Crosstalk Between Mitochondrial(dys)Function and MitochondrialAbundanceSEBASTIEN MICHEL,1 ANAIS WANET,1 AURELIA DE PAUW,2 GUILLAUME ROMMELAERE,1

THIERRY ARNOULD,1 AND PATRICIA RENARD1*1Laboratory of Biochemistry and Cell Biology (URBC), NARILIS (Namur Research Institute for Life Sciences),

University of Namur (FUNDP), Namur, Belgium2Institute of Experimental and Clinical Research (IREC), Pole of Pharmacology and Therapeutics (FATH 5349),

University of Louvain (UCL) Medical School, Brussels, Belgium

A controlled regulation of mitochondrial mass through either the production (biogenesis) or the degradation (mitochondrial qualitycontrol) of the organelle represents a crucial step for proper mitochondrial and cell function. Key steps of mitochondrial biogenesis andquality control are overviewed, with an emphasis on the role of mitochondrial chaperones and proteases that keep mitochondria fullyfunctional, provided the mitochondrial activity impairment is not excessive. In this case, the whole organelle is degraded by mitochondrialautophagy or ‘‘mitophagy.’’ Beside the maintenance of adequate mitochondrial abundance and functions for cell homeostasis,mitochondrial biogenesis might be enhanced, through discussed signaling pathways, in response to various physiological stimuli, likecontractile activity, exposure to low temperatures, caloric restriction, and stem cells differentiation. In addition, mitochondrialdysfunction might also initiate a retrograde response, enabling cell adaptation through increased mitochondrial biogenesis.J. Cell. Physiol. 227: 2297–2310, 2012. ! 2011 Wiley Periodicals, Inc.

Although the examples cited above suggest that the control ofmitochondrial mass, through biogenesis or quality control, isbeneficial to the cell/organism demands, the data generatedfrom diverse pathologies suggest that modulations inmitochondrial abundance/functions may participate to thepathogenesis. Increased mitochondrial abundance is generallydescribed to characterize mitochondrial myopathies as well asmost cancers, and is generally accompanied by qualitativemodifications of mitochondria, thereby affecting programmedcells death susceptibility. On the contrary, in ageing, obesity andtype 2 diabetes, mitochondrial biogenesis and functions aregenerally down-regulated. The development of insulinresistance, favored by an impaired mitochondrial function,tends to reduce the abundance of the organelle, through avicious circle.

Modern View of the Mitochondrion

Mitochondria are composed of approximately 1,500 proteins,of which 13 are (still) encoded by the mitochondrial DNA(mtDNA) in mammalians. The integrity (of) and crosstalkbetween both nuclear and mitochondrial genomes are thusessential to ensure the normal and various functions ofmitochondria. Indeed, beside oxidative ATP production,mitochondria assume other functions such as heme synthesis,b-oxidation of free fatty acids, metabolism of certain aminoacids, production of free radical species, formation and exportof Fe/S clusters, iron metabolism, and play a crucial role incalcium homeostasis (Duchen, 2004). In addition, firstlydescribed as a key checkpoint of intrinsic programmed celldeath, accumulating data point mitochondria as a centralplatform involved in many cellular pathways, such as thoserecently highlighted participating to innate immune response(West et al., 2011) or its lipidic contribution to autophagosomalmembrane genesis during starvation-induced autophagy (Haileyet al., 2010).

Since its first description by R. A. Von Kolliker in 1857(Liesa et al., 2009), our understanding of mitochondria has alsotremendously evolved regarding dynamic of the organelle.Indeed, the static bean-shaped view of isolated mitochondria,

while still observed in many handbooks, has now been replacedby a highly branched and dynamic network that movesthroughout the cell and undergoes structural transitions,changing the length, morphology/shape, and size of theorganelle depending on the particular cell type. In addition, themitochondrial morphology can change according to the cellcycle, the cellular differentiation status or themetabolic activityand energy needs (Liesa et al., 2009). Interestingly, a crosstalkdoes exist between dynamics and function of mitochondria asdynamics affect the activity of the organelle while dysfunction ofmitochondrion does also impact on the morphology anddynamics of this sub-cellular structure (Kuznetsov et al., 2009).These changes in shape are tightly regulated by the balancebetween ‘‘fusion’’ and ‘‘fission’’ events that regulate theappearance of the dynamic organelle, its composition, and thusits activity and functions (Kuznetsov et al., 2009).

In mammalian cells, the core of the mitochondrial fusionmachinery consists of two large GTPases located at the outermitochondrial membrane (OMM), the mitofusins 1 and 2(MFN1 and MFN2), that form homo- and hetero-oligomericcomplexes between apposing mitochondria (Koshiba et al.,2004;Meeusen et al., 2004; Chen et al., 2005;Detmer andChan,2007a). While mitofusins 1 and 2 are mainly involved in thefusion of OMM and the tethering of mitochondria toendoplasmic reticulum (ER) (de Brito and Scorrano, 2009),the dynamin-like GTPase protein OPA1, localized in theintermembrane space (IMS) and associated with the inter

Contract grant sponsor: Association Belge Contre les MaladiesNeuro-Musculaires (ABMM, Belgium).

*Correspondence to: Patricia Renard, Laboratory of Biochemistryand Cell Biology (URBC), NARILIS (Namur Research Institute forLife Sciences), University of Namur (FUNDP), 61 rue de Bruxelles,5000 Namur, Belgium. E-mail: patsy.renard@fundp.ac.be

Received 4 September 2011; Accepted 7 September 2011

Published online in Wiley Online Library(wileyonlinelibrary.com), 16 September 2011.DOI: 10.1002/jcp.23021

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mitochondrial membrane (IMM), mediates the fusion of theinner membranes (Cipolat et al., 2004; Chen et al., 2005).Mitochondrial fission requires the recruitment of Drp1(a GTPase belonging to the dynamin family members) fromthe cytosol to the OMM by a mitochondrial integral outermembrane protein called Fis1 (fission protein 1 homolog)(Liesa et al., 2009). The subsequent oligomerization of Drp1,finally leading to fission, is regulated by post-translationalmodifications such as phosphorylation, S-nitrosylation,ubiquitination, and sumoylation (for a review: Chang andBlackstone, 2010). Moreover, it is worth mentioning that bothfusion and fission processes mediate a partial mitochondrialcomplementation by determining intra-mitochondrialexchange of mtDNA, proteins, and lipid membranes, thuscausing a dynamic flux allowing segregation of mitochondrialmolecules (Detmer and Chan, 2007b). For extended reviewsonmitochondrial dynamics, seeWestermann (2010) andOteraand Mihara (2011).

Finally, mitochondrion does not work as an isolatedorganelle, but rather interacts and communicates with othercell components such as cytoskeleton for organelle movement,ER at specific domain called mitochondria-associatedmembranes (MAMs) (Vance, 1990) and transports cargo toperoxisomes by mitochondria-derived vesicles (MDVs)(Neuspiel et al., 2008).

Because mitochondrial abundance, integrity, activity, andappropriate morphology is fundamental for cell life, thedisturbance/alterations of the organelle can disrupt many cellfunctions (and in some conditions, impairs cell survival leadingto cell death), an initiating event in the onset of numerousdiseases. Indeed, mitochondrial dysfunction is associated, as acause or a consequence, with a wide panel of humanpathologies, ranging fromneuromuscular degenerative diseasesto some types of cancers, unhealthy aging, and metabolicdiseases (Stark and Roden, 2007; Dimauro, 2011; Jezek et al.,2010). Thus, a controlled regulation of mitochondrial massthrough either the production (biogenesis) or the degradation(mitochondrial quality control) of the organelle represents oneof the crucial steps for proper mitochondrial and cell function.Before discussing the existing crosstalk betweenmitochondrial

abundance, function, and dysfunction, key steps ofmitochondrial biogenesis and quality control will be firstdescribed hereafter.

Highlights on Mitochondrial Abundance RegulationKey steps of mitochondrial biogenesis

The definition of ‘‘mitochondrial biogenesis’’ refers to both theformation of mitochondria during the cell cycle, the genesis ofthe organelle for normal turnover (Attardi and Schatz, 1988),as well as the phylogenesis and ontogenesis of mitochondria(Shepard et al., 1998). More recently, Nisoli and co-workershave classified the proposed theories on the biogenesis ofmitochondria into three categories: (i) de novo synthesis of theorganelle from sub-microscopic precursors present in thecytoplasm; (ii) formation fromothermembranous structures ofthe cell; (iii) growth and division of pre-existing mitochondria.The bulk of experimental evidences performed onmitochondrial biogenesis process is in favor of the concept thatpre-existing mitochondria may grow and divide. However, theother two possibilities have not been ruled out conclusively yet(Nisoli et al., 2004).

A tightly coordinated regulation of mitochondrialbiogenesis in response to external stimuli. Whendealing with the biogenesis of mitochondria, at least threecoordinated aspects related to the composition, structure,and function of the organelle require special attention: (i) thetranscription, the import, targeting, and potential assemblyof nuclear genes-encoded proteins within mitochondria;(ii) the maintenance, replication, and the transcription ofmitochondrial genome; and (iii) the biogenesis of mitochondrialmembranes. To be coordinated, all these aspects need to betightly orchestrated at the transcriptional level in response tovarious external stimuli and downstream signaling pathways.

In response to various external stimuli, including coldexposure, energy deprivation, caloric restriction, or physicalexercise, several cell signaling pathways, mainly dependenton cAMP/PKA, NO/cGMP, Ca2!/CaMKIV, or AMPK, regulatethe expression and/or the activation of the co-activatorPGC-1a that, in turn, modulates the transcriptional activity of

BOX 1. Main transcription factors involved in mitochondrial biogenesis

Transcriptionfactor Regulated genes Refs.

NRF-1 Respiratory subunits Scarpulla (2008b)Mitochondrial heme synthesisMitochondrial importation system and assemblyMitochondrial translation (ribosomal proteins, tRNA synthetases)MtDNA replication, transcription and translation

NRF-2 Respiratory subunits, Tfam, TFB1M, TFB2MTFAM mtDNAERRaa Respiratory subunits, mitochondriale importation system,

oxydative metabolism, mitochondrial dynamics-associated genesMootha et al. (2004), Schreiber et al. (2004)

PPARaa Oxydative metabolism Vega et al. (2000)PPARga Uncoupling proteins Puigserver et al. (1998)Thyroid hormone Weitzel and Alexander Iwen (2011)CREBa Cytochrome c, PGC-1a Franko et al. (2008), Vercauteren et al. (2006)YY1 COX genes Basu et al., (1997), Cunningham et al. (2007)C-MYCa MtDNA polymerase, NRF-1 target genes, PGC-1b Li et al. (2005), Morrish et al. (2003)MEF-2a PGC-1a, muscle-specific COX subunits Handschin et al. (2003), Ramachandran et al. (2008),

Wan and Moreadith (1995)SP1 Tfam, TFB1M, TFB2M, POLg2, ANT2 Evans and Scarpulla (1989), Li et al. (1996a,b)ATF2a PGC-1a, UCP1 Cao et al. (2004)NFATa PGC-1a Olson and Williams (2000)

PGC-1a, Peroxisome-proliferator activated receptorGammaCo-activator-1 alpha;NRF1/2, nuclear respiratory factor 1/2; TFAM,mitochondrial transcription factorA; ERRa, estrogen-related receptor alpha; PPAR, peroxisome proliferator-activated receptor; CREB, cAMP response-element binding protein; YY1, yin-yang1; MEF-2, myocyte enhancer factor-2; SP1,specificity protein 1; HNF4, hepatocyte nuclear factor 4; ATF2, activating transcription factor-2; FoxO1, forkhead box protein O1; NFAT, nuclear factor of activated T-cells;COX, cytochrome c oxidase; POLg, DNA polymerase gamma ANT2, adenine nucleotide translocator.aRegulate PGC-1 expression.

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nuclear-encoded mitochondrial genes by interacting andactivating nuclear respiratory factors 1 and 2 (NRF-1/2)and the estrogen related receptor alpha (ERRa). In addition,PGC-1a is also regulated at the post-translational level byphosphorylation, acetylation, ubiquitination, methylation, andN-acetylglucosamination (Fernandez-Marcos and Auwerx,2011).

PGC-1a, which was firstly discovered as an interactingpartner of the nuclear receptor PPARg in brown adipose tissue(Puigserver et al., 1998), is the founding member of thePeroxisome-proliferator activated receptor GammaCo-activator (PGC) family, acting as master coordinators ofmitochondrial biogenesis by functional interaction with varioustranscription factors (listed in Box 1) that regulate theexpression of nuclear genes encoding mitochondrial proteins(Puigserver et al., 1998; Andersson and Scarpulla, 2001;Kressler et al., 2002; Lin et al., 2002). PGC-1 family is composedof three members: PGC-1a, PGC-1b, and PGC-relatedco-activator (PRC) (Scarpulla, 2011). Like PGC-1a, PGC-1bstrongly activates mitochondrial biogenesis and enhancescellular respiration in many cell types such as myotubes andhepatocytes (Lin et al., 2003; St-Pierre et al., 2003; Shao et al.,2010). However, conversely to PGC-1a, the mechanism bywhich PGC-1b modulates mitochondrial biogenesis remainsunclear. If results first suggested major redundant andoverlapping function of PGC-1a and PGC-1b, both co-activators now appear to play distinct and specific roles in theregulation of mitochondrial biogenesis and function (Scarpulla,2011). Indeed, PGC-1b is unable to completely compensate theloss of PGC-1a, but appears to play a more specific role duringbrown and white adipocyte differentiation (Lin et al., 2004;Uldry et al., 2006; Nie and Wong, 2009). The third member ofPGC-1 family, PRC, is an ubiquitously expressed co-activator ofwhich function is poorly understood, except for its role inmitochondrial biogenesis in dividing cells (Scarpulla, 2011). PRCis an immediate early gene that is rapidly induced by serum inquiescent fibroblasts in response to mitogenic signals. SpecificshRNA-mediated knock-down of PRC expression in U2OS cellline results in the inhibition of cell growth, suggesting that PRCserves to integrate the expression of the respiratorymachinerywith a program of cell expression modifications involved in cellproliferation (Scarpulla, 2008a).

MtDNA replication and mtDNA-encoded genestranscription. It is assumed that mtDNA replication isan essential component of the mitochondrial biogenesis evenif biogenesis of the organelle dissociated from mtDNAamplification has been described/reported.

The DNA polymerase identified in animal mitochondria isDNA polymerase g (POLG), an enzyme that performs allactivities necessary to carry out mtDNA replication and repairmitochondrial genome to some extent (Clay Montier et al.,2009). In mitochondria, both replication and transcription ofmtDNA are coupled, as mtDNA synthesis-related proteins arealso involved in genome transcription (Clayton, 1992). Invertebrates, the non-coding D-loop sequence contains theorigin of replication for the H-strandDNA but is also the site ofbidirectional transcription from opposing heavy- and light-strand promoters (Clayton, 2000). The protein components ofnucleoids such as mitochondrial single strand binding protein(mtSSBP) and Twinkle work together to perform helixdestabilization during replication, while mtTFA/Tfam isnecessary for the initiation of both replication and transcription(Korhonen et al., 2003). mtDNA transcription is performed bya single RNA polymerase (POLRMT), assisted by proteinsresponsible for promoter recognition (TFB1M, TFB2M),activation (Tfam), and transcription termination (mTERF)(Ghivizzani et al., 1994; Scarpulla, 2008b). The mammalianmtDNA encodes for 2 rRNAs, 20 tRNAs, and 13 polypeptideswhich are all coding for respiratory chain subunits (Diaz and

Moraes, 2008). The other subunits of the electron transportchain (ETC) are encoded by nuclear genes, translated in thecytosol, and then actively imported into the mitochondriawhere complexes are assembled such as well described forcomplex 1 (Koopman et al., 2010). Different import pathwayshave been characterized depending on the mitochondriallocation of the protein (matrix, IMM, OMM, IMS) (Diaz andMoraes, 2008). Multimeric complexes such as, translocase ofthe outer mitochondrial membrane (TOM) and translocaseof the inner mitochondrial membrane (TIM) complexes areessential for the import and compartment sorting ofmitochondrial proteins into the organelle.

Biogenesis of mitochondrial membranes. In additionto correct/appropriate expression and importation ofmitochondrial proteins, mitochondria are also heavilydependent on the successful incorporation of phospholipidsinto both OMM and IMM for their biogenesis and themaintenance of their functions, as phospholipids form a barrierto maintain mitochondrial membrane potential but are alsoinvolved in diverse mitochondrial functions, including theassembly of mitochondrial respiratory chain and fusion andfission events (Gebert et al., 2011). Most mitochondrialphospholipids, such as phosphatidylcholine,phosphatidylserine, phosphatidylinositol, and sterols, aresynthesized in the smooth endoplasmic reticulum (SER) andtransported from mitochondria-associated membranes(MAMs) to mitochondria. Imported phosphatidylserine israpidly decarboxylated within the mitochondrial innermembrane to form phosphatidylethanolamine (Shiao et al.,1995; Voelker, 1997). Phosphatidylethanolamine can then betransferred back to the SER through MAMs to be convertedinto phosphatidylcholine that requires an extra methylationstep (Voelker, 2005). In addition, mitochondria is also the siteof the synthesis of several acidic phospholipids, includingphosphatidic acid, CDP-diacylglycerol, phosphatidylglycerol,and cardiolipin (van Meer et al., 2008). While the transportsystems for the import of mitochondrial proteins andphospholipids were first thought to act independently fromeach other, accumulating data are now showing a strongcooperation between them as illustrated by the participationof Tam4, a crucial mitochondrial protein for assembly andmaintenance of translocase complexes, in the synthesis ofcardiolipin (Kutik et al., 2008). For recent and extended reviewson connection between mitochondrial lipids and proteinsimport in the biogenesis of the organelle, see Gohil andGreenberg (2009), Gebert et al. (2011), and Osman et al.(2011).

Overview of mitochondrial turnover and quality control

The abundance of mitochondrial population in cells does notonly result from the biogenesis of the organelle but is alsodependent on its turnover that involves the degradation ofinternal mitochondrial components as well as the autophagicdigestion of the entire organelle by a process called‘‘mitophagy’’ (Kanki and Klionsky, 2010). The total replacementof the mitochondrial population in liver, heart, and brain hasbeen estimated to 9.3, 17.5, and 24.4 days, respectively(Menzies and Gold, 1971). However, the basal turnover ofmitochondria can gradually be enhanced in response todifferent stresses of various intensities such as the accumulationof unfolded or aggregated proteins that induce a specific andadaptive mitochondrial unfolded protein response (mtUPR)(Haynes and Ron, 2010). However, severe mitochondrialdysfunction such as activity impairment caused by defectiveoxidative phosphorylation will lead to the entire organelledegradation. The gradated response to increasingmitochondrial stress are crucial in a cell attempt to recoverproper mitochondrial homeostasis, which if not, would lead to

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cell dysfunction or even cell death by and the initiation ofapoptosis (Tatsuta and Langer, 2008; Luce et al., 2010).

MtDNA copy number regulation. Mitochondriaquality control implies a tight control of mtDNA copy number,a process that is not only cell-type specific but also largelydependent on developmental stages. While studies performedon the turnover rates of mtDNA have demonstrated that thehalf-life time of mtDNA molecules is tightly regulated (Grosset al., 1969; Bai et al., 2000; Cao et al., 2007), mechanisms thatregulate the abundance of mtDNA are still poorly understoodand debated in the literature. However, different hypotheses toexplain the control of the mtDNA copy number have beenproposed, such as the control by the availability of nucleotides(Tang et al., 2000b), the number of replication sites (Tang et al.,2000a) or the detection by still unknown sensors of a loweror a higher threshold of mtDNA copy number leading tothe activation of either the replication machinery or thedegradation machinery of mtDNA, respectively (Clay Montieret al., 2009). Even if additional actors do probably still need tobe identified, accumulating data point Tfam/mtTFA as one ofthe crucial regulators that control mtDNA copy numbers as itinduces mtDNA replication (Korhonen et al., 2003), maintainsmtDNA integrity (Larsson et al., 1998), negatively regulates themitochondrial base excision repair (Canugovi et al., 2010), andmight stabilize mtDNA within nucleoids (Kang et al., 2007).

Mitochondrial chaperones and proteases: Sentriesof mitochondrial proteins homeostasis. To keepmitochondria fully functional, mitochondrial proteins have tobe and kept properly folded by chaperones and damaged oraggregated proteins have to be rapidly degraded by proteases.Mitochondrial chaperones and proteases implicated in thisquality control are listed in Box 2, a classification based on theirtarget location and function and recently reviewed (Baker et al.,2011). In addition to basal/constitutive quality control tomaintain the mitochondrial activity, mitochondria are also ableto respond to an accumulation of unfolded proteins within theorganelle through a mtUPR analogous to the ER UPR. Indeed, acoordinated transcriptional activation for the expression ofmitochondrial chaperones (HSPD1, HSPE1) and proteases(ClpP, YME1L1) has been described in mtDNA-depleted r0

cells (Martinus et al., 1996) and in cells that overexpress atruncated form of a mitochondrial enzyme that accumulates inthe matrix (Zhao et al., 2002). However, detailed mechanismsand actors involved in the mitochondrial stress response arestill poorly understood in mammals. Even if the implicationof CHOP-10 as an important mtUPR regulator has beendescribed, 3,899 nuclear genes in the human genome arepredicted to contain the 7-bp consensus element for CHOP-

10-C/EBPb transcription factors, suggesting that additionalregulatory mechanisms/effectors must be involved in thespecific activation of the mtUPR (Ryan and Hoogenraad, 2007).Conserved sequences surrounding the CHOP-10 consensuselement, named mitochondrial unfolded protein responseelement 1/2 (MURE1/2), in the mtUPR-responsive genespromoter have also been identified but binding proteins tothese motifs still need to be discovered (Zhao et al., 2002;Aldridge et al., 2007). So far, data suggest that the mtUPR is atwo-stage regulatory process (Ryan and Hoogenraad, 2007).First, the sensing of unfolded proteins in mitochondria wouldtrigger a retrograde signaling to the nucleus and subsequentinduction of CHOP-10 (Horibe and Hoogenraad, 2007). Then,the CHOP-10-C/EBPb heterodimer likely in cooperation withadditional factors, would bind to target promoters and activatethe transcription of mtUPR-responsive genes such asmitochondrial chaperones (HSPD1, HSPE1) and proteases(ClpP, YME1L1), allowing an adaptive response.

Organelle degradation by mitophagy. Whenmitochondrial activity impairment becomes excessive, themitochondrion can be degraded as a whole organelle bymitochondrial autophagy or ‘‘mitophagy’’ (Elmore et al., 2001;Klionsky, 2007). Autophagy is the process by which organellesand fragments of cytoplasm are sequestered by an isolationmembrane, and subsequently delivered into lysosomes forhydrolytic digestions and recycling (Klionsky, 2007). Mitophagyis a selective autophagy, which has been reported to targetdepolarized mitochondria, notably during apoptosis, a processknown to be dependent on the permeability transition pore(PTP) opening (Elmore et al., 2001; Suen et al., 2008; Twig et al.,2008). As for mitochondrial complementation, fusion andfission events can also be involved in the generation ofdepolarized mitochondria. Indeed, it has been shown thatfission can produce unequal daughter mitochondria that differin membrane potential, a phenomenon also observed inasymmetric division (Rivolta and Holley, 2002). In addition,fusion events probability is decreased for depolarizedmitochondria, which will be targeted and degraded byautophagy (Twig et al., 2008). Key actors of mitophagy initiationhave been described such as the E3-ubiquitin ligase Parkin,the PTEN-induced putative kinase 1 (PINK1), and the pro-apoptotic proteins Bnip3 and Nix (for a review see Narendraand Youle (2011) and Thomas et al. (2011), respectively). Theseeffectors act as links between mitochondrial dysfunction,depolarization of mitochondrial membrane potential, andautophagic machinery recruitment. Briefly, mitophagy isinitiated following PINK1 stabilization at the OMM andrecruitment of cytosolic Parkin upon inner mitochondrial

BOX 2. Mitochondrial quality control actors at the protein level

NameTargetlocation Activity Target Refs.

mtHSP70 Matrix Chaperone Imported unfolded peptides and misfolded proteins Young et al. (2003)Cpn60, Cpn10 Matrix Chaperone Imported unfolded peptides and misfolded proteins Bender et al. (2011)Lon Matrix AAA-Ser-Protease Denatured and oxidized proteins, TFAM Bender et al. (2011)ClpP Matrix AAA-Ser-Protease Aggregated and misfolded proteins Hansen et al. (2005), Haynes et al. (2007)i-AAA (YME1L1) IMS AAA! metalloprotease Misfolded proteins, OPA1 Song et al. (2007)m-AAA (Afg3l2,

paraplegin)Matrix AAA! metalloprotease Non-assembled proteins Korbel et al. (2004)

Htra2/Omi IMS Serine-Protease IMS proteins Moisoi et al. (2009)OMA1 IMM Zinc-metalloprotease OPA1 Ehses et al. (2009), Head et al. (2009)PARL IMM Rhomboid protease PINK1 Meissner et al. (2011), Shi et al. (2011)UPS OMM Protease Mitochondrial proteins facing cytoplasm Livnat-Levanon and Glickman (2011)

mtHSP70, mitochondrial heat shock protein 70; Cpn60, chaperonin 60; Cpn10, chaperonin 10; ClpP, caseinolytic peptidase; i-AAA, intermembrane space AAA (ATPase associated withdiverse cellular activities) protease; m-AAA, matrix AAA protease; Htra2/Omi, high temperature requirement protein serine peptidase 2; PARL, presenilin-associated rhomboid-like;UPS, ubiquitin–proteasome system; IMS, intermembrane space; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; PINK1, PTEN-induced putative kinase 1;OPA1, optic atrophy 1.

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membrane potential collapse (Narendra et al., 2008, 2010). Thedirect or indirect nature of interaction between both proteinsand how the interaction between the two protein partnersinducesmitophagy is not fully understood but data tend to showa rapid ubiquitination of mitochondrial proteins, such asVDAC1 inmammals, after Parkin recruitment toOMM(Geisleret al., 2010). As p62 has been shown to interact both withubiquitinated proteins and the autophagosome markermicrotubule-associated light chain 3-II (LC3-II), it has beenpostulated that this adaptor protein could mediate therecruitment of autophagic machinery to depolarizedmitochondria as described for other forms of specificautophagy such as pexophagy (Kim et al., 2008; Geisler et al.,2010). On the other hand, Bnip3 has also been described asa potential initiator of mitophagy by enhancing proteosomaldegradation of altered ETC proteins, responsible formitochondrial membrane depolarization without OMMpermeabilization, in MEFs lacking Bax/Bak (Koopman et al.,2010). Thus, even if the mechanism of action is still largelyunknown, Bnip3 could act as a molecular switch betweenmitochondrial turnover and programmed cell death, dependingon the severity of mitochondrial activity impairment (Thomaset al., 2011).

Number Matters: Crosstalk Between Cell Functionand Mitochondrial MassMitochondrial abundance during the cell cycle

As cell division is a biological process that consumes energy, anincrease in mitochondrial biogenesis has been suggested toencounter the high energetic demand during cell proliferation.Nevertheless, mitochondrial population has been reportedto divide during mitosis, providing daughter cells with acomplement of mitochondria. Whereas, for a while, it wasconsidered that there was no link between cell cycle andbiogenesis of mitochondria (absence of synchronization), it hasbeen recently reported that mitochondrial biogenesis beginsfrom early G1 phase. The biogenesis is characterized by anincrease in mitochondrial mass and a higher mitochondrialmembrane potential observed between G1 and G1/S.Accumulation of mitochondria is now known to progressthroughout the mitotic phase. The mtDNA content and thelevel of the transcription factor NRF-1 also appear to increasefrom G1/S to G2 phase (Lee et al., 2007). While a link betweencell cycle and the biogenesis of mitochondria has now beenclearly established, the timing of the biosynthesis of mtDNA inmammalian cells during progression of cell cycle is still a matterof debate. Previous studies reported thatmtDNAmolecules doundergo rounds of replication and segregation of sequencevariants in a randomly dependent manner during cell division(Bogenhagen and Clayton, 1977; Shoubridge, 2000), whileother reports show that mtDNA replication occurs at specificstages/steps of the cell cycle (late S-G2M) after most nDNAsynthesis has been completed (Posakony et al., 1977; Radsakand Schutz, 1978). However, the lack of significant changes inthe mtDNA content in liver cells during cellular proliferationsuggests a current view that both mtDNA and nDNA synthesisare coordinated and supports a concerted trans-activation ofthe replication of both genomes (Ruiz De Mena et al., 2000).

Context-specific modulation of mitochondrialabundance

If biogenesis of the organelle is tied up to the cell cycle, it is alsomodulated in response to various physiological stimuli suchas contractile activity, exposure to low temperatures, caloricrestriction, and stem cells differentiation.

Mitochondrial biogenesis in response to contractileactivity. In skeletal muscle, contractile activity (which mimicsthe effect of endurance training) induces mitochondrial

biogenesis through an increase in cytosolic calciumconcentration ([Ca2!]c) and ATP consumption leading to AMPaccumulation and AMPK activation (Ljubicic et al., 2010). Theincrease in [Ca2!]c during muscle contraction also activatesCaMKIV (calcium/calmodulin-dependent protein kinase IV),which in turn, phosphorylates and triggers the activation of bothCREB and MEF-2 that bind to PGC-1a promoter, inducingthe expression of the crucial co-activator (McKinsey et al.,2000, 2002; Corcoran and Means, 2001; Michael et al., 2001;Wu et al., 2002). Furthermore, the elevated cytosolic calciumconcentration activates the calcineurin A (CnA), a proteinphosphatase that is able to remove a phosphate group onmembers of the nuclear factor of activated T-cell (NFAT)family, thereby stimulating a cytosolic-nuclear translocationof these proteins and their binding to the PGC-1a promoter(Olson and Williams, 2000). Besides inducing the expressionof PGC-1, contractile activity also activates this transcriptionalco-activator through AMPK activation. Indeed, AMPKphosphorylates PGC-1a at Thr17 and Ser538, thus promotingPGC-1a dissociation from its repressor protein p160Myb(Hardie and Pan, 2002), and/or allows SIRT1-dependentdeacetylation and subsequent activation of PGC-1a (Hardieand Pan, 2002; Canto et al., 2009). Additionally, the activation ofAMPK also increases PGC-1a expression in muscle (Baar et al.,2002), likely through a positive feedback loop that involvesPGC-1a auto-regulation on its ownpromoter (Handschin et al.,2003; Amat et al., 2009; Canto et al., 2009). In muscle cells,AMPK could also indirectly stimulate mitochondrial biogenesis,by triggering the translocation of MEF-2 in the nucleus allowingthe binding to its target promoters (Al-Khalili et al., 2004).Finally, subsequent PGC-1a expression to p38 MAPKactivation has been described in a study performed on timecourse of the adaptive response of muscle to exercise (Wrightet al., 2007).

Mitochondrial biogenesis in response to exposureto low temperatures. In order to maintain body energybalance and core temperature, exposure of mammals to lowtemperatures for prolonged periods of time induces a markedincrease in mitochondrial mass in brown adipocytes (Klauset al., 1991). Indeed, brown adipose tissue (BAT) has beendescribed as a specialized tissue responsible for thethermoregulatory mechanism, the so-called non-shiveringthermogenesis, a process that occurs in most mammalianhibernators and also more recently identified in adult humans(Cypess et al., 2009; Virtanen et al., 2009; van MarkenLichtenbelt et al., 2009). Adaptive thermogenesis occursthrough mitochondrial OXPHOS uncoupling by theendogenous uncoupling protein 1 (UCP1) (Lowell andSpiegelman, 2000). During cold acclimation, the differentiationof brown adipocytes is stimulated through the binding ofnoradrenaline to b1-adrenergic receptors (b1-AR) (Himms-Hagen et al., 1994; Bronnikov et al., 1999), whereas theincreased thermogenic capacity of BAT is mediated by thebinding of noradrenaline to b3-AR (Nisoli et al., 1995; Zhaoet al., 1998). The thermogenic signaling cascade results inenhanced adenylate cyclase activity, the increased productionof cAMP and is accompanied by an increase in [Ca2!]c. Thesesecondary messengers also induce mitochondrial biogenesisand thermogenic capacity of brown adipocytes via eitherelevated cAMP levels leading to CREB phosphorylation byprotein kinaseA (PKA), orNO-dependent organelle biogenesisthrough guanylate-cyclase-dependent signaling pathway thatactivates PGC-1a (Nisoli et al., 2003). The NO-dependentmitochondrial biogenesis is not restricted to brown adipocytesas it has also been demonstrated in 3T3-L1, U937, and HeLacells (Nisoli et al., 2003).

Mitochondrial biogenesis induced by calorie restriction.Calorie restriction, which is well known to increase life span innumerous organisms ranging from yeast to mammals (Lin et al.,

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2000; Ingram et al., 2004), has also been reported to inducemitochondrial biogenesis through eNOS expression and cGMPformation in brown andwhite adipose tissues as well as in brain,liver, and heart (Nisoli et al., 2005).Note that caloric restrictionhas also been reported to induce mitochondrial biogenesisin skeletal muscle through the activation of SIRT1 and AMPK(Civitarese et al., 2007).However, the role of calorie restrictionas a stimulator of mitochondrial biogenesis is still controversialas Hancock et al. (2011) did not find any cue of an inducedmitochondrial biogenesis after a 30% calorie restriction for3 months as shown by measuring PGC-1a level and keymitochondrial proteins in heart, brain, liver, adipose tissue, andskeletal muscle. However, among the studies devoted to theconnection between calorie restriction and biogenesis ofmitochondria, few are doubting about the role of CR in theprocess.

Stem cells differentiation-associated mitochondrialbiogenesis. Recently, the role of mitochondria in stemness/differentiation has also drawn attention, as mitochondriadisplay different phenotypes in pluripotent and differentiatedcells and because the biogenesis ofmitochondria is suspected tocontribute and/or be regulated by cell differentiation (Rehman,2010) Stem cells are currently defined by two criteria:pluripotency and self-renewal. While embryonic stem cells(ESC) retain the ability to propagate indefinitely and todifferentiate into any cell type, somatic stem cells (also calledadult stem cells) are more limited in their self-renewal capacityand the number of cell lineages they can generate (Zhang andWang, 2008). Somatic stem cells include hematopoietic stemcells (HSC), located in the bone marrow, mesenchymal stemcells (MSC), located in various tissues, and progenitor cells,characterized by a more restricted proliferation potentialthat only generate in vivo the cell type of their tissuelocation. More recently, a new stem cells category emerged:induced pluripotent stem cells (iPSC) resulting from thereprogrammation of somatic differentiated cells by forcedexpression of transcription factors like Oct4, Nanog, and Sox2,identified as master ‘‘stemness’’ regulators that control self-renewal and pluripotency (Wang et al., 2010). Stem cells areknown to mainly rely on glycolysis for their energy supply(Mohyeldin et al., 2010). Their mitochondrial content is thuslow and characterized by an ‘‘immature phenotype’’ consistingof a reduced oxygen consumption capacity, a lowmitochondrialmembrane potential, and an immature network of round-shapeperi-nuclear mitochondria with poorly developed cristae(Rehman, 2010; Wilkerson and Sankar, 2011). This immaturemitochondrial phenotype seems to be a hallmark of all types ofstem cells studied for this criteria as it was found in murine(Zeuschner et al., 2010) and human (Cho et al., 2006; Prigioneet al., 2010) ESC, in primate adult stromal cells (MSC derivedfrom adipose tissue) (Lonergan et al., 2006), in human bonemarrow MSC (Chen et al., 2008). On the opposite,differentiation and/or commitment of stem cells into aspecific cell lineage is accompanied by a strong biogenesisof mitochondria and a metabolic shift towards oxidativephosphorylation (Rehman, 2010). Surprisingly, it was reportedthat pluripotent cells (both human ESCs and iPS cell lines)display a higher content of enzymes catalyzing reactions of theTCA cycle when compared with differentiated cells as well asa higher expression of the mitochondrial electron transportcomplexes III and V. While these observations may appearcontradictory with the above-mentioned reduced OXPHOS instem cells, the authors suggested that the increased hexokinaseII and pyruvate dehydrogenase kinase 1 (PDHK1) protein levelsmay be involved in the overall lower OXPHOS activity inpluripotent cells. Indeed, whereas the higher expression ofhexokinase II may favor glycolytic metabolism, the increase inPDHK1protein levelsmay lead, through the phosphorylation ofPDHE1a, to a reduction of PDH complex activity resulting in a

reduced availability of substrates entering the TCA cycle andtherefore to reduced oxidative phosphorylation (Varum et al.,2011).

Interestingly, the immature phenotype of mitochondria doesnot only correlate with stemness but the impairment ofmitochondrial activity could also alter stem cell differentiation.Indeed, mitochondrial inhibitors can retard the osteogenicdifferentiation of hMSC (Chen et al., 2008) and dramaticallyreduce cardiac differentiation of both mESC (Spitkovsky et al.,2004; Chung et al., 2007) and cardiac mesangioblasts (SanMartin et al., 2011). In addition, these inhibitors enhance hESCpluripotency (Varum et al., 2009). Let us also mention that thereprogramming of differentiated cells, with a maturemitochondrial network, into iPSC, is accompanied by theemergence of mitochondrial structures of immaturephenotype, as shown both in murine (Zeuschner et al., 2010)and human iPSC (Prigione et al., 2010). Moreover, human MSCalso reprogram adult cardiomyocytes into progenitors throughpartial cell fusion and mitochondria transfer (Acquistapaceet al., 2011).

These data suggest the existence of a bidirectional causal linkbetween stem/progenitor cell differentiation andmitochondrialbiogenesis (reviewed in Rehman, 2010). Although themolecular mechanisms linking mitochondria biogenesis and celldifferentiation are poorly understood, recent data indicate thatat least some well-known actors of mitochondrial biogenesis,like PGC-1 family, PPARb and nitric oxide, could be involved inmitochondria biogenesis induced during differentiation. Indeed,deficiency in both PGC-1a and PGC-1b totally inhibitsmitochondrial biogenesis induced by brown fat differentiationof murine preadipocyte lines (Uldry et al., 2006). Regardinghepatogenic differentiation, a PPARb agonist significantlyincreases mitochondrial abundance and the number ofalbumine-positive cells differentiated from mESC. It is alsointeresting to mention that both hepatogenic differentiationandmitochondrial biogenesis are hampered in the presence of aPPARb antagonist (Zhu et al., 2010). In addition, S-NitrosoAcetylPenicillamine (SNAP), a NO donor, stimulatesmitochondrial biogenesis but also enhances the hepatogenicdifferentiation of mESC (Sharma et al., 2009) and thecardiogenic differentiation of cardiac mesangioblasts (SanMartin et al., 2011).

However, a large-scale study performed by Prigione andAdjaye comparing fibroblasts to ES and iPS cell lines, leftundifferentiated or differentiated either in vitro or in vivo,depicts a complex picture of the main mitochondrial biogenesisregulators transcript abundance. A number of genes involved inmitochondrial biogenesis (PGC-1a, PGC-1b, Tfam, NRF1,POLG, and POLG2) are upregulated in human ES and iPS celllines compared to somatic HFF1 fibroblasts (Prigione andAdjaye, 2010). According to these authors, this phenomenonmay represent a nuclear response to a decreased content inmtDNA in pluripotent cells. Moreover, a consensual down-regulation of PGC-1a, PGC-1b, Tfam POLG, and POLG2 wasobserved during the spontaneous in vitro differentiation intoembryoid bodies, except in the iPS2 cell line, while most ofthese actors were upregulated in fully in vivo differentiatedteratomas. This is still complexified by the recent observationthat mtDNA can be altered during the induction ofpluripotency, leading to iPS cell lines harboring homoplasmic orheteroplasmic mtDNA mutations, although this apparentlydoes not modify the stem cell-associated metabolicreprogramming (Prigione et al., 2011).

As mitochondrial biogenesis is tightly linked to stem cellcommitment/differentiation, it is tempting to speculate thatstemness regulators might also control mitochondrialbiogenesis, while currently, to our knowledge, no molecularlink has been established between stemness and mitochondrialbiogenesis master regulators. Future work in this direction is

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thus necessary to characterize the crosstalk between stem cellsdifferentiation and biogenesis of the mitochondria.

Beside mitochondria abundance, peri-nuclear location andlimited networking of stem cells mitochondria could also havesome functional significance, although this is currently notknown. One can suppose that mitochondrial dynamicsregulators like OPA1, Mfn1/2, and Drp1 are probablyresponsible, at least partly, for this particular mitochondriaphenotype. Although no causal relationship has beenevidenced, it is interesting to note that OPA1 mRNAabundance is modified in hESC silenced for Oct4, Nanog, orSox2 and that binding sites for these transcription factors arepredicted in the promoter of OPA1 (Jung et al., 2010).

Although very promising, considering the huge therapeuticpotential offered by stem/progenitor cells, the field ofmitochondrial maturation during cell differentiation is still in itsearly stages and many questions remain to be answered. Whatare the mechanisms restricting mitochondrial biogenesis instem cells, in which high proliferation rate requires energy?What are the signals controlling mitochondrial biogenesis uponstem cell differentiation? How an activated mitochondrialbiogenesis can favor various differentiation processes likecardiogenic, hepatic, or osteogenic differentiation? Is there adirect or indirect link between the bona fide master stemnessregulators (like Oct4, Nanog, and Sox2) and the mainmitochondrial biogenesis regulators of the PGC-1 familymembers?

Crosstalk Between Mitochondrial Mass, OrganelleDysfunction, and/or Associated Pathologies

So far, we have described anterograde signaling in the sense thatexternal cues trigger signaling pathways leading tomodifications in the expression of nuclear-encodedmitochondrial proteins affecting the nuclear control onmitochondrial activity and function. However, whenmitochondrial function is impaired, mitochondria to nucleuscommunication might also initiate from the organelle to impactnuclear genes expression by a retrograde response, enablingcell adaptation to altered mitochondrial metabolism. Inmammals, this mitochondrial stress response has been firstidentified inC2C12myoblasts treatedwith ethidium bromide orcarbonylcyanide-3-chlorophenylhydrazone (CCCP), triggeringmtDNA depletion or a disruption of Dcm, respectively (Biswaset al., 1999). Calcium released from mitochondria as secondmessengers leads to the activation of Ca2!/calmodulin-responsive calcineurin and their related factors stress to inducethe up-regulation of several target genes involved in Ca2!

transport and storage, as well as the activation of the Ca2!-dependent transcription factors NFAT, ATF2, and NFkB/Relfactors, which collectively alter the expression of nuclearOXPHOS genes (Biswas et al., 1999). Ca2!-dependentretrograde signaling was also confirmed later on in totallyor partially mtDNA-depleted A549 (Amuthan et al., 2002),143B (Arnould et al., 2002), and L929 (Mercy et al., 2005).Mechanistically, our group showed that in r0 humanosteosarcoma 143B cells and in a myoclonic epilepsy withragged red fibers (MERRF) cybrid cell line carrying the mutatedmitochondrial tRNALys (A8344G), an increase in cytosolicfree Ca2! leads to the activation of CaMKIV, which in turnactivates CREB by protein phosphorylation (Arnould et al.,2002). Furthermore, partially (r"L929) or totally (r0143B)mtDNA-depleted cells also harbor a slight increase inmitochondrial biogenesis depending on intracellular Ca2! leveland CREB activation (Mercy et al., 2005). Finally, CREB hasalso been implicated in the regulation of nuclear-encodedmitochondrial proteins, such as cytochrome c, duringmyogenesis (Franko et al., 2008). In addition to Ca2!, othersecondary messengers such as ROS, NO, ATP, NAD!/NADH

and proteins such as mitochondrial DNA absence sensitivefactor (MIDAS) (Nakashima-Kamimura et al., 2005) have alsobeen associated with mitochondrial retrograde response (foran extended reviewon the implication of thesemembers duringretrograde response, please see Finley and Haigis (2009).

If all these mitochondrial stress responses can lead to PGC-1-dependent mitochondrial biogenesis, the MIDAS protein,whose expression is increased by the absence of mtDNA, hasbeen shown to regulate mitochondrial abundance, in a PGC-1-independent manner, and the expression of enzymes thatcatalyze the synthesis of the mitochondria-specificphospholipid cardiolipin in HeLa cells (Nakashima-Kamimuraet al., 2005). One should also mention that the mtUPR,described here above, can also be considered as a retrograderesponse since it is initiated by the formation of proteinaggregates in the mitochondria and triggers the activation ofnuclear transcription factors, among which CHOP-10, thatspecifically controls the expression of mitochondrialchaperones and proteases (Haynes and Ron, 2010).

Increased mitochondrial mass-associated pathologies

Mitochondrial myopathies and increased mitochon-drial abundance. In human tissues, energy balance requiresadaptation of mitochondrial density and quantity (as well asquality) of the OXPHOS apparatus to energy needs. Thisequilibrium is challenged in patients with mitochondrialdiseases, prompting bioenergetic adaptations that are usuallyassociated with adaptive compensatory mechanisms thatinvolved a boost of mitochondria biogenesis. A pathologicalaccumulation of abnormal mitochondria under thesarcolemma, known as ‘‘ragged red fibers,’’ is typically observedin skeletal muscle cells of patients affected by respiratory chaindisorders, or in transgenic mice models developed to studythese diseases (Jezek et al., 2010). Furthermore, the presenceof apoptotic markers, such as caspase3 activation, DNAfragmentation and Bax overexpression, in patient musclebiopsies has been shown to correlate with ragged red fibers,while underlying mechanisms connecting these observationsare still missing (Aure et al., 2006). Even though the origin of thisragged-red fibers phenotype remains unclear, it is generallyaccepted that mitochondrial pathologies result primarily from areduced capacity of mitochondria to produce energy in theform of ATP (McKenzie et al., 2004). However, Fisher and co-workers have demonstrated that the cytopathologiesassociated with mitochondrial diseases in Dictyostelium, such asa slower growth, an alteredmulticellular morphogenesis and animpaired phototaxis, are induced by the chronic activation ofAMPK an energy sensor, rather than by a lack of ATP itself(Bokko et al., 2007). Furthermore, an impairment ofmitochondrial energy supply through the inhibition of thephosphocreatine circuit has been shown to stimulatemitochondrial gene expression and proliferation of alteredmitochondria in rat heart cells (Wiesner et al., 1999). Besidemodifications in mitochondria abundance, mitochondriadysfunction also affects the mitochondrial network asmitochondrial fragmentation, correlating with processing oflarge isoforms ofOPA1 (L-OPA1), has been reported in severalmodels of mitochondrial dysfunction like cybrid cells fromMERRF patients, mouse embryonic fibroblasts harboring anerror-pronemtDNA POLg, in heart tissue derived from heart-specific Tfam knock-out mice suffering from mitochondrialcardiomyopathy, and in skeletal muscles biopsies from patientssuffering frommitochondrial myopathies such as mitochondrialmyopathy, encephalopathy, lactic acidosis, and stroke (MELAS)(Duvezin-Caubet et al., 2006).

A fundamental question to address would be: what isthe functional significance and meaning of an increasedmitochondrial abundance induced by mitochondrial

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dysfunction? This can be intuitively interpreted as a form ofquantitative compensation of a qualitatively deficient organelle,in order to maintain cell homeostasis containing less efficientmitochondria. Indeed, Moraes and his collaborators havedemonstrated that a stimulation of mitochondrial biogenesisin mice with mitochondrial myopathy caused by a COXdeficiency results in a longer lifespan as a result of a functionalimprovement at the muscle level and a delayed onset of themyopathy (Wenz et al., 2008). However, an excessivemitochondrial mass induced by mitochondrial dysfunction mayalso have deleterious effects, as it has been shown thatapoptosis markers are associated with ragged red fibers, asevidenced in a large-scale analysis conducted in individualmuscle fibers from patients suffering from differentmitochondrial myopathies (Aure et al., 2006). The question toknowwhether these apoptoticmarkers occur spontaneously inresponse to mitochondrial dysfunction or results from a highersensitivity to pro-apoptotic molecules present in the cellenvironment and challenging cells that display mitochondrialdysfunction is not completely resolved as some evidence thatmtDNA mutation and depletion increase resistance of humancells to apoptosis (Park et al., 2004) while we, and others, haveshown that cells lacking mtDNA or harboring point mutationsare more sensitive to pro-apoptotic molecules (Liu et al., 2004;Rommelaere et al., 2011). Cell type, stimuli and nature of themitochondrial dysfunction might explain these differences incell behavior.

Finally, one should mention that mitochondrial biogenesisinduced by mitochondrial dysfunction has been shown to beassociated with a reduced mitochondrial import for matrixproteins in partially (r"L929) or totally (r0143B) mtDNA-depleted cells (Mercy et al., 2005), although mitochondrialprotein import decrease is not a compulsory responseassociated with mitochondrial biogenesis in mtDNA-depletedcells as Hood and his collaborators have shown that differentialcompensatory adaptations reflected by divergent changes innuclear gene expression patterns of Tom20, Tom34, Tim23,mtHsp70, and Tfam occurred in r" C2C12 cells and infibroblasts from MELAS patients to maintain normal rates ofprotein import in response to mtDNA defects (Joseph et al.,2004).

Increased mitochondrial abundance in cancer cells.The impact of aberrant mitochondrial mass was also largelystudied in cancer. Indeed, key mitochondrial actors have beenassociated with cancer progression such as increased mtDNAcontent with histopathologic grade in malignant head and necklesions (Kim et al., 2004), Tfam expression with endometrialcarcinomas progression (Toki et al., 2010) and enhancedmitochondrial biogenesis has been linked to different types ofcancers (breast, gastric, lung, esophageal) (Isidoro et al., 2004).In addition, neoplasmic transformation is always accompaniedwith metabolic reprogramming, giving proliferative advantageto cancer cell (Zhou et al., 2010). If Otto Warburg’s theoryof enhanced glycolysis in tumor cells has been first extendedto all type of cancer, more recent data are now showing howcomplex and heterogeneous is metabolism in malignant cellsas well as its crosstalk with mitochondrial mass and function.Indeed, increased organelle abundance in transformed cells hasbeen associated with enhanced glycolysis (Shapovalov et al.,2011), increased OXPHOS (Lee et al., 2011), or both (Samperet al., 2009). For instance, mitochondrial mass is increased inosterosarcoma 143B cells with abnormally enlarged organelles.In addition, 143B cells mtDNA content and mtSSB expressionare higher in comparison with osteoblastic hFOB cell line andeven if ETC complexes expression is also unregulated, activity isdecreased as well as oxygen consumption. Interestingly, knock-down of mtSSB is sufficient to reduce mtDNA content, torestore oxygen consumption and to reduce proliferation ofaggressive 143B cells in vitro and in vivo (Shapovalov et al.,

2011). Thus, excessive mitochondrial proliferation may be,at least in part, responsible of organelle dysfunction andacquisition of malignant phenotype in highly glycolytic 143Bcells. On the other hand, increased mtDNA content andexpression of mitochondrial biogenesis regulators such asPGC-1a, Tfam and NRF-1 has been described in biopsies frompatient with cutaneous carcinoma [arsenic-induced Bowen’sdisease (As-BD)] (Lee et al., 2011). Furthermore, keratinocytestreated with low concentration of arsenic show increasedmitochondrial mass, OXPHOS activity and cell proliferation,advantages that are lost upon Tfam knock-down but not inresponse to PGC-1a invalidation (Lee et al., 2011). Increasedmitochondrial biogenesis and respiration have also been shownin thymic lymphomas fromp53"/"mice, which are, surprisingly,also accompanied by enhanced glycolytic activity (Samper et al.,2009). Thus, crosstalk between mitochondrial mass and cancercell metabolism is not clear and most likely depends on severalfactors such as tumor grade, tumormicro-environment or levelof hypoxia, among others. Indeed, it has been proposed thatATP can be produced at different level either by glycolysis orOXPHOS depending on the class of cancers (Moreno-Sanchezet al., 2007). In addition, depending on heterogeneous hypoxiclevel within tumor and micro-environment, Sonveaux et al.(2008) have described a metabolic coupling between hypoxic/glycolytic and oxygenated/oxidative cancer cells through lactateuptake via the monocarboxylate transporter 1 (MCT1) incervix squamous carcinoma cells. The so-called ‘‘reverseWarburg effect’’ model postulates that oxidative cells recyclemetabolites, such as lactate, provide by highly glycolyticneighboring cells to generate large amount of ATPwithin tumoroxygenated area (Pavlides et al., 2009). Finally, it has recentlybeen suggested that waves of expression occur duringcarcinogenesis and that as tumor develops mitochondrialrevival might occur and stimulate OXPHOS-dependent ATPproduction after being mainly reliant on glycolysis (Smolkovaet al., 2011).

So far, different effectors and mediators have already beendescribed to be responsible for mitochondrial mass regulationwithin cancer cells such as c-Myc, HIF-1, PNC1, Ca2!, and NO.NO and Ca2! have been shown to induce mitochondrialbiogenesis as part of a retrograde signaling activated in responseto respiratory dysfunction in follicular thyroid carcinoma cells(Le Pennec et al., 2011). In cancer cells, NO would triggerorganelle biogenesis through PGC-1a and PRC-dependentERRa and NRF-1 expression and, surprisingly, a decrease incytosolic Ca2! concentration would induce PRC and ERRaexpression, which is different from the classical Ca2!-mediatedretrograde response described for non-transformed cells (LePennec et al., 2011). We also know that the mitochondrialpyrimidine nucleotide carrier (PNC1), an IGF-1 and insulin-responsive protein (Floyd et al., 2007), regulates mitochondriafunction and biogenesis in MCF-7 andHeLa cells. Indeed, PNC1over-expression and gene expression silencing experimentsclearly demonstrate its participation in the maintenance oforganelle function to prevent excessive ROS production andinvasive phenotype acquisition (Favre et al., 2010). Theoncogenic transcription factor c-Myc has also been reported tobe up-regulated in many cancer cell type. This factor wouldpromote proliferation, genome instability and self-renewal(Vafa et al., 2002; Chang et al., 2008). As c-Myc is also knownto increase the expression of genes encoding mitochondrialproteins and to stimulate mitochondrial biogenesis, Dang andco-workers proposed that it may potentially link c-Myc-stimulated ROS production and genome instability (Wonseyet al., 2002; Dang et al., 2005; Li et al., 2005). Finally, HIF-1 hasbeen shown to indirectly inhibit mitochondrial biogenesis inrenal cell carcinoma by repressing c-Myc activity either bystimulating expression of its repressor MIX1 or by enhancingits degradation by the proteasome (Zhang et al., 2007).

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Keeping the importance of mitochondria in the regulationof apoptosis in mind (Kroemer, 2003) and knowing thatchemotherapy-induces apoptotic resistance of cancer cells tochemical drugs (independently of MDR over-expression), itwould be interesting to study the impact of abnormalmitochondria mass on programmed cells death susceptibility.As both pro- and anti-apoptotic regulators reside inmitochondria, how is this equilibrium affected when theabundance of mitochondrial population becomes too high andwhat are the consequences on apoptotic sensitivity? The impactof increased mitochondrial abundance and abnormal OXPHOScomplexes expression and activity described in some cancersshould also be better understood regarding ROS production interms of tumorigenicity as mutated mtDNA-induced ROSproduction is sufficient to acquire metastatic phenotype(Ishikawa et al., 2008). Finally, it would also be interesting tofurther investigate the mitochondrial quality control inmitochondrial population not only during boostedmitochondrial biogenesis but also during mitophagy as thepro-apoptotic Bnip3 expression is induced under hypoxia(Bruick, 2000).

Conversely, a decrease in mitochondrial biogenesis has alsobeen associated with lung cancer and other types of diseasessuch as obesity, insulin resistance orwith aging. As described forlung tumors, where PGC-1a and Tfam as well as ETCcomplexes are down-regulated (Bellance et al., 2009), thesemodifications are usually correlated with the lower expressionof transcriptional regulators such as PGC-1a, PGC-1b, andNRF-1, whereas the positive signaling pathways that inducemitochondrial biogenesis inmitochondrial retrograde responseare generally inhibited (Hock and Kralli, 2009).

Decreased mitochondrial abundance and dysfunction

Studies of obese individuals or patients with type 2 diabeteshave revealed that mitochondrial biogenesis and function aregenerally down-regulated in these conditions (Semple et al.,2004; Richardson et al., 2005; Sparks et al., 2005; Rong et al.,2007), but a crucial question remains to address: does insulinresistance result form impairedmitochondrial activity or do thealterations of mitochondrial function appear as a consequenceof the onset of insulin resistance as many genes and regulatorsinvolved in the biogenesis of mitochondria are also controlledby insulin-dependent signaling pathways (Cheng et al., 2010).

Crosstalk between reduced mitochondrial contentand insulin resistance. Numerous studies, based on human,mouse, or cell models, highlight a strong and positivecorrelation between high fat load and PGC-1 down-regulation,accompanied by a decrease in mitochondrial abundance.Indeed, PGC-1a and/or PGC-1b expression are/is decreased insubcutaneous fat of morbidly obese subjects (Semple et al.,2004), in high-fat diet fed humans (Richardson et al., 2005;Sparks et al., 2005) and mice (Rong et al., 2007) and indifferentiated 3T3-L1 adipocytes treated with highconcentrations of glucose and free fatty acids that eventuallyexhibit insulin resistance (Gao et al., 2010). One of the linksbetween high fat accumulation and down-regulation ofmitochondrial biogenesis may rely on the modifications of theadipokine expression ratio between sensitizing adipokines suchas adiponectin (Kadowaki and Yamauchi, 2005) and adipokinestriggering insulin resistance such as resistin, among others(Banerjee et al., 2004). Indeed, adipocytes from visceral adiposetissue of obese individuals release reduced amounts ofadiponectin, an ‘‘insulin-sensitizing adipokine,’’ which leads to areduced activation of the AMPK/PGC-1a pathway in muscletissue, and subsequently reduces the abundance ofmitochondria and thus the capacity for OXPHOS (Kadowakiand Yamauchi, 2005). Furthermore, adipocyte is far from beingthe only cell type concerned by high fat load, as the exposure

of C2C12 myotubes to palmitate down-regulates PGC-1a,mitochondrial genes expression and oxygen consumption(Crunkhorn et al., 2007). However, these observations havebeen challenged by more recent data indicating that increasingplasma free fatty acids and high-fat diets in rat induce PGC-1aand PPARb/d expression leading to an increase inmitochondrial content (Garcia-Roves et al., 2007; Hancocket al., 2008). These apparent conflicting data might result fromdifferences in fatty acid and diet composition, suggesting that itis the type rather than simply the quantity of lipids that mainlyaffects mitochondrial gene expression (Hock and Kralli, 2009).

The fact that high fat-loads are correlated in somecircumstances with a reduction of mitochondrial abundancesuggests that the onset of insulin resistancemight be caused by adecrease inmitochondrial abundance. Indeed, the abundance ofmtDNA and mitochondrial proteins as well as themitochondrial biogenesis are both considerably reduced inadipocytes of diabetic (db/db) mice (Choo et al., 2006; Ronget al., 2007). In human skeletal muscles, both PGC-1a and PGC-1b expression are reduced by about 50% in diabetic patients(Patti et al., 2003), as confirmed by a full transcriptome analysisdemonstrating that a major difference between diabeticpatients and healthy individuals is that PGC-1a-responsivegenes involved in OXPHOS are dramatically down-regulated(Mootha et al., 2003). In addition, insulin resistance in healthyaged individuals with no diabetic family history correlates with adecline in mitochondrial OXPHOS (Petersen et al., 2003).

It is important to emphasize that, beside mitochondriaabundance, one should also consider mitochondrialmorphology. In insulin-resistant differentiated 3T3-L1adipocytes treated with high glucose concentration and freefatty acids, the decreased mitochondrial biogenesis wasaccompanied by a remodeling of mitochondrial morphologyreflected by smaller mitochondria with condensed cristae, aswell as a decrease in MFN1 expression and an increase in Drp1protein levels (Gao et al., 2010). Moreover, skeletal muscles oftype 2 diabetic patients exhibit reduced expression of themitochondrial fusion protein MFN2 (Bach et al., 2003, 2005)and reduced mitochondrial size caused by an increase inmitochondria fragmentation (Kelley et al., 2002; Toledo et al.,2006). MNF2 also controls mitochondrial metabolism, asMfn2loss-of-function reduces glucose oxidation, decreasesDcm, andlowers oxygen consumption and mitochondrial proton leak incultured cells (Bach et al., 2003; Chen et al., 2005; Pich et al.,2005). Several lines of evidence indicate that PGC-1a and PGC-1b stimulate Mfn2 expression through ERRa transcriptionfactor (Soriano et al., 2006; Liesa et al., 2008). Based on thesedata, Palacin’s research group proposed that alterations on thePGC-1/MFN2 axe confers susceptibility to the development ofmetabolic diseases (Zorzano et al., 2009).

If a decreased abundance/function of mitochondria has oftenbeen reported in high fat load and/or (pre)diabetic contexts,conversely, mitochondria dysfunction can also impair insulinsignaling. Mitochondrial dysfunction induced either genetically(r0) or pharmacologically by metabolic inhibitors (oligomycin)in C2C12 myotubes causes aberrant insulin signaling, especiallyregarding IRS-1 (insulin-receptor substrate 1) activation andexpression, and abnormal utilization of glucose, as observed inmany insulin resistance states (Lim et al., 2006). The depletionof mtDNA also causes insulin resistance through the alterationof IRS-1 in rat myocytes (Park and Lee, 2007). The importanceof mitochondria in insulin sensitivity is also highlighted by thefact that many of the drugs used for type 2 diabetes, such asmetformin and thiazolidinediones (TZD), increase AMPKactivity in skeletal muscle. The activated AMPK can thusstimulate the biogenesis of mitochondria, GLUT4translocation, and fatty acid oxidation while reducing fatty acidsynthesis (Hardie, 2008). Moreover, as exercise-inducedmitochondrial biogenesis in both animals and humans rescues,

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at least partly, insulin resistance-induced mitochondrialdysfunction (Hawley and Lessard, 2008). Furthermore, thedecrease in PGC-1a expression observed in the (pre)diabeticpatients might be a cause of lipid-induced insulin resistance(Russell, 2005; Benton et al., 2008; Earnest, 2008). However,recent studies challenged the hypothesis that a decrease inPGC-1a expression plays a causal role in the development ofdiabetes, as PGC-1a skeletal muscle-specific knockout miceshow normal peripheral insulin sensitivity and conversely,transgenic mice over-expressing PGC-1a in muscle are moreprone to insulin resistance (Handschin et al., 2007; Choi et al.,2008).

In conclusion, as we have seen, it thus seems that a viciouscircle does exist between the development of insulin resistanceand the onset of modifications inmitochondrial abundance and/or activity as crosstalk does exist. Insulin does affect thebiogenesis of mitochondria while mitochondrial activity is alsocontrolling the insulin resistance and the activity of downstreamsignaling.

Decreased mitochondrial mass during aging. Areduction of mitochondrial biogenesis has also been associatedwith ‘‘healthy’’ aging. Indeed, gene expression analysis (wholetranscriptome analyses) of skeletal muscle, white adiposetissue, kidney, brain, and heart consistently show a decrease inthe expression of nuclear genes encoding mitochondrialproteinswith age (Lu et al., 2004; Zahn et al., 2006; Linford et al.,2007; Melov et al., 2007). Alternatively, mitochondrial turnovermay also be reduced with age, resulting in an age-relatedaccumulation of old, damaged, or non-functional mitochondria(Terman, 2006). Many studies show that fatty acid oxidationcapacity is also decreased in a variety of tissues of elderlyhumans and older rodents (Melanson et al., 1997; Tucker andTurcotte, 2002; Park et al., 2006; Solomon et al., 2008). Theexpression of PGC-1a also declines with age in rodent skeletalmuscle but such a decline can be blunted by CR (Baker et al.,2006; Hepple et al., 2006). Indeed, although the mechanisms bywhich CR exerts its anti-ageing effects remain unclear, itsbeneficial impact on mitochondrial biogenesis and function iswell established (Gredilla et al., 2001; Sanz et al., 2005; Lopez-Lluch et al., 2006). CR might also increase the organelleturnover (by mitophagy) in liver, facilitating the removal ofdamaged mitochondria (Miwa et al., 2008). Decreasedmitochondrial biogenesis has also been associated withtelomere dysfunction. In telomerase-deficient mice, activatedp53 binds and represses PGC-1a/b promoters, therebyimpairing mitochondrial biogenesis and function, increasingROS production and cardiomyopathy (Sahin et al., 2011).Telomeres shortening may be one of the explanations of thedecreased response of PGC-1a to stimuli, such as exercise,observed in aged rat (Derbre et al., 2011). In addition, genes-profiling analysis of mice expressing a proofreading-deficientversion of the mitochondrial DNApolg (mutator mice) hasshown a general down-regulation of genes associated withmitochondrial function and a reduced content of complexes ofthe respiratory chain (Hiona et al., 2010). Endurance exercise-induced mitochondrial biogenesis of the mutator mice issufficient to improve the aging phenotype observed in thismodel and reverse mitochondrial dysfunction has shown byincreased oxidative capacity (Safdar et al., 2011). The growingevidence also suggest that reductions in AMPK activity maybe an important factor associated with the decreasedmitochondrial biogenesis associated with ageing (Winder et al.,2000; Bergeron et al., 2001; Zong et al., 2002; Garcia-Roveset al., 2008).

Conclusion

As we have seen in this review, the net result of organelleproduction and degradation determine the abundance of

mitochondrial population within the cell by a fine tuningbetween mitochondrial biogenesis and quality control/mitophagy, respectively. On one hand, mitochondrialbiogenesis is mainly regulated by the PGC-1 family ofco-activators, which coordinate nuclear and mtDNA-encodedmitochondrial genes expression in cooperation with specifictranscription factors (Box 1). On the other hand, qualitycontrol operates at least at two different levels: proteases andchaperones prevent accumulation of misfolded proteins andtoxic aggregates (Box 2) and mitophagy target dysfunctionalorganelle for entire degradation and turnover.

The equilibrium/balance between biogenesis anddegradation of mitochondria is essential in maintainingappropriate mitochondrial activity (bioenergetic but not only)and change in mitochondrial mass is crucial for cell adaptationunder various conditions such physical exercise, cold, caloricrestriction, or during differentiation of stem cells. Thus,aberrant mitochondrial abundance might lead to organelledysfunction (and vice versa) and development of pathologiessuch as illustrated with mitochondrial myopathies, cancers, anddiabetes. Decipher signaling pathways responsible of aberrantmitochondrial mass will help to better understand thedevelopment of such pathologies and to discover potentialtargets for therapy.

Mitochondrial biogenesis stimulation can be beneficial toprevent or adapt to organelle dysfunction. Indeed, enduranceexercise-induced mitochondrial biogenesis is beneficial inmutator mice, characterized by accelerated aging phenotype,leading to systemic improvement of mitochondrial function andmorphology (Safdar et al., 2011). However, exacerbatedaccumulation of dysfunctional organelle can also lead topathology development such as observedwith ragged red fibersin mitochondrial diseases, which points the necessity to have aparallel increase of the mitochondrial quality control to targetaltered organelle and preserve a functional pool of themitochondrial population. It is interesting to mention thatcellular stresses-induced AMPK activation can also trigger bothautophagy and mitochondrial biogenesis via mTOR inhibitionand SIRT1-dependent deacetylation of PGC-1a, respectively(Deshmukh et al., 2008; Canto et al., 2010). Thusmitochondrialbiogenesis and mitophagy might be tightly regulated in acoordinated manner (Gottlieb and Carreira, 2010). In addition,to better understand the etiology of mitochondrial diseases perse and the wide variety of pathologies associated withmitochondrial dysfunction, it might be necessary to invest in theresearch devoted to better understand the crosstalk betweenmitochondrial dynamic, biogenesis and mitophagy as manyeffectors, regulators, and markers are shared by theseinterconnected biological processes (Ehses et al., 2009; Headet al., 2009; Gomes et al., 2011).

Acknowledgments

S.Michel is recipient of doctoral fellowship from the Fonds pourla Recherche dans l’Industrie et l’Agriculture (FRIA, Belgium).A.Wanet is recipient of doctoral fellowship from Le Fonds de laRecherche Scientifique (FNRS, Belgium). This work wassupported by the Association Belge Contre les MaladiesNeuro-Musculaires (ABMM, Belgium).

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