Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction

The International Journal of Biochemistry & Cell Biology 41 (2009) 1899–1906

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Review

Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction Robert W. Gilkerson Department of Neurology, College of Physicians & Surgeons, Columbia University, Russ Berrie Pavilion 307, 1150 St. Nicholas Ave., New York, NY 10032, United States

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Article history: Available online 9 April 2009 Keywords: Mitochondria Mitochondrial DNA Nucleoids Heteroplasmy Bioenergetics

a b s t r a c t Mitochondrial DNA plays a crucial role in cellular homeostasis; however, the molecular mechanisms underlying mitochondrial DNA inheritance and propagation are only beginning to be understood. To ensure the distribution and propagation of the mitochondrial genome, mitochondrial DNA is packaged into macromolecular assemblies called nucleoids, composed of one or more copies of mitochondrial DNA and associated proteins. We review current research on the mitochondrial nucleoid, including nucleoid-associated proteins, nucleoid dynamics within the cell, potential mechanisms to ensure proper distribution of nucleoids, and the impact of nucleoid organization on mitochondrial dysfunction. The nucleoid is the molecular organizing unit of mitochondrial genetics, and is the site of interactions that ultimately determine the bioenergetic state of the cell as a whole. Current and future research will provide essential insights into the molecular and cellular interactions that cause bioenergetic crisis, and yield clues for therapeutic rescue of mitochondrial dysfunction. © 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mitochondrial nucleoid: DNA packaging and protein composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nucleoids in mitochondrial dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nucleoids as determinants of mitochondrial pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A nucleoid-based model of the mitochondrial threshold effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Human mitochondrial (mtDNA) is a 16,569 bp circular DNA, and is typically present in approximately 1000 copies per cell, although copy number appears to vary among cell types. Since the discovery of the mitochondrial genome (Nass and Nass, 1963a; Nass and Nass, 1963b), it has been apparent that mtDNA plays by its own set of rules, both genetically and cell biologically. MtDNA is inherited maternally and displays non-Mendelian inheritance patterns: instead of a phenotype resulting from two gene copies, per the nuclear genome, mtDNA displays population genetics, as the mitochondrial phenotype is determined by the many mtDNA copies

Abbreviations: mtDNA, mitochondrial DNA; MERFF, myoclonus epilepsy with ragged-red fibers; MELAS, mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes. E-mail address: [email protected]. 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.03.016

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within the cell. MtDNA also has its own genetic code, differing in several particulars from the universal genetic code. In order to protect, maintain, and propagate the mitochondrial genome accurately, mtDNA is packaged into protein–DNA assemblies called nucleoids. This review will focus on the mechanisms of mtDNA packaging, the protein factors associated with mtDNA in the nucleoid, mtDNA copy number control at the nucleoid, the integration of nucleoids into mitochondrial dynamics within the cell, and nucleoid mediation of mtDNA genetic patterns in human and mammalian systems. Current research is providing a better understanding of the molecular mechanisms governing the often-confusing patterns of mitochondrial genetics, as well as providing clues for therapeutic methods to rescue mitochondrial dysfunction. Although much of the most in-depth mechanistic research regarding mtDNA nucleoids was first done in model organisms such as Saccharomyces cerevisiae, this review will be largely limited to discussion of the current state of research in human and mammalian systems. However, it must be emphasized that examples of

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pioneering research on nucleoid organization (Boldogh et al., 2003; Meeusen and Nunnari, 2003), composition (Kaufman et al., 2000; Miyakawa et al., 1995), and mobility (Okamoto et al., 1998) in model systems paved the way for researchers addressing the mammalian nucleoid.

2. The mitochondrial nucleoid: DNA packaging and protein composition While mtDNA has long been presumed to be ‘naked’ due to its relatively high rate of mutation, it is increasingly apparent that nucleoids provide an important mtDNA packaging function. The mitochondrial protein TFAM (transcription factor A, mitochondrial), an abundant high mobility group (HMG) DNA-binding protein which was initially identified as an mtDNA transcription factor, has been revealed to be a major nucleoid organizing protein through its packaging of mtDNA (Alam et al., 2003; Kaufman et al., 2007) and regulation of mtDNA copy number (Ekstrand et al., 2004). TFAM binds mtDNA at an average of one TFAM per 10 bases of mtDNA (Alam et al., 2003), acting in an architectural, as well as transcriptional, role for mtDNA maintenance (Kanki et al., 2004). TFAM actively packages and cooperatively binds DNA in vitro: the affinity of TFAM for DNA is further enhanced by the presence of previouslybound TFAM (Kaufman et al., 2007). Thus, the traditional concept of mtDNA as ‘naked’ is not accurate: the mitochondrial nucleoid provides an effective packaging of mtDNA for the maintainance and propagation of the mitochondrial genome. Indeed, the typical dimensions of mitochondrial nucleoids observed within the cell, by a variety of methods, are approximately 70 nm diameter (Iborra et al., 2004). However, a ‘naked’ relaxed circle of the 16.6 kb mtDNA would actually extend up to 2 ␮m throughout the mitochondrion. This would be a most inefficient way to maintain mtDNA and would render mtDNA highly susceptible to faulty segregation of mtDNA, not to mention higher rates of mtDNA mutation and damage. Thus, the compact, discrete nature of the mitochondrial nucleoid provides an efficient packaging of mtDNA for maintenance and propagation. The number of mtDNAs contained within a single nucleoid remains a somewhat controversial issue. The majority of research on nucleoids in human systems has been in rapidly dividing, tumor-derived cultured cells. Several studies report that mitochondrial nucleoids in these cells carry approximately five mtDNAs per nucleoid (Gilkerson et al., 2008; Iborra et al., 2004; Legros et al., 2004). These studies quantified the mtDNA copy number per cell and divided that number by the number of nucleoid foci within a cell, generating estimates from 2 to 10 mtDNAs per nucleoid. However, one of the groundbreaking studies of human nucleoids, using ethidium bromide fluorescence of individual nucleoids calibrated against the fluorescence from known quantities of phage DNA, found that nucleoids carry either one or two mtDNAs (Satoh and Kuroiwa, 1991). It remains to be determined whether copy number per nucleoid is a constant, or actually varies among tissue and cell types. Nucleoids in postmitotic tissues may carry higher numbers of mtDNAs within a single nucleoid, consistent with findings of mtDNA duplication events in heart tissue, indicative of intermolecular recombination (Fromenty et al., 1997; Kajander et al., 2001): postmitotic cells maintaining mtDNA in nucleoids with a high copy number may allow for a greater opportunity for recombination events than in rapidly dividing cells, where intermolecular recombination can occur (D’Aurelio et al., 2004), but does not appear to happen frequently (Gilkerson et al., 2008). In cells carrying duplications of mtDNA (i.e. a single molecule composed of a WT molecule and a deletion, or -mtDNA), the duplication was eventually resolved to two monomeric WT and -mtDNA molecules (Tang et al., 2000). It appears that the molecular machinery necessary for

recombination is thus present in mitochondria, as intramolecular recombination occurs with some frequency, while intermolecular recombination is quite rare. Similarly, when the yeast Holliday junction resolvase CCE1 was introduced into human cells carrying duplications of mtDNA, WT molecules were restored to the cell (Sembongi et al., 2007), providing further support for a somewhat frequent occurrence of intramolecular mtDNA recombination in human cells, with intermolecular recombination remaining a very rare event. Nucleoid organization probably has a great deal to do with mtDNA copy number control, as the nucleoid is the organizing body of mtDNA, and it is clear that TFAM is an essential modulator of mtDNA copy number. The genetic pathways controlling mitochondrial biogenesis have been shown to affect both mtDNA and nucleoid-associated proteins such as TFAM (Gleyzer et al., 2005); however, it remains to be seen how nucleoids mediate copy number control. It has been demonstrated that cells modulate mtDNA mass, rather than copy number per se (Tang et al., 2000); however, the molecular mechanisms behind these observations remain to be determined. A key question of nucleoid biology has been to determine which proteins are bona fide components of the nucleoid. While a number of proteins identified as nucleoid-associated factors are, logically enough, proteins required for mtDNA transcription, maintenance, and replication, other protein components are being identified which provide clues to how mtDNA is maintained within the mitochondrion, revealing potential higher-order domain structuring of the mitochondrial network to ensure the proper placement and distribution of mtDNA. Further, some intriguing nucleoid-associated factors are being uncovered which suggest that mtDNA’s impact on cellular signaling and metabolism may extend well beyond the mitochondrial network. The following is a look at the proteins associated with the mitochondrial nucleoid, including several proteins with important signaling functions in the cell at large. Biochemical purification of mitochondrial nucleoids, in concert with other experimental methods, has revealed many of the principal components of the nucleoid. TFAM has been identified as a major protein component of human and mammalian nucleoids (Bogenhagen et al., 2008; Garrido et al., 2003; Kasashima et al., 2008; Wang and Bogenhagen, 2006). Additional mtDNA-associated components include the Twinkle helicase, mitochondrial polymerase ␥, and mitochondrial single-stranded binding (mtSSB) protein (Garrido et al., 2003). These proteins have been identified as participants in mtDNA maintenance (Garrido et al., 2003; Tyynismaa et al., 2004), and so their presence in the nucleoid is not particularly surprising. However, a number of mitochondrial inner membrane proteins have been identified in purified nucleoid preparations, such as the adenine nucleotide translocase (ANT), as well as subunits of Complex I of the mitochondrial respiratory chain (Wang and Bogenhagen, 2006) and subunits of the ATP synthase (Bogenhagen et al.). While these factors may be non-specific components found at the nucleoid due to their relative abundance within the organelle, it is also possible that specific interactions with these proteins serve to link mtDNA to the inner membrane, acting to hold the nucleoid in place, rather than have it drift with a higher degree of freedom through the matrix. The presence of an mtDNA ‘tether’ protein, which would link the nucleoid with the mitochondrial inner membrane, has been postulated for many years (Albring et al., 1977; Iborra et al., 2004); however, no such protein has yet been identified. As noted by Bogenhagen et al. (2008), it will be necessary to examine the specific interaction of the different proteins in order to reveal the links between mtDNA and its nucleoid organization definitively. Such studies in S. cerevisiae were among the first to demonstrate the dynamic importance of mitochondrial nucleoids (Boldogh et al., 2003; Meeusen and Nunnari, 2003; Okamoto et al., 1998); as mammalian genetics ‘comes of age’,

R.W. Gilkerson / The International Journal of Biochemistry & Cell Biology 41 (2009) 1899–1906

these issues will be clarified in human and mammalian systems as well. In addition to straightforward components such as transcription and replication factors, as well as potential inner membrane proteins, a number of rather surprising proteins have been identified as nucleoid components that may have signaling roles in the cell at large, linking mtDNA maintenance to other cellular processes. Chief among them is human prohibitin 1, which is involved in mtDNA organization through its stabilization of the TFAM-mtDNA interaction as well as in TFAM-independent interactions; prohibitin 1 knockdown results in partial release of mtDNA and destabilization of nucleoids (Kasashima et al., 2008). Both prohibitins 1 and 2 have been identified as nucleoid components in biochemical purifications (Bogenhagen et al., 2008; Wang and Bogenhagen, 2006). Prohibitins participate in a wide variety of cellular functions, including apoptosis, cell cycle regulation, signal transduction, and lifespan regulation (Czarnecka et al., 2006); within the mitochondrion, prohibitins appear to mediate the structure of the cristae, helping to organize the inner membrane, as well as participating in the regulation of proteolytic processing in the inner membrane (Merkwirth and Langer, 2008). These findings place a broadly important cellular signaling factor at the nucleoid, suggesting that the nucleoid is involved in essential cellular processes beyond the mitochondrion and illustrating the importance of mtDNA maintenance and inheritance for the homeostasis of the cell as a whole. Other potential nucleoid-associated proteins include ATAD3 (He et al., 2007) and p53 (Yoshida et al., 2003). The mitochondrial inner membrane protein ATAD3 was found to bind in vitro to a synthetic version of the mtDNA D-loop, a key region for transcription and mtDNA replication (Clayton, 1982); further, RNAi gene silencing of ATAD3 resulted in destabilization of mitochondrial nucleoid organization (He et al., 2007). However, ATAD3 was not found in biochemical nucleoid isolations, suggesting that ATAD3 may be associated with nucleoids, albeit not in direct contact with mtDNA (Bogenhagen et al., 2008). p53 was found to coprecipitate with TFAM during immunoprecipitation experiments, displaying protein–protein interactions requiring specific regions of both proteins. Moreover, p53 enhanced TFAM’s binding affinity for damaged DNA in vitro (Yoshida et al., 2003). In addition, human mtDNA contains a region predicted to be a p53 binding sequence (Heyne et al., 2004). However, as with ATAD3, p53 was not detected in nucleoid purifications (Bogenhagen et al., 2008). p53, as an important tumor suppressor, plays a role in numerous cell signaling pathways, including mediation of mitochondrial apoptosis in response to DNA damage (Marchenko et al., 2000) and hypoxia (Sansome et al., 2001). Despite the absence of these proteins in biochemical nucleoid purifications, it is possible that they may nevertheless participate in crucial signaling events at the nucleoid. Bogenhagen et al. (2008) have proposed that mitochondrial nucleoids have a ‘layered’ structure consisting of mtDNA, a core of proteins for replication and transcription, with translation and complex assembly proteins in the outer portion of the nucleoid. This ‘layered’ model is consistent with our own observation that nucleoid foci of mtDNA, observed by in situ hybridization, are smaller than nucleoid foci seen by anti-TFAM immunolabeling (unpublished data), supporting the idea of a protective layer of protein around the DNA at the core of the nucleoid. We speculate that proteins such as the prohibitins, ATAD3, and p53 may indeed mediate important signaling events at the nucleoid, but that these interactions necessarily occur at the outer ‘shell’ of the nucleoid by the nature of their communication with the rest of the cell. Thus, mitochondrial nucleoids seem to maintain mtDNA within a core of TFAM and factors necessary for transcription, replication, and translation, interacting with key signaling factors at the periphery of the nucleoid, thus protecting mtDNA and facilitating function and signaling at a single suborganellar site.

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3. Nucleoids in mitochondrial dynamics The mitochondrial nucleoid is a macromolecular structure specialized to provide a submitochondrial organization of mtDNA, allowing for efficient maintenance of mtDNA in discrete segregating units located at intervals throughout the mitochondrial network. The packaging of mtDNA into nucleoids is readily apparent through numerous microscopic visualization methods, in which punctate nucleoid structures are distributed at regular intervals throughout the mitochondrial network (Iborra et al., 2004; Kang and Hamasaki, 2005; Legros et al., 2004; Margineantu et al., 2002). The nucleoid’s position in the matrix lends it access to the most interior continuous compartment of the mitochondrial network, allowing transcripts and proteins produced at the nucleoid to diffuse through the mitochondrial network, facilitating the interaction and transcomplementation of mitochondria carrying different mtDNAs (Hayashi et al., 1994; Sato et al., 2004). While nucleoids do not appear to exchange mtDNAs among each other, heterologous nucleoids have been shown to complement each other to restore bioenergetic function, apparently due to the free diffusion of mtDNA transcripts through the mitochondrial matrix (Gilkerson et al., 2008). Beyond the diffusion of mtDNA-derived mRNAs and proteins, however, it has been demonstrated that mtDNA nucleoids themselves can efficiently repopulate the mitochondria of mtDNA-deficient 0 cells within 12 h (Legros et al., 2004), indicating that nucleoids have a certain mobility within the matrix, given sufficient time. A major recent advance in mitochondrial research has seen the emergence of mitochondrial fission and fusion as a balance of two genetically regulated processes which are connected to mitochondrial function (reviewed in-depth in Chan, 2006). The identification of genes responsible for fission and fusion has resulted in a reappraisal of mitochondrial morphology and organization, as it illustrates the ways in which mitochondrial structure is highly adaptable and genetically regulated, allowing for conformational changes in response to variations in cellular bioenergetic demand, signaling cues, and environmental insults. While it has been well established that mitochondria have an elaborate multimembrane internal structure, optimized for maximal bioenergetic function, it is increasingly clear that the overall structure of mitochondria within the cell is also very elaborate and sensitive. The mammalian proteins FIS1 and DLP1 (also known as DRP1) have been shown to mediate mitochondrial fission: FIS1 is an integral mitochondrial protein, while DLP1 is largely cytosolic, but is recruited to the mitochondria, where it cooperatively acts with FIS1 to ‘pinch off’ the mitochondrial membranes and elicit fission events (Lee et al., 2007; Pitts et al., 1999; Smirnova et al., 2001). It is likely that an additional protein factor or factors participates in this recruitment of DLP1, as knocking out FIS1 does not disrupt DLP1 recruitment to the mitochondria (Lee et al., 2007). The mammalian proteins MFN1 and MFN2, as well as OPA1, are required for mitochondrial fusion. These two opposing processes maintain mitochondria in a balance between a completely interconnected mitochondrial network, and a population of disconnected individual organelles. However, it is unknown how the fusion and fission machinery interact with mtDNA maintenance and propagation to ensure the proper distribution and inheritance of the mitochondrial genome. Although it remains unclear how mitochondrial nucleoids are parceled out in the opposing processes of fusion and fission, several lines of evidence suggest that definite, though uncharacterized, mechanisms exist to ensure the efficient, even distribution of nucleoids during mitochondrial fission and fusion. There have been numerous observations to provide clues to how mitochondrial nucleoids are partitioned. It is clear from multiple microscopic methods that mtDNA nucleoids are organized as discrete punctae, present at regular intervals along the mitochondrial network. Moreover, it has been shown that, upon fragmentation of the

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mitochondrial network, each individual organelle has at least one nucleoid (Margineantu et al., 2002). Chen et al. (2007) have found that knockout of MFN1 and MFN2 results in a loss of mtDNA nucleoids, such that numerous mitochondria have no apparent mtDNA nucleoids. In addition, downregulating DLP1 to prevent mitochondrial fission results in a loss of mtDNA (Parone et al., 2008). These findings suggest coordination between mitochondrial fission/fusion and mtDNA maintenance, indicating that molecular mechanisms exist to ensure an even distribution of mtDNA throughout the mitochondrial network, regardless of its morphological state at any given time. Accordingly, we describe two possible models of mitochondrial ‘subdomains’ which may provide a theoretical basis for exploring molecular mechanisms of nucleoid distribution in human and mammalian systems: One possible mechanism to explain nucleoid inheritance places the nucleoid within a submitochondrial ‘organizing center’, composed of protein–protein interactions linking mtDNA through the mitochondrial inner and outer membranes to cytoskeletal and endoplasmic reticulum (ER) contacts outside the mitochondrion. These ‘organizing centers’ would coordinately include mtDNA and nucleoid-associated factors tethered to the inner membrane, where they associate with protein import and translational machinery (coupling mtDNA- and nuclear-encoded proteins at one site), as well as mitochondrial fission and fusion machinery and cytoskeletal and ER proteins at the mitochondrial outer membrane. Thus, mtDNA could be directly associated with protein import and translational machinery, as well as the machinery mediating both mitochondrial fission/fusion and extramitochondrial attachments, ensuring that all necessary mitochondrial components are parceled out. Such a model is consistent with the emerging picture of nucleoid organization in yeast (Chen and Butow, 2005). Alternatively, it may be useful to reexamine how nucleoids are propagated and maintained in bacteria. While bacterial nucleoids give mitochondrial nucleoids their name, due to similarities in the cellular packaging of small circular DNAs, they may also provide clues as to how mitochondria efficiently distribute their nucleoids, given the endosymbiontic origin of mitochondria. Importantly, bacteria maintain distinct mechanisms to ensure proper distribution of nucleoids following cell division. Bacterial cell division proceeds by the FtsZ protein forming ‘Z rings’, which then mediate septation at the Z ring site (Sun and Margolin, 2004). In E. coli, the Min protein system ensures that cell division occurs at the proper site via septation inhibition, while the ‘nucleoid occlusion’ system ensures that cell division events do not occur over a nucleoid, thus preventing the loss of a nucleoid due to bacterial fission events. The Noc (in B. subtilis) and SlmA (in E. coli) nucleoid occlusion proteins, associate with the nucleoid and prevent Z ring formation and subsequent cell division events at the nucleoid (Rothfield et al., 2005). It is possible that mitochondria may utilize a similar scheme, in which nucleoids and fission/fusion machinery occupy different submitochondrial domains, to ensure that mtDNAs are not lost during mitochondrial fission and fusion events. This would necessitate a nucleoid-associated protein in the inner mitochondrial membrane acting like a bacterial nucleoid-occlusion protein to define the ‘fission-free’ zone, preventing the formation of the DLP1–FIS1 mitochondrial fission ring. Moreover, it is likely that the fission, and possibly fusion, machinery is assembled into organizing centers for attachment to the cytoplasm and ER contacts, providing an organizing center for mitochondrial movement, fission/fusion, and Ca2+ signaling; excluding the nucleoid from these organizing centers would allow these important mitochondrial functions to be accomplished without the risk of losing mtDNA nucleoids. This is consistent with microscopy showing that mitochondrially localized DLP1 does not colocalize with nucleoids, but is usually found on either side of nucleoids (Garrido et al., 2003), and additional findings that mitochondrial fission events occur next to, but not at,

nucleoid foci (Iborra et al., 2004). Research into the compartmentation of various mitochondrial functions is necessary to determine which of the above models (if either) is more accurate.

4. Nucleoids as determinants of mitochondrial pathology The mitochondrial nucleoid, as the organizing unit of mtDNA, is a fundamental component of bioenergetic homeostasis. As such, mutations in nuclear factors associated with the nucleoid result in defects in mtDNA maintenance and propagation, while the organization of mtDNA variants within a cell’s nucleoids will determine to a large extent the mitochondrial bioenergetic status of the cell. The highly variable nature of mtDNA genetics and the resulting effects on cellular bioenergetic status lead us to propose that the study of mtDNA genotype/phenotype relationships at the suborganellar level will be essential to understand mitochondrial dysfunction, as well as to develop therapeutic methods to rescue pathogenic states stemming from high levels of mtDNA mutations. While we have discussed above the proteins associated with the mitochondrial nucleoid, many studies have revealed that mutations in a number of these proteins cause either the loss of mtDNA content from the cell, or appear to generate mtDNA mutations. The loss of TFAM, the mitochondrial transcription factor and principal packaging protein, is correlated in many studies with loss of mtDNA. Knockout of TFAM in mice results in mtDNA depletion and mitochondrial dysfunction (Ekstrand et al., 2004). The mitochondrial helicase Twinkle and mitochondrial DNA polymerase ␥ both appear to increase the age-dependent accumulation of mtDNA mutations (Wanrooij et al., 2004). In addition, mutations in polymerase ␥ have been shown to cause depletion of mtDNA (Davidzon et al., 2005). The exact molecular mechanisms at the nucleoid by which these nuclear mutations cause mtDNA mutations or depletion remain unclear; however, it is abundantly demonstrated that faulty nucleoid proteins results in damage or loss of mtDNA, indicating that ‘tampering’ with mtDNA’s packaging results in instability and mutation of mtDNA. Mutations of mtDNA, both point mutations and -mtDNAs have been shown to cause a host of tissue-specific and systemic diseases. Classical mitochondrial diseases, such as myoclonus epilepsy with ragged-red fibers (MERFF), mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS), and Kearns–Sayre syndrome, have been shown to result from the expansion of a mtDNA mutation to high levels within a particular tissue or cell (reviewed in DiMauro and Schon, 2003). It has recently been shown that high levels of -mtDNAs are found in neurons of the substantia nigra associated with aging and Parkinson’s disease, indicating a loss of mitochondrial enzymatic function and neuronal death (Bender et al., 2006; Kraytsberg et al., 2006). Whether maternally inherited or sporadic, it is clear that mtDNA mutations can have devastating effects on a cell via bioenergetic dysfunction, either causally or by predisposing cells to dysfunction and premature cell death. Nucleoid organization presents a molecular mechanism to explain patterns of mtDNA dynamics and the bioenergetic phenotypes resulting from them. MtDNA frequently exists in multiple forms within the same cell and tissue, a situation called heteroplasmy. It is well established that most cell types can tolerate a high proportion of mutant mtDNA and maintain effective mitochondrial function; this ‘threshold effect’ (reviewed in DiMauro and Schon) is variable, depending on the type of mtDNA mutation and cell type. Cells can usually withstand a majority of mutant mtDNA, which can be compensated for by a small proportion of remaining WT mtDNA. However, once the ‘threshold’ is crossed, the remaining WT mtDNAs can no longer adequately maintain mitochondrial function, and the cell is rendered bioenergetically defunct due to mitochondrial dysfunction.

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Within a heteroplasmic cell, WT and mutant mtDNAs are distributed throughout the mitochondrial network. Heterologous nucleoids, carrying different mtDNAs, do not appear to exchange genetic material via stable fusion at any appreciable frequency, indicating that individual nucleoids remain genetically autonomous, at least in rapidly dividing cell types (Gilkerson et al., 2008). The ‘faithful nucleoid’ model, first proposed by Jacobs et al. (2000), accurately predicted that the heteroplasmic state of a cell will be determined by how the WT and mutant mtDNAs are organized into the cell’s nucleoids: if the two mtDNA variants are maintained in separate nucleoid populations, the cell’s overall heteroplasmy will be fluid, subject to selective pressure, replicative advantage, and other variables, while if the two mtDNAs are maintained within the same nucleoid population, the cell’s overall heteroplasmy will remain relatively static.

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locales. In support of this concept, Fig. 1B shows the intramitochondrial distribution of WT mtDNA and RNA transcripts within two cybrid cell lines. WT cells reveal a concentration of WT mtDNA and RNA fluorescence (green) at the nucleoids, with RNAs diffusing away throughout the mitochondrial network (visualized with Mitotracker (red)). However, in a cybrid carrying 93% -mtDNA and 7% WT mtDNA (FLP6b25.27), WT mtDNA and RNA transcripts are found in only a few isolated mitochondria throughout the cell. Moreover, the mitochondria are organized as a collection of individual organelles, rather than the interconnected network observed in the WT (Fig. 1B). Uncovering the precise molecular mechanisms linking the mitochondrial fission and fusion pathways with bioenergetic function will determine whether this model is correct.

6. Therapeutic prospects 5. A nucleoid-based model of the mitochondrial threshold effect When a cell carries a below-threshold level of mutant mtDNAs, WT mtDNAs produce sufficient amounts of transcripts to maintain WT-like levels of OXPHOS proteins, membrane potential, oxygen consumption, respiratory activity (DiMauro and Schon, 2003), and WT-like mitochondrial morphology (Santra et al., 2004). However, above the threshold of mutation load (for example, cells carrying -mtDNAs have a threshold of 80% mutation load), cells cannot maintain WT levels of mitochondrial function, lacking sufficient mtDNA-encoded transcripts to adequately produce the necessary OXPHOS proteins to maintain membrane potential and respiratory activity. These cells typically have the fragmented, blebbed mitochondrial morphology associated with homoplasmy for mutant mtDNAs, or 100% mutation load (Gilkerson et al., 2008; Santra et al., 2004). This loss of WT-like mitochondrial morphology indicates that the mitochondria are no longer a united network, instead existing as a population of individual organelles. This morphological transition is often a useful marker of bioenergetic status in cultured cells. These observations suggest that the threshold effect is determined, at least in part, by the ability of WT transcripts to diffuse through the matrix, become translated into respiratory complex subunits inserted in the inner membrane, and provide a basal level of normal mitochondrial function to maintain effective respiratory function and a reticular mitochondrial morphology, as in WT cells (Fig. 1A). These suborganellar genotype/phenotype relationships suggest that the maximal diffusible distance of WT transcripts may define the threshold of mitochondrial function: if the intramitochondrial distance between two WT mtDNA-containing nucleoids is farther than their transcripts/finished proteins can travel in any appreciable quantity, a ‘dead zone’ of respiratory deficiency will result, leading to mitochondrial fission at that point (Fig. 1A). Interestingly, it has been shown that cells with only mild mitochondrial dysfunction can occasionally be seen to have an increase in mitochondrial branching and filamentation (Bratic et al., 2009; Eisenberg et al., 2008; Koopman et al., 2007), suggesting that sub-threshold levels of mitochondrial dysfunction may cause a compensatory increase in mitochondrial ‘networking’ to facilitate enhanced diffusion of WT mtDNA transcripts throughout the network. However, once the threshold is breached, the resulting respiratory ‘dead zones’ within the mitochondrial network cause the collapse of membrane potential and a concomitant fragmentation of the mitochondrial network, resulting in a mixed population of individual organelles, most with little or no respiratory activity. The respiratory profile of the entire cell will in fact be dictated in large degree by the collective ability of individual nucleoids to provide efficient respiratory function in their particular submitochondrial

At present, rational genetic therapies for patients suffering from mitochondrial disorders do not exist. Due to the mitochondrial threshold effect, it is likely that the introduction of a small number of WT mtDNAs into a cell carrying high levels of mutant mtDNA would result in a restoration of mitochondrial function. However, unlike nuclear genetic mutations, which can typically be compensated for by the addition of a WT copy of the gene incorporated into the chromosomal DNA, no methods currently exist for the stable introduction of exogenous mtDNA into cells. Efforts to rescue mitochondrial dysfunction via genetic manipulation to date have thus fallen into one of two categories: strategies attempting to overcome this technological hurdle and stably introduce exogenous WT mtDNA into cells, and strategies involved in attempting to induce cells to either rid themselves of mutant mtDNA, or selectively amplify their existing WT mtDNAs. The only successful strategy for the genetic transformation of mitochondria was developed in S. cerevisiae, in which Johnston et al. bombarded respiratory-deficient cells with microprojectiles coated with DNA. Transformants were obtained with the introduced DNA integrated into the mitochondrial genome (Johnston et al., 1988). However, no corresponding mammalian or human study has been published. Several strategies have been published attempting to ‘get around’ this obstacle in human and mammalian systems by various molecular methods. The mtDNA-encoded ATP6 subunit of the mitochondrial ATP synthase was allotopically expressed as a recoded nuclear gene, rescuing an ATP synthesis defect in cultured cybrid cells carrying the pathogenic T8993G mutation in the mitochondrial ATP6 gene (Manfredi et al., 2002), while a nucleus-encoded ATP6 gene from the algae Chlamydomonas reinhardtii rescued ATP synthesis in the same T8993G cybrid cell system (Ojaimi et al., 2002). It has been shown that cytosolic tRNAs can be imported into human mitochondria, rescuing an mtDNA-encoded tRNA defect, using a Leishmania-based caveolin-dependent RNA import complex (Mahata et al., 2006). A mitochondrially targeted restriction endonuclease, which specifically cut only mutant mtDNA, was found to rapidly shift mtDNA heteroplasmy in mice (BayonaBafaluy et al., 2005). While these methods each use a cunning molecular strategy to rescue mitochondrial dysfunction, demonstrating potential mechanisms for therapeutic use, all remain far removed from therapeutic use in patients. Thus, until exogenous mtDNA can be stably introduced into human cells, true mitochondrial gene therapy remains beyond our reach. Strategies to stably introduce WT mtDNA should likely incorporate the nucleoid packaging of mtDNA: any introduced mtDNA would necessarily need to be complexed with TFAM in order to be stably maintained within the cell. TFAM levels correlate with mtDNA copy number in patients (Larsson et al., 1994), transgenic mice (Ekstrand et al., 2004), and cell culture systems (Pohjoismaki et al., 2006). TFAM-mediated DNA packaging is therefore likely to be the bare minimum of nucleoid

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Fig. 1. (a) A nucleoid-based model of the mitochondrial threshold effect. In a heteroplasmic cell containing high levels of mutant mtDNA and a small percentage of WT mtDNA, our model predicts that the distance between WT mtDNA-containing nucleoids will determine whether or not mitochondrial dysfunction occurs. In both the ‘below threshold’ (i.e. below the critical mtDNA mutation load, top) and ‘above threshold’ (bottom) scenarios, two WT mtDNA nucleoids are seen as large red circles. Their RNA transcripts (arrows) diffuse away from their respective nucleoids and are inserted into the inner membrane, where they are incorporated into fully functional OXPHOS complexes (small red ovals). The distance D is defined as the maximal distance that the transcripts can effectively diffuse from the nucleoid through the matrix. In the ‘below threshold’ scenario, the distance D for each nucleoid overlaps with that of the other, ensuring complete ‘coverage’ of bioenergetic capability with active OXPHOS complexes (light red shading). In the ‘above threshold’ scenario (bottom), the distance D for each nucleoid does not overlap with that of its nearest neighbor. This creates a bioenergetic ‘dead zone’ (gray shading), which is respiratory-deficient and mitochondrially dysfunctional. This ‘dead zone’ will then lead to fragmentation of the mitochondrial network. In such a heteroplasmic system, many more mutant mtDNA-containing nucleoids than WT nucleoids will be present; however, as they will be non-functional, they have been omitted from the model, for simplicity’s sake. (b) Distribution of WT mtDNA and mtRNA visualized by in situ hybridization. WT mtDNA and mtRNA was visualized in human cybrid cells carrying 100% WT mtDNA (WT) or heteroplasmic 93% -mtDNA : 7% WT mtDNA (FLP6b25.27) by fluorescence in situ hybridization (FISH) per Gilkerson et al., 2008, omitting RNase treatment to allow visualization of mtRNA. A probe corresponding to nt 7909–9417 of human mtDNA allowed specific visualization of WT mtDNA but not -mtDNA. In the WT cells, mitochondria exist predominantly as a reticular network, as visualized with MitoTracker, while the WT mtDNA + RNA are apparent as bright nucleoid punctae joined by slightly less intense signal from the mtRNA diffusing away from the nucleoids. In contrast, the FLP6b25.27 heteroplasmic line (above threshold for mitochondrial dysfunction (Santra et al., 2004)) has a more fragmented mitochondrial morphology, with only a handful of mitochondria showing signal for WT mtDNA + mtRNA. Size bar = 0.5 ␮m.

complexing necessary for the stable propagation of exogenous mtDNA introduced into mitochondria; other nucleoid-associated factors may prove beneficial or necessary. Due to the mitochondrial threshold effect, small changes in mtDNA heteroplasmy can have profound effects in altering mitochondrial bioenergetics, as discussed above. To this end, numerous efforts have been made to rescue mitochondrial dysfunction in heteroplasmic cells by selectively amplifying WT mtDNA, or inhibiting mutant mtDNAs. Synthetic peptide nucleic acids, which preferentially recognize mutant mtDNAs, have been developed which can inhibit replication of the A8344G pathogenic mtDNA mutation in vitro (Taylor et al., 1997), as well as bind preferentially to the sequence-repeat breakpoint of the “common deletion” of mtDNA (McGregor et al., 2003); however, like the molecular methods described above, this method is not yet ready for use in patients.

It has been shown that respiratory-deficient cybrid cells heteroplasmic for a -mtDNA can be shifted to a greater percentage of WT mtDNA by treatment with ketogenic media supplements, thereby rescuing mitochondrial dysfunction. Interestingly, following the shift, the investigators observed larger-than-normal ‘supernucleoids’, possibly aggregates of new WT mtDNAs (Santra et al., 2004). It is likely that the existence of WT and -mtDNAs in different nucleoid populations allows the observed flux in mtDNA heteroplasmy, providing a molecular basis for further efforts to shift heteroplasmy in vivo. Moreover, ketogenic treatment may be mimicked in patients via the ketogenic diet, which is already an established therapeutic method. Depending on the molecular mechanism of the heteroplasmic shift observed by Santra et al., it may be possible to elicit a similar heteroplasmic shift pharmacologically. Similarly, heteroplasmy has been seen to shift in muscle

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fibers, in which satellite cells fused with postmitotic muscle fibers, increasing the proportion of WT mtDNA and respiratory activity in the tissue as a whole (Taivassalo et al., 1999). These studies collectively demonstrate that it is in fact possible to shift mtDNA heteroplasmy and rescue mitochondrial dysfunction, whether by selecting for nucleoids carrying WT mtDNAs, or against nucleoids carrying mutant mtDNAs. More research on the precise mechanisms of heteroplasmic shifting may yield efficient pharmacological methods to effect changes in mtDNA heteroplasmy and rescue mitochondrial dysfunction. 7. Summary The combination of mtDNA and numerous protein components within a tightly packaged segregating unit provides a mechanism for mtDNA maintenance and propagation, with profound implications for human disease, particularly in mitochondrial genotype-phenotype relationships, beginning at the suborganellar level and cumulatively for the cell as a whole. Although therapeutic methods for mitochondrial dysfunction are lacking, understanding the fundamental organization of mtDNA within the mitochondrion provides essential mechanistic knowledge that will facilitate the development of methods to rescue mitochondrial dysfunction in human disease. Acknowledgements We thank Eric Schon for helpful comments on the manuscript. The experiment shown in Fig. 1B was supported by a Muscular Dystrophy Association Development Grant (MDA3869 to R.W.G.). References Alam TI, Kanki T, Muta T, Ukaji K, Abe Y, Nakayama H, et al. Human mitochondrial DNA is packaged with TFAM. Nucleic Acids Res 2003;31(6):1640–5. Albring M, Griffith J, Attardi G. Association of a protein structure of probable membrane derivation with HeLa cell mitochondrial DNA near its origin of replication. Proc Natl Acad Sci U S A 1977;74(4):1348–52. Bayona-Bafaluy MP, Blits B, Battersby BJ, Shoubridge EA, Moraes CT. Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc Natl Acad Sci U S A 2005;102(40):14392–7. Bender A, Krishnan KJ, Morris CM, Taylor GA, Reeve AK, Perry RH, et al. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet 2006;38(5):515–7. Bogenhagen DF, Rousseau D, Burke S. The layered structure of human mitochondrial DNA nucleoids. J Biol Chem 2008;283(6):3665–75. Boldogh IR, Nowakowski DW, Yang HC, Chung H, Karmon S, Royes P, et al. A protein complex containing Mdm10p, Mdm12p, and Mmm1p links mitochondrial membranes and DNA to the cytoskeleton-based segregation machinery. Mol Biol Cell 2003;14(11):4618–27. Bratic I, Hench J, Henriksson J, Antebi A, Burglin TR, Trifunovic A. Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development. Nucl Acids Res 2009. Chan DC. Mitochondria: dynamic organelles in disease, aging, and development. Cell 2006;125(7):1241–52. Chen H, McCaffery JM, Chan DC. Mitochondrial fusion protects against neurodegeneration in the cerebellum. Cell 2007;130(3):548–62. Chen XJ, Butow RA. The organization and inheritance of the mitochondrial genome. Nat Rev Genet 2005;6(11):815–25. Clayton DA. Replication of animal mitochondrial DNA. Cell 1982;28(4):693–705. Czarnecka AM, Campanella C, Zummo G, Cappello F. Mitochondrial chaperones in cancer: from molecular biology to clinical diagnostics. Cancer Biol Ther 2006;5(7):714–20. D’Aurelio M, Gajewski CD, Lin MT, Mauck WM, Shao LZ, Lenaz G, et al. Heterologous mitochondrial DNA recombination in human cells. Hum Mol Genet 2004;13(24):3171–9. Davidzon G, Mancuso M, Ferraris S, Quinzii C, Hirano M, Peters HL, et al. POLG mutations and Alpers syndrome. Ann Neurol 2005;57(6):921–3. DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med 2003;348:2656–68. Eisenberg I, Novershtern N, Itzhaki Z, Becker-Cohen M, Sadeh M, Willems PH, et al. Mitochondrial processes are impaired in hereditary inclusion body myopathy. Hum Mol Genet 2008;17(23):3663–74. Ekstrand MI, Falkenberg M, Rantanen A, Park CB, Gaspari M, Hultenby K, et al. Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum Mol Genet 2004;13(9):935–44.

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