Early-onset liver mtDNA depletion and late-onset
proteinuric nephropathy in Mpv17 knockout mice
Carlo Viscomi1, Antonella Spinazzola1, Marco Maggioni3, Erika Fernandez-Vizarra1,
Valeria Massa1, Claudio Pagano4, Roberto Vettor4, Marina Mora2and Massimo Zeviani1,?
1Unit of Molecular Neurogenetics – Pierfranco and Luisa Mariani Center for the Study of Mitochondrial Disorders in
Children and2Unit of Neuromuscular Diseases, IRCCS Foundation Neurological Institute ‘C. Besta’, Milan, Italy,
3Service of Pathology, San Paolo University Hospital, Milan, Italy and4Department of Medical and Surgical Sciences,
University of Padova School of Medicine, Padova, Italy
Received August 2, 2008; Revised and Accepted September 22, 2008
In humans, MPV17 mutations are responsible for severe mitochondrial depletion syndrome, mainly affecting
the liver and the nervous system. To gain insight into physiopathology of MPV17-related disease, we inves-
tigated an available Mpv17 knockout animal model. We found severe mtDNA depletion in liver and, albeit to a
lesser extent, in skeletal muscle, whereas hardly any depletion was detected in brain and kidney, up to 1 year
after birth. Mouse embryonic fibroblasts did show mtDNA depletion, but only after several culturing pas-
sages, or in a serumless culturing medium. In spite of severe mtDNA depletion, only moderate decrease in
respiratory chain enzymatic activities, and mild cytoarchitectural alterations, were observed in the
Mpv172/2livers, but neither cirrhosis nor failure ever occurred in this organ at any age. The mtDNA transcrip-
tion rate was markedly increased in liver, which could contribute to compensate the severe mtDNA depletion.
This phenomenon was associated with specific downregulation of Mterf1, a negative modulator of mtDNA
transcription. The most relevant clinical features involved skin, inner ear and kidney. The coat of the
Mpv172/2mice turned gray early in adulthood, and 18-month or older mice developed focal segmental glo-
merulosclerosis (FSGS) with massive proteinuria. Concomitant degeneration of cochlear sensory epithelia
was reported as well. These symptoms were associated with significantly shorter lifespan. Coincidental
with the onset of FSGS, there was hardly any mtDNA left in the glomerular tufts. These results demonstrate
that Mpv17 controls mtDNA copy number by a highly tissue- and possibly cytotype-specific mechanism.
The term mitochondrial DNA depletion syndrome (MDS) des-
ignates a group of autosomal recessive traits characterized by
low mtDNA copy number in specific tissues (1).
Three clinical presentations are known: myopathic, ence-
phalomyopathic and hepatocerebral. This condition is geneti-
cally heterogeneous; in ?80% of the cases the gene remains
elusive. In the remaining 20%, several disease genes have
been identified, that are responsible for different clinical
presentations (2). For instance, mutations in thymidine
kinase 2 (TK2) (3) and deoxyguanosine kinase (dGK) (4),
two enzymes involved in deoxynucleotide recycling in mito-
chondria, cause muscle- or liver-specific forms of MDS,
reductase subunit 2 (p53-R2) and thymidine phosphorylase
(TP), two cytosolic enzymes controlling the de novo biosyn-
thesis of deoxynucleotides (p53-R2), or the catabolism of
nucleotide precursors (TP), are responsible for severe
myopathic MDS with proximal tubule insufficiency (5), and
myoneurogastrointestinal encephalomyopathy (MNGIE) (6),
respectively. Also certain recessive mutations in the catalytic
subunit of mtDNA-specific polymerase g (7,8), and Twinkle,
the mtDNA helicase (9,10), can cause different forms of
?To whom correspondence should be addressed at: Unit of Molecular Neurogenetics, IRCCS Foundation Neurological Institute ‘C. Besta’, Via Temolo
4, 20126 Milan, Italy. Tel: þ39 0223942630; Fax: þ39 0223942619; Email: firstname.lastname@example.org
# 2008 The Author(s)
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Human Molecular Genetics, 2009, Vol. 18, No. 1
Advance Access published on September 24, 2008
hepatocerebral MDS. These proteins are all involved in the
maintenance of mtDNA, either by controlling the supply of
deoxynucleotides to, or by carrying out the synthesis of
mtDNA. However, for other MDS-associated genes, the mech-
anism that links mutations to mtDNA depletion is unclear, and
the very function of the corresponding proteins remains elusive.
This uncertainty is well illustrated by SUCLA2 and SUCLG1,
encoding two isoforms of Krebs-cycle succinyl–CoA ligase,
which are involved in encephalomyopathic MDS (11,12), and
by MPV17, a gene on human chromosome 2 that we showed
to be responsible for a peculiar form of hepatocerebral MDS
(13). Additional MPV17 mutant patients have later been
reported in different ethnic backgrounds (14–16). The onset
of the MPV17-associated disease is typically heralded by
severe hypoglycemic crises in early infancy, associated with
rapidly progressive deterioration of hepatic function, leading
to liver cirrhosis and failure. Prevention of hypoglycemic epi-
sodes by frequent meals, based on prompt-release carbo-
hydrates such as corn starch, can be effective in limiting
hepatic damage (13). Nevertheless, in spite of the possibility
to obtain partial control of fatal metabolic accidents, the surviv-
ing patients invariably progress in their liver degeneration, and
develop neurological symptoms in their childhood. The full-
blown syndrome has been thoroughly investigated in subjects
affected by Navajo neurohepatopathy (NNH), a disease that
has been known for decades to afflict the Navajo people of
South-western USA. NNH was recently attributed to a specific
mutation in the MPV17 protein, the p.R50Q, which originated
from a single ancestral carrier common to all NNH patients
(17). Clinical features of NNH/MPV17 syndrome include
sensory motor neuropathy with ataxia, leukoencephalopathy,
corneal ulcerations, acral mutilation, poor weight gain, short
stature, sexual infantilism, serious systemic infections, and of
course liver derangement (18). The same p.R50Q mutation
responsible for NNH was previously found in a large
MPV17-associated MDS family from Southern Italy (13), but
the mutational event was proven to have occurred indepen-
dently in the two family sets (19).
The Mpv17 gene was first identified in laboratory mice
created by random insertion of a retroviral construct in
the genome of mouse embryonic stem cells (20). The
Mpv172/2mice showed severe kidney dysfunction dominated
by proteinuria due to focal segmental glomerulosclerosis
(FSGS) (21). The onset was in early mouse adulthood (2–3
months after birth) and the course was progressive, leading
to systemic hypertension but no renal failure (22). In combi-
nation with the kidney phenotype, the Mpv172/2mice dis-
played degeneration of the inner ear structures, particularly
of the organ of Corti and stria vascularis, determining pro-
found hearing loss (23,24). However, after a few generations
the renal phenotype was no longer reported in Mpv172/2
mice, whereas the cochlear defect persisted.
to the peroxisomal compartment (25), we clearly demonstrated
that this ubiquitously expressed, relatively small (?19.5 kDa)
polypeptide is exclusively present in, and tightly anchored to,
the inner membrane of mitochondria by four transmembrane
domains (13). We also showed that in the Mpv172/2mice as
in the MPV17 mutant patients, liver mtDNA content was
profoundly reduced relative to Controls (13).
In spite of these discoveries, the very function of Mpv17 in
mitochondrial biogenesis remains unknown, as well as the
mechanism leading to tissue-specific mtDNA depletion in
both mutant mice and men. To gain insight into this matter,
we have further characterized the clinical features, course,
and organ-specific physiopathology of the Mpv172/2mouse
Clinical and morphological findings
Animal studies were approved by the animal welfare ethics
committee of the ‘Carlo Besta’ Neurological Institute, in
accordance with the Institutional Animal Care and Use Com-
mittee guidelines. Standard food and water were given ad
libitum except for ad hoc experiments.
The Mpv172/2mice did not develop overt hepatic failure at
any age. Light microscopy examination showed neither
massive necrosis nor cirrhosis in mice as old as 2 years, in
contrast to what is observed in MPV17 mutant infants.
However, morphological alterations were present in the liver
parenchyma starting from 5 months of age, consisting of swel-
ling of hepatocytes with collapse of sinusoidal spaces, shrink-
ing of nuclei in several cells, scattered degeneration of discrete
areas of hepatic lobules, and inflammatory infiltrates concen-
trated in the portal triads (Fig. 1A). Neither accumulation of
lipid droplets, nor fibrosis, nor proliferation of the bile ductular
cytochrome-c-oxidase (COX)-specific reaction was detected
by histochemical staining (Fig. 1B). These lesions were pro-
gressive, starting from 5 months of age onward. AST, ALT
and CK enzyme levels were constantly high in blood of
2/2 mice (Supplementary Material, Table S1), ostensibly
indicating subclinical hepatic and muscular damage. The
blood lactate levels were moderately elevated as well,
suggesting partial impairment of mitochondrial respiration
(Supplementary Material, Table S1).
Although a Rotarod test failed to reveal significant impair-
ment of motor skills (data not shown), in skeletal muscle sec-
tions of 1-year-old Mpv172/2mice several fibers (5–6%)
stained negative to the histochemical reaction to COX,
indicating the presence of a subclinical mitochondrial
myopathy (Fig. 1C). No alterations in muscle were present
in 5-month-old Mpv172/2mice (data not shown).
Electron microscopy examination was normal in hepato-
cytes of 15- and 60-day-old Mpv172/2livers (data not
shown), but mitochondrial ballooning and disappearance of
the internal cristae was consistently observed in older
Mpv172/2mice, with proliferation of membranes surrounding
the altered organelles (Fig. 1D). These abnormalities were not
present in the mitochondria of other tissues (data not shown).
In infants, hypoglycemic episodes occur after 3–4 h of
fasting. However, 48 h fasting failed to induce hypoglycemia
in 2-month-old Mpv172/2as well as Mpv17þ/þmice (Sup-
plementary Material, Fig. S1). An intra-peritoneal glucose
tolerance test was normal in 2-month-old Mpv172/2mice
(Supplementary Material, Fig. S2). Prolonged exposure to
low temperature is a stress condition that stimulates activation
of heat production by mitochondrial respiration. We failed to
Human Molecular Genetics, 2009, Vol. 18, No. 1 13
Figure 1. Histological and histochemical analysis. (A–D) Refer to Mpv172/2mice; (E–H) refer to Mpv17þ/þmice. (A and E) Liver H&E (?20): 2 years of
age; (B and F) liver COX (?20): 1 year of age; (C and G) muscle COX (?20): 1 year of age; (D and H) electron microscopy ultrastructure of liver hepatocytes
(?10 400): 1 year of age (see text for details).
14Human Molecular Genetics, 2009, Vol. 18, No. 1
observe any clinical symptom referable to hypothermia in both
2-month-old Mpv172/2and Mpv17þ/þlittermate mice main-
tained at 48C for 72 h. Lastly, valproic acid is an anti-epileptic
drug which is highly hepatotoxic, and may be fatal in some
forms of hepatocerebral MDS, e.g. POLG-associated Alpers–
Huttenlocher syndrome (8). Intra-peritoneal administration
of 30 mg/kg valproate for 5 days failed to induce either
morphological or biochemical abnormalities in the liver of
Taken together, these results demonstrate a remarkable
refractoriness of the Mpv172/2mice to liver damage, in
spite of the extremely reduced levels of mtDNA documented
previously (13) and reconfirmed in this work (discussed later).
Clinical time course
Next, we asked whether the ablation of Mpv17 could produce
age-dependent symptoms later in life, and affect lifespan. Two
large series of Mpv172/2and Mpv17þ/þlittermates were
maintained under observation in standard captivity conditions
for over 2 years.
We first observed that the coat of Mpv172/2mice invariably
turnedgray5–6monthsafterbirth,whilethatof þ/þ littermates
remained uniformly black, as typical of the C57B/6 strain back-
ground of our mice (Fig. 2A). We then measured a significantly
lower body weight of Mpv172/2versus Mpv17þ/þmice after
1 year of life for both males (Fig. 2B) and females (Fig. 2C).
reduced in a group of Mpv172/2mice (n. 14) versus a group of
þ/þ littermates (n. 12), both reared and kept in identical con-
ditions (Kaplan–Meier log-rank test P ¼ 0.002). The median
lifespan (in days) of the Mpv172/2group was 645 days. This
result prompted us to re-evaluate morphological, biochemical
and clinical features of our mice at late age.
When the kidney of 18-month-old Mpv172/2mice was
examined, we observed progressive FSGS, which at 2 years
Figure 2. Clinical features. (A) A 2-year-old Mpv172/2individual. The inset shows 2/2 (gray) and þ/þ (black) littermates at 1 year of age; (B and C) body
weight at different ages in 2/2 (gray line) and þ/þ (black line) male and female individuals. A single asterisk indicates a Student’s t-test P ¼ 0.01; a double
asterisk indicates P ¼ 0.002; (D) Kaplan–Meier survival probability, P ¼ 0.002 is the value of the log-rank test.
Human Molecular Genetics, 2009, Vol. 18, No. 115
ofageevolved intofrank degenerativechanges oftheglomeruli
(Fig. 3A), accompanied by degenerative abnormalities in the
enchymal loss and interstitial fibrosis (Fig. 3B). Accordingly,
Mpv172/2mice showed the presence of massive proteinuria
from 18 months of age onwards, but, surprisingly, creatinine
and urea levels in blood remained within the normal
range (Supplementary Material, Table S2) indicating that
overt renal failure did not occur. Robust histoenzymatic
reaction to COX was preserved in the epithelial cells of
Bowman capsule, cortical tubules and medullary pyramids of
Mpv172/2kidneys at any age, although it appeared somewhat
reduced in aged Mpv172/2mice compared with Mpv17þ/þ
individuals. As shown in Figure 3C, the COX reactivity was
rather uneven in 2-year-old Mpv172/2tubules, some of
which stained normally, while others displayed reduced reac-
tivity. In contrast, the cells of the glomerular tufts, which are
mostly composed of endothelial cells, mesangial cells and
podocytes, were much less reactive to COX in both groups of
samples at any age; nevertheless, COX-specific staining of
Mpv17þ/þglomeruli was generally more intense than that
glomeruli, especially in 2-year-old mice
Lastly, because of the early change in coat pigmentation
and late-onset thinning of the skin, we also examined skin
histology of 2-year-old Mpv172/2versus Mpv17þ/þmice.
As shown in Figure 4A, the Mpv172/2mice displayed
severe atrophy of the skin layers, with thinning of the
epidermis, loss of subcutaneous fat, and disorganization of
the muscle layer. The hair follicles were also severely
hypotrophic in Mpv172/2skin samples with concomitant
reduction in number and size of the sebaceous glands and
Characterization of mtDNA depletion
and biochemical phenotype
In an earlier report we showed that the amount of mtDNA in
liver of 7-month-old Mpv172/2mice was as low as 5% as that
of Mpv17þ/þlittermates, it was 25% in muscle, and normal or
slightly reduced down to 70% in brain and kidney.
In order to evaluate whether the mtDNA copy number is
dependent on Mpv17 gene dosage, we measured mtDNA
content in livers of Mpv172/þheterozygous and Mpv17þ/þ
homozygous littermates at 7 months of age. There was no stat-
istically significant difference between the two groups (data
not shown), indicating that Mpv17 is indeed associated with
a strictly recessive trait.
In order to establish the time course of the depletion during
extrauterine life, we measured the mtDNA copy number in
liver, muscle and brain of Mpv172/2and Mpv17þ/þlitter-
mates at different ages. As shown in Figure 5, the mtDNA
content of Mpv17þ/þ
mice showed a concordant age-
dependent variation in the three tissues, with a steady increase
from birth levels during the first 60–90 days of life, and a pro-
gressive reduction afterwards. However, the mtDNA content
remained constantly very low in liver and low in muscle of
mice, while a variation similar to that of
Mpv17þ/þlittermates was found in the Mpv172/2brains. In
liver, the mtDNA copy number per diploid nuclear genome,
equivalent to one cell nucleus, was 1366+384 molecules/
nucleus in 2-month-old Mpv17þ/þmice versus 93+73
in Mpv172/2littermates. In muscle of the same groups of
mice the mtDNA copy number/nucleus was 3321+835 in
Mpv17þ/þversus 611+191 in Mpv172/2samples.
In order to see whether the mtDNA abnormalities observed
in tissues could also be reproduced in a cell culture system,
Figure 3. Histology and histochemistry of kidney in 2-year-old mice. (A–C) Refer to Mpv172/2mice; (D–F) refer to þ/þ mice. (A and D) H&E (?20); (B
and E) H&E (?10); (C and F) COX (?10) (see text for details).
16 Human Molecular Genetics, 2009, Vol. 18, No. 1
we characterized Mpv172/2and Mpv17þ/þmouse embryonic
fibroblasts (MEFs) at different passages. We found that
in Mpv17þ/þMEFs, as in Mpv17þ/þtissues, the mtDNA
content increased over time, whereas it progressively declined
in Mpv172/2MEFs from passage 1 to 10 (Fig. 5). As a conse-
quence, the relative mtDNA content, which was similar in
Mpv172/2versus Mpv17þ/þcell lines at the first passage
(P1), decreased by 60–80% at P5 and remained low in later
passages (Fig. 6A).
Marked reduction in mtDNA was also observed in confluent
(non-proliferating) MEFs, cultured in the absence of fetal calf
serum (serum deprivation) (Fig. 6B). These data indicate that
mtDNA depletion can occur in MEFs by inducing aging or
stress conditions that require an efficient OXPHOS (oxidative
Because of the late renal phenotype manifested by .-
18-month-old Mpv172/2mice, we measured the mtDNA
content in the renal parenchyma 1-year and 2-year-old mice.
The amount of mtDNA was virtually identical in whole
kidneys of three Mpv17þ/þversus three Mpv172/2mice at 1
year of age. In particular, the mtDNA copy number was
728+52 mtDNA/nucleus in
mtDNA/nucleus in Mpv172/2samples. However, the mtDNA
content was reduced to ?47% in the Mpv172/2versus
Mpv17þ/þ2-year-old groups, each consisting of three mice. In
particular, the mtDNA copy number per cell was 1012+156
mtDNA/nucleus in Mpv17þ/þversus 470+170 mtDNA/
nucleus in Mpv172/2samples (Student’s t-test P ¼ 0.015).
Since the predominant kidney abnormality consisted of
severe proteinuria, i.e. an alteration of the glomerular filtering
Figure 5. Real-time PCR analysis of mtDNA copy number from 2 weeks to 1 year of age. The gray line refers to Mpv172/2mice; the black line refers to
Mpv17þ/þlittermates. A single asterisk indicates a Student’s t-test P ? 0.01; a double asterisk indicates P ? 0.002.
Figure 4. Histology of 2-year-old skin. H&E (?10). (A and B) Refer to Mpv172/2and Mpv17þ/þmice respectively (see text for details).
Human Molecular Genetics, 2009, Vol. 18, No. 117
proficiency, we also measured the mtDNA content in pools
of glomeruli and cortical tubules microdissected from kidneys
of two 6-month-old and two 2-year-old littermates (Table 1).
The mtDNA molecules/diploid nucleus was significantly less
in Mpv172/2versus Mpv17þ/þglomeruli (P , 0.025), but the
difference was much higher in 2-year-old mice (P , 0.00007).
Contrariwise, no significant differences were observed in
Mpv172/2tubules versus Mpv17þ/þtubules at 6 months
(P ¼ 0.66) and 2 years. (P ¼ 0.56).
The biochemical activities of respiratory chain complexes I and
IV declined rapidly after birth, and remained stably low, in
Mpv172/2liver homogenates, to ?30–40% of those measured
in Mpv17þ/þlittermates (Table 2). No significant differences
were detected for the same activities measured in other tissues
of the two groups of mice, including muscle, brain and kidney.
Therefore, the impairment of mtDNA-related respiratory chain
Figure 6. Immunofluorescence studies on MEF cultures. The green fluorescence refers to nuclear and mitochondrial DNA visualized with an anti-DNA antibody.
(A) MEF cultures at passage 1 (P1) and passage 7 (P7); (B) MEF cultures with (þFCS) or without (2FCS) 10% fetal calf serum (see text for details).
18 Human Molecular Genetics, 2009, Vol. 18, No. 1
activities was disproportionately mild when compared with the
profound decrease in the mtDNA copy number detected in
Mpv172/2liver, as well as Mpv172/2muscle, tissues.
In order to evaluate whether compensatory mechanisms were
transcription and translation of mtDNA genes in the liver of
2/2 and þ/þ mice at different ages. Figure 7A shows that
were only moderately reduced in Mpv172/2mice compared
with the Mpv17þ/þlittermates at all examined ages. An even
lesssignificant decrease was found insome of the corresponding
proteins, for instance COX1, visualized by western blotting
These results suggested the existence of a compensatory
mechanism at the level of mtDNA transcription and possibly
translation as well. To test this hypothesis, we carried out pulse-
chase in organello transcription experiments in isolated mito-
chondria of 6-month-old Mpv172/2versus Mpv17þ/þmice.
Transcription was analyzed after 2, 4, 5 and 6 h of incubation.
As shown in Figure 7C, the amount of mtDNA-specific tran-
scripts in Mpv172/2mice was lower than in Mpv17þ/þlitter-
mates in absolute terms, but the efficiency of transcription was
much higher, considering that the content of mtDNA was extre-
mely reduced, in fact virtually undetectable. 1n both Mpv172/2
the fifth hour, to then decrease at the sixth. Taken together, these
results indicate the existence of vigorous transcriptional com-
pensation of mtDNA depletion in Mpv172/2liver.
We next investigated whether this compensatory effect could
drial biogenesis, mtDNA maintenance or mtDNA transcription.
To this aim, we analyzed by real-time PCR the expression levels
of a host of suitable genes in Mpv172/2versus Mpv17þ/þ
6-month-old livers. As shown in Figure 8A, the analysis
included factors activating mitochondrial biogenesis, such as
Pgc1a, Pgc1b and Nrf1 (26,27), and factors involved in
mtDNA replication (Pol gA, Twinkle) and/or transcription
that was expressed differently in the two groups of mice was
Mterf1, which was consistently and significantly reduced by
?40% in Mpv172/2liver samples. Interestingly, identical or
similar results were obtained for Mterf1 in muscle but not in
brain of Mpv172/2mice, in agreement with the amount of
mtDNA measured in the same tissues (Fig. 8B).
The reduction in Mterf1 transcript was accompanied by
reduced amount of the corresponding protein in liver mitochon-
dria (?60–70%), as shown by western blot analysis using an
anti-Mterf1 polyclonal antibody (Fig. 8C). In order to evaluate
whether reduced expression of Mterf1 was sufficient to deter-
minea change inthe ratio
protein-encoding mtDNA transcripts, we measured the band
intensity of the in organello transcribed cytochrome c oxidase
subunit I (COI) and 16S rRNA species shown in Figure 7C at
2 and 4 h. The COI/16S rRNA ratio at the second hour was
2.05 in the Mpv172/2versus 1.49 in the Mpv17þ/þliver mito-
chondria; at the fourth hour it was 2.43 versus 1.09.
The Mpv172/2mice are the first example of an animal model
of MDS. TK2 knockout (29) and knockin (30) mice have more
recently been reported.
We showed that in young mice, as in humans, the ablation
of Mpv17 determines a profound reduction in mtDNA content
in liver and, to a lesser extent, in skeletal muscle, but neither in
brain nor in kidney. Measurement at different ages revealed
that, in keeping with previous data in Mpv17þ/þmice the
mtDNA content varied over time in liver and muscle (31),
while it remained constantly low throughout life in the same
tissues of Mpv172/2mice. This was not the case for
Mpv172/2brain tissue, in which both the absolute mtDNA
content and its age-dependent variation were similar to those
of Mpv17þ/þbrains. Although the mechanistic role of
Mpv17 remains elusive, these results demonstrate that the
absence of this protein impairs a dynamic control on
mtDNA copy number, in a tissue-specific and possibly
developmentally regulated manner. This conclusion is also
supported by studies in cell cultures, since MEFs from
Mpv172/2mice failed to show any mtDNA depletion at the
beginning, but did develop it after several passages, a situation
that simulates an aging process, or after serum deprivation, a
paradigm of cellular stress.
The low mtDNA content in liver was associated with sur-
prisingly mild morphological alterations of its cytoarchitec-
ture, whereas, at the ultrastructural level, mitochondria of
Mpv172/2hepatocytes were profoundly altered, especially
in 5-month and older mice. Mitochondria became ballooned,
the cristae disappeared, an electron-dense amorphous material
accumulated in the matrix, and the organelles were surrounded
by membranous structures, probably derived from a prolifer-
ation of endoplasmic reticulum. Although similar ultra-
structural changes were also described in MPV17 mutant
patients (14), they are not specific to this condition, having
been reported in other forms of hepatocerebral MDS (32).
Table 1. mtDNA copy number in tubules and glomeruli of Mpv17þ/þand
Mpv172/2mice at 6 months and 2 years of age
?P , 0.025 (Student’s t-test);??P , 0.00007.
Table 2. Biochemical activities of complexes I and IV in liver homogenate
(each group was composed of 4–6 individuals)
Values are expressed as ratio between specific activities expressed as
(nmol/min/mg non-collagen protein); CS, citrate synthase.
?P , 0.05 (Student’s t-test);??P , 0.008.
Human Molecular Genetics, 2009, Vol. 18, No. 119
In addition, no such changes were found in extrahepatic
tissues of our mice, which were also lacking Mpv17 but
have normal or moderately reduced amount of mtDNA.
Taken together, these results suggest that the mitochondrial
abnormalities seen in Mpv172/2liver are secondary to func-
tional impairment of the organelles, rather than reflecting a
role of Mpv17 in controlling the shape of the cristae and the
structure of the inner mitochondrial compartment.
The very low content of mtDNA in liver was associated
with milder decrease in mtDNA-related respiratory chain
activities, including those of complexes I and IV. Incidentally,
the activity of an mtDNA-independent respiratory chain
complex such as succinate dehydrogenase (SDH) was also
found normal in a previous study on 6-month-old Mpv172/2
livers (13), as was the SDH histochemistry in the same
tissue (data not shown). These data explain the overall preser-
vation of functional liver proficiency and the lack of the dra-
matic morphological changes that were documented in
human patients. In contrast with the human condition,
the absence of Mpv17 was not associated with metabolic
fragility of the Mpv172/2mice, which failed to develop
fasting-induced hypoglycemia, increased sensitivity to hepato-
toxic valproate, or hypothermia by prolonged exposure to
cold. Based on analysis of transcription and translation of
mtDNA in liver mitochondria, we demonstrated a remarkable
compensatory mechanism that maintains cellular respiration of
Mpv172/2mouse liver (and muscle as well) at levels compa-
tible with virtually normal life in captivity. In particular, both
northern blotting and in organello transcription analyses
showed that one compensatory key point is at the level of tran-
scription, since the ratio between mtDNA transcripts and
mtDNA content in Mpv172/2organelles was several fold
higher than in Mpv17þ/þorganelles. As a result, the amount
of mtDNA-specific translation products was comparable in
livers of 2/2 versus þ/þ mice.
Functional compensation was recently reported in several
mtDNA-depleted tissues of Tk22/2mice (30), and transcrip-
tional compensation was demonstrated in heterozygous Tfam
knockout mice (33), and in skeletal muscle of patients with
TK2 mutations (34). Thus, compensation at the transcriptional
or translational level may well be a general phenomenon in
response to mtDNA depletion.
These results prompted us to evaluate the expression level
of several factors involved in mtDNA transcription/mainten-
ance. A 40% reduction was consistently found in the
steady-state transcript level of Mterf1 in liver, while all
other genes examined were not differentially expressed
between þ/þ and 2/2 mice. A concordant reduction in
Mterf1 protein was immunodetected using a specific antibody.
Mterf1 is a transcriptional termination factor that blocks the
Figure 7. RNA and protein blot analyses of Mpv172/2and Mpv17þ/þlittermates at different ages. (A) Northern blot analysis of mtDNA transcripts. The nuclear
multicopy gene encoding 18S ribosomal RNA was used for standardization, preND5 refers to the ND5 þ ND4 pre-mRNA; (B) protein blot analysis using an
antibody specific to COX1 subunit. An antibody against the 30kDa subunit of SDH was used for standardization; (C) pulse-chase in organello mitochondrial
RNA synthesis. Notice the reduction in band intensity at 6 h in both 2/2 and þ/þ samples. The ethidium-bromide staining of the mtDNA-specific band is
shown on top: in contrast with the þ/þ samples, the 2/2 samples have hardly any detectable mtDNA.
20Human Molecular Genetics, 2009, Vol. 18, No. 1
heavy-strand mtDNA polycistronic transcription by looping
mtDNA across two binding sites: the heavy-strand promoter
2, HSP2, and a termination site within the tRNALeu(UUR)
gene, adjacent to the gene encoding 16S rRNA (35). As a con-
sequence, Mterf1 decreases the HSP2-dependent expression of
the mtDNA structural genes (and of many tRNA genes as
well), which are all located downstream from the 16S rRNA
gene, whereas transcription of the 12S and 16S rRNA genes,
which can proceed from both HSP2 and from a second
heavy strand promoter, HSP1, remains unaffected. As a
result, the amount of rRNAs is 15–60-fold higher relative to
mRNA (36,37). Thus, one of the physiological roles attributed
to Mterf1 is to maintain mitochondrial 12S and 16S rRNA
transcript levels in excess relative to mRNA transcripts, so
as to prevent that the endowment in ribosomes of mitochon-
dria becomes a limiting factor to protein translation. The
increase in organello transcription of COI mRNA relative to
16S rRNA that we observed in Mpv172/2liver mitochondria
does indeed support this hypothesis. Likewise, the observation
that Mterf1 transcript was also reduced in Mpv172/2muscle,
another tissue with significant mtDNA depletion, whereas it
was normal in Mpv172/2brain, which had virtually no or
very little mtDNA depletion, suggests that this variation is
functionally relevant. Taken together, our data suggest for
Mterf1 a role in the transcriptional (and translational) compen-
satory response to reduced mtDNA copy number in specific
Mpv172/2organs. Interestingly, a general increase in tran-
scription of mitochondrial RNAs is associated with ablation
of mouse Mterf3, which is part of the same Mterf protein
family that includes Mterf1 (38).
Some of the clinical features of the Mpv172/2mouse were
not observed in the human equivalent, possibly because their
MPV17-MDS is an early-onset disorder, which usually leads
patients to death in infancy or childhood.
For instance, Mpv172/2mice showed a progressive loss of
the coat color, starting several months after birth. Hair graying
is not related to normal aging in the mouse, as also demon-
strated by our own observation that Control littermate mice
remained uniformly black well after 2 years of age. The
knockout mouse for Bcl2, a mitochondrial anti-apoptotic
factor, turns gray because of loss of the melanocyte precursor
stem cells in the hair follicle niche (39,40). Given the mito-
chondrial localization of Mpv17, a pro-apoptotic mechanism
could also be acting in the Mpv172/2model. For instance,
reactive oxygen species (ROS) can damage melanocytes
either directly or by inducing mitochondrial apoptosis (41),
and increased production of ROS was previously reported in
fibroblasts of Mpv172/2mice (25,42). In addition, the skin
of aged 2/2 individuals showed severe atrophy, particularly
affecting the subcutaneous fat and muscular layer. These fea-
tures can be the result of the compromised clinical conditions
of these mice; however, they could also reflect a more specific
pathogenic process, since similar findings were recently
reported in Tk2 knockout mice (29), another model of
Figure 8. Real-time PCR quantification of mRNA transcripts from genes involved in mtDNA biogenesis. (A) Liver transcripts of 2-month-old 2/2 mice.
PGC1-a and PGC1-b, peroxisome proliferative activated receptor gamma coactivator 1, isoforms a and b; NRF1, nuclear respiratory factor 1; POLG, polymer-
ase g subunit A; TFAM, transcription factor A mitochondrial; TFBIM and TFBIIM, transcription factors BI and BII mitochondrial; MTERF1, mitochondrial
termination factor 1; Twinkle, mitochondrial helicase; POLRMT, RNA polymerase mitochondrial; (B) Mterf1 transcript levels in different tissues of 2-month-old
Mpv172/2mice. The dotted lines indicate the mean levels obtained in Control littermates, taken as 100%; (C) Mterf1 western blot analysis. The SDH-specific
band was used as a standard for protein content.
Human Molecular Genetics, 2009, Vol. 18, No. 1 21
The most striking organ pathology of Mpv172/2mice con-
sisted of FSGS, followed by hyaline degeneration and loss,
degenerative changes of the tubular system, and fibrosis. As
a consequence, the Mpv172/2mice developed massive protei-
nuria, due to abnormal leakiness of the glomerular filtering
system. Most likely as a consequence of the kidney disease,
they showed progressive downhill of their health conditions
starting from 18 months after birth, with highly significant
reduction in average and maximum lifespan, compared with
the þ/þ littermates.
The occurrence of FSGS, associated with extensive flatten-
ing of the foot processes of podocytes, was reported in the first
papers on the Mpv172/2mouse model (20,42). The ultrastruc-
tural alterations occurred after the maturation of podocytes,
i.e. were not due to arrest in the full development of arborized
foot processes that takes place after birth (21). The morpho-
logical alterations were associated with proteinuria starting
in mouse early adulthood (?50 days after birth). However,
the kidney phenotype was no longer detected after a few gene-
rations, at least within the lifetime considered by the exami-
ners. We ourselves failed to find any trace of proteinuria or
renal pathology in Mpv172/2mice as long as 12 months
after birth (13). Our present data show that glomerular
damage still occurs in Mpv172/2mice but starts much later
in life. The reason for such a remarkable age-dependent time-
shift of disease onset is unknown, but it could depend on a
modification of the genetic background of the mice. For
instance, the mouse strain of our own colony, C57/B6, is
Mpv172/2mice reported in the first papers.
Concomitant lesions of the membranous and cellular struc-
tures of the cochlear receptor were also reported in Mpv172/2
mice (23). Although in the present study we did not examine
the inner ear, recent publications by others have confirmed the
presence of severe cochlear lesions in the very same mouse
colony used for our work (24).
The development of severe, progressive lesions in the renal
glomeruli is indeed a remarkable finding, considering that in
the bulk of kidney parenchyma we found hardly any
mtDNA depletion in young mice, and moderate reduction in
aged individuals, not in the least comparable with that found
in the liver. In order to further investigate the molecular
basis of this apparent discrepancy, we quantified the mtDNA
content of cells in the glomerular tufts versus tubular epithelial
cells. Glomeruli isolated by laser-microdissection from protei-
nuric Mpv172/2kidneys displayed marked reduction in
mtDNA content compared with those obtained from non-
proteinuric þ/þ kidneys of the same age, i.e. 2 years old.
More than the absolute amount of mtDNA, we observed the
same phenomenon in glomeruli as that found in liver,
muscle, and MEFs, that is, the failure of Mpv172/2cells to
undergo the physiological increase (and variations) in
mtDNA copy number detected in Mpv17þ/þorgans during
post-natal lifetime. In addition, a significant trend toward
reduction in glomerular mtDNA content was already detected
in non-proteinuric, 6-month-old Mpv172/2mice, implicating
that proteinuria is the result of a chronic pathogenic process
ultimately damaging glomerular structure and function. In
contrast, no difference in mtDNA content was detected in
the renal tubules of both young and old kidneys, consistent
with the absence of tubular insufficiency in Mpv172/2mice
at any age.
Taken together, these results suggest that glomerular podo-
cytes are exquisitely prone to marked mtDNA depletion,
which become clinically relevant in aged Mpv17-less mouse
kidneys. Although the mtDNA content of normal podocytes
is already low, being associated with hardly any COX-specific
histochemical reactivity (43), further reduction in mtDNA
amount is likely to have functional consequences.
Glomerular filtering per se is an essentially passive
phenomenon, but it requires the formation and maintenance
of an intact and highly selective glomerular basement mem-
brane (GBM). Anatomical and functional features of GBM
are the results of interaction between endothelial cells of
glomerular capillaries with podocytes. The latter are highly
differentiated post-mitotic cells that maintain various energy-
demanding functions including the synthesis and organization
of cytoskeletal and extracellular matrix proteins. Earlier
reports suggested that podocyte lesions of Mpv172/2mice
were associated with increased ROS production (25,42), and
administration of ROS scavengers was also effective in pre-
venting, or ameliorating, the proteinuria and focal nephro-
sclerosis in these mice (42).
FSGS is a major renal complication of mitochondrial cyto-
pathies in humans (43), including mtDNA mutations such as
the 3243A.G ‘MELAS’ transition in tRNALeu(UUR), and
single, large-scale mtDNA deletions (see reference 44 for a
review on this issue). Mutant mice carrying a large deletion
of mtDNA develop FSGS lesions, leading to death within 6
months due to renal failure (45). Rrm2b-knockout mice,
equivalent to p53-R2 mutant patients, suffer both mtDNA
depletion and glomerular hyaline degeneration with pro-
teinuria (46). Finally, puromycin aminoglucoside nephrosis
(PAN) is the best-described animal model of glomerular
disease, evolving into FSGS. Kidney of PAN rats show
reduction in respiratory chain enzymatic activities and
oxygen consumption, with swelling of renal tubular mitochon-
dria (43). PAN has been associated with decrease in mtDNA
content to as low as 34% in rat kidney glomeruli at the
FSGS stage (43). Early reports on Mpv172/2mice have
indeed shown that these mice were exquisitely sensitive to
PAN (42). Again, PAN-induced proteinuria and glomerular
lesions can be attenuated by exposure to antioxidants,
suggesting that excessive ROS production is a pathogenic
mechanism also in this condition (42).
In the light of the earlier considerations and of our own
results, an excess of ROS could well occur as a consequence
of OXPHOS failure in Mpv172/2glomeruli, leading to podo-
cyte damage and degeneration. These effects could in turn com-
promise the anatomical and functional integrity of the GBM,
and determine both the proteinuria and the progressive morpho-
logical changes seen in the kidney of old 2/2 individuals.
Neither the renal nor the inner ear pathologies were reported
in MPV17 mutant patients, including those affected by NNH.
These symptoms are in fact reminiscent of a group of human
steroid-resistant proteinuric nephrosclerosis and perceptive
hearing loss, known as Alport syndrome (OMIM #301050).
Alport syndrome is caused by mutations in COL4A3,
COL4A4 or COL4A5 genes, which encode the alpha-3,
by theassociation of
22Human Molecular Genetics, 2009, Vol. 18, No. 1
alpha-4 and alpha-5 chains of type IV collagen. These
cysteine-rich isoforms of type IV collagen are essential com-
ponents of mature GBM. As a consequence, the GBM of
Alport syndrome patients retains a fetal distribution of the
cysteine-poor alpha-1 and alpha-2 isoforms of type IV col-
lagen, which confer GBM a higher susceptibility to proteolytic
attack by collagenases and cathepsins (47).
The renal and inner-ear damages are often accompanied
by lesions in other organs, notably the epidermis and the
eye. The latter include lesions of the retina (retinal flecks),
lens (lenticonus and spherophakia) and cornea (posterior
corneal dystrophy and recurrent corneal erosion). Interest-
ingly, corneal ulcerations and corneal scarring are late
manifestations of NNH that are allegedly attributed to, but
not convincingly proven to derive from, anesthesia due to
sensory peripheral neuropathy.
A mechanism similar to that acting in Alport syndrome has
Mpv17-associated FSGS, since the lack of this protein in
2/2 mice induces the overexpression of MMP-2, a metallo-
proteinase of the extracellular matrix present in both kidney
and inner ear (48).
Although Alport syndrome has never been connected to
mitochondrial dysfunction, other genetic disorders of collagen
have been. For instance, missense mutations of COL6A,
encoding the collagen-6 protein isoform, are responsible for
relatively benign Bethlem myopathy (OMIM #158810),
while non-sense mutations in the same gene cause the much
more severe Ullrich myopathy (OMIM #254090). In mito-
chondria of both human patients and a COL6A-gene knockout
mouse, malfunctioning or absence of collagen-6 is associated
with a mitochondrial defect linked to dysregulation of the
mitochondrial permeability transition pore (PTP) (49). This
defect is corrected by cyclosporine A, an inhibitor of PTP
Other human conditions resembling Alport syndrome are
Epstein (OMIM #153650) and Fechtner (OMIM #153640)
syndromes. These entities are variants of the same genetic
defect, that is, mutations in the myosin heavy chain-9 non-
muscle isoform (52). This protein has recently been shown
to have a double localization: in the cytoskeletal framework,
for instance in the stereocilia of the cochlear hair cells, as
well as in the inner compartment of mitochondria (53).
Taken together, these considerations foster the interesting
hypothesis that besides a role in controlling mtDNA copy
number and ROS production, Mpv17 may be functionally
linked to components of the extracellular matrix or of the
cytoskeleton, which are involved in the integrity of the
GBM and possibly other epithelial basement membranes in
vertebrates, notably mammals.
in the pathogenesisof
MATERIALS AND METHODS
Diagnosis of the recombinant and wild-type alleles of the
Mpv17 gene was performed by PCR-based length polymorph-
ism analysis on tail genomic DNA using suitable primers
(Supplementary Material, Table S3). The recombinant allele
corresponds to a PCR band of 234 bp, whereas the wild-type
allele corresponds to a PCR band of 217 bp. The two bands
were separated electrophoretically through a 2% agarose gel
in TE and visualized under a UV screen.
For standard histology, 10 mm sections from different tissues
were post-fixed in 4% paraformaldehyde; for histochemistry,
samples were frozen in liquid-nitrogen cooled isopentane
and 10 mm cryostat sections were used to visualize COX reac-
tivity (54). For standard ultrastructural studies, fixation was by
2% glutaraldehyde in PBS.
Total DNA was extracted from tissues following standard pro-
cedures. For mtDNA content analysis, SYBR-GREEN real-
time PCR was performed using primers specific to a mouse
mtDNA fragment of the COI gene, and primers specific to
RNaseP, a single copy gene taken as a nuclear gene reference
(Supplementary Material, Table S4). Relative copy number
was calculated from threshold cycle value (DCt), and the
mtDNA copy number/cell was calculated as 2 ? 22DCtto
account for the two copies of RNaseP in each nucleus (55).
Real-time quantitative PCR was carried out using an ABI
PRISM 7000 Sequence Detection System. The amplification
profile was according to a two-step protocol: one cycle at
508C for 2 min, one cycle at 958C for 10 min, and then 40
cycles of 958C for 15 s and 608C for 1 min. A final dissociation
step (958C for 15 s, 608C for 20 s, 958 for 15 s) was added to
assess for unspecific primer–dimer amplifications.
For the analysis of transcripts, total RNA was extracted from
liquid-nitrogen snap-frozen liver and muscle specimens by
Trizol, according to manufacturer instructions (Invitrogen,
Carlsbad, CA, USA). Two microgram of total RNA was
treated with RNase-free DNase and retro-transcribed by using
the ‘cDNA cycle’ kit (Invitrogen). Approximately 2–5 ng of
cDNA was used for real-time PCR assay using primers specific
for amplification of several genes, according to the ABI-Primer
Express software (Supplementary Material, Table S3). In some
experiments, we microdissected discrete areas of 10 mm H&E
stained cryostat sections under a laser-equipped Nikon Eclipse
TE2000-S. Total DNA was extracted using the MicrolysisTM
kit (Microzone Limited, UK).
Total RNA was extracted from liver and muscle samples, as
described, run on a formaldehyde agarose gel, and capillary
blotted onto a nylon membrane (Nþ, Amersham) according
to manufacturer instructions. Hybridization was carried out
according to standard procedures, using probes specific for
mouse COI, ND5, 18S, obtained by PCR amplification with
suitable primers (Supplementary Material, Table S5).
In organello RNA synthesis
Mouse liver mitochondria were isolated as described (56), and
in organello experiments were carried out as described (57).
Briefly, mitochondria from one liver were suspended in
Human Molecular Genetics, 2009, Vol. 18, No. 123
0.6 ml of incubation buffer (25 mM sucrose, 75 mM sorbitol,
100 mM KCl, 10 mM K2HPO4, 0.05 mM EDTA, 5 mM
MgCl2, 1 mM ADP, 10 mM glutamate, 2.5 mM malate, 10 mM
Tris–HCl pH 7.4 and 1 mg/ml of free fatty acid bovine
serum albumin). For in organello transcription analysis,
20 mCi of [k-32P]-UTP was added and incubated at 378C for
60 min in a rotary shaker. For pulse chase experiments, mito-
chondria were preincubated for 2 h with [k-32P]-UTP, precipi-
tated by centrifuging at 13000 r.p.m., and resuspended in
fresh incubation medium in the presence of 200-fold excess
of cold UTP, and incubated for several periods of time
before harvesting. Mitochondrial RNA was extracted by Pro-
tease IX (Sigma) digestion and run on methyl-mercury
hydroxide agarose gel. The gels were then stained with ethi-
dium bromide, photographed under a UV screen and dried
for autoradiography (Phosphorimaging System, Biorad).
on tissue homogenates, and normalized to citrate synthase
activity, an indicator of the number of mitochondria (58).
Analysis in body fluids
AST, ALT, CK, Creatinine, glucose and urea were determined
in blood samples by standard methods. Lactate levels were
measured by the Lactate Pro kit (Arkray, Kyoto, Japan).
Urinary protein was measured by Bayer Multistix 10 SG
Primary MEF cultures were prepared from individual E13.5
embryos obtained from an intercross of heterozygous Mpv17
mice. MEFs were cultured in complete DMEM with high
glucose with or without 10% fetal bovine serum (see Results).
For immunofluorescence analysis, cells were plated on
coverslips, preincubated with 100 nM MitoTracker Red dye
(Molecular Probes) for 30 min at 378C, followed by fixation
for 20 min with 4% paraformaldehyde in PBS at 378C, and
permeabilized with 0.5% Triton X in PBS for 5 min at room
temperature. The cells were then incubated with primary
Anti-DNA antibody (Progen) for 1 h (diluition anti-DNA
1:25). After incubation, the cells were rinsed in PBS and
primary antibodies were visualized using a fluorescent-tagged
secondary antibody (Alexa Fluor 488 goat anti-mouse IgG) at
1:1000 dilution in PBS.
Protein blot analysis
Ten percent w/v homogenates in 10 mM phosphate buffer pH
7.4 were prepared from different tissues and centrifuged at
800g for 10 min to eliminate cellular debris. Total protein
extracts were run through a 12% SDS–polyacrylamide gel
filters. The filters were immunostained with specific antibodies
against complex IV subunit I (COX1) and the 30 kDa subunit
of SDH (Molecular probes, Invitrogen) and protein bands were
visualized using the ECL chemiluminscence kit (Amersham).
and electroblottedonto nitrocellulose
Twenty microgram of proteins from isolated liver mito-
chondria were run on a 12% SDS–polyacrylamide gel for
Mterf1 expression analysis and the nitrocellulose filter was
immunostained with a Mterf1 polyclonal antibody (kindly
provided by Dr Aleksandra Trifunovic, Karolinska Institute,
Two-tailed, unpaired Student’s t-test was used for statistical
analysis. Survival analysis was carried out using the Kaplan–
Meier estimate and log-rank test for survival probability.
Supplementary Material is available at HMG Online.
This work was supported by the Pierfranco and Luisa
Mariani Foundation, the Telethon-Italy Foundation grant no
GGP07019, the Italian Ministry of University and Research
(FIRB 2003 – project RBLA038RMA), the Italian Ministry
of Health (RF2006 ex 56/05/21), MITOCIRCLE and EUMI-
from the European Union framework program 6. E.F.-V. is a
Marie Curie intra-European fellowship (FP6-2005-Mobility-
5) number 040140-MAD.
The authors are grateful to Professor Hans Weiher for the gen-
erous gift of the Mpv17 knockout mice, and to Mr Roberto
Bellavia, Mrs Flavia Blasevich, Dr Paola Cavalcante, and
Dr Cristina Cappelletti for skillful technical assistance.
Funding to pay the open access charges was provided by
Telethon Foundation, Italy.
Conflict of Interest statement. None declared.
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