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The Journal of Neuroscience, 2002, 22:RC215:1-6
RAPID COMMUNICATION
Mutant Cu, Zn Superoxide Dismutase that Causes Motoneuron
Degeneration Is Present in Mitochondria in the CNS
Cynthia M. J.
Higgins1,
Cheolwha
Jung1,
Hongliu
Ding1, and
Zuoshang
Xu1, 2, 3
1 Department of Biochemistry and Molecular
Pharmacology, 2 Cell Biology, and
3 Neuroscience Program, University of Massachusetts Medical
School, Worcester, Massachusetts 01655
 |
ABSTRACT |
Mutations in Cu, Zn superoxide dismutase (SOD1) cause a fraction of
amyotrophic lateral sclerosis (ALS), which involves motoneuron degeneration, paralysis, and death. An acquired activity by mutant SOD1
is responsible for the cellular toxicity, but how mutant SOD1 kills
motoneurons is unclear. In transgenic mouse models of ALS,
mitochondrial degeneration occurs early, before disease onset, raising
the question of how mutant SOD1 damages mitochondria. Here we
investigate the intracellular localization of SOD1 in the CNS to
determine whether SOD1 is present in mitochondria, where it could
directly damage this organelle. We show that endogenous mouse SOD1,
wild-type human, and mutant human SOD1 (G93A), when expressed as
transgenes, are colocalized with mitochondria in spinal cord by
immunofluorescence confocal microscopy. By immunoelectron microscopy,
we show that SOD1 is present within mitochondria at similar
concentrations as in the cytoplasm. Thus SOD1, in addition to being a
cytosolic enzyme, is present inside mitochondria in the CNS.
Key words:
motor neuron disease; neurodegenerative disease; neurodegeneration; spinal cord; ALS; aging
| |
INTRODUCTION |
Superoxide
dismutase (SOD1) is a 17 kDa protein, known to be present in
cytoplasm as a homodimer (Fridovich, 1986 ). Its function is superoxide
dismutation. Mutations in SOD1 cause a fraction of amyotrophic lateral
sclerosis (ALS), a fatal neurodegenerative disease in which motoneurons
degenerate, resulting in paralysis and death (Brown, 1995). Several
experiments have demonstrated that mutant SOD1 causes motoneuron
degeneration by gaining a toxic property, rather than a loss of
superoxide dismutation function (Dal Canto and Gurney, 1995; Ripps et
al., 1995; Wong et al., 1995). There are two major hypotheses regarding
the toxic properties gained by the mutant SOD1. The first suggests that
mutant SOD1 damages cells by producing peroxynitrite (Estevez et al.,
1999), whereas the second proposes that mutant SOD1 damages cells by its enhanced peroxidase activity (Wiedau-Pazos et al., 1996; Yim et
al., 1996, 1997). The validity of either hypothesis remains to be
proven (Xu, 2000).
An important question in understanding the mechanism of motoneuron
degeneration is what the cellular target for this toxicity is, or what
is the cellular pathway that leads eventually to cell death. Previous
studies have observed mitochondrial vacuolation in transgenic mice
expressing mutant SOD1 (Dal Canto and Gurney, 1995; Wong et al., 1995),
and the peak of this vacuolation correlates with the onset of clinical
disease (Kong and Xu, 1998). Furthermore, widespread mitochondrial
abnormalities are present before the peak vacuolation and clinical
disease (Kong and Xu, 1998). The evidence suggests that mitochondrial
damage is an early step in the mutant SOD1-induced motoneuron
degeneration pathway, raising the question of how mutant SOD1 damages mitochondria.
SOD1 has been thought to be a cytoplasmic protein (Crapo et al., 1992).
Some studies, however, have suggested the presence of a small fraction
of the total cellular SOD1 in the intermembrane space of liver
mitochondria (Weisiger and Fridovich, 1973; Tyler, 1975), although this
has been controversial (Geller and Winge, 1982). In light of the new
evidence that mitochondria are damaged in mice expressing mutant SOD1
and develop ALS, we revisited this issue and investigated whether
mutant SOD1 is associated with mitochondria in the CNS. We present
double immunofluorescence and ultrastructural evidence that a fraction
of endogenous mouse SOD1 and transgenic wild-type and mutant human SOD1
in the mouse is present within mitochondria in the CNS. Thus, mutant
SOD1 could exert its toxicity directly on mitochondria, leading to
mitochondrial damage and onset of motoneuron degeneration.
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MATERIALS AND METHODS |
Transgenic mice. Two transgenic mouse lines (The
Jackson Laboratory, Bar Harbor, ME) were used. One expresses human SOD1
mutant G93A
(C57BL/6J-TgN(SOD1-G93A)1Gurdl) (G93A
mice), and the other expresses wild-type human SOD1 (WS mice) (Gurney
et al., 1994). Nontransgenic littermates of G93A transgenic mice were
used as wild-type controls (WT mice). All transgenic mice were
identified using PCR according to Gurney et al. (1994). Mice were
maintained at the University of Massachusetts Medical School animal
facility according to the guidelines set forth by the Institutional
Animal Care and Use Committee.
Double immunofluorescence. Mice were killed by decapitation
under anesthesia, and spinal cords were dissected quickly. Lumbar and
cervical spinal cords were cut out, frozen immediately in OCT freezing
media (Sakura, Torrance, CA) on dry ice, and stored at  80°C. Eight
micrometer sections were cut using a cryostat and mounted on slides
treated with Vectabond (Vector Laboratories, Burlingame, CA). The
slides were incubated in PBS at room temperature for 30 min, followed
by fixation with 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.5, for 10 min. After washing, the sections were
doubly stained with anti-SOD1 (Pardo et al., 1995) and anti-cytochrome C oxidase subunit 1 (COX1) antibodies (Molecular Probes, Eugene, OR)
according the protocol described previously (Kong et al., 1998; Levine
et al., 1999). The stained sections were examined and digitized using a
confocal microscope (TCS-SP; Leica, Mannheim, Germany). Imaging
analysis and three-dimensional reconstruction were conducted using
MetaMorph (Universal Imaging Corporation, West Chester, PA).
Immunoelectron microscopy. Mice (60- to 80-d-old) were
perfused under anesthesia with fixative (4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH
7.5). Lumbar spinal cords from fixed mice were dissected out and
dehydrated through an ethanol series to 100% ethanol and embedded in
hard grade LR White resin (Electron Microscopy Sciences, Fort
Washington, PA) by polymerization overnight at 60°C. Sixty-five
nanometer sections were cut from the embedded tissue using a
Reichert-Jung Ultracut E microtome and collected onto gold grids (200 mesh) (SPI Inc., Westchester, PA). Sections on grids were processed for
immunogold electron microscopy according to recommendations by the
manufacturer of the gold-conjugated antibodies (Amersham Biosciences,
Piscataway, NJ) with some modification. Sections were etched in 0.1N
HCl for 5 min, rinsed three times for 5 min each in TBS (in
mM: 25 Tris, 140 NaCl, and 2.7 KCl, pH 8.0),
placed in blocking buffer for 30 min (0.1% gelatin, 1% normal goat
serum, and 0.3% Triton-X-100 in TBS), placed in primary antibody for 2 hr at room temperature, rinsed three times for 5 min each in TBS,
placed in gold-conjugated secondary antibodies (10 nm gold anti-mouse
and 5 nm gold anti-rabbit) for 1 hr, rinsed three times in TBS, fixed
in 2% glutaraldehyde, rinsed three times in TBS, rinsed in water,
stained with Reynold's lead citrate followed by aqueous 2% uranyl
acetate, and dried on filter paper. The following antibodies and sera
were used: normal rabbit serum (Vector Laboratories), rabbit polyclonal
anti-SOD1 (Pardo et al., 1995), a second rabbit polyclonal anti-SOD1
(Biodesign, Saco, ME), mouse monoclonals against cytochrome oxidase
subunit 1 (COX1) and against COX subunit 4 (Molecular Probes), and
rabbit polyclonal anti-neurofilament light chain (Xu et al., 1993). All antibodies and serum were used at a 1:10 dilution.
The stained sections were viewed and photographed using a Philips CM10
transmission electron microscope. The negatives were digitized, and
immunogold density was determined using MetaMorph software as follows.
The number of gold particles were counted inside and outside
mitochondria and then normalized to the areas of mitochondria and
nonmitochondria, respectively. Myelin and extracellular areas are
excluded from the measurement. However, it is not practical to exclude
all other cytoplasmic organelles because the identity and the boundary
of these organelles are often not unequivocal. Therefore, organelles
other than mitochondria are included as nonmitochondrial area. Results
were summarized as the average per mitochondrion and average per
extramitochondrial space in a micrograph.
| |
RESULTS |
To determine whether SOD1 is associated with mitochondria, we
examined whether SOD1 was present in mitochondrial fractions from mouse
brain homogenates. SOD1 was detected in the mitochondrial fractions
from WT mice and in increasing amounts from mice expressing wild-type
human SOD1 (WS) and mice expressing mutant SOD1 (G93A) (data not
shown). This result gave us preliminary evidence that SOD1 was
associated with mitochondria in the CNS as was shown for liver
(Weisiger and Fridovich, 1973; Tyler, 1975). As in the previous reports
using liver, it was impossible to purify mitochondria using biochemical
fractionation to the extent that other organelles could be eliminated
as sources of SOD1 (Geller and Winge, 1982). Therefore, we decided to
resolve this issue using morphological means.
Double immunofluorescence staining was performed on spinal cord
sections from G93A mice using anti-SOD1 and anti-COX1 (mitochondrial marker) antibodies. Because of the high concentration of cytoplasmic SOD1 (which generates a high SOD1 staining background), it was difficult to demonstrate colocalization of SOD1 and COX1 signals in
fixed mouse spinal cords. To reduce the level of extraorganelle cytosolic SOD1, unfixed frozen spinal cord sections from G93A mice were
incubated in PBS for 30 min to allow unassociated SOD1 to diffuse away.
Sections were then fixed with 4% paraformaldehyde and stained using
routine double immunofluorescent staining method.
Widespread overlapping signals from mitochondria and SOD1 were observed
(Fig. 1A) (notice the
abundant yellow structures in the right panel; some examples are
pointed out by arrows). All COX1-immunoreactive structures
overlap with SOD1-positive structures, although many SOD1-positive
structures are negative for COX1 (Fig. 1A). Because
mitochondria are small, it was possible that overlaps seen in the XY
dimensions were not real in the Z dimension. To rule out this
possibility, serial sections were recorded using a confocal microscope
and reconstructed in three dimensions. In all cases, the overlaps
between the COX1 and SOD1 signals were confirmed (Fig.
1B,C) (also see supplemental videos at
www.jneurosci.org). The same results were obtained in
digitonin-permeablized cultured pheochromocytoma cell line and
HeLa cells when their endogenous SOD1 was detected by double
SOD1 and COX1 immunofluorescence (data not shown).
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Figure 1.
Double immunofluorescent staining of spinal cord
sections from G93A mice using anti-COX1 (green)
and SOD1 (red) antibodies. A,
Low-magnification view of an area covering part of the ventral column
motor axon exit zone. The panel on the
right shows the superimposition of COX1 and SOD1
staining. B, C, High-magnification view of two small
groups of mitochondria. The three panels on the
right illustrate the same mitochondria as shown in the
left panels except that they are rotated 40 and 90° in
B and C, respectively. The
numbers mark the same mitochondria viewed from the two
different angles. The bottom two panels are
superimposition of the top two green and red
panels (for a complete three-dimensional view, see the
supplemental videos at www.jneurosci.org).
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To determine whether the SOD1 is inside or attached to the outside of
mitochondria, SOD1 was further localized at the ultrastructural level
using immunoelectron microscopy (Fig. 2).
Lumbar spinal cord sections from the three different mice (G93A, WS,
and WT) were stained by immunogold for SOD1 (small particles) and
mitochondrial enzyme cytochrome oxidase (large particles). Mitochondria
were identified by their characteristic structure and by their positive staining for cytochrome oxidase (Fig. 2A,C,E, large
particles). All mitochondria are positively stained for SOD1 (Fig.
2A,C,E, small particles). Far more small particles
are present in G93A and WS spinal cords than those in the WT spinal
cord, consistent with overexpression of SOD1 in the transgenic animals.
Very few, if any, small particles are present in the matrix (Fig.
2A,C, asterisks), indicating that SOD1 is unlikely to
be in the matrix compartment. Sections incubated with a second
anti-SOD1 antibody (Biodesign) yielded the same staining pattern (data
not shown). In control staining in which an anti-neurofilament subunit
NF-L antibody was substituted for the anti-SOD1 antibody, small
particles are largely confined to the cytoplasm and are segregated from the large particles that reside inside mitochondria (Fig.
2B,D,F). In two additional controls in which
the sections were incubated with nonimmune rabbit serum followed by the
secondary antibody or the sections were incubated with the secondary
antibody alone, only sparse background particles were occasionally
detected (data not shown).
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Figure 2.
Immunogold localization of SOD1 in the spinal cord
from G93A (A, B), WS (C, D), and WT
(E, F) mice. A, C, E, Sections
reacted with rabbit polyclonal anti-SOD1 (5 nm particles) and mouse
monoclonals against COX subunit 1 and 4 (10 nm particles). B, D,
F, Sections incubated with rabbit polyclonal anti-neurofilament
light chain (5 nm particles) and mouse anti-COX1 and 4 (10 nm
particles). Scale bars, 100 nm. Each asterisk indicates
significantly less than the concentration within mitochondria
(p = 0.02 using two-tailed Student's t
test).
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To estimate the relative concentration of SOD1 inside mitochondria when
compared with that in the intracellular space outside of mitochondria,
we counted the small gold particles inside and outside mitochondria.
These counts confirm that the gold particle densities representing SOD1
in WS and G93A mice are several fold higher than those in the WT mice
(Fig. 3). The density of SOD1 in
mitochondria is slightly higher inside mitochondria than that in the
extramitochondrial cytosolic space, although this difference is only
significant in G93A mice (Fig. 3) (p = 0.02 using two-tailed Student's t test). In contrast to a high
density of SOD1 both inside and outside of mitochondria, staining
with a rabbit polyclonal antibody against neurofilament light chain
shows a high density only outside of mitochondria and essentially no
counts of gold particles within mitochondria (Fig. 3).
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Figure 3.
Density of immunogold particles staining for SOD1
(using the antibody from Biodesign) in mitochondrial and
extramitochondrial cytosolic areas in the spinal cord of WT, WS, or
G93A mice. The number of 5 nm gold spots within individual mitochondria
(mito) and in the cytosolic area outside mitochondria
(non-mito) in a micrograph were counted and divided by
the mitochondrial area and cytosolic area outside mitochondria,
respectively (see Materials and Methods). A, SOD1
density. Five electron micrographs were counted for each genotype. The
asterisk indicates that the concentration of G93A SOD1
outside of mitochondria is significantly less than the concentration in
mitochondria (two-tailed Student's t test).
B, Neurofilament light chain density. Three electron
micrographs were counted for each genotype.
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DISCUSSION |
This study presents immunofluorescence and ultrastructural
evidence that a fraction of wild-type and mutated SOD1 is localized within mitochondria in the CNS. First, double immunofluorescence and
three-dimensional reconstruction reveal that all mitochondria are
colocalized with SOD1 signals in the spinal cord of mutant SOD1
transgenic mice (Fig. 1). Second, abundant SOD1 signals are detected
within the mitochondria by immuno-EM (Fig. 2). In addition, quantification of immunogold staining against SOD1 reveals that the
gold particle density inside mitochondria is similar to that in the
cytosol outside of mitochondria, suggesting that SOD1 concentration inside mitochondria is at comparable levels with the cytosol.
The presence of SOD1 in mitochondria was reported by several previous
studies, which suggested that SOD1 is in the intermembrane space
(Weisiger and Fridovich, 1973; Peeters-Joris et al., 1975; Tyler, 1975;
Henry et al., 1980). However, doubts were raised because a later study
found that SOD1 in mitochondrial fractions could be accounted for by
contamination with lysosomes (Geller and Winge, 1982). Further doubts
were raised when some laboratories reported the presence of SOD1 in
peroxisomes (Keller et al., 1991; Dhaunsi et al., 1992; Wanders and
Denis, 1992). These controversies are derived from the difficulty in
making pure mitochondria completely devoid of contamination by other
cellular organelles. Our current approach circumvented this difficulty
by double immunofluorescence and immunogold staining on prefixed CNS
spinal cords and cultured cells. The results demonstrate that a
fraction of SOD1 is inside mitochondria in the CNS.
In conjunction with previous data showing that mitochondria are an
early target for damage by mutant SOD1 in transgenic mice (Dal Canto
and Gurney, 1995; Wong et al., 1995; Kong and Xu, 1998), these results
raise the possibility that mutant SOD1 might damage mitochondria
directly. This is supported by morphological observation in human ALS,
which showed abundant abnormal mitochondria (Hirano et al., 1984;
Nakano and Hirano, 1987). Although not all transgenic mouse models for
ALS (e.g., G85R) (Bruijn et al., 1997) exhibit morphologically
abnormal mitochondria, functional abnormalities cannot be ruled out.
Indeed, mutant SOD1, when introduced into cells, causes mitochondrial
dysfunction (Carri et al., 1997; Kruman et al., 1999). Using cybrid
technology, Swerdlow et al. (1998) demonstrated mitochondrial
dysfunction in platelet cells isolated from humans with sporadic ALS.
The efficacy of creatine treatment in slowing the disease progression
in mice expressing mutant SOD1 further supports that mitochondrial
damage plays a role in mutant SOD1-induced motoneuron degeneration
(Klivenyi et al., 1999).
Our results agree with recently published data demonstrating that SOD1
is in the intermembrane space of yeast mitochondria (Sturtz et al.
2001) and a report showing that mutant SOD1 was associated with
vacuolated mitochondria in the CNS of ALS transgenic mice (Jaarsma et
al., 2001). Our data show that endogenous SOD1 in wild-type mice is in
morphologically normal mitochondria, and that both normal and
ALS-causing mutant human SOD1 in transgenic mice are in mitochondria,
probably at concentrations similar to that in the cytoplasm. Although
our antibody does not distinguish between mouse and human SOD1 (it
detects both), it is unlikely that normal and mutant SOD1 are
segregated. This is because when either is overexpressed, signal
intensity is proportionally increased in both the mitochondrial and
cytoplasmic compartments (Fig. 3).
Mitochondria are known to be a major, and probably the most
predominant, source of oxidative free radicals in cells, and thus, may
provide a fertile environment for the effects of the mutant SOD1
toxicity. For instance, if either of the two major hypotheses regarding
the nature of the mutant SOD1 toxicity (see introductory remarks) is
correct, mitochondria could provide abundant substrates for the mutant
SOD1 to further generate toxic free radicals, which in turn damage
mitochondria from within. Some possible downstream effects have been
reported in literature, including impairment in energy metabolism
(Hatazawa et al., 1988), elevated oxidative stress (Beal, 1998; Hall et
al., 1998), increased sensitivity to excitotoxicity (Ikonomidou and
Turski, 1996; Rothstein, 1996; Bittigau and Ikonomidou, 1997),
deficient axonal transport (Cleveland, 1999), and deregulation of
apoptosis (Kostic et al., 1997; Li et al., 2000). Of particular
interest is a recent observation that the vulnerability to
excitotoxicity in motoneurons appears to be selectively enhanced when
mitochondrial function is impaired (Kaal et al., 2000). Taken together,
our results indicate that mitochondrial damage is an important area of
investigation in the causes of ALS.
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FOOTNOTES |
Received Dec. 17, 2001; revised Dec. 17, 2001; accepted Jan. 9, 2002.
This work is supported by grants from the Amyotrophic Lateral Sclerosis
Association, National Institute of Neurological Disorders and Stroke
(NINDS) Grant RO1 NS35750, and Worcester Foundation for Biomedical
Research (Z.S.X.). C.M.J.H. is supported by National Institutes of
Health Training Grant 5 T32 NS07366-05. The EM work was performed with
the support of the Core Electron Microscopy Facility of the University
of Massachusetts Medical School. We thank Dr. Gregory Hendricks for his
expert advice with electron microscopy and Charlene Baron for
assistance with image production. The contents of this report are
solely the responsibility of the authors and do not necessarily
represent the official views of NINDS. We thank Wenli Ding and Laura
Fenton for maintaining transgenic colonies and Ninfa Gatha for
technical assistance.
Correspondence should be addressed to Zuoshang Xu, Department of
Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail: Zuoshang.Xu{at}umassmed.edu.
This article is published in
The Journal of Neuroscience, Rapid Communications Section,
which publishes brief, peer-reviewed papers online, not in print. Rapid
Communications are posted online approximately one month earlier than
they would appear if printed. They are listed in the Table of Contents
of the next open issue of JNeurosci. Cite this article as:
JNeurosci, 2002, 22:RC215 (1-6). The
publication date is the date of posting online at
www.jneurosci.org.
| |
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ABSTRACT
INTRODUCTION
