Neurobiology of Aging 34 (2013) 248 –262

www.elsevier.com/locate/neuaging

Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing

factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis
Cristian A. Droppelmanna, Brian A. Kellera, Danae Campos-Meloa, Kathryn Volkeninga,b,
Michael J. Stronga,b,*
a
Molecular Brain Research Group, Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
b
Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

Received 6 December 2011; received in revised form 26 May 2012; accepted 24 June 2012

Abstract

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder of unknown etiology characterized by the selective degen-
eration of motor neurons. Recent evidence supports the hypothesis that alterations in RNA metabolism in motor neurons can explain the
development of protein inclusions, including neurofilamentous aggregates, observed in this pathology. In mice, p190RhoGEF, a guanine
nucleotide exchange factor, is involved in neurofilament protein aggregation in an RNA-triggered transgenic model of motor neuron disease.
Here, we observed that rho guanine nucleotide exchange factor (RGNEF), the human homologue of p190RhoGEF, binds low molecular
weight neurofilament mRNA and affects its stability via 3= untranslated region destabilization. We observed that the overexpression of
RGNEF in a stable cell line significantly decreased the level of low molecular weight neurofilament protein. Furthermore, we observed
RGNEF cytoplasmic inclusions in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TAR (trans-active
regulatory) DNA-binding protein 43 (TDP-43). Our results provide further evidence that RNA metabolism pathways are integral to ALS
pathology. This is also the first described link between ALS and an RNA binding protein with aggregate formation that is also a central cell
signaling pathway molecule.
© 2013 Elsevier Inc. All rights reserved.

Keywords: Amyotrophic lateral sclerosis; Rho guanine nucleotide exchange factor; RGNEF; p190RhoGEF; NFL; RNA stability; RNA binding protein;
Protein aggregates

1. Introduction Hays, 2004), TAR DNA binding protein of 43 kDa (TDP-

Amyotrophic lateral sclerosis (ALS) is an adult-onset
progressive disorder characterized by the selective degen-
eration of motor neurons resulting in paralysis and death 3
to 5 years after onset in most patients (Strong et al., 2005).
There are no treatments that will arrest disease progression
and the cause remains elusive. Protein aggregate formation
in motor neurons is a neuropathological hallmark of this
disease. These aggregates contain proteins such as neuro-
filament (NF) (Kondo et al., 1986), peripherin (He and
* Corresponding author at: Room C7-120, University Hospital, LHSC,
339 Windermere Road, London, Ontario N6A 5A5, Canada. Tel.: ⫹1 519
663 3874; fax: ⫹1 519 663 3609.
E-mail address: Michael.Strong@schulich.uwo.ca (M.J. Strong).
43) (Arai et al., 2006; Neumann et al., 2006), fused in
sarcoma/translocated in liposarcoma (FUS/TLS) (Kwiat-
kowski et al., 2009; Vance et al., 2009), copper/zinc super-
oxide dismutase 1 (SOD1) (Shaw et al., 2008; Stieber et al.,
2000), ubiquitin (Arai et al., 2006; Neumann et al., 2006),
and 14-3-3 (Kawamoto et al., 2004). Additionally, motor
neurons also show a selective decrease in the levels of
polyadenylated mRNA, low molecular weight neurofila-
ment (NFL) mRNA, ␣-internexin mRNA, and peripherin
mRNA (Bergeron et al., 1994; Wong et al., 2000). These
data, together with the participation of TDP-43 and FUS/
TLS as RNA binding proteins (Buratti et al., 2001; Crozat et
al., 1993), supports the hypothesis that alterations in RNA
metabolism in motor neurons can lead to the development

0197-4580/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.neurobiolaging.2012.06.021
 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262
of protein aggregates in ALS (Strong, 2010). This hypoth-
esis has been supported with a recent report showing a
pathological association of an expanded hexanucleotide re-
peat in the noncoding region of C9ORF72 with ALS (Deje-
sus-Hernandez et al., 2011; Renton et al., 2011).
The NF proteins are a highly conserved family of neu-
ronal intermediate filament proteins. There are 3 members
of the NF family, a 68-kDa form (NFL), a 160-kDa form
(middle molecular weight NF), and a 200-kDa form (high
molecular weight NF). NF subunits assemble as heteropo-
lymers in which the initial polymerization of NFL subunits
is required for normal NF architecture formation (for review
see Strong, 1999). In transgenic mouse models overexpress-
ing peripherin or high molecular weight NF in which there
is targeted disruption of the NFL gene, selective death of
motor neurons is observed, along with motor dysfunction
and the formation of intermediate filament aggregates, in-
cluding intraneuronal NF aggregates (Beaulieu et al., 1999,
2000). These results highlight the importance of NF stoi-
chiometry in motor neurons and the importance of NFL
mRNA stability as a possible cause of the protein aggre-
gates observed in ALS (Szaro and Strong, 2010; Thyagara-
jan et al., 2007).
In mice, it has been observed that p190RhoGEF, a gua-
nine nucleotide exchange factor (GEF) (van Horck et al.,
2001) interacts with a destabilizing region of NFL mRNA
providing stability to the transcript (Cañete-Soler et al.,
2001). This protein is involved in NF protein aggregation
observed in an RNA-triggered transgenic model of motor
neuron disease (Lin et al., 2005; Nie et al., 2002). Previ-
ously, we showed that the RNA binding domain of the
human homologue of p190RhoGEF, called rho guanine
nucleotide exchange factor (RGNEF), can interact with
NFL mRNA (Volkening et al., 2010). Here, we show that
full length RGNEF is an RNA binding protein that acts as
an NFL mRNA stability factor via 3= untranslated region
(UTR) destabilization and reduces NFL protein levels when
overexpressed in HEK293T cells. Moreover, we show that
RGNEF also presents a pathogenic phenotype including
aggregate formation in motor neurons of ALS patients.
2. Methods
2.1. Cases
Postmortem frozen and formalin-fixed, paraffin-embed-
ded tissues were collected as part of the ALS protocol at
London Health Sciences Centre (London, Ontario, Canada).
Six control cases (4 male, 2 female, age range 61–74 years),
13 sporadic ALS (sALS; 8 male, 5 female, age range 45– 80
years) cases, and 3 familial ALS (fALS; 2 male, 1 female,
age range 56 –71 years) cases without known mutations in
the coding sequence of SOD1, FUS/TLS, and TDP-43 and
without C9ORF72 expanded repeats were used in these
studies.
249
2.2. Anti-RGNEF antibody production
A polyclonal goat antibody against RGNEF that recog-
nizes a sequence on the RNA binding domain of the protein
was produced by Twenty-First Century Biochemicals (Mar-
lborough, MA, USA). The specificity of the antibody was
analyzed by competition assay with a blocking peptide
identical to the antigenic peptide used to produce the anti-
body (Supplementary Fig. 1).
2.3. RGNEF and p190RhoGEF plasmids
The full RGNEF coding sequence (GenBank BC157846)
was cloned by polymerase chain reaction (PCR) using Pfu
DNA polymerase (Fermentas Canada, Inc., Burlington,
Canada). The commercial RGNEF clone MHS1768-
99865695 (Open Biosystems Lafayette, Colorado, USA)
was used as a template. The RGNEF coding sequence was
ligated to pcDNA3.1-myc-His (Invitrogen, Life Technolo-
gies Inc., Burlington, Canada) between the KpnI/XhoI sites
(pcDNA-RGNEF-myc; Supplementary Fig. 2A). A deletion
in the 3= end of the RGNEF sequence (from nucleotide 3769
to stop codon) including the entire region that encodes the
RNA binding domain was generated. The product was also
incorporated into pcDNA3.1-myc-His between the KpnI/
XhoI sites to create pcDNA-RGNEF-⌬CT-myc (Supple-
mentary Fig. 2B). In addition, a fragment of an alternatively
spliced form of RGNEF with a shorter RNA binding do-
main lacking exons 29 and 33 (BD-⌬exon29/33) was cloned
from HEK293T cDNA using primers that recognize coding
exons 25 to 34 of RGNEF. To generate glutathione S-trans-
ferase (GST) fusion proteins, the sequences corresponding
to the RNA binding domain of RGNEF (RGNEF-BD) and
BD-⌬exon29/33 were cloned into pGEX-5X-3 (GE Health-
care Life Sciences, Baie d’Urfe, Quebec, Canada) between
EcoRI/XhoI sites (Supplementary Fig. 2C and D). The plas-
mid pcDNA-p190RhoGEF was kindly provided by Dr.
Wouter H. Moolenaar (Amsterdam, Netherlands).
2.4. RGNEF stable cell line
A stable cell line of HEK293T cells expressing full
length RGNEF-myc (HEK293T-RGNEF(⫹)) was created
using the Jump-in Fast Gateway Targeted Integration Sys-
tem (Invitrogen) according to the manufacturer’s instruc-
tions. This technology uses PhiC31 integrase-mediated recom-
bination to stably integrate DNA sequences of choice at
specific genomic locations called pseudo-attP sites in mamma-
lian cells (Thyagarajan et al., 2001,Thyagarajan et al. 2008).
The cells, with the gene of interest successfully incorporated
into their genome, were selected using hygromycin B.
2.5. Western blots and immunocytochemistry
HEK293T cells were transfected with pcDNA-RGNEF-
myc, pcDNA-RGNEF-⌬CT-myc, or pcDNA3.1-myc-His
(control) using Lipofectamine 2000 (Invitrogen) according
to the manufacturer’s protocols. After 24 hours, the cells
250 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262
were lysed in NP-40 buffer and SDS-PAGE was performed
using 100 ␮g of protein extracts and then transferred to a
nitrocellulose membrane. Mouse anti-myc (1:2500; Cedar-
lane Laboratories, Burlington, NC, USA), rabbit anti-NFL
(1:1000; Invitrogen), mouse anti-actin (1:400; Santa Cruz
Biotechnologies, Santa Cruz, CA, USA), and rabbit anti-
GAPDH (1:2500; Abcam, Cambridge, UK) antibodies were
used for Western blotting. For immunocytochemistry,
HEK293T-RGNEF(⫹) cells were fixed in 4% paraformal-
dehyde for 15 minutes and washed with phosphate-buffered
saline (PBS). Cells were incubated with mouse anti-myc
(1:1000; Cedarlane Laboratories) for 1 hour at room tem-
perature and then with anti-mouse Alexa Fluor 543 (1:1000;
Invitrogen). Finally, the cells were incubated with Hoechst
to visualize nuclei.
2.6. GST fused protein purification
GST-RGNEF-BD, GST-BD-⌬exon29/33, and GST pro-
teins were purified from BL21DE3 E. coli (Invitrogen)
using the GSTrap 4B purification system (GE Healthcare
Life Sciences) according to the manufacturer’s instructions.
The purified protein was confirmed by SDS-PAGE stained
with Coomassie blue and verified by molecular size by
comparison with molecular weight standards.
2.7. RNA-immunoprecipitation-PCR (RNA-IP-PCR)
HEK293T cells were transfected with pcDNA-RGNEF-
myc, pcDNA-RGNEF-⌬CT-myc, or pcDNA3.1-myc-His
(control) vector using Lipofectamine 2000 (Invitrogen). Af-
ter 24 hours, whole cell homogenates were prepared using
NP-40 buffer with proteinase inhibitors (Complete Protease
Inhibitor; Roche, Mississauga, Ontario, Canada) and RNase
inhibitors (RNAaseOUT; Invitrogen). Cell lysate (1 mg)
was mixed with 10 ␮L Pansorbin (Calbiochem-Millepore,
Billerica, MA, USA) and 1 ␮g of antibody (mouse anti-myc
or IgG as control) and incubated for 1 hour at room tem-
perature. After centrifugation at 10,000g, pellets were
washed 3 times in 1 mL cold RNase-free NP-40 buffer (PBS
containing 1% NP-40 and proteinase inhibitors and RNase
inhibitors as above), and then 3 times in cold PBS (with
proteinase inhibitors and RNase inhibitors as above). The
RNA was resuspended in 40 ␮L of nuclease-free water and
then reverse transcription (RT)-PCR was performed to de-
tect immunoprecipitated NFL mRNA. Alternatively, RNA-
IP-PCR was performed by mixing 2 ␮g of purified GST-
RGNEF-BD, GST-BD-⌬exon29/33, or GST protein and 2
␮g of proteinase K-treated total RNA isolated from control
and ALS patients using anti-GST antibody (Amersham-GE
Healthcare Life Sciences). For all experiments, a negative
RT reaction (without reverse transcriptase) and negative
PCR (without cDNA template) were used as negative con-
trols. HEK293T cDNA from total RNA was used as tem-
plate for positive controls.
2.8. RNA isolation, relative quantitative PCR
and RT-PCR
Total RNA from transfected HEK293T cells was isolated
using Trizol reagent (Invitrogen). Reverse transcription was
performed using the Superscript II reverse transcriptase
system (Invitrogen). Relative quantitative RT-PCR for lu-
ciferase mRNA and NFL mRNA stability experiments were
performed by coamplifying firefly luciferase and renilla
luciferase or RGNEF and 18S (as internal standard) during
the linear range of both reactions. Every experiment was
performed in triplicate. Both luciferases are expressed from
the same vector (pmirGLO vector; Promega, Madison, WI,
USA) and renilla luciferase works as the control for trans-
fection efficiency. The experiment and data analysis for the
luciferase mRNA stability experiment was performed in the
same way as the luciferase reporter assays. For the detection
of NFL mRNA in HEK293T cells and RNA- immunopre-
cipitation experiments, standard RT-PCR protocols were
followed without quantification. PCR primers for firefly
luciferase were forward 5=-CAA GAC TAT AAG ATT
CAA TCT GCC CTG CTG-3= and reverse 5=-GAT GTT
GGG GTG TTG CAG CAG GAT-3= (Amplicon: 561 base
pair [bp]). PCR primers for renilla luciferase were forward
5=-GAG CAA CGC AAA CGC ATG ATC ACT G-3= and
reverse 5=-TTC AGC AGC TCG AAC CAA GCG GT-3=
(Amplicon: 302 bp). PCR primers for NFL were forward
5=-CAA GAC CCT GGA AAT CGA AG-3= and reverse
5=-TCT TGG ACA TGG CTG GTG TA-3= (Amplicon: 405
bp) and PCR. primers for 18S were forward 5= AGT TGG
TGG AGC GAT TTG TC3= and reverse 5= TTC CTC GTT
CAT GGG GAA TA3= (Amplicon: 297 bp.).
2.9. GEF activity
The GEF activity in HEK293T cells transfected with
pcDNA-RGNEF-myc and HEK293T transfected with
pcDNA3.1-myc-His (control) cells was determined using the
“Rho Activation Assay Biochem Kit” (Cytoskeleton Inc., Den-
ver, CO, USA) according to the manufacturer’s instructions.
2.10. Luciferase reporter assay
The reporter plasmid was constructed by inserting the 3=
UTR of human or mouse NFL mRNA between the NheI/
SalI sites downstream of the firefly luciferase coding se-
quence in the pmirGLO vector after PCR amplification
using Pfu DNA polymerase. Five constructs were made:
pmirGLO-mouse NFL 3= UTR (GenBank NM_010910;
bases 1–286); pmirGLO-NFL 3= UTR-Short (UTR-S; en-
coding a segment of the human NFL 3= UTR analogous to
the murine NFL 3= UTR; bases 1–286 of human 3= UTR);
pmirGLO-NFL 3= UTR-Medium (UTR-M; GenBank;
BC039237; bases 1–1380); pmirGLO-NFL 3= UTR-Long
(UTR-L; GenBank; NM_006158; bases 1–1838); and pmir-
GLO-NFL 3= UTR M⌬S (3= UTR-M with bases 1–286
deleted, resulting in a sequence of 1094 bp) (Supplementary
 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262
Fig. 3). HEK293T or Neuro2A cells were plated 24 hours
prior to transfection in 96-well plates at 9 ⫻ 103 cells per
well. Transfections were performed with Lipofectamine
2000 according to the manufacturer’s instructions. Cells
were cotransfected with 2.83 fmol of pcDNA-RGNEF-
myc, pcDNA-p190RhoGEF, pcDNA-RGNEF-⌬CT-myc, or
pcDNA-TDP-43 (Strong et al., 2007) and 3.47 fmol of
pmirGLO with the corresponding NFL 3= UTR. Luciferase
activity was measured 24 hours after transfection using the
Dual-Glo Luciferase Assay System (Promega) in a Turner
Biosystems Luminometer Modulus (Promega). The pmir-
GLO vector expresses both luciferases with different pro-
moters eliminating the necessity of transfecting 2 vectors
for normalizing luciferase activity. Thus firefly luciferase
activity was normalized to renilla luciferase activity to ad-
just for variations in transfection efficiency among experi-
ments. Data were also normalized to the RGNEF,
p190RhoGEF, or TDP-43 effect on pmirGLO without NFL
3= UTR (empty vector) to obtain the specific effect over
NFL 3= UTR. To present destabilization as negative values
and stabilization as positive values, the data were presented
as 1-([NFL*]/[Vector*]), where: [NFL*] ⫽ (normalized
luciferase activity of pmirGLO coupled NFL 3= UTR
alone)/(normalized luciferase activity pmirGLO-NFL cou-
pled NFL 3= UTR in presence of protein) and [Vector*] ⫽
(normalized luciferase activity pmirGLO alone)/(normal-
ized luciferase activity pmirGLO in presence of protein).
Each experiment was performed in triplicate.
2.11. siRNA experiments
An small inhibitory RNA (siRNA) against RGNEF
(SASI_HS01_00264015; Sigma) was transfected in HEK293T-
RGNEF(⫹) cells using Lipofectamine 2000 according to
the manufacturer’s instructions. To maintain the siRNA
expression for 7 days, 3 consecutive transfections were done
before lysing the cells and performing the Western blot. In
parallel, as control, HEK293T cells and HEK293T-RG-
NEF(⫹) cells were transfected using a control siRNA
(Misssion siRNA Universal Negative Control No. 1; Sigma,
Oakville, Ontario, Canada). Each experiment was per-
formed in triplicate.
2.12. Immunohistochemistry
Archival paraffin-embedded sections of lumbar spinal
cord from control and ALS cases were serially sectioned at
6-␮m thickness. For 3,4 diaminobenzide (DAB)-immuno-
histochemistry, tissue sections were deparaffinized using
standard protocols and then immunolabeled using a goat
anti-RGNEF (1:500; optimum pH: 7.4) antibody. The anti-
gen–antibody complex was visualized using the Vectastain
Elite ABC kit (Vector, Burlington, Ontario, Canada) and an
Olympus BX45 microscope with Image-Pro Plus software,
version 7 (Leeds, Minneapolis, MN, USA). For confocal
immunofluorescence microscopy the sections were simi-
larly processed but double-labeled. Goat anti-RGNEF
251
(1:100), mouse anti-ubiquitin (1:5000; Chemicon, Temecula,
CA, USA), mouse anti-TDP-43 (1:500; Abnova, Walnut, CA,
USA), rabbit anti-p62/Sequestosome-1 (1:1000; Enzo Life Sci-
ences, Brockville, Ontario, Canada), and rabbit anti-14-3-3
(1:250; Santa Cruz Biotechnology) were used as primary an-
tibodies. Anti-RGNEF was detected using secondary anti-
body conjugated to Alexa Fluor 488 (1:500; Invitrogen).
Anti-TDP-43, anti-p62/Sequestosome-1, anti-ubiquitin, and
anti-14-3-3 were detected using secondary antibody conju-
gated to Alexa Fluor 546 (1:500; Invitrogen). Slides were
viewed with a Nikon 510 Confocal microscope and local-
ization determined by capture and examination of single
plane images in sequential mode for colocalization.
2.13. Colocalization images and schematic diagram
of proteins
An intensity correlation analysis (Li et al., 2004) using
ImageJ software was performed for the colocalization im-
ages. The colocalized pixels are shown as product of the
differences from the mean (PDM) images. PDM ⫽ (red
intensity⫺mean red intensity) ⫻ (green intensity⫺mean
green intensity). In the colocalization images blue color
indicates a low level of colocalization while yellow and
white indicate a high degree of colocalization. The sche-
matic representation of proteins was performed using the
Dog 2.0 software (Ren et al., 2009).
2.14. Statistics
For the luciferase reporter assays experiments and NFL
protein quantification, analysis of variance with Newman–
Keuls post hoc test was performed. The results are presented
as mean ⫾ standard error of the mean and p values corre-
spond to comparison with the control. Student t tests were
performed for GEF activity analysis, the effect of RGNEF
over firefly luciferase mRNA and the NFL mRNA levels in
HEK293T-RGNEF(⫹). Data are presented as mean ⫾ stan-
dard error of the mean and the p values correspond to the
comparison with the control. A value of p ⬍ 0.05 was
considered statistically significant for all experiments.
3. Results
3.1. Human RGNEF shows a high similarity with
murine p190RhoGEF
An amino acid (aa) sequence comparison analysis was
performed to determine the similarity between the murine
p190RhoGEF (GenBank U73199) and its human homolog,
RGNEF (GenBank BC157846). These proteins share the
same conserved domains (Fig. 1A). After Basic Local
Alignment Search Tool analysis (BLAST utility; blast.
ncbi.nlm.nih.gov), we observed an 80% identity between
p190RhoGEF and RGNEF at the protein level. The GEF
domain (mouse: aa 847–1039; human: aa 850 –1042 accord-
ing the conserved domains utility; www.ncbi.nlm.nih.gov/
Structure/cdd/wrpsb.cgi) showed 92% identity (Fig. 1B).
252 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262

Fig. 1. Mouse p190RhoGEF and human rho guanine nucleotide exchange factor (RGNEF) show a high level of identity in their sequences. (A) Schematic
of the conserved domains shared between p190RhoGEF and RGNEF. Domains include a cystein rich zinc finger domain (yellow), RhoGEF domain (blue),
Pleckstrin homology (PH) domain (green), and RNA binding domain (red). These proteins share the same conserved domains. (B) Alignment of the GEF
domain (between brackets) of RGNEF (human; H) and p190RhoGEF (mouse; M). Conserved amino acids are shown in blue. (C) Alignment of the RNA
binding domain (between brackets) of RGNEF (human; H) and p190RhoGEF (mouse; M). Conserved amino acids are shown in red.

The RNA binding domain (mouse: aa 1254 –1610; Cañete-
Soler et al., 2001; human: aa 1257–1614; Volkening et al.,
2010) showed 76.2% identity (Fig. 1C). These results show
a great similarity in the functional domains of p190RhoGEF
and RGNEF; thus, it is reasonable to predict similar func-
tional properties of both proteins.
3.2. RGNEF binds to NFL mRNA
We previously showed that the RNA binding domain of
RGNEF (RGNEF-BD) interacts with NFL mRNA (Volk-
ening et al., 2010). Here, we analyzed the interaction of the
full length RGNEF protein with human NFL transcript. Full
length myc-tagged RGNEF was transiently expressed in
HEK293T cells (Supplementary Figs. 2A andSupplemen-
tary Fig 4A) and RNA-IP-PCR was performed to detect
interaction with the NFL mRNA that is endogenously ex-
pressed in these HEK293T cells (Supplementary Fig. 4B).
An RGNEF version without the RNA binding domain
(RGNEF-⌬CT-myc) (Supplementary Figs. 2B and Supple-
mentary Fig 4C) was used as a control for interaction
specificity. Our results showed specific interaction of full
length RGNEF (containing the RNA binding domain) with
NFL mRNA in transfected cells using an anti-myc antibody
IP (Fig. 2A, lane 2), while cells transiently expressing
RGNEF-⌬CT-myc do not show any interaction with NFL
mRNA (Fig. 2A, lane 3). The same was observed for control
cells (transfected with the empty vector; Fig. 2A, lane 1)
and cells expressing RGNEF-myc or RGNEF-⌬CT-myc
with control IgG IP (Fig. 2A, lanes 4 and 5). To evaluate if
this interaction was direct or mediated by other proteins, we
performed RNA-IP using purified GST-RGNEF-BD (Sup-
plementary Fig. 2C and Supplementary Fig 3D) and purified
proteinase K-treated total RNA from spinal cord of control,
sALS and fALS patients. Purified GST-BD-⌬exon29/33,
which expresses a GST fusion protein with a shorter RNA
binding domain but with an equivalent mass than GST-
RGNEF-BD (Supplementary Fig. 2B and Supplementary
Fig 3D), and purified GST alone, were used to confirm
specificity. We observed interaction only with GST-
RGNEF-BD in control, sALS, and fALS patients (Fig. 2B).
These results clearly show that full length RGNEF interacts
with NFL mRNA and suggest that the basic interaction of
this mRNA with the RNA binding domain of RGNEF does
not appear to require any intermediate proteins.
3.3. RGNEF has GEF activity and acts as an NFL mRNA
destabilizing factor
To analyze the cellular roles of RGNEF, we examined
whether RGNEF exhibits GEF activity as well as whether it
can act as a human NFL mRNA stability factor. As RGNEF
 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262 253

Fig. 2. Rho guanine nucleotide exchange factor (RGNEF) binds to neurofilament (NFL) mRNA. (A) RNA-immunoprecipitation-polymerase chain reaction
(RNA-IP-PCR) experiment analyzing the interaction of full length RGNEF (RGNEF-myc) and RGNEF-⌬CT-myc (RGNEF lacking RNA binding domain),
with endogenous NFL mRNA in HEK293T cells. Lysates of cells transfected with plasmids pcDNA-RGNEF-myc, pcDNA-RGNEF-⌬CT-myc and
pcDNA3.1-myc-His (control) were immunoprecipitated using anti-myc antibody and IgG (control). NFL mRNA was detected in the immunoprecipitated
pellets by RT-PCR. Interaction was only observed in cells expressing RGNEF-myc (lane 2). There was no interaction detected with RGNEF-⌬CT-myc (lane
3), empty vector (lane 1) or in cells expressing RGNEF-myc and BD-⌬exon-myc and immunoprecipitated with IgG (lanes 4, 5). RT-PCR reaction controls
were no RT negative control, no cDNA negative control, and positive control using HEK293T cDNA. (B) RNA-IP-PCR experiment analyzing the interaction
of purified GST-RGNEF-BD, GST-BD-⌬exon29/33 and GST with NFL mRNA using purified total RNA obtained from control, sporadic amyotrophic lateral
sclerosis (sALS) and familial amyotrophic lateral sclerosis (fALS) spinal cords. Anti-GST antibody was used for the immunoprecipitation and NFL mRNA
was detected in the pellet by RT-PCR. Interaction was observed with the complete RNA binding domain of RGNEF (GST-RGNEF-BD) but not with GST
alone or GST-BD-⌬exon29/33. RT-PCR reaction controls were no RT negative control, no cDNA negative control, and positive control using HEK293T
cDNA.

has a conserved GEF domain, we predicted that RGNEF
would have the capacity to activate RhoA protein as GEF
activity in the cell is required for activation of RhoA (Nal-
bant et al., 2009). We used a RhoA activation assay that
determines the amount of activated RhoA (GTP-RhoA) in a
cellular lysate based on the capacity of GTP-RhoA to bind
to Rhotekin binding domain (RBD) (Ren et al., 1999). We
found that HEK293T cells transiently expressing RGNEF-
myc showed increased GTP-RhoA interaction with RBD,
and consequently, more GEF activity, than control cells
transfected with the empty vector (3.4-fold; p ⬍ 0.05; Fig.
3). These data show that RGNEF has the ability to function
as a GEF protein as would be predicted.
Next, we evaluated the effect of RGNEF on the stability
of human NFL via the 3= UTR by using a firefly luciferase
reporter gene coupled to the 3= UTR of NFL mRNA. The
pmirGLO system was used so that the 2 luciferases (firefly
and renilla) were expressed from 1 construct avoiding ex-
pression alterations with transfection efficiency variability.
Four constructs were screened, pmirGLO-NFL 3= UTR-S
(286 bp), pmirGLO-NFL 3= UTR-M (1380 bp), pmirGLO-
NFL 3= UTR-L (1838 bp) and pmirGLO-NFL 3= UTR-
M⌬Short (3= UTR-M⌬S; 1094 bp). The purpose of 3= UTR-
M⌬S construct was to eliminate putative determinants of
stabilization that could be present in the 3= UTR-S (Cañete-
Soler et al., 1998). There was specific downregulation of
luciferase activity, suggesting destabilization of the lu-
ciferases’ mRNAs, in HEK293T cells cotransfected with
pcDNA-RGNEF-myc and pmirGLO fused to NFL 3= UTR
constructs (3= UTR-S: ⫺0.0834 ⫾ 0.0194; 3= UTR-M:
⫺0.1027 ⫾ 0.0174; 3= UTR-L: ⫺0.1333 ⫾ 0.0429: 3=
UTR-M⌬S: ⫺0.1827 ⫾ 0.0383; all p ⬍ 0.05; negative
values indicate downregulation; Fig. 4A). To determine if
the presence of the RNA binding domain is critical in the
observed effect over the NFL 3= UTR, a cotransfection of
pcDNA-RGNEF-⌬CT-myc and pmirGLO fused to NFL 3=
UTR (Short, Medium, or Long) was performed. Using this
mutant RGNEF, the downregulating effect previously ob-
served with the full-length RGNEF was completely abol-
ished, and an upregulation of luciferase activity could even
be observed (Supplementary Fig. 5). For a control of the
model, we examined the interaction of TDP-43 with human
NFL-3= UTR as we previously described (Strong et al.,
2007). HEK293T cells were cotransfected with pmirGLO-
NFL 3= UTR-S and pcDNA-TDP-43 and an increase in the
luciferase activity was detected (0.0656 ⫾ 0.0095, p ⬍
0.05; positive value indicates upregulation; Fig. 4A), sug-
gesting stabilization of the NFL 3= UTR-S, which is con-
sistent with our previous reports (Strong et al., 2007; Volk-
ening et al., 2009).
Because of the apparent contradiction with the data re-
garding mouse p190RhoGEF, which was found to stabilize
NFL mRNA (Cañete-Soler et al., 2001), we decided to
evaluate the effect of p190RhoGEF over the luciferase re-
254 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262

Fig. 3. Rho guanine nucleotide exchange factor (RGNEF) shows guanine nucleotide exchange factor (GEF) activity in HEK293T cells. (A) The GEF activity
in HEK293T transiently expressing RGNEF-myc was evaluated using the capacity of activated RhoA (RhoA-GTP) to bind to Rhotekin binding domain
(RBD). A pull-down experiment using RBD-agarose beads in lysates of cells transfected with pcDNA-RGNEF-myc (RGNEF) and pcDNA3.1-myc-His
(control) was performed. The upper panel of (A) shows the presence of RGNEF in cells transfected with pcDNA-RGNEF-myc to confirm expression. In the
middle panel (pull down experiment), cells overexpressing RGNEF show a higher amount of active RhoA than the empty vector control. The lower panel
shows the input control for the experiment (1/6 of the protein amount used in the pull-down experiment). (B) Quantification of 3 GEF activity experiments
calculated as the RhoA in the pull-down against the input amount of RhoA. The results are presented as mean ⫾ standard error of the mean (* ⫽ p ⬍ 0.05).

porter gene coupled to mouse NFL 3= UTR in our experi-
mental model. We observed an upregulation of the lucifer-
ase activity, suggesting stabilization of the mRNA, in
HEK293T cells cotransfected with pcDNA-p190RhoGEF
and pmirGLO-mouse NFL 3= UTR (0.0922 ⫾ 0.0524, p ⬍
0.05; Fig. 4A), totally in agreement with the published data.
Interestingly, when we performed the same experiment us-
ing pmirGLO-NFL 3= UTR-S (91% of identity with mouse
NFL 3= UTR) the result was practically identical (0.0971 ⫾
0.0532; p ⬍ 0.05; Fig. 4A). These results suggest that the
destabilizing and stabilizing capacity of RGNEF and
p190RhoGEF respectively are intrinsic properties of these
proteins.
Because we conducted the experiments in a nonneural
cell line, we used mouse Neuro2A cells to evaluate whether
the RGNEF effect over the luciferase coupled to NFL 3=
UTR could be observed in a neuronal cell line. Further, we
aimed to determine whether RGNEF acts analogously to
p190RhoGEF and therefore has a heterologous effect on the
mouse NFL 3= UTR. We observed that RGNEF can down-
regulate the luciferase reporter gene coupled to mouse NFL
3= UTR (⫺0.1059 ⫾ 0.0563; p ⬍ 0.01; Fig. 4B). As a
control, we evaluated the effect of p190RhoGEF on the
mouse 3= UTR. As expected, we observed upregulation of
luciferase activity (0.1057 ⫾ 0.0556; p ⬍ 0.01; Fig. 4B).
These results support the above conclusion about the intrin-
sic properties of these proteins, demonstrated that the de-
stabilizing effect of RGNEF can be observed in neuronal
cell lines, and that RGNEF can act in a heterologous way.
The decrease in luciferase activity in the reporter gene
assay and the interaction of RGNEF with NFL mRNA both
suggest, but do not prove, a direct effect of RGNEF on NFL
mRNA stability. To clarify this, we measured the effect of
RGNEF on the levels of firefly luciferase coupled NFL 3=
 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262 255

Fig. 4. Rho guanine nucleotide exchange factor (RGNEF) acts as a destabilizing factor of neurofilament (NFL) mRNA. (A) Reporter gene assay showing
decreased normalized luciferase activity (see Methods) in HEK293T cells cotransfected with pcDNA-RGNEF-myc and pmirGLO linked to 1 of 4 different
lengths of human 3= untranslated region (UTR) NFL mRNA (S ⫽ 3= UTR Short; M ⫽ 3= UTR Medium; L ⫽ 3= UTR Long; M⌬S ⫽ 3= UTR M⌬S). Also,
we observed an increase in the normalized luciferase activity in cells cotransfected with pcDNA- TAR DNA binding protein of 43 kDa (TDP-43) and
pmirGLO NFL 3= UTR-S or with pcDNA-p190RhoGEF and pmirGLO-mouse NFL 3= UTR or pmirGLO NFL 3= UTR-S. p values (* p ⬍ 0.05; ** p ⬍ 0.01;
*** p ⬍ 0.001) refer to comparison with control experiment (luciferase activity of the vector without NFL 3= UTR in presence of RGNEF-myc or
p190RhoGEF or TDP-43). (B) Reporter gene assay showing decreased normalized luciferase activity in Neuro2A cell cotransfected with pcDNA-RGNEF-
myc and pmirGLO-mouse NFL 3= UTR and increased luciferase activity in cells cotransfected with pcDNA-p190RhoGEF and pmirGLO-mouse NFL 3=
UTR. p values (** p ⬍ 0.01) refer to comparison to control experiment (luciferase activity of the vector without NFL 3= UTR in presence of RGNEF-myc
or p190RhoGEF). (C) The level of normalized luciferase mRNA (see Methods) in HEK293T cells cotransfected with pmirGLO-NFL 3= UTR-M⌬S and
pcDNA-RGNEF-myc. We observed a significant reduction of luciferase-3= UTR-M⌬S mRNA in the presence of RGNEF-myc. p value (** p ⬍ 0.01) refer
to comparison with control experiment (luciferase mRNA levels of the vector without NFL 3= UTR in presence of RGNEF-myc).
256 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262
UTR-M⌬S mRNA. We used the 3= UTR-M⌬S construct
because it showed the highest reduction in luciferase activ-
ity (Fig. 4A), allowing easier detection of variation in the
firefly luciferase mRNA levels. We observed that in the
presence of RGNEF, there was a significant reduction in
the mRNA levels of luciferase-coupled NFL 3= UTR-M⌬S
(⫺0.3465 ⫾ 0.1116, p ⬍ 0.01; Fig. 4C).
These results clearly show that RGNEF acts as an NFL
mRNA destabilizing factor through a seemingly direct ef-
fect on the NFL 3= UTR. Taken together, RGNEF functions
as a GEF protein and it can also act as an mRNA binding
protein, imparting instability to human NFL mRNA.
3.4. NFL protein levels are decreased in a stable cell line
overexpressing full length RGNEF
To evaluate the NFL mRNA destabilizing activity
of RGNEF on NFL protein levels, we created the
HEK293T-RGNEF(⫹) stable cell line that overexpresses
full length RGNEF-myc (Fig. 5A). We obtained a clone
with a highly significant decrease in NFL protein levels
(18.28-fold; p ⬍ 0.001) (Fig. 5B and C). This effect over
NFL protein levels was seen in other clones generated by
the same manner (Supplementary Fig. 6). To determine
whether the decrease of NFL protein levels was due to an
effect of RGNEF on the NFL mRNA levels, we analyzed
the NFL mRNA levels in HEK293T-RGNEF(⫹) cells. We
observed a significant decrease of the NFL mRNA levels in
this cell line (Fig. 5D).
To evaluate if the decrease of NFL proteins levels was
caused specifically by RGNEF, we performed a rescue exper-
iment using a specific siRNA against RGNEF. After treating
HEK293T-RGNEF(⫹) cells with the siRNA against RGNEF,
we observed a significant increase of NFL proteins levels
together with a decrease of RGNEF protein levels due to the
siRNA (Fig. 5E and F). The percentage of NFL protein recov-
ered after the siRNA treatment is in agreement with the pre-
viously reported recovery rate of NFL protein levels observed
after an inducible shutdown of its expression (Millecamps et
al., 2007).
These results clearly indicate that the NFL mRNA de-
stabilizing activity of RGNEF can impact NFL protein lev-
els, suggesting that RGNEF may have an important impact
on NF homeostasis in motor neurons.
3.5. RGNEF forms cytoplasmic inclusions in motor
neurons from ALS patients
To analyze the intracellular localization of RGNEF, we
examined the presence of this protein in motor neurons of
ALS and control spinal cords by immunohistochemistry.
We observed homogeneous cytoplasmic RGNEF immuno-
reactivity in spinal motor neurons of control tissue (Fig.
6A). In contrast, intensely immunoreactive intracytoplasmic
accumulations of RGNEF were clearly observed in every
case of ALS examined, regardless of ALS variant (sALS or
fALS). Two morphological variants of RGNEF-immunore-
active aggregates were observed: skein-like (Fig. 6B and D)
and punctate (Fig. 6C and E). There was no apparent rela-
tionship between the type of aggregate observed and ALS
variant. When quantification was performed comparing
RGNEF and TDP-43 aggregates there was no statistically
significant difference between the number of RGNEF and
TDP-43 protein inclusions in ALS patients (Table 1). These
results strongly suggest a pathogenic phenotype for RGNEF
in ALS motor neurons as has been postulated for TDP-43.
3.6. RGNEF colocalizes with ubiquitin, p62/
Sequestosome-1, and TDP-43 in motor neurons from
ALS patients
To determine if RGNEF could coaggregate with other
proteins known to be present in pathogenic aggregates, we
evaluated the colocalization of RGNEF with ubiquitin,
TDP-43 and p62/Sequestosome-1 (a marker of neurodegen-
eration that participates in the ubiquitin pathway) (Kuusisto
et al., 2001, 2008; Pikkarainen et al., 2008, 2010; Seppänen
et al., 2009). We observed a high degree of colocalization
between RGNEF and ubiquitin in ALS patients (Fig. 7A).
The same was observed for RGNEF and p62/Sequesto-
some-1 (Fig. 7B). In control motor neurons, RGNEF did not
colocalize with ubiquitin and p62/Sequestosome-1 (Fig. 7A
and B). Some RGNEF inclusions colocalized with TDP-43
in ALS, while others did not (Fig. 7C). Interestingly, a weak
but consistent colocalization of RGNEF and TDP-43 was also
observed in the nucleus of healthy motor neurons (Fig. 7C).
Because 14-3-3 is known to interact with p190RhoGEF (Zhai
et al., 2001) and NFL mRNA (Ge et al., 2007), we aimed to
determine whether it colocalizes with RGNEF. We observed a
very weak colocalization of 14-3-3 with RGNEF aggregates in
the ALS cases that were analyzed (Supplementary Fig. 7).
However we did observe colocalization between RGNEF and
14-3-3 in Nissl body-like structures, where RGNEF is present
in some motor neurons of control patients (Supplementary
Fig. 7).
When coupled with data suggesting that the presence of
aggregates is pathological, particularly those that are
TDP-43 and/or FUS/TLS immunoreactive (Arai et al.,
2006; Deng et al., 2010; Kwiatkowski et al., 2009; Neu-
mann et al., 2006; Vance et al., 2009), these results strongly
suggest that RGNEF aggregates observed in ALS are also
pathological. Further, this suggests that a common pathway
of aggregate formation may exist in ALS-affected motor
neurons.
4. Discussion
The major finding in this study is that RGNEF, the
human homolog of the mouse protein p190RhoGEF, is a
bifunctional protein that appears to be involved in the pa-
thology of ALS. This manifests as aggregates and skeins
that colocalize with other proteins that have been described
as pathological in ALS (TDP-43, ubiquitin, p62/Sequesto-
 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262 257

Fig. 5. Neurofilament (NFL) protein levels are decreased in a stable cell line expressing full length RGNEF. (A) Rho guanine nucleotide exchange factor
(RGNEF)-myc was detected using an anti-myc antibody in the stable cell line HEK293-RGNEF(⫹) and observed through confocal microscopy. RGNEF (red)
was localized almost exclusively in the cytoplasm, however, a very low amount of RGNEF was observed in the nucleus. Hoechst (blue) was used as a nuclear
marker (scale bar ⫽ 5 ␮m). (B) The amount of NFL protein in HEK293T-RGNEF(⫹) clone A was analyzed by Western blot and compared with the level
of NFL in HEK293T cells (control). A clear reduction in NFL protein levels was observed in the stable cell line compared with control cells. (C)
Quantification of 3 Western blot experiments comparing the NFL protein levels between control and HEK293T-RGNEF(⫹) clone A cells. The results are
presented as mean ⫾ standard error of the mean. p value compares HEK293T-RGNEF(⫹) cells with control, standardized against GAPDH (***p ⬍ 0.001).
(D) The amount of NFL mRNA in HEK293T-RGNEF(⫹) clone A was analyzed by relative quantitative polymerase chain reaction (PCR) and compared with
the level of NFL in HEK293T cells (control). A clear reduction in NFL mRNA levels was observed in the stable cell line compared with control cells as
observed in the quantification of 3 experiments (***p ⬍ 0.001). (E) Western blot experiment showing the effect of an siRNA against RGNEF over the NFL
protein levels. HEK293T and HEK293T-RGNEF(⫹) clone A cells were treated with a control siRNA and also HEK293T-RGNEF(⫹) clone A cells were
treated with an siRNA against RGNEF. The amount of NFL is clearly increased in the presence of the siRNA against RGNEF, while the levels of RGNEF
are significantly decreased. (F) Quantification of 3 Western blot experiments comparing the NFL protein levels between HEK293T and HEK293T-
RGNEF(⫹) clone A cells treated with control siRNA and HEK293T-RGNEF(⫹) clone A treated with siRNA against RGNEF (***p ⬍ 0.001).

some-1). We have shown that RGNEF is an RNA binding
protein that acts as an RNA destabilizing factor for human
NFL mRNA via its 3= UTR, and can regulate NFL protein
levels in cells. We have also determined that RGNEF can
actively function as a GEF protein.
We have demonstrated that full length RGNEF interacts
with NFL mRNA and that this interaction does not appear to
require a protein mediator. This builds upon our initial
observation that the RNA binding domain of RGNEF could
interact with NFL mRNA in whole cell lysates from spinal
258 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262
Fig. 6. Rho guanine nucleotide exchange factor (RGNEF) forms cytoplasmic inclusions in motor neurons of amyotrophic lateral sclerosis (ALS) patients.
Immunohistochemistry of RGNEF in motor neurons of control (A), sporadic ALS (sALS) (B, C) and familial ALS (fALS) (D, E) spinal cords. RGNEF forms
extensive skein-like aggregates (B, D) and punctate aggregates (D, E) in ALS motor neurons (Scale bar ⫽ 10 ␮m).
cord (containing all protein and RNA) in ALS but not
control cases (Volkening et al., 2010). Taken together, these
data strongly suggest that the presence of an RNA binding
protein complex-based control mechanism in control spinal
cord, or its absence in ALS spinal cord, prevents RGNEF
interaction with NFL mRNA. Interestingly, using HEK293T
as cellular model, interaction between RGNEF and NFL
mRNA was observed, including regulation of NFL mRNA
levels via the 3= UTR, as well as regulation of NFL protein
Table 1
Quantification of RGNEF and TDP-43 neuronal cytoplasmic inclusions
in motor neurons from control, sALS and fALS patients
Neuronal cytoplasmic inclusions (%)
Control sALS fALS
RGNEF 0 8.83 ⫾ 3.43 8.15 ⫾ 1.61
TDP-43 0 16.23 ⫾ 9.16 9.52 ⫾ 3.99
Neuronal cytoplasmic inclusions from spinal cord motor neurons of con-
trol, sALS, and fALS patients (n ⫽ 3) were quantified. For the quantifi-
cation, only cells with a visible nucleus were considered. No statistically
significant difference between the number of RGNEF and TDP-43 neuro-
nal cytoplasmic inclusions in sALS and fALS was observed. Note that the
error in the TDP-43 quantification is higher than for RGNEF due to the fact
that not all ALS cases quantified in this study display TDP-43-positive
aggregates.
Key: ALS, amyotrophic lateral sclerosis; fALS, familial ALS; RGNEF, rho
guanine nucleotide exchange factor; sALS, sporadic ALS; TDP-43, TAR
DNA binding protein of 43 kDa;
levels. This suggests that this cellular model could share
several characteristics with ALS tissues at the level of NFL
mRNA regulation involving RGNEF. Analyzing the putative
proteins that could participate in the regulation of this interac-
tion with NFL mRNA in this cellular model will be crucial to
understand the role of this protein in ALS pathology.
The effect of RGNEF on NFL mRNA stability was
analyzed using the NFL mRNA 3= UTR. This was previ-
ously reported as a widely described target for RNA binding
proteins that regulates the stability of NFL in mice and
humans (Cañete-Soler et al., 1998, 2001; Ge et al., 2005,
2007; Lin et al., 2004; Strong et al., 2007; Volkening et al.,
2009). We used 3 different NFL mRNA 3= UTRs: 3=
UTR-S, analogous to the mouse NFL 3= UTR, and 3=
UTR-M and 3= UTR-L reported in sequence databases. We
observed a destabilizing effect on all of these NFL 3= UTRs
with an effect directly proportional to the length of the 3=
UTR. This result might indicate the presence of more than
1 binding motif for RGNEF on NFL 3= UTR-M and -L. The
destabilizing effect of RGNEF over NFL 3= UTR was com-
pletely abolished when an RGNEF version was used that
did not contain the RNA binding domain (RGNEF-⌬CT-
myc). This version of RGNEF does not interact with NFL
mRNA, but a stabilizing effect can be observed. This could
be explained by the presence of positive regulating factors
that compete with RGNEF now gaining access to NFL 3=
UTR in absence of the RNA binding domain of RGNEF.
 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262 259

Fig. 7. Rho guanine nucleotide exchange factor (RGNEF) colocalizes with ubiquitin, p62/Sequestosome-1, and partially with TAR DNA binding protein of
43 kDa (TDP-43). (A) Immunofluorescence using antibodies against RGNEF (green) and ubiquitin (red) in amyotrophic lateral sclerosis (ALS) and control
motor neurons was performed. The merge and colocalization analysis (product of the differences from the mean [PDM] image) show that RGNEF aggregates
show significant colocalization with ubiquitin (yellow). In control motor neurons, RGNEF does not colocalize with ubiquitin. (B) Immunofluorescence using
antibodies against RGNEF (green) and p62/Sequestosome-1 (red) in ALS and control motor neurons was performed. The merge and colocalization analysis
(PDM image) show that RGNEF aggregates show significant colocalization with p62/Sequestosome-1 in ALS, but not in controls. (C) Immunofluorescence
using antibodies against RGNEF (green) and TDP-43 (red) in ALS and control motor neurons was performed. The merge and colocalization analysis (PDM
image) show that RGNEF aggregates show partial colocalization with TDP-43. In control motor neurons RGNEF shows a weak colocalization with TDP-43
in the nucleus (scale bar ⫽ 10 ␮m). In the PDM images, blue color indicates low level of colocalization while yellow and white indicates high colocalization.
260 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262
Interestingly, removal of the first 286 bases of the 3=
UTR-M (3= UTR-M⌬S) resulted in greater downregulation of
luciferase signal. Together with the stabilizing effect of
TDP-43 on 3= UTR-S, this result supports the hypothesis that
there is a presence of alternate stability determinants within
this region, which is consistent with our previous reports for
14-3-3 (Ge et al., 2007) and TDP-43 (Volkening et al., 2009).
Considering these results, 2 very important future directions
will be to determine the regions of NFL 3= UTR that interact
with RGNEF, and to determine the levels of in vivo NFL
expression of each mRNA bearing different 3= UTR variants.
It was interesting to observe that the destabilizing effect
of RGNEF on human NFL 3= UTR was opposite to the
effect described for murine p190RhoGEF (Cañete-Soler et
al., 2001). We first hypothesized that the observed differ-
ence between our work and that previously described for
p190RhoGEF was technical in nature—a protection assay
versus our luciferase assay using live cells. Due to this, we
decided to analyze the effect of p190RhoGEF over the
mouse NFL 3= UTR using our system. Notably, we con-
firmed the results of Cañete-Soler et al. (2001) and we also
observed that p190RhoGEF can act to stabilize human NFL 3=
UTR. Further, we observed that RGNEF can act to destabilize
mouse NFL 3= UTR in mouse neuronal cells. Consequently,
these results strongly suggest that p190RhoGEF is intrinsically
a stabilizing protein and RGNEF is a destabilizing protein. The
destabilizing effect of RGNEF was confirmed using a stable
cell line expressing RGNEF-myc. In this cell line, the NFL
protein levels were greatly decreased, an effect produced by
the downregulation of NFL mRNA levels and specifically
dependent on the presence of RGNEF because when RG-
NEF levels were diminished by an siRNA, the NFL levels
increased.
Considering that ALS is not a natural pathology in ro-
dents, the differences between p190RhoGEF and RGNEF
emphasize the importance of understanding the molecular
mechanism of NFL mRNA regulation in humans. The mo-
tor neuron death in the reported mouse model involving
p190RhoGEF was explained by its resulting from the disas-
sembly of a putative stabilizing complex where p190RhoGEF
is participating when NFL mRNA or protein are overexpressed
(Lin et al., 2005). In humans, however, we hypothesize that the
cause of the pathology is a result of the aggregation of RGNEF
that could sequester NFL mRNA, induce neurofilamentous
aggregates, and subsequently, motor neuron death. It is note-
worthy that RGNEF has been shown to interact with NFL
mRNA only in ALS cases (Volkening et al., 2010). We do
not yet know whether NFL protein is involved in ALS
pathogenesis involving RGNEF; however this will be an
area of focus for future studies. Therefore, it is possible that
the mouse model provided accurate clues relating to the
involvement of p190RhoGEF in motor neuron cell death,
but we speculate that the mechanism involved is different in
humans due to the opposing roles of RGNEF and
p190RhoGEF in NFL mRNA stabilization.
A remarkable finding in this study is the observation of
extensive RGNEF protein aggregates in all cases of ALS
studied. The immunoreactivity was consistent with that ob-
served for proteins such as FUS/TLS (Kwiatkowski et al.,
2009; Vance et al., 2009) and TDP-43 (Arai et al., 2006;
Neumann et al., 2006), which strongly suggests that
RGNEF is a new element in the pathophysiology of ALS.
The colocalization of RGNEF with ubiquitin and p62/Se-
questosome-1 aggregates strongly supports the pathological
nature of these aggregates. Notably, RGNEF showed some
colocalization with TDP-43 in ALS affected motor neurons.
This is interesting because RGNEF and TDP-43 both inter-
act with NFL mRNA (Strong et al., 2007; Volkening et al.,
2009). TDP-43 can interact with multiple RNA targets (Co-
lombrita et al., 2012; Sephton et al., 2011; Tollervey et al.,
2011; Xiao et al., 2011) and multiple proteins (Freibaum et
al., 2010). It is probable that RGNEF can display this same
attribute. Therefore, it is possible that when different inter-
acting partners (RNAs and/or proteins) are involved,
RGNEF and TDP-43 form spatially separated aggregates
and when similar interacting partners are involved, both
proteins aggregate in the same compartment. While the
mechanism by which RGNEF is induced to form cytosolic
aggregates remains to be determined, we can hypothesize a
function for RGNEF in RNA transport because a similar
mechanism has been proposed for TDP-43 (Strong, 2010;
Strong et al., 2007). Additionally, RGNEF and TDP-43
show weak colocalization in the nucleus of healthy control
motor neurons, suggesting a physiological nuclear role for a
small pool of RGNEF that may be related with its RNA
binding protein function.
Our results put RGNEF into the small group of RNA
binding proteins that form aggregates in ALS, along with
TDP-43 (Arai et al., 2006; Neumann et al., 2006), FUS/TLS
(Deng et al., 2010; Kwiatkowski et al., 2009; Vance et al.,
2009), and mutant SOD1 (Bruijn et al., 1997, 1998; Rosen
et al., 1993; Stieber et al., 2000). In this form, RGNEF is
another important component of the cellular phenotype ob-
served in ALS, and may be a prominent contributor to the
disease pathogenesis given its effect on NFL mRNA. It
is noteworthy that the murine homologue to RGNEF,
p190RhoGEF, can act as an antiapoptotic factor in neuronal
cells (Wu et al., 2003), suggesting that the sequestration of
RGNEF into aggregates in ALS may contribute to neuronal
death. The presence of aggregates of RGNEF, an RNA binding
protein that can bind and destabilize NFL transcripts, could
also explain the lower levels of NFL mRNA detected in ALS
motor neurons (Wong et al., 2000), as well as provide a
possible mechanism for NF aggregate formation together with
a link between ALS and Rho signaling pathways.
Disclosure statement
The authors declare that they have no competing inter-
ests.
 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262
Acknowledgements
The authors thank Dr. Rosa Rademakers for the
C9ORF72 expanded repeats genotyping of ALS patients
and Dr. Wouter H. Moolenaar for kindly providing pcDNA-
p190RhoGEF plasmid. This work was supported by the
Canadian Institute of Health Research (CIHR), The McFeat
Family Fund, and the ALS Society of Canada.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, http://dx.doi.org/10.1016/j.
neurobiolaging.2012.06.021.
References
Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H.,
Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y., Oda, T., 2006.
TDP-43 is a component of ubiquitin-positive tau-negative inclusions in
frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
Biochem. Biophys. Res. Commun. 351, 602– 611.
Beaulieu, J.M., Jacomy, H., Julien, J.P., 2000. Formation of intermediate
filament protein aggregates with disparate effects in two transgenic
mouse models lacking the neurofilament light subunit. J. Neurosci. 20,
5321–5328.
Beaulieu, J.M., Nguyen, M.D., Julien, J.P., 1999. Late onset of motor
neurons in mice overexpressing wild-type peripherin. J. Cell Biol. 147,
531–544.
Bergeron, C., Beric-Maskarel, K., Muntasser, S., Weyer, L., Somerville,
M.J., Percy, M.E., 1994. Neurofilament light and polyadenylated
mRNA levels are decreased in amyotrophic lateral sclerosis motor
neurons. J. Neuropathol. Exp. Neurol. 53, 221–230.
Bruijn, L.I., Becher, M.W., Lee, M.K., Anderson, K.L., Jenkins, N.A.,
Copeland, N.G., Sisodia, S.S., Rothstein, J.D., Borchelt, D.R., Price,
D.L., Cleveland, D.W., 1997. ALS-linked SOD1 mutant G85R medi-
ates damage to astrocytes and promotes rapidly progressive disease
with SOD1-containing inclusions. Neuron 18, 327–338.
Bruijn, L.I., Houseweart, M.K., Kato, S., Anderson, K.L., Anderson, S.D.,
Ohama, E., Reaume, A.G., Scott, R.W., Cleveland, D.W., 1998. Ag-
gregation and motor neuron toxicity of an ALS-linked SOD1 mutant
independent from wild-type SOD1. Science 281, 1851–1854.
Buratti, E., Dörk, T., Zuccato, E., Pagani, F., Romano, M., Baralle, F.E.,
2001. Nuclear factor TDP-43 and SR proteins promote in vitro and in
vivo CFTR exon 9 skipping. EMBO J. 20, 1774 –1784.
Cañete-Soler, R., Schwartz, M.L., Hua, Y., Schlaepfer, W.W., 1998. Sta-
bility determinants are localized to the 3’-untranslated region and
3’-coding region of the neurofilament light subunit mRNA using a
tetracycline-inducible promoter. J. Biol. Chem. 273, 12650 –12654.
Cañete-Soler, R., Wu, J., Zhai, J., Shamim, M., Schlaepfer, W.W., 2001.
p190RhoGEF Binds to a destabilizing element in the 3’ untranslated
region of light neurofilament subunit mRNA and alters the stability of
the transcript. J. Biol. Chem. 276, 32046 –32050.
Colombrita, C., Onesto, E., Megiorni, F., Pizzuti, A., Baralle, F.E., Buratti,
E., Silani, V., Ratti, A., 2012. TDP-43 and FUS RNA-binding proteins
bind distinct sets of cytoplasmic messenger RNAs and differently
regulate their post-transcriptional fate in motoneuron-like cells. J. Biol.
Chem. 287, 15635–15647.
Crozat, A., Aman, P., Mandahl, N., Ron, D., 1993. Fusion of CHOP to a
novel RNA-binding protein in human myxoid liposarcoma. Nature 363,
640 – 644.
Dejesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., Boxer, A.L., Baker,
M., Rutherford, N.J., Nicholson, A.M., Finch, N.A., Flynn, H., Adam-
son, J., Kouri, N., Wojtas, A., Sengdy, P., Hsiung, G.Y., Karydas, A.,
261
Seeley, W.W., Josephs, K.A., Coppola, G., Geschwind, D.H., Wszolek,
Z.K., Feldman, H., Knopman, D.S., Petersen, R.C., Miller, B.L.,
Dickson, D.W., Boylan, K.B., Graff-Radford, N.R., Rademakers, R.,
2011. Expanded GGGGCC hexanucleotide repeat in noncoding region
of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72,
245–256.
Deng, H.X., Zhai, H., Bigio, E.H., Yan, J., Fecto, F., Ajroud, K., Mishra,
M., Ajroud-Driss, S., Heller, S., Sufit, R., Siddique, N., Mugnaini, E.,
Siddique, T., 2010. FUS-immunoreactive inclusions are a common
feature in sporadic and non-SOD1 familial amyotrophic lateral sclero-
sis. Ann. Neurol. 67, 739 –748.
Freibaum, B.D., Chitta, R.K., High, A.A., Taylor, J.P., 2010. Global
analysis of TDP-43 interacting proteins reveals strong association with
RNA splicing and translation machinery. J. Proteome Res. 9, 1104 –
1120.
Ge, W.W., Volkening, K., Leystra-Lantz, C., Jaffe, H., Strong, M.J., 2007.
14-3-3 protein binds to the low molecular weight neurofilament (NFL)
mRNA 3’ UTR. Mol. Cell. Neurosci. 34, 80 – 87.
Ge, W.W., Wen, W., Strong, W., Leystra-Lantz, C., Strong, M.J., 2005.
Mutant copper-zinc superoxide dismutase binds to and destabilizes
human low molecular weight neurofilament mRNA. J. Biol. Chem.
280, 118 –124.
He, C.Z., Hays, A.P., 2004. Expression of peripherin in ubiquinated inclu-
sions of amyotrophic lateral sclerosis. J. Neurol. Sci. 217, 47–54.
Kawamoto, Y., Akiguchi, I., Nakamura, S., Budka, H., 2004. 14-3-3
proteins in Lewy body-like hyaline inclusions in patients with sporadic
amyotrophic lateral sclerosis. Acta Neuropathol. 108, 531–537.
Kondo, A., Iwaki, T., Tateishi, J., Kirimoto, K., Morimoto, T., Oomura, I.,
1986. Accumulation of neurofilaments in a sporadic case of amyo-
trophic lateral sclerosis. Jpn. J. Psychiatry Neurol. 40, 677– 684.
Kuusisto, E., Kauppinen, T., Alafuzoff, I., 2008. Use of p62/SQSTM1
antibodies for neuropathological diagnosis. Neuropathol. Appl. Neuro-
biol. 34, 169 –180.
Kuusisto, E., Salminen, A., Alafuzoff, I., 2001. Ubiquitin-binding protein
p62 is present in neuronal and glial inclusions in human tauopathies
and synucleinopathies. Neuroreport 12, 2085–2090.
Kwiatkowski, T.J., Jr, Bosco, D.A., Leclerc, A.L., Tamrazian, E., Vander-
burg, C.R., Russ, C., Davis, A., Gilchrist, J., Kasarskis, E.J., Munsat,
T., Valdmanis, P., Rouleau, G.A., Hosler, B.A., Cortelli, P., de Jong,
P.J., Yoshinaga, Y., Haines, J.L., Pericak-Vance, M.A., Yan, J.,
Ticozzi, N., Siddique, T., McKenna-Yasek, D., Sapp, P.C., Horvitz,
H.R., Landers, J.E., Brown, R.H., Jr., 2009. Mutations in the FUS/TLS
gene on chromosome 16 cause familial amyotrophic lateral sclerosis.
Science 323, 1205–1208.
Li, Q., Lau, A., Morris, T.J., Guo, L., Fordyce, C.B., Stanley, E.F., 2004.
A syntaxin 1, Galpha(o), and N-type calcium channel complex at a
presynaptic nerve terminal: analysis by quantitative immunocolocal-
ization. J. Neurosci. 24, 4070 – 4081.
Lin, H., Zhai, J., Cañete-Soler, R., Schlaepfer, W.W., 2004. 3’ untranslated
region in a light neurofilament (NF-L) mRNA triggers aggregation of
NF-L and mutant superoxide dismutase 1 proteins in neuronal cells.
J. Neurosci. 24, 2716 –2726.
Lin, H., Zhai, J., Schlaepfer, W.W., 2005. RNA-binding protein is involved
in aggregation of light neurofilament protein and is implicated in the
pathogenesis of motor neuron degeneration. Hum. Mol. Genet. 14,
3643–3659.
Millecamps, S., Gowing, G., Corti, O., Mallet, J., Julien, J.P., 2007.
Conditional NF-L transgene expression in mice for in vivo analysis of
turnover and transport rate of neurofilaments. J. Neurosci. 27, 4947–
4956.
Nalbant, P., Chang, Y.C., Birkenfeld, J., Chang, Z.F., Bokoch, G.M., 2009.
Guanine nucleotide exchange factor-H1 regulates cell migration via
localized activation of RhoA at the leading edge. Mol. Biol. Cell 20,
4070 – 4082.
Neumann, M., Sampathu, D.M., Kwong, L.K., Truax, A.C., Micsenyi,
M.C., Chou, T.T., Bruce, J., Schuck, T., Grossman, M., Clark, C.M.,
262 C.A. Droppelmann et al. / Neurobiology of Aging 34 (2013) 248 –262
McCluskey, L.F., Miller, B.L., Masliah, E., Mackenzie, I.R., Feldman,
H., Feiden, W., Kretzschmar, H.A., Trojanowski, J.Q., Lee, V.M.,
2006. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and
amyotrophic lateral sclerosis. Science 314, 130 –133.
Nie, Z., Wu, J., Zhai, J., Lin, H., Ge, W., Schlaepfer, W.W., Cañete-Soler,
R., 2002. Untranslated element in neurofilament mRNA has neuro-
pathic effect on motor neurons of transgenic mice. J. Neurosci. 22,
7662–7670.
Pikkarainen, M., Hartikainen, P., Alafuzoff, I., 2008. Neuropathologic
features of frontotemporal lobar degeneration with ubiquitin-positive
inclusions visualized with ubiquitin-binding protein p62 immunohisto-
chemistry. J. Neuropathol. Exp. Neurol. 67, 280 –298.
Pikkarainen, M., Hartikainen, P., Alafuzoff, I., 2010. Ubiquitinated p62-
positive, TDP-43-negative inclusions in cerebellum in frontotemporal
lobar degeneration with TAR DNA binding protein 43. Neuropathol-
ogy 30, 197–199.
Ren, J., Wen, L., Gao, X., Jin, C., Xue, Y., Yao, X., 2009. DOG 1.0:
illustrator of protein domain structures. Cell Res. 19, 271–273.
Ren, X.D., Kiosses, W.B., Schwartz, M.A., 1999. Regulation of the small
GTP-binding protein Rho by cell adhesion and the cytoskeleton.
EMBO J. 18, 578 –585.
Renton, A.E., Majounie, E., Waite, A., Simón-Sánchez, J., Rollinson, S.,
Gibbs, J.R., Schymick, J.C., Laaksovirta, H., van Swieten, J.C., Myl-
lykangas, L., Kalimo, H., Paetau, A., Abramzon, Y., Remes, A.M.,
Kaganovich, A., Scholz, S.W., Duckworth, J., Ding, J., Harmer, D.W.,
Hernandez, D.G., Johnson, J.O., Mok, K., Ryten, M., Trabzuni, D.,
Guerreiro, R.J., Orrell, R.W., Neal, J., Murray, A., Pearson, J., Jansen,
I.E., Sondervan, D., Seelaar, H., Blake, D., Young, K., Halliwell, N.,
Callister, J.B., Toulson, G., Richardson, A., Gerhard, A., Snowden, J.,
Mann, D., Neary, D., Nalls, M.A., Peuralinna, T., Jansson, L., Isoviita,
V.M., Kaivorinne, A.L., Holtta-Vuori, M., Ikonen, E., Sulkava, R.,
Benatar, M., Wuu, J., Chio, A., Restagno, G., Borghero, G., Sabatelli,
M., Heckerman, D., Rogaeva, E., Zinman, L., Rothstein, J.D., Sendt-
ner, M., Drepper, C., Eichler, E.E., Alkan, C., Abdullaev, Z., Pack,
S.D., Dutra, A., Pak, E., Hardy, J., Singleton, A., Williams, N.M.,
Heutink, P., Pickering-Brown, S., Morris, H.R., Tienari, P.J., Traynor,
B.J., Traynor, B.J., 2011. A hexanucleotide repeat expansion in
C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron
72, 257–268.
Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hen-
tati, A., Donaldson, D., Goto, J., O’Regan, J.P., Deng, H.X., etAl, etA.l,
1993. Mutations in Cu/Zn superoxide dismutase gene are associated
with familial amyotrophic lateral sclerosis. Nature 362, 59 – 62.
Sephton, C.F., Cenik, C., Kucukural, A., Dammer, E.B., Cenik, B., Han,
Y., Dewey, C.M., Roth, F.P., Herz, J., Peng, J., Moore, M.J., Yu, G.,
2011. Identification of neuronal RNA targets of TDP-43-containing
ribonucleoprotein complexes. J. Biol. Chem. 286, 1204 –1215.
Seppänen, A., Pikkarainen, M., Hartikainen, P., Hofmann, S.C., Majamaa,
K., Alafuzoff, I., 2009. Expression of collagen XVII and ubiquitin-
binding protein p62 in motor neuron disease. Brain Res. 1247, 171–
177.
Shaw, B.F., Lelie, H.L., Durazo, A., Nersissian, A.M., Xu, G., Chan, P.K.,
Gralla, E.B., Tiwari, A., Hayward, L.J., Borchelt, D.R., Valentine, J.S.,
Whitelegge, J.P., 2008. Detergent-insoluble aggregates associated with
amyotrophic lateral sclerosis in transgenic mice contain primarily full-
length, unmodified superoxide dismutase-1. J. Biol. Chem. 283, 8340 –
8350.
Stieber, A., Gonatas, J.O., Gonatas, N.K., 2000. Aggregates of mutant
protein appear progressively in dendrites, in periaxonal processes of
oligodendrocytes, and in neuronal and astrocytic perikarya of mice
expressing the SOD1(G93A) mutation of familial amyotrophic lateral
sclerosis. J. Neurol. Sci. 177, 114 –123.
Strong, M.J., 1999. Neurofilament metabolism in sporadic amyotrophic
lateral sclerosis. J. Neurol. Sci. 169, 170 –177.
Strong, M.J., 2010. The evidence for altered RNA metabolism in amyo-
trophic lateral sclerosis (ALS). J. Neurol. Sci. 288, 1–12.
Strong, M.J., Kesavapany, S., Pant, H.C., 2005. The pathobiology of
amyotrophic lateral sclerosis: a proteinopathy? J. Neuropathol. Exp.
Neurol. 64, 649 – 664.
Strong, M.J., Volkening, K., Hammond, R., Yang, W., Strong, W., Ley-
stra-Lantz, C., Shoesmith, C., 2007. TDP43 is a human low molecular
weight neurofilament (hNFL) mRNA-binding protein. Mol. Cell. Neu-
rosci. 35, 320 –327.
Szaro, B.G., Strong, M.J., 2010. Post-transcriptional control of neurofila-
ments: New roles in development, regeneration and neurodegenerative
disease. Trends Neurosci. 33, 27–37.
Thyagarajan, A., Strong, M.J., Szaro, B.G., 2007. Post-transcriptional
control of neurofilaments in development and disease. Exp. Cell Res.
313, 2088 –2097.
Thyagarajan, B., Liu, Y., Shin, S., Lakshmipathy, U., Scheyhing, K., Xue,
H., Ellerström, C., Strehl, R., Hyllner, J., Rao, M.S., Chesnut, J.D.,
2008. Creation of engineered human embryonic stem cell lines using
phiC31 integrase. Stem Cells 26, 119 –126.
Thyagarajan, B., Olivares, E.C., Hollis, R.P., Ginsburg, D.S., Calos, M.P.,
2001. Site-specific genomic integration in mammalian cells mediated
by phage phiC31 integrase. Mol. Cell. Biol. 21, 3926 –3934.
Tollervey, J.R., Curk, T., Rogelj, B., Briese, M., Cereda, M., Kayikci, M.,
König, J., Hortobágyi, T., Nishimura, A.L., Zupunski, V., Patani, R.,
Chandran, S., Rot, G., Zupan, B., Shaw, C.E., Ule, J., 2011. Charac-
terizing the RNA targets and position-dependent splicing regulation by
TDP-43. Nat. Neurosci. 14, 452– 458.
van Horck, F.P., Ahmadian, M.R., Haeusler, L.C., Moolenaar, W.H.,
Kranenburg, O., 2001. Characterization of p190RhoGEF, a RhoA-
specific guanine nucleotide exchange factor that interacts with micro-
tubules. J. Biol. Chem. 276, 4948 – 4956.
Vance, C., Rogelj, B., Hortobágyi, T., De Vos, K.J., Nishimura, A.L.,
Sreedharan, J., Hu, X., Smith, B., Ruddy, D., Wright, P., Ganesal-
ingam, J., Williams, K.L., Tripathi, V., Al-Saraj, S., Al-Chalabi, A.,
Leigh, P.N., Blair, I.P., Nicholson, G., de Belleroche, J., Gallo, J.M.,
Miller, C.C., Shaw, C.E., 2009. Mutations in FUS, an RNA processing
protein, cause familial amyotrophic lateral sclerosis type 6. Science
323, 1208 –1211.
Volkening, K., Leystra-Lantz, C., Strong, M.J., 2010. Human low molec-
ular weight neurofilament (NFL) mRNA interacts with a predicted
p190RhoGEF homologue (RGNEF) in humans. Amyotroph. Lateral
Scler. 11, 97–103.
Volkening, K., Leystra-Lantz, C., Yang, W., Jaffee, H., Strong, M.J., 2009.
Tar DNA binding protein of 43 kDa (TDP-43), 14-3-3 proteins and
copper/zinc superoxide dismutase (SOD1) interact to modulate NFL
mRNA stability. Implications for altered RNA processing in amyo-
trophic lateral sclerosis (ALS). Brain Res. 1305, 168 –182.
Wong, N.K., He, B.P., Strong, M.J., 2000. Characterization of neuronal
intermediate filament protein expression in cervical spinal motor neu-
rons in sporadic amyotrophic lateral sclerosis (ALS). J. Neuropathol.
Exp. Neurol. 59, 972–982.
Wu, J., Zhai, J., Lin, H., Nie, Z., Ge, W.W., García-Bermejo, L., Muschel,
R.J., Schlaepfer, W.W., Cañete-Soler, R., 2003. Cytoplasmic retention
sites in p190RhoGEF confer anti-apoptotic activity to an EGFP-tagged
protein. Brain Res. Mol. Brain Res. 117, 27–38.
Xiao, S., Sanelli, T., Dib, S., Sheps, D., Findlater, J., Bilbao, J., Keith, J.,
Zinman, L., Rogaeva, E., Robertson, J., 2011. RNA targets of TDP-43
identified by UV-CLIP are deregulated in ALS. Mol. Cell. Neurosci.
47, 167–180.
Zhai, J., Lin, H., Shamim, M., Schlaepfer, W.W., Cañete-Soler, R., 2001.
Identification of a novel interaction of 14 –3-3 with p190RhoGEF.
J. Biol. Chem. 276, 41318 – 41324.