PGC-1 / induced expression partially compensates for respiratory chain defects in cells from patients with mitochondrial disorders

Article (PDF Available)inHuman Molecular Genetics 18(10):1805-12 · April 2009with38 Reads
DOI: 10.1093/hmg/ddp093 · Source: PubMed
Members of the peroxisome proliferator-activated receptor gamma coactivator (PGC) family are potent inducers of mitochondrial biogenesis. We have tested the potential effect of increased mitochondrial biogenesis in cells derived from patients harboring oxidative phosphorylation defects due to either nuclear or mitochondrial DNA mutations. We found that the PGC-1alpha and/or PGC-1beta expression improved mitochondrial respiration in cells harboring a complex III or IV deficiency as well as in transmitochondrial cybrids harboring mitochondrial encephalomyopathy lactic acidosis and stroke A3243G tRNA((Leu)UUR) gene mutation. The respiratory function improvement was found to be associated with increased levels of mitochondrial components per cell, although this increase was not homogeneous. These results reinforce the concept that increased mitochondrial biogenesis is a promising venue for the treatment of mitochondrial diseases.
PGC-1a/b induced expression partially
compensates for respiratory chain defects in cells
from patients with mitochondrial disorders
Sarika Srivastava
, Francisca Diaz
, Luisa Iommarini
, Karine Aure
Anne Lombes
and Carlos T. Moraes
Department of Neurology and
Department of Cell Biology and Anatomy, University of Miami School of Medicine,
1095 NW 14th Terrace, Miami, FL 33136, USA,
Dipartimento di Scienze Neurologiche, Universita
di Bologna,
Bologna, Italy and
Inserm 582; UPMC-Paris6; AP/HP, Paris F-75013, France
Received August 20, 2008; Revised February 3, 2009; Accepted February 24, 2009
Members of the peroxiso me proliferator-activated receptor
coactivator (PGC) family are potent inducers of
mitochondrial biogenesis. We have tested the potential effect of increased mitochondrial biogenesis in cells
derived from patients harboring oxidative phosphorylation defects due to either nuclear or mitochondrial
DNA mutations. We found that the PGC-1a and/or PGC-1b expression improved mitochondrial respiration
in cells harboring a complex III or IV deficiency as well as in transmitochondrial cybrids harboring mitochon-
drial encephalomyopathy lactic acidosis and stroke A3243G tRNA
gene mutation. The respiratory
function improvement w as found to be ass ociated with increased levels of mitochondrial compo nents per
cell, although this increase was not homogeneous. These results reinforce the concept that increased mito-
chondrial biogenesis is a promising venue for the treatment of mitochondrial diseases.
Oxidative phosphorylation (OXPHOS) dysfunctions play a
critical pathogenic role in several human diseases (1). To
date, more than 200 different mitochondrial DNA (mtDNA)
mutations and probably a similar number of nuclear DNA
(nDNA) mutations have been identified in patients with mito-
chondrial diseases. Defects in mitochondrial OXPHOS func-
tion affect preferentially high-energy demand tissues such as
brain, skeletal muscle, heart, retina, renal tubules and endo-
crine glands. Almost invariably, the OXPHOS defect in
patients is partial, suggesting that a complete defect would
be incompatible with life. Therefore, patients either have
mutant mtDNA coexisting with the wild-type mtDNA
(mtDNA heteroplasmy) or a homoplasmic mtDNA mutation
causing a partial impairment of OXPHOS. Likewise, nDNA
mutations associated with diseases commonly cause partial
defects with OXPHOS residual activity (1). Tissues of many
patients with mitochondrial disorders may show massive
mitochondrial proliferation, which has been assumed to be a
metabolic response to the decreased OXPHOS function (1).
The potential mechanisms of mitochondrial proliferation are
now being better understood, thanks to the recent discoveries
of transcriptional control of metabolic pathways and mito-
chondrial biogenesis.
Transcriptional coactivators of the PGC-1 gene family are
master regulators of mitochondrial biogenesis and oxidative
metabolism (2). There are three family members namely
PGC-1a, PGC-1b and PGC-1 related coactivator (PRC).
PGC-1a and PGC-1b are expressed in tissues with high-energy
demand such as brown fat, skeletal muscle, heart, brain and
kidney (3,4), whereas PRC is expressed ubiquitously (5).
Studies have shown that PGC-1a/b are potent regulators of
mitochondrial function and biogenesis (3,69). PGC-1a/b
co-activates transcription factors that in turn stimulate the
expression of a large number of nuclear genes involved in mito-
chondrial respiration and biogenesis. These transcription factors
include the nuclear receptors peroxisome proliferator-activated
receptors (PPARs), the NRF-1 and -2 (nuclear respiratory
factors) and ERRa (estrogen-related receptor alpha) (10,11).
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Present address: Harvard Medical School, Boston, MA 02115, USA.
To whom correspondence should be addressed. Tel: þ1 3052435858; Fax: þ1 3052433914; Email:
# The Author 2009. Published by Oxford University Press. All rights reserved.
For Permissions, please email:
Human Molecular Genetics, 2009, Vol. 18, No. 10 18051812
Advance Access published on March 18, 2009
The present study demonstrated that induced PGC-1a/b
upregulation improves mitochondrial respiration and
OXPHOS function in cells with partial OXPHOS defects
caused by either nDNA or mtDNA mutations.
We have studied three fibroblast cell lines obtained from pedi-
atric patients with mitochondrial disorders (see Patients
section). Two of these patients had a defect in complex IV
and one in complex III activities. Fibroblasts were isolated
and transduced with a retrovirus expressing hTERT (human
telomerase). MtDNA sequencing and family history indicated
that they had a nDNA defect, which was identified as located
in the Surf1 gene in Patient D (Pat.D) (see Patients section).
The molecular defects in the other patients remain unknown.
In addition, we also studied human osteosarcoma cells harbor-
ing the A3243G tRNA
mtDNA mitochondrial ence-
phalomyopathy lactic acidosis and stroke (MELAS) mutation
which have been shown to have a respiratory defect due to
partial defects in enzyme complexes I and IV (12).
Biochemical characterization of the patient fibroblasts
showed OXPHOS complex defects
We performed the biochemical characterization of the cell
lines obtained after immortalization of the patients’ fibroblasts
and confirmed that the cell lines from Pat.D and Pat.C had iso-
lated complex IV defects, whereas that from Pat.N had an iso-
lated complex III defect (Fig. 1A). Steady-state levels of
representative subunits of the involved respiratory complex
were shown by western blot analysis (Fig. 1B). The levels
of cytochrome oxidase COXII and COXIV subunits of
complex IV in Pat.D and Pat.C fibroblasts and those of
ironsulfur protein (ISP) and core-1 subunit of complex III
in Pat.N fibroblasts were severely decreased (Fig. 1B).
PGC-1a/b expression improved mitochondrial
respiration in OXPHOS deficient patient fibroblasts
and MELAS cybrids
To investigate if an increase in mitochondrial biogenesis could
compensate for partial OXPHOS defects, we transduced the
patients’ fibroblasts and the MELAS A3243G cybrids with
recombinant adenovirus (rAd) expressing PGC-1a and/or
PGC-1b. Successful expression of the PGC-1a vector was
monitored by green fluorescent protein (GFP) expression
(from its own cytomegalovirus promoter). Transductions at
titers conferring .70% GFP positive cells were used for
analysis. Similar titers were used for rAd expressing
PGC-1b. At these concentrations of rAd, we could not
observe toxicity. The increased expression of PGC-1a after
rAdPGC-1a transduction could be documented by western
blot (Supplementary Material, Fig. S1).
Induced expression of PGC-1a/b in patient fibroblasts sig-
nificantly increased respiration both from the endogenous sub-
strates (electrons entering at complexes I and II) and from the
ascorbate/TMPD where electrons are donated to cytochrome c
and subsequently to cytochrome c oxidase and oxygen.
Interestingly, the two fibroblasts with defective complex IV,
caused by distinct nuclear gene defects, showed different
response to PGC-1 isoforms overexpression. Pat.D fibroblasts
showed 50% increase both in endogenous and ascor bate/
TMPD driven respiration after PGC-1b expression but not
after PGC-1a expression. In contrast, Pat.C fibroblasts
showed 50% increase after PGC-1a expression (Fig. 2A).
On the other hand, Pat.N fibroblasts showed . 2-fold increase
in endogenous as well as ascorbate/TMPD driven respiration
with expression of either PGC-1a or PGC-1b isoforms
(Fig. 2A). Interestingly, the wild-type fibroblasts did not
show increase in respiration with PGC-1a nor PGC-1b
expression (Fig. 2A), suggesting that in wild-type cells there
may be limiting factors that were not increased by the
Induced expression of PGC-1a/b in MELAS A3243G trans-
mitochondrial cybrids showed similar results. The endogenous
and ascorbate/TMPD driven respiration were significantly
increased ( 2-fold) after PGC-1a or PGC-1b expression in
MELAS A3243G mutant cybrids compared with the GFP
expressing wild-type controls (Fig. 2B). The wild-type
control cybrids did not show an increase in respiration after
PGC-1a/b expression (Fig. 2B). MELAS cells were also trans-
duced with PGC-1aþPGC-1b, but a synergistic effect was not
observed (data not shown).
b expression enhanced mitochondrial
enzyme complexes activity in patient fibroblasts
and MELAS cybrids
To search for the mechanisms of increased mitochondrial res-
piration in patient fibroblasts and MELAS A3243G cybrids
overexpressing PGC-1a/b, we determined whether it was
associated with increase in the pre viously defective mitochon-
drial activity. There were modest increases in the OXPHOS
enzymes. Pat.D showed the greater increases, followed by
Pat.C and finally by Pat.N who essentially showed no signifi-
cant increases in complexes IþIII or IV. Complex IþIII was
increased in MELAS mutant cells (Fig. 3). These results
showed that the improvement in respiration associated with
PGC-1a/b expression was not necessarily associated with an
increase of respiratory chain levels, including the defective
enzyme complexes. The activity of the TCA cycle enzyme,
citrate synthase (CS), was significantly increased in all the
cells: both the patient fibroblasts and the MELAS A3243G
cybrids, after PGC-1a/b expression (Fig. 3C). An increase
in the TCA cycle enzyme activity would generate an increased
number of reducing equivalents that in turn might stimulate
the electron transport chain. We also observed that the activity
of complex II (succinate dehydrogenase) was increased after
PGC-1a/b expression both in patient fibroblasts and
MELAS A3243G cybrids (data not shown).
PGC-1a/b upregulation increased expression of various
OXPHOS subunits in the patients’ fibroblasts and the
MELAS A3243G cybrids
We further investigated whether the expressi on of PGC-1a/b
isoforms altered the steady-state levels of OXPHOS proteins.
The endogenous levels of several (but not all) OXPHOS
1806 Human Molecular Genetics, 2009, Vol. 18, No. 10
subunits were significantly increased after PGC-1a/b
expression both in homogenates from patient fibroblasts and
MELAS A3243G cybrids. The steady-state levels of ND39,
SDH, Cyt c, Core-1, ISP, COXIV, COXVb and ATPase a sub-
units were significantly increased after PGC-1a/b induced
expression both in patient fibroblasts and the MELAS
A3243G cybrids (Fig. 4AC), suggesting that an increase in
the steady-state levels of various OXPHOS subunits could
help enhance the electron transport function via the enzyme
complexes that in turn may lead to increase in respiration.
PGC-1a/b upregulation increased the steady-state levels of
OXPHOS complexes in the patients’ fibroblasts and the
MELAS A3243G cybrids, but did not alter the fraction
included in supercomplexes
The levels of assembled OXPHOS complexes were also
increased upon the expression of PGC-1a/b (Fig. 5A).
MELAS cells showed a higher increase in complex III than
in complexes I and IV, a pattern that was also observed with
the patients’ fibroblasts. However, complexes I and IV were
also increased in most samples. Complex IV in Pat.D and
complex III in Pat.N were very defective and essentially
absent in these samples. There was a very small increase in
their levels after treatment, observed only in overexposed
films (data not shown). We also investigated whether the
effect on respiration was due to a preferential increase in
assembled supercomplexes (Fig. 5B). Samples solubilized in
digitonin, to preserve the supercomplex structure were ana-
lyzed by blue native PAGE (BN-PAGE). Western blots of
these gels showed that supercomplexes were not preferentially
increased relative to the levels of the individual complexes. In
these cell lines, a very small fraction of complex IV was
observed in supercomplexes (data not shown).
We have previously described the peculiar features of a color-
ectal tumor cell line with severe mtDNA mutations (null
mutations) affecting complexes I and IV activity. Surprisingly,
these cells respired at close to normal levels and had extremely
high levels of PGC-1a and PGC-1b (13). When this mtDNA
was transferred to a different nuclear background, the cells
were unable to respire. PGC-1 family members are master
regulators of mitochondrial biogenesis and there is strong
evidence that mitochondrial proliferation is a natural compen-
satory phenomenon in mitochondrial diseases. Unfortunately,
in diseases associated with heteroplasmic mtDNA mutations,
mitochondria proliferation is usually observed in small
domains of extreme OXPHOS deficiency where mtDNA
mutation focal levels reach close to 100% (14,15). Increasing
PGC-1 family members’ expression in all cells could allow
healthy mitochondria to also proliferate, thus increasing the
ATP generating capacity of the cell. This initial observation
led us to investigate whether the overexpression of PGC-1 iso-
forms could have a beneficial effect for cells with an OXPHOS
defect caused by either mtDNA or nDNA abnormalities. This
concept was supported by our recent observations showing
that increased expression of PGC-1a improved a mitochon-
drial myopathy associated with a COX10 conditional gene del-
etion in mice (16). Moreover, Bastin et al. (17) found that
bezafibrate, a PPAR pan-agonist improved respiration of
OXPHOS defective cells in culture. Their data suggested
that not only PPAR was being activated but also PGC-1a.
The use of more specific PPAR agonists showed that the acti-
vation of PPARd was mostly responsible for the effect (17).
In the present study, we found that increasing expression of
PGC-1a or PGC-1b leads to an improvement in the respirat-
ory capacity of cell lines with OXPHOS defects due to a
mtDNA or a nDNA gene alteration. Interestingly, control
cells showed increased components of mitochondria without
a significant increase in their respiration rate. This observation
suggests the presence of factors regulating the respiration rate
that are independent from the amount of OXPHOS com-
ponents, not necessarily induced by PGC-1 family members.
Likely, candidates are the ratio ADP/ATP and the flux of pro-
duction of substrates for the respiratory chain. In contrast,
defective cells consistently showed improvement in respir-
ation, but with different features. Overexpression of PGC-1b
was efficient in improving Pat.D’s respiration defect due to
a Surf1 mutation, whereas PGC-1a was more efficient for
Pat.C’s defect. The increase in both these patients was not
Figure 1. Characterization of fibroblast lines from patients with mitochondrial
disorders. Three fibroblast lines from patients previously diagnosed with mito-
chondrial disorders associated with complex IV (Pat.C and Pat.D) and
complex III (Pat.N) deficiencies were analyzed for their respiratory complexes
function as a ratio to citrate synthase (A)(n ¼ 3 independent measurements,
bars ¼ SD) and steady-state levels of OXPHOS components by western blot
(B). The western blot on (B) was probed with antibodies against two
complex III subunits (ISP and core-1) and two complex IV subunits (COXII
and COXIV). Error bars represent SD of at least three independent measure-
P , 0.05 in comparisons with the enzyme activities observed in the
Human Molecular Genetics, 2009, Vol. 18, No. 10 1807
as strong as in Pat.N (complex III deficiency) or the MELAS
mutant cybrids. Therefore, respiration can be improved by
PGC-1a and/or PGC-1b overexpression, but at different
levels depending on the host cell, the defect and the PGC-1
The increase in the steady-state levels of OXPHOS com-
ponents observed with both PGC-1 isoforms involved numer-
ous proteins but it was heterogeneous. Increase in
mitochondrial biogenesis was not accompanied by a stoicheo-
metric increase in all mitochondrial components. Curiously,
the increases did not correlate well with the specific defect.
In other words, patients with a COX deficiency did not have
COX subunits preferentially increased when compared with
other mitochondrial proteins. The reason for this heterogeneity
is not well understood, but not surprising, as target genes
respond differently to PGC-1 activation (18). This heterogen-
eity could also be observed in the analyses of assembled
complexes and supercomplexes. Complex III appears to be
Figure 2. PGC-1a/b overexpression improves respiration of patients’ fibroblasts. Fibroblasts were transduced with Ad-PGC-1a or Ad-PGC-1b. After 72 h, cells
were collected and oxygen consumption measured both with endogenous substrates (E) and with ascorbate/TMPD (A/T), which donates electrons to cytochrome
c. Infections with adGFP were used as controls. (A) Shows the results with the fibroblasts lines. (B) Shows the results for the MELAS mutant cybrids, whereas
(C) shows the wild-type cybrids. Error bars represent SD of at least three independent measurements.
P , 0.01;
P , 0.001.
1808 Human Molecular Genetics, 2009, Vol. 18, No. 10
very responsive to induction by PGC-1 isoforms, and that may
explain the increased respiration observed in Pat.N. The
reason for the increase in respiration in Pat.N and in
complex III levels in most samples is unknown, but as men-
tioned above, it may be due to the responsiveness of
complex III genes to PGC-1 activation. Cytochrome c, for
example, is highly induced by PGC-1a, whereas other genes
coding for mitochondrial proteins are less sensitive (18).
Because the levels of complex III were very low and increased
very little upon expression of PGC-1 isoforms in Pat.N, it is
likely that other adaptations have a role in the increased respir-
ation. A similar argument can be made for complex IV in
Pat.D. In fact, the samples with the greatest increase in respir-
ation were the ones with the most severe defects (Pat.D, Pat.N
and MELAS). These observations suggest that the respiratory
rescue is due to an increase of not only specific subunits, but
also electron donors, transporters, assembly factors, transla-
tional activators, etc.
The improved respiration of OXPHOS defective cells when
mitochondrial biogenesis is induced has important impli-
cations for the treatment of mitochondrial disorders. Although
overexpression of PGC-1 isoforms may be difficult to achi eve,
the work of Bastin et al. (17). and ours (16) suggest that
similar effects can be achieved pharmacologically (17). In
summary, we now showed that PGC-1a/b overexpression
can lead to a marked improvement in OXPHOS defects
caused by mutations in nDNA or mtDNA. The mechanisms
appear to be related to an increase in both the levels of
OXPHOS limiting factors and the overall mitochondrial
environment, thereby increasing the electron transfer and
ATP production per cell.
Pat.C was a boy, second child of consanguineous parents (first
cousins). One older sibling was subjected to therapeutic abor-
tion for renal and cerebral malformations. One younger
brother had a similar disease with hypotonia, encephalopathy,
hypertrophic cardiomyopathy and COX deficiency. He also
had one healthy sister. He presented with hypotonia and
repeated apneic episodes since birth necessitating mechanic
ventilation. Hypertrophic myocardiopathy was detected at 1
month of age. Hyporeactivity, pyramidal syndrome, monoto-
nous EEG, altered evoked visual potentials and abnormal
brain imaging with leukodystrophy, basal ganglia lucencies
and progressive cortical atrophy were observed at 2 months
of age. Lactate levels were consistently increased in blood
and CSF since birth (37 m
M in blood, 6 mM in CSF). He
also had increased intermediates of Krebs cycle in the urine.
Muscle biopsy at 1 month of age showed mild accumulation
of lipids and isolated respiratory COX deficiency (25%
residual activity). Cultured skin fibroblasts showed 35%
residual COX activity. MtDNA had normal amount and
sequence. The patient died at 3 months of age; post-mortem
muscle and liver samples showing isolated respiratory
Figure 3. PGC-1a/b-related increase in respiration is associated with
increased OXPHOS enzyme activities. Complexes I þ III (A), IV (B) and
citrate synthase (C) activities were measured spectrophotometrically in total
cell homogenates (per mg/protein) obtained 72 h after recombinant adenovirus
transduction. Although there was an overall increase in citrate synthase
activity, OXPHOS enzyme increases were more variable.
Human Molecular Genetics, 2009, Vol. 18, No. 10 1809
complex IV defect (20 and 2% residual activity in muscle and
liver, respectively). Transfection of the child’s skin fibroblasts
with the wild-type cDNA of Surf1, COX10, Sco1 or Sco2
genes did not restore complex IV histochemical activity thus
demonstrating that none of these genes was responsible for
the complex IV defect.
Pat.D was a girl, second child of consanguineous parents
(second cousins). She presented with normal development
up to 15 months of age, fo llowed by progressive onset of anor-
exia, recurrent vomiting, motor difficulties with ataxia and
pyramidal syndrome. At 3 years of age, brain imaging
showed typical lesions of Leigh syndrome with basal
ganglia, brainstem and cerebellum alterations. At 5 years of
age, she presented with major failure to thrive (height at 28
SD), severe hypotonia, ataxia, pyramidal syndrome and
muscle weakness. Lactate was constantly increased in blood
and CSF. Muscle biopsy showed mild accumulation of lipids
and severe defect of respiratory complex IV by histochemistry
and biochemistry (5% residual activity). Cultured skin fibro-
blasts were also deficient in complex IV activity (20% residual
activity). mtDNA had normal amount and sequence. Transfec-
tion of the child’s skin fibroblasts with the wild-type Surf1
cDNA restored complex IV histochemical activity thus
demonstrating that Surf1 gene alterations were responsible
for the complex IV defect. Sequencing of the Surf1 gene
showed several alterations including an homozygous 7 bp
deletion in the 85 bp long intron between exons 1 and 2
(þ30deltgcgggg), an heterozygous G . A mutation in exon
5 (gly124arg), an heterozygous T . C mutation in exon 9
(leu281pro) and an heterozygous C . T mutation in exon 6
creating a synonymous change (phe181phe).
Pat.N was a girl, third child of consanguineous parents (first
cousins). She had three episodes of coma with hyperlactatemia
and ketoacidosis between 2 and 3.5 years of age. Each episode
was triggered by infection (urinary infection, chicken pox).
During the third episode, recurrent vomiting preceded the
onset of coma and polypnea. Biological investigations dis-
closed severe metabolic acidosis (pH 6.94, bicarbonates
M), hyperlactatemia (11 mM, n , 2mM) with high
lactate/pyruvate ratio (31, n , 15), hyperammonemia
(300 m
M, n , 50 mM), a 2-fold increase of blood transaminase
levels and moderate hepatic insufficiency (factor V 49%).
Muscle biopsy at 3 years of age showed normal morphology
and isolated respiratory complex III defect (50% residual
activity). Cultured skin fibroblasts also disclosed complex III
defect (50% residual activity). MtDNA had normal levels
and the cytochrome b gene had a normal sequence.
Fibroblasts obtained from a patient with typical MELAS
syndrome were enucleated and the mtDNA transferred to a
Figure 4. PGC-1a/b-related increase in respiration is associated with
increased levels of OXPHOS components. Western blot analyses showed
that PGC-1a/b overexpression-related increase in respiration in cells from
patients with OXPHOS defects was associated with increased expression of
OXPHOS components. Lanes were loaded with 100 mg of cell lysates. (A)
Shows protein levels in Pat.C and Pat.D. (B) Shows the analyses for Pat.N
and CTRL. (C) Describes similar analyses for MELAS and WT control.
The antibodies used were the ones against: SDH (flavoprotein of complex
II), ND39 (complex I), COXI, IV, Vb (complex IV), core-1 and Rieske
iron sulfur protein (complex III), cytochrome c, ATPase subunit
(complex V), VDAC1 (outer membrane protein) and tubulin.
1810 Human Molecular Genetics, 2009, Vol. 18, No. 10
osteosarcoma rho zero cell as described (19). Clones with
different levels of A3243G mutated mtDN A were obtained
and a clone containing only mutated mtDNA and a clone con-
taining exclusively the wild-type mtDNA were selected and
used in this study.
Cell culture
Fibroblasts derived from the above described patients (C, D
and N) were immortalized under cell culture conditions by
infecting the cells with a recombinant retrovirus encoding
hTERT (human telomerase) (20). Immortalized patient fibro-
blasts and the MELAS A3243G tRNA
drial cybrids were grown in Dulbecco’s modified Eagle’s
medium supplemented with 1 m
M pyruvate, 50 mg/ml uridine
and 10% fetal bovine serum.
Adenoviral infection
The patient fibroblasts and MELAS transmitochondrial
cybrids were transduced with adenovirus expressing GFP,
PGC-1a or PGC-1b constructs. Ad-GFP was obtained from
the viral vector core facility at Colorado State University
(Fort Collins, CO, USA). Ad-PGC-1a and Ad-PGC-1b were
a gift from Dr Bruce M. Spiegelman (Dana-Farber Cancer
Institute, Harvard Medical School, Boston, MA, USA) and
also contained a GFP marker expressed from an independent
promoter. Serial dilutions of the virus were made for
Ad-GFP (titer 9.2 10
virus particles/ml), Ad-PGC-1 a
(titer 6.9 10
virus particles/ml) and Ad-PGC-1 b (titer
8.5 10
virus particles/ml) to determine the amount of
virus needed for 100% infection. Approximately 5 7
virus particles/ml were used to infect patient fibroblasts
or MELAS transmitochondrial cybrid cell lines. Twenty-four
hours after infection, the cell culture medium was replaced
with fresh medium. Cell s were analyzed 72 h after infection.
Polarographic studies
Cell respiration (endogenous and ascorbate/TMPD) was
measured in Ad-GFP, Ad-PG C-1a or Ad-PGC-1b transduced
patient fibroblast or MELAS cybrids. Respiration was
measured in buffer containing 25 m
M TrisHCl, 10 mM
and 150 mM sucrose (pH 7.4) using a Clark oxygen
electrode in a water-jacketed cell, magnetically stirred at
378C (Hansatech Instruments, Norfolk, UK) as described (21).
Western blot analysis
Total cell extracts (100 mg) prepared from GFP or PGC-1a/b
transduced patient fibroblasts and MELAS A3243G transmito-
chondrial cybrids were run on SDSPAGE and transferred to
polyvinylidene difluoride membranes (Bio-Rad). Blots were
blocked overnight in 5% milk and incubated with the
primary antibody for 2 h followed by a secondary anti-mouse
IgG conjugated to horseradish peroxidase (HRP). The chemi-
luminescent signal was detected using Phototope-HRP western
blot detection kit (New England Biolabs, Beverly, MA).
Monoclonal antibodies against ND39, SDH(Fp) (succinate
dehydrogenase flavoprotein subunit), core-1 of complex III,
ISP subunit of complex III, cytochrome c, COXI, COXIV,
COXVb, ATPase a subunit, VDAC1 and tubulin were used
for western detection. The ND39, SDH(Fp), core-1, ISP,
COXIV, COXVb and VDAC1 antibodies were obtained
from Molecular Probes (Eugene, OR, USA). The COXI and
cytochrome c antibodies were obtained from Mitosciences
(Eugene, OR, USA). Separation and western blot of
BN-PAGE were performed as described (22).
Spectrophotometric analysis
Mitochondrial enzyme complex activities were measured
spectrophotometrically in cells using a DU-640 spectropho-
tometer (Beckman Instruments Inc., Fullerton, CA, USA) as
described elsewhere (23). The activities of NADHcyto-
chrome c oxidoreductase (complex I þ III), cytochrome c
oxidase (complex IV) and CS were determined by following
the cytochrome c reduction (complexes I þ III) or oxidation
(complex IV) at 550 nm (23). All assays were per formed at
378C (except the CS at 308C). Total cell lysates were norm al-
ized by protein content and aliquots used for the different
Figure 5. PGC-1a/b expression increases the levels of assembled OXPHOS
complexes but does not change the relative fraction incorporated into super-
complexes. Cell samples were solubilized in either lauryl maltoside (A)or
digitonin (B) to preserve the complex or supercomplex structure, respectively.
Samples were separated in a 4 16% PAGE as described in Materials and
Methods, transferred to a PVDF membrane and probed with antibodies repre-
sentative of OXPHOS complexes. COXI (complex IV), core-2 (complex III),
ND39 (complex I), VDAC1 and tubulin.
Human Molecular Genetics, 2009, Vol. 18, No. 10 1811
Supplementary Material is available at HMG online.
We are grateful to Dr Bruce M. Spiegelman (Dana-Farber
Cancer Institute, Harvard Medical School, Boston, MA) for
the recombinant adenoviruses.
Conflict of Interest statement. None declared.
This work was supported by Muscular Dystrophy Association
and PHS grants NS041777, CA085700 and EY10804 (to
C.T.M.). F.D. work was supported by the James & Esther
King Biomedical Research Program.
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1812 Human Molecular Genetics, 2009, Vol. 18, No. 10
    • "Briefly, total respiration was measured in a buffer containing 0.3 M mannitol, 10 mM KCl, 5 mM MgCl 2 , 10 mM KH 2 PO 4 and 1 mg/ml BSA (pH 7.4) in a water-jacketed cell magnetically stirred at 37 °C (Hansatech instruments, Norfolk , UK). Oxygen consumption rates were measured both in the presence and absence of potassium cyanide (KCN) to assess the rate of KCN sensitive respiration .Oxygen consumption measurements from HEK 293 cells were done as previously described [58, 59]. Briefly, HEK 293 cells were trypsinized and harvested in a sucrose-containing buffer (25 mM Tris–HCl, 10 mM K 2 HPO 4 and 150 mM sucrose (pH 7.4)). "
    [Show abstract] [Hide abstract] ABSTRACT: The phenotypic spectrum among girls with heterozygous mutations in the X-linked intellectual disability (XLID) gene CASK (calcium/calmodulin-dependent serine protein kinase) includes postnatal microcephaly, ponto-cerebellar hypoplasia, seizures, optic nerve hypoplasia, growth retardation and hypotonia. Although CASK knockout mice were previously reported to exhibit perinatal lethality and a 3-fold increased apoptotic rate in the brain, CASK deletion was not found to affect neuronal physiology and their electrical properties. The pathogenesis of CASK associated disorders and the potential function of CASK therefore remains unknown. Here, using Cre-LoxP mediated gene excision experiments; we demonstrate that deleting CASK specifically from mouse cerebellar neurons does not alter the cerebellar architecture or function. We demonstrate that the neuron-specific deletion of CASK in mice does not cause perinatal lethality but induces severe recurrent epileptic seizures and growth retardation before the onset of adulthood. Furthermore, we demonstrate that although neuron-specific haploinsufficiency of CASK is inconsequential, the CASK mutation associated human phenotypes are replicated with high fidelity in CASK heterozygous knockout female mice (CASK(+/-)). These data suggest that CASK-related phenotypes are not purely neuronal in origin. Surprisingly, the observed microcephaly in CASK(+/-) animals is not associated with a specific loss of CASK null brain cells indicating that CASK regulates postnatal brain growth in a non-cell autonomous manner. Using biochemical assay, we also demonstrate that CASK can interact with metabolic proteins. CASK knockdown in human cell lines cause reduced cellular respiration and CASK(+/-) mice display abnormalities in muscle and brain oxidative metabolism, suggesting a novel function of CASK in metabolism. Our data implies that some phenotypic components of CASK heterozygous deletion mutation associated disorders represent systemic manifestation of metabolic stress and therefore amenable to therapeutic intervention. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0295-6) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016
    • "However, elevated ROS levels can induce the expression of PPARGC1˛ (Hoffmann et al., 2013). Activation of PPARGC1˛ has also been reported to promote the partial rescue of an OXPHOS defect (Botta et al., 2013; Mootha et al., 2003; Srivastava et al., 2007 Srivastava et al., , 2009 Viscomi et al., 2011) and induce the mitochondrial encoded transcript ND5, which is associated with the up regulation of PPARGC1˛ (Srivastava et al., 2007Srivastava et al., , 2009). Therefore, the up regulation of PPARGC1˛ in combination with the induction of ND5 observed in the MCCA transcriptome is consistent with the observed genetic profile indicative of elevated levels of ROS and the activation of a rescue pathway to compensate for impaired OXPHOS activity. "
    [Show abstract] [Hide abstract] ABSTRACT: Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive inherited metabolic disease of leucine catabolism with a highly variable phenotype. Apart from extensive mutation analyses of the MCCC1 and MCCC2 genes encoding 3-methylcrotonyl-CoA carboxylase (EC, molecular data on MCC deficiency gene expression studies in human tissues is lacking. For IEMs, unbiased '-omics' approaches are starting to reveal the secondary cellular responses to defects in biochemical pathways. Here we present the first whole genome expression profile of immortalized cultured skin fibroblast cells of two clinically affected MCC deficient patients and two healthy individuals generated using Affymetrix(®)HuExST1.0 arrays. There were 16191 significantly differentially expressed transcript IDs of which 3591 were well annotated and present in the predefined knowledge database of Ingenuity Pathway Analysis software used for downstream functional analyses. The most noticeable feature of this MCCA deficient skin fibroblast transcriptome was the typical genetic hallmark of mitochondrial dysfunction, decreased antioxidant response and disruption of energy homeostasis, which was confirmed by mitochondrial functional analyses. The MCC deficient transcriptome seems to predict oxidative stress that could alter the complex secondary cellular response that involve genes of the glycolysis, the TCA cycle, OXPHOS, gluconeogenesis, β-oxidation and the branched-chain fatty acid metabolism. An important emerging insight from this human MCCA transcriptome in combination with previous reports is that chronic exposure to the primary and secondary metabolites of MCC deficiency and the resulting oxidative stress might impact adversely on the quality of life and energy levels, irrespective of whether MCC deficient individuals are clinically affected or asymptomatic.
    Full-text · Article · Jul 2016
    • "Similarly, transgenic overexpression of PGC-1í µí»¼ ameliorated the phenotype and increased the activity of mitochondrial respiratory complexes in POLG1 mutant " Mutator " mice [205]. Furthermore, adenoviral overexpression of PGC-1í µí»¼ partially restored respiratory deficits in fibroblasts obtained from patients with mitochondrial disease of various origin (though to different efficacy) and in MELAS cybrids [206]. These altogether suggest a potential therapeutic relevance of boosting mitochondrial biogenesis via PGC-1í µí»¼-mediated approaches in diseases with genetic mitochondrial disorder. "
    [Show abstract] [Hide abstract] ABSTRACT: Impaired function of certain mitochondrial respiratory complexes has long been linked to the pathogenesis of chronic neurodegenerative disorders such as Parkinson’s and Huntington’s diseases. Furthermore, genetic alterations of mitochondrial genome or nuclear genes encoding proteins playing essential roles in maintaining proper mitochondrial function can lead to the development of severe systemic diseases associated with neurodegeneration and vacuolar myelinopathy. At present, all of these diseases lack effective disease modifying therapy. Following a brief commemoration of Professor Albert Szent-Györgyi, a Nobel Prize laureate who pioneered in the field of cellular respiration, antioxidant processes, and the roles of free radicals in health and disease, the present paper overviews the current knowledge on the involvement of mitochondrial dysfunction in central nervous system diseases associated with neurodegeneration including Parkinson’s and Huntington’s disease as well as mitochondrial encephalopathies. The review puts special focus on the involvement and the potential therapeutic relevance of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1 α ), a nuclear-encoded master regulator of mitochondrial biogenesis and antioxidant responses in these disorders, the transcriptional activation of which may hold novel therapeutic value as a more system-based approach aiming to restore mitochondrial functions in neurodegenerative processes.
    Full-text · Article · Aug 2015
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