A JOURNAL OF NEUROLOGY
SOD1 targeted to the mitochondrial
intermembrane space prevents motor
neuropathy in the Sod1 knockout mouse
Lindsey R. Fischer,1,* Anissa Igoudjil,2,* Jordi Magrane ´,2Yingjie Li,1Jason M. Hansen,3
Giovanni Manfredi2and Jonathan D. Glass1
1 Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA
2 Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY 10065, USA
3 Department of Paediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
*These authors contributed equally to this work.
Correspondence to: Jonathan D. Glass,
Emory Centre for Neurodegenerative Disease,
101 Woodruff Circle,
Suite 6000, Atlanta,
GA 30322, USA
Motor axon degeneration is a critical but poorly understood event leading to weakness and muscle atrophy in motor neuron
diseases. Here, we investigated oxidative stress-mediated axonal degeneration in mice lacking the antioxidant enzyme, Cu,Zn
superoxide dismutase (SOD1). We demonstrate a progressive motor axonopathy in these mice and show that Sod1?/?primary
motor neurons extend short axons in vitro with reduced mitochondrial density. Sod1?/?neurons also show oxidation of mito-
chondrial—but not cytosolic—thioredoxin, suggesting that loss of SOD1 causes preferential oxidative stress in mitochondria, a
primary source of superoxide in cells. SOD1 is widely regarded as the cytosolic isoform of superoxide dismutase, but is also
found in the mitochondrial intermembrane space. The functional significance of SOD1 in the intermembrane space is unknown.
We used a transgenic approach to express SOD1 exclusively in the intermembrane space and found that mitochondrial SOD1 is
sufficient to prevent biochemical and morphological defects in the Sod1?/?model, and to rescue the motor phenotype of these
mice when followed to 12 months of age. These results suggest that SOD1 in the mitochondrial intermembrane space is
fundamental for motor axon maintenance, and implicate oxidative damage initiated at mitochondrial sites in the pathogenesis
of motor axon degeneration.
Keywords: SOD; axon; neuromuscular junction; motor neuron disease; mitochondria
Abbreviations: O2??= superoxide; SOD1 = Cu,Zn superoxide dismutase
Motor axons are the anatomical and functional link between spinal
motor neurons and skeletal muscles. Degeneration of motor axons
at the neuromuscular junction is an early feature of motor neuron
disease in animal models (reviewed in Fischer and Glass, 2007)
and in humans (Bjornskov et al., 1984; Maselli et al., 1993).
Moreover, axonal degeneration is sufficient to cause weakness
doi:10.1093/brain/awq314 Brain 2011: 134; 196–209 |
Received June 27, 2010. Revised August 18, 2010. Accepted September 11, 2010. Advance Access publication November 14, 2010
? The Author (2010). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
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and muscle atrophy, even in the absence of motor neuron cell
death (Gould et al., 2006; Rouaux et al., 2007; Suzuki et al.,
2007). Axon degeneration is therefore a key pathologic event
and therapeutic target in motor neuron disease, although the
pathogenic mechanisms that initiate axon degeneration are
Evidence from Cu,Zn superoxide dismutase (SOD1) knockout
mice suggests that this antioxidant enzyme is essential for motor
axon maintenance. Sod1?/?mice develop normally and lack
behavioural deficits up to 6 months of age (Reaume et al.,
1996), but ageing Sod1?/?mice perform poorly on the Rotarod
and exhibit accelerated skeletal muscle atrophy (Muller et al.,
2006). Spinal motor neuron and ventral root axon numbers are
normal at 17 and 19 months, respectively (Flood et al., 1999;
Shefner et al., 1999), but muscle fibre atrophy and fibre-type
grouping, suggestive of chronic denervation and reinnervation,
are detectable by 6 months (Flood et al., 1999). EMG recordings
also show spontaneous activity and progressive loss of motor units
(Shefner et al., 1999). Thus, genetic deletion of SOD1 spares
motor neurons and proximal axons, but may be detrimental to
distal motor axons.
SOD1 is one of three superoxide dismutases in mammalian cells
that catalyse the conversion of superoxide (O2??) to H2O2
(McCord and Fridovich, 1969). SOD1 is traditionally considered
to be the cytoplasmic isoform, SOD2 the mitochondrial isoform
and SOD3 the secreted form (Zelko et al., 2002). However, SOD1
is also found in the mitochondrial intermembrane space (Weisiger
and Fridovich, 1973; Sturtz
Vijayvergiya et al., 2005). The functional significance of SOD1
in the intermembrane space remains to be defined, although a
protective role is likely. Mitochondria isolated from Sod1?/?
mouse muscle (Muller et al., 2007; Jang et al., 2010) and
Sod1?/?Caenorhabditis elegans (Yanase et al., 2009) exhibit
increased generation of reactive oxygen species including O2??
and H2O2. Similarly, antisense knockdown of SOD1 in vitro
causes preferential oxidation of mitochondrial—not cytosolic—
proteins, loss of mitochondrial membrane potential and decreased
ATP production (Aquilano et al., 2006). Thus, loss of SOD1 may
result in an accumulation of mitochondrial reactive oxygen species,
leading to oxidative damage and mitochondrial dysfunction.
Here, we demonstrate that targeted replacement of SOD1 only
in the mitochondrial intermembrane space rescues motor axon
outgrowth and normalizes the mitochondrial redox state in
Sod1?/?neurons in vitro, and prevents weakness and neuro-
muscular junction denervation in Sod1?/?mice followed up to
12 months of age. These data suggest that mitochondrial oxida-
tive stress is an underlying cause of distal motor axonopathy, and
demonstrate that localization of SOD1 in the intermembrane
space is sufficient for the survival of motor axons.
et al., 2001; In ˜arrea,2002;
Materials and methods
Sod1?/?mice, generated by Huang and colleagues (1997), were
obtained from Marie Csete (Emory University) (Muller et al., 2006).
Sod1?/?males were crossed with thy1-YFP16 females (Feng et al.,
2000) to obtain Sod1+/?, thy1-YFP16 breeders, expressing yellow
fluorescent protein (YFP) in all motor axons. Both transgenic lines
were in a C57BL/6 background. For ease of description, thy1-YFP16
is omitted from the genotype and expression should be assumed. Mice
were housed in microisolator cages on a 12h light–dark cycle and
given free access to food and water. Genotyping was by standard
polymerase chain reaction analysis on tail-snip DNA. YFP status was
determined by fluorescent examination of epidermal nerve fibres in ear
To generate mitoSOD1 transgenic mice, human SOD1 complemen-
tary DNA was inserted in a prion promoter vector (MoPrP.Xho)
(Borchelt et al., 1996) at the XhoI site, between PrP exons 2 and 3.
The start codon of human SOD1 was removed and substituted by an
in-frame DNA linker of 6 nt, containing a BamHI site and coding for
Gly–Ser. We then inserted a 561nt complementary DNA encoding the
first 187 amino acids of mouse mitofilin (GenBank accession code:
NM_029673) (John et al., 2005) in frame with the 50-end of the
human SOD1 plus linker. The mitofilin complementary DNA was ob-
tained by polymerase chain reaction amplification of a mouse comple-
mentary DNA library using primer sequences derived from the mouse
The plasmid was microinjected into fertilized eggs of B6CBF1 mice.
Offspring harbouring the transgene were identified by polymerase
chain reaction using the following primers: CCGCTCGAGATGCTGCG
GGCGTGTCAG (sense) and CCGCTCGAGTTATTGGGCGATCCCAAT
(antisense) to generate a 1027bp product. Five lines of mitoSOD1
transgenic mice were identified. Male founders were crossed with
B6SJLF1/J females (Jackson).
A two-step mating scheme was used to generate mice expressing
only mitoSOD1. Sod1?/?males were crossed with mitoSOD1 females
to generate F1 heterozygotes (mitoSOD1,Sod1+/?). Sod1+/?and
mitoSOD1,Sod1+/?mice were then crossed to generate the target
genotype (mitoSOD1,Sod1?/?) and littermate controls.
Neuromuscular junction morphology
Tibialis anterior muscle was dissected, pinned in mild stretch and
immersion fixed for 30min in 4% paraformaldehyde at room tempera-
ture. Fixed muscles were cryoprotected in 30% sucrose/phosphate-
buffered saline (48–72h at 4?C), flash frozen in supercooled isopentane
and 35mm frozen sections were cut longitudinally through the entire
muscle. Acetylcholine receptors at the motor endplate were labelled
with Alexa Fluor 555-conjugated a-bungarotoxin (Invitrogen), 1:5000
in phosphate-buffered saline (30min, room temperature). Motor axon
terminals were identified by YFP fluorescence. Innervated, intermediate
and denervated endplates were defined by complete, partial or absent
overlap between nerve terminal and endplate, respectively. All endplates
were evaluated in every fourth section. No difference was seen between
innervation in male and female mice, therefore the data were pooled.
Mitochondrial isolation from mouse
Tissues (brain and spinal cord) were homogenized in a Dounce hom-
ogenizer in MS-EGTA buffer (225mM mannitol, 75mM sucrose,
5mM HEPES, 1mM EGTA, pH 7.4), and centrifuged at 2000g for
5min (4?C). The supernatant was centrifuged at 15000g for 20min to
generate the cytosolic fraction (supernatant) and crude mitochondrial
fraction (pellet). Thecytosolic
(22000g for 20min) to eliminate membrane contamination. The crude
fraction wascentrifuged twice
Mitochondrial SOD1 protects motor axons Brain 2011: 134; 196–209 |
mitochondrial fraction was resuspended and washed twice in MS-EGTA.
To prepare purified mitochondria, the crude mitochondrial pellet
was layered onto 9ml of 23% Percoll in MS-EGTA and centrifuged
at 25000g for 11min. The pellet was resuspended in MS-EGTA
and centrifuged three times at 14000g for 14min. The final purified
mitochondrial pellet was resuspended in MS-EGTA at ?10mg/ml. All
reagents were from Sigma.
Western blot analyses
The expression and mitochondrial localization of mitoSOD1 were
tested by western blot of crude mitochondria prepared from spinal
cord. Proteins in whole homogenates (50mg), cytosol (20mg) and
mitochondria (20mg) were separated on a 12% sodium dodecyl
sulphate polyacrylamide gel, transferred to polyvinylidene difluoride
membranes (BioRad) and immunoblotted with sheep anti-SOD1
(1:5000, Calbiochem), mouse anti-Tim23 (1:5000, Stressgen), goat
Blue native gel electrophoresis
Spinal cord mitochondrial proteins (50mg) were separated on a
10–16% gradient blue native gel as previously described (Schagger
and von Jagow, 1991). After electrophoresis, proteins were transferred
to polyvinylidene difluoride and immunoblotted for SOD1 and core II
subunit of complex III (mouse anti-CIII, 1:2500, Molecular Probes). A
high molecular weight ladder (Amersham) was used to estimate
Alkaline extraction of mitochondrial
Alkaline extraction of mitochondrial proteins was performed as
described (Vijayvergiya et al., 2005). Briefly, mitochondria (50mg)
were incubated in the presence or absence of 0.1M Na2CO3
(pH 11.5), with or without Triton X-100 (1%), for 30min at 4?C,
then centrifuged at 91000g for 25min. The pellet was saved.
Supernatant proteins were precipitated with ice-cold 12% trichloro-
acetic acid and centrifuged at 18000g for 15min, followed by an
ice-cold acetone wash. Pellet and precipitated supernatant proteins
were analysed by western blot as described above.
Mitoplasts were prepared as previously described (Acin-Perez et al.,
2009). Briefly, purified mitochondria (300mg) were suspended in
MS-EGTA, water (1/10 volume) and digitonin (1mg digitonin/5mg
mitochondrial protein), and the mixture was incubated on ice for
45min. KCl (150mM) was then added, followed by incubation for
2min on ice and centrifugation at 18000g for 20min. The mitoplast
pellet was resuspended to 0.5mg/ml in MS-EGTA. The supernatant
containing the post-mitoplast fraction was subjected to trichloroacetic
acid precipitation as described above.
Proteinase K treatment of mitoplasts
Proteinase K treatment of mitoplasts was performed as previously
described (Vijayvergiya et al., 2005). Briefly, mitoplasts (25mg) were
incubated in the presence or absence of proteinase K (20mg/ml), with
or without Triton X-100 (0.1%), for 1h on ice. Proteolysis was
stopped by adding 2mM phenylmethylsulphonyl fluoride.
SOD1 activity: spectrophotometric
SOD1 activity was assessed as previously described (Vives-Bauza et al.,
2007) with minor modifications. The assay measures the reduction of
acetylated cytochrome c by O2??generated by xanthine/xanthine oxi-
dase. All reagents were from Sigma except for xanthine oxidase
(Calbiochem). Spinal cord mitochondria (50mg) and cytosolic fractions
(10mg) were incubated in 1ml of reaction buffer (50mM phosphate
buffer, pH 7.8, 0.1mM EDTA, 1mM NaN3, 100mM xanthine, 2U
xanthine oxidase and 25mM partially acetylated cytochrome c).
Cytochrome c reduction was followed spectrophotometrically at
550nm, at 25?C for 3min. The activity of SOD1 (KCN-insensitive)
was determined by adding 2mM KCN, and subtracting residual activ-
ity from total SOD activity. One unit of SOD activity was defined as
the amount of enzyme required to inhibit the rate of reduction of
cytochrome c by 50%. Note that in measuring SOD1 activity in mito-
chondria it is necessary to minimize the interference associated with
the interaction of cytochrome c with cytochrome c oxidase and cyto-
chrome c reductases, using partially acetylated cytochrome c as
described (Azzi et al., 1975; Kuthan et al., 1986). Nevertheless, we
found some residual cytochrome c reduction in the mitochondria of
Sod1?/?samples (Fig. 5E). This can be explained by the fact that the
preparation of acetylated cytochrome c actually contains up to 40%
non-acetylated cytochrome c, which can react with mitochondrial
oxidases and reductases.
Primary motor neuron culture
Spinal motor neurons were enriched from E12.5 mouse embryos by
density centrifugation (Zhang et al., 2006). Spinal cords were isolated,
incubated in 0.05% trypsin (Worthington, 37?, 10min) and dissociated
by pipetting up and down in neurobasal medium (Invitrogen) contain-
ing 0.1% trypsin inhibitor (Sigma), 100U/ml DNase (Worthington)
and 0.4% bovine serum albumin. Cells were centrifuged through
4% bovine serum albumin (400g, 5min), resuspended and centrifuged
through 10% (v/v) Optiprep (Axis Shield, 700g, 10min). The interface
was aspirated, centrifuged through a second bovine serum albumin
cushion, and resuspended in growth medium consisting of neurobasal
with 2% B27 supplement minus antioxidants (Invitrogen), 2% horse
serum (Invitrogen), 0.5 mM Glutamax (Invitrogen), BDNF, CNTF,
GDNF and NT-3 (10ng/ml, Peprotech). Cells were plated on coverslips
coated with polyornithine (10mg/ml, Sigma) and Matrigel (1:25, BD
Bioscience). By this method, 84.7 ? 4.8% of cells at 24h were immu-
noreactive for the motor neuron marker, Hb9 (1:1000, Abcam).
Individual spinal cords were kept separate during cell isolation and
genotype subsequently determined by polymerase chain reaction.
Axon length was determined by systematic random sampling of cells
along a pre-marked grid. Motor neurons were identified morpho-
logically under phase contrast and photographed at ?20 on an
Olympus IK51 inverted microscope using an Olympus Qcolor3 digital
camera. Axons were manually traced and measured using ImageJ soft-
ware (http://rsb.info.nih.gov/ij/). In cells with multiple processes, the
axon was considered to be the longest process. The average number
of neurons analysed in each of four trials was 130 ? 41 (Sod1+/+),
158 ? 34 (Sod1+/?) and 163 ? 19 (Sod1?/?), or 500–600 neurons
per group (n=4 was used for statistical analysis).
Brain 2011: 134; 196–209L. R. Fischer et al.
Mitochondrial density in axons was evaluated using Mitotracker Red
CM-H2XRos (Invitrogen). Dye was added to cells at 250nM in
serum-free medium (30min, 37?C). Cells were returned to growth
medium for 10min and fixed in pre-warmed 4% paraformaldehyde
(15min, room temperature). Coverslips were inverted onto slides
using anti-fade mounting medium (Vectashield+DAPI, Vector Labs),
and cells were examined under standard fluorescence microscopy.
In some experiments, Mitotracker staining was followed by immuno-
labelling with an antibody specific for human SOD1 (Sigma, 1:500).
Twenty-five neurons per coverslip were selected and photographed
at ?40 magnification by unbiased coverslip scanning. Mitochondrial
density was evaluated morphologically using ImageJ software. Lengths
of individual mitochondria were measured, combined and divided by
the length of the axon. At least three replicates per genotype were
obtained over the course of multiple motor neuron preparations.
Primary cortical neuron cultures
Primary cortical neurons were isolated from E15.5 mouse embryos.
Brains were removed in ice-cold dissecting buffer (2.5mM HEPES,
4mM NaHCO3and 35mM glucose in Hanks’ balanced salt solution)
and cortices were dissected free of subcortical structures and meninges
and incubated in 0.25% Trypsin/EDTA (Sigma), for 15min at 37?C.
Tissue was dissociated in dissection buffer containing 0.1mg/ml tryp-
sin inhibitor (Sigma) and 200U/ml DNase (Worthington). Cells were
pelleted by centrifugation at 218g for 5min and resuspended in
growth medium consisting of neurobasal (Invitrogen), 5% foetal
bovine serum (Atlanta Biologicals), 200mM GlutaMAX (Invitrogen)
and 2% B27 minus antioxidants (Invitrogen). Cells were plated in
poly-L-lysine coated six-well plates at a density of 2?106cells/well.
Individual brains were kept separate during cell isolation and genotype
subsequently determined by polymerase chain reaction on tail-
Thioredoxin redox western analysis
The redox state of thioredoxin-1 (cytosolic) and thioredoxin-2 (mito-
chondrial) was determined by redox western analysis (Halvey et al.,
2005). Primary cortical neurons were washed once with ice-cold
phosphate-buffered saline, incubated in 10% trichloroacetic acid at
4?C for 20min, and removed with a cell scraper. Following centrifu-
gation at 16000g for 2min, the protein pellet was washed in 100%
acetone and resuspended by sonication in derivatization buffer:
acid) (Invitrogen) in 100mM Tris, pH 7.6, 1% sodium dodecyl sul-
phate. Derivatization was allowed to proceed for 1h at room tempera-
ture. Samples were diluted 1:1 in non-reducing sodium dodecyl
sulphate sample buffer (Bio-Rad), boiled and separated on two 15%
polyacrylamide gels run in parallel. Immunoblotting was carried out by
standard methods using goat anti-thioredoxin-1 (1:2500, American
Diagnostica,Stamford, CT, USA)
(1:7500, Invitrogen). Membrane scanning and band densitometry
were performed on an Odyssey infrared imaging system (Li-COR)
using Odyssey software. The redox potential Eh(in mV) was calculated
using the Nernst equation: Eh=E0+(2.303 RT/nF)?log(ox/red),
where E0=?256mV (thioredoxin-1) or ?330mV (thioredoxin-2).
Note that protein levels were not equalized between samples. Only
relative changes between reduced and oxidized bands are of quanti-
Images were captured on a Zeiss LSM 510 NLO META system,
coupled to a Zeiss Axiovert 100M inverted microscope. Neuromuscular
junction z-stacks were obtained with a Plan-Neofluar ?40 (NA 1.3) oil
objective with optical slice thickness of 1mm. Motor neurons were
imaged with a Plan-Neofluar ?20 (NA 0.3) objective or Plan-Neofluar
?100 (NA 1.3) oil objective. Z-stacks were compressed and images
exported using LSM Image Examiner software (Zeiss).
Results are expressed as mean ? SD, and comparisons were made by
ANOVA with Tukey post hoc analysis (a=0.05), unless otherwise spe-
cified, using Prism software (GraphPad).
Loss of SOD1 causes a progressive
Previous studies ofthe Sod1?/?mouseprovide compelling, although
indirect, evidence for a motor axonopathy (Flood et al., 1999;
Shefner et al., 1999). To facilitate morphological analysis of neuro-
muscular junctions, we first crossed Sod1?/?mice (Muller et al.,
2006) with thy1-YFP16 mice (Feng et al., 2000) to generate
mice expressing YFP in all motor axons. Absence of SOD1 protein
and enzymatic activity was verified in Sod1?/?, thy1-YFP16 off-
spring by western blot and SOD1 zymography (Supplementary
We observed progressive hind-limb weakness in Sod1?/?mice,
evidenced by significant loss of grip strength by 12 months
(Fig. 1C). Whereas most Sod1+/+
remain suspended from a wire grid for at least 300s, the mean
latency to fall for Sod1?/?animals was 134s (P 5 0.001).
Sod1?/?mice had obvious difficulty gripping the wire with their
hind limbs, and hind limb grip was typically lost first, followed by
forelimb grip (Supplementary Videos 1–4).
To determine how loss of SOD1 affects distal motor axons,
we examined neuromuscular junction morphology in the tibialis
anterior muscle, which undergoes significant (?50%) atrophy in
20-month-old Sod1?/?mice (Muller et al., 2006). Neuromuscular
junction innervation at 1, 4, 12 and 18 months of age was eval-
uated by the overlap between YFP-positive motor axon terminals
and motor endplates labelled with Alexa Fluor 555-conjugated
a-bungarotoxin (Fig. 1A and B). At 1 month, the tibialis anterior
muscle was fully innervated in Sod1?/?mice. At 4 months,
69.5 ? 6.2% of endplates were innervated in Sod1?/?mice, com-
pared to 97.1 ? 0.9% and 99.6 ? 0.3% in Sod1+/+and Sod1+/?
mice, respectively (P 5 0.001). By 18 months, only 33.9 ? 8.5%
of Sod1?/?endplates were innervated compared to 92.2 ? 2.8%
in Sod1+/+mice and 79.9 ? 15.5% in Sod1+/?mice (P 5 0.001).
This demonstrates that SOD1 is required for maintenance of motor
Mitochondrial SOD1 protects motor axonsBrain 2011: 134; 196–209 |
axons in vivo, and suggests that distal motor axons are sensitive to
oxidative stress-mediated injury.
Axon defects in primary motor neuron
To examine axonal defects intrinsic to Sod1?/?motor neurons, we
cultured primary motor neurons from E12.5 mice in medium free
of standard antioxidant supplements (Fig. 2A). Sod1?/?motor
neurons were short-lived compared with controls. No viable
Sod1?/?cells remained at 72h in culture. At 24h, Sod1?/?
motor neurons had markedly shorter axons compared with con-
trols (Fig. 2C). Mean axon length in Sod1?/?cells (?SEM) was
50?3mm at 24h, compared with 93?5 and 83?3mm for
Sod1+/+and Sod1+/?cells, respectively (P50.001).
Given a previous report of mitochondrial damage in Sod1?/?
cells (Aquilano et al., 2006), we tested whether mitochondrial
density was altered in Sod1?/?axons (Fig. 2B). At 24h in culture,
mitochondria were labelled with Mitotracker Red-CM-H2XRos,
the motor neurons were fixed and individual mitochondria along
the length of the axon were measured. Mitochondrial density
was expressed as the cumulative length of all mitochondria
in the axon, divided by the total length of the axon (Fig. 2D).
Figure 1 Genetic deletion of SOD1 causes progressive denervation at the neuromuscular junction. (A) Confocal projections of tibialis
anterior neuromuscular junctions at 1, 4, 12 and 18 months of age, with motor axons in green (yellow fluorescent protein) and endplates
in red (bungarotoxin). Examples of innervated (1), intermediate (2) and denervated (3) endplates are shown. By 18 months, many
endplates are vacant and show a fragmented morphology consistent with chronic denervation. Fragmentation was not observed in
innervated endplates. Scale bar=20mm. (B) Percent innervated, intermediate and denervated endplates in tibialis anterior. Endplates
(898 ? 203) were assessed per muscle. (**P 5 0.01, ***P 5 0.001 for Sod1?/?versus Sod1+/+; ANOVA with Tukey post hoc test;
n=3–7 animals per group). (C) Latency to fall (s) on grip strength test at 12 months of age. The best performance out of three trials, to a
maximum of 300s, was recorded for each animal (***P 5 0.001; ANOVA with Tukey post hoc test; n=6–9 animals per group).
Brain 2011: 134; 196–209L. R. Fischer et al.