THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 24, pp. 20652–20663, June 8, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Proteomic and Metabolomic Analyses of Mitochondrial
Complex I-deficient Mouse Model Generated by
Spontaneous B2 Short Interspersed Nuclear Element (SINE)
Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S
Protein 4 (Ndufs4) Gene*□
S
Received for publication, November 25, 2011, and in revised form, April 5, 2012 Published, JBC Papers in Press, April 25, 2012, DOI 10.1074/jbc.M111.327601
Background: Mitochondrial complex I deficiency is a common inherited metabolic disease.
Results: B2 transposable element insertion into Ndufs4 in mice causes loss of the “N assembly module” of complex I, alterations
in cellular metabolites, and neurological symptoms.
Conclusion: NDUFS4 subunit is required for complex I stability.
Significance: Understanding the effects of oxidative phosphorylation defects is essential for the development of treatments.
Eukaryotic cells generate energy in the form of ATP, through a
network of mitochondrial complexes and electron carriers known
as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and
nuclear DNA. Humans diagnosed with mutations in the gene
NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH
dehydrogenase ubiquinone Fe-S protein 4), typically suffer from
Leigh syndrome, a neurodegenerative disease with onset in infancy
or early childhood. Mitochondria from NDUFS4 patients usually
lack detectable NDUFS4 protein and show a CI stability/assembly
* This work was supported by an Australian Postgraduate Award (to S. A. K.),
National Health and Medical Research Council (NHMRC) fellowships (to H. H. S,
D. R. T., G. K. S., and A. K. V.), NHMRC career development awards (to M. M. and
M. B.), NHMRC Program Grant 257501, and NHMRC Project Grants 607403 and
602530, the Australian Research Council (to M. T. R.), the Victorian Government’s Operational Infrastructure Support Program, and the Nossal Leadership
Award from the Walter and Eliza Hall Institute of Medical Research (to H. S. S.).
□
S
This article contains supplemental Tables 1– 8 and Figs. 1–3.
1
Both authors contributed equally to this work.
2
To whom correspondence may be addressed: Murdoch Childrens Research
Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia. Tel.: 61-38341-6235; Fax: 61-3-8341-6212; E-mail: [email protected].
3
To whom correspondence may be addressed: Dept. of Molecular Pathology,
Centre for Cancer Biology, SA Pathology, Box 14 Rundle Mall Post Office,
Adelaide, South Australia 5000, Australia. Tel.: 61-8-8222-3651; Fax: 61-88222-3146; E-mail: [email protected].
20652 JOURNAL OF BIOLOGICAL CHEMISTRY
defect. Here, we describe a recessive mouse phenotype caused by
the insertion of a transposable element into Ndufs4, identified by a
novel combined linkage and expression analysis. Designated
Ndufs4fky, the mutation leads to aberrant transcript splicing and
absence of NDUFS4 protein in all tissues tested of homozygous
mice. Physical and behavioral symptoms displayed by Ndufs4fky/fky
mice include temporary fur loss, growth retardation, unsteady gait,
and abnormal body posture when suspended by the tail. Analysis of
CI in Ndufs4fky/fky mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was
shown to lack subunits of the “N assembly module”, which contains
the NADH binding site, but contained two assembly factors not
present in intact CI. Metabolomic analysis of the blood by tandem
mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/
NADⴙ ratio that inhibits mitochondrial fatty acid ␤-oxidation.
The oxidative phosphorylation (OXPHOS)4 system in mitochondria consists of five protein complexes (CI–CV) and is
4
The abbreviations used are: OXPHOS, oxidative phosphorylation; CI–CV,
complex I–V, respectively; OMIM, Online Mendelian Inheritance in Man姞;
df, degree of freedom; SNP, single nucleotide polymorphism; BN, blue
native; Pn, postnatal day n; nt, nucleotide(s); SINE, short interspersed
nuclear element; LINE, long interspersed nuclear element.
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Dillon W. Leong,a1 Jasper C. Komen,b1 Chelsee A. Hewitt,a Estelle Arnaud,c Matthew McKenzie,d Belinda Phipson,e
Melanie Bahlo,e,f Adrienne Laskowski,b Sarah A. Kinkel,a,g,h Gayle M. Davey,g William R. Heath,g Anne K. Voss,a,h
René P. Zahedi,i James J. Pitt,j Roman Chrast,c Albert Sickmann,i,k Michael T. Ryan,l Gordon K. Smyth,e,f,h
David R. Thorburn,b2 and Hamish S. Scotta,h,m,n3
From the aMolecular Medicine Division, gImmunology Division, and eBioinformatics Division, Walter and Eliza Hall Institute of
Medical Research, Parkville, Victoria 3052, Australia, the bMurdoch Childrens Research Institute, Royal Children’s Hospital and
Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the cDépartement de Génétique Médicale,
Université de Lausanne, 1005 Lausanne, Switzerland, the dCentre for Reproduction and Development, Monash Institute of Medical
Research, Clayton, Victoria 3168, Australia, the hDepartment of Medical Biology and fDepartment of Mathematics and Statistics,
University of Melbourne, Parkville, Victoria 3052, Australia, the iLeibniz-Institut für Analytische Wissenschaften e.V.,
44227 Dortmund, Germany, jVCGS Pathology, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria
3052, Australia, the kMedizinisches Proteom Center, Ruhr-Universität-Bochum, 44780 Bochum, Germany, the lDepartment of
Biochemistry, La Trobe University, Bundoora, Victoria 3086, Australia, and the mDepartment of Molecular Pathology, Centre for
Cancer Biology, SA Pathology, Box 14 Rundle Mall Post Office, Adelaide, South Australia 5000, Australia, and the nSchools of
Medicine and Molecular and Biomedical Science, University of Adelaide, South Australia 5005, Australia
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
JUNE 8, 2012 • VOLUME 287 • NUMBER 24
mouse model presented here arose due to a spontaneous B2
SINE retroviral insertion into the Ndufs4 gene (Ndufs4fky/fky
mice). We used a novel strategy involving genetic linkage based
on single nucleotide polymorphisms (SNPs) and microsatellites, together with expression exon arrays, which identified not
only the affected gene but the affected exon. The phenotype of
our Ndufs4fky/fky mice is similar to that described by Kruse et al.
(13) for the conventional knockout. However, we show here
that in Ndufs4fky/fky mice, certain metabolites in the blood are
significantly different compared with unaffected controls,
which may potentially be used as biomarkers for disease in the
future. Furthermore, we demonstrate a clear CI assembly defect
in Ndufs4fky/fky mice, which is similar to that found in patients
with mutations in NDUFS4.
EXPERIMENTAL PROCEDURES
Animal Handling—The spontaneous Ndufs4fky mutation
originated in a transgenic C57BL/6 (B6) strain. For genetic
mapping, B6 Ndufs4fky/⫹ males were crossed with BALB/c (BC)
females. Experiments were carried out on backcross (N2) and
intercross (Fn) offspring, as well as progeny from the founding
B6 colony. Ethics approval was granted by the Walter and Eliza
Hall Institute Animal Ethics Committee (Project 2005.041) and
an approved protocol by the Murdoch Childrens Research
Institute Animal Ethics Committee (A526). Mice were housed
on 12-h light/dark cycles and provided ad libitum access to
food and water. Ndufs4fky/fky mice were euthanized around
postnatal day 40, when they lost more than 10% of their body
weight and/or showed hind limb clasping or forward curling
when suspended by the tail according to our ethical guidelines.
Growth Curves and Organ Weights—Mice were weighed
from the age of 5 to 45 days postnatal every afternoon. Differences between control and affected mice were assessed using a
permutation test designed for growth curves (available on the
Walter and Eliza Hall Institute of Medical Research Bioinformatics Web site) with 10,000 permutations. p values were computed as recommended by Phipson and Smyth (15).
Genetic Linkage—Genetic linkage to locate the fky locus was
performed by outcrossing onto BALB/c, and there was complete penetrance in the resulting progeny (81 of 370 ⫽ 0.22,
␹2 ⫽ 1.91, df ⫽ 1, p ⫽ 0.17) with no sex bias (␹2 ⫽ 0.62, df ⫽ 1,
p ⫽ 0.43). Penetrance of the mutant phenotype in founding and
outcross colonies was determined using Pearson’s ␹2 goodness
of fit test. Sex bias was examined using Pearson’s test of independence with Yates correction. A total of 99 offspring, comprising 20 backcross (N2) and 79 intercross (F2) animals, were
analyzed at 309 SNP markers using the SEQUENOM威
iPLEXTM assay and a custom panel of 15 microsatellites (document number 8876-006, R01, April 2005). Briefly, each SNP
region was PCR-amplified, followed by a clean-up step to
dephosphorylate unincorporated dNTPs. A second amplification was performed using primers that stop just before each
SNP nucleotide, with the reaction terminating after a single
base extension. Therefore, the final amplification products for
each polymorphic marker would differ by only one nucleotide,
which was the SNP itself. Amplicons were analyzed using mass
spectrometry (SpectroCHIP威 bioarrays) with a MassARRAY
work station (version 3.3). Single point analysis was done on the
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responsible for the generation of the majority of the energy for
the cell in the form of the energy-rich molecule ATP (1). Complex I (CI; NADH-ubiquinone oxidoreductase) is a major entry
point of electrons into the OXPHOS system via the enzymatic
conversion of NADH into NAD⫹, the transfer of the released
electrons to ubiquinone, and the consequent enzymatic reduction of the latter. Simultaneous with these actions, CI pumps
protons out of the mitochondrial matrix, thereby contributing
to the generation of the mitochondrial membrane potential
(⌬␺M), which can be used by complex V for ATP synthesis or
alternatively for the generation of heat. In mammals, CI
requires the correct assembly of 45 subunits encoded by both
the nuclear and mitochondrial DNA in order to function correctly, making it the largest of the OXPHOS complexes.
Defects in the OXPHOS system are the most common cause
of inherited metabolic diseases affecting 1 in 5000 live births (2).
CI deficiency is the most common of the OXPHOS disorders
and has been shown to be caused by mutations in at least 20
subunit genes plus at least nine other genes encoding CI assembly factors (3), with many other affected individuals yet to have
their genetic cause identified. As for any of the OXPHOS disorders, CI deficiency may affect any organ at any age, but most
often organs with a high energy demand, such as the heart and
brain, are affected. Accordingly, patients with isolated CI deficiency most often present with the early onset neurodegenerative disease Leigh syndrome (OMIM number 256000) (4, 5).
There is currently no effective treatment available for patients
with CI deficiency, as is generally the case for all patients with
OXPHOS disorders. Up until now, patient management has
largely been limited to the treatment of symptoms (6) and
genetic counseling for those families where mutations have
been identified with the possibility of prenatal testing.
One problem in developing new treatment approaches has
been the lack of relevant animal models for OXPHOS disorders,
in particular mouse models, to test promising drugs. Until
recently, the only mouse models with CI deficiency were the
spontaneous mutant “Harlequin” mouse with a proviral insertion in intron 1 of the Aif (apoptosis-inducing factor) gene,
resulting in a reduced overall expression of AIF protein and the
subsequently generated conditional Aif knockouts (7–10).
These models have some limitations because the exact role of
AIF in CI maintenance and activity is still unclear. This was
emphasized by the recent discovery of patients with an isolated
CIV deficiency caused by mutations in the orthologous AIF
gene (11). Furthermore, the variability of the phenotype of Harlequin mice may complicate interpretation of the treatment
outcomes.
The Ndufs4 gene encodes a matrix arm subunit of CI.
Patients with mutations in the NDUFS4 gene develop Leigh
syndrome and have a CI assembly/stability defect and a severe
defect in CI activity. Homozygous expression of a truncated
form of NDUFS4 results in embryonic lethality (12), whereas
mice with a complete knock-out of Ndufs4 (Ndufs4⫺/⫺ mice)
develop severe neurological impairment, leading to death at ⬃7
weeks (13). Ndufs4⫺/⫺ mice develop severe neurological
impairment, leading to death at ⬃7 weeks. Additional studies
showed that the majority of the symptoms of Ndufs4⫺/⫺ mice
were due to the CI defect in the brain (14). Our CI-deficient
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
5
D. Wu and G. K. Smyth, manuscript in preparation.
20654 JOURNAL OF BIOLOGICAL CHEMISTRY
tion, and sequence reads were examined using SeqMan
software (DNASTAR威 Lasergene). Ndufs4 reference sequences
were downloaded from the University of California, Santa Cruz,
Genome Browser Mouse Feb 2006 assembly.
SDS-PAGE, BN-PAGE, and Immunoblotting—For SDSPAGE, fresh frozen tissues were homogenized in lysis buffer
(100 mM Tris/HCl pH 8, 100 mM NaCl, 0.1% Triton X-100, 2
mM EDTA, and protease inhibitor mixture), briefly sonicated,
and centrifuged at 4 °C. The resulting supernatant was collected, and 20 ␮g of protein extract was resolved using SDSPAGE on a 15% acrylamide gel. NDUFS4 was detected using a
monoclonal antibody (MitoSciences, MS104).
For BN-PAGE, mitochondria were isolated from mouse
heart as described previously for cultured cells (21) and solubilized in either 1% n-dodecyl-␤-D-maltoside or 1% Triton X-100
before separation of complexes by BN-PAGE (22). Proteins
were either stained with 0.5% Coomassie Blue R (Sigma) in 40%
ethanol, 7% acetic acid or transferred to PVDF membrane
(Immobilon, Millipore) for immunodecoration (22). Membranes were probed with antibodies against the 70-kDa subunit
of CII (Invitrogen) or against the CI subunit NDUFA9 (23).
For mass spectrometric analysis of holo-CI and “crippled” CI,
mitochondria were solubilized in 1% Triton X-100 followed by
separation on a 4 –10% BN-PAGE gel. Complex I was further
purified by another round of BN-PAGE. To this end, strips of
the first dimension BN-polyacrylamide gel were excised and
placed and run on a second BN-polyacrylamide gel.
The resolved gel was fixed in 50% methanol, 2% phosphoric
acid; washed in distilled H2O; incubated in 34% methanol, 2%
phosphoric acid, 17% ammonium sulfate; and stained with Brilliant Blue G colloidal Coomassie. Complexes were then excised
from the gel for mass spectrometric analysis.
Nano-LC-MS/MS Analysis—Excised gel bands were washed,
reduced, carbamidomethylated, and subjected to tryptic in-gel
digestion as described previously (24). Generated peptides were
extracted with 15 ␮l of 0.1% trifluoroacetic acid (TFA). NanoLC-MS/MS analyses were conducted using an LTQ-Orbitrap
XL (Thermo Scientific, Bremen, Germany) mass spectrometer,
online-coupled to an inert U3000 nano-HPLC system (Dionex,
Amsterdam, The Netherlands).
Briefly, peptides were preconcentrated in 0.1% TFA on a
100-␮m inner diameter reverse phase trapping column (Synergi HydroRP, 4-␮m particle size, 80-Å pore size, 2-cm length,
Phenomenex) and afterward separated on a 75-␮m inner diameter reverse phase column (Synergi HydroRP, 2-␮m particle
size, 80-Å pore size, 30-cm length, Phenomenex) main column
by applying a 40-min binary gradient (solvent A, 0.1% formic
acid; solvent B, 0.1% formic acid, 84% acetontrile) ranging from
5 to 50% solvent B in 40 min and from 50 to 95% B in 1 min, at
a flow rate of 270 nl/min and 60 °C.
MS survey scans from 350 to 2000 m/z were acquired in the
Orbitrap with a resolution of 60,000 using the polysiloxane at
m/z 445.120030 as lock mass. The five most intense signals were
subjected to collision-induced dissociation-based MS/MS in
the LTQ using a dynamic exclusion. AGC target values were set
to 106 and 104 for the Orbitrap and the LTQ, respectively.
Raw data were transformed into mgf (Mascot generic file)
format, and generated peak lists were searched against the IPI
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SNP genotypes to check segregation distortion and determine a
threshold for genome-wide significance. A custom binomial
test implemented in R was then used to look for bias to B6
homozygosity.
Microsatellite genotyping was performed using markers
from public databases (supplemental Table 1). Amplification
was done under standard conditions using fluorescently labeled
primers (Applied Biosystems), and PCR products were separated by capillary electrophoresis on the AB3730 DNA analyzer. Fluorescent signals were translated into fragment sizes
using Genemapper (version 3.7). Microsatellite genotypes were
examined by visual comparison of haplotypes to identify
regions of B6 homozygosity.
Genotyping—The novel Ndufs4 mutation is genotyped using
PCR under standard conditions with one forward primer in
intron 3 (5⬘-TAGGAAGGGAGAGACGAGCA-3⬘) and two
reverse primers located in the B2 SINE insert (5⬘-TTACCCACTGAGCCATCTCAC-3⬘) and exon 3 (5⬘-GATGCCCAACCCATCAAAG-3⬘), respectively.
Microarray Analysis—Total RNA was isolated from whole
brain of two unaffected and two affected P37 mice using the
Qiagen RNeasy威 Lipid Tissue Midi Kit according to the manufacturer’s instructions, including DNase treatment. RNA was
processed and hybridized onto an Affymetrix GeneChip威
Mouse Exon 1.0ST Array according to the manufacturer’s
instructions (GeneChip威 Whole Transcript Sense Target
Labeling Assay Manual 701880 Rev.2). Fluorescent signals were
detected using a GeneChip威 Scanner 3000.
Intensity values were background corrected, log2-transformed, and quantile-normalized using the robust multiarray
average algorithm (16). Expression values were summarized
and analyzed at both the gene and the exon levels. In each case,
differential expression was assessed using empirical Bayes
moderated t tests (17). Benjamini and Hochberg adjustment
was applied to control the false discovery rate for the 30 genes in
the linkage region. A custom RefSeq-based CDF file was used
for the gene level analysis (18). Pathway analysis was run on the
genome-wide differential expression results using ROAST (19),
Camera,5 and gene sets from the Broad Institute’s Molecular
Signatures Database (MSigDB) (20) mapped to mouse
orthologs (see the Walter and Eliza Hall Institute of Medical
Research Bioinformatics Web site).
Cloning and Sequencing—Genomic Ndufs4 primers for each
exon were designed to include 200 –300 bp of adjacent splice
boundaries, whereas primers for cDNA amplification were
designed to the middle of each exon (primer sequence available
on request). Cloning of cDNA amplicons was carried out using
the Promega pGEM威-T Vector System with JM109 competent
cells according to the manufacturer’s instructions. Sequencing
reactions were prepared using the Applied Biosystems Prism威
BigDyeTM Terminator (version 3.1) as recommended by the
Australian Genome Research Facility. Sequencing of clones
was done using M13 primers 5⬘-GGAAACAGCTATGACCATG-3⬘ and 5⬘-GTAAAACGACGGCCAGT-3⬘. Fluorescently labeled amplicons were analyzed using capillary separa-
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
JUNE 8, 2012 • VOLUME 287 • NUMBER 24
and analyzed by electrospray tandem mass spectrometry as
described previously with minor modifications (28). Multiple
reaction monitoring was used to target a selected panel of
amino acids and acylcarnitines. Quantitation was performed
using added stable isotope internal standards. Significant
changes between Ndufs4fky/fky mice and control were determined by t test. Differences were considered significant at p ⬍
0.05.
Acylcarnitine Analysis of Tissues—Approximately 10 mg of
heart or liver tissue was analyzed according to van Vlies et al. (29)
with minor modifications. The amounts of acylcarnitine species
were semiquantified using the following internal standards:
[2H9]carnitine for C0–C2 carnitines, [2H3]isovalerylcarnitine for
C3–C6 carnitines, [2H3]octanoylcarnitine for C8–C10 carnitines,
and [2H3]tetradecanoylcarnitine for C12–C20 carnitines. Butylated acylcarnitine species were measured in duplicate by electrospray tandem mass spectrometry using a Waters Quattro LC
instrument.
RESULTS
Spontaneous B2 SINE Insertion (fky) into Ndufs4 Results in
Progressive Neurological Disorder—During a breeding strategy
with heterozygous male Dnmt3L mice (30) and female RIPmOVA transgenic C57BL/6 mice (31) a spontaneous “funky”
mutation (fky) occurred. This mutation segregates independently of the parental transgenes, and inheritance is consistent
with an autosomal recessive locus. The fky/fky phenotype presents from around postnatal day 16 (P16) with fur loss in the
neck area, which subsequently spreads caudally along the trunk
toward the tail (Fig. 1A). This fur loss is transient and regrows
after a week in a similar pattern from the neck down the trunk.
Homozygous mice show a failure to thrive from postnatal week
2 and by P40 are, on average, 26% smaller than their normal
littermates (p ⫽ 10⫺5 for males; p ⫽ 10⫺5 for females; Fig. 1B).
From week 5 onward, the affected mice develop neurological
symptoms, including tilting of the head (16 of 75 ⫽ 0.21), walking in circles (17 of 75 ⫽ 0.23), and forward curling in combination with twisting of the body when suspended by the tail (51
of 75 ⫽ 0.68). From P40 onward, the symptoms progress rapidly, resulting in euthanasia according to ethical guidelines.
Despite the severity of the mutation in the homozygous state,
there was no evidence of prenatal lethality, as determined for a
cohort of 465 progeny, of which 107 displayed the recessive
phenotype (107 of 465 ⫽ 0.23, ␹2 ⫽ 0.98, df ⫽ 1, p ⫽ 0.32) with
no sex bias (51 males and 56 females, df ⫽ 1, ␹2 ⫽ 0.48, p ⫽
0.49). We performed a novel combination of both linkage and
expression analysis to rapidly identify the gene responsible for
the fky/fky phenotype. Genetic linkage to locate the fky locus
was initiated by outcrossing the C57BL/6 fky with BALB/c wildtype mice and performing a genome-wide interrogation of 309
SNPs in 20 backcross and 79 intercross offspring. Analysis of
these SNPs led to a subregion of chromosome 13, where a custom panel of 15 microsatellites further fine-mapped the recessive locus to a 6-megabase interval on mouse chromosome 13
between D13Mit196 and rs3708633 (supplemental Fig. 1A and
Table 2), which contained 30 annotated RefSeq genes (Ensembl
Genome Browser; mouse build 35). Expression analysis of these
30 genes using whole brain RNA on the Affymetrix GeneChip威
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mouse database version 3.52 (55,303 sequences) using Mascot
2.2.2 with the following settings: trypsin as enzyme with a maximum of one missed cleavage, carbamidomethylation of Cys
(⫹57.0214 Da) as fixed and oxidation of Met (⫹15.9949 Da) as
well as phosphorylation of Ser, Thr, and Tyr (⫹79.9663 Da) as
variable modifications, a peptide mass tolerance of 10 ppm, and
an MS/MS tolerance of ⫾0.5 Da. Only proteins with at least two
different, manually checked peptide MS/MS spectra above a
Mascot score of 35 (p ⬍ 0.05 at 28) were taken into account.
Mitochondrial CI Activity—OXPHOS enzyme activities
were determined spectrophotometrically as described previously (5), except that assays were performed at 25 °C. Activities
of respiratory chain complexes of tissues from Ndufs4fky/fky and
wild type mice were compared by a t test. Differences were
considered significant at p ⬍ 0.05.
Mitochondrial ATP Synthesis Assay—Mitochondria were
immediately isolated from tissues of mice euthanized by cervical dislocation according to Pallotti and Lenaz (25) for heart
and according to Sims (26) for brain. ATP synthesis capacity of
isolated mitochondria (6.25 ␮g/ml end concentration) was
measured as described previously with digitonin omitted (27).
CI-dependent ATP synthesis was achieved with the substrates
malate (10 mM) and glutamate (10 mM), whereas CII-dependent
ATP synthesis was achieved with succinate (10 mM) in the presence of rotenone (2.5 ␮M). ATP synthesis capacity was
expressed as the ratio between CI-dependent ATP synthesis
normalized against CII-dependent ATP synthesis. Data
obtained with mitochondria from Ndufs4fky/fky and control
mice were compared by t test. Differences were considered significant at p ⬍ 0.05.
Histology—Skeletal muscle (quadriceps) was cryosectioned
to 8 ␮m and stained with oil red O. Oil red O stock solution was
prepared by solubilizing 300 mg of dye in 100 ml of isopropyl
alcohol. Cryosections were fixed in paraformaldehyde for 10
min and incubated in 60% oil red O stock solution for 10 min.
Slides were washed three times with distilled water, mounted,
and examined. For the analysis of the optic nerve the mice were
perfused with 1% paraformaldehyde and 2% glutaraldehyde in
0.1 M cacodylate buffer (pH 7.3) for 5 min. Optic nerves were
accurately dissected and postfixed by immersion in the fixative
solution for 2 h at 4 °C, washed in 0.1 M cacodylate buffer, and
osmicated for 4 h in 1% OsO4 (Fluka). Nerves were rinsed in
water, dehydrated, and embedded in Epon 812 substitute
(Fluka). One-micrometer semithin sections were stained with
1% toluidine blue and examined by light microscopy.
Mouse brains were cryosectioned in a 1:4 series at 20 ␮m.
Spinal cords were sectioned in a 1:24 series at 20 ␮m. All cut
sections were mounted onto gelatin-coated slides. One series
from each cut tissue was stained with cresyl violet. Images
(⫻10) of sections were obtained using an Olympus Bx51 microscope and then analyzed using Neurolucida software. ImagePro
Plus software was used to measure the motor cortex, sensory
cortex, corpus collosum, ventricle, striatum, hippocampus
(dentate gyrus and CA1), substantia nigra, pontine nucleus,
ventral posterior thalamic nucleus (VP), cerebellar layers, and
the areas of entire hemispheres.
Metabolic Analysis of Blood from Dried Bloodspot Cards—
Blood drawn from the heart was placed on Whatman 903 paper
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
Mouse Exon 1.0ST Array found Ndufs4 to be down-regulated
in homozygous mice (p ⫽ 4 ⫻ 10⫺24; supplemental Table 2).
Moreover, the expression of Ndufs4 showed relatively uniform
down-regulation of all five exons with the exception of exon 3,
which was at least 2.5-fold more down-regulated than the other
exons (supplemental Fig. 1C and Table 3).
Sequencing of all five Ndufs4 exons and neighboring splice
boundaries revealed a 258-nucleotide (nt) insertion within
exon 3 (Fig. 2A). The insert sequence was queried against
Repbase using RepeatMasker (A. F. A. Smit, R. Hubley, and
P. Green, RepeatMasker Open-3.0, 1996 –2004; available on
the World Wide Web), and a 97% match (171 of 176 nt
excluding the 69-nt poly(A) tail) to a known B2 short interspersed nuclear element (SINE) was found (Fig. 2, B and C).
Genetic screening of our mouse colony found that this
mutant Ndufs4 variant, Ndufs4fky, segregates with the recessive phenotype.
B2 SINE Insertion Results in Depletion of NDUFS4 Protein in
Ndufs4fky/fky Mice—RT-PCR analysis of whole brain RNA
showed a truncated canonical Ndufs4 transcript in Ndufs4fky/fky
mice, and sequencing revealed that exon 3 was absent (Fig. 3A),
which is concordant with the exon level microarray analysis.
The absence of exon 3 from the canonical Ndufs4 transcript
is predicted to cause a frameshift resulting in the formation
of premature termination codons within exons 4 and 5.
Canonical transcripts containing both exon 3 and the B2
insert were present in lower levels, but positioning of the B2
element in exon 3 also results in premature termination
codon formation within the exon. Western blot analysis of
brain, heart, liver, and skeletal muscle failed to detect
NDUFS4 protein in Ndufs4fky/fky mouse tissues compared
with wild type and heterozygous littermates (n ⫽ 3) (Fig. 3B),
consistent with post-transcriptional degradation of the
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FIGURE 1. Mice with a homozygous “funky” mutation have transient hair
loss (A) and a failure to thrive (B). A, funky mice present with baldness
behind the neck around P17, which progresses down the trunk (P21) toward
the tail (P24) after which the hair grows back in the same order. B, growth
curves comparing homozygous funky males and females to normal littermates. Homozygous mice show a reduction in growth rate from the age of 15
days. n ⫽ 6 for affected males (M, Affected); n ⫽ 33 for control males (M,
Control); n ⫽ 10 for affected females (F, Affected); n ⫽ 20 for control females (F,
Control); p ⫽ 10⫺5 for males; p ⫽ 10⫺5 for females. Error bars, S.E.
Ndufs4 product due to premature termination codon-induced nonsense-mediated decay (32).
Ndufs4 encodes the 18-kDa structural subunit of mitochondrial CI. In humans mutations in NDUFS4 cause mitochondrial
CI deficiency (OMIM number 252010) (33–35), whereas a conventional knockout of the Ndufs4 gene in mouse was recently
also shown to cause CI deficiency (13). Measurement of CI
activity in brain, heart, muscle, liver, and kidney tissues from
Ndufs4fky/fky mice, normalized against citrate synthase activity,
showed decreased CI activity levels compared with wild type
(Fig. 3C). The highest residual activity was detected in heart
tissue (22.1 ⫾ 1.3% of wild type level), whereas liver had the
lowest residual activity compared with wild type (10.4% ⫾
1.1%). Additionally, no difference in CI activity was found
between wild type and heterozygous mice (data not shown).
This corresponds with the phenotype of heterozygous animals,
which we found indistinguishable from wild type animals.
The activity of the other OXPHOS complexes (CII, CIII, and
CIV) was not affected by the depletion of NDUFS4, as shown
for brain, indicating an isolated effect on CI in Ndufs4fky/fky
mice (n ⫽ 4, p ⬎⬎ 0.05, unpaired t test; Fig. 3D).
In order to determine the effect of the NDUFS4 depletion on
the functionality of the OXPHOS system, we studied the capacity of isolated mitochondria to produce ATP when respiring on
the substrates malate and glutamate, which feed electrons into
the system at the level of CI. As a control, we measured ATP
synthesis with succinate, which feeds electrons in via CII, plus
rotenone to inhibit CI activity. Because no difference in CI
activity and phenotype was found between the wild type and
heterozygous genotypes, they were combined and referred to as
the “control” group. As a result of the defect in CI activity,
mitochondria isolated from heart and brain tissue of
Ndufs4fky/fky show a decreased capacity to generate ATP compared with control mitochondria (ratio of ATP generated by
respiring on malate and glutamate versus succinate and rotenone; Fig. 3E). Despite the large reduction measured in the CI
activity assays for both the heart and brain, there was only a
small reduction of 18% in the capacity to generate ATP in the
heart, with a somewhat greater reduction of 36% in the brain
(Fig. 3E).
Depletion of NDUFS4 Protein Leads to Loss of N Module of
CI—In humans, nonsense mutations, deletions, and duplications within NDUFS4 have been demonstrated to disrupt
NDUFS4 protein synthesis, leading to abnormal mitochondrial
CI formation and causing reduced activity (33–37). We examined mitochondrial CI assembly state in Ndufs4fky/fky mice
using BN-PAGE separation followed by Coomassie staining.
Ndufs4fky/fky heart mitochondria solubilized in 1% n-dodecyl␤-D-maltoside for BN-PAGE have a clear defect in CI assembly/
stability, as shown by disappearance of the band corresponding
to the correct molecular weight for CI as visible in the control
lane (Fig. 4A, lanes 1 and 2). Instead, there is a band with a lower
molecular weight corresponding to a protein complex of ⬃800
kDa appearing in Ndufs4fky/fky heart mitochondria (lane 2),
indicating a crippled CI. We next used milder solubilization
conditions to allow for detection of the supercomplex of CI and
CIII (CI-CIII2) on BN-PAGE (Fig. 4A, lanes 3 and 4). The CICIII2 supercomplexes present as a lower molecular weight spe-
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
cies, indicating a supercomplex of crippled CI with CIII in the
Ndufs4fky/fky mitochondria. Immunoblotting with antibody
against the CI subunit NDUFA9 subsequent to BN-PAGE of 1%
Triton X-100-solubilized mitochondria confirmed the crippled
state of CI and the CI-CIII2 supercomplex from Ndufs4fky/fky
mitochondria (Fig. 4B). The amount of the crippled CI and
CI-CIII2 supercomplex was also decreased compared with wild
type mice. Furthermore, the assembly state and amount of CI
from heterozygous Ndufs4⫹/fky mitochondria did not differ
from wild type mitochondria, consistent with the results
obtained for the CI activity measurements (data not shown).
The composition of the crippled CI found in the BN-PAGE
experiments with Ndufs4fky/fky mitochondria was subsequently
determined using nano-liquid chromatography tandem mass
spectrometry (nano-LC-MS/MS) with CI from wild type and
Ndufs4fky/fky mitochondria. This resulted in the detection of 38
of the 45 subunits in wild type CI, whereas in the crippled complex, 30 subunits were detected (Table 1 and supplemental
Table 4). Five subunits remained undetected in both wild type
and crippled CI, indicating that these subunits may be difficult
to detect. Strikingly, in addition to NDUFS4, the subunits
NDUFA12, NDUFS1, NDUFS6, NDUFV1, and NDUFV2 were
JUNE 8, 2012 • VOLUME 287 • NUMBER 24
not detected in the crippled CI. These subunits, including
NDUFS4, make up the N module responsible for NADH oxidation and transfer of electrons to iron sulfur clusters (Fig. 5).
These data confirm that NDUFS4 is essential for the assembly
and/or stability of the N module component of the CI matrix
arm.
Not only is the crippled CI missing the N module, there were
additional proteins detected that specifically associated with
the crippled complex, namely NDUFAF1 (CIA30) and NDUFAF2 (B17.2L) (supplmental Table 4 and Fig. 5). These proteins
are known to be CI assembly factors and have previously been
found to associate with assembly intermediates in multiple CIdeficient patients (38 – 40). Moreover, NDUFAF1 was found
associated with the crippled CI in patients with pathogenic
NDUFS4 mutations (39, 41).
Metabolomic Analysis of Ndufs4fky/fky Mice Shows Increased
Hydroxyacylcarnitines—Having characterized the effect of
NDUFS4 loss on CI assembly, we next sought to assess its
impact on broader aspects of cellular metabolism. Tandem
mass spectrometric targeted analysis of acylcarnitines and
amino acids in the blood of Ndufs4fky/fky mice (mean age 40.1 ⫾
4.4 days for Ndufs4fky/fky mice and 40.1 ⫾ 4.4 days for control
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FIGURE 2. The “funky” mutation is a B2 SINE insertion into exon 3 of Ndufs4. A, sequencing chromatograph comparing Ndufs4 exon 3 in wild type and
homozygous mice; the inserted sequence begins 52 nt after the 5⬘ start of the exon. B, RepeatMasker output showing alignment of insert sequence with
B2_Mm1a; 171 of 176 nt ⫽ 97% match. Transition and transversion substitutions are indicated by i and v, respectively. C, Ndufs4fky locus showing the B2 insert
and target site duplication (TSD) sequences.
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
mice, mean ⫾ S.D.) showed only a few specific differences in
metabolite levels when compared with control animals (Fig. 6
and supplemental Table 5). Interestingly, hydroxyacylcarnitines with a carbon chain of C4:0, C16:0, and C18:1 were all
increased compared with control animals (Fig. 6, A–C).
Although the absolute levels of these metabolites in the blood
are low and probably will not directly influence disease outcome, the increased hydroxyacylcarnitines may indicate a block
in the third NAD⫹-dependent step of the mitochondrial fatty
acid ␤-oxidation pathway caused by the CI defect. However,
additional acylcarnitine analysis of liver and heart of
Ndufs4fky/fky mice revealed no large differences in carnitine species compared with control mice (supplemental Tables 6 and
7). Furthermore, oil red O staining of skeletal muscle failed to
detect lipid accumulation in Ndufs4fky/fky mice (supplemental
Fig. 2), which is occasionally found in patients with CI deficiency (5). These results suggest that the defect may result in
altered ratios of fatty acyl species but does not cause a gross
defect in fatty acid oxidation.
Tandem MS analysis of the blood amino acid levels showed
only significant changes in the levels of glycine, phenylalanine,
and homocitrulline, which were all elevated in Ndufs4fky/fky
blood (Fig. 6, D–F).
20658 JOURNAL OF BIOLOGICAL CHEMISTRY
DISCUSSION
Linkage in combination with expression profiling has been
successful in identifying autosomal recessive disease genes in
both mice and humans (42, 43). Here we performed an analysis
of expression data focused solely on the critical interval identified by the customized linkage analysis and fine mapping of the
susceptibility locus. This reduces the multiple testing penalty
for the differential expression analysis. Additionally, as far as we
are aware, this is the only example of using exon arrays, where
in hindsight, due to the nature of the mutation (see below), the
expression analyses identified not only the gene but the exon of
the gene mutated in the fky/fky phenotype. SINEs belong to a
family of endogenous retrovirus-like sequences known as
transposable elements, which are known to cause disease
through insertional mutagenesis (44 – 46). The spontaneous
occurrence of the fky mutation not only highlights the detrimental effects of insertional mutagenesis but also prompts us to
question if either of the parental transgenes Dnmt3L (DNA
methyltransferase 3-like gene) or RIP-mOVA (membranebound ovalbumin under the control of the rat insulin promoter)
had played a part in promoting mutagenic activity. Although
RIP-mOVA is an unlikely candidate (31), DNMT3L has been
found to be required for DNA methylation and genetic represVOLUME 287 • NUMBER 24 • JUNE 8, 2012
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FIGURE 3. B2 SINE insertion into Ndufs4 results in isolated CI deficiency in all tissues tested. A, RT-PCR showing truncated Ndufs4 transcripts in whole brain
RNA of Ndufs4fky/fky mice; wild type transcript variant (V1) was identified through the sequencing of clones; the schematic on the right describes the different
amplicons visible on the gel; white arrows indicate the locations of stop codons. B, NDUFS4 protein was not detected in whole brain, heart, liver, and skeletal
muscle of Ndufs4fky/fky mice via Western blot. C, CI activity, normalized against citrate synthase (CS), was reduced in all tissues tested of Ndufs4fky/fky mice (n ⫽
3–5). D, mitochondrial CII, CIII, and CIV activities were normal in brain tissues of Ndufs4fky/fky mice (n ⫽ 4). E, ATP synthesis capacity of heart and brain
mitochondria was determined as the CI-dependent ATP production (with substrate combination malate and glutamate) normalized against CII-dependent
ATP production (with substrate succinate in the presence of CI inhibitor rotenone) and is shown as a percentage of control mitochondria (age range P30 –P41,
heart n ⫽ 13, brain n ⫽ 5). C and D, black bars, wild type mice; white bars, Ndufs4fky/fky mice. E, control, Ndufs4⫹/f⫹ and Ndufs4⫹/fky; fky/fky, Ndufs4fky/fky. *, p ⬍ 0.05;
**, p ⬍ 0.01; ***, p ⬍ 0.005; #, p ⬍ 0.001; ##, p ⬍ 0.0005; ###, p ⬍ 0.0001; ####, p ⬍ 0.00005. Error bars, S.E.
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
FIGURE 5. Schematic representation of the results from nano-LC-MS/MS
analysis of the crippled CI in mitochondria from Ndufs4fky/fky mice. Left,
the different modules present in CI in wild type mice. Right, crippled complex
I from Ndufs4fky/fky mice misses all subunits that make up the N module,
whereas the assembly factors Ndufaf1 and Ndufaf2 were found associated
with the crippled complex I. Representation of the outcome of nano-LCMS/MS analysis was based on the assembly model described by McKenzie
and Ryan (64).
FIGURE 4. Depletion of NDUFS4 changes the assembly status of CI. A, representative BN-polyacrylamide gel showing crippled CI monomer when
Ndufs4fky/fky heart mitochondria are solubilized in 1% n-dodecyl ␤-maltoside
(DDM; lane 2). When Ndufs4fky/fky heart mitochondria are solubilized in 1%
Triton X-100 (TX100), the CI-CIII2 supercomplex (SC) can be observed (lanes
3 and 4). A large proportion of this CI-CIII2 SC is detected in the crippled
state in Ndufs4fky/fky mitochondria (lane 4). B, BN-PAGE followed by Western blot with antibody against CI subunit NDUFA9 shows weaker staining
and crippled status of CI (CI) monomer and CI-CIII2 (CI/CIII2) supercomplex
in 1% Triton X-100-solubilized mitochondria from heart tissues of
Ndufs4fky/fky mice.
TABLE 1
Representative MALDI-TOF results of isolated wild type and “crippled”
Ndufs4fky/fky complex I
Detected subunits
Not detected subunits
N module subunits detected
Detected assembly factors
a
b
Wild type
holocomplex
Ndufs4fky/fky
crippled complex
38
7a
6
0
30
15a
0b
2
Includes 5 subunit that were not detected in both.
None of the N module subunits were detected.
JUNE 8, 2012 • VOLUME 287 • NUMBER 24
sion of transposable elements in spermatogonia (30).
Dnmt3L⫺/⫺ male mice are sterile due to spermatogenic arrest,
whereas Dnmt3L⫹/⫺ males are phenotypically normal and fertile. Considering that the fky/fky phenotype arose from breeding Dnmt3L⫹/⫺ males, this suggests that the loss of one functional Dnmt3L allele may be sufficient to increase the frequency
of transposable element-related mutagenesis in the male germ
line.
The Ndufs4fky locus has a 13-nt target site duplication
5⬘-AAAACCAGAAAGG-3⬘ and a putative target sequence
5⬘-TCAAAA-3⬘, which are characteristic of documented insertions (47, 48) mediated by long interspersed nuclear elements
(LINEs) (49, 50) (Fig. 2C). The B2 SINE insertion introduced a
premature termination codon in Ndufs4, which most likely
triggered nonsense-mediated decay of mutant mRNA, resulting in a complete depletion of NDUFS4 protein as shown in Fig.
3, A and B (32).
Ndufs4 encodes an 18-kDa subunit of mitochondrial CI and
is highly expressed in energy-demanding tissues, such as the
heart, skeletal muscle, and various components of the central
nervous system, such as the amygdala, cerebellum, and dorsal
root ganglion (51). Patients diagnosed with homozygous
NDUFS4 mutations suffer from mitochondrial CI deficiency
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FIGURE 6. NDUFS4 depletion results in significant changes in blood
metabolite levels as measured by tandem MS. A–C, tandem MS analysis of
blood from dried bloodspot cards showed significant increases in hydroxyacylcarnitine species with a mass corresponding to hydroxy-C4:0 acylcarnitine,
hydroxy-C16:0 acylcarnitine, and hydroxy-C18:1 acylcarnitine in Ndufs4fky/fky
mice. D–F, the only amino acid levels that were significantly changed were
those of glycine, phenylalanine, and homocitrulline (all increased). n ⫽ 27 for
controls; n ⫽ 26 for Ndufs4fky/fky. Error bars, S.E.
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
20660 JOURNAL OF BIOLOGICAL CHEMISTRY
explanation is that because mice mature to adulthood in just 6
weeks, their longer survival in developmental terms and milder
neuropathology are due to the time required for pathology to
result from accumulated tissue damage.
At first glance, the predominantly neurological phenotype of
Ndufs4fky/fky mice may seem surprising, considering that the
Ndufs4 subunit is expressed ubiquitously and that CI activity
was decreased substantially in all tissues studied. However, the
selective vulnerability of the nervous system to mitochondrial
dysfunction has been noted previously (54). This is partly
explained by its high energy requirement, with the brain
accounting for ⬃20% of oxygen consumption and ⬃25% of glucose consumption despite representing only ⬃2% of body
weight (55). In Ndufs4fky/fky mice, a contributing factor may lie
in the observation that CI-linked ATP synthesis capacity was
also decreased more markedly in the brain than in the heart (64
and 82% of control values, respectively). Our studies of brain
homogenate also probably underestimate the functional consequences of decreased CI activity in some neural cells. Previous
studies found that ATP synthesis in rat synaptic mitochondria
began to fall when CI activity was decreased by 25%, whereas in
non-synaptic mitochondria, there was no effect until CI activity
was decreased by 72% (56). Hence, the neurological susceptibility of Ndufs4fky/fky mice probably relates to heterogeneity at the
level of CI activity within and between tissues, differing functional thresholds for CI activity loss between and within tissues,
and variation in downstream pathways related to cellular function and cell death (57).
Although the phenotypes of Ndufs4⫺/⫺ (13) and Ndufs4fky/fky
mice bear many similarities to the clinical features seen in
patients with CI deficiency, there are also differences. Distinctly, the transient fur loss seen in both models does not have
an equivalent human symptom, although hair and skin abnormalities have been observed in patients with mitochondrial disease (58 – 61). Progressive fur loss has been reported in a different mouse model of mitochondrial disease, the Polg “mutator”
mouse, which accumulate mitochondrial DNA mutations with
increasing age (62, 63). Ndufs4 does not appear to be highly
expressed in the skin, but age-specific onset of fur loss suggests
temporal regulation of Ndufs4. Future expression analysis of
Ndufs4 in skin samples before, during, and after the onset of fur
loss may provide further insight.
Analysis of the assembly status of Ndufs4fky/fky CI using BNPAGE revealed the presence of an ⬃830-kDa crippled CI species that could associate into supercomplexes (Fig. 4, A and B).
Earlier studies using fibroblasts of patients with Ndufs4 mutations showed the presence of a crippled species that is believed
to be an accumulated assembly intermediate of CI (36, 37, 41).
As with the low complex I activity found in all tissues, the small
amounts of assembled complex I and CI-CIII2 contrast with the
relatively high level of ATP synthesis. The latter is measured in
fibroblasts whose plasma membrane has been permeabilized
with a low concentration of digitonin and which retain an intact
mitochondrial inner membrane. CI activity is measured in sonicated tissue homogenates, and BN-PAGE studies use relatively
mild detergent conditions but nonetheless solubilize the
OXPHOS complexes from the mitochondrial inner membrane.
Given that NDUFS4 is involved in CI assembly and stability,
VOLUME 287 • NUMBER 24 • JUNE 8, 2012
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(OMIM number 252010). Symptomatically, these patients suffer from Leigh syndrome (OMIM number 256000), an early
onset progressive neurodegenerative disorder often seen in
congenital mitochondrial disorders, and exhibit symptoms
such as failure to thrive, lethargy, hypotonia, and neurodegenerative disease (4, 52). This spectrum of symptoms was recapitulated in a conventional knock-out mouse of Ndufs4 in which
exon 2 was excised using the Cre recombinase-loxP (Cre-loxP)
system (13). The Ndufs4fky/fky mice described here show essentially the same phenotype as the conventional knockout,
including the transient hair loss, failure to thrive, and development of neurological defects from P35, which usually resulted
in euthanasia of affected mice by P40 according to our ethical
guidelines.
Leigh syndrome is characterized by lesions in various parts of
the central nervous system, such as the brainstem, basal ganglia,
cerebellum, and spinal cord (4). Preliminary neuropathology
revealed no gross morphological abnormalities in the brain of
41- and 48-day-old Ndufs4fky/fky mice (supplemental Table 8).
In agreement with this observation, genome-wide microarray
analysis of the total RNA between whole brain of unaffected
and affected P37 mice showed only two differentially expressed
genes, both down-regulated, Ndufs4 and Ppdpf. Intensive and
sophisticated bioinformatic attempts failed to find even subtly
altered relevant biological pathways both with (e.g. keywords
brain, neuro, oxidative, ampk, and fatty) and without (MSigDB)
prior knowledge.
However, in agreement with the visual loss observed in
Ndufs4⫺/⫺ mice (13), axonal damage was present in optic nerve
of Ndufs4fky/fky mice by P58 (supplemental Fig. 3). Initial light
microscopic studies in brain regions of Ndufs4⫺/⫺ mice also did
not show differences between wild type and knock-out mice
(13). However, a more detailed study published subsequently by
the same group revealed neuronal loss in particular brain
regions of mice with a late stage disease phenotype, which was
suggested to result from progressive glial activation (14). Due to
our ethical guidelines, our mice were typically euthanized
before the onset of the late stage of disease as defined by Quintana et al. (14). This probably explains why we did not detect
significant neuropathological changes in the brain.
Measurement of CI activity, normalized against citrate synthase, detected a 78 –90% reduction in brain, heart, liver, and
muscle tissues from Ndufs4fky/fky mice (Fig. 3C), which is similar
to the levels found in muscle tissue of patients and initially
reported in tissues of the conventional Ndufs4⫺/⫺ mice (13, 52).
A subsequent study found somewhat higher activity in the
heart, where CI activity was only 56% reduced (53), which is
probably due to different sample preparation and assay methods. Similar assays of the other major mitochondrial complexes
(CII, CIII, and CIV) showed no differences in activity in
Ndufs4fky/fky mice (Fig. 3, C and D). These results provide convincing evidence that the homozygous Ndufs4 mutation has an
isolated effect on CI. Reduced CIII activity, which has been
found in some NDUFS4 patients (33), was not detected in
Ndufs4fky/fky mice. Ndufs4fky/fky mice survive to adulthood at 6
weeks of age, whereas most patients with homozygous Ndufs4
mutations die in infancy (33). This could suggest some functional redundancy of NDUFS4 in mice. However, a more likely
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
JUNE 8, 2012 • VOLUME 287 • NUMBER 24
detachment of the NADH oxidation module with the remaining crippled CI associated with the assembly factors NDUFAF1
and NDUFAF2.
Acknowledgments—Efficient animal management and data collection were facilitated by the Walter and Eliza Hall Institute of Medical
Research (WEHI) and Murdoch Childrens Research Institute animal
technicians. Processing of histological samples was aided by WEHI
Histology and the Integrative Neuroscience Facility. Affymetrix exon
arrays and linkage genotyping were performed by the Australian
Genome Research Facility, which was established through the Commonwealth-funded Major National Research Facilities Program.
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these observations together suggest that the amount of fully
assembled CI in the intact inner membrane may be higher than
that predicted by enzyme assay and BN-PAGE analyses.
Nano-LC-MS/MS analysis was performed to identify the
subunits that make up the crippled CI and showed the complete
lack of subunits belonging to the NADH oxidation (N) module.
This module is believed to be attached as a whole to an intermediate containing all other subunits at the final stage of the
assembly into mature CI (Fig. 5) (for a review, see Ref. 64). A CI
species lacking the N module has been identified previously in
in vitro subunit import studies using control and patient mitochondria (lacking NDUFS4 or NDUFS6) and is considered to be
a genuine CI assembly intermediate (41, 64). Supporting this,
our study here showed the association of CI assembly factors
NDUFAF1 and NDUFAF2 with the crippled complex species,
suggesting that the complex does not dissociate under the conditions of BN-PAGE. Although NDUFAF2 has been found
associated with the 830-kDa assembly intermediate present in
patients with mutations in NDUFS4, this has not been shown
previously for NDUFAF1 (39, 40). However, NDUFAF1 has
been detected in the 830-kDa assembly intermediate of control
mitochondria (38). We note that crippled CI was not detected
in the initial report on Ndufs4⫺/⫺ mice (13) but was described
recently in a study that also identified a small complex of ⬃200
kDa retaining NADH oxidation activity (53). Those observations are consistent with our results of mass spectrometric
analysis of crippled CI.
Measurement of acylcarnitine species is a well characterized
method of detecting defects in fatty acid oxidation in humans
and mice (65). Our metabolic screen of the blood of
Ndufs4fky/fky mice showed an increase in multiple hydroxyacylcarnitine species (Fig. 6, A–C). The exact structure of these
hydroxyacylcarnitine species cannot be determined via the tandem mass spectrometry method used in this study. However,
3-hydroxyacylcoenzyme A species are formed during mitochondrial ␤-oxidation and converted into ␤-ketoacylcoenzyme
A in the NAD⫹-dependent step of the ␤-oxidation spiral (for a
review, see Ref. 66). Hence, the increase of hydroxyacylcarnitines suggests a block in ␤-oxidation at the conversion of
3-hydroxyacylcoenzyme A into the corresponding acylcarnitine species. In Ndufs4fky/fky mice, CI appears to lack the module
responsible for the conversion of NADH into NAD⫹. This may
cause an imbalance in the NAD⫹/NADH ratio inside mitochondria and be the reason for the block in fatty acid ␤-oxidation. Studies in patients suffering from OXPHOS disorders
have shown the presence of increased serum alanine levels
and/or decreases in citrulline levels (67–70). This was not
found in the blood of Ndufs4fky/fky mice, which only had levels of
glycine, phenylalanine, and homocitrulline significantly elevated (Fig. 6, D–F). However, these levels were not dramatically
changed, and they probably do not impact greatly on the
phenotype.
In summary, we have characterized an animal model of CI
deficiency that spontaneously arose due to the insertion of a B2
SINE transposable element, using a novel combination of linkage and exon arrays. Biochemically, the Ndufs4fky/fky mice have
a CI defect similar to that observed in fibroblasts of patients
with mutations in NDUFS4. CI is unstable, resulting in the
Pathogenic B2 SINE Insertion Causing Complex I Deficiency in Mice
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SUPPLEMENTARY DATA
SUPPLEMENTARY FIGURE LEGENDS
SUPPLEMENTARY FIGURE 1. Genetic linkage analysis to locate the recessive C57BL/6 locus.
(A) Fine-mapping to mouse chromosome 13. Haplotypes across the critical region are shown with
grey boxes (F) representing B6/BC heterozygous loci and filled boxes (J) representing B6
homozygous loci; each column represents one mouse, each row is a genomic marker and critical
markers are highlighted. (n.d. = not done). (B) Microarray analysis moderated t-statistic barplot for 30
RefSeq genes between D13Mit196 and rs3708633; each bar represents one gene; the list of genes is
given in Supplementary Table 1. Significance threshold (P = 0.05) is shown by the red broken line (--). (C) Barplot of moderated t-statistics for individual exons of Ndufs4; each bar represents one exon;
significance threshold (P = 0.05) is shown by the red broken line (---).
SUPPLEMENTARY FIGURE 2. Oil red O staining of quadriceps muscle. Staining patterns,
restricted to the periphery of muscle fibres, are similar between wild type and Ndufs4fky/fky
cryosections. Lipid accumulation is not evident in Ndufs4fky/fky mice.
SUPPLEMENTARY FIGURE 3. Optic nerve histology of wild type and Ndufs4fky/fky mice.
Semithin sections of optic nerve stained with toluidine blue show damaged axonal structure (black
arrow heads) in Ndufs4fky/fky optic nerve at P58 (n=2). Moreover, less axons are present in the section
of Ndufs4fky/fky optic nerve, illustrating axonal loss which is more severe in the last case (right panel).
3DWKRJHQLF%6,1(LQVHUWLRQFDXVLQJFRPSOH[,GHILFLHQF\LQPLFH
Supplementary Figure 1
A
Mouse chromosome 13q
62.0cM
B
Affected (homozygous) progeny
Chr Position(bp) Mar ker
215F 744F 887F 701M 327M 567M 212M
n.d.
13 112734483 rs29568773
65.0cM
13
113486588 D13MIT260
67.5cM
13
113757142 rs13482018
68.0cM
13
113882768 D13MIT196
74.9cM
13
119862123
rs3708633
75.0cM
13
120094608
rs6412462
BW
n.d.
B
n.d.
n.d.
n.d.
n.d.
C
4.0
2.0
Ndufs4 exon #
1
2
3
4
0.0
0.0
-5.0
t-statistic
t-statistic
-2.0
-4.0
-6.0
-8.0
-10.0
-10.0
-15.0
-12.0
-14.0
-16.0
-18.0
-20.0
Ndufs4
P-value = 4x10-24
-25.0
P-value = 5x10-5
5
3DWKRJHQLF%6,1(LQVHUWLRQFDXVLQJFRPSOH[,GHILFLHQF\LQPLFH
Supplementary Figure 2
A
wild type
B
Ndufs4
fky/fky
3DWKRJHQLF%6,1(LQVHUWLRQFDXVLQJFRPSOH[,GHILFLHQF\LQPLFH
Supplementary Figure 3
wild type
Ndufs4
fky/fky
3DWKRJHQLF%6,1(LQVHUWLRQFDXVLQJFRPSOH[,GHILFLHQF\LQPLFH
Supplementar y Table 1: Oligonucleotide primers used in microsatellite fine-mapping of mouse
chromosome 13. C57BL/6 alleles can be distinguished from BALB/c alleles based on the size of
pcr products
Micr osatellite
Pr imer
dir ection
Sequence (5'-3')
Forward
CAATCCTGAGTGGAACAAAGTG
Reverse
GATGTGCTGAGTTGTGAACCA
Forward
ACTTTGACACCTTTTCTCAGTTCC
Reverse
TGGGATTAAAGCAGACATACAGG
Forward
TCAGCCAATATACCACTCTAATAAA
Reverse
GCCAAACAGCCAAAAATATAC
Forward
ACTGCCCATTCATGTGTGTG
Reverse
AGGAAGAAAAATTAGAAGAGAAAAAAA
Forward
AGAGACCCTGACTCAAACAAGG
Reverse
TTTTTCTTTTATTTTGGTGATTTGG
Forward
TCCTCAGACCTCTGCTTTGA
Reverse
CATGCATAAAGGTAATCCCCA
Forward
CATCCAGGAAGGCAATAAGG
Reverse
CAAATGCACAGTGCCGAG
Forward
TTGACTCTTTAAAAGACTGGTGTCT
Reverse
CCTGGGAAGTGTATCCTAGTGG
Forward
GCTCAAAGCCCTGGGTTC
Reverse
CACTGTGATGATGGCTAGTTCTG
Forward
TCCTGCAAAAGTGGAGCC
Reverse
TGGAAACAAGCTCTTGGAGG
Forward
ACCAATCTCCCAGGTAACCC
Reverse
GGTGGGAGGAGAATGCAATA
Forward
CTGGTAACGATGTATTATCAGTGTTT
Reverse
CGACAATGAGATATTTTTTGTTAATC
Forward
AAAATTATGAATGCAAGTGCACA
Reverse
GGGTATCTGATACCAGTGCCC
Forward
TAAATTTGGATGCAGACAATGG
Reverse
TTAAAAATAGAAATGGCTCTGTGTG
Forward
TGCAGATAGGGTCTGAAGGC
Reverse
GAGCCACAATGTCAAGCAAA
D13MIT97
D13MIT126
D13MIT27
D13MIT193
D13MIT105
D13MIT320
D13MIT147
PCR pr oduct (bp)^
C57BL/6 BALB/c
152
160
142
132
202
198
126
124
149
151
279
275
108
93
142
144
148
150
146
140
164
156
201
193
100
88
115
109
133
137
D13MIT36
D13MIT72
D13MIT51
D13MIT227
D13MIT170
D13MIT291
D13MIT260
D13MIT196
^ Based on DNA sequences downloaded from UCSC genome browser
3DWKRJHQLF%6,1(LQVHUWLRQFDXVLQJFRPSOH[,GHILFLHQF\LQPLFH
6XSSOHPHQWDU\ 7DEOH 0LFURDUUD\ DQDO\VLV VWDWLVWLFV IRU WKH JHQHV LQ WKH FULWLFDO fky OLQNDJH UHJLRQ One row represents one gene (except for Ppap2a which
has two separate probesets). Genes are ordered by genomic location. FC: fold change, t: moderated t-statistic, FDR: false discovery rate.
5HI6HT,'
NM_029898
NM_010560
NM_139299
NM_010029
NM_178746
NM_008247
NM_008903
NM_028151
NM_172594
NM_001037914
NM_027127
NM_010370
NM_008196
NM_023612
NM_130796
NM_019960
NM_172595
NM_177004
NM_010887
NM_008046
NM_175218
NM_013826
NM_008396
NM_001033228
NM_134058
NM_021459
NM_010330
NM_010408
NM_021556
NM_008002
*HQRPLFSRVLWLRQES
113409697 - 113504880
113584986 - 113627719
113643684 - 113701540
113719213 - 113773179
113781653 - 113853776
113921669 - 113988558
113921800 - 113988520
113988659 - 114048258
114048671 - 114090065
114114745 - 114121272
114163640 - 114167266
114214702 - 114221857
114292752 - 114301775
114330536 - 114338982
114713060 - 114739423
114783773 - 114784537
114915436 - 115278307
115245225 - 115272470
115409528 - 115508921
115574224 - 115579464
115728114 - 115777702
115939163 - 115949602
115956791 - 116052919
116080956 - 116222842
116209233 - 116211036
117418962 - 117430367
118339912 - 118393379
118721980 - 119096083
119499390 - 119506533
119833906 - 119910984
*HQHQDPH
ankyrin repeat domain 55
interleukin 6 signal transducer
interleukin 31 receptor A
DEAD (Asp-Glu-Ala-Asp) box polypeptide 4
RIKEN cDNA 9130023D20 gene
phosphatidic acid phosphatase 2a
phosphatidic acid phosphatase 2a
superkiller viralicidic activity 2-like 2 (S. cerevisiae)
DEAH (Asp-Glu-Ala-His) box polypeptide 29
similar to geminin (LOC622408), mRNA
RIKEN cDNA 2310016C16 gene
granzyme A
granzyme K
endothelial cell-specific molecule 1
sorting nexin associated golgi protein 1
heat shock protein 3
ADP-ribosylation factor-like 15
RIKEN cDNA A430090L17 gene
NADH dehydrogenase (ubiquinone) Fe-S protein 4
follistatin
RIKEN cDNA 4930544M13 gene
molybdenum cofactor synthesis 2
integrin alpha 2
integrin alpha 1
pelota homolog (Drosophila)
ISL1 transcription factor, LIM/homeodomain
embigin
hyperpolarization-activated, cyclic nucleotide-gated K+ 1
mitochondrial ribosomal protein S30
fibroblast growth factor 10
*HQHV\PERO
Ankrd55
Il6st
Il31ra
Ddx4
Slc38a9
Ppap2a
Ppap2a
Skiv2l2
Dhx29
NP_001033003.1
Gpx8
Gzma
Gzmk
Esm1
Snx18
Hspb3
Arl15
A430090L17Rik
Ndufs4
Fst
4930544M13Rik
Mocs2
Itga2
Itga1
Pelo
Isl1
Emb
Hcn1
Mrps30
Fgf10
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1.00
1.06
0.97
0.90
1.09
0.84
0.89
0.92
0.93
0.78
1.03
1.06
1.13
1.02
0.93
0.94
1.04
1.03
0.87
1.00
0.92
0.97
1.07
0.89
1.02
1.05
0.95
0.97
0.96
W
-0.01
0.72
-0.37
-1.21
1.02
-2.01
-1.43
-1.00
-0.87
-2.91
0.30
0.66
1.50
0.23
-0.83
-0.75
0.48
0.32
-1.68
0.05
-1.02
-0.39
0.76
-1.33
0.20
0.53
-0.55
-0.32
-0.42
3YDOXH
0.99
0.48
0.71
0.23
0.31
0.05
0.16
0.32
0.39
0.01
0.76
0.51
0.14
0.82
0.41
0.46
0.64
0.75
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0.10
0.96
0.31
0.70
0.45
0.19
0.84
0.60
0.59
0.75
0.67
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0.99
0.88
0.88
0.86
0.87
0.49
0.78
0.87
0.88
0.08
0.88
0.88
0.78
0.90
0.88
0.88
0.88
0.88
(
0.73
0.99
0.87
0.88
0.88
0.81
0.90
0.88
0.88
0.88
0.88
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4646896
4657135
5314511
5190989
5511583
4811229
5014211
5201771
115409545 - 115409601
115409604 - 115409640
115409672 - 115409699
115428698 - 115428747
115437743 - 115437853
115437876 - 115437905
115472334 - 115472403
115508842 - 115508880
115508896 - 115508923
Exon 5
Exon 5
Exon 5
Exon 4
Exon 3
Exon 3
Exon 2
Exon 1
Exon 1
4
4
4
4
4
4
4
4
4
2
1
1
2
3
1
2
1
4
7.8E-03
2.7E-02
9.5E-03
3.8E-03
1.2E-04
1.5E-03
1.7E-02
1.8E-02
1.8E-02
-6.55
-10.36
-3.12
-8.34
-21.46
-8.30
-9.62
-6.16
-6.74
3.8E-03
7.6E-04
4.0E-02
1.6E-03
5.5E-05
1.7E-03
9.9E-04
4.7E-03
3.4E-03
5.4E-03
3.3E-03
4.0E-02
3.3E-03
5.5E-04
3.3E-03
3.3E-03
5.8E-03
5.4E-03
Pathogenic B2 SINE insertion causing complex I deficiency in mice
Supplementary Table 4: Subunits of wildtype and Ndufs4fky/fky complex I detected by MALDITOF. One row represents one complex I subunit.
Subunits
Location
Subcomplex
(Lazarou,
Thorburn et al.
2009)
mt-Nd1
Membrane
Ιγ
mt-Nd2
Membrane
Ιγ
mt-Nd3
Membrane
Ιγ
mt-Nd4
Membrane
Ιγ
mt-Nd4L
Membrane
Ιγ
mt-Nd5
Membrane
Ιβ
mt-Nd6
Membrane
Ια
Ndufa1
Peripheral matrix arm
Ιλ,α
Ndufa2
Peripheral matrix arm
Ιλ,α
Ndufa3
Membrane
Ια
Ndufa4
Membrane
Ια
Ndufa5
Peripheral matrix arm
Ndufa6
Membrane
Ndufa7
Peripheral matrix arm
Ndufa8
Membrane
Ια
Ndufa9
Membrane
Ια
Ndufa10
Membrane
Ια
Ndufa11
Peripheral matrix arm
Ιλ,α
Ndufa12
Peripheral matrix arm
Ιλ,α
Ndufa13
Membrane
Ιλ,α
Ndufs1
Peripheral matrix arm
Ιλ,α
Ndufs2
Peripheral matrix arm
Ιλ,α
Ndufs3
Peripheral matrix arm
Ιλ,α
Ndufs4
Peripheral matrix arm
Ιλ,α
Ndufs5
Peripheral matrix arm
Ιλ,α
Ndufs6
Peripheral matrix arm
Ιλ,α
Ndufs7
Peripheral matrix arm
Ιλ,α
Ndufs8
Peripheral matrix arm
Ιλ,α
Ndufb1
Missing from mitocarta
Ndufb2
Membrane
Ιβ
Ndufb3
Membrane
Ιβ
Ndufb4
Membrane
Ιβ
Ndufb5
Membrane
Ιβ
wild type
Ndufs4fky/fky
holo
complex I
"crippled"
complex I
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Ιλ,α
Ια
N.D.
Ιλ,α
N.D.
8
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Pathogenic B2 SINE insertion causing complex I deficiency in mice
Ndufb6
Membrane
Ιβ
Ndufb7
Membrane
Ιβ
Ndufb8
Membrane
Ιβ
Ndufb9
Membrane
Ιβ
Ndufb10
Membrane
Ιβ
Ndufb11
Membrane
Ιβ
Ndufv1
Peripheral matrix arm
Ιλ,α
N.D.
Ndufv2
Peripheral matrix arm
Ιλ,α
N.D.
Ndufv3
Peripheral matrix arm
Ιλ,α
Ndufab1
Peripheral matrix arm
Ια,β
Ndufc1
Membrane
Ιγ
Ndufc2
Membrane
Ιβ
Total
45
detected
N.D.
N.D.
N.D.
N.D.
38
30
Ndufaf1
N.D.
Ndufaf2
N.D.
Dark grey boxes, detected subunits. Light grey boxes, detected N module subunits (except Ndufv3).
N.D., not detected subunits.
Reference: Lazarou, M., D. R. Thorburn, et al. (2009). "Assembly of mitochondrial complex I and
defects in disease." Biochim Biophys Acta 1793(1): 78-88.
9
Pathogenic B2 SINE insertion causing complex I deficiency in mice
Supplementary Table 5: Blood metabolite levels analysed by tandem mass spectrometry. Each
row shows the levels of amino acids or acylcarnitines in the blood of control and Ndufs4fky/fky mice.
=
Control (n 27)
Ndufs4fky/fky (n=27)
Metabolite
Mean (µM) ± SEM Mean (µM) ± SEM P
GUANIDINOACETIC ACID
2.92
±
0.34
2.91
±
0.26
9.8E-01
OH PROLINE
9.09
±
1.26
6.81
±
1.00
1.6E-01
GLYCINE
254.43
±
10.07
317.50
±
11.82
1.6E-04#
ALANINE
533.19
±
34.95
603.44
±
47.64
2.4E-01
VALINE
132.98
±
8.00
134.49
±
10.40
9.1E-01
ORNITHINE
38.42
±
2.95
41.13
±
3.29
5.4E-01
METHIONINE
44.58
±
3.15
45.95
±
4.19
7.9E-01
PHENYLALANINE
50.85
±
2.79
60.40
±
3.00
2.3E-02**
GLYCYL-PROLINE
2.16
±
0.14
2.39
±
0.15
2.7E-01
ARGININE
48.72
±
3.32
51.55
±
4.06
5.9E-01
CITRULLINE
65.96
±
5.20
56.67
±
2.94
1.3E-01
TYROSINE
71.65
±
4.12
87.74
±
7.19
5.9E-02
HOMOCITRULLINE
1.03
±
0.10
1.56
±
0.14
3.1E-03***
ARGININOSUCCINIC ACID
0.45
±
0.11
0.65
±
0.13
2.7E-01
FREE CARNITINE
31.20
±
1.25
26.44
±
0.93
3.7E-03***
C2 ACYLCARNITINE
78.28
±
4.53
81.49
±
4.61
6.2E-01
C3 ACYLCARNITINE
1.06
±
0.07
0.74
±
0.06
1.7E-03***
C4 ACYLCARNITINE
0.59
±
0.08
0.61
±
0.08
8.9E-01
C5:1 ACYLCARNITINE
0.04
±
0.01
0.02
±
0.00
1.0E-01
C5 ACYLCARNITINE
0.12
±
0.01
0.12
±
0.01
5.8E-01
OH C4 ACYLCARNITINE
0.19
±
0.02
0.58
±
0.06
6.0E-07##
C6 ACYLCARNITINE
0.10
±
0.01
0.07
±
0.01
6.0E-02
OH C5 ACYLCARNITINE
0.15
±
0.01
0.14
±
0.01
4.8E-01
C8 ACYLCARNITINE
0.06
±
0.01
0.05
±
0.00
2.0E-01
C10 ACYLCARNITINE
0.04
±
0.00
0.04
±
0.00
3.3E-01
C12 ACYLCARNITINE
0.09
±
0.02
0.12
±
0.02
4.7E-01
C6 DC ACYLCARNITINE
0.00
±
0.00
0.00
±
0.00
2.6E-01
OH C5 DC ACYLCARNITINE
0.01
±
0.00
0.01
±
0.00
1.3E-01
C14:1 ACYLCARNITINE
0.07
±
0.01
0.06
±
0.01
3.0E-01
C14 ACYLCARNITINE
0.13
±
0.01
0.12
±
0.01
8.0E-01
C16 ACYLCARNITINE
0.98
±
0.06
1.02
±
0.05
6.4E-01
OHC16 ACYLCARNITINE
0.04
±
0.00
0.12
±
0.02
3.2E-03***
C18 ACYLCARNITINE
0.44
±
0.02
0.36
±
0.02
1.0E-02*
OH C18:1 ACYLCARNITINE
0.04
±
0.00
0.11
±
0.02
8.6E-03**
fky/fky
Significant changes between Ndufs4
mice and control as by t-test are indicated in the last
column. * P < 0.05; ** P < 0.01; *** P < 0.005; # P < 0.0005; ## P < 0.00005.
10
Pathogenic B2 SINE insertion causing complex I deficiency in mice
Supplementary Table 6: Heart acylcarnitine levels analysed by tandem mass spectrometry. Each
row shows the levels of acylcarnitines in the liver of control and Ndufs4fky/fky mice expressed as pmole
acylcarnitine species per mg of heart weight (wet weight, WW).
Control (n=20)
Ndufs4fky/fky (n=13)
Mean
Mean
Metabolite
± SEM (pmole/mg
± SEM P
(pmole/mg
WW)
WW)
FREE CARNITINE
521.42
±
36.35
711.18
±
63.46
1.01E-02*
C2 ACYLCARNITINE
80.89
±
4.08
83.57
±
7.08
7.27E-01
C3 ACYLCARNITINE
2.45
±
0.28
2.93
±
0.54
3.95E-01
C4 ACYLCARNITINE
5.02
±
0.58
3.28
±
0.71
7.62E-02
C5:1 ACYLCARNITINE
0.11
±
0.01
0.14
±
0.01
1.02E-01
C5 ACYLCARNITINE
0.57
±
0.08
0.36
±
0.03
2.90E-02*
1.27
±
1.03
±
0.17
2.58E-01
0.62
±
0.68
±
0.07
5.03E-01
1.40
±
2.22
±
0.36
6.15E-02
2.79
±
1.64
±
0.65
1.82E-01
2.65
±
2.92
±
0.86
8.46E-01
3.86
±
1.72
±
1.04
1.41E-01
6.26
±
3.57
±
1.55
1.73E-01
12.92
±
7.74
±
2.17
3.42E-02*
C18:1 ACYLCARNITINE
21.91
±
2.26
10.61
±
3.23
7.67E-03**
C18 ACYLCARNITINE
7.32
±
1.12
3.60
±
1.24
4.56E-02*
OH C4 ACYLCARNITINE
8.69
±
0.82
8.72
±
1.07
9.81E-01
OH C5 ACYLCARNITINE
1.82
±
0.14
2.36
±
0.19
3.08E-02*
OH C5 DC ACYLCARNITINE
0.10
±
0.02
0.10
±
0.02
9.96E-01
OH C6 ACYLCARNITINE
2.07
±
0.64
2.11
±
0.76
9.70E-01
OH C8 ACYLCARNITINE
5.31
±
1.03
8.41
±
1.48
9.27E-02
OH C10 ACYLCARNITINE
1.00
±
0.24
1.36
±
0.34
3.96E-01
2.46
±
2.62
±
1.08
8.96E-01
1.31
±
1.29
±
0.24
9.54E-01
3.02
±
4.29
±
0.73
1.34E-01
10.99
±
8.95
±
3.45
6.74E-01
0.80
±
0.63
±
0.17
3.26E-01
0.09
±
0.07
±
0.02
5.05E-01
C6 ACYLCARNITINE
C8 ACYLCARNITINE
C10 ACYLCARNITINE
C12 ACYLCARNITINE
C6 DC ACYLCARNITINE
C14:1 ACYLCARNITINE
C14 ACYLCARNITINE
C16 ACYLCARNITINE
OH C12 ACYLCARNITINE
OH C14 ACYLCARNITINE
OHC16 ACYLCARNITINE
OH C18:1 ACYLCARNITINE
OH C18 ACYLCARNITINE
OH C20 ACYLCARNITINE
0.12
0.05
0.16
0.51
0.93
0.87
1.14
1.22
0.72
0.20
0.46
3.02
0.08
0.01
Significant changes between Ndufs4fky/fky mice and control as by t-test are indicated in the last
column. * P < 0.05; ** P < 0.01.
11
Pathogenic B2 SINE insertion causing complex I deficiency in mice
Supplementary Table 7: Liver acylcarnitine levels analysed by tandem mass spectrometry. Each
row shows the levels of acylcarnitines in the liver of control and Ndufs4fky/fky mice expressed as pmole
acylcarnite species per mg of liver weight (wet weight, WW).
Control (n=20)
Ndufs4fky/fky (n=13)
Mean
Mean
Metabolite
± SEM (pmole/mg
± SEM P
(pmole/mg
WW)
WW)
FREE CARNITINE
214.40
±
9.09
175.92
±
15.00
2.64E-02*
C2 ACYLCARNITINE
35.15
±
2.01
29.29
±
3.31
1.18E-01
C3 ACYLCARNITINE
2.08
±
0.26
2.30
±
0.41
6.35E-01
C4 ACYLCARNITINE
4.64
±
0.69
4.81
±
0.95
8.79E-01
C5:1 ACYLCARNITINE
0.02
±
0.00
0.03
±
0.00
9.98E-02
C5 ACYLCARNITINE
0.69
±
0.10
1.03
±
0.19
1.02E-01
0.89
±
1.24
±
0.34
3.28E-01
0.37
±
0.75
±
0.10
3.18E-03***
0.16
±
0.22
±
0.03
6.33E-02
0.47
±
0.76
±
0.13
5.85E-02
1.07
±
0.67
±
0.15
3.58E-02*
0.29
±
0.42
±
0.08
1.64E-01
0.49
±
0.49
±
0.07
9.51E-01
0.95
±
1.11
±
0.26
5.78E-01
C18:1 ACYLCARNITINE
1.27
±
0.15
2.36
±
0.95
2.82E-01
C18 ACYLCARNITINE
0.42
±
0.06
0.58
±
0.18
4.06E-01
OH C4 ACYLCARNITINE
3.59
±
0.30
4.94
±
0.63
6.73E-02
OH C5 ACYLCARNITINE
0.43
±
0.02
0.43
±
0.03
8.39E-01
OH C5 DC ACYLCARNITINE
0.09
±
0.01
0.09
±
0.02
9.30E-01
OH C6 ACYLCARNITINE
0.27
±
0.02
0.45
±
0.09
8.57E-02
OH C8 ACYLCARNITINE
0.49
±
0.06
0.37
±
0.04
1.27E-01
OH C10 ACYLCARNITINE
0.78
±
0.07
0.79
±
0.14
9.56E-01
1.31
±
0.84
±
0.24
3.35E-01
0.23
±
0.37
±
0.08
9.29E-02
0.11
±
0.14
±
0.02
1.70E-01
0.14
±
0.31
±
0.08
6.22E-02
0.05
±
0.09
±
0.01
1.88E-02**
0.01
±
0.02
±
0.00
9.57E-02
C6 ACYLCARNITINE
C8 ACYLCARNITINE
C10 ACYLCARNITINE
C12 ACYLCARNITINE
C6 DC ACYLCARNITINE
C14:1 ACYLCARNITINE
C14 ACYLCARNITINE
C16 ACYLCARNITINE
OH C12 ACYLCARNITINE
OH C14 ACYLCARNITINE
OHC16 ACYLCARNITINE
OH C18:1 ACYLCARNITINE
OH C18 ACYLCARNITINE
OH C20 ACYLCARNITINE
0.17
0.04
0.01
0.05
0.11
0.03
0.05
0.10
0.33
0.02
0.01
0.02
0.00
0.00
Significant changes between Ndufs4fky/fky mice and control as by t-test are indicated in the last
column. * P < 0.05; ** P < 0.01; *** P < 0.005.
12
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Supplementary Table 8: Comparisons of anatomical brain regions between Ndufs4 fky/fky
and wildtype mice. Measurement of brain regions with Image Pro Plus. Measurements are
depicted in microns.
Brain region
(fky/fky)
n=3
(+/+)
n=2
P
Motor cortex
1473 ± 13.65
1364 ± 3.646
0.0088**
Sensory cortex 1
1439 ± 30.22
1616 ± 6.492
0.0207*
Sensory cortex 2
1140 ± 22.05
1379 ± 77.57
0.0339*
Striatum
2370 ± 94.56
2504 ± 25.75
0.3589
Ventricle
394.0 ± 31.75
516.3 ± 194.0
0.4755
Dentate gyrus length
1854 ± 153.7
2017 ± 97.71
0.4966
Dentate gyrus width
125.1 ± 7.238
123.0 ± 5.383
0.8387
CA1 length
1778 ± 302.4
2032 ± 157.5
0.5336
CA1 width
79.42 ± 16.1 7
75.81 ± 8.023
0.8601
Substantia nigra
4211 ± 546.7
3673 ± 68.19
0.4319
Pons
2344 ± 214.5
2471 ± 117.6
0.6929
2Cb length
1166 ± 92.92
996.8 ± 135.7
0.3590
2Cb width
905.6 ± 35.19
817.1 ± 295.0
0.7196
Area of hemispheres
(section 1)
33.07 x10
-6
± 1.93 x10
-6
3.37 x10
-6
± 1.8 x10
-6
Area of hemispheres
(section 1)
43.33 x10
-6
± 3.48 x10
-5
41.65 x 10
-6
± 6.15 x 10
-6
0.7390
Area of hemispheres
(section 1)
23.37 x10
-6
± 8.17 x10
-5
27.85 x 10
-6
± 4.05 x 10
-6
0.2555
0.8388
Mean ± SD for Ndufs4 fky/fky mice (fky/fky), mean ± spread between 2 samples for wildtype
mice (+/+). Significant changes between Ndufs4 fky/fky mice and wildtype as by t-test are indicated
in last column. * P < 0.05; ** P < 0.01.
Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse
Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE)
Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene
Dillon W. Leong, Jasper C. Komen, Chelsee A. Hewitt, Estelle Arnaud, Matthew
McKenzie, Belinda Phipson, Melanie Bahlo, Adrienne Laskowski, Sarah A. Kinkel,
Gayle M. Davey, William R. Heath, Anne K. Voss, René P. Zahedi, James J. Pitt,
Roman Chrast, Albert Sickmann, Michael T. Ryan, Gordon K. Smyth, David R.
Thorburn and Hamish S. Scott
J. Biol. Chem. 2012, 287:20652-20663.
doi: 10.1074/jbc.M111.327601 originally published online April 25, 2012
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