Cisd2 deficiency drives premature aging
and causes mitochondria-mediated defects
Yi-Fan Chen,1Cheng-Heng Kao,2Ya-Ting Chen,1,3Chih-Hao Wang,4Chia-Yu Wu,1Ching-Yen Tsai,1
Fu-Chin Liu,5Chu-Wen Yang,6Yau-Huei Wei,4Ming-Ta Hsu,4Shih-Feng Tsai,1,3and Ting-Fen Tsai1,3,7
1Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan;2Center of
General Education, Chang Gung University, Taoyuan 333, Taiwan;3Division of Molecular and Genomic Medicine, National
Health Research Institutes, Zhunan, Miaoli County 350, Taiwan;4Institute of Biochemistry and Molecular Biology, National
Yang-Ming University, Taipei 112, Taiwan;5Institute of Neuroscience, National Yang-Ming University, Taipei 112, Taiwan;
6Department of Microbiology, Soochow University, Taipei 111, Taiwan;
CISD2, the causative gene for Wolfram syndrome 2 (WFS2), is a previously uncharacterized novel gene.
Significantly, the CISD2 gene is located on human chromosome 4q, where a genetic component for longevity
maps. Here we show for the first time that CISD2 is involved in mammalian life-span control. Cisd2 deficiency in
mice causes mitochondrial breakdown and dysfunction accompanied by autophagic cell death, and these events
precede the two earliest manifestations of nerve and muscle degeneration; together, they lead to a panel of
phenotypic features suggestive of premature aging. Our study also reveals that Cisd2 is primarily localized in the
mitochondria and that mitochondrial degeneration appears to have a direct phenotypic consequence that triggers
the accelerated aging process in Cisd2 knockout mice; furthermore, mitochondrial degeneration exacerbates with
age, and the autophagy increases in parallel to the development of the premature aging phenotype. Additionally,
our Cisd2 knockout mouse work provides strong evidence supporting an earlier clinical hypothesis that WFS is in
part a mitochondria-mediated disorder; specifically, we propose that mutation of CISD2 causes the mitochondria-
mediated disorder WFS2 in humans. Thus, this mutant mouse provides an animal model for mechanistic
investigation of Cisd2 protein function and help with a pathophysiological understanding of WFS2.
[Keywords: Cisd2; Wolfram syndrome 2; autophagy; knockout mice; mitochondria; premature aging]
Supplemental material is available at http://www.genesdev.org.
Received January 8, 2009; revised version accepted March 31, 2009.
CISD2 is the second member of the gene family contain-
ing the CDGSH iron sulfur domain. There are currently
three members in this gene family: CISD1 (synonyms
ZCD1, mitoNEET), CISD2 (synonyms ZCD2, Noxp70,
and Miner1) and CISD3 (synonym Miner2). CISD1 is an
outer mitochondrial membrane protein that was origi-
nally identified as a target protein of the insulin sensitizer
drug pioglitazone used to treat type 2 diabetes (Colca
et al. 2004). CISD1 protein contains a transmembrane
domain, a CDGSH domain, and a conserved amino acid
sequence for iron binding; biochemical experiments sug-
gest that CISD1 is involved in the control of respiratory
rates and regulates oxidative capacity (Wiley et al. 2007).
However, CISD2 and CISD3 are novel genes with pre-
viously uncharacterized functions. The only molecular
documentation for CISD2 is that CISD2 was one of the
markers for early neuronal differentiation in a cell culture
study (Boucquey et al. 2006).
Recently, the CISD2 gene has been identified as the
second causative gene (Amr et al. 2007) associated with
Wolfram syndrome (WFS; MIM 222300), which is an
autosomal recessive neurodegenerative disorder. WFS is
highly variable in its clinical manifestations, which in-
clude diabetes insipidus, diabetes mellitus, optic atrophy,
and deafness; thus, it is also known as the ‘‘DIDMOAD
syndrome’’ (Barrett and Bundey 1997). Positional cloning
and mutation studies have revealed that WFS is a genet-
ically heterogeneous disease with a complex molecular
basis involving more than one causative gene in humans
(Domenech et al. 2006). A portion of WFS patients
belonging to the WFS1 group (MIM 606201) carried loss-
of-function mutations in the WFS1 (wolframin) gene,
which encodes a transmembrane protein primarily local-
ized in the endoplasmic reticulum (ER) (Inoue et al. 1998;
Strom et al.1998; Takeda et al. 2001). In addition to this,
a homozygous mutation of the CISD2 gene has been
identified in three consanguineous families with WFS
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1779509.
GENES & DEVELOPMENT 23:1183–1194 ? 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org 1183
(Amr et al. 2007), and these patients have been classified
as WFS2 (MIM 604928). However, the function of the
CISD2 protein in these patients and in all other organ-
isms remains unknown, and its physiological role has not
Significantly, the CISD2 gene is located within the
region on human chromosome 4q where a genetic com-
ponent for human longevity has been mapped. Previ-
ously, Puca et al. (2001) studied 137 sets of extremely old
siblings (308 individuals in all) and conducted a genome-
wide scan search for predisposing loci that might confer
longevity; this linkage study revealed a single region on
chromosome 4q and suggests that there may be at least
one master gene contributing to life-span control; how-
ever, the responsible gene has not been identified.
In this study, we apply a mouse genetics approach
and demonstrate that Cisd2 is involved in mammalian
life-span control and plays an essential role in mitochon-
drial integrity. Cisd2 deficiency causes mitochondria-
mediated phenotypic defects in mice. Furthermore, cell
culture and biochemical investigations revealed that
Cisd2 is a mitochondrial protein. Additionally, Cisd2
knockout mice exhibit many clinical manifestations of
WFS patients including early-onset degeneration of cen-
mature death, as well as impaired glucose tolerance. This
study therefore provides an animal model for mechanistic
understanding of WFS, specifically WFS2, pathogenesis.
Shortened life span in Cisd2?/?mice
CISD2 is an evolutionarily conserved gene localized on
human chromosome 4q24 (Supplemental Table 1; Sup-
plemental Figs. 1, 2); the mouse syntenic region is on
chromosome 3G3. Northern blot analysis showed that
Cisd2 is a widely expressed gene in mice. Interestingly,
quantitative real-time RT–PCR revealed that expression
levels of Cisd2 decrease in an age-dependent manner in
naturally aged mice (Supplemental Fig. 3). To study the
role of Cisd2 involvement in development and patho-
physiology, we generated Cisd2 knockout mice. Southern
and Northern blot analyses demonstrated that the Cisd2
expression in the homozygous knockout (Cisd2?/?) mice
(Supplemental Fig. 4). Growth retardation and a smaller
somatotype are clearly evident; it appears that there is
almost no growth after 5 wk old in theCisd2?/?mice (Fig.
1A). Early senescence is accompanied by a shortened life
span when survival of the various genotypes is examined
and there appears to be signs of haploinsufficiency for
Cisd2 in view of the slightly lower survival rate for the
heterozygous (Cisd2+/–) mice (Fig. 1B).
Premature aging phenotype
Starting at 8 wk old, Cisd2?/?mice begin to acquire a set
of aged appearance phenomena remarkably similar to
those of premature aging syndrome (Hasty et al. 2003;
Kipling et al. 2004). These include prominent eyes and
protruding ears (Fig. 1C). Ocular abnormalities were ob-
served as the Cisd2?/?mice developed opaque eyes and
blindness, which was accompanied by cornea damage at
20 wk old (Fig. 1D). Histopathological examination re-
vealed that the opacity of the cornea was due to debris
deposition in the scar tissue outside the cornea (Fig. 1E).
In addition, corneal neovascularization was observed in
the Cisd2?/?mice; this can impair vision and is usually
associated with pathogenesis due to eye trauma or the
presence of a degenerative disorder (Supplemental Fig. 5).
There was also early depigmentation in the fur at ~48 wk
old (Fig. 1F; Supplemental Fig. 6); furthermore, hair fol-
licle atrophy and a decreased hair density could be de-
tected in Cisd2?/?mice (Fig. 1G,H). A decrease in the hair
regrowth rate was also observed in the Cisd2?/?mice
(Supplemental Fig. 7A,B). Additionally, the skin of 48-
wk-old Cisd2?/?mice exhibits a phenotype with a no-
ticeably thickened dermis, an expanded surface, and
a significant decrease in subcutaneous adipose tissue
and muscle (Fig. 1I–K).
showed that the trabeculae of the femur are noticeably
thinner in Cisd2?/?mice (Fig. 2A). Dual energy X-ray
absorpitometer (DEXA) detected a decrease in femur
density after 8 wk old; interestingly, the decrease of
femur density also started to emerge in heterozygous
Cisd2+/?mice, but at 24 wk old, while a progressively
more severe phenotype was observed at the same age
with Cisd2?/?mice (Fig. 2B). This shows, in addition to
what was observed in terms of life-span evaluation, that
there is also an apparent Cisd2 haploinsufficiency with
respect to femur density. The results from the gross
anatomy viewpoint, from the X-ray radiography, and
using micro-CT reveal a significant lordokyphosis phe-
notype after 12 wk old (Fig. 2C,D; Supplemental Fig.
7C,D); consequently, this seems to lead to a decrease in
mean thoracic volume (Fig. 2E) and thence pulmonary
function abnormalities. Indeed, we observed decreases in
various respiratory parameters as measured by plethys-
mography after 20 wk old in the Cisd2?/?mice (Supple-
mental Fig. 8). Muscle degeneration was detectable at 3
wk old in the Cisd2?/?mice. There was a progres-
sive degeneration of muscle fibers and the magnitude of
the degeneration exacerbated with age (Fig. 2F–J); mus-
cle degeneration was further confirmed by transmis-
sion electron microscopy (TEM) (Supplemental Fig. 9).
In addition, angular fibers, which are an indicator of
muscle atrophy caused by neuron degeneration, could
be observed in the Cisd2?/?mice (Fig. 2H).
One possible mechanism for the accelerated aging
phenotypes is a defect in cellular proliferation in the
Cisd2?/?mice. To test this possibility, we created several
primary mouse embryonic fibroblast (MEF) cell lines
from individual embryos with different genotypes. Our
results revealed no significant difference in the doubling
time and MEF cell growth (Supplemental Fig. 10), sug-
gesting that accelerated aging in the Cisd2?/?mice is not
due to an intrinsic defect in cellular proliferation.
A summary of theaging-relatedphenotypesin Cisd2?/?
mice is provided in Supplemental Table 2. These mutant
Chen et al.
1184GENES & DEVELOPMENT
mice exhibit a premature aging phenotype with 100%
penetrance for both sexes using either a C57BL/6 (B6) or
a 129Sv/B6 mixed background.
Mitochondrial degeneration and autophagy
The observation of prematureaging phenotypesinvolving
muscle degeneration prompted a detailed examination of
the tissue ultrastructure of the homozygous knockout
mice. A TEM study revealed that mitochondrial degen-
eration occurs in the axons of sciatic nerves, brain cells
(Fig. 3A–C), cardiac muscle cells, and skeletal muscle
cells (Fig. 3D–F) in the Cisd2?/?mice. Notably, the
mitochondrial outer membrane (OM) appeared to have
broken down prior to the destruction of the inner cristae
(Fig. 3B,E). In wild-type mice, the myelinated axons are
enveloped with a myelin sheath formed by the fusion of
many layers of plasma membrane from Schwann cells
(Fig. 3G). However, considerable disintegration of the
myelin sheath and degeneration of axon was detected in
the Cisd2?/?sciatic nerves (Fig. 3H,I). Importantly, these
mitochondrial abnormalities, involving destruction of
mitochondria, myelin sheath disintegration, and axonal
lesions, are already present to a certain extent in 2-wk-old
Cisd2?/?mice (Fig. 3J–L; Supplemental Fig. 11), a stage
prior to the first premature aging phenotype of muscle
and nerve degeneration in these mice. Interestingly,
the damaged mitochondria appear to induce autophagy
to eliminate the dysfunctional organelles (Kim et al.
2007) because we identified morphologically distinct
autophagic vacuoles (Eskelinen 2008) in muscle, sciatic
nerve, optic nerve, and brain tissue (Fig. 3J–L; Supple-
mental Fig. 12). The general term autophagic vacuole
refers to an autophagosome, amphisome, or autolyso-
some. Morphologically, autophagic vacuoles can be clas-
sified into two categories: (1) early or initial autophagic
vacuoles (AVis)—i.e., autophagosomes, which are double-
membraned structurescontaining undigestedcytoplasmic
material or organelles; (2) late or degradative autophagic
vacuoles (AVds), including amphisomes and autolyso-
somes, which contain partially degraded cytoplasmic
material (Eskelinen 2008; Fader and Colombo 2009).
Remarkably, mitochondrial degeneration exacerbates
with age, and the magnitude of the autophagy increases
in parallel to the development of premature aging pheno-
type (Fig. 3M,N). We measured the thickness of myelin
span, and the ocular and cutaneous symptoms of aging in
Cisd2?/?mice. (A) Growth curves of the different
genotypes. (B) Decreased survival rate of the Cisd2?/?
mice. (C) The prominent eyes and protruding ears of the
Cisd2?/?mice. (D) The Cisd2?/?mice go blind at ~6 mo
old. (E) The opacity of cornea was analyzed by histolog-
ical examination. Masson’s trichrome staining indicated
debris deposition in the scar tissue outside the cornea. (F)
Early depigmentation and gray hair are present on top of
the head and on the shoulders. The representative photo
was taken from a 12-mo-old Cisd2?/?female. (G) Hair
follicle atrophy in the Cisd2?/?mice was demonstrated
by Masson’s trichrome staining. (H) A decreased density
of hair follicles containing hair in the Cisd2?/?mice was
detected compared with wild-type skin. (I,J) Histological
analyses of the skin from 12-mo-old wild-type and
Cisd2?/?mice, respectively. The Cisd2?/?skin exhibits
a phenotype involving a hyperplastic epidermis, hair
follicle atrophy, a decrease in subcutaneous fat and
muscle, and an increased thickness of the dermis layer.
(K) Quantification of the subcutaneous muscle tissue,
adipose tissue, and dermis for the histological sections of
the wild-type and Cisd2?/?skins. (*) P < 0.05 was
considered statistically significant.
The decreased body weight, the shortened life
Cisd2 deficiency drives premature aging
GENES & DEVELOPMENT 1185
sheaths and counted the numbers of myelinated axons in
sciatic nerve; our results revealed no significant differ-
ences between different genotypes (Supplemental Fig. 13),
indicating that these two factors are not involved in the
nerve degeneration of Cisd2?/?mice. We also examined
the autophagosome marker LC3-II (Kabeya et al. 2000) in
skeletal and cardiac muscles, which are the most sensi-
tive tissues toin vivoautophagic degradation(Mizushima
et al. 2004); indeed, the ratio of LC3-II/LC3-I was signif-
icantly higher in Cisd2?/?mice than in their wild-type
littermates. This biochemical evidence confirms the
TEM results and provides a quantitative basis for the
autophagy induction (Fig. 3O,P).
Autophagy can lead to cell death by directly activating
autophagic (type II programmed) cell death that produces
self-degradation of the dying cells (Shimizu et al. 2004;
Mizushima et al. 2008); alternatively, autophagy might
cause cell death through activation of apoptosis (Scott
et al. 2007). To determine whether there is an increased
level of apoptosis in the Cisd2?/?mice, we performed
TUNEL assays on various mouse tissues to detect apo-
ptotic cells in situ and found no evidence of increased
apoptosis in Cisd2?/?mice (Supplemental Fig. 14). In
addition, it has been reported that starvation can induce
muscle autophagy (Mizushima et al. 2004). To test this
possibility, we measured the metabolic indices including
intake of foodand water andgeneration of urine andstool.
Our results revealed no significant difference in these
metabolic indices between Cisd2?/?and wild-type mice
at 6 wk old (Supplemental Fig. 15A); this is 4 wk after the
detection of autophagic activation at 2 wk old. This
excludes starvation/malnutrition as the cause of auto-
phagic induction in Cisd2?/?mice. A decrease in the
metabolic index becomes evident after 12 wk old (Sup-
plemental Fig. 15B), and this is likely to be a consequence
of the aging phenotype.
Cisd2 is probably a mitochondrial OM protein
The annotated characteristics of Cisd2 protein are very
similar to Cisd1, which is an outer mitochondrial mem-
brane protein (Supplemental Fig. 16A; Wiley et al. 2007).
To address the subcellular localization, we expressed the
EGFP-tagged Cisd2 protein in NIH3T3 cells. Our result
indicated that Cisd2 was colocalized with the mitochon-
drial marker (Supplemental Fig. 16B). However, deletion
of the N-terminal 58 amino acids completely abolished
the mitochondrial localization; furthermore, when the
N-terminal 58 amino acids were fused to EGFP, this
construct was able to redirect EGFP from a nuclear and
cytoplasmic localization to the mitochondria (Fig. 4A),
suggesting that Cisd2 is a nucleus-encoded mitochondrial
protein and its N-terminal 58 amino acids are both
necessary and sufficient to direct mitochondrial localiza-
tion. To confirm the subcellular localization of the Cisd2
protein, the cytosolic and mitochondrial fractions were
prepared from skeletal muscle of wild-type mice. Anti-
bodies against Cisd1 and Cisd2 were generated. Western
blot analysis revealed that Cisd2 protein, like the mito-
chondrial proteins Cisd1 and Hsp60 (Samali et al. 1999;
thoracic volume in the Cisd2?/?mice. (A) Micro-CT imaging of
observed in the Cisd2?/?mouse. (B) Osteopenia was analyzed by
Cisd2?/?mice. At 24wk old, a decrease in femur densityalso was
detected with the Cisd2+/?mice and, at 24 wk old, the phenotype
mouse displays a lordokyphosis (curvature of the spinal column)
phenotype. (D) Micro-CT scanning allowed three-dimensional
reconstruction ofthe thoracic and spinal column. (E) Lordokypho-
siswas evident and had led toa decreaseinthoracic volumeinthe
Cisd2?/?mice compared with their wild-type littermates. (F,G)
and 28-mo-old wild-type mice.(H,I) Muscle degeneration of 4-wk-
transverse sections of the skeletal muscle. Black arrows indicate
degenerated transverse fibers that are present in the Cisd2?/?and
also in spontaneously aged mice. The blue arrow indicates an
angular fiber, which is an indicator of muscle atrophy caused by
neuron degeneration. (J) Quantification of the degenerating fibers
in the skeletal muscles. (*) P < 0.05; (**) P < 0.005.
Abnormalities of skeleton and muscle and decrease of
Chen et al.
1186 GENES & DEVELOPMENT
induction in the muscles and neurons of the Cisd2?/?
mice. (A) Wild-type mitochondria in the brain (hippo-
campus). (B) A Cisd2?/?mitochondrion in the brain
(hippocampus). Note that the outer mitochondrial mem-
brane has broken down (arrowhead), while the inner
cristae appear to be intact. (C) Cisd2?/?mitochondria
in sciatic nerve. One mitochondrion (arrowhead) has
a destroyed OM, but with cristae still visible; the other
mitochondrion (arrow) has destroyed OMs and IMs. (D)
Wild-type mitochondria in cardiac muscle. (E) Cisd2?/?
mitochondria in cardiac muscle. This micrograph shows
one mitochondrion (arrowhead) with a destroyed OM
and two degenerated mitochondria consisting of debris
(arrows). (F) A cluster of autophagic vacuoles and abnor-
mal mitochondria was observed between the myofibrils
of Cisd2?/?skeletal muscle (white arrows). (G) A wild-
type myelinated axon of the sciatic nerve. (N) Nucleus of
Schwann cell; (MS) myelin sheath. (H) A myelinated
axon of sciatic nerve from a Cisd2?/?mouse. An ovoid
with a disintegrating myelin sheath and a degenerating
axonal component are shown. (I) Debris from an axon
undergoing degeneration in the Cisd2?/?sciatic nerve.
(J–L) Early or AVis enclosing mitochondria (arrows) and
late or AVds were detected in the axonal component and
cytoplasm of a Schwann cell from a 2-wk-old Cisd2?/?
sciatic nerve. (M,N) Percentage of myelinated axons
present in the sciatic nerves showing disintegration of
their myelin sheaths and autophagic vacuoles, including
AVi and AVd, in their axonal component. There were
three mice for each group. (O) Western blotting to
detected the presence of the proteins LC3-I and LC3-II.
(P) Ratios of the LC3-II to LC3-I. There were three mice
for each group. (*) P < 0.05; (**) P < 0.005. Mouse age in
A–I is 4 wk old.
Mitochondrial degeneration and autophagy
Cisd2 deficiency drives premature aging
GENES & DEVELOPMENT 1187
Colca et al. 2004; Wiley et al. 2007), is primarily localized
in the mitochondrial fraction (Fig. 4B). To further define
the submitochondrial localization of Cisd2, we separated
mouse liver mitochondria into the following fractions:
OM, mitoplasts (MP, inner membrane [IM] and matrix),
and intermembrane space (IMS, soluble material between
the IM and OM). Immunoblotting each fraction with
antibodies against Cisd2 and known markers revealed
that Cisd2 was highly enriched in the OM fraction, as was
the OM marker VDAC-1; this result strongly suggests
that Cisd2 is a mitochondrial OM protein (Fig. 4C).
Previously, Amr et al. (2007) reported that the Flag-
tagged CISD2 protein colocalized with the ER marker
calnexin in the transfected mouse P19 and human
HEK293 cells. We sought to determine if there is a small
portion of the Cisd2 protein sorted into the ER/sarcoplas-
mic reticulum (SR) using subcellular fractions prepared
from skeletal muscles of wild-type mice. Our data indeed
revealed a weak signal indicating the presence of Cisd2
protein in the post-mitochondrial supernatant, and this
colocalized with the ER markers in the microsomal
fractions. The ratio of the Cisd2 protein present in the
mitochondria versus ER was estimated to be about 5.8:1
(Supplemental Fig. 17).
Mitochondria are the cellular energy factories that gen-
erate ATP via oxidative phosphorylation. To investigate
outer mitochondrial membrane, and Cisd2 de-
ficiency leads to mitochondrial dysfunction. (A)
EGFP-tagged Cisd2 protein is directed to the
mitochondria by an N-terminal signal sequence.
The EGFP-Cisd2 proteins were expressed in
NIH3T3 cells. EGFP-tagged full-length Cisd2 pro-
tein was colocalized with MitoTracker Red,
whereas deletion of the N-terminal 58 amino
acids completely abolished mitochondria locali-
zation. When the N-terminal 58-amino-acid se-
quence was fused to EGFP, this construct was
able to redirect EGFP from a nuclear and cyto-
plasmic localization to the mitochondria. (B) Sub-
cellular localization of the Cisd2 and Cisd1
proteins analyzed by Western blotting using pro-
tein extracts of the mitochondrial (Mito) and
cytosolic (Cyto) fractions prepared from skeletal
muscles of 12-wk-old mice. Polyclonal antibody
(Ab) against Cisd2 protein (15 kDa) was gener-
ated; this antibody cross-reacts with Cisd1 pro-
tein (12 kDa). Antibodies against mitochondrial
proteins Cisd1 and Hsp60 were used as controls.
(C) Ten micrograms of each submitochondrial
fraction prepared from the livers of 4-wk-old mice
were analyzed by Western blot using antibodies
against Cisd2 and known mitochondrial marker
proteins. OM marker: (VDAC-1) voltage-depen-
dent anion channel-1; IM marker: (ATP5B) com-
plex V b subunit; matrix marker: (PDH) pyruvate
dehydrogenase. (MP) Microplast (IM and matrix);
(IMS) intermembrane space. (D) Impaired mito-
chondrial respiration in the skeletal muscle of
4-wk-old Cisd2?/?mice. Representative oxy-
graphs of the mitochondria after adding first
glutamate-malate and then ADP into the closed
chamber of the oxygen meter. (E) Respiratory
activity was expressed as oxygen consumption
rate (nanomoles of O2per minute per milligram
of mitochondria) in the resting state, for gluta-
mate-malate supported respiration, and for ADP
activated respiration. A significant decrease in ox-
ygen consumption was detected in the Cisd2?/?
mitochondrial samples (n = 4) compared with wild-type samples (n = 3). (F) The RCR (O2consumption rate after ADP addition/O2
consumption rate after glutamate-malate addition) was significantly lower in the Cisd2?/?mitochondria. (G) Comparison of electron
transport activities of the respiratory enzyme complexes of mitochondria prepared from the skeletal muscles of 4-wk-old Cisd2?/?(n =
4) and wild-type mice (n = 4). (NCCR activity) Measurement of NCCR activity, which represents complexes I–III; (SCCR activity)
measurement of SCCR activity, which represents complexes II and III; (CCO activity) cCCO activity, which represents complex IV.
(*) P < 0.05; (**) P < 0.005.
Cisd2 is primarily localized in the
Chen et al.
1188GENES & DEVELOPMENT
whether the mitochondrial degeneration detected in this
study has a direct functional consequence leading to
a respiratory dysfunction, we assessed mitochondrial
aerobic respiration using isolated mitochondria prepared
from skeletal muscle. This was done by measuring the
oxygen consumption after stimulating the mitochondria
with glutamate-malate and ADP to activate the respira-
tory chain reactions. Our results revealed a significant
decrease in the oxygen consumption and the respiratory
control ratio (RCR) in the Cisd2?/?mitochondria (Fig.
4D–F). To further expand this investigation, we explored
the iron-sulfur proteins, which are essential electron
carriers in the mitochondrial respiratory chain; there
are up to 12 different iron-sulfur clusters that shuttle
electrons through complex I–III (Rouault and Tong 2008).
We measured the activities of the various iron-sulfur
proteins of complex I–III (NADH cytochrome c reduc-
tase, NCCR) and complex II–III (succinate cytochrome c
reductase, SCCR). In addition, we also measured the
activity of complex IV (cytochrome c oxidase, CCO),
which contains hemes and copper centers for electron
transport (Rouault and Tong 2008). Our results showed
that there was an average 30% decrease in the electron
transport activities of complex I–III, complex II–III, and
complex IV in the Cisd2?/?mitochondria compared with
wild-type mitochondria (Fig. 4G). Together with the
oxygen consumption experiment, these results reveal
a respiratory dysfunction in the Cisd2?/?mitochondria.
To test whether an increased level of reactive oxygen
species (ROS), which is a by-product of mitochondrial
oxidative phosphorylation, may contribute to the pheno-
types of Cisd2?/?mice, we monitored the intracellular
ROS, mainly H2O2, in MEF cells and primary cells
obtained from the brains and livers of different genotypes
of mice. There was no significant difference in the ROS
levels in these primary cells between the different geno-
types (Supplemental Fig. 18A). In addition, the mRNA
levels of the enzymes that scavenge ROS were unaffected
in brain, heart, liver, and skeletal muscle, suggesting that
there was no ROS-induced stress response present in the
Cisd2?/?mice (Supplemental Fig. 18B).
WFS and Cisd2?/?mice
In order to evaluate the usefulness of Cisd2?/?mice as an
animal model for WFS2 and gain insight into the mech-
anistic basis of WFS pathogenesis, we compared the
clinical manifestations of this disease and the phenotype
of Cisd2?/?mice. WFS is a clinically heterogeneous
disease; only juvenile-onset diabetes mellitus and optic
atrophy are necessary criteria for WFS diagnosis. Impor-
tantly, Cisd2?/?mice exhibit a progressive neurodegen-
erative phenotype that includes optic nerve defects (Fig.
5A,B; Supplemental Fig. 12). Regarding glucose homeo-
stasis, we found that Cisd2?/?mice display a milder
phenotype, namely, impaired glucose tolerance and de-
creased insulin secretion, which was revealed by the oral
glucose tolerance test (Fig. 5C,D). In addition, insulin
tolerance tests did not show insulin resistance in the
Cisd2?/?mice; in fact, these mutant mice were some-
what more sensitive to insulin (Fig. 5E). Furthermore,
immunohistochemistry (IHC) staining of the pancreatic
islets revealed no obvious difference in insulin expression
within the b cells between Cisd2?/?and wild-type mice
(Fig. 5F). Taken together, these results indicate impaired
glucose homeostasis in the Cisd2?/?mice, which seems
to have an insulin secretory defect rather than insulin
tolerance in Cisd2?/?mice. (A) A representative TEM micro-
graph showing a late or AVd detected in the axonal component
of a myelinated axon of the optic nerve in 24-wk-old Cisd2?/?
mice. The white arrow indicates a disintegrating myelinated
axon. (B) Percentage of myelinated axons of the optic nerves
containing autophagic vacuoles, including AVi and AVd, in the
axonal component. There were three mice for each group; (wk)
week. (C,D) Blood glucose levels and plasma insulin levels,
respectively, before (0 min) and after the glucose load at the
indicated time points. Oral glucose (1.5 g/kg body weight)
tolerance tests were performed on 12-wk-old Cisd2?/?and
wild-type mice, all of which had a C57BL/6 genetic background.
Blood samples were collected to determine the mice’s blood
glucose levels and plasma insulin levels. (E) Insulin (0.75 U/kg
body weight) tolerance tests were performed on 12-wk-old
Cisd2?/?and wild-type mice. There were three mice in each
group, and three independent measurements were carried out on
each mouse. (*) P < 0.05; (**) P < 0.005. (F) IHC staining of
insulin in the b cells of pancreatic islets using tissue sections
prepared from 12-wk-old Cisd2?/?and wild-type mice.
Optic nerve degeneration and impaired glucose
Cisd2 deficiency drives premature aging
GENES & DEVELOPMENT1189
resistance. The importance of mitochondrial dysfunction
in b-cell insulin secretion defects has been previously
confirmed in other mouse models, which demonstrated
that mitochondrial ATP production is a critical part of
the b-cell signaling system and allows insulin release
(Wallace 2001; Torraco et al. 2009). However, there was
no overt diabetes observed in the Cisd2?/?mice with the
C57BL/6 congenic background. This is consistent with
a previous observation that C57BL/6 background confers
a more diabetes-resistant phenotype (Coleman 1992); a
similar finding of a genetic background effect also had
been reported for WFS1 (wolframin) knockout mice
(Ishihara et al. 2004). In addition to optic atrophy and
glucose intolerance, the phenotypic features of Cisd2?/?
mice reflect other aspects of the clinical manifestations of
WFS patients including early (juvenile) onset and pre-
mature death (Supplemental Table 3). Thus, this mutant
mouse may also provide an animal model for mechanistic
investigation of Cisd2 protein function and help with the
pathophysiological understanding of WFS2.
For more than a decade, physicians and researchers have
fiercely debated as to whether WFS is associated with
mitochondria and a defect in ATP supply. Most WFS
patients die prematurely with severe neurological dis-
abilities involving the central nervous system and pe-
ripheral nerves (Barrett and Bundey 1997; Domenech
et al. 2006). In 1993, Bu and Rotter proposed a dual
genome defect model and hypothesized that mitochon-
drial DNA mutation and nuclear genetic defects that
interfere with the normal function of mitochondria can
independently lead to WFS (Bu and Rotter 1993). This
hypothesis was based on the clinical observations
that the affected tissues and organs in WFS patients
have a high metabolic demand and most of the clinical
manifestations of WFS are consistent with an ATP supply
defect, which is often seen in mitochondria-mediated
disorders. There were several studies supporting this
hypothesis (Ro ¨tig et al. 1993; Vora and Lilleyman 1993;
Barrientos et al. 1996). Notably, Bundey et al. (1992)
described a WFS patient having morphologically and
biochemical abnormal mitochondria in the muscle bi-
opsy; this finding indicated that a mitochondrial defect
may be involved in the pathogenesis of WFS. However,
other clinical studies revealed no evidence supporting the
hypothesis of mitochondrial deficiency (Hofmann et al.
1997; Barrett et al. 2000). This controversy seems to have
been resolved by the identification of different causative
genes for WFS, and this hypothesis is supported by the
mouse works carried out in this study.
WFS1 is associated with an ER defect
Previous studies in patients had identified WFS1 (wolf-
ramin) as the causative gene for WFS1 (Inoue et al. 1998;
Strom et al. 1998). Biochemical and cell culture inves-
tigations revealed that wolframin is a transmembrane
protein primarily localized in the ER and may be involved
in the regulation of ER stress and calcium homeostasis
(Takeda et al. 2001; Fonseca et al. 2005; Zatyka et al.
2008). In animal studies, a pancreatic phenotype related
to glucose intolerance and impaired insulin secretion, but
not the neurodegenerative phenotype, has been reported
in wolframin knockout mice. Wolframin deficiency in
mice leads to progressive loss of b cells and impaired
glucose homeostasis (Ishihara et al. 2004), which appears
to be caused by increased ER stress and apoptosis in
the pancreatic b cells (Riggs et al. 2005; Yamada et al.
2006). Obviously, the pathogenesis of WFS1 patients with
wolframin mutations is mechanistically related to an ER
rather than a mitochondrial defect. This provides an ex-
planation for the discrepancy as to why there were con-
tradictory observations in some WFS (specifically WFS1)
patients who do not have any detectable abnormality in
WFS2 is a mitochondria-mediated disorder
Recently, Amr et al. (2007) identified CISD2 homozygous
mutations in WFS patients and suggested that CISD2 is
the causative gene responsible for WFS2. Our Cisd2 gene
knockout mouse work provides strong evidence support-
ing the hypothesis that WFS is a mitochondria-mediated
disorder; thus, specifically, WFS2, which is caused by
a CISD2 mutation, is a mitochondria-mediated disorder.
Previous clinical studies in WFS patients suggested that
optic atrophy probably represents a degeneration of the
optic nerve (Mtanda et al. 1986; Barrett et al. 1997).
Indeed, our mouse work has revealed that progressive
degeneration of the optic nerve is one of the earliest
phenotypic features detected at 2–3 wk of age, which is
before weaning; this phenotype exacerbates with age in
the Cisd2?/?offspring (Fig. 5; Supplemental Fig. 12).
Regarding glucose homeostasis, although the phenotype
is relatively milder and only glucose intolerance was
observed in the diabetes-resistant C57BL/6 background,
in the future, it will be of great interest to introduce the
Cisd2 mutant allele into C57BLKS/J (Mao et al. 2006),
129/Sv (Terauchi et al. 2003), or other diabetes-prone
strains of mice, which may contain genetic modifier(s)
that increase susceptibility to diabetes. This will allow
the effect of the genetic background on the severity of
diabetes to be examined.
Our present study reveals that Cisd2 is primarily
localized in the mitochondria. Cisd2 deficiency causes
mitochondrial dysfunction accompanied by autophagic
cell death, and these events precede neuron and muscle
degeneration; together, they lead to a panel of phenotypic
features suggestive of premature aging. Since muscles and
nerves have the highest energy needs and are therefore
the most dependent on mitochondrial function, this ex-
plains why neuronal lesions and muscle abnormalities
are the two earliest manifestations and why they precede
the gross premature aging phenotype. Accordingly, mito-
consequence that triggers the accelerated aging process in
Cisd2?/?mice (Fig. 6). Our results thus provide strong
evidence for the causal involvement of mitochondrial
Chen et al.
1190 GENES & DEVELOPMENT
dysfunction in driving mammalian aging as suggested
previously by other studies in mitochondrial DNA muta-
tor mice (Trifunovic et al. 2004; Kujoth et al. 2005;
Vermulst et al. 2008). There are many genetic factors
that have the potential to shorten life span (Kuro-o
et al. 1997; Hasty et al. 2003; Mounkes et al. 2003;
Niedernhofer et al. 2006). However, the human genetic
factor that has been specifically identified as present on
human chromosome 4q is present in the same region as
Cisd2, and this is highly suggestive. Nonetheless, experi-
ments that shorten life span might be less informative
than those that prolong a healthy life span (Kurosu et al.
2005; Schriner et al. 2005; Pinton et al. 2007). Accord-
ingly, it will be of great interest to evaluate the life history
of transgenic mice expressing elevated levels of Cisd2
protein to see whether Cisd2 is the genetic determinant
on human chromosome 4 that may regulate mammalian
longevity and allow an unusually long life span to be
Materials and methods
Generation of the Cisd2 knockout mouse
Mouse Cisd2 genomic DNA was obtained by screening a BAC
library (Research Genetics, Inc.) derived from the C57BL/6 (B6)
mouse strain. A SpeI–BamHI 6.4-kb DNA fragment, which
contains part of intron 1, exon 2, and part of exon 3 of the Cisd2
gene, was used as the homologous recombination arms for
construction of an insertion-type targeting vector (Supplemental
Fig. 4). The Cisd2 targeting vector, containing the puromycin
selection cassette, was linearized with ApaI and transfected into
AB2.2 ES cells using electroporation. Targeted ES cell clones
were screened by Southern blot analysis using a 39-flanking
probe, specifically a 1.7-kb BamHI–EcoRI fragment from exon
3. Targeted ES cells were injected into B6 blastcysts. Chimeric
male mice were bred with B6 females. Germline transmission
was obtained from their agouti progeny. The mice were bred in
a specific pathogen-free facility and treated according to the
National Research Council’s Guide for the Care and Use of
Laboratory Animals. Heterozygous males were backcrossed with
B6 females for nine successive generations to introduce the
Cisd2 targeted allele onto the B6 congenic background.
Various mouse tissues were collected, fixed with 10% formalin
buffered with phosphate, and embedded in paraffin. Tissue
sections (3–4 mm) were subjected to hematoxylin-eosin (H&E)
and Masson’s trichome staining by standard procedures (Young
and Heath 2003).
Skeleton and bone density analyses
The bone specimens were fixed in 10% formalin buffered with
phosphate and stored in 70% ethanol. The bone density per
square centimeter was quantified from two-dimensional images
of the bones using a DEXA apparatus (Northern Radiology). For
micro-CT scanning, knockout and wild-type specimens were
fixed in 10% formalin buffered with phosphate, stored in 70%
ethanol, and then examined by eXplore Locus SP Preclinical
Specimen Micro-CT (GE Healthcare). Whole-body and femur
scans were performed in the axial plane with the specimens
mounted in a cylindrical sample holder. Three-dimensional
images of the skeletons were reconstructed from the micro-CT
scanningslices and used for analysesof the skeletalstructure and
morphology. Quantitative data were calculated by eXplore
MicroView version 2.0 Software Guide (GE Healthcare).
Cutaneous and skin analyses
Tissue sections of the dorsal skin were stained with H&E and
Masson’s trichome staining. The thicknesses of the dermal,
adipose, and muscle layers were quantified by random measure-
ments of the length of individual skin samples using SPOT
Imaging Software Advance (Diagnostic Instruments, Inc.).
Various mouse tissues were fixed in a mixture of glutaraldehyde
(1.5%) and paraformaldehyde (1.5%) in phosphate buffer (pH 7.3).
They were post-fixed in 1% OsO4 and 1.5% potassium hexano-
ferrate, then rinsed in cacodylate and 0.2 M sodium maleate
buffers (pH 6.0), and block-stained with 1% uranyl acetate.
Following dehydration, the various tissues were embedded in
Epon and sectioned for TEM as described previously (Kao et al.
Western blotting and IHC staining
Tissue samples were homogenized in lysis buffer (20 mM Tris at
pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100 with
Complete protease inhibitor cocktail [(Roche]) and denatured by
boiling for 5 min. The extracted proteins were separated on
a 13% SDS–polyacrylamide gel (Bio-Rad) and electro-transferred
to an Amersham Hybond N+ membrane (GE Healthcare). The
membranes were blocked with 5% (w/v) nonfat dry milk, in-
cubated with primary antibody, washed, and then detected using
a Visualizer Kit (Upstate Biotechnologies, 64-201BP). The fol-
lowing antibodies were used for Western blotting: LC3B (1:1000;
Cell Signaling, 2755); Gapdh (1:5000; Abcam, ab9482); Hsp60
(1:2000; Chemicon, AB3497); Hsp70 (1:2000; BD Transduction
Laboratories, 610608); VDAC-1 (1:1000; Calbiochem, 529532);
ATP5B (1:2000; Molecular Probe, A21351); PDH (1:1000; Santa
Cruz Biotechnologies, sc65242). IHC staining of insulin protein
tion of age in the Cisd2?/?mice. The timing of the onset of each
phenotype approximates the average age of onset for that phe-
notype; (wk) week. The onset age for each mouse for each
phenotype shows variation around the average onset age to
a limited degree.
Summary of the aging-related phenotypes as a func-
Cisd2 deficiency drives premature aging
GENES & DEVELOPMENT1191
was performed using paraffin-embedded pancreas sections (3
mm). Pancreas sections were soaked in antigen retrieval buffer
containing 10 mM sodium citrate (pH 6.0) and heated in a
microwave oven twice for 10 min (Sunpentown, SM-1220,
650W). The sections were then incubated with primary antibody
against insulin (1:100; Abcam, ab7842 guinea pig polyclonal
antibodies) for 18–24 h at 4°C, detected by biotinylated second-
ary antibodies (1:500; Abcam, ab6907), and visualized by the
LSAB Kit (DakoCytomation, K0690).
Rabbit anti-mouse Cisd1 and Cisd2 polyclonal antibodies
Mouse cDNA fragments of Cisd1 (corresponding to amino acids
27–108) and Cisd2 (corresponding to amino acids 52–135) were
amplified by PCR and cloned into the pQE-31 (Qiagen) vector,
which contains a His tag sequence. The expression plasmids for
His-Cisd1 and His-Cisd2 were transformed into M15 bacteria,
fied using Nickel-resin (Novagen). These proteins were injected
into rabbits to generate antisera containingpolyclonal antibodies
against the mouse Cisd1 and Cisd2, respectively.
The EGFP-tagged Cisd2 expression plasmids were transfected
into NIH/3T3 cells using lipofectamin 2000 (Invitrogen, 11668-
019). Cells transiently expressing EGFP-Cisd2 fusion proteins
were plated on gelatin-coated glass coverslips, stained with
various organelle probes including mitochondria (MitoTracker
Red CMXRos; Invitrogen Life Technologies), ER (anti-calnexin;
Sigma, C7617), and Golgi apparatus (anti-Golgi 97; Molecular
Probe). The coverslips were then fixed and visualized by confocal
microscopy (Olympus FluoView FV300). Nuclei were counter-
stained with DAPI (49-6-diamidino-2-phenylindole; Sigma).
Isolation of mitochondria from skeletal muscle
Fresh skeletal muscles were washed twice with PBS and homog-
enized immediately in ice-cold SEH buffer (0.25 M sucrose, 1
mM EGTA, 3 mM HEPES, protease inhibitor cocktail at pH 7.2).
The mitochondrial pellet was obtained by low-speed centrifuga-
tion (800g) of the homogenate, followed by high-speed centrifu-
gation (10,000g) of the supernatant. Finally, the mitochondrial
pellet was resuspended in an appropriate volume of SEH buffer.
The isolated mitochondria were used immediately for evaluation
of mitochondrial respiration and oxidative phosphorylation.
Mitochondrial subfractionation was performed according to the
method described by Pagliarini et al. (2005) with some modifi-
cations. Briefly, 0.5 mL of mitochondrial suspension (10 mg/mL)
was incubated with 10 mg of purified digitonin on ice for 30 min,
and the mixture was gently inverted every 10 min. After high-
speed centrifugation (12,000g), the mitoplast (containing the IM
and matrix) was pelleted, and the supernatant was subjected to
ultracentrifugation (150,000g) to separate the OM and IMS of the
Measurement of oxygen consumption
The oxygen consumption rate was measured using a 782 Oxygen
Meter (Strathkelvin Instruments). An aliquot of 300 mL of assay
buffer (125 mM sucrose, 65 mM KCl, 2 mM MgCl2, 20 mM
Na+,K+-phosphate buffer at pH 7.2) containing ~0.2~0.5 mg of
mitochondria was delivered into the closed chamber of the
oxygen meter at 37°C to measure the steady-state oxygen
consumption rate of the mitochondria (Chen et al. 2008). In
order to further estimate the respiratory function of mitochon-
dria, we measured the glutamate-malate-supported respiration
and RCR of mitochondria. First, we used a Hamilton syringe
(Strathkelvin) to add 10 mM glutamate and 10 mM malate
(Sigma-Aldrich) into the chamber as the electron donor and
recorded the glutamate–malate-supported oxygen consumption
rate. After 5 min, we injected 3 mL of 100 mM ADP to attain
a final ADP concentration of 1 mM in the assay medium. The
rate of activated respiration was recorded to measure the RCR of
Respiratory enzyme complex activity
The following activity assays were performed according to the
method described by Wei et al. (1998). The activities of NCCR
(which represents complex I–III activity) and SCCR (which
represents complex II–III activity) were measured by following
the reduction of exogenous oxidized cytochrome c. An aliquot
of 20~50 mg of submitochondrial particles (SMP) was preincu-
bated with the assay buffer (1.5 mM KCN, 50 mM K2HPO4at
pH 7.4) containing b-NADH or succinate for 15 min at 37°C.
After addition of cytochrome c to the mixture, the change in the
absorbance at 550 nm was recorded on a UV/visible spectropho-
tometer. CCO (which represents complex IV) activity was de-
termined by following the oxidation of exogenous reduced
cytochrome c. An aliquot of 20~50 mg SMP was preincubated
in the assay buffer (5 mM K2HPO4at pH 7.4) for 10 min at 30°C.
After addition of ferrocytochrome c to the assay mixture, the
change in absorbance at 550 nm was recorded on a UV/visible
Oral glucose tolerance test and insulin tolerance test
Mice after a 10-h fast (10 p.m. to 8 a.m.) were orally adminis-
trated with glucose solution (1.5 g/kg body weight) using
a feeding needle (Juan et al. 2004). Blood samples were collected
fromtailtips before(0 min) andafterglucoseload atthe indicated
time points. The blood glucose levels were measured using
glucose test strips (LifeScan; Johnson & Johnson) and SureStep
Brand Meter. Serum insulin levels were determined by an ELISA
kit (Mercodia). The insulin tolerance test was performed after
a 2-h fast (9 a.m. to 11 a.m.) and involved an intraperitoneal
injection of insulin (0.75 U/kg body weight; Novolin human
regular insulin; Novo Nordisk) (Tran et al. 2008). There were
three mice for each group and three independent measurements
for each mouse.
Results are presented as means 6 SD. Differences among mul-
tiple groups were analyzed by a one-way ANOVA (SPSS 14.0
statistical software). Comparisons between two groups were
done using a Student’s t-test. Mouse survival rates were calcu-
lated by the Kaplan-Meier method, and differences in the
survival of different groups of mice were determined by the
log-rank (Mental-Cox) test. When analyzing statistical differ-
ences between the knockout and wild-type mice, P < 0.05 was
We thank Dr. Lian-Fu Deng (Affiliated Ruijin Hospital of
Shanghai Second Medical University, Shanghai Institute of
Traumatology and Orthopeadics); Dr. An-Guor Wang (Taipei
Chen et al.
1192 GENES & DEVELOPMENT
Veterans General Hospital); Dr. Ming-Ling Kuo (Chang Gung
University); Dr. Chih-Cheng Chen (Academia Sinica); and Dr.
Alan M. Lin, Dr. Hen-Li Chen, Dr. Chun-Ming Chen, Dr. Chi-
Chang Juan, Yi-Shin Lai, Ching-Wen Cheng, and Hui-Wen
Zhuang (National Yang-Ming University) for their insight and
technical assistance. We thank the Microarray and Gene Expres-
Genome Research Center. The Core Facility is supported by the
National Research Program for Genomic Medicine (NRPGM),
tional Science Council (NRPGM 95HC007, NSC96-2752-B-010-
004-PAE, and NSC97-2320-B-010-015-MY3) and a grant from the
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