Debska-Vielhaber, G.

Kudin, A.P.

Kunz, W.S.

Peeva, V.

Schöler, S.

Vielhaber, S.

Zsurka, G.

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cytosolic area with ﬂuorescence staining also could be observed and
their unique pattern of mitochondrial dynamics represented was
discussed.
doi:10.1016/j.bbabio.2012.06.202
8P2
Hepatic mitochondrial bioenergetic and dynamic behaviour
adaptations in response to high-fat feeding
L. Lionetti, M.P. Mollica, R. Sica, I. Donizzetti, G. Gifuni, A. Pignalosa,
G. Cavaliere, R. Putti
Department of Biological Sciences, University of Naples “Federico II”,
Naples, Italy
E-mail: lilla.lionetti@unina.it
Introduction: High-fat feeding induces hepatic lipid accumulation
associated with concomitant mitochondrial fat oxidation enhance-
ment and electron-chain impairment that result in excessive for-
mation of reactive oxygen species. Little is known about hepatic
mitochondrial dynamic behaviour adaptation to high-fat feeding.
Mitochondria are dynamic organelles that frequently undergo ﬁssion
and fusion processes, imbalances in which have recently emerged
as important etiological factors in obesity and insulin-resistance.
Bearing in mind that ﬁsh-oil feeding has anti-steatotic effects, the
present work aimed to evaluate the effects of high-lard (mainly
saturated fatty acids) and high-ﬁsh-oil (mainly omega-3 polyunsat-
urated fatty acids) diets on both hepatic mitochondrial bioenergetic
and dynamic behaviour.
Methods: Hepatic lipid accumulation was monitored in rats fed a
high-lard or high-ﬁsh-oil (40% J/J) diet for 6 weeks. Mitochondrial
functions were assessed by evaluating FADH2 linked respiratory rates,
fatty acid oxidation rates, energetic efﬁciency (by measuring basal
and fatty acid induced proton leak kinetics), and oxidative stress
(by measuring H2O2 release and aconitase activity). Mitochondrial
dynamic behaviour was assessed by analysing the proteins relevant
to the processes of fusion (MFN2, OPA1) and ﬁssion (DRP1, Fis1)
(by western blot and immunohistochemical analysis).
Results: Hepatic lipid accumulation, electron chain impairment
and oxidative stress induced by high-lard diet were associated with
both decreased fatty acid induced proton leak and a shift toward
mitochondrial ﬁssion processes (decreased MFN2 content and in-
creased DRP1 and Fis1 content). On the other hand, the anti-steatotic
effect of high-ﬁsh oil feeding was associated with mild uncoupling
(increases in both basal and fatty acid induced proton leak kinetics),
oxidative stress prevention and increasedmitochondrial fusion processes.
Conclusions: Fission phenotype and increased energetic efﬁciency
are associated with the steatotic effect of high-lard diet, whereas
fusion phenotype and decreased energetic efﬁciency contributed to
the anti-steatotic effect of high-ﬁsh oil diet.
doi:10.1016/j.bbabio.2012.06.203
8P3
Mitophagy mediated by Rheb and the maintenance of
mitochondrial activity
S. Melser, E. Hebert Chatelain, J. Lavie, E. Giraud, C. Jose,
R. Rossignol, G. Bénard
MRGM Maladies Rares: Génétique et Metabolism, EA 4576,
Université de Bordeaux, France
Service de Génétique Médicale, CHU de Bordeaux, Ecole de Sages Femmes,
Hopital Pellegrin, 1 Place Amélie Raba-Léon, France
Neurocentre Magendie, INSERM U862, Physiopathologie de la Plasticité
Neuronale, Université Bordeaux, 2 146 rue Léo Saignat, 33077 Bordeaux,
France
E-mail: su.melser@inserm.fr
Cells constantly adapt the amount of mitochondria to supply
energy in accordance to physiological demand. The amount of mito-
chondria depends on the ﬁne balance between biogenesis and degra-
dation processes. The relationship between mitochondrial biogenesis
and bioenergetics has been broadly investigated but it is not clear
how mitochondrial degradation and energetics may be linked.
Mitophagy is a conserved degradation process in which the autoph-
agic machinery delivers mitochondria to the lysosomes. We found that
increasing mitochondrial activity by supplying cells exclusively with
oxidative substrates such as glutamine or dimethyl-alpha ketoglutarate
triggered mitophagy.
Using gradient partition analysis and ﬂuorescence microscopy we
found that Ras-homologue enriched in brain (Rheb) can be recruited to
mitochondria preferentially in cells with forced mitochondrial activity.
Interestingly, silencing of Rheb inducesmitochondrial outgrowth,while
ectopic expression increases formation of autophagosomes and mito-
chondrial degradation. Furthermore, mitophagy induced by Rheb over-
expression also triggered the enhancement of OXPHOS efﬁciency;
respiration measured with a Seahorse XF 24 analyzer was increased in
these cells despite a lower amount of mitochondria. In contrast, Rheb
silencing decreased routine oxygen consumption and coupled mito-
chondrial respiration.
In mitochondria, Rheb is able to regulate mitophagy triggered by
mitochondrial activity through physical interaction with the mito-
chondrial mitophagy receptor Nix and the autophagosome related
protein LC3, as shown by immunoprecipitation and Blue Native-
PAGE. Rheb likely promotes interaction between Nix and LC3 and
therefore Nix silencing inhibits Rheb-induced mitophagy.
In summary, our ﬁndings indicate that mitochondrial degradation
through mitophagy mediated by Rheb participates in the regulation
of cellular energy homeostasis.
doi:10.1016/j.bbabio.2012.06.204
8P4
Disrupted mitochondrial fusion affects oxidative phosphorylation
by mtDNA depletion
V. Peeva1, G. Zsurka1, S. Schöler1, A.P. Kudin1, S. Vielhaber2,
G. Debska-Vielhaber2, W.S. Kunz1
1Department of Epileptology and Life & Brain Center, University of Bonn,
Sigmund-Freud-Str. 25, D-53105 Bonn, Germany
2Department of Neurology, University of Magdeburg, Leipziger Str. 44,
D-39120 Magdeburg, Germany
E-mail: Viktoriya.Peeva@ukb.uni-bonn.de
The mitofusin 2 gene (MFN2) encodes a mitochondrial outer
membrane protein crucial for mitochondrial fusion machinery [1].
Mutations inMFN2 are associated with Charcot–Marie–Tooth disease
type 2A (CMT2A), which is an autosomal dominant axonal neurop-
athy [2].
It is still unclear how the mitochondrial fusion dynamics alter the
mitochondrial function. Here,we investigated themitochondrial function
in skeletal muscle and cultured ﬁbroblasts from four CMT2A patients,
all carrying different MFN2 mutations (p.Met376Val, p.Arg707Pro,
p.Val226_Ser229del, and p.Gln74Arg).
Histology of the muscle biopsies showed altered mitochon-
drial distribution in type 2A skeletal muscle ﬁbers, gathering of
Abstracts S73
subsarcolemmal mitochondria and rarefaction of intermyoﬁbrillar mito-
chondria. Although the maximal activities of respiration of saponin-
permeabilized muscle ﬁbers and digitonin-permeabilized ﬁbroblasts
were only slightly affected by theMFN2 mutations, their sensitivity to
the cytochrome c oxidase (COX) inhibitor azide was increased, which
indicates a decrease of in vivo activity of COX.
In comparison to controls, the MFN2 ﬁbroblast samples showed a
decrease in the mitochondrial DNA copy number, which explains the
observed mitochondrial respiratory chain dysfunction. Additionally, an
increased amount of deletions was observed. However, the deletions are
unlikely to contribute signiﬁcantly to the detected respiratory impair-
ment, because of their minor overall amounts in these patients.
Our ﬁndings support the viewpoint that impairment of mitochon-
drial fusion causesmild respiratory chain dysfunction through defective
mitochondrial DNA replication.
References
[1] T. Koshiba, S.A. Detmer, J.T. Kaiser, H. Chen, J.M. McCaffery, D.C.
Chan, Science (5685) (2004) 858–862.
[2] S. Züchner, I.V. Mersiyanova, M. Muglia, N. Bissar-Tadmouri, J.
Rochelle, E.L. Dadali, M. Zappia, E. Nelis, A. Patitucci, J. Senderek,
Y. Parman, O. Evgrafov, P.D. Jonghe, Y. Takahashi, S. Tsuji, M.A.
Pericak-Vance, A. Quattrone, E. Battaloglu, A.V. Polyakov, V.
Timmerman, J.M. Schröder, J.M. Vance, Nat Genet (5) (2004)
449–451.
doi:10.1016/j.bbabio.2012.06.205
8P5
A lung cancer model linking apoptotic resistance and metastatic
potential via defects in mitochondrial ﬁssion protein
Dynamin-related protein 1
Kelly Jean Thomas, Marty R. Jacobson
ColoradoMesaUniversity, Department of Biological Sciences, Grand Junction,
CO, USA
Saccomanno Research Institute, St. Mary's Hospital and Regional Medical
Center, Grand Junction, CO, USA
E-mail: kjcraig@coloradomesa.edu
Resistance to apoptosis is a hallmark of cancer. Evasion of apoptosis is
implicated in almost all aspects of cancer progression, as well as
treatment resistance. Apoptosis is regulated in part by mitochondria,
which control tissue homeostasis by eliminating damaged cells. In this
study, resistance to apoptosiswas identiﬁed in lung epithelial (A549) cells
as a consequence of defects in mitochondrial and autophagic function.
Mitochondrial function is determined in part by mitochondrial
morphology, a process regulated by mitochondrial dynamics whereby
the joining of twomitochondria, fusion, inhibits apoptosis while ﬁssion,
the division of a mitochondrion, initiates apoptosis. Mitochondrial
length correlated with metastatic potential; lung epithelial cells with
increased metastatic potential had mitochondria with an elongated
phenotype—mimicking cells deﬁcient in mitochondrial ﬁssion protein,
Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1mito-
chondrial recruitment and decreased Drp1-dependent ﬁssion. Cyto-
chrome c release, caspase-3 and PARP cleavage were impaired both
basally and with apoptotic stimuli in A549 cells.
Metastatic potential positively correlated with mitochondrial mass,
suggesting defects in mitophagy (mitochondrial selective autophagy).
A549 cells had decreased LC3-II lipidation and lysosomal inhibition
suggesting that defects in autophagy occur upstream of lysosomal
degradation. Immunostaining also indicated that mitochondrial local-
ized LC3 punctae in A549 cells increased aftermitochondrial uncoupling
orwith a combination of mitochondrial depolarization and ectopic Drp1
expression. Increased inhibition of apoptosis in A549 cells is correlated
with impeded mitochondrial ﬁssion and mitophagy. We suggest that
mitochondrial ﬁssion defects contribute to apoptotic resistance in lung
cancer cells with a high propensity for metastasis.
doi:10.1016/j.bbabio.2012.06.206
8P6
Mitochondrial fusion/ﬁssion proteins in NARP and Rho0 human
osteosarcoma cells
J. Walczak, J. Duszyński, J. Szczepanowska
Nencki Institute of Experimental Biology, Polish Academy of Sciences,
Warsaw, Poland
E-mail: jwalczak@nencki.gov.pl
Dysfunctions of mitochondria are usually associated with numerous
diseases likemetabolic disorders, cancer and neurodegenerative diseases.
Changes caused by the chronic mitochondrial stress include defects
in respiratory chain complexes, morphology and organization of the
mitochondria, mitochondrial membrane potential (Δψ), cytosolic Ca2+
concentration, ATP and ROS levels. These parameters are involved in
the retrograde signaling from mitochondria to nucleus that triggers
mitochondrial stress response (MSR) of the cell and its subsequent
adaptation to altered mitochondrial functions [1,2].
Although knowledge about components involved in the mitochon-
drial retrograde signal transduction is still incomplete, it is likely that
mitochondrial morphology and positioning within the cell can play
an important role in mitochondrial–nuclear communication. Proteins
implicated in dynamics of mitochondria were investigated in cells with
chronic mitochondrial stress:
1) Rho0 human osteosarcoma cells, lacking mitochondrial DNA,
2) Cybrid NARP human osteosarcoma cells with point mutation
T8993G in subunit 6 of ATP synthase (98% of heteroplasmy).
We have previously shown that many aspects of physiology
(calcium homeostasis, ROS metabolism) as well as mitochondrial
network and cytoskeleton organization in cells with chronic mitochon-
drial stress (NARP and Rho0) differs from that in WT cells [3]. Our new
results indicate that the proﬁle of proteins responsible for the dynamics
ofmitochondria (Drp1, Opa1,Mfn1 and Fis1) is different in investigated
cell lines. The observations carried out in the confocal microscope show
changes in the organization of mitochondria within these cells.
References
[1] M. Wojewoda, J. Duszyński, M.R. Więckowski, J. Szczepanowska,
Mitochondrion 12 (2012) 149–155.
[2] M. Wojewoda, J. Duszyński, J. Szczepanowska, Biochim. Biophys.
Acta 1797 (2010) 890–896.
[3] Szczepanowska J, Malinska D, Wieckowski MR, Duszynski J (2012)
Biochim. Biophys. Acta In press.
doi:10.1016/j.bbabio.2012.06.207
8P7
The MINOS complex: Keeper of mitochondrial
membrane architecture
Ralf M. Zerbes1,2, Maria Bohnert1, Karina von der Malsburg1,
Bettina Warscheid3,4, Ida J. van der Klei5,
AbstractsS74


Disrupted mitochondrial fusion affects oxidative phosphorylation by mtDNA depletion 

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Disrupted mitochondrial fusion affects oxidative phosphorylation by mtDNA depletion

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