Different Human Copper-Zinc Superoxide Dismutase Mutants, SOD1G93A and SOD1H46R, Exert Distinct Harmful Effects on Gross Phenotype in Mice
Distinct Harmful Effects on Gross Phenotype in Mice. PLoS ONE 7(3): e33409. doi:10.1371/journal.pone.0033409
Different Human Copper-Zinc Superoxide Dismutase G93A H46R Mutants, SOD1 and SOD1 , Exert Distinct Harmful Effects on Gross Phenotype in Mice
Lei Pan 0
Yasuhiro Yoshii 0
Asako Otomo 0
Haruko Ogawa 0
Yasuo Iwasaki 0
Hui-Fang Shang 0
Shinji Hadano 0
Maya Koronyo-Hamaoui, Cedars-Sinai Medical Center, Maxine-Dunitz Neurosurgical Institute, United States of America
0 1 Department of Molecular Life Sciences, Tokai University School of Medicine , Isehara, Kanagawa , Japan , 2 The Institute of Medical Sciences, Tokai University , Isehara, Kanagawa , Japan , 3 Department of Neurology, Toho University Omori Medical Center, Ota-ku, Tokyo, Japan, 4 Research and Development Division, Teaching and Research Support Center, Tokai University , Isehara, Kanagawa , Japan , 5 Department of Neurology, West China Hospital, Sichuan University , Chengdu, Sichuan , China , 6 Research Center for Brain and Nervous Diseases, Tokai University Graduate School of Medicine , Isehara, Kanagawa , Japan
Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of fatal neurodegenerative diseases characterized by a selective loss of motor neurons in the brain and spinal cord. Creation of transgenic mice expressing mutant Cu/Zn superoxide dismutase (SOD1), as ALS models, has made an enormous impact on progress of the ALS studies. Recently, it has been recognized that genetic background and gender affect many physiological and pathological phenotypes. However, no systematic studies focusing on such effects using ALS models other than SOD1G93A mice have been conducted. To clarify the effects of genetic background and gender on gross phenotypes among different ALS models, we here conducted a comparative analysis of growth curves and lifespans using congenic lines of SOD1G93A and SOD1H46R mice on two different genetic backgrounds; C57BL/6N (B6) and FVB/N (FVB). Copy number of the transgene and their expression between SOD1G93A and SOD1H46R lines were comparable. B6 congenic mutant SOD1 transgenic lines irrespective of their mutation and gender differences lived longer than corresponding FVB lines. Notably, the G93A mutation caused severer disease phenotypes than did the H46R mutation, where SOD1G93A mice, particularly on a FVB background, showed more extensive body weight loss and earlier death. Gender effect on survival also solely emerged in FVB congenic SOD1G93A mice. Conversely, consistent with our previous study using B6 lines, lack of Als2, a murine homolog for the recessive juvenile ALS causative gene, in FVB congenic SOD1H46R, but not SOD1G93A, mice resulted in an earlier death, implying a genetic background-independent but mutation-dependent phenotypic modification. These results indicate that SOD1G93A- and SOD1H46R-mediated toxicity and their associated pathogenic pathways are not identical. Further, distinctive injurious effects resulted from different SOD1 mutations, which are associated with genetic background and/or gender, suggests the presence of several genetic modifiers of disease expression in the mouse genome.
Funding: This work was supported by Research and Study Project of Tokai University Educational System General Research Organization (SH), a Grant-in-Aid for
Scientific Research from the Japan Society for the Promotion of Science (SH), Daiichi-Sankyo Foundation of Life Science (SH), and Japan China Medical Association
(SH). LP receives support for a Tokai University School of Medicine Research Aid. 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.
Amyotrophic lateral sclerosis (ALS) is an inexorable
neuromuscular disorder characterized by progressive loss of motor neurons in
the spinal cord, brainstem and motor cortex . Most ALS patients
become severely paralyzed and die within 35 years after diagnosis.
The majority of patients are sporadic (sALS), while 510% are
familial cases (fALS), among which approximately 1520% are
associated with mutations in the gene encoding Cu/Zn superoxide
dismutase (SOD1) . Patients with sALS and mutant
SOD1linked fALS share many clinical and pathological features [3,4].
Importantly, recent studies have highlighted that not only mutant
SOD1 in fALS but also wild-type SOD1 can be pathogenic in sALS
patients [5,6], illuminating a possible SOD1-dependent pathogenic
mechanism common to sALS and fALS.
To date, more than 160 mutations scattered throughout the
SOD1 protein have been identified in fALS (http://alsod.iop.kcl.
ac.uk/als/). Although mutant SOD1-mediated neuronal toxicity
appears to account for disease expression , the exact mechanism
by which mutant SOD1 impairs neuronal function leading to
motor neuron death remains unclear, let alone the clinical
heterogeneity; e.g. age at onset and disease duration, seen within
and/or among SOD1-linked families [3,8,9]. Thus far, it is
generally thought that in addition to the toxic entities associated
with different SOD1 mutations, a complex interplay between other
genetic and environmental factors results in symptomatic
variability in SOD1-linked fALS.
Genetically engineered mice have played a pivotal role not only
in the molecular pathogenesis but also in the development of
therapeutics for many genetic as well as non-genetic diseases.
Indeed, the creation of ALS animal models, namely transgenic
mice expressing a mutant SOD1, has made an enormous impact
on progress of the ALS studies [4,10,11]. More than 10 different
lines of transgenic mouse carrying a different SOD1 mutation, all
of which show a selective degeneration of spinal motor neurons,
have been established [4,11]. Interestingly, the onset and
progression of disease phenotypes appear to vary from line to
line [12,13]. Among these lines, animals carrying a G93A SOD1
mutation (SOD1G93A)  were most widely used and extensively
characterized. Although a copy number of the SOD1G93A
transgene, and thus expression level of the SOD1G93A protein, is
a major determinant of disease severity , genetic background
and gender may also affect the SOD1G93A-linked symptoms in mice
. It is conceivable that there are some common genetic
modifiers affecting to disease expression in mutant
SOD1expressing ALS mouse models [15,16]. However, with the use of
only a limited number of lines; i.e. SOD1G93A, such notions still
remain inconclusive. Further, although it is generally thought that
the different mutant SOD1 proteins are likely to cause motor
neuron disease by a similar mechanism, this idea has also yet to be
Recently, we have generated two congenic mouse lines carrying
either SOD1G93A or a H46R mutation (SOD1H46R) on a C57BL/6N
(B6) background , and crossed those mice to B6 congenic mice
lacking Als2, a murine homolog for the causative gene for a
number of recessive juvenile motor neuron diseases (MNDs) ,
generating B6 congenic ALS2-deficient SOD1G93A and SOD1H46R
mice . The SOD1H46R mutation accounts for a mild form of
familial ALS that was originally identified in Japanese kindred
[20,21] and characterized by unusually extended disease duration
after onset [3,21]. Surprisingly, lack of Als2 in B6 congenic
SOD1H46R, but not SOD1G93A, mice results in an earlier death .
In addition, loss of Gfap, a gene encoding glial fibrillary acidic
protein (GFAP), in B6 congenic SOD1H46R, but not SOD1G93A,
mice also accelerates the disease progression . Thus, it is
possible that the SOD1G93A and SOD1H46R-mediated pathogenic
mechanisms are not the same. However, at this stage, we could not
formally exclude the possibility that these phenomena are unique
to mice on a B6 background.
In this study, to further clarify the phenotype variability among
different mutant SOD1-expressing ALS mouse models on different
genetic backgrounds, we newly generated congenic lines of
SOD1G93A and SOD1H46R mice as well as those lacking Als2 on a
FVB/N (FVB) background, and conducted a comparative analysis
of gross phenotypes in these mutants with different genetic
Copy numbers of the transgene in SOD1G93A and
SOD1H46R transgenic mouse lines with different genetic
In this study, we generated four independent congenic mouse
lines expressing the human mutated SOD1 gene; i.e., C57BL/6N
congenic SOD1G93A (B6_G93A) and SOD1H46R (B6_H46R), and
FVB/N congenic SOD1G93A (FVB_ G93A) and SOD1H46R (FVB_
H46R). The transgene in each mouse line was transmitted in the
expected Mendelian ratio of an autosomal gene (data not shown).
The previous studies have demonstrated that the estimated copy
numbers of SOD1G93A and SOD1H46R in original transgenic lines
are approximately 24  and 20 , respectively. Since it has
been shown that a copy number of the mutated SOD1 transgene
affects the disease severity , we first analyzed the copy
numbers of the transgene in our mouse lines by quantitative PCR.
The relative number of transgenes copy was estimated by the
difference in threshold cycle (DCT, delta CT) between the
transgene (SOD1G93A or SOD1H46R) and control (mouse Sod1).
There were no significant differences in the DCT values among all
four lines with different transgenes, genetic backgrounds, and/or
genders (Figure 1A and Table 1). These results indicate that each
transgene locus retains comparable number of the mutated SOD1
gene that is stably transmitted in the course of generating our
congenic lines, and that the copy numbers of the transgene
between SOD1G93A and SOD1H46R are almost equal.
Levels of the transgene transcripts in SOD1G93A and
SOD1H46R transgenic mouse lines with different genetic
To investigate whether the differences in mutations, genetic
backgrounds, and/or genders affect the expression levels of the
mutated SOD1 transcript, we performed a quantitative reverse
transcriptase (qRT)-PCR using total RNA from the spinal cord of
mice at a pre-clinical stage (12 weeks of age). Although the levels of
transcript for SOD1H46R relative to the b-actin mRNA (Actb)
showed a higher tendency when compared to those for SOD1G93A,
there were no significant differences in the levels of mutated SOD1
mRNA among different transgenic lines used in this study
(Figure 1B and Table 1). These data indicate that expression
levels of the mutated SOD1 transcripts are affected neither by
difference in mutations, genetic backgrounds, nor genders in mice.
Levels of the mutant SOD1 protein in SOD1G93A and
SOD1H46R transgenic mouse lines with different genetic
To examine whether differences in mutations, genetic
backgrounds, and/or genders affect the expression levels of the mutant
SOD1 protein, we next performed western blot analysis of the spinal
cord extracts obtained from mice at a pre-clinical stage (12 weeks of
age) using anti-human SOD1 antibody. Although the levels of the
mutant SOD1 proteins slightly varied (Figure 2A and 2B), a
quantitative analysis revealed no statistical differences in the mean
values among all tested mouse lines (Figure 2B and Table 1). Since
the detection efficiency between different SOD1 mutants with
antibody used in this study (polyclonal antibody raised against
fulllength SOD1 of human origin) may not necessarily be exactly
equivalent, we could not completely exclude the possibility that
expression levels of SOD1G93A and SOD1H46R are different.
Nonetheless, considering comparable levels of both transcripts
(Figure 1B and Table 1), it seems fair to conclude that their protein
levels are also comparable. The results indicate that the expressions
of the mutant SOD1 proteins are not affected by differences in
mutations, genetic backgrounds, and/or genders in mice.
Effects of different mutations on growth curves in SOD1
transgenic mice with different genetic backgrounds
As all four congenic mouse lines generated in this study showed
comparable levels of mutant transgene and protein expressions
(Figure 1, Figure 2, and Table 1), it is assumed that these mutant
SOD1-expressing ALS mouse models could be an appropriate
means to analyze the effects of mutations, genetic backgrounds,
and/or genders on gross phenotypes in vivo. In this study, we first
focused on body weight, as it has been widely accepted that onset
of disease in mutant-SOD1 transgenic ALS mouse models can be
estimated by the reduction of body weight [24,25].
During the experimental periods, both B6 and FVB congenic
wildtype animals showed a constant increase in their body weight,
whereas all the mutant SOD1 mouse lines started losing weight in the
middle (Figure 3AD). Indeed, both SOD1G93A and SOD1H46R mice
with different genetic background exhibited progressive motor
dysfunction and paralysis particularly on the phase of weight
reduction (data not shown). The mean values of body weight for
wild-type animals at each time point were significantly higher than
those for mutant litters, except for those at earlier ages (59 weeks) of
male B6_G93A (Figure 3B). The peak mean value of the body weight
in each experimental group ranged from 11 to 18 weeks; 15 week in
female B6_G93A and 15 week in female B6_H46R (Figure 3A), 15
week in male B6_G93A and 18 week in male B6_H46R (Figure 3B),
13 week in female FVB_G93A and 16 week in female FVB_H46R
(Figure 3C), and 11 week in male FVB_G93A and 16 week in male
FVB_H46R (Figure 3D). Notably, SOD1G93A mice showed more
extensive reduction when compared to SOD1H46R animals
irrespective of gender and genetic background (Figure 3AD). Further, both
female and male FVB_G93A exhibited an earlier body weight loss
than FVB_H46R counterparts (Figure 3C and D). These results
support the notion that the H46R mutation in SOD1 results in a
milder disease phenotype than does the G93A mutation in mouse
[18,22] and human . On the other hand, visual inspection of
animal behavior revealed that the onset of disease and the turning
point of the growth curves were closely matched in B6 congenic
mutant SOD1 transgenic mice, as previously reported [24,25].
However, it was obvious that the body weight were persistently
increased at an early phase of disease progression, where animals
clearly showed gait abnormalities, in FVB congenic mutant animals,
particularly in FVB_H46R. Thus, the peak of the growth curve in
mice on a FVB background may not define an earliest sign of disease.
Effects of gender on survival in SOD1G93A and SOD1H46R
transgenic mouse lines with different genetic backgrounds
We next focused on lifespan (survival), which is thought to be
one of the most important gross phenotype in ALS mouse models.
The mean values of survival varied in mice with different SOD1
mutations, genetic backgrounds, and/or genders (Table 2),
consistent with previous reports [11,16]. Kaplan-Meier survival
analysis revealed that female FVB_G93A mice lived longer than
male counterpart (Figure 4C). By contrast, no obvious gender
effects on survival in other mouse lines, including B6_G93A
(Figure 4A), B6_H46R (Figure 4B), and FVB_H46R mice
(Figure 4D), were observed. These data suggest that gender
differently affects survival in mice carrying the different SOD1
mutation on a different genetic background.
Effects of genetic background on survival in SOD1G93A
and SOD1H46R transgenic mouse lines
We investigated whether genetic background affected on
survival in our congenic mouse lines. Kaplan-Meier survival
analysis revealed that all FVB congenic lines carrying mutant
SOD1 gene irrespective of their mutation and gender differences
showed a significant shorter lifespan when compared to
corresponding B6 lines (Figure 5AD). Namely, compared to the H46R
mutation (Figure 5C and D), devastating effects of the G93A
mutation were more prominent in FVB congenic mice (Figure 5A
and B). These results strongly indicate that difference in genetic
background strongly affects lifespan in mutant SOD1-expressing
ALS mouse models, and suggest that FVB congenic mice are more
susceptible to mutant SOD1-mediated toxic insults than B6 lines.
Effects of different types of the SOD1 mutation on
survival in transgenic mice with different genetic
We next examined whether the different SOD1 mutations
affected survival in mice on the same genetic background and
gender. Kaplan-Meier survival analysis revealed that the G93A
mutation resulted in a shorter lifespan than did the H46R
mutation in both B6 and FVB lines irrespective of gender
(Figure 6AD). It is noted that such toxic effects of the G93A
mutation were more obvious in mice on a FVB background
(Figure 6C and D). The data suggest that the G93A mutation in
SOD1 causes a severer disease phenotype than does the H46R
mutation, and support the notion that FVB congenic mice are
more susceptible to the SOD1G93A-mediated toxic insults than B6
Effect of ALS2 loss on growth curves in FVB congenic
SOD1G93A and SOD1H46R transgenic mouse lines
Loss of function mutation in the ALS2 gene accounts for a
number of juvenile recessive forms of ALS/MNDs [19,26,27].
Previously, we demonstrated that genetic ablation of Als2 in
SOD1H46R, but not SOD1G93A, mice on a B6 background
aggravated the mutant SOD1-associated disease symptoms and
led to the earlier death , suggesting distinctive susceptibilities
to ALS2 loss in different mutant SOD1-expressing ALS mouse
models. In this study, to clarify whether such functional
interactions in vivo between ALS2 and mutant SOD1 depended
on genetic background of mouse lines, we crossed FVB congenic
SOD1G93A and SOD1H46R mice to FVB congenic Als2-null mice
, generating FVB congenic ALS2-deficient SOD1G93A (Als22/2;
SOD1G93A) and SOD1H46R (Als22/2;SOD1H46R) mice (FVB_
G93A_Als22/2 and FVB_H46R_Als22/2), respectively, and
analyzed their body weight and lifespan.
During the experimental periods, the mean values of body
weight for FVB congenic wild-type animals at each time point
were significantly higher than those for all mutant litters
(Figure 7AD), consistent with the previous findings observed in
B6 congenic lines . There were no significant differences in
body weight between both mutant SOD1 mice and their
ALS2deficient counterparts (Figure 7AD), except that male
FVB_H46R_Als22/2 showed a lower value than male
FVB_H46R at 21 weeks of age (Figure 7D). The results suggest
a limited role of ALS2 in growth curve in FVB congenic mutant
Effect of ALS2 loss on survival in FVB congenic SOD1G93A
and SOD1H46R transgenic mouse lines
To further investigate the effect of ALS2 loss on survival in FVB
congenic SOD1G93A and SOD1H46R transgenic mouse lines, we
analyzed lifespan by Kaplan-Meier analysis. Consistent with the
observation in FVB_G93A and FVB_H46R mice (Figure 4C and
D), there was a small but significant gender difference in
FVB_G93A_Als22/2 (p = 0.0215) (Figure 8A), while no
differences in FVB_H46R_Als22/2 mice were observed (Figure 8B).
Importantly, as in the case for B6 lines , FVB_H46R_Als22/2
mice died earlier than FVB_H46R mice in both gender (Figure 8E
and F), while no differences in lifespan between FVB_
G93A_Als22/2 and FVB_G93A mice were observed (Figure 8C
and D). These results combined with previous findings  suggest
a distinctive susceptibility to ALS2 loss in different mutant
SOD1expressing mice irrespective of their genetic background, and
indicate that the pathogenic pathways associated with ALS2 loss
and SOD1H46R, but not SOD1G93A, -mediated neuronal toxicity
might act synergistically in vivo.
In the present study, we generated four congenic ALS mouse
model lines; SOD1G93A and SOD1H46R mice on two different
genetic backgrounds; B6 and FVB, and showed that the expression
levels of the mutant SOD1 proteins among these different lines
were comparable. This allows us to conduct a comparative
analysis of growth curve and survival using these mice,
demonstrating some concrete evidence indicating that two
different SOD1 mutations exerts a distinct harmful effect on gross
phenotypes in mice.
Multiple epidemiological surveys have indicated that gender
affects the incidence, age at onset, and disease duration in sALS
patients [29,30]. However, the male-to-female ratio in the
SOD1linked fALS patients is 1:1 , rather suggesting the absence of
gender effects in human ALS patients with the SOD1 mutations.
Nonetheless, it has been reported that female mice lived longer
than male in several different congenic SOD1G93A lines on a SJL/J
(SJL), C3H/HeJ (C3H), BALB/cByJ (BALB), and FVB
backgrounds, while neither B6 nor DBA/2J (DBA) congenic mice
showed any gender effects on lifespan [15,16]. Consistently, we
here showed that female FVB, but not B6, congenic SOD1G93A
mice lived longer than corresponding male counterpart,
confirm*Values are mean 6 SD.
ing that the G93A mutation does exert a genetic
backgrounddependent gender-specific harmful effect in mice. Interestingly,
unlike SOD1G93A mice, both B6 and FVB congenic mice
expressing SOD1H46R showed no observable gender effect on
survival. Together, not only genetic background but also
difference in the SOD1 mutations may affect the gender-associated
phenotypic modification, at least, in mice.
In addition to gender effects as above, it is widely appreciated
that genetic background affects many phenotypes including
lifespan in mutant SOD1-expressing ALS mouse models
[15,16,31,32]. Likewise, Als2-null mice, another type of ALS/
MND model, on a FVB but not B6 background shows shorter
lifespan than do wild-type litters . Consistently, we here
revealed that both FVB congenic SOD1G93A and SOD1H46R lines
exhibited a significant shorter lifespan when compared to B6
counterparts. It is noted that the mutant SOD1-mediating
devastating effects on life span appear more prominent in
SOD1G93A than SOD1H46R mice, particularly those on a FVB
background. FVB mice are known to be more vulnerable to
glutamate receptor-mediated excitotoxic cell death [33,34], as well
as to mitochondrial toxin-mediated metabolic cell death ,
when compared to B6 mice. These findings suggest that FVB
congenic mice may have certain properties involving in the
preferential vulnerability to cellular-toxicities including mutant
SOD1-mediated insults. Conversely, it is equally likely that B6
mouse carries the gene conferring the resistance to these insults.
One of the important findings obtained from this study was that
the different SOD1 mutations showed distinct adverse effects on
gross phenotypes in ALS/MND mouse models. Comparative
analysis of growth curves and lifespans revealed that the G93A
mutation in SOD1 caused a severer disease phenotype than did the
H46R mutation, where SOD1G93A mice showed more extensive
body weight loss and earlier death. These trends were more
striking in FVB congenic mice. Remarkably, lack of Als2, a murine
homolog for the recessive juvenile ALS causative gene , in
FVB congenic SOD1H46R, but not SOD1G93A, mice resulted in an
earlier death. We have previously demonstrated similar results
using B6 congenic Als2-deficient SOD1H46R but not SOD1G93A mice
, indicating a genetic background-independent but
mutationdependent phenotypic modification. Although the possibility that
phenotypic variances observed are due to locus-specific effects of
each transgene and/or adjacent genes in a particular genomic
region should not be excluded, these findings lend credence to the
notion that SOD1G93A- and SOD1H46R-mediated toxicity and
their associated pathogenic pathways are not identical. Recent
findings that loss of Gfap, a gene encoding glial fibrillary acidic
protein (GFAP), in B6 congenic SOD1H46R, but not SOD1G93A,
mice slightly accelerates the disease progression , also support
Provided that the SOD1G93A and SOD1H46R mutants exert the
distinctive harmful effects on symptoms associated with ALS/
MNDs, how does different mutation in the same gene result in the
distinctive phenotypic modification? It has been reported that
although age at onset among families carrying different SOD1
mutations with high penetrance is less variable with the mean
value ranging from 45 to 50 years , the mean disease duration
after onset varies depending on mutation type, with a range of 0.9
to 18.7 years among SOD1-linked families . Indeed, the disease
durations after onset in patients with the G93A and H46R
mutations are considerably different (G93A vs H46R; 2.261.5 vs
17.0611.0 years) [3,21]. It is noted that our congenic ALS mouse
models partly recapitulate such differences. Currently, although
exact molecular basis for the mutation-dependent effect remains
unclear, it is appreciated that the SOD1-mediated dismutase
enzymatic activity is not a major determinant for the phenotypic
modification, since there is no correlation between disease
severities and the SOD1 dismutase activities [36,37]. Rather, the
differences in the propensity for the aggregate formation among
the different mutant SOD1 proteins might be related [7,38]. In
cultured cells, SOD1H46R mutant forms fewer insoluble aggregates
and inclusions when compared with SOD1G93A . Further,
unlike in SOD1G93A mice , no obvious SOD1-positive
inclusions are detected in the spinal cord of SOD1H46R mice
. These results indicate that SOD1H46R is less prone to form
aggregates than SOD1G93A. Additionally, large vacuolar structures
originated from distended mitochondria are evident in SOD1G93A
, while such pathological features are barely observed in
SOD1H46R mice. Instead, a widespread axonal pathology and
degeneration considerably precede motor neuron loss in the spinal
cord of SOD1H46R mice [18,23]. Thus, it is conceivable that
molecular basis for the pathogenesis in each mutant
SOD1expressing mouse model may not be the same.
It is generally thought that symptomatic heterogeneity observed
in patients with ALS/MNDs may reflect varied etiology and/or
results from a complex interplay between other genetic and
environmental factors [8,9,40]. Some epidemiological studies
suggest that traumatic injury and exercise are risk factors for the
development of ALS [41,42], but these results were not widely
confirmed . Animal studies using a SOD1G93A ALS model have
demonstrated that extensive endurance exercise hastens a decrease
in motor performance and death following onset of disease in male
mice , while moderate exercise seems to be beneficial .
Further, it has recently been shown that environmental
enrichment significantly improves motor performance in SOD1G93A mice
in a gender-specific manner . However, it is still unclear as to
whether and how such environmental factors affect disease
symptoms. Future studies using different congenic ALS mouse
models are warranted to clarify not only genetic but also such
environmental factors associated with ALS/MNDs.
Preclinical animal studies are prerequisite for the development
of therapeutic agents for the treatment of ALS/MNDs. Thus far, a
large number of successful therapeutic interventions in preclinical
animal studies have failed to translate into human applications
Figure 4. Effect of gender on survival in different mutant SOD1 transgenic mouse lines. (A) Survival curves for C57BL/6 (B6) congenic
SOD1G93A transgenic mice (B6_G93A) [female (F); closed circle: n = 19, male (M); open square: n = 15]. (B) Survival curves for B6 congenic SOD1H46R
transgenic mice (B6_H46R) (F; closed circle: n = 31, M; open square: n = 63). (C) Survival curves for FVB/N (FVB) congenic SOD1G93A transgenic mice
(FVB_G93A) (F; closed circle: n = 20, M; open square: n = 17). (D) Survival curves for FVB congenic SOD1H46R transgenic mice (FVB_H46R) (F; closed
circle: n = 20, M; open square: n = 27). Kaplan-Meier analysis with Log-rank (Mantel-Cox) test reveals a significant gender difference in FVB_G93A
(p = 0.003), but not in B6_G93A (p = 0.7344), B6_H46R (p = 0.3723), and FVB_H46R (p = 0.4439).
. To solve these issues, standard operation procedures (SOPs)
for preclinical animal studies for ALS/MNDs has recently been
proposed , in which in addition to the minimum experimental
requirements for any proof of concept or preclinical study, the use of
not only SOD1G93A model but also other potential ALS models such
as transgenic mice expressing either mutant dynactin, TAR
DNAbinding protein (TDP43), or fused in sarcoma (FUS) mutants are
recommended. However, the trend is still largely biased toward the
use of single mutant SOD1 ALS mouse model; i.e., SOD1G93A .
We here propose that mice expressing SOD1 mutants other than
SOD1G93A should also be included within this list. It seems logical
that convincing evidences could be obtained by replicated
demonstrations of efficacy and effectiveness of interventions with
different ALS models in preclinical animal studies.
In conclusion, the findings presented in this study strongly
support the notion that the different mutant SOD1 proteins cause
motor neuron dysfunction and death by a similar but not identical
mechanism, suggesting the presence of different genetic modifiers
of disease expression, which are associated with a combination
with particular SOD1 mutation, genetic background, and/or
gender. Thus, congenic ALS mouse models with different SOD1
mutations generated in this study should provide a useful means
not only for the identification of modifier genes as well as
environmental factors associated with ALS/MNDs, but also for
preclinical animal studies in ALS/MNDs.
Materials and Methods
All animal experimental procedures were carried out in accord
with the Fundamental Guidelines for Proper Conduct of Animal
Experiment and Related Activities in Academic Research
Institutions under the jurisdiction of the Ministry of Education,
Culture, Sports, Science and Technology, Japan, and reviewed
and approved by The Institutional Animal Care and Use
Committee at Tokai University.
Figure 5. Effect of genetic background on survival in different mutant SOD1 transgenic mouse lines. (A) Survival curves for C57BL/6 (B6)
congenic SOD1G93A transgenic female mice (B6_G93A F) (closed circle: n = 19) and FVB/N (FVB) congenic SOD1G93A transgenic female mice (FVB_G93A
F) (open square: n = 20). (B) Survival curves for B6 congenic SOD1G93A transgenic male mice (B6_G93A M) (closed circle: n = 15) and FVB congenic
SOD1G93A transgenic male mice (FVB_G93A M) (open square: n = 17). (C) Survival curves for B6 congenic SOD1H46R transgenic female mice (B6_H46R F)
(closed circle: n = 31) and FVB congenic SOD1H46R transgenic female mice (FVB_H46R F) (open square: n = 20). (D) Survival curves for B6 congenic
SOD1H46R transgenic male mice (B6_H46R M) (closed circle: n = 63) and FVB congenic SOD1H46R transgenic male mice (FVB_H46R M) (open square:
n = 27). Kaplan-Meier analysis with Log-rank (Mantel-Cox) test identifies significant differences in survival for mutant SOD1 transgenic lines between
B6 and FVB backgrounds (p,0.0001).
We generated C57BL/6N (B6) congenic SOD1H46R mice by
crossing original SOD1H46R-tg males (C57BL/66DBA/2)  to
B6 females for more than 16 generations (.N16). Then, B6
congenic SOD1H46R-tg males were backcrossed to FVB/NJcl (FVB)
females for more than 10 generations (.N10), generating FVB
congenic SOD1H46R lines. Congenic SOD1G93A mice on two different
genetic backgrounds were also generated by crossing
B6SJLTgN(SOD1-G93A)1Gur males (C57BL/6J6SJL)  derived from
Jackson Laboratories to either B6 or FVB females for more than 10
generations (.N10). In addition, we newly generated two FVB
congenic lines of double mutants; Als22/2;SOD1H46R and Als22/2;
SOD1G93A. FVB congenic lines of Als2+/2 mice generated by
crossing F2 Als2+/2 mice (129P26B6)  to FVB mice for more
than 10 generations (.N10)  were utilized to produce FVB
congenic Als2+/2;SOD1H46R and Als2+/2;SOD1G93A mice. We
crossed FVB congenic SOD1H46R or SOD1G93A males to FVB
congenic Als2+/2 female mice, generating mice with nine different
genotypes; Als2+/+ (wild-type), Als2+/2, Als22/2, Als2+/+;SOD1H46R,
Als2+/2;SOD1H46R, Als22/2;SOD1H46R, Als2+/+;SOD1G93A, Als2+/2;
SOD1G93A, and Als22/2;SOD1G93A, by crossing male Als2+/2;
SOD1H46R or Als2+/2;SOD1G93A to female Als2+/2 mice. Among
these animals, wild-type, Als2+/+;SOD1H46R, Als22/2;SOD1H46R,
Als2+/+;SOD1G93A, and Als22/2;SOD1G93A mice were used in this
study. Mice were genotyped by PCR using genomic DNA from tail
tissues as described [18,48]. Mice were housed at 2223uC with a
12 hr light/dark cycle. Food and water were fed ad libitum. Body
weight of each animal was weekly monitored. Their lifespan
(endpoint) was determined by the observation that mice were
unable to move by themselves.
Analysis of the copy number of the SOD1 transgene
The copy numbers of the human SOD1 transgene in the mouse
genome was estimated using real time quantitative PCR by
determining the difference in threshold cycle (DCT) between the
human SOD1 transgene and a reference mouse gene (Sod1).
Primers used in this study were as follows: human SOD1; forward
(F): 59-TGCCAGCAGAGTACACAAG-39, reverse (R):
59-ATCAAAGCCCAGTTTTGTGG-39, mouse Sod1; F:
59-GCTCAACAATGCAGCAAGTC-39. The real time PCR was performed in a 20 ml
reaction mixture containing genomic DNA (10 ng), primers mix
(final primer concentration of 500 nM each), and SYBR Green
PCR Master Mix (QuantiFast SYBR Green PCR Kit; Qiagen) in
a MicroAmp 96 well plate (Applied Biosystems) using 7500 Fast
Real-Time PCR System (Applied Biosystems). The thermal
conditions were the initial denaturation at 95uC for 5 min
followed by 40 cycles of 95uC for 10 sec and 60uC for 30 sec.
Preparation protein and total RNA samples
Spinal cord tissues were weighed and homogenized in 2
weightvolume (mg/ml) of phosphate buffer saline (PBS). A fraction (25 ml)
of the homogenates was subjected to RNA extraction, and the
remaining of them was used for protein extraction as described
. In brief, total RNA was extracted from tissue homogenates
using Sepazole RNA I super G (Nacalai Tesque), and purified by
SV Total RNA Isolation System (Promega) according to the
manufacturers instructions. Protein samples were obtained from
the remaining tissue homogenates by lysing with buffer A [25 mM
Tris-HCl (pH 7.5), 50 mM NaCl, 1% (w/v) Triton X-100 (TX),
Complete Protease Inhibitor Cocktail (Roche)], followed by
centrifugation at 23,0006g for 20 min at 4uC. The resultant
supernatant was collected as a TX-soluble protein fraction. Protein
concentration of each fraction was determined by the Micro BCA
or Pierce 660 nm Protein Assay system (Thermo Scientific).
Quantitative reverse transcriptase-PCR
The quantitative reverse transcriptase (qRT)-PCR was
performed on a 0.5 ng of total RNA using QuantiFast SYBR Green
RT-PCR (Qiagen) with specific primers (0.6 mM each) as follows;
human SOD1; F: 59-AGGGCATCATCAATTTCGAG-39, R:
ACATTGCCCAAGTCTCCAAC-39, mouse Actb (b-actin); F:
59-TCTCAGCTGTGGTGGTGAAG-39. The thermal cycling conditions consisted of a
30 min of reverse transcription step at 50uC, 10 min of initial
denaturation at 95uC, followed by 40 cycles of amplification steps
of 95uC for 15 sec and 60uC for 30 sec. The levels of the SOD1
transcripts were analyzed by determining the difference in DCT
between the human SOD1 and the mouse Actb transcripts in each
Antibodies and western blot analysis
Primary antibodies used for western blot analysis included
rabbit polyclonal anti-SOD1 (1:50,000, Santa Cruz, FL-154) and
anti-b-actin (1:1000, Sigma, A-5060) antibodies. Secondary
antibody was horseradish peroxidase (HRP)-conjugated donkey
anti-rabbit IgG (1:5000, Amersham Bioscience). Equal amount of
proteins (12 mg) were electrophoretically separated by a
SDSpolyacrylamide gel (SuperSep Ace, 520% WAKO) and
transferred onto a polyvinylidine difluride membrane (Bio-Rad). The
membranes were blocked with Blocking One (Nacalai Tesque) for
2 hr at room temperature, and incubated with the appropriate
primary antibody in TBST [20 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 0.1% (w/v) Tween 20] containing 5% Blocking One
(Nacalai Tesque). After washing with TBST, membranes were
incubated with HRP-conjugated secondary antibody at room
temperature for 2 hr, followed by a repeated wash with TBST.
Signals were visualized by Immobilon Western (Millipore) and
Xray film (Amersham Bioscience), and were quantified by analyzing
the digitally captured images using CS Analyzer ver3 (ATTO).
Statistical analyses were conducted using PRISM5 (GraphPad).
Statistical significance was evaluated by ANOVA followed by
appropriate post hoc tests for multiple comparisons between groups.
Survival data were compared using Kaplan-Meier survival analysis
with Log-rank (Mantel-Cox) test. A p-value,0.05 was considered
as reaching statistical significance.
We thank Profs. Masashi Aoki (Tohoku University) and Yasuto Itoyama
(National Center of Neurology and Psychiatry) for generously sharing
Conceived and designed the experiments: YI HFS SH. Performed the
experiments: LP YY HO AO SH. Analyzed the data: LP SH. Contributed
reagents/materials/analysis tools: AO SH. Wrote the paper: LP YY AO
Dion PA , Daoud H , Rouleau GA ( 2009 ) Genetics of motor neuron disorders: new insights into pathogenic mechanisms . Nat Rev Genet 10 : 769 - 782 .
Rosen DR , Siddique T , Patterson D , Figlewicz DA , Sapp P , et al. ( 1993 ) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis . Nature 362 : 59 - 62 .
Cudkowicz ME , McKenna-Yasek D , Sapp PE , Chin W , Geller B , et al. ( 1997 ) Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis . Ann Neurol 41 : 210 - 221 .
SOD1H46R transgenic mice, and Prof. Joh-E Ikeda (Tokai University) for his invaluable and generous support .
Kato S ( 2008 ) Amyotrophic lateral sclerosis models and human neuropathology: similarities and differences . Acta Neuropathol 115 : 97 - 114 .
Bosco DA , Morfini G , Karabacak NM , Song Y , Gros-Louis F , et al. ( 2010 ) Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS . Nat Neurosci 13 : 1396 - 1403 .
Haidet-Phillips AM , Hester ME , Miranda CJ , Meyer K , Braun L , et al. ( 2011 ) Astrocytes from familial and sporadic ALS patients are toxic to motor neurons .
Nat Biotechnol 29 : 824 - 828 .
7. Pasinelli P , Brown RH ( 2006 ) Molecular biology of amyotrophic lateral sclerosis: insights from genetics . Nat Rev Neurosci 7 : 710 - 723 .
8. Al-Chalabi A , Andersen PM , Chioza B , Shaw C , Sham PC , et al. ( 1998 ) Recessive amyotrophic lateral sclerosis families with the D90A SOD1 mutation share a common founder: evidence for a linked protective factor . Hum Mol Genet 7 : 2045 - 2050 .
9. Andersen PM , Nilsson P , Keranen ML , Forsgren L , Hagglund J , et al. ( 1997 ) Phenotypic heterogeneity in motor neuron disease patients with CuZnsuperoxide dismutase mutations in Scandinavia . Brain 120(Pt 10 ): 1723 - 1737 .
10. Gurney ME , Pu H , Chiu AY , Dal Canto MC , Polchow CY , et al. ( 1994 ) Motor neuron degeneration in mice that express a human Cu ,Zn superoxide dismutase mutation. Science 264 : 1772 - 1775 .
11. Turner BJ , Talbot K ( 2008 ) Transgenics, toxicity and therapeutics in rodent models of mutant SOD1-mediated familial ALS . Prog Neurobiol 85 : 94 - 134 .
12. Dal Canto MC , Gurney ME ( 1995 ) Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu,Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS) . Brain Res 676 : 25 - 40 .
13. Bruijn LI , Becher MW , Lee MK , Anderson KL , Jenkins NA , et al. ( 1997 ) ALSlinked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions . Neuron 18 : 327 - 338 .
14. Alexander GM , Erwin KL , Byers N , Deitch JS , Augelli BJ , et al. ( 2004 ) Effect of transgene copy number on survival in the G93A SOD1 transgenic mouse model of ALS . Brain Res Mol Brain Res 130 : 7 - 15 .
15. Heiman-Patterson TD , Deitch JS , Blankenhorn EP , Erwin KL , Perreault MJ , et al. ( 2005 ) Background and gender effects on survival in the TgN(SOD1- G93A)1Gur mouse model of ALS . J Neurol Sci 236 : 1 - 7 .
16. Heiman-Patterson TD , Sher RB , Blankenhorn EA , Alexander G , Deitch JS , et al. ( 2011 ) Effect of genetic background on phenotype variability in transgenic mouse models of amyotrophic lateral sclerosis: a window of opportunity in the search for genetic modifiers . Amyotroph Lateral Scler 12 : 79 - 86 .
17. Acevedo-Arozena A , Kalmar B , Essa S , Ricketts T , Joyce P , et al. ( 2011 ) A comprehensive assessment of the SOD1G93A low-copy transgenic mouse, which models human amyotrophic lateral sclerosis . Dis Model Mech 4 : 686 - 700 .
18. Hadano S , Otomo A , Kunita R , Suzuki-Utsunomiya K , Akatsuka A , et al. ( 2010 ) Loss of ALS2/Alsin exacerbates motor dysfunction in a SOD1H46Rexpressing mouse ALS model by disturbing endolysosomal trafficking . PLoS ONE 5 : e9805 .
19. Hadano S , Hand CK , Osuga H , Yanagisawa Y , Otomo A , et al. ( 2001 ) A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2 . Nat Genet 29 : 166 - 173 .
20. Aoki M , Ogasawara M , Matsubara Y , Narisawa K , Nakamura S , et al. ( 1993 ) Mild ALS in Japan associated with novel SOD mutation . Nat Genet 5 : 323 - 324 .
21. Aoki M , Ogasawara M , Matsubara Y , Narisawa K , Nakamura S , et al. ( 1994 ) Familial amyotrophic lateral sclerosis (ALS) in Japan associated with H46R mutation in Cu/Zn superoxide dismutase gene: a possible new subtype of familial ALS . J Neurol Sci 126 : 77 - 83 .
22. Yoshii Y , Otomo A , Pan L , Ohtsuka M , Hadano S ( 2011 ) Loss of glial fibrillary acidic protein marginally accelerates disease progression in a SOD1H46R transgenic mouse model of ALS . Neurosci Res 70 : 321 - 329 .
23. Sasaki S , Nagai M , Aoki M , Komori T , Itoyama Y , et al. ( 2007 ) Motor neuron disease in transgenic mice with an H46R mutant SOD1 gene . J Neuropathol Exp Neurol 66 : 517 - 524 .
24. Boillee S , Yamanaka K , Lobsiger CS , Copeland NG , Jenkins NA , et al. ( 2006 ) Onset and progression in inherited ALS determined by motor neurons and microglia. Science 312 : 1389 - 1392 .
25. Yamanaka K , Chun SJ , Boillee S , Fujimori-Tonou N , Yamashita H , et al. ( 2008 ) Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis . Nat Neurosci 11 : 251 - 253 .
26. Hadano S , Kunita R , Otomo A , Suzuki-Utsunomiya K , Ikeda JE ( 2007 ) Molecular and cellular function of ALS2/alsin: implication of membrane dynamics in neuronal development and degeneration . Neurochem Int 51 : 74 - 84 .
27. Otomo A , Kunita R , Suzuki-Utsunomiya K , Ikeda JE , Hadano S ( 2011 ) Defective relocalization of ALS2/alsin missense mutants to Rac1-induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation . FEBS Lett 585 : 730 - 736 .
28. Hadano S , Yoshii Y , Otomo A , Kunita R , Suzuki-Utsunomiya K , et al. ( 2010 ) Genetic background and gender effects on gross phenotypes in congenic lines of ALS2/alsin-deficient mice . Neurosci Res 68 : 131 - 136 .
29. Traynor BJ , Codd MB , Corr B , Forde C , Frost E , et al. ( 1999 ) Incidence and prevalence of ALS in Ireland, 1995-1997: a population-based study . Neurology 52 : 504 - 509 .
30. Haverkamp LJ , Appel V , Appel SH ( 1995 ) Natural history of amyotrophic lateral sclerosis in a database population . Validation of a scoring system and a model for survival prediction . Brain 118 (Pt 3): 707 - 719 .
31. Kunst CB , Messer L , Gordon J , Haines J , Patterson D ( 2000 ) Genetic mapping of a mouse modifier gene that can prevent ALS onset . Genomics 70 : 181 - 189 .
32. Wooley CM , Sher RB , Kale A , Frankel WN , Cox GA , et al. ( 2005 ) Gait analysis detects early changes in transgenic SOD1(G93A) mice . Muscle Nerve 32 : 43 - 50 .
33. Schauwecker PE , Steward O ( 1997 ) Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches . Proc Natl Acad Sci U S A 94 : 4103 - 4108 .
34. Schauwecker PE ( 2002 ) Modulation of cell death by mouse genotype: differential vulnerability to excitatory amino acid-induced lesions . Exp Neurol 178 : 219 - 235 .
35. Schauwecker PE ( 2005 ) Susceptibility to excitotoxic and metabolic striatal neurodegeneration in the mouse is genotype dependent . Brain Res 1040 : 112 - 120 .
36. Ratovitski T , Corson LB , Strain J , Wong P , Cleveland DW , et al. ( 1999 ) Variation in the biochemical/biophysical properties of mutant superoxide dismutase 1 enzymes and the rate of disease progression in familial amyotrophic lateral sclerosis kindreds . Hum Mol Genet 8 : 1451 - 1460 .
37. Reaume AG , Elliott JL , Hoffman EK , Kowall NW , Ferrante RJ , et al. ( 1996 ) Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury . Nat Genet 13 : 43 - 47 .
38. Strom AL , Shi P , Zhang F , Gal J , Kilty R , et al. ( 2008 ) Interaction of amyotrophic lateral sclerosis (ALS)-related mutant copper-zinc superoxide dismutase with the dynein-dynactin complex contributes to inclusion formation . J Biol Chem 283 : 22795 - 22805 .
39. Bruijn LI , Houseweart MK , Kato S , Anderson KL , Anderson SD , et al. ( 1998 ) Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1 . Science 281 : 1851 - 1854 .
40. Mitchell JD , Borasio GD ( 2007 ) Amyotrophic lateral sclerosis . Lancet 369 : 2031 - 2041 .
41. Strickland D , Smith SA , Dolliff G , Goldman L , Roelofs RI ( 1996 ) Physical activity, trauma, and ALS: a case-control study . Acta Neurol Scand 94 : 45 - 50 .
42. Haley RW ( 2003 ) Excess incidence of ALS in young Gulf War veterans . Neurology 61 : 750 - 756 .
43. Veldink JH , Kalmijn S , Groeneveld GJ , Titulaer MJ , Wokke JH , et al. ( 2005 ) Physical activity and the association with sporadic ALS . Neurology 64 : 241 - 245 .
44. Mahoney DJ , Rodriguez C , Devries M , Yasuda N , Tarnopolsky MA ( 2004 ) Effects of high-intensity endurance exercise training in the G93A mouse model of amyotrophic lateral sclerosis . Muscle Nerve 29 : 656 - 662 .
45. McCrate ME , Kaspar BK ( 2008 ) Physical activity and neuroprotection in amyotrophic lateral sclerosis . Neuromolecular Med 10 : 108 - 117 .
46. Stam NC , Nithianantharajah J , Howard ML , Atkin JD , Cheema SS , et al. ( 2008 ) Sex-specific behavioural effects of environmental enrichment in a transgenic mouse model of amyotrophic lateral sclerosis . Eur J Neurosci 28 : 717 - 723 .
47. Ludolph AC , Bendotti C , Blaugrund E , Chio A , Greensmith L , et al. ( 2010 ) Guidelines for preclinical animal research in ALS/MND: A consensus meeting . Amyotroph Lateral Scler 11 : 38 - 45 .
48. Hadano S , Benn SC , Kakuta S , Otomo A , Sudo K , et al. ( 2006 ) Mice deficient in the Rab5 guanine nucleotide exchange factor ALS2/alsin exhibit agedependent neurological deficits and altered endosome trafficking . Hum Mol Genet 15 : 233 - 250 .