in SOD1 gene cause a subset of FALS
has attracted great attention, though studies to date have been limited
to alterations in the coding region and investigations at protein level.
Recently, the importance of SOD1 expression regulation at multiple
levels has become clear, as the alteration of SOD1 induction may be in-
volved in ALS. Indeed, our previous data demonstrated that the increase
of SOD1 mRNA level may be a specic biomarker for the sporadic form
of ALS pathology (Gagliardi et al., 2010). In particular, analyses on the
spinal cord tissues conducted in the same study also evidenced an
enhanced expression of SOD1 in terms of both mRNA and protein. The
Table 1
Characteristic of sporadic ALS patients (SALS) and control (CTR) subjects.
Sample N Mean age/age of onset Disease duration Limb/bulbar
(male/female) (mean SE, years) (mean SE, month) onset
SALS 15 (9/6) 62.14 2.52 34.29 7.21 8/7
CTR 14 (9/5) 55.36 3.07
genetic investigation on SOD1 promoter failed to identify relevant func-
tional changes within DNA sequence (Milani et al., 2012); furthermore,
an epigenetic study reported the lack of particular SOD1 promoter
methylation patterns in both white cells and brain tissues from ALS
patients (Oates and Pamphlett, 2007). These ndings suggested that
alterations in SOD1 mRNA amounts are more likely attributed to the
activation of specic intracellular molecular cascades, triggered by
disease-linked conditions, such as oxidative stress and inammation,
rather than to genetic or epigenetic changes in the regulatory region.
Although SOD1 has been often considered a housekeeping gene due
to its high and ubiquitous expression, increasing evidence reveals that
SOD1 mRNA and protein amounts are modulated by complex signaling
pathways. Over the past decade, much effort has been directed to the
transcriptional-dependent events that control intracellular SOD1
mRNA content. However, besides gene induction, there is increasing
attention directed to posttranscriptional regulation of gene expression
patterns in response to pathological stimuli (Abdelmohsen et al.,
2008). Therefore, given the lack of data on this subject, we decided to
focus the present study on the modulation of SOD1 mRNA levels by
posttranscriptional mechanisms, in particular via ELAV proteins. As ox-
idative stress is one of the major actors involved in sporadic ALS, and it is
a trigger of ELAV activation, we treated human neuroblastoma SH-SY5Y
cells with H2O2 as a starting point for our study, nding that this stressor
induced a signicant up-regulation of SOD1 mRNA levels. Notably, in
the same in vitro model we previously highlighted the role of ELAV
proteins in the regulation of another stress-related gene (Amadio
et al., 2008). H2O2-treated neuronal cells are employed as a reliable
and adequate cellular model to thoroughly study oxidative-induced
SOD1 protein modications with relevance to SALS (Ezzi et al., 2007).
Subsequently, we comprehensively analyzed the 3UTR region of
SOD1 mRNA in an attempt to reveal the cis-acting sequences and
predict the corresponding trans-acting factors that can regulate SOD1
and whose potential activation in SALS patients could account for the
observed enhanced SOD1 expression. By performing bioinformatic
analysis, we identied within SOD1 3UTR primary sequence the
presence of AREs, that are putative ELAV-binding sites. Furthermore,
the secondary structure analysis of the 3UTR comprising the
computationally-identied ARE predicted that this candidate region is
inserted in a stemloop structure, consistently with the results reported
by Lpez de Silanes et al. (2004). REMSA experiment validated the in
silico prediction, demonstrating that the recombinant HuR protein
bound specically and directly to the ARE-bearing probe of SOD1.
ELAV protein capability to bind to SOD1 mRNA was further conrmed
by REMSA performed with cellular extracts and, more importantly, by
immunoprecipitation coupled with real time qPCR experiments. In
particular, the isolation of the mRNAs co-immunoprecipitated with en-
dogenous ELAV proteins showed that after H2O2 stimulation the
amount of SOD1 mRNA bound by ELAV proteins was signicantly
higher than in control cells. In parallel, under the same experimental
condition, we observed ELAV accumulation in the cytoplasm; this intra-
cellular relocation is in line with the role of ELAV/HuR as a nuclear
cytoplasmic shuttling protein. Indeed, ELAV inuence on target
mRNAs is strongly dependent upon their localization in the cytoplasm,
where these proteins can stabilize target mRNAs and modulate their
translation (Colombrita et al., 2013). Our results are in agreement
with other observations reporting that oxidative stress agents deter-
mine ELAV/HuR accumulation in the cytoplasm, where it promotes
mRNA stability (Abdelmohsen et al., 2008; Kuwano et al., 2008; Viiri
et al., 2013). Furthermore, after 60 min of H2O2 treatment, we found a
consistent increase of SOD1 protein in mRNP complexes, which are
discrete cytoplasmic loci enriched in RBPs and mRNAs. By protein syn-
thesis inhibition experiment we could assert that this effect was caused
by enhanced translational status rather than by a translocation of an
already present SOD1 protein from an intracellular compartment to
another one. The observation that at 30 min no variation in SOD1 pro-
tein amount could be detected suggests that this time is too short for
 P. Milani et al. / Neurobiology of Disease 60 (2013) 5160 57

Fig. 5. In PBMCs HuR protein phosphorylation, but not its expression, is affected by sporadic ALS. (A) HuR protein level in the cytoplasmic fraction of PBMCs from healthy controls (CTR)
and sporadic ALS patients (SALS). Representative immunoblotting (left) and relative mean gray level ratios (mean + S.E.M.) of HuR/LDH immunoreactivities (right) measured in the
cytoplasm of PBMCs from SALS patients (n = 15) and sex- and age-matched controls (n = 14). (B) HuR protein localization in the nucleus of PBMCs from both CTR and SALS subjects.
Representative confocal uorescence microscopy images show HuR protein (red) in PBMCs from SALS patients and controls. Nuclei were visualized using the uorescent nuclear dye DAPI
(blue). Scale bar: 7 m. (C) Increased phosphorylation of HuR protein in PBMCs from SALS patients. Representative Western blotting (left) and densitometric analysis (right panel) of
phospho-Threonine (p-Thr) residues after immunoprecipitation with either anti-HuR antibody or an irrelevant IgG1 isotype antibody (as negative control) in the cytoplasmic fractions
of PBMCs from SALS patients (n = 7) and sex- and age-matched controls (CTR; n = 7). Results are expressed as mean S.E.M.; the level of phosphorylated threonine residues of
HuR protein in control subjects was taken as 100%. Paired Student's t-test, *p b 0.05. Ab I.P. = antibody used for the immunoprecipitation.

SOD1 protein translation; however, we cannot exclude that a slight
change occurred already at this time, but WB technique is not sensitive
enough to enable the detection of a pelting increase of such a highly
abundant protein.
Collectively, these data suggest that, in our in vitro model, following
oxidative stress SOD1 induction is inuenced by posttranscriptional
processes, plausibly mediated by ELAV-dependent mechanisms. Given
that until now transcriptional events were supposed to mainly control
SOD1 levels in response to stressful stimuli (Milani et al., 2011), these
ndings are particularly noteworthy and open new perspectives in the
understanding of SOD1 gene regulation mechanisms. Moreover, these
observations prompted us to check whether ELAV-mediated pathways
can be also detected in ALS pathology where we previously demonstrat-
ed enhanced SOD1 expression (Gagliardi et al., 2010). To this purpose,
we rstly examined ELAV/HuR localization and phosphorylation-
dependent activation in PBMCs from both SALS patients and healthy
subjects. Notably, PBMCs from sporadic patients were shown to reect
many of the pathological alterations occurring in the nervous system
(Cereda et al., 2006; Cova et al., 2006; Guareschi et al., 2012; Mougeot
et al., 2011), including the increased ROS levels that we reported here;
these peripheral cells can therefore be useful to test hypotheses on the
primary pathological events leading to ALS, thus providing an insight
into disease-causing mechanisms in vivo. Even if we did not reveal
changes in intracellular localization, we detected a signicant increase
in ELAV/HuR phosphorylation in patients' PBMCs compared to controls,
suggesting that this protein is more activated in SALS. Indeed, a growing
body of evidence is showing that kinase-triggered phosphorylation of
ELAV/HuR is crucial for the modulation of its function and allows it
to connect extracellular signals to specic posttranscriptional events
elicited by this RBP (Amadio et al., 2010; Doller et al., 2008, 2010;
Nagai et al., 1995).
Finally, we revealed enhanced perinuclear and cytoplasmic expres-
sion of ELAV in the cerebral motor cortex of SALS patients compared
to that of healthy subjects. These data suggest that the up-regulated
RNA-binding proteins may be more available and massively enrolled
in the positive regulation of target mRNAs, such as SOD1. Remarkably,
positivity for cytoplasmic ELAV was paralleled by increased levels of
both SOD1 mRNA and protein. Similar results were found in the spinal
cord either for ELAV proteins (see Supplementary data) and for SOD1
mRNA/protein (Gagliardi et al., 2010). Additional experiments will be
necessary to test the direct interactions between ELAV proteins and
SOD1 mRNA in vivo and to fully elucidate the exact role of SOD1 post-
transcriptional regulation in SALS. Although we do not know the exact
contribution of the post-transcriptional control mechanisms in the
SOD1 gene expression, an intriguing hypothesis is that a positive regu-
lation of gene expression by RBPs, in particular ELAV, may contribute
to building up with time SOD1 and other stress-related proteins playing
a role in chronic pathologies.
58 P. Milani et al. / Neurobiology of Disease 60 (2013) 5160

Fig. 6. The expression of ELAV proteins and SOD1 mRNA is increased in the cerebral motor cortex of SALS patients. (A) Immunohistochemical localization of ELAV proteins in the cerebral
motor cortex of SALS patients. Control section in which the primary antibody was substituted with non-immune serum shows an absence of labeling (NEG). In the motor cortex of healthy
controls (CTR) and sporadic ALS patients (SALS) the labeling was intense in the nuclei. In SALS patients a diffuse signal was also observed in the perinuclear region and in the cytoplasm as
compared with the motor cortex of control subjects, as better disclosed by the higher magnication of the insets. Our primary antibody could detect all the ELAV proteins, both the neuron-
specic ELAV and the ubiquitous one. Scale bar: 100 m. (B) mRNA SOD1 expression is increased in the cerebral motor cortex of sporadic ALS patients. Expression of SOD1 mRNA
measured by real time qPCR in the cerebral motor cortex of control subjects (CTR; n = 6) and sporadic ALS patients (SALS; n = 10). The values obtained from SOD1 mRNA have been
normalized to the levels of GAPDH mRNA and expressed as means S.E.M.; **p b 0.01, Student's t-test. (C) Immunohistochemical localization of SOD1 protein in the cerebral motor
cortex of SALS patients. Labeling of a control (CTR) and a patient section (SALS) showed a higher immunoreactivity in SALS case evident both at lower and at higher magnication. Control
section in which the primary antibody substituted with non-immune serum showed an absence of labeling (NEG).

Previous evidence from our group showed that neuronal ELAV pro- between ELAVs and SOD1 mRNA and suggests that this binding posi-
teins are down-regulated in the hippocampus of Alzheimer's disease tively inuences SOD1 expression under oxidative stress, a condition
patients, with a parallel decrease in the amount of the target mRNA observed in ALS. Consistently, besides an up-regulation of SOD1 expres-
ADAM10 (Amadio et al., 2009); here we report an opposite prole of sion, we reveal the activation of ELAVs in both peripheral cells and
ELAV levels and their SOD1 target transcript. These observations suggest nervous tissues from SALS patients compared to controls, suggesting
that ELAV proteins can interfere at different levels on the pathological that this event likely represents a mechanism relevant to the disease
process considering the involved central nervous system area and the as well as a novel putative pharmacological target.
down-stream mRNA targets, nally producing peculiar pathological
features. Our in vitro studies suggest that SOD1 mRNA up-regulation is List of abbreviations
a direct consequence of the oxidative insult but we cannot exclude
AREs adenine/uracil-rich elements
that other events participate to the alteration in SOD1 expression on
CTR control subjects
the development of the disease in vivo.
ELAV embryonic lethal abnormal visual RNA-binding proteins
Clearly, signicant gaps still remain in understanding the pathologi-
FALS familial amyotrophic lateral sclerosis
cal implications of SOD1 mRNA expression and regulation; however the
H2O2 hydrogen peroxide
study of ELAV protein pathways could provide new insights for the
IHC immunohistochemistry
comprehension of motor neuron diseases.
mRNP messenger-RiboNucleoProteic complexes
PBMCs peripheral blood mononuclear cells
Conclusion qPCR quantitative polymerase chain reaction
RBPs RNA-binding proteins
ELAV proteins bind to an array of mRNAs and mainly lead to gene ex- REMSA RNA electrophoretic mobility shift assay
pression up-regulation by increasing the stability and/or translation of RNA-IP RNA-immunoprecipitation
target mRNAs. Our in vitro study demonstrates the physical association SALS sporadic amyotrophic lateral sclerosis
 P. Milani et al. / Neurobiology of Disease 60 (2013) 5160
SOD1 Cu/Zn superoxide dismutase
TNF tumor necrosis factor alpha
UTR untranslated region
WB Western blotting
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.nbd.2013.08.005.
Acknowledgments
We thank the Neurological & Neuromuscular Biobank of the Neuro-
logical Institute C. Mondino for providing us with blood samples of
ALS patients and the Human Brain and Spinal Fluid Resource Center
(Los Angeles, CA) for the brain samples. We are grateful to the ALS pa-
tients and their families that participated in this study. We thank Dr.
Matteo Salina for his help in data analysis and the facility Grandi
Strumenti of the University of Pavia for confocal image acquisition
and particularly Dr. Patrizia Vaghi for her helpful assistance. This work
was supported by Italian Ministry of Health (Grants Ricerca Corrente
20092011).
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