RNA-Seq Profiling of Spinal Cord Motor Neurons from
a Presymptomatic SOD1 ALS Mouse
Urmi Bandyopadhyay1,2., Justin Cotney1., Maria Nagy1,2, Sunghee Oh1¤a, Jing Leng1,4,
Milind Mahajan1¤b, Shrikant Mane1, Wayne A. Fenton1, James P. Noonan1,3,4, Arthur L. Horwich1,2,3*
1Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, United States of America, 2Howard Hughes Medical Institute, Yale University
School of Medicine, New Haven, Connecticut, United States of America, 3Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut,
United States of America, 4Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut, United States of America
Mechanisms involved with degeneration of motor neurons in amyotrophic lateral sclerosis (ALS; Lou Gehrig’s Disease) are
poorly understood, but genetically inherited forms, comprising ,10% of the cases, are potentially informative. Recent
observations that several inherited forms of ALS involve the RNA binding proteins TDP43 and FUS raise the question as to
whether RNA metabolism is generally disturbed in ALS. Here we conduct whole transcriptome profiling of motor neurons
from a mouse strain, transgenic for a mutant human SOD1 (G85R SOD1-YFP), that develops symptoms of ALS and paralyzes
at 5–6 months of age. Motor neuron cell bodies were laser microdissected from spinal cords at 3 months of age, a time
when animals were presymptomatic but showed aggregation of the mutant protein in many lower motor neuron cell
bodies and manifested extensive neuromuscular junction morphologic disturbance in their lower extremities. We observed
only a small number of transcripts with altered expression levels or splicing in the G85R transgenic compared to age-
matched animals of a wild-type SOD1 transgenic strain. Our results indicate that a major disturbance of polyadenylated RNA
metabolism does not occur in motor neurons of mutant SOD1 mice, suggesting that the toxicity of the mutant protein lies
at the level of translational or post-translational effects.
Citation: Bandyopadhyay U, Cotney J, Nagy M, Oh S, Leng J, et al. (2013) RNA-Seq Profiling of Spinal Cord Motor Neurons from a Presymptomatic SOD1 ALS
Mouse. PLoS ONE 8(1): e53575. doi:10.1371/journal.pone.0053575
Editor: Huaibin Cai, National Institute of Health, United States of America
Received August 27, 2012; Accepted December 3, 2012; Published January 3, 2013
Copyright: ? 2013 Bandyopadhyay et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Howard Hughes Medical Institute (A.L.H.), NIH GM094780 (J.P.N.), and an Andersen Foundation Fellowship (J.C.). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤a Current address: Division of Human Genetics, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of
¤b Current address: Genomics Core Facility, Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York, United States of
. These authors contributed equally to this work.
The mechanisms involved with degeneration of motor neurons
in amyotrophic lateral sclerosis (ALS, Lou Gehrig’s Disease) are
poorly understood. Inherited forms, accounting for ,10% of
cases, are potentially informative, however. They include muta-
tions in SOD1 (superoxide dismutase 1) [1,2], ubiquilin , VCP
(valosin-containing protein; p97) , optineurin , TDP43 (TAR
DNA binding protein 43) [6,7], and FUS (fused in sarcoma) [8,9],
as well as a recently identified heritable hexanucleotide repeat
expansion within an intron of a gene on chromosome 9 [10,11]. It
appears that protein misfolding is involved in the gain of function
associated with mutant SOD1-linked disease [2,12,13] and that,
correspondingly, protein quality control may be affected by
mutations in ubiquilin, a protein lying in the pathway of
proteasomal degradation [3,14], in VCP, a AAA+ hexameric ring
assembly that acts on ubiquitinated proteins [15,16], or in
optineurin, which encodes an autophagy receptor protein .
Although TDP43 and FUS, like SOD1, ubiquilin, and optineurin,
are found in aggregates in the cytosol of motor neurons of patients
with mutations in these genes, they are RNA binding proteins in
which the mutations may lead not only to their misfolding and
mislocalization but also to perturbed RNA metabolism, as
documented by recent studies [18,19]. This raises the question
of whether RNA metabolism is disturbed in motor neurons as
a more general manifestation of disease in other forms of ALS, e.g.
in mutant SOD1-associated disease. Here, we have addressed this
issue by profiling RNA from laser captured spinal cord motor
neurons of an SOD1 mutant transgenic mouse strain using RNA-
seq, comparing it with a corresponding wild-type SOD1 trans-
genic. The SOD1 mutant mice were analyzed at a presymptomatic
time point when significant pathologic changes were already
occurring, including SOD1 aggregation in many lower motor
neuron cell bodies, astrogliosis in the vicinity of such neurons, and
morphologic changes in neuromuscular junctions (NMJs) in
gastrocnemius muscle. We observe minimal effects on mRNA in
the mutant SOD1 transgenic mice, with altered levels of only
a small number of transcripts and only rare splicing differences,
relative to wild-type SOD1 transgenic mice. Thus, in contrast to
the substantial changes in RNA levels and splicing reported for
TDP43 and FUS mutant conditions, SOD1-linked disease is not
PLOS ONE | www.plosone.org1 January 2013 | Volume 8 | Issue 1 | e53575
associated with major mRNA disturbance as part of the
Laser capture microdissection of motor neurons
To measure transcriptional differences between motor neurons
of mice transgenic for G85R SOD1-YFP and wild-type SOD1-
YFP, we carried out laser capture microdissection of cell bodies of
large ventral horn motor neurons from spinal cords, followed by
RNA-seq. The G85R SOD1-YFP strain carries over 200 copies of
the transgene, being homozygous for an insertion on mouse
chromosome 4, and most of these animals develop lower extremity
paralysis between 4 and 6.5 months of age (Fig. 1A). The wild-type
SOD1-YFP strain exhibits a similar steady-state level of fusion
protein in spinal cord despite a lower transgene copy number,
a function of the .10-fold more rapid turnover of the G85R
SOD1-YFP as compared with wild-type . The wild-type
transgenic animals do not develop motor disease. We studied
animals at 3 months of age, at which time nearly all G85R animals
are presymptomatic, albeit that most manifest yellow fluorescent
cytosolic aggregates in a fraction of motor neuron cell bodies in
spinal cord (Fig. S1). At this time, there is also astrogliosis
surrounding these neurons and morphologic alteration of a signif-
icant fraction of NMJs in gastrocnemius muscle (unpublished).
Freshly obtained cords were rapidly frozen in OCT embedding
solution and 20 mm cryosections were stained with 1% Azure B
dye in 70% ethanol (Fig. 1B; see Methods), then subjected to laser
capture microdissection of large cell bodies in the ventral horn
(Fig. 1E). The large cell bodies were motor neuron cell bodies,
verified by anti-ChAT antibody staining carried out on the same
section (Fig. 1C), and exhibited diffuse YFP fluorescence from the
expressed fusion protein (Fig. 1D). Some sections from G85R
animals also contained cells with large, intensely fluorescent
aggregates that exhibited a darker Azure B staining pattern
(Fig. S1); these were specifically excluded from laser capture.
Laser-dissected neuron cell bodies were collected directly into
guanidine thiocyanate (Fig. 1E) from each of two mutant and two
wild-type animals (,4000 cells/animal), and RNA was prepared
from them. The quality and quantity of RNA were determined
using a Pico RNA chip on an Agilent 2100 Bioanalyzer. A typical
yield from 4,000 cell bodies was ,50 ng of total RNA, with an
RNA Integrity Number (RIN) $8.5.
To test the purity of motor neuron RNA from laser captured
cell bodies, we assessed for the presence of RNA for GFAP,
a protein specific to astrocytes, which surround motor neurons and
are abundant in the ventral horn. We carried out qRT-PCR on
RNA from laser captured neurons and on an identical amount of
RNA similarly prepared from total ventral horn. The amount of
GFAP RNA in the motor neuron RNA preparation was ,8% that
in the total ventral horn RNA (Fig. S2). We thus conclude that
RNA from motor neuron cell bodies was minimally contaminated
with RNA from neighboring astrocytes.
RNA-seq and differential mRNA expression in motor
Total RNA recovered from motor neuron cell bodies was
subjected to polyA selection, fragmentation, cDNA synthesis,
adaptor ligation, and library amplification according to the
standard Illumina mRNA-Seq protocol (Fig. 1E and see Methods).
Seventy-five bp single-end reads were obtained from an Illumina
GA IIx for two animals each of the G85R and wild-type strains.
The raw data have been deposited in the GEO database, accession
number GSE38820. Reads were mapped and gene expression was
quantified as previously described . RNA-Seq generated
greater than 40 million reads for each sample, with a minimum
of 65% of the reads mapping to the mouse genome at a single
location for each replicate. We detected expression of 17,237 genes
on average from the four samples (Table S1). Overall, gene
expression was highly reproducible between the sample replicates
as well as between G85R and wild-type samples (Fig. S3). Using
a log-linear model coupled with likelihood ratio test (log-linear
LRT), we identified only 62 genes as differentially expressed
between G85R and wild-type motor neurons after multiple
hypothesis testing correction (BHP ,0.05) [21,22], out of a total
of 352 genes meeting the less-stringent criterion of raw p-value
,0.05 (Table S2).
Using the 352-gene list, we found that genes showing reduced
mRNA levels in G85R were enriched for gene ontology terms
related to several aspects of neuronal function. These included
neuronal cytoskeletal components involved in neurite outgrowth
and axon formation such as Nefl, Nefm (neurofilament light and
medium chains, respectively), and Prph (peripherin) (Table S3).
Also in this category were genes associated with calcium
metabolism and sensing, such as the calcitonin gene-related
peptide (Cgrp) precursors, Calca and Calcb, calcineurin (Ppp3ca),
and hippocalcin-like 1 (Hpcal1). Genes whose expression was
increased in G85R were enriched for gene ontology terms related
to mitochondrial function, ion homeostasis, and cytoplasmic
vesicle formation (Table S4). These included nuclear genes
encoding mitochondrial proteins primarily associated with OX-
PHOS complexes I, IV, and V. Overall, however, there was not
a general increase in RNA levels for the majority of mitochondrial
proteins encoded in the nuclear genome.
To confirm the RNA-Seq results, we subjected a number of
differentially expressed genes to validation by qRT-PCR. We
chose genes that had at least a 1.5-fold change in expression in the
mutant with a BHP value ,0.05 or a raw p-value ,0.05 (Fig. 2A).
For each gene, multiple sets of primers were chosen that spanned
exon junctions to eliminate any signal from amplification of
contaminating genomic DNA; two exceptions are noted in the
Fig. 2B legend. As in the RNA-Seq experiments, RNA prepared
from laser captured motor neurons was employed as template for
first strand cDNA synthesis. We selected 44 genes for validation by
qRT-PCR using 0.19 ng of total RNA per reaction. At this level of
starting material, we were only able to validate nine genes as
significantly differentially expressed. However, we noted that
many of the genes that failed had very high Ctvalues for both
wild-type and mutant samples due to low amounts of input RNA.
Therefore, a second round of validation was carried out for seven
genes that had failed to validate initially, now using 1.5 ng of total
RNA per reaction. This resulted in confirmed differential
expression for six additional genes. In total, 15 genes were
validated as significantly differentially expressed between mutant
and wild-type strains by all primer sets across multiple qRT-PCR
experiments (Fig. 2B). The largest changes identified by both
RNA-Seq and qRT-PCR affected an Hsp110 (Hsph1) and b2
microglobulin (B2m) RNA, both .2-fold elevated, and the
phospholipase A2, Pla2g4e, which was reduced by approximately
8-fold. Notably absent were changes affecting the other two
Hsp110 orthologs (Hspa4 and Hspa4l) or other quality control
components, e.g. from the proteasome, UPR, or mitochondrial
UPR. Consistently, no significant change was detected in the RNA
level of the major heat-shock transcription factor, Hsf1 (Table S1).
Hsc70 (Hspa8), the major chaperone associating with G85R
SOD1 in spinal cord , could not be evaluated because of the
presence of processed pseudogenes in the mouse genome. From
these data, it appears that, at this presymptomatic time when
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RNA-Seq Profiling of an SOD1 ALS Mouse
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protein aggregation and gliosis, as well as effects on NMJs, are
already occurring (unpublished observations), there is no global
heat shock or unfolded protein response in the motor neurons. By
contrast, we have validated several components involved with
metabolizing or sensing calcium, such as Hpcal1, Calca, Pcp4, and
Pla2g4e, suggesting a role for calcium-regulated pathways in the
pathogenesis of this model system. The nature of the large
diminution of Pla2g4e RNA remains unclear. Using an available
antibody, we observed punctate staining for Pla2g4e in the cell
bodies of wild-type motor neurons, whereas mutant cells exhibited
only diffuse staining (Fig. S4). The Pla2g4e staining in wild-type
cell bodies largely co-localized with the lysosomal marker Lamp2
(not shown), as would have been predicted by an earlier
localization study .
Lack of major disturbances of mRNA processing in the
G85R mutant strain
Recent studies using a Tdp43 knockdown model for ALS have
suggested that misregulation of RNA-splicing is a major contrib-
utor to ALS onset and progression in that setting . In contrast,
the effect here of transgenic G85R SOD1-YFP on motor neuron
RNA splicing was minimal. We detected only 8 of the 287
aberrant splicing events identified in the Tdp43 study in either
wild-type or G85R transcriptomes. Of these, only three genes,
Eif4h, Hisppd2a, and Spp1, showed evidence of differential
splicing exclusively in the G85R strain (validated by qRT-PCR;
Table S5), suggesting that mutant SOD1 is associated with
a different mechanism of disease causation.
In addition, RNA editing has been implicated in neuronal cell
death in human ALS patients . In particular, under normal
conditions, nearly all GLUR2 transcripts (Gria2 in mouse) have
been shown to be edited in normal motor neurons and other
neuronal tissue, resulting in the change of a glutamine codon to an
arginine codon. On the other hand, some spinal motor neurons of
end-stage sporadic ALS patients exhibited significantly decreased
editing at this position . Analysis of Gria2 transcripts in the
RNA-Seq data sets from our transgenic mice confirmed extensive
editing of the transcript, but we detected no differences in editing
efficiencies between wild-type and G85R motor neurons. Similar-
ly, such differences in editing have not been observed in individual
neurons of rats expressing H46R or G93A mutant forms of human
RNA-Seq also allows the detection of regions of the genome not
previously annotated as being transcribed (novel TARs). We
detected 4860 novel TARs in the two strains. As was observed for
mRNA expression differences, only a small fraction of these were
differentially expressed in G85R, 23 reduced and 44 increased
(Table S6). The collective of novel TARS likely represents a variety
of RNA types, including long noncoding RNAs, enhancer RNAs,
and novel UTRs of known mRNAs . In order to better
evaluate these sequences, we associated novel TARs upregulated
in G85R with the nearest two genes. These genes were then
grouped according to gene ontology using all novel TARS as
a background set. We found a single gene ontology molecular
function category that was enriched, that of heat shock protein
binding, containing three genes (Table S7). It was not clear that
the novel TAR in the neighborhood of one of these genes, Dnajb1,
was actually part of that gene’s transcript. In contrast, the other
two genes in this category, Limk1 (Lim domain-containing kinase
1) and Gak (cyclin G-associated kinase; auxilin 2), had novel 39-
UTRs clearly associated with a fraction of the transcripts and
present almost exclusively in G85R (Fig. 3A). There are no
annotated ESTs or antisense transcripts in these regions, and the
sequence in these locations was found to be uniquely mappable,
precluding these UTRs from being overlapping transcripts or
arising from artifacts in the alignment. The extensions for Limk1
and Gak are approximately 1700 nt and 1500 nt, respectively, and
do not alter the coding potential of the original transcript nor
create additional open reading frames. We confirmed the presence
and overabundance of these novel 39-UTRs via qRT-PCR
In sum, RNA-Seq analysis of motor neurons in transgenic
G85R SOD-YFP ALS animals at a presymptomatic timepoint
identifies a small degree of change of only a few mRNAs, even
though aggregation is already occurring in some motor neuron cell
bodies, with surrounding astrogliosis, and there are morphologic
NMJ abnormalities. Although it cannot be excluded that one or
more of these changes, for example depression of the mRNA for
Pla2g4e or other calcium-related genes identified here, could be
a primary driver of mutant SOD1-linked disease, it seems more
likely that the few changes at the RNA level observed here are
secondary responses to the effects of high level expression of the
misfolded protein in motor neurons. For example, the elevation of
mRNA for the cytosolic molecular chaperone, Hsp110, is likely
a stress response to the misfolded cytosolic mutant SOD1. The
profile observed here contrasts with the substantial changes in
mRNA biogenesis reported in the setting of TDP43 or FUS-linked
pathogenesis, where both levels and splicing of many RNAs are
An earlier microarray study of RNA from laser captured motor
neurons from presymptomatic G37R SOD1, as well as G85R
transgenic mice likewise observed a relatively small number of
genes that exhibited altered transcript levels . Changes were
observed in the D/L serine biosynthesis pathway, but these were
specific to G37R. Changes in complement components were
observed in both strains. In the present study, we detected these
RNAs, but only C1qa differed significantly between G85R and
wild-type animals. A microarray study of RNA from laser
dissected spinal motor neurons of G93A SOD1 mice at three
time points in the development of disease also found a relatively
small number of changes relative to non-transgenic litter mates,
particularly at later stages of disease . No significant
commonalities between either of the two previous studies [28,29]
and the study here were apparent, except that all found one or
more components of the complement system upregulated. We
validated this change in C1qa via qRT-PCR, suggesting that this
Figure 1. RNA-Seq of laser capture microdissected spinal cord motor neurons from wtSOD1-YFP and G85R SOD1-YFP transgenic
mice. A. Survival curve of G85R SOD1-YFP mice with copy number greater than 200. 80% of the mice were paralyzed (and euthanized) between
,115 days and 205 days (red bar). N=226. For the present study of motor neuron RNA, presymptomatic mice at ,90 days of age were used. B.-D.
Spinal cord from a 3 month old G85R SOD1-YFP mouse. Frozen section of right ventral horn region is shown, stained with Azure B dye, panel B (see
Methods); incubated with anti-ChAT antibodies, panel C; or directly examined for YFP fluorescence, panel D. The large blue-stained cell bodies in
panel B are motor neurons as indicated by anti-ChAT staining in panel C. Note that the same cells have YFP fluorescence in panel D. E. Large Azure B-
stained cell bodies were laser captured directly into a guanidine thiocyanate solution (see Methods) and subsequent steps carried out as diagrammed
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difference might be involved in pathogenesis. It is not clear,
however, whether this is a motor neuron derived transcript or
a contaminant from inadvertently dissected surrounding activated
glia, given that C1q has been implicated in neuroinflammation.
Another microarray study, using laser captured motor neurons
from cranial nerve nuclei and cervical spinal cord of pre-
symptomatic G93A rats, focused on transcription differences
between these regions that might account for the sparing of cranial
nerves 3/4 from disease . Notably, IGF-II and guanine
deaminase RNAs were preferentially expressed in cranial nerve 3/
4 motor neurons of G93A rats, but no comparisons were made
with expression patterns in wild-type animals.
The previous laser capture studies used early versions of mouse
microarrays with only single probes to several genes we have
identified here, such as Pla2g4e, making them relatively insensitive
to changes in such genes. In addition, these array experiments
would not have been able to detect any previously unannotated
transcripts such as those we observed here at the 39-end of Limk1
and Gak, both of which could be of further interest. For example,
loss of Limk1 results in the regression of presynaptic motor neuron
termini in Drosophila and altered dendritic spines in mice [31,32].
Moreover, Limk1 transcripts are translated locally in dendrites of
cultured hippocampal neurons and regulated through the 39-UTR
by miR134 . The extension of the 39-UTR we have observed
could introduce additional regulatory sequences affecting the
maintenance of neuromuscular junctions and/or dendritic spines.
The role of Gak (auxilin 2) in neurons is less well defined, but it
contains an auxilin-type J-domain and interacts with Hsc70 to
support clathrin uncoating and vesicle cycling in non-neuronal
tissues, where it is the only auxilin . It is upregulated in brains
of auxilin knockout mice and supports sufficient uncoating activity
Rep1 Rep 2
Figure 2. Differential expression in G85R motor neurons. A. Heatmap of selected genes from Table S2 significantly differentially expressed
(raw p-value ,0.005) between wild-type and G85R SOD1-YFP mice. For each gene listed, the ratio of RPKM values (G85R/WT) for individual pairs of
biological replicates (Rep1, Rep2) is plotted according to the color code below. B. Validation of differentially expressed genes in G85R by qRT-PCR.
Shown are box plots representing relative expression values of each gene in G85R versus wild-type motor neurons. Upper whisker represents top
25% of values, box represents the middle 50% of values, and lower whisker represents bottom 25% of values. Median value is indicated by horizontal
dashed line. Statistical significance calculated by REST 2009  is indicated by * = p,0.05, ** = p,0.005, *** = p,0.0005. RNAs from at least three
different mouse pairs were compared for each gene. Note that the Hsp110 and B2m validations used one exon-junction-spanning and one non-
spanning primer set; minus reverse transcriptase controls for these samples were negative for DNA contamination. The expression changes in the left
nine genes were validated with 0.19 ng of total RNA, while the remaining six were validated with 1.5 ng of total RNA.
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to permit some animals to survive ; in contrast, Gak knockout
is an early post-natal lethal .
Given the small number of changes observed here in a mutant
SOD1-linked setting, it seems more likely that the primary effect(s)
of the mutant SOD1 protein either lie at the level of translation or
are post-translational. Post-translational effects, in particular,
could involve interaction between the misfolded mutant SOD1
and cellular cytosolic or membrane proteins, which could affect
their roles in macromolecular traffic, organellar function, and/or
synaptic function. Further translational, morphologic, and bio-
chemical analyses may be able to address how the mutant SOD1
protein drives motor neuron pathology.
Materials and Methods
This study was performed in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. All
animal experiments were conducted according to a protocol
approved by Yale University Institutional Animal Care and Use
Committee (protocol #2011–10931).
The transgenic mouse strains, wild-type SOD1-YFP (strain 592)
and mutant G85R SOD1-YFP (strain 737), have been described
. The transgene copy numbers of all animals were determined
by qPCR using primer sets for human genomic SOD1 and mouse
genomic ApoB, as recommended by Jackson Laboratories.
Animals of the 737 strain that were used for RNA isolation had
apparent copy numbers between 210 and 300; copy numbers for
the wild-type 592 strain were 6–8.
Wild type (strain 592) and mutant (strain 737) transgenic mice
were sacrificed with a lethal dose of ketamine, followed by cardiac
perfusion with PBS for 2 min. Spinal cords were dissected within
5 min, divided transversely into 9 to 10 pieces, embedded in OCT,
then frozen in 2-methybutane cooled with liquid nitrogen. They
were stored at 280uC until used for RNA preparation. Twenty
micron frozen tissue slices, each containing 9–10 individual
sections, were produced using a Leica CM3050S cryostat at
220uC and mounted on RNase-free PEN-membrane 2 mm slides
(Leica); they were kept at 280uC until used. Individual slides were
thawed and dried for 30–40 sec, followed by washing for 30–
45 sec in RNase-free 70% ethanol for OCT removal. The
following steps were then performed in order: 30–45 sec in-
cubation in 1% Azure B (MP Biomedicals) in 70% ethanol, two
30–40 sec washes in 70% ethanol, and 40 sec air-drying. Azure B
stained motor neuron cell bodies from the ventral horn from each
section on the slide were laser dissected (Leica LMD6000, 20X
objective) and collected in 30 ml guanidine thiocyanate buffer +
DTT (RLT buffer; RNeasy Micro kit, Qiagen) into the cap of
a 0.6 ml microfuge tube. Recovery of all identifiable motor
neurons from the 9–10 sections on each slide (,150 total) required
,30 min. Individual collection tubes were stored at 220uC until
a sufficient number had been collected. Samples were thawed and
pooled, and total RNA was extracted from 3000 to 4000 motor
neuron cell bodies from each animal using the RNeasy Micro kit
(Qiagen) according to the protocol provided for LMD tissue. The
quality of the RNA was determined on an Agilent 2100
BioAnalyzer using an RNA Pico chip. Total RNA preparations
with an RNA Integrity Number (RIN) above 8.5 were used for
RNA-Seq and qRT-PCR. RNA from two animals from each
strain was analyzed separately by RNA-Seq, and RNA from at
least 3 animals per strain was analyzed by qRT-PCR.
Transcriptome and splicing analysis
Seventy-five bp single-end reads were mapped using Bowtie (v
0.12.3) to the mouse genome (mm9), and a custom splicing
database generated from UCSC Known Gene annotation as
previously described [21–22]. Gene expression was quantified
using Cufflinks (v 0.8.3), and differential expression was de-
termined as previously described [21–22].
135,135,000135,140,000135,145,000 135,150,000135,155,000135,160,000 135,165,000
Fold Increase in G85R
Gak 3' UTR
Limk1 3' UTR
Figure 3. Differential expression of novel TARs in G85R motor neurons. A. RNA-Seq signal plots in reads per million mapped reads at the
Limk1 gene from wild-type (black) and G85R (red) motor neurons. The canonical Limk1 gene map is shown at the top. The enlarged region shows
a clear 39-UTR extension in G85R motor neuron RNA. Similar data were observed for Gak (not shown). B. Validation of novel 39-UTRs of Gak and Limk1
by qRT-PCR. Values are relative expression for each 39-UTR in G85R versus wild-type motor neuron RNA.
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Quantitative reverse transcriptase PCR
Multiple sets of primers (each spanning an exon-exon junction)
were designed for each mRNA of interest using Primer Blast
(NCBI) against the Mus musculus mRNA RefSeq database
(Table S8). In preliminary experiments with cDNA prepared
from isolated mouse brain RNA, amplified products from each
primer pair were subjected to DNA sequencing to confirm their
specificity; those sets that produced incorrect or mixed sequences
were not used. In general, at least two satisfactory pairs were
identified for each RNA; exceptions are noted in the figure
legends. cDNA from total RNA from each sample was generated
using SuperScript III First Strand Synthesis System (Invitrogen)
with a mixture of random hexanucleotide and oligo-dT primers.
qRT-PCR reactions were performed in triplicate on a volume of
cDNA corresponding to 0.19 ng or 1.5 ng of input motor neuron
RNA in a 10 mL reaction volume per well using Power SYBR
Green PCR Master Mix (Applied Biosystems) on an ABI PRISM
7900 (Applied Biosystems) (Fig. 2B legend). One set of mouse
Gapdh primers and two sets of mouse Hprt1 primers were used as
reference for all samples. The reference mRNAs showed no
difference in expression between mutant and wild-type. Because of
the small amount of RNA available, controls without reverse
transcriptase in the first strand synthesis reaction were carried out
randomly with only a few primer sets. An exception was in the
study of the novel TARs for Limk1 and Gak (Fig. 3B), where the
size of the extended RNA segment precluded using an exon-exon
junction pair and necessitated the inclusion of this control. No
amplification products were observed in any of the minus reverse
transcriptase reactions, including those performed for the experi-
ments in Fig. 3B. Fold-change between mutant and wild-type mice
were determined using the DDCtmethod as implemented in the
Applied Biosystems software; calculations using any of the
reference sets gave the same results within experimental error.
old G85R SOD1-YFP mouse. Image of right ventral horn of
the spinal cord of a 3 month old G85R SOD1-YFP animal at the
lumbar level showing: left, YFP fluorescence and, right, Azure B
staining. A number of the motor neuron cell bodies have very
strong local YFP fluorescence, indicative of aggregation, which has
been confirmed by EM analysis (unpublished observations). The
corresponding Azure B-stained cell bodies (red arrow heads) are
much darker than the other cell bodies. Such motor neurons were
not laser captured for this study.
Aggregation in motor neurons of a 3 month
vested motor neurons. Relative expression of GFAP, an
astrocyte-specific marker, in wild-type and mutant motor neurons
compared to total ventral horn.
Contamination by astrocyte RNA in har-
log2(RPKM) values from wild-type and G85R replicates. Pearson
correlation coefficients for each comparison are indicated by R.
section with anti-Pla2g4e antibody. A) Representative
sections are shown for 3-month old wild-type SOD1-YFP (top
row) and G85R SOD1-YFP animals (bottom row); 20 mm sections
from perfused animals were subjected to immunohistochemistry as
described in Methods S1. Left panels, YFP fluorescence (green)
with DAPI staining (blue); in the wt animal, a motor neuron in the
Antibody staining of lumbar spinal cord
center of the panel is strongly YFP fluorescent and, likewise,
a neuron at the lower right in the mutant animal is fluorescent.
Middle panels, after anti-Pla2g4e antibody staining using an
Alexafluor 555 (red) secondary antibody, the wild-type motor
neuron exhibits cytosolic puncta, whereas the mutant fails to show
similar staining in the neuron. The wild-type did not exhibit such
staining in the absence of the primary antibody (not shown). Right
hand panels, merge. Magnification 100x with oil immersion
objective. B) Quantification of Pla2g4e immunofluorescence.
Images such as those in Fig. S4A were quantitated by counting
the red Pla2g4e puncta associated with YFP-positive (green)
ventral motor neurons. The percent of cells with .30 puncta (blue
bars), 10–30 puncta (red bars), and ,10 puncta (yellow bars) is
shown for three sets of wild-type (WT) and G85R mutant animals.
The number of cells evaluated for each animal is shown in
parentheses. Note the large number of mutant motor neurons with
,10 Pla2g4e-staining puncta compared to wild-type, where most
show .30 puncta. This reduced antibody staining correlates well
with the reduced mRNA levels for this protein detected by RNA-
Seq (Table S2) and qRT-PCR (Fig. 2B).
reads for wild-type and G85R motor neurons. Total genes
detected as expressed for each sample are defined as any gene
with an RPKM greater than zero from original read mapping.
The total level of gene expression for each sample was determined
as the sum of RPKM for all genes.
Sequencing Statistics. Total and uniquely mapped
ential gene expression. Log-linear analysis with LRT-statistic
of wild-type and G85R motor neuron RPKMs. Columns are:
UCSC cluster id, UCSC transcript id, MGI gene symbol, Entrez
Gene accession number, wild-type replicate 1 RPKM, wild-type
replicate 2 RPKM, G85R replicate 1 RPKM, G85R replicate 2
RPKM, log2fold change, LRT statistic, raw p-value, Bonferroni
corrected p-value, and Benjamani-Hochberg corrected p-value
(BHP). All RPKMs were calculated by adding one read to all genes
to eliminate zero values.
Wild-type versus G85R motor neuron differ-
in G85R. Full DAVID output [S2,S3] for genes identified as
having increased levels in G85R from Table S2 (BHP #0.05 and/
or raw p-value #0.05 and log2 fold change $2).
DAVID output for genes with increased levels
G85R. Full DAVID output [S2,S3] for genes identified as having
reduced levels in G85R from Table S2 (BHP #0.05 and/or raw
p-value #0.05 and log2 fold change $2).
DAVID output for genes with reduced levels in
motor neurons. Identification and number of reads supporting
each novel splice in each RNA-Seq replicate. Columns are: UCSC
transcript id, MGI gene symbol, Entrez Gene accession number,
junction coordinate, Reads mapping to junction from wild-type
replicate 1, wild-type replicate 2, G85R replicate 1, and G85R
Splice identification in wild-type and G85R
Identification and log-linear analysis with LRT-statistic of novel
Differentially expressed novel TARs in G85R.
RNA-Seq Profiling of an SOD1 ALS Mouse
PLOS ONE | www.plosone.org7January 2013 | Volume 8 | Issue 1 | e53575
transcriptionally active regions in wild-type and G85R motor Download full-text
neurons. Columns are: novel TAR id, wild-type replicate 1
RPKM, wild-type replicate 2 RPKM, G85R replicate 1 RPKM,
G85R replicate 2 RPKM, log2fold change, LRT statistic, raw p-
value, Bonferroni corrected p-value, and Benjamani-Hochberg
corrected p-value (BHP).
ed by novel TARs with increased levels in G85R motor
neurons. Full GREAT output for gene ontology enrichments of
genes assigned to novel TARS compared to a background set of all
novel TARs identified in wild-type and G85R expression data.
Gene ontology enrichment for genes associat-
Primers used in qRT-PCR.
We thank George Farr for technical assistance and A. Gulhan Ercan-
Sencicek and the laboratory of M. State (Yale School of Medicine) for help
with PCR validation studies.
Conceived and designed the experiments: UB JC WAF JPN ALH.
Performed the experiments: UB JC MN MM SM. Analyzed the data: UB
JC SO JL WAF JPN. Wrote the paper: UB JC WAF JPN ALH.
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