The implications of mitochondrial DNA copy number regulation during embryogenesis

Mitochondrion 11 (2011) 686–692

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Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o

Review

The implications of mitochondrial DNA copy number regulation during embryogenesis Phillippa J. Carling, Lynsey M. Cree, Patrick F. Chinnery ⁎ Mitochondrial Research Group, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK

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Article history: Received 6 January 2011 received in revised form 20 April 2011 accepted 18 May 2011 Available online 26 May 2011 Keywords: Genetic bottleneck Inheritance Mitochondrial biogenesis mtDNA replication Copy number Transcription

a b s t r a c t Mutations of mitochondrial DNA (mtDNA) cause a wide array of multisystem disorders, particularly affecting organs with high energy demands. Typically only a proportion of the total mtDNA content is mutated (heteroplasmy), and high percentage levels of mutant mtDNA are associated with a more severe clinical phenotype. MtDNA is inherited maternally and the heteroplasmy level in each one of the offspring is often very different to that found in the mother. The mitochondrial genetic bottleneck hypothesis was first proposed as the explanation for these observations over 20 years ago. Although the precise bottleneck mechanism is still hotly debated, the regulation of cellular mtDNA content is a key issue. Here we review current understanding of the factors regulating the amount of mtDNA within cells and discuss the relevance of these findings to our understanding of the inheritance of mtDNA heteroplasmy. © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mtDNA genetic bottleneck: An evolutionary perspective . . . . . . . 2.1. Experimental approaches to defining the bottleneck mechanism . . 3. Factors controlling the maintenance of mtDNA . . . . . . . . . . . . . . 3.1. mtDNA replication and transcription factors as molecular regulators 3.2. Mitochondrial biogenesis . . . . . . . . . . . . . . . . . . . . . 3.3. Fission, fusion and mitochondrial nucleoid complexes . . . . . . . 3.4. Nucleoside pools and mtDNA maintenance . . . . . . . . . . . . 3.5. Reactive oxygen species . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Mitochondria are the primary intracellular organelles responsible for the synthesis of adenosine triphosphate (ATP) by o xidative phosphorylation. Human mitochondria contain their own 16.6 kb genome

Abbreviations: mtDNA, mitochondrial DNA; PGCs, primordial germ cells; d.p.c, days post coitum; ALP, alkaline phosphatase. ⁎ Corresponding author at: Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK. Tel.: + 44 191 241 8611; fax: + 44 191 241 8666. E-mail addresses: [email protected] (P.J. Carling), [email protected] (L.M. Cree), [email protected] (P.F. Chinnery).

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(mtDNA) which codes for 13 essential proteins of the respiratory chain, along with 22 tRNAs and 2 rRNAs specific to mitochondria and essential for the translation of mtDNA genes. In mammals, mtDNA is strictly maternally inherited. As a consequence, mammalian mtDNA undergoes negligible inter-molecular recombination manifesting at the population level (Elson et al., 2001). MtDNA molecules are thought to exist in clusters, called nucleoids, tethered to the inner mitochondrial membrane. MtDNA nucleoids are adjacent to the mitochondrial respiratory chain which is a potent source of oxygen free radicals. Lacking protective histones and with rudimentary DNA repair mechanisms, mtDNA has a higher rate of oxidative nucleotide base damage when compared to nuclear DNA (Brown et al., 1979). These mutations are manifested both at the population level and in the aged organism through somatic mutagenesis (Taylor and Turnbull, 2005).

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P.J. Carling et al. / Mitochondrion 11 (2011) 686–692

Each nucleated mammalian cell contains numerous copies of mtDNA. When all of the molecules are identical, the situation is known as homoplasmy. However, a mixture of mutated and wild-type mtDNA is called heteroplasmy, and the percentage level can vary between 0 and 100%. The low propensity for inter-molecular recombination both in vitro and in vivo, means that relatively stable heteroplasmy can exist in mammalian cells (Schon et al., 1997). Pathogenic mutations are generally well tolerated, and do not cause a phenotypic effect until the percentage level of the mutation (mutation load) exceeds a specific threshold level. If exceeded, this leads to a bioenergetic defect within the cell. If a tissue or organ contains many cells expressing a biochemical defect, this can cause organ dysfunction and result in mitochondrial disease. Affected individuals present with a wide array of clinical phenotypes, particularly affecting organs with high energy demands such as the brain and skeletal muscle (Drachman, 1968; Fukuhara et al., 1980; Olson et al., 1972; Pavlakis et al., 1984; Petty et al., 1986). In general, individuals who harbour high heteroplasmy levels are more likely to be affected by disease, and also to have a more severe phenotype (Chinnery et al., 1997). Heteroplasmy levels often vary markedly between a mother and each one of her children. This means that asymptomatic mothers with medium to low heteroplasmy levels can have children with higher levels of the mutation which causes mitochondrial disease (Blok et al., 1997; Holt et al., 1989; Larsson et al., 1992). The mitochondrial bottleneck hypothesis was proposed to explain the change in heteroplasmy levels observed within one generation. In the early 1980s, Hauswirth, Olivio and Laipis observed marked differences in the mitochondrial genotypes between different Holstein cows within the same lineage, with some being homoplasmic wild-type and others harbouring a homoplasmic polymorphism. Given the highly unlikely possibility of recurrent mutation at exactly the same site in different branches of the same small pedigree, they proposed an alternative mechanism to explain their findings: the random segregation or unequal amplification of different mitochondrial genotypes during oocyte development in a heterogeneous population of mitochondria (Hauswirth and Laipis, 1982; Laipis et al., 1988; Olivo et al., 1983). Since they observed rapid shifts in genotype from one allele to another within one generation, this suggested that the founder cow was actually heteroplasmic, containing both sequence variations, and only a sub-population of the available mtDNA molecules re-populated the offspring. This would lead to a sampling effect, and dramatic changes in the inherited level of heteroplasmy during each transmission, thus enabling the switch from one genotype to another. Understanding the biological basis of the mtDNA bottleneck has been the focus of several recent studies, each providing evidence supporting one of three possible mechanisms (which are not mutually exclusive). Firstly, dramatic reduction in mitochondrial copy number during early germ-line development (Cree et al., 2008). Secondly, aggregation of mtDNA into clusters of molecules which are identical and segregate together (homoplasmic segregating units). Packaging mtDNA into these groups reduces the effective population size and in itself can lead to more rapid segregation of mtDNA (Cao et al., 2007; Cao et al., 2009). Thirdly, the preferential replication of a subpopulation of the available genomes (Wai et al., 2008). If this subpopulation is randomly selected from the overall pool, this will also lead to a bottleneck effect similar to that proposed by Hauswirth, Olivio and Laipis. All of the above mechanisms are based on the notion that either single mtDNA molecules or clusters of identical mtDNA molecules (ie homoplasmic segregating units) are the basic units of inheritance of mtDNA. However, alternatives have been proposed, including heterogeneous clusters of molecules (heteroplasmic segregating units). This could explain why, in some pedigrees, the level of heteroplasmy does not appear to change between the generations (Howell et al., 1992; Meirelles and Smith, 1997). It is possible that these segregating units are actually “mitochondrial nucleoids”, which are clusters of mtDNA associated with relevant replication and transcription proteins tethered

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to the inner mitochondrial membrane. Current evidence suggests that nucleoids are probably homoplasmic and rarely exchange genetic material between themselves (Gilkerson et al., 2008; Poe et al., 2010), but this has not been demonstrated in germ cells or oocytes. Finally, it is possible that selective destruction of mtDNA or mtDNA nucleoids could explain the bottleneck, possibly through the process of mitophagy (Twig et al., 2008). However, direct experimental evidence pointing towards this mechanism is currently lacking. Although the precise mechanism of transmission is still hotly debated, consistent experimental evidence has shown a dramatic reduction of mtDNA levels in the germ line during embryonic development. Thus, each is critically dependent on the processes regulating the amount of mtDNA within the cell, and the rate of replication of mtDNA. In this review we will consolidate what is known about the regulation and maintenance of the mitochondrial genome, specifically with relation to the mitochondrial bottleneck. 2. The mtDNA genetic bottleneck: An evolutionary perspective The mitochondrial bottleneck may, at first, seem to be primarily a deleterious process, causing mitochondrial disease in the children of healthy mothers. However, from an evolutionary perspective, the opposite is the case. Reducing the mutational load during transmission protects the human species from the mutational meltdown predicted by Muller's Ratchet in asexual populations (Bergstrom and Pritchard, 1998; Felsenstein, 1974; Muller, 1964). The mitochondrial genome has a higher mutation rate than nuclear DNA and is situated close to the respiratory chain, a potent source of damaging free radicals (Lenaz, 2001). Without the mitochondrial bottleneck, any mutation acquired would never be lost from the maternal lineage, leading to the relentless accumulation of deleterious mutations. MtDNA-encoded proteins would have progressively reduced function, leading to human extinction. However, this same mechanism leads to apparently random inheritance patterns in human pedigrees transmitting pathogenic mutations (Howell et al., 1992). In essence, the bottleneck accelerates the rate of selection against deleterious alleles at the level of the organism. In the short term (over a few generations), this can cause mitochondrial diseases within families, but in the longer term (over many more generations), this removes adverse mutations from the maternal germ line, and thus “cleanses” the population. Understanding this process will hopefully lead to more reliable genetic counselling for families with these diseases, and to the development of new treatments which prevent transmission, thus preventing mitochondrial disorders affecting subsequent generations. 2.1. Experimental approaches to defining the bottleneck mechanism Jenuth et al. studied heteroplasmy levels in the cells of a developing mouse embryo using an animal model transmitting a mixture of polymorphic variants derived from different inbred laboratory mouse strains (BALB and NZB), making measurements in primordial germ cells (PGCs, the precursor cells for oocytes), primary oocytes, mature oocytes, and in the offspring of heteroplasmic female mice (Jenuth et al., 1996). This work suggested that the production of heteroplasmy variance, generated by the mtDNA bottleneck, occurred after the initial formation of the PGCs, but before differentiation of primary oocytes. In both the primary oocytes and the offspring, the heteroplasmy levels were evenly distributed above and below the mean value. This suggested that the underlying mechanism of inheritance was principally determined by random genetic drift for these polymorphic variants, although intriguingly these same variants showed a non-random distribution during post-natal tissue segregation (Battersby and Shoubridge, 2001; Jenuth et al., 1997), under nuclear genetic control (Jokinen et al., 2010). Several studies of heteroplasmy transmission in human pedigrees also implicate random genetic drift in the mechanism of inheritance of heteroplasmy (Brown et al., 2001; Wonnapinij et al., 2008), although others point

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towards a selective effect in favour of mutant mtDNA (discussed in (Chinnery et al., 2000)). However, studies in human pedigrees are difficult to interpret because ascertainment bias through a clinically affected individual can influence the experimental observations. Recent mouse studies of homoplasmic mtDNA variants do implicate selection during transmission (Fan et al., 2008; Stewart et al., 2008). However, the issue of random drift, selection or both mechanisms operating during the transmission of mtDNA heteroplasmy has yet to be resolved (Chinnery et al., 2000; Wonnapinij et al., 2010), and it may differ from mutation to mutation. When considering the biology of mtDNA inheritance, a number of recent technical advances have made it possible to dissect out the mechanism in more detail. In mice, pre-implantation development occurs between 0 and 4.5 days post coitum (d.p.c) where a fertilised oocyte develops into a blastocyst prepared for implantation (Fig. 1a). Individual blastomeres can be isolated from the 2–32 cell embryos and assessed for mtDNA content (copy number) using quantitative real-time PCR. Two groups used this technique to show that during the pre-implantation phase, cells split by binary fission without any increase in mtDNA content within the whole embryo, showing no net replication of mtDNA before implantation. As a consequence, each subsequent cell division reduced the amount of mtDNA within the daughter cells by ~50% (Cao et al., 2007; Cree et al., 2008). This finding was supported by studies using the whole embryo and a measurement of the number of mtDNA molecules per cell using real-time quantitative PCR to compare levels of mitochondrial and nuclear genes (Aiken et al., 2008). Assaying copy number in the complete blastocyst showed the total mtDNA remains constant (Cree et al., 2008), consistent with earlier Southern blot data (Piko and Taylor, 1987). Shortly after implantation (7.25 days, d.p.c, in mice) the PGCs first appear (Ginsburg et al., 1990). The PGC population is initially just 40 cells which are induced from cells within the embryonic endoderm. However,

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Fig. 1. Embryonic development in mice and the formation of the germ line. a. Development of a fertilised oocyte into an implanted blastocyst between 0 and 4.5 days post conception (d.p.c.). b. Formation and migration of the primordial germ cell (PGC) population during post-implantation development.

by 13 d.p.c the PGC population has migrated to the genital ridges and exponentially expanded to approximately 25,000 cells in mice (McLaren and Lawson, 2005; Tam and Snow, 1981) (Fig. 1). The first step in determining copy number involves the unambiguous isolation of PGCs at several stages of development. This is experimentally demanding, and several approaches have been used (Cao et al., 2007; Cao et al., 2009; Cree et al., 2008; Wai et al., 2008). The first technique used to identify PGCs was alkaline phosphatase (ALP) staining (Ginsburg et al., 1990; Jenuth et al., 1996). Using this approach, Cao et al. did not detect a dramatic reduction in copy number in early post implantation development. The authors interpreted these findings to indicate that the bottleneck is due to the segregation of homoplasmic multi-copy nucleoids, rather than independent mtDNA molecules (Fig. 2) (Cao et al., 2007). However, ALP interferes with the quantitative real-time PCR reaction used to measure copy number in these experiments, raising concerns about the data derived from ALP-stained cells. Alternative approaches involve transgenic mice with fluorescently-tagged PGC-specific protein constructs, including: Stella (DPPA3) (Cree et al., 2008; Payer et al., 2006), Blimp1 (PRDM1) (Cao et al., 2009; Sugimoto and Abe, 2007) and Oct4 (POU5F1) (Cao et al., 2009; Wai et al., 2008; Yeom et al., 1996). Cells expressing the fluorescent proteins can be isolated manually under a fluorescent microscope (Cao et al., 2007; Cao et al., 2009; Wai et al., 2008) or using flow cytometry (Cree et al., 2008). Cao et al. reassessed copy number in PGCs at varying stages of development following isolation with Blimp1. Although these experiments revealed a lower copy number than previously, the values were still considerably higher than results from other groups (Cree et al., 2008; Wai et al., 2008). Cree et al. and Wai et al. independently demonstrated a severely reduced copy number in early PGCs. However, the results were interpreted differently by each group. Cree et al. used a mathematical simulation to predict that the approximate 200 mtDNA molecules measured at 7.5 d.p.c were sufficient to produce the heteroplasmy variation seen in the offspring of heteroplasmic mice. Seventy percent of the heteroplasmy variance was attributed to the physical restriction of mtDNA levels in early PGCs, and a further 30% variance was produced during rapid proliferation in PGCs. They concluded that the mitochondrial bottleneck could be produced through the unequal segregation of the dramatically reduced mitochondrial genome population in early post implantation development (Fig. 2). Wai et al. (2008) studied the heteroplasmy levels within PGCs in the growing population. Despite detecting the most dramatically reduced copy number at 8.5 d.p.c, a limited number of heteroplasmy measurements did not appear to show an increase in heteroplasmy variance within the early PGC population. This led Wai et al. to suggest that variation in heteroplasmy does not develop until maturation in the primary follicles. At this point, the mtDNA copy number exceeds 10,000. They therefore attributed the rapid segregation of genotypes to selective replication of a subgroup of genomes within these postnatal cells, rather than the reduction in copy number (Wai et al., 2008) (Fig. 2). It is generally accepted that heteroplasmy variance needs to be normalised to account for variation between the heteroplasmy levels of the mothers sampled at different time points. When a mother's heteroplasmy level is closer to 50% there is generally larger variation seen than from a mother who has heteroplasmy level closer to either 0 or 100%. Since, by chance, mothers with heteroplasmy levels around 50% were sampled at later stages of development, once normalised, the change in heteroplasmy variance during post-natal folliculogenesis was not as dramatic as first reported (Samuels et al., 2010). There is therefore a clear need to confirm the precise timing of the bottleneck using an independent validated technique. Despite these differences of opinion, there is general agreement that mtDNA replication is limited before blastocyst implantation, leading to a key issue: when and how does mitochondrial genome replication resume?

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Fig. 2. Proposed mechanisms for the mtDNA genetic bottleneck. a. Variation in heteroplasmy levels are generated through the unequal partitioning (segregation) of mutant and wildtype genotypes during cell division. This leads to accelerated drift in heteroplasmy levels which occurs principally as a consequence of the dramatic reduction in mtDNA copy number immediately prior to primordial germ cell (PGC) population expansion (Cree et al., 2008). b. Variation in heteroplasmy generated through the unequal segregation of homoplasmic nucleoids in PGCs (Cao et al., 2007). Each nucleoid contains multiple copies of mtDNA which are identical. c. Variation in heteroplasmy generated through the replication of a subpopulation of mitochondrial genomes during oocyte maturation in post-natal life (Wai et al., 2008). Increasing severity refers to the clinical consequences of a higher percentage level of mutant mtDNA.

3. Factors controlling the maintenance of mtDNA Several key functions need to be addressed if we are to understand the regulation of replication during embryonic and germ-cell development. What proteins are involved? How are the mtDNA molecules dispersed throughout the cell? And what are the processes implicated in the regulation and maintenance of mtDNA? 3.1. mtDNA replication and transcription factors as molecular regulators Two models of mtDNA replication have been proposed: the strand asynchronous model and simultaneous (synchronous) model (Clayton, 1982; Holt et al., 2000). The asynchronous model proposes that replication begins at the heavy strand promoter, and replicates twothirds of the heavy strand, exposing a second promoter site for the light strand where replication for the light strand begins (Clayton, 1982). The simultaneous replication model suggests that both strands replicate at the same time, using 5′ to 3′ leading- and lagging-strand synthesis, a process similar to the replication of nuclear DNA (Holt et al., 2000). Extensive study of the replication fork showed that seemingly singlestranded DNA intermediates in the asynchronous model of replication were actually RNA–DNA hybrid strands. This finding lead to a modified model known as RITOLS (RNA incorporated throughout the lagging strand) (Holt, 2009).The proteins necessary for successful replication of the mtDNA remain the same, regardless of the model, and their functions have been extensively studied. Polymerase gamma (polγ) is the enzyme responsible for mtDNA replication. Polγ has two subunits: a catalytic subunit (encoded by POLG) and an accessory subunit (or p55 subunit, encoded by POLG2) (Olson et al., 1995; Ropp and Copeland, 1996). Studies in Drosophila melanogaster indicate that over-expression of the catalytic subunit does not increase the amount of mtDNA within individual cells. In contrast, increased expression of the accessory subunit does increase mtDNA content (Lefai et al., 2000). Experiments in human tissue have shown that the catalytic subunit is expressed at equal levels in the different tissues of the body (Schultz et al., 1998) and that over-expression of the catalytic subunit in cultured cells does not increase copy number (Spelbrink et al., 2000). This suggests that the accessory unit acts as a major regulator of polymerase activity. The promoter controlling expression of the p55 accessory subunit contains DNA replication-related elements (DREs),

bound by DREF (Zbed1 in humans) (Hirose et al., 1996; Lefai et al., 2000). Interestingly, recent studies have found that DREF affects the promoter activity of the Drosophila homologue of p53, dmp53 (Trong-Tue et al., 2010). Despite low sequence homology between Drosophila and human p53 proteins, the protein function is conserved and p53 has implicated roles in copy number regulation (Lebedeva et al., 2009). Transcription factors are also essential for the replication of mtDNA because mtDNA replication is initiated by an RNA primer. One key protein is the mitochondrial RNA polymerase, the enzyme responsible for the assembly of RNA sequences. The RNA polymerase works in conjunction with several transcription factors, including mitochondrial transcription factor A (TFAM) and mitochondrial transcription factors B1 and B2 (TFB1M and TFB2M) (Falkenberg et al., 2002; Gaspari et al., 2004). TFAM is responsible for the initiation of replication and transcription, and also acts as a structural support, facilitating protein binding to the mtDNA. Several studies have shown that the overexpression of TFAM in mouse tissue can increase copy number, and gene knockdown of TFAM in cultured cells causes mtDNA depletion (Ekstrand et al., 2004; Larsson et al., 1998). Without mtDNA, TFAM protein appears structurally unstable, with depletion of mtDNA causing reduced levels of TFAM protein, but not the mRNA (Ekstrand et al., 2004; Larsson et al., 1994). Estimates of the ratio between mtDNA copy number and TFAM molecules vary dramatically between experiments, ranging from one TFAM molecule per 10 bp to one molecule per 1000 bp (Ekstrand et al., 2004; Maniura-Weber et al., 2004). It has been shown that TFAM expression is regulated by PGC-1α (peroxisome proliferators-activated receptor gamma coactivator 1 alpha) and nuclear respiratory factors (NRF-1 and -2) (Wu et al., 1999), important regulatory factors in mitochondrial biogenesis. Twinkle is encoded by PEO1 and is a 5′-3′ helicase involved in unwinding mitochondrial DNA in preparation for replication (Korhonen et al., 2003; Spelbrink et al., 2001). Twinkle has also been shown to increase mtDNA copy number when over-expressed, and knock down experiments result in reduced mtDNA content (Tyynismaa et al., 2004). Mitochondrial single stranded binding protein (mtSSBP, encoded by SSBP1) and Twinkle increase the processivity of polγ (Farr et al., 1999). Experiments have shown a correlation between copy number increase and an increase in the level of mtSSBP, suggesting that this protein might also have an important role in copy number regulation (Schultz et al., 1998). The promoter region of the Drosophila melanogaster homologue

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(mtSSB) also contains two DRE sites for binding DREF transcription factor (Ruiz De Mena et al., 2000). 3.2. Mitochondrial biogenesis PGC-1α is the major factor controlling mitochondrial biogenesis and can be stimulated by many different pathways. It is induced by cold to allow adaptive thermogenesis, converting more of the energy produced by the respiratory chain into heat (Puigserver et al., 1998). It is also inducible by hypoxic conditions, via the AMPK signalling pathway, predicted to boost ATP production and protect the heart from damage (Zhu et al., 2010). PGC-1 α is known to induce the expression and transcriptional activity of NRF-1 (and to a lesser extent, NRF-2). The presence of functional NRF-1 has been shown to be essential for PGC-1α induced mitochondrial biogenesis (Wu et al., 1999). The proximal promoter of the TFAM gene contains binding sites for both NRFs and disruption of these binding sites severely reduces the expression of the TFAM promoter (Virbasius and Scarpulla, 1994). The TFAM protein can then localise to the mitochondria to bind mtDNA where it is essential for initiation of mtDNA transcription (Falkenberg et al., 2002). PGC-1α also activates thyroid hormone receptors which bind response elements on the mitochondrial genome and increase the transcription of mitochondrial genes (Pillar and Seitz, 1997; Puigserver et al., 1998; Weitzel et al., 2003). A recent microarray study indicates that PGC-1α induces expression of approximately 600 genes, whose protein products function within the mitochondria (Pagliarini et al., 2008). Most of these proteins are not directly involved with copy number regulation because biogenesis requires production of structural and respiratory chain proteins. However, approximately 5% have roles in mitochondrial transcription, replication and fission and fusion which all may influence the control of copy number within the cell. 3.3. Fission, fusion and mitochondrial nucleoid complexes Mitochondrial fission and fusion enable the exchange of genetic material and replication factors. Knockdown of fission proteins encoded by DNM1L and FIS1 in cultured rhabdomyosarcoma cells (Malena et al., 2009), and knockdown of fusion protein products from MFN1, MFN2 and OPA1, cause mtDNA depletion in skeletal muscle (Chen et al., 2010). The rate of fusion and fission appears to influence the access of nucleoid complexes (containing mtDNA and replication factors including TFAM, polγ, Twinkle and mtSSBP) to other replication factors within the mitochondria (Bogenhagen et al., 2008). Reduced fusion and fission will inevitably reduce the availability of replication factors and thus compromise replication, leading to a lower copy number in cells. Initially it was thought that nucleoids underwent ‘faithful’ replication, whereby each nucleoid would produce identical copies of itself, replicating each genome just once (Jacobs et al., 2000). This would imply heteroplasmy within each nucleoid, and thus allow functional complementation within the cell as a whole. However, subsequent studies have shown that, at least under certain circumstances, nucleoids do not exchange genetic material between themselves, and are usually homoplasmic (Cavelier et al., 2000; Gilkerson et al., 2008; Poe et al., 2010). 3.4. Nucleoside pools and mtDNA maintenance MtDNA replication is also critically dependent on a sufficiently large pool of nucleotides within mitochondria (Rampazzo et al., 2004). Mitochondrial nucleotide pools are maintained through two major pathways, the cytosolic import pathway and mitochondrial salvage pathway. The cytosolic import pathway imports nucleotides made by cytosolic ribonucleotide reductase (RNR) into the mitochondria. The mitochondrial salvage pathway converts nucleosides to nucleotides within the mitochondria. Both pathways are critical for the maintenance of mtDNA. Mutations have been identified in several genes involved in nucleotide maintenance, including TYMP (Thymidine

Phosphorylase) (Nishino et al., 1999) and RRM2B (alternative secondary subunit to RNR) (Bourdon et al., 2007) in the cytosolic import pathway and TK2 (dGK) (Saada et al., 2001) and DGUOK (dGK) (Mandel et al., 2001) in the mitochondrial salvage pathways. Pathological mutations in these genes and several others can cause mitochondrial DNA depletion syndrome (MDS), and present with varying phenotypes typical of mitochondrial disease (Rotig and Poulton, 2009).

3.5. Reactive oxygen species Reactive oxygen species (ROS) are produced by the respiratory chain during oxidative phosphorylation. ROS appear to play a role in the pathophysiology of specific mtDNA diseases and may contribute to the ageing process (Lenaz, 2001). It may be a critical signal in the regulation of cellular mtDNA content (Moreno-Loshuertos et al., 2006). In mice, polymorphic mtDNA haplotypes have been found to be associated with different levels of endogenous ROS production. Mouse cell lines with higher ROS production had an increased mtDNA copy number, thought to compensate for a mild respiratory chain deficiency. This suggests that the upregulation of mitochondrial biogenesis and mtDNA copy number acts as a defence mechanism against the damage caused by ROS production (Moreno-Loshuertos et al., 2006). Measuring ROS is difficult, but levels can be estimated indirectly through tissue markers for oxidative stress (TMOS) and levels of MnSOD (manganese superoxide dismutase) which increase in response to ROS levels (Aiken et al., 2008). NRF2 is tethered to the cytoskeleton in the cytoplasm by KEAP1 where it is unable to activate its transcriptional targets in the nucleus. Increased ROS has been shown to release NRF2 from KEAP1 in a phosphorylation dependent manner, allowing movement of NRF2 to the nucleus and activation of detoxifying and antioxidant enzymes (Motohashi and Yamamoto, 2004). Since NRF2 has been identified as a transcriptional activator of TFAM (Virbasius and Scarpulla, 1994), this pathway provides a possible link between ROS production an increase in mitochondrial biogenesis and mtDNA copy number. Increased ROS production also causes p53 to migrate into mitochondria, where it interacts with polγ to stimulate binding to damaged DNA (Achanta et al., 2005; Yoshida et al., 2003). p53 knockout mouse models and iRNA knockdown of p53 in cultured human primary fibroblasts both led to a dramatic reduction in copy number. Downstream effects of this knockdown included a reduction of mitochondrial membrane potential and mitochondrial mass, and reduced expression of both TFAM and p53R2 proteins, the alternative subunit to ribonucleotide reductase (RNR). This implicates p53 as an essential protein in the maintenance of mtDNA copy number in cells, even in the absence of mutations (Lebedeva et al., 2009).

4. Conclusion Given the complexity of the many factors known to influence mtDNA replication, it is remarkable that the cellular mtDNA content remains tightly regulated within defined limits, carefully matched to the energetic requirements of the cell. Ostensibly independent pathways involving transcription, biogenesis, alterations in fusion and fission frequencies, nucleotide pool regulation and ROS production have all been shown to influence mtDNA copy number and may thus be important in controlling the mtDNA bottleneck. At present these different mechanisms do not appear to be completely integrated, although patterns are emerging to suggest that all operate in a coordinated manner. Understanding these processes will be an essential step in elucidating the molecular and cellular mechanisms underpinning mtDNA transmission in mammals, and thus the mtDNA bottleneck. This will hopefully reveal mechanisms amenable to therapeutic manipulation, which may be applicable in clinical practice.

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Acknowledgements PFC is a Wellcome Trust Senior Fellow in Clinical Science and a UK NIHR Senior Investigator who also receives funding from the Medical Research Council (UK), the UK Parkinson's Disease Society, and the UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust. PJC is funded by Fight MERRF.

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