SEMINARIO SEMANA Nº 10 BIOLOGIA CELULAR Y MOLECULAR FACULTAD DE MEDICINA HUMANA

ADN MITOCONDRIAL. APLICACIONES EN MEDICINA FORENSE. MITOCONDRIOPATÍAS
1) Mencione las diferencias estructurales y funcionales que existen entre la membrana mitocondrial interna y externa. 2) ¿Por qué se dice que las mitocondrias son estructuras dinámicas? 3) ¿Qué ocurre con los componentes mitocondriales después de la fusión? 4) ¿Qué enfermedades son producidas por mutaciones en genes esenciales para la dinámica mitocondrial? 5) ADN mitocondrial y ADN nuclear: semejanzas, diferencias y cómo se heredan. 6) Mitocondriopatías: Características comunes, clasificación. Investigue las bases moleculares de dos enfermedades producidas por estos defectos. 7) ¿Por qué es importante la matriz mitocondrial? 8) Si existe algún daño en las mitocondrias de una persona ¿cuál de los siguientes procesos se verían directamente afectados?, explique brevemente cada uno de los procesos afectados: a. Glucolisis. b. Ciclo de Krebs. c. Transporte de electrones. d. Fosforilación oxidativa. e. Fermentación. 9) ¿Qué utilidad tiene el ADN mitocondrial en la medicina forense? Mencione algunas aplicaciones.

Lecturas:  Detmer S and Chan D. Functions and dysfunctions of mitochondrial dynamics. Nature Reviews Mol Cell Bio 2007; 8: 870-879.  Solano A, Playán A, López-Pérez M y Montoya M. Enfermedades genéticas del ADN mitocondrial humano. Salud Pública de México 2001; 43:151-161.

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Functions and dysfunctions of mitochondrial dynamics
Scott A. Detmer and David C. Chan

Abstract | Recent findings have sparked renewed appreciation for the remarkably dynamic nature of mitochondria. These organelles constantly fuse and divide, and are actively transported to specific subcellular locations. These dynamic processes are essential for mammalian development, and defects lead to neurodegenerative disease. But what are the molecular mechanisms that control mitochondrial dynamics, and why are they important for mitochondrial function? We review these issues and explore how defects in mitochondrial dynamics might cause neuronal disease.
Cristae
Invaginations of the  mitochondrial inner  membrane.

Nebenkern structure
A cytosolic structure, found in  some insect spermatids, that is  formed by the fusion of  mitochondria.

Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. Correspondence to D.C.C. e-mail: dchan@caltech.edu doi:10.1038/nrm2275 Published online   10 October 2007

In the 1950s, seminal electron microscopy studies led to the canonical view of mitochondria as bean-shaped organelles. These studies revealed the ultrastructural hallmarks of mitochondria, which include double lipid membranes and unusual inner membrane folds termed cristae . Recent studies have led to renewed appreciation for the fact that the mitochondrial structure is highly dynamic1,2. Mitochondria have drastically different morphologies depending on the cell type and, even in the same cell, mitochondria can take on a range of morphologies, from small spheres or short rods to long tubules. In fibroblasts, for example, mitochondria visualized with fluorescent proteins or specific dyes typically form tubules with diameters of ~0.5 mm, but their lengths can range from 1–10 mm or more. Even more remarkably, imaging studies in live cells indicate that mitochondria constantly move and undergo structural transitions. Mitochondrial tubules move with their long axes aligned along cytoskeletal tracks3. Individual mitochondria can encounter each other during these movements and undergo fusion, resulting in the merging of the double membranes and the mixing of both lipid membranes and intramitochondrial content (BOX 1). Conversely, an individual mitochondrion can divide by fission to yield two or more shorter mitochondria. What are the molecular mechanisms that underlie these unusual behaviours, and do they have consequences for mitochondrial function and cell physiology? In this Review, we discuss the dynamic nature of mitochondria and summarize the mechanisms that drive mitochondrial fusion and fission. In addition, we discuss recent insights into how these processes affect the function of mitochondria. As well as controlling the

shape of mitochondria, fusion and fission are crucial for maintaining the functional properties of the mitochondrial population, including its respiratory capacity. Moreover, mitochondrial dynamics has key roles in mammalian development, several neurodegenerative diseases and apoptosis.

Mitochondria as dynamic organelles By several criteria, mitochondria are dynamic organelles. First, the shape and size of mitochondria are highly variable and are controlled by fusion and fission. Second, mitochondria are actively transported in cells and they can have defined subcellular distributions. Finally, the internal structure of mitochondria can change in response to their physiological state.
Dynamic shape. The length, shape, size and number of mitochondria are controlled by fusion and fission (FIG. 1a). At steady state, the frequencies of fusion and fission events are balanced 4 to maintain the overall morphology of the mitochondrial population. When this balance is experimentally perturbed, dramatic transitions in mitochondrial shape can occur. Genetic studies in yeast and mammals indicate that cells with a high fusion-to-fission ratio have few mitochondria, and that these mitochondria are long and highly interconnected5–8 (FIG. 2). Conversely, cells with a low fusionto-fission ratio have numerous mitochondria that are small spheres or short rods — these are often referred to as ‘fragmented mitochondria’. In vivo, such changes in dynamics control mitochondrial morphology during development. For example, during Drosophila melanogaster spermatogenesis, many mitochondria synchronously fuse to form the Nebenkern structure, which is required for sperm motility9.
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Box 1 | What happens to mitochondrial components after fusion?

+

Wild type

+

Fusion mutant

In time-lapse movies of labelled mitochondria in living cells, mitochondria are observed to undergo cycles of fusion and fission. With each fusion event, two mitochondria are Nature Reviews | Molecular Cell Biology merged into one. Intuitively, true fusion would be expected to involve outer membrane and inner membrane fusion, which would also result in mixing of the matrix contents. Indeed, these expectations have been experimentally confirmed. Apparent fusion events that have been visualized in cells can be confirmed by a cell-hybrid mitochondrial fusion assay6,32 (see figure). In this assay, mitochondria in two distinct cell lines are differentially labelled with mitochondrially targeted green fluorescent protein (GFP) and DsRed. The cell lines are co-plated onto cover slips and exposed briefly to polyethylene glycol, a chemical that induces adjacent cells to fuse into cell hybrids. After a recovery period, the cell hybrids are examined for mitochondrial fusion. In cell hybrids from normal cells, mitochondrial fusion results in mitochondria that carry both GFP and DsRed (see figure, top). Cells that are defective for mitochondrial fusion form cell hybrids with distinct red and green mitochondria (see figure, bottom). A conceptually similar assay can be performed with yeast cells by allowing labelled yeast strains to mate and form zygotes4. By using matrix-targeted fluorophores, these assays show that mitochondrial fusion results in mixing of the matrix contents. Moreover, by using mitochondrial markers that are localized to the outer or inner membranes, fusion of the individual membranes can be experimentally demonstrated; under normal conditions, outer and inner membrane fusion appear to be closely synchronized. An important question is what happens to mitochondrial DNA (mtDNA) after fusion. Each mitochondrion contains multiple copies of the mtDNA genome that are organized into one or more nucleoids. After fusion, these nucleoids appear to be motile and can potentially interact with each other73. In mammalian cells, mtDNA recombination has been documented, but its extent and importance is unclear.

lines of evidence from neuronal studies suggest that mitochondrial transport is regulated. First, mitochondria are recruited to regions with high energy demands, including active growth cones, presynaptic sites and postsynaptic sites13,16,17. Such recruitment is regulated by neuronal activation, further arguing that the recruitment of mitochondria is responsive to the local metabolic state. Second, neuronal mitochondria pause most often at sites that lack other mitochondria, resulting in a well-spaced axonal mitochondrial distribution14. Third, studies with the membrane-potential indicator dye JC-1 suggest that mitochondria with high membrane potential preferentially migrate in the anterograde direction, whereas mitochondria with low membrane potential move in the retrograde direction14. These migration patterns suggest that active mitochondria are recruited to distal regions with high energy requirements, whereas impaired mitochondria are returned to the cell soma, perhaps for destruction or repair. Finally, mitochondrial transport along axons responds to local concentrations of nerve growth factor (nGF), suggesting that specific signalling pathways control mitochondrial recruitment and retention16,18. Dynamic internal structure. In addition to changes in the overall shape of mitochondria, the internal structures of mitochondria are also dynamic. Three-dimensional tomography of cryopreserved samples has provided new views of inner membrane organization and plasticity19. The inner membrane can be divided into distinct regions: the inner boundary membrane, the cristae membrane and the cristae junctions (FIG. 1c). The inner boundary membrane comprises the regions in which the inner membrane is in close proximity to the outer membrane. These regions are probably important for protein import and might be the sites of coupled outer and inner membrane fusion. The cristae junctions are narrow ‘neck’ regions that separate the inner boundary membrane from the involuted cristae membrane. Cytochrome c, an intermembrane-space protein, is enriched in the space that is encased by cristae membranes, and the regulated opening of cristae junctions might be important for its relocalization during apoptosis20. These regions of the mitochondrial inner membrane are not only morphologically distinct but also appear to constitute separate functional domains. Proteins that are involved in the translocation of proteins through the inner membrane, such as the TIM23 complex, are enriched in the inner boundary membrane, whereas proteins that are involved in oxidative phosphorylation are enriched in the cristae membranes21–23. In addition, the structure of mitochondrial membranes is linked to the metabolic state of mitochondria (FIG. 1c). Purified mitochondria placed in low ADP conditions have limited respiration and have an ‘orthodox’ morphology, characterized by narrow cristae and few cristae junctions per cristae compartment. under high ADP and substrate conditions, isolated mitochondria have high respiratory activity and a ‘condensed’ morphology, characterized by larger cristae and several cristae junctions per cristae compartment19. It is unknown how inner membranes convert between these states, but inner membrane fusion
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Anterograde
The direction from the cell  body towards the periphery.

Retrograde
The direction from peripheral  regions towards the cell body.

Oxidative phosphorylation
A biochemical pathway for ATP  production that results in  oxygen consumption and is  localized to the mitochondrial  cristae.

Dynamic subcellular distribution. Mitochondrial transport is required to distribute mitochondria throughout the cell (FIG. 1b). In most cells, mitochondria are highly motile and travel along cytoskeletal tracks. Mitochondrial transport depends on the actin cytoskeleton in budding yeast10 and on both actin and microtubules in mammalian cells3,11,12. Depending on the cellular context, these transport processes can ensure proper inheritance of mitochondria or can recruit mitochondria to active regions of the cell. For example, in budding yeast, mitochondria are transported into and retained in the developing bud to ensure mitochondrial inheritance to the daughter cell10. This regulation of mitochondrial distribution is particularly evident in neurons. Quantitative measurements of neuronal mitochondrial transport have reported rates ranging from 0.4 mm min–1 (ReF. 13) to 0.1–1 mm sec–1 (ReFs 11,14,15). Such directed movements are not continuous; rather, they are saltatory, with pauses often followed by a reversal of direction. These patterns might reflect the attachment and detachment of cytoskeletal motors. Although these movements can appear chaotic, several

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a
Fusion + Fission

b
Anterograde movement Nucleus

member of the mitofusin family of GTPases. The yeast orthologue, Fzo1, has a conserved role in mitochondrial fusion24, and genetic screens in yeast have identified additional modulators of mitochondrial fusion and fission2,25 (FIG. 3b). The core machineries that mediate mitochondrial fusion and fission are best understood in yeast. Several of these components have functionally conserved mammalian homologues. More comprehensive discussions of the molecular mechanisms of mitochondrial fusion and fission have been presented in recent reviews (for example, see ReF. 1). Mitochondrial fusion. In yeast, the core mitochondrial fusion machinery consists of two GTPases: Fzo1 and Mgm1 (FIG. 3). Fzo1 is located on the mitochondrial outer membrane and is essential for fusion of the outer membranes24,26. The mammalian homologues of Fzo1 are the mitofusins MFn1 and MFn2. These two related proteins form homo-oligomeric and hetero-oligomeric complexes that are essential for fusion6,27,28. Mitofusins are required on adjacent mitochondria during the fusion process, implying that they form complexes in trans between apposing mitochondria26,29. A heptad repeat region of MFn1 has been shown to form an antiparallel coiled  coil that is probably involved in tethering mitochondria during fusion29. Mgm1 is a dynamin-related protein that is essential for fusion of the mitochondrial inner membranes in yeast30, a function that is consistent with its localization to the intermembrane space and its association with the inner membrane. The mammalian orthologue oPA1 is also essential for mitochondrial fusion28,31. In yeast, the outer membrane protein ugo1 physically links Fzo1 and Mgm1, but no mammalian orthologue has yet been discovered2. The membrane potential across the mitochondrial inner membrane is maintained by the electron transport chain and is essential for mitochondrial fusion26,32. Ionophores that dissipate the mitochondrial membrane potential cause mitochondrial fragmentation, owing to an inhibition of mitochondrial fusion32,33. In an in vitro fusion assay, both the proton and the electrical gradient components of the membrane potential are important26. The mechanistic link between membrane potential and fusion remains to be resolved, but one factor appears to be the dependence of post-translational processing of oPA1 on the membrane potential34. Recent work has also identified mitochondrial lipids as important factors in fusion. Mitochondrial morphology screens in yeast identified members of the ergosterol  synthesis pathway as being required for normal mitochondrial morphology35,36. Recently, mitochondrial phospholipase D has been identified as a protein that is important for mitochondrial fusion37. This mitochondrial outer membrane enzyme hydrolyses cardiolipin to generate phosphatidic acid. Interestingly, ergosterol has been associated with yeast vacuole fusion38, and phosphatidic acid is thought to play a part in generating the membrane curvature that is required for sNARe-mediated fusion39. Thus, specific lipids might have similar roles in distinct types of membrane fusion.
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Retrograde movement

c
IMS CJ IBM CM ‘Condensed’ morphology ‘Orthodox’ morphology OM IM

Low [ADP] High [ADP]

Figure 1 | mitochondria as dynamic organelles. a | Mitochondrial fusion and fission control mitochondrial number and size. With fusion, Nature Reviews | Molecular Cell Biology two mitochondria become a single larger mitochondrion with continuous outer and inner membranes. Conversely, a single mitochondrion can divide into two distinct mitochondria by fission. b | In mammalian systems, mitochondria are distributed throughout the cytoplasm by active transport along microtubules and actin filaments. Distinct molecular motors transport the mitochondria in anterograde or retrograde directions. c | Inner membrane dynamics. The diagram indicates the different regions of the inner membrane. The bottom panels show electron microscopy (EM) tomograms of two mitochondria under different metabolic conditions (red, outer membrane; yellow, inner boundary membrane; green, cristae membrane). Cristae organization can vary widely, often in response to the bioenergetic state of the cell: an ‘orthodox’ cristae morphology, with narrow cristae and few cristae junctions per cristae compartment, is found in low ADP conditions, whereas a ‘condensed’ morphology, with larger cristae and several junctions per cristae compartment, is found in high ADP conditions19. EM images reproduced with permission from ReF. 19  (2006) Elsevier. CJ, cristae junction; CM, cristae membrane; IBM, inner boundary membrane; IM, inner membrane; IMS, intermembrane space; OM, outer membrane.

Coiled coil
A structural motif that is  formed by polypeptide  sequences that contain  hydrophobic heptad repeats.

and fission might be involved19. Taken together, these observations indicate that inner membrane morphology is intimately related to bioenergetics, although the causal relationship remains unclear.

Dynamin
A large GTPase that is thought  to mediate vesicle scission  during endocytosis.

Mediators of fusion and fission Molecular analysis of mitochondrial morphology began with the discovery in 1997 of the D. melanogaster fusion factor fuzzy onions (FZo), a mitochondrial outer membrane GTPase that is required for the fusion of mitochondria during spermatogenesis9. FZo is the founding

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No fusion Mfn-null Wild type No fission DRP1 K38A

Figure 2 | mitochondrial fusion and fission regulate morphology. Mitochondrial length, size and connectivity are determined by theNature Reviews | Molecular Cell Biology relative rates of mitochondrial fusion and fission. In wild-type cells (shown in the central panel), mitochondria form tubules of variable length. In the absence of mitochondrial fusion (for example, in mitofusin (Mfn)null cells (shown in the left panel), which lack MFN1 and MFN2), unopposed fission results in a population of mitochondria that are all fragmented. Conversely, decreased fission relative to fusion (for example, in DRP1 K38A cells (shown in the right panel), which have a dominant-negative form of dynamin-related protein-1 (DRP1)) results in elongated and highly interconnected mitochondria. Scale bar represents 10 mm.

Mitochondrial membrane potential
The electrochemical gradient  that exists across the  mitochondrial inner  membrane.

Ergosterol
A steroid compound that is a  component of yeast cell  membranes and which might  have a role similar to that of  cholesterol in mammalian cell  membranes.

SNARE
(soluble N‑ethylmaleimide‑ sensitive fusion protein (NsF)  attachment protein (sNAP)  receptor). A highly α‑helical  protein that mediates the  specific fusion of vesicles with  target membranes. 

F-box protein
A protein containing an F‑box  motif, a small domain that is  used for protein interactions.  The best‑characterized F‑box  proteins are components of an  e3 ubiquitin ligase, and help in  ubiquitin‑dependent protein  degradation by recognizing  specific substrates.

Mitochondrial fission. Mitochondrial fission requires the recruitment of a dynamin-related protein (Dnm1 in yeast and DRP1 in mammals) from the cytosol (FIG. 4). Both Dnm1 and DRP1 assemble into punctate spots on mitochondrial tubules, and a subset of these complexes lead to productive fission events5,7,8. By analogy with the classical function of dynamin in endocytosis, Dnm1 and DRP1 are thought to assemble into rings and spirals that encircle and constrict the mitochondrial tubule during fission25. Consistent with this model, purified Dnm1 can indeed form helical rings and spirals in vitro, with dimensions that are similar to those of constricted mitochondria40. Moreover, Dnm1 assembly is required for fission activity41. The recruitment of Dnm1 to yeast mitochondrial fission sites involves three other components. one of these is Fis1, a mitochondrial integral outer membrane protein that is essential for fission42–44. Fis1 binds indirectly to Dnm1 through one of two molecular adaptors, Mdv1 or Caf4 (ReF. 45) (FIG. 4b). Either Mdv1 or Caf4 is sufficient to allow the Fis1-dependent recruitment of Dnm1, although Mdv1 has a more important role in mediating fission. FIS1, the mammalian homologue of Fis1, is also essential for mitochondrial fission46, but no homologues of Mdv1 or Caf4 are currently known. FIS1 and DRP1 are also required for the fission of peroxisomes47,48.

This modification of DRP1 might affect its association with mitochondrial membranes. Mitochondrial fission is also regulated by the cell cycle. For example, mitochondria in Hela cells are usually tubular, but they become more fragmented during mitosis, a phenomenon that might facilitate the partitioning of mitochondria to daughter cells during cytokinesis. This regulated fragmentation of mitochondria is due to increased mitochondrial fission, and phosphorylation of DRP1 during mitosis has been implicated55. In addition to the genes that encode core fusion and fission components, other genes can affect mitochondrial morphology. large-scale visual screens for aberrant mitochondrial morphology in mutant yeast have yielded numerous genes of interest and provided general insights into the control of mitochondrial morphology35,36. These screens suggest that several cellular pathways influence mitochondrial morphology and inheritance, including ergosterol biosynthesis, mitochondrial protein import, actin dynamics, vesicular fusion and ubiquitin-mediated protein degradation. The close interplay between mitochondrial protein import and morphology has been emphasized by the recent finding that the mitochondrial morphology genes MMM1, MDM10 and MDM12 have a direct role in the assembly of β‑barrel proteins in the outer mitochondrial membrane56. Proteins that are required for mitochondrial transport. Energy-dependent molecular motors transport mitochondria along cytoskeletal filaments. Along microtubules, multiple kinesin family members and cytoplasmic dynein have been implicated in anterograde and retrograde mitochondrial transport, respectively3. Recent work has clarified the linkage between mitochondria and kinesin-1. Genetic screens in D. melanogaster identified milton and Miro, both of which are required for anterograde mitochondrial transport in neurons57,58. Milton interacts directly with kinesin and Miro, which is a mitochondrial outer membrane protein that has GTPase and Ca2+-binding eF‑hand domains59. In yeast, disruption of the Miro orthologue Gem1 results in abnormalities in mitochondrial morphology and poor respiratory activity60. Both GTP-binding and Ca2+-binding motifs are essential for Gem1 function, which appears not to be involved in fusion or fission. Depending on the cell type, mitochondria can also travel along actin filaments under the control of myosin motors3. Proteins that mediate inner membrane morphology. Studies of mitochondrial inner membrane structure are complicated by the intimate link between mitochondrial bioenergetics and cristae structure. As a result, disruption of the proteins that are important for bioenergetics can lead to a secondary effect on inner membrane structure. nevertheless, several proteins probably have a specific role in controlling cristae structure. In addition to their roles in mitochondrial fusion, Mgm1 and oPA1 are important for cristae structure. loss of Mgm1 in yeast or knockdown of oPA1 in mammalian cells results in disorganized inner membrane structures30,61–64. In both cases, homo-oligomeric interactions are involved30,64.
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β-barrel protein
A protein composed of a   β‑sheet that is rolled up   into a cylinder. One such  mitochondrial β‑barrel protein  is VDAC (voltage‑dependent  anion channel), which forms a  pore in the outer membrane.

Kinesin
A microtubule‑based molecular  motor protein that is most  often directed towards the plus  end of microtubules. 

Other regulators of dynamics Mitochondrial fusion and fission activities are probably coordinated with cellular physiology. In yeast, the steadystate levels of Fzo1 are controlled by the F‑box protein Mdm30, which negatively regulates Fzo1 levels in a proteasome-independent manner49,50. In mammalian cells, post-translational modification of DRP1 regulates its function in mitochondrial fission. The mitochondrial E3 ubiquitin ligase MARCH5 is essential for mitochondrial fission51. This requirement is probably related to the ability of MARCH5 to promote DRP1 ubiquitylation and to associate physically with ubiquitylated DRP1 (ReFs 52,53). Furthermore, during apoptosis, sumoylation of DRP1 is activated in a BAX- and/or BAK-dependent manner54.

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a
OM fusion IM fusion

b

Fzo1

mitofilin in mammalian cells causes dramatic abnormalities of the cristae, resulting in the formation of complex layers of inner membrane69. Depletion of Mmm1, Mdm31 and Mdm32 — yeast proteins implicated in mitochondrial (mt) DNA maintenance — also cause aberrant cristae morphologies70,71.

Ugo1 s-Mgm1 I-Mgm1 IMS OM

IM

Cristae

Dynein
A microtubule‑based molecular  motor that is directed towards  the minus end of microtubules.

EF-hand domain
A helix‑loop‑helix protein motif  that can bind a Ca2+ ion.

Figure 3 | mitochondrial fusion. a | Mitochondrial fusion consists of outer membrane (OM) fusion followed by inner membrane (IM) fusion. Normally these events occur Nature Reviews | Molecular Cell Biology coordinately. b | The dynamin-related proteins Fzo1 and Mgm1 are key molecules in the yeast mitochondrial fusion machinery. Fzo1 is an integral outer membrane protein with GTPase and heptad repeat domains that face the cytoplasm. All of the domains are required for the fusion activity of Fzo1. Mgm1 is present on the inner membrane, facing the intermembrane space (IMS), and is proteolytically processed by a rhomboid protease. Both long (l-Mgm1) and short (s-Mgm1) forms are required for mitochondrial fusion. In addition to inner membrane fusion, Mgm1 is required for the maintenance of cristae structures. Ugo1 binds to both Fzo1 and Mgm1 and probably coordinates their function. All components are encoded by nuclear DNA. The mitofusin proteins MFN1 and MFN2 are the mammalian homologues of Fzo1; OPA1 is the mammalian homologue of Mgm1. No mammalian homologue of Ugo1 has been identified so far.

Biological functions of mitochondrial dynamics Why do mitochondria continually fuse and divide? Recent studies show that these processes have important consequences for the morphology, function and distribution of mitochondria. First, fusion and fission control the shape, length and number of mitochondria. The balance between these opposing processes regulates mitochondrial morphology. Second, fusion and fission allow mitochondria to exchange lipid membranes and intramitochondrial content. Such exchange is crucial for maintaining the health of a mitochondrial population. Third, the shape of mitochondria affects the ability of cells to distribute their mitochondria to specific subcellular locations. This function is especially important in highly polarized cells, such as neurons. Finally, mitochondrial fission facilitates apoptosis by regulating the release of intermembrane-space proteins into the cytosol. As a result of these cellular functions, mitochondrial dynamics has consequences for development, disease and apoptosis.
Maintaining a healthy mitochondrial population. Mitochondrial fusion is required to maintain a functional mitochondrial population in the cell. Fibroblasts that lack both MFn1 and MFn2 have reduced respiratory capacity, and individual mitochondria show great heterogeneity in shape and membrane potential28. Cells that lack oPA1 show similar defects, with an even greater reduction in respiratory capacity. How does fusion protect mitochondrial function? It is probable that a primary function of mitochondrial fusion is to enable the exchange of contents between mitochondria (BOX 1). As a result, mitochondria should not be considered autonomous organelles; instead, the hundreds of mitochondria in a typical cell exist as a population of organelles that cooperate with each other through fusion and fission. The heterogeneous properties of mitochondria in fusion-deficient cells are consistent with this model28. In normal cells, a few mitochondria might be non-functional owing to the loss of essential components. However, this dysfunction is transient because mitochondrial fusion provides a pathway for these defective mitochondria to regain essential components (FIG. 5a). An essential component of mitochondrial function is mitochondrial DnA (mtDnA), which is organized into compact particles termed nucleoids. The mtDnA genome encodes essential subunits of the respiratory complexes I, III and Iv, and is therefore essential for oxidative phosphorylation. When mitochondrial fusion is abolished, a large fraction of the mitochondrial population loses mtDnA nucleoids72. During mitochondrial division in normal cells, most daughter mitochondria inherit at least one mtDnA nucleoid73. However, in cases where a
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Mitochondrial F1F0 ATP synthase
A large, multisubunit enzyme  embedded in the  mitochondrial cristae that uses  the proton gradient across the  inner membrane to synthesize  ATP.

Mitochondrial DNA
(mtDNA). A circular genome  (~16 kb in mammals) located  in the mitochondrial matrix  that encodes 13 polypeptides  of the electron transport chain,  22 tRNAs and 2 rRNAs.

Nucleoid
A compacted mass of DNA.  Mitochondrial DNA is  organized into nucleoids, each  consisting of several  mitochondrial genomes.

Mitochondrial  F 1FoATP  synthase , a rotary enzyme embedded in the inner membrane that couples proton pumping to ATP synthesis, is essential for normal cristae structure65. This role in inner membrane structure involves a dimeric form of ATP synthase that contains the additional subunits e and g. As visualized by electron microscopy, the ATP synthase dimer has a dimeric interface with a sharp angle that could distort the local lipid membrane. This distortion might contribute to the high membrane curvature that characterizes cristae tubules66,67. Mgm1 is required for the oligomerization of ATP synthase, providing a link between these two modulators of cristae structure63. Additional proteins modulate inner membrane dynamics. In yeast, Mdm33 is required for normal mitochondrial morphology and its overexpression leads to septation and vesiculation of the inner membranes68. Because of these phenotypes, Mdm33 has been suggested to have a role in inner membrane fission. Knockdown of

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a
Dnm1

b
Dnm1

Adaptor poteins (Mdv1, Caf4)

Fis1 OM IM

Figure 4 | mitochondrial fission. a | In yeast, mitochondrial fission is mediated by the dynamin-related protein Dnm1. Cytoplasmic Dnm1Nature Reviews | mitochondrial outer localizes to the Molecular Cell Biology membrane (OM), where it oligomerizes into a ring structure that constricts and severs the mitochondrion. In this model, Dnm1 functions in an analogous manner to the way dynamin functions in endocytosis. b | The localization of Dnm1 on the mitochondrial outer membrane is mediated by Fis1 and the adaptor proteins Mdv1 and Caf4. Fis1 is an integral outer membrane protein that interacts with the N termini of Mdv1 and Caf4. Both Mdv1 and Caf4 have C-terminal WD-40 repeats that bind Dnm1. Fis1 and Dnm1 have mammalian homologues (FIS1 and DRP1, respectively), but no Mdv1 or Caf4 homologues have been identified so far. IM, inner membrane.

daughter fails to inherit a nucleoid, mitochondrial fusion would enable restoration of mtDnA. In fusion-deficient cells, the lack of content exchange prevents restoration of mtDnA nucleoids and probably accounts for the heterogeneity in membrane potential and the reduced respiratory capacity. It should be noted that fusiondeficient cells still maintain significant numbers of mtDnA nucleoids; however, due to ongoing mitochondrial fission, these nucleoids are encased by a small mitochondrial mass, and therefore the functional mitochondrial mass (at least in terms of bioenergetics) in such cells is greatly reduced (FIG. 5b). In addition to mtDnA, it is also possible that other components, such as substrates, metabolites or specific lipids, can be restored in defective mitochondria by fusion. Further studies will determine whether content exchange is the primary function of mitochondrial fusion. The importance of mitochondrial fusion in development and disease might be a consequence of this function. Essential developmental functions. Perturbations in mitochondrial dynamics result in specific developmental defects. Mice that lack either MFn1, MFn2 or oPA1 fail to survive past mid-gestation6,74,75. MFn2 has a highly specific function in the development of the trophoblast giant cell layer of the placenta6. likewise, MFn1 appears to have an essential placental function72. Mitochondrial fission is also an essential process. Worms that are deficient in mitochondrial division die before adulthood76. An infant patient with a dominantnegative DRP1 allele has been reported. This patient died at ~1 month of age and had a wide range of abnormalities, including reduced head growth, increased lactic acid and optic atrophy. Fibroblasts from this patient showed elongated mitochondria and peroxisomes 77. It is unclear how the developmental defects are related to these organellar shape changes.

Mitochondrial distribution and recruitment in neurons. Given the importance of mitochondrial dynamics in maintaining bioenergetics, these dynamics are probably a ubiquitous phenomenon that is important for all cells. However, certain cells, particularly neurons, seem to be especially dependent on its proper control. This dependence of neurons probably stems from their high energy demands and the special importance of proper mitochondrial distribution: mitochondria are concentrated in several neuronal regions, including pre- and postsynaptic sites13,17. To achieve this non-uniform distribution, neurons rely heavily on active transport to recruit mitochondria and other organelles to nerve terminals3. The proper localization of mitochondria to axon terminals depends on mitochondrial dynamics. neurons that lack Milton, Miro or DRP1 show defective mitochondrial transport and have sparse mitochondria at axon terminals. Such distribution defects lead to reduced capacity for synaptic transmission57,58,78. It seems likely that mitochondria that are localized to synapses are primarily required to drive ATP-dependent processes. The synapses of neurons that express mutant DRP1 show defective mobilization of the reserve vesicle pool (an ATPdependent process), and the defects in synaptic transmission can be rescued by experimentally filling synapses with ATP. In addition, synapse-localized mitochondria help to regulate Ca2+ homeostasis, although this function appears to be crucial only during intense synaptic activity. Synapses that lack Miro or DRP1 have elevated resting Ca2+ levels, but normal Ca2+ dynamics are maintained except under sustained nerve stimulation57,78. Both mitochondrial fusion and fission affect the mitochondrial distribution in dendrites. In hippocampal neurons, mitochondria accumulate at dendritic spines following neuronal stimulation13. Inhibition of mitochondrial fission causes elongation of the mitochondria and decreases the abundance of dendritic mitochondria and the density of dendritic spines. Conversely, increased fission facilitates the mobilization of dendritic mitochondria and leads to an increased spine number13. In the cerebellum, the distribution of mitochondria in the dendritic processes of Purkinje neurons is highly dependent on mitochondrial fusion72 (see below). Lymphocyte chemotaxis. Mitochondrial dynamics appears to be important for proper mitochondrial redistribution in lymphocytes during chemotaxis 79. Mitochondria are concentrated in the trailing edge in lymphocyte cell lines that migrate in response to chemical attractants. Modulation of mitochondrial fusion or fission affects both mitochondrial redistribution and cell migration. Fragmentation enhances mitochondrial redistribution and cell migration, whereas conditions that promote fusion have the opposite effect. Therefore, as in neurons, mitochondrial shape in lymphocytes can affect the recruitment of mitochondria to local cellular areas. Regulation of apoptosis. In apoptosis, several structural changes occur in mitochondria during the early phase of cell death (FIG. 6). The mitochondria become fragmented owing to increased fission activity. At approximately the
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Chemotaxis
The directed movement of cells  in response to a chemical  stimulus.

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a Wild-type cells b Fusion-deficient cells

Fusion

Defective mitochondrion

Fission

Rescued mitochondrion

Figure 5 | mitochondrial dynamics protects mitochondrial function. a | In wild-type Nature Reviews | Molecular Cell Biology cells, the vast majority of mitochondria are functional (shown in green). In this simplified diagram, one mitochondrion is depicted as non-functional (shown in orange). One of several possible reasons for dysfunction is a lack of mitochondrial DNA (mtDNA) nucleoids (shown as black circles). The dysfunctional mitochondrion can regain its function and mtDNA by fusing with a neighbouring mitochondrion. The fused mitochondrion then undergoes fission, with both daughter mitochondria receiving mtDNA nucleoids. It should be noted that the identities of the daughter mitochondria are distinct from the parental mitochondria, owing to content exchange and the fact that the fission point is typically distinct from the fusion point. b | In fusion-deficient cells, mitochondria are fragmented due to ongoing fission in the absence of fusion. Mitochondria that lack mtDNA nucleoids accumulate because there is no pathway for defective mitochondria to regain mtDNA. Fusion-deficient cells can maintain mtDNA nucleoids, but such nucleoids serve a much smaller mitochondrial mass.

can occur in the absence of mitochondrial fission. An important issue to resolve in future studies is how fission is related to the permeabilization of mitochondria. Surprisingly, the apoptotic proteins BAX and BAK, which have well-established pro-apoptotic roles in mitochondrial membrane permeabilization, also appear to regulate mitochondrial morphology. BAX and BAK double-knockout cells have fragmented mitochondria due to reduced mitochondrial fusion87, although the extent of this effect depends on the experimental system88. little is known about how BAX and BAK mediate their effects on mitochondrial morphology, but BAX influences MFn2 distribution on the mitochondrial outer membrane87 and BAK associates with MFn1 and MFn2 (ReF. 88). In conjunction with MoMP, remodelling of the cristae membranes is required for the rapid and efficient release of cytochrome c20,89. Most cytochrome c is localized to cristae compartments20; oPA1 appears to regulate the diameter of cristae junctions and therefore regulates cytochrome c release 64,90. overexpression of oPA1 blocks cytochrome c release following the induction of apoptosis by maintaining narrow cristae junctions64. DRP1 has also been proposed to play a part in cristae remodelling during apoptosis91.

Role in human disease Several human diseases are caused by mutations in genes that are essential for mitochondrial dynamics (TABLe 1). Each of these diseases causes degeneration of specific nerves, reinforcing the notion that neurons are particularly prone to defects in mitochondrial dynamics.
OPA1 and autosomal dominant optic atrophy. Heterozygous mutations in oPA1 cause autosomal dominant optic atrophy (ADoA), the most common heritable form of optic neuropathy92,93. This disease is characterized by the degeneration of retinal ganglion cells, the axons of which form the optic nerve. More than 100 pathogenic oPA1 mutations have been reported, with most occurring in the GTPase domain94. Half of the mutants encode a truncated protein owing to a nonsense mutation. A few nonsense mutations abolish nearly the entire coding sequence, suggesting that haploinsufficiency of oPA1 can cause ADoA. It remains possible that other, less severe, truncations might have dominant-negative activity. How these oPA1 mutations cause the clinical symptoms of ADoA remains to be clarified. non-neuronal cells from patients with ADoA can have aggregated, fragmented or normal mitochondria93,95; however, because data from only a few patients have been reported, it is not clear whether these findings are the norm. In addition, oPA1 mutations have been associated with reduced ATP production and reduced mtDnA content96,97. The defects that have been documented in human ADoA diseased tissue are not as severe as those observed in experimental cells in which oPA1 is depleted. Fibroblasts that are deficient for oPA1 have fragmented mitochondria, defects in respiration, aberrant cristae structure and increased susceptibility to apoptosis28,31,62,98.
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Mitochondrial outer membrane permeabilization
(MOMP). The opening of pores  in the mitochondrial outer  membrane — an early event  during apoptosis that releases  apoptotic factors from the  mitochondrial intermembrane  space.

Haploinsufficiency
A genetic state in diploids in  which a single functional copy  of a gene is insufficient to  maintain a normal phenotype.

same time, mitochondrial outer membrane permeabilization (MoMP) causes the release of contents of the intermembrane space, such as cytochrome c and second mitochondria-derived activator of caspase (SMAC)/ Diablo, into the cytoplasm. Because cytochrome c is preferentially sequestered in cristae compartments, it is thought that the opening of cristae junctions is a vital step in facilitating its efficient release. once in the cytosol, cytochrome c activates a cascade of caspases that propagate and execute the apoptotic programme. These three structural changes — fragmentation, MoMP and cristae remodelling — occur at similar times, but their temporal sequence and causative links are still controversial80,81. Mitochondrial fragmentation during apoptosis is associated with dynamic changes in the mitochondrial localization of several proteins, including BAX, BAK, MFn2, endophilin and DRP1 (ReF. 81). Inhibition of fission activity blocks mitochondrial fragmentation, reduces cytochrome c release and can reduce or delay cell death depending on the experimental system46,82,83. In Caenorhabditis elegans and D. melanogaster, disruption of DRP1 reduces the number of cell deaths84–86. In multiple systems, it seems that fission is important for rapid and efficient cell death, although apoptosis

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Cytochrome c

Apoptotic stimuli

Cristae junctions

Figure 6 | mitochondrial dynamics during apoptosis. At an early stage of apoptosis, Nature Reviews | Molecular Cell Biology three structural changes occur in mitochondria. Fragmentation takes place as a result of increased fission mediated by dynamin-related protein-1 (DRP1) and the mitochondrial fission-1 protein (FIS1). Mitochondrial outer membrane permeabilization (MOMP; indicated by dashed outlines) is induced by the pro-apoptotic BCL2-family members BAX and BAK. MOMP enables the release of cytochrome c (shown as red dots) and other soluble proteins from the intermembrane space. However, release of cytochrome c is efficient only if the cristae junctions are widened to allow escape from the cristae compartments. The dynamin-related proteins OPA1 and DRP1 have been implicated in cristae remodelling.

Mouse models of ADoA that contain oPA1 mutations develop the features of ADoA in an age-dependent manner74,75. Heterozygous mice show a progressive decline in retinal ganglion cell number and aberrations of axons in the optic nerve. Mice that are homozygous for the oPA1 mutation die at mid-gestation74,75, which is consistent with an essential requirement for mitochondrial fusion during embryonic development6. MFN2 and Charcot-Marie-Tooth 2A. Charcot-MarieTooth (CMT) disease, one of the most common hereditary neuropathies, is caused by mutations in at least 30 different genes99. Affected individuals have progressive distal motor and sensory impairments that start in the feet and hands as a result of the degeneration of the long peripheral nerves. Depending on the type of CMT, these diseases are caused by either a primary defect in the Schwann cells that myelinate the peripheral nerves or by a defect in the neurons themselves99. CMT2A is an axonopathy that is caused by the latter type of defect, and it has been associated with >40 mutations in MFn2. nearly all of these disease alleles contain missense mutations or short, in-frame deletions100. Most mutations cluster in or near the GTPase domain, but some also

Sural nerve
A sensory nerve innervating  the calf and foot that is  commonly investigated by  biopsy for the evaluation of  peripheral neuropathies.

Table 1 | Disorders associated with mitochondrial perturbations
Disease
CMT2A ADOA CMT4A Unnamed

mitochondrial function
Fusion Fusion Fission? Fission

gene
MFN2 OPA1 GDAP1 DRP1

Description
Autosomal dominant peripheral neuropathy Autosomal dominant optic atrophy (ADOA) Autosomal recessive peripheral neuropathy Neonatal lethality

CMT, Charcot-Marie-Tooth; DRP1, dynamin-related protein-1; GDAP1, ganglioside-induced differentiation-associated protein-1; MFN2, mitofusin-2; OPA1, optic atrophy-1.

occur in each of the heptad repeat domains of MFn2. In addition to the loss of peripheral nerve function, a subset of patients with CMT2A have optic atrophy, suggesting that oPA1 and MFn2 mutations can lead to overlapping clinical outcomes101,102. Because of the difficulties in studying nerve tissue from patients, the pathogenic mechanisms that lead to peripheral nerve degeneration in CMT2A are not well understood. only one study has reported ultrastructural defects in mitochondria from the nerves of patients with CMT2A. Mitochondria in the sural nerve of two patients showed structural aberrations in their outer and inner membranes, along with swelling that is suggestive of mitochondrial dysfunction103. Aggregation of mitochondria was also observed. Interestingly, CMT2A alleles of MFN2 (ReFs 27,104) cause mitochondrial aggregation and subsequent mitochondrial transport defects in neurons104. However, the mitochondrial aggregation phenotype is dependent on significant overexpression27, and therefore its relevance to disease pathogenesis remains to be clarified. Several perplexing issues remain to be resolved concerning the molecular genetics of CMT2A. How does mutation of one copy of MFN2 lead to disease? Why are long peripheral neurons selectively affected, given that MFn2 is a broadly expressed protein? Clues to these issues have come from analysis of CMT2A alleles in mice27. Many CMT2A alleles of Mfn2 are non-functional for fusion when expressed alone. However, the fusion activity of these non-functional alleles can be efficiently complemented by wild-type MFn1 (but not MFn2). This complementation is due to the ability of MFn1 and MFn2 to form hetero-oligomeric complexes that are functional for fusion. In a patient with CMT2A, therefore, cells that express MFn1 are protected from gross loss of fusion activity. By contrast, cells with little or no MFn1 expression suffer a greater relative loss of fusion activity. In part, these properties of the CMT2A alleles might underlie the selective loss of sensory and motor neurons. Consistent with this model, MFn2 seems to be more highly expressed in central and peripheral nervous tissue than MFn1 (S.A.D. and D.C.C., unpublished observations). Even in the peripheral nerves, it appears that mitochondrial fusion defects are only partial because only the longest nerves are affected. Most probably, the extreme dimensions of the long peripheral nerves make them most vulnerable to changes in mitochondrial dynamics. How might perturbations in mitochondrial fusion lead to neurodegeneration? Clues to the pathogenic mechanisms have come from the finding that mice that lack MFn2 show highly specific degeneration of Purkinje neurons in the cerebellum, resulting in cerebellar ataxia72. Purkinje cells are the sole efferent neurons of the cerebellum, and they have exquisitely formed dendritic processes. Both developing and mature Purkinje cells that lose MFn2 fail to support dendritic outgrowth, particularly that of dendritic spines, which are the sites of synaptic connections. In normal Purkinje cells, abundant tubular mitochondria reside in dendritic processes. By contrast, mutant Purkinje cells have fragmented mitochondria that fail to distribute effectively along dendritic
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processes. In addition, the Purkinje cells show a loss of respiratory activity, probably owing to an accumulation of mitochondria that lack mtDnA nucleoids. Therefore, loss of mitochondrial fusion in Purkinje neurons impairs respiratory activity and mitochondrial localization. GDAP1 and Charcot-Marie-Tooth 4A. Another form of CMT is associated with defects in mitochondrial dynamics. Ganglioside-induced differentiation-associated protein-1 (GDAP1) is mutated in CMT4A, one of the few recessive forms of CMT disease. CMT4A has both demyelinating and axonal features and, consistent with this mixed clinical presentation, GDAP1 is expressed in both Schwann cells and neurons105. GDAP1 is an integral outer membrane protein that probably affects mitochondrial division105. Disease alleles either fail to localize to mitochondria or are defective in stimulating mitochondrial fission when overexpressed105. If GDAP1 disease alleles disrupt normal mitochondrial fission, they might cause mitochondrial distribution defects similar to those that are induced by the DRP1 mutations discussed above13,78.
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Perspectives The study of mitochondrial dynamics has undergone great advances in the past few years. It is now clear that mitochondrial dynamics is important for the functional state of mitochondria. By enabling content exchange between mitochondria, fusion and fission prevent the accumulation of defective mitochondria. These opposing processes also control mitochondrial shape, which affects the distribution of mitochondria as well as their participation in apoptosis. As a result, mitochondrial dynamics is particularly important in cells and tissues that have a special dependence on mitochondrial function. Defects in mitochondrial dynamics can manifest in mammalian development, apoptosis and disease. As our knowledge of mitochondrial dynamics increases, we can expect to learn about its involvement in other processes. The link between defects in mitochondrial fusion and neurodegenerative disease is particularly intriguing. In future studies, the pathophysiological mechanisms that underlie neurodegenerative diseases such as ADoA and CMT2A will hopefully be further dissected in appropriate animal models.
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Structure of dimeric mitochondrial ATP synthase: novel F0 bridging features and the structural basis of mitochondrial cristae biogenesis. Proc. Natl Acad. Sci. USA 102, 12356–12358 (2005). 67.  Dudkina, N. V., Heinemeyer, J., Keegstra, W., Boekema, E. J. & Braun, H. P. Structure of dimeric ATP synthase from mitochondria: an angular association of monomers induces the strong curvature of the inner membrane. FEBS Lett. 579, 5769–5772 (2005). 68.  Messerschmitt, M. et al. The inner membrane protein Mdm33 controls mitochondrial morphology in yeast. J. Cell Biol. 160, 553–564 (2003). 69.  John, G. B. et al. The mitochondrial inner membrane protein mitofilin controls cristae morphology. Mol. Biol. Cell 16, 1543–1554 (2005). 70.  Hobbs, A. E., Srinivasan, M., McCaffery, J. M. & Jensen, R. E. Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability. J. Cell Biol. 152, 401–410 (2001). 71.  Dimmer, K. S., Jakobs, S., Vogel, F., Altmann, K. & Westermann, B. Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast. J. Cell Biol. 168, 103–115 (2005). 72.  Chen, H., McCaffery, J. M. & Chan, D. C. Mitochondrial fusion protects against neurodegeneration in the cerebellum. Cell 130, 548–562 (2007). This study showed that mitochondrial fusion is essential for respiratory function and that it protects neurons in the cerebellum from degeneration — findings that will probably be relevant for understanding CMT2A pathogenesis. 73.  Legros, F., Malka, F., Frachon, P., Lombes, A. & Rojo, M. Organization and dynamics of human mitochondrial DNA. J. Cell Sci. 117, 2653–2662 (2004). 74.  Alavi, M. V. et al. A splice site mutation in the murine Opa1 gene features pathology of autosomal dominant optic atrophy. Brain 130, 1029–1042 (2007). 75.  Davies, V. J. et al. Opa1 deficiency in a mouse model of autosomal dominant optic atrophy impairs mitochondrial morphology, optic nerve structure and visual function. Hum. Mol. Genet. 16, 1307–1318 (2007). 76.  Labrousse, A. M., Zappaterra, M. D., Rube, D. A. & van der Bliek, A. M. C. elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane. Mol. Cell 4, 815–826 (1999). 77.  Waterham, H. R. et al. A lethal defect of mitochondrial and peroxisomal fission. N. Engl. J. Med. 356, 1736–1741 (2007). This study provided clinical evidence for the essentiality of mitochondrial fission. 78.  Verstreken, P. et al. Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron 47, 365–378 (2005). 79.  Campello, S. et al. Orchestration of lymphocyte chemotaxis by mitochondrial dynamics. J. Exp. Med. 203, 2879–2886 (2006). 80.  Arnoult, D. Mitochondrial fragmentation in apoptosis. Trends Cell Biol. 17, 6–12 (2007). 81.  Youle, R. J. & Karbowski, M. Mitochondrial fission in apoptosis. Nature Rev. Mol. Cell Biol. 6, 657–663 (2005). 82.  Frank, S. et al. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev. Cell 1, 515–525 (2001). This study provided the first evidence that mitochondrial fission has a significant role in apoptosis. 83.  Parone, P. A. et al. Inhibiting the mitochondrial fission machinery does not prevent Bax/Bak-dependent apoptosis. Mol. Cell. Biol. 26, 7397–7408 (2006). 84.  Abdelwahid, E. et al. Mitochondrial disruption in Drosophila apoptosis. Dev. Cell 12, 793–806 (2007). 85.  Goyal, G., Fell, B., Sarin, A., Youle, R. J. & Sriram, V. Role of mitochondrial remodeling in programmed cell death in Drosophila melanogaster. Dev. Cell 12, 807–816 (2007). 86.  Jagasia, R., Grote, P., Westermann, B. & Conradt, B. DRP-1-mediated mitochondrial fragmentation during EGL-1-induced cell death in C. elegans. Nature 433, 754–760 (2005). 87.  Karbowski, M., Norris, K. L., Cleland, M. M., Jeong, S. Y. & Youle, R. J. Role of Bax and Bak in mitochondrial morphogenesis. Nature 443, 658–662 (2006). 88.  Brooks, C. et al. Bak regulates mitochondrial morphology and pathology during apoptosis by interacting with mitofusins. Proc. Natl Acad. Sci. USA 104, 11649–11654 (2007). 89.  Goldstein, J. C., Waterhouse, N. J., Juin, P., Evan, G. I. & Green, D. R. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nature Cell Biol. 2, 156–162 (2000). 90.  Cipolat, S. et al. Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 126, 163–175 (2006). 91.  Germain, M., Mathai, J. P., McBride, H. M. & Shore, G. C. Endoplasmic reticulum BIK initiates DRP1-regulated remodelling of mitochondrial cristae during apoptosis. EMBO J. 24, 1546–1556 (2005). 92.  Alexander, C. et al. OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nature Genet. 26, 211–215 (2000). 93.  Delettre, C. et al. Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nature Genet. 26, 207–210 (2000). References 92 and 93 showed that dominant optic atrophy is caused by mutations in OPA1. 94.  Ferre, M., Amati-Bonneau, P., Tourmen, Y., Malthiery, Y. & Reynier, P. eOPA1: an online database for OPA1 mutations. Hum. Mutat. 25, 423–428 (2005). 95.  Olichon, A. et al. Effects of OPA1 mutations on mitochondrial morphology and apoptosis: relevance to ADOA pathogenesis. J. Cell Physiol. 211, 423–430 (2006). 96.  Kim, J. Y. et al. Mitochondrial DNA content is decreased in autosomal dominant optic atrophy. Neurology 64, 966–972 (2005). 97.  Lodi, R. et al. Deficit of in vivo mitochondrial ATP production in OPA1-related dominant optic atrophy. Ann. Neurol. 56, 719–723 (2004). 98.  Griparic, L., van der Wel, N. N., Orozco, I. J., Peters, P. J. & van der Bliek, A. M. Loss of the intermembrane space protein Mgm1/OPA1 induces swelling and localized constrictions along the lengths of mitochondria. J. Biol. Chem. 279, 18792–18798 (2004). 99.  Zuchner, S. & Vance, J. M. Mechanisms of disease: a molecular genetic update on hereditary axonal neuropathies. Nature Clin. Pract. Neurol. 2, 45–53 (2006). 100.  Zuchner, S. et al. Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nature Genet. 36, 449–451 (2004). This study showed that mutations in MFN2 cause the peripheral neuropathy CMT2A. 101.  Zuchner, S. et al. Axonal neuropathy with optic atrophy is caused by mutations in mitofusin 2. Ann. Neurol. 59, 276–281 (2006). 102. Chung, K. W. et al. Early onset severe and late-onset mild Charcot-Marie-Tooth disease with mitofusin 2 (MFN2) mutations. Brain 129, 2103–2118 (2006). 103. Verhoeven, K. et al. MFN2 mutation distribution and genotype/phenotype correlation in Charcot-Marie-Tooth type 2. Brain 129, 2093–2102 (2006). 104. Baloh, R. H., Schmidt, R. E., Pestronk, A. & Milbrandt, J. Altered axonal mitochondrial transport in the pathogenesis of Charcot-Marie-Tooth disease from mitofusin 2 mutations. J. Neurosci. 27, 422–430 (2007). 105. Niemann, A., Ruegg, M., La Padula, V., Schenone, A. & Suter, U. Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease. J. Cell Biol. 170, 1067–1078 (2005).

45. 

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47.  48. 

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57.  58. 

59. 

60. 

61. 

Acknowledgements

62. 

This work was supported by grants from the National Institutes of Health. D.C.C. is an Ellison Medical Foundation Senior Scholar in Aging.

DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene BAK | BAX | Dnm1 | DRP1 | FIS1 | FZO | Fzo1 | GDAP1 | Gem1 | MARCH5 |MDM10 | MDM12 | Mdm30 | Mdm31 | Mdm32 | Mdm33 | MFN1 | MFN2 | Mgm1 | milton | Miro | MMM1 | NGF | OPA1 | SMAC | TIM23 | Ugo1 OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=OMIM CMT2A | CMT4A

63. 

64.  65.  66. 

FURTHER INFORMATION
David C. Chan’s homepage: http://www.its.caltech.edu/~chanlab

all links are active in the online pDf

nATuRE REvIEWS | molecular cell biology
© 2007 Nature Publishing Group

voluME 8 | novEMBER 2007 | 879

Enfermedades del mtDNA

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Enfermedades genéticas del ADN mitocondrial humano
Abelardo Solano, Q. F. B.,(1) Ana Playán, Ph.D.,(1) Manuel J. López-Pérez, Ph.D.,(1) Julio Montoya, Ph.D.(1)
Solano A, Playán A, López-Pérez MJ, Montoya J. Enfermedades genéticas del ADN mitocondrial humano. Salud Publica Mex 2001;43:151-161. El texto completo en inglés de este artículo está disponible en: http://www.insp.mx/salud/index.html Solano A, Playán A, López-Pérez MJ, Montoya J. Genetic diseases of the mitochondrial DNA. Salud Publica Mex 2001;43:151-161. The English version of this paper is available at: http://www.insp.mx/salud/index.html

Resumen Las enfermedades mitocondriales son un grupo de trastornos que están producidos por un fallo en el sistema de fosforilación oxidativa (sistema Oxphos), la ruta final del metabolismo energético mitocondrial, con la consiguiente deficiencia en la biosíntesis del trifosfato de adenosina (ATP, por sus siglas en inglés). Parte de los polipéptidos que componen este sistema están codificados en el ácido desoxirribonucleico (DNA) mitocondrial y, en los últimos años, se han descrito mutaciones que se han asociado con síndromes clínicos bien definidos. Las características genéticas del DNA mitocondrial, herencia materna, poliplasmia y segregación mitótica, confieren a estas enfermedades propiedades muy particulares. Las manifestaciones clínicas de estas enfermedades son muy heterogéneas y afectan a distintos órganos y tejidos por lo que su correcto diagnóstico implica la obtención de datos clínicos, morfológicos, bioquímicos y genéticos. El texto completo en inglés de este artículo está disponible en: http://www.insp.mx/salud/ index.html Palabras clave: ADN mitocóndrico; enfermedades mitocondriales; España

Abstract Mitochondrial diseases are a group of disorders produced by defects in the oxidative phosphorylation system (Oxphos system), the final pathway of the mitochondrial energetic metabolism, resulting in a deficiency of the biosynthesis of ATP. Part of the polypeptide subunits involved in the Oxphos system are codified by the mitochondrial DNA. In the last years, mutations in this genetic system have been described and associated to well defined clinical syndromes. The clinical features of these disorders are very heterogeneous affecting, in most cases, to different organs and tissues and their correct diagnosis require precise clinical, morphological, biochemical and genetic data. The peculiar genetic characteristics of the mitochondrial DNA (maternal inheritance, polyplasmia and mitotic segregation) give to these disorders very distinctive properties. The English version of this paper is available at: http://www.insp.mx/salud/ index.html

Key words: DNA, mitochondrial; mitochondrial diseases; Spain

subcelulares L as mitocondriaselson organelosde las célulasque se encuentran en citoplasma eucariotas, cuya función principal es la producción de la energía celular en forma de trifosfato de adenosina

(ATP, por sus siglas en inglés). Una de las particularidades de estos organelos es la de poseer un sistema genético propio con toda la maquinaria necesaria para su expresión, es decir, para replicar, transcribir y

Este trabajo ha sido subvencionado por la Dirección General de Enseñanza Superior e Investigación Científica (PB97-1019), el Fondo de Investigaciones Sanitarias (FIS 98-0049-01), la Diputación General de Aragón (P24/97) de España, así como por el Consejo Nacional de Ciencia y Tecnología (Conacyt) de México. (1) Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, España. Fecha de recibido: 22 de mayo de 2000 • Fecha de aprobado: 13 de noviembre de 2000 Solicitud de sobretiros: Dr. Julio Montoya. Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Universidad de Zaragoza. Miguel Servet 177, E-50013 Zaragoza, España. Correo electrónico: jmontoya@posta.unizar.es salud pública de méxico / vol.43, no.2, marzo-abril de 2001 151

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Solano A y col.

traducir la información genética que contiene. El ácido desoxirribonucleico mitocondrial (mtDNA, por sus siglas en inglés) humano es una molécula circular compuesta por 16 569 pares de bases1 que contiene información para 37 genes: dos ácidos ribonucleicos ribosómicos (rRNA), componentes de los ribosomas específicos mitocondriales, 22 de transferencia (tRNA), que son capaces de leer todo el código genético, y 13 polipéptidos que forman parte de cuatro de los cinco complejos multienzimáticos del sistema de fosforilación oxidativa (sistema Oxphos), etapa terminal de la ruta de producción de ATP. Estos péptidos corresponden a siete subunidades (ND1, 2, 3, 4, 4L, 5, 6) del dinucleótido de nicotinamida y adenina reducido (NADH): ubiquinona óxido-reductasa (complejo I); una subunidad (cyt b) de la ubiquinol: citocromo c óxido-reductasa (complejo III); tres subunidades (CO I, II, III) de la citocromo c oxidasa (complejo IV), y dos subunidades de la ATP sintetasa (complejo V)2

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(figura 1). El resto de los polipéptidos componentes de estos complejos, así como el complejo II completo, están codificados en el DNA nuclear. La biogénesis de este sistema constituye un caso único en la célula ya que para su formación se requiere la expresión coordinada de los dos sistemas genéticos. Los caracteres moleculares básicos y peculiares del sistema genético mitocondrial se descubrieron al inicio de los años ochenta,1,3-6 y en 1988 se encontraron las primeras mutaciones asociadas a enfermedades.7-9 Desde entonces, el número de mutaciones en el mtDNA y de enfermedades asociadas ha crecido de modo espectacular y ha generado lo que hoy se podría llamar como una “medicina mitocondrial”.10,11 Se designa con el nombre de enfermedades mitocondriales a un grupo de trastornos cuya característica común es un defecto en la producción de ATP. Sin embargo, frecuentemente este término se aplica a trastornos producidos por daños en el sistema Oxphos, debido a que durante muchos años sólo se habían encontrado mutaciones en el mtDNA relacionados con los mismos. Hoy en día, se han comenzado a identificar genes nucleares codificantes de proteínas de los complejos del sistema Oxphos o responsables de su ensamblaje. En este trabajo nos limitaremos a describir las enfermedades debidas a daños en el sistema genético mitocondrial por ser las más conocidas y por presentar un modo de herencia muy particular. Caracteres específicos de la genética mitocondrial El tipo de herencia del sistema genético mitocondrial, su localización en un organelo citoplasmático, la disposición contínua de los genes sin nucleótidos intermedios ni intrones y la poliplasmia (alto número de copias en cada célula) proporcionan caracteres genéticos que los diferencian claramente de los del DNA nuclear. Cada célula contiene entre unas 1 000 y 10 000 copias de mtDNA dependiendo del tejido, pasando por unos cuantos cientos en los espermatozoides y hasta unas 100 000 en el oocito. Cada mitocondria contiene entre 2 y 10 moléculas. Herencia materna. El mtDNA se hereda por vía materna con un patrón vertical no mendeliano. La madre trasmite su genoma mitocondrial a todos sus hijos, pero solamente las hijas lo pasarán a todos los miembros de la siguiente generación y así sucesivamente. Esto se debe al elevado número de moléculas de mtDNA que existe en los óvulos (entre 100 000 y 200 000 copias) en comparación con unos pocos cientos que hay en los espermatozoides. Además, las mitocondrias
salud pública de méxico / vol.43, no.2, marzo-abril de 2001



>

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MITOCONDRIAL

Enfermedades del mtDNA

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que puedan entrar en el óvulo fecundado se eliminan por un proceso activo.12 Segregación mitótica. El fenotipo de una línea celular puede variar durante la división celular debido a que las mitocondrias se distribuyen al azar entre las células hijas por lo que si en una célula coexisten dos poblaciones de mtDNA, una normal y otra mutada (heteroplasmia), a lo largo de las divisiones se podrán originar tres genotipos diferentes: homoplásmico para el DNA mitocondrial normal, homoplásmico para el DNA mutado y heteroplásmico. Por tanto, el fenotipo de una célula con heteroplasmia dependerá del porcentaje de DNA mutado que contenga. Si el número de moléculas de mtDNA dañado es relativamente bajo se produce una complementación con las moléculas de DNA normal y no se manifestará el defecto genético. Cuando el DNA mutado sobrepasa un umbral determinado se manifestará un fenotipo patogénico (efecto umbral), es decir, si la producción de ATP llega a estar por debajo de los mínimos necesarios para el funcionamiento de los tejidos, debido a la producción defectuosa de proteínas codificadas en el mtDNA, se produce la aparición de la enfermedad. El número de moléculas de DNA es diferente en cada órgano y tejido según la cantidad de energía requerida para su funcionamiento. Por ello, los tejidos que preferentemente se afectan son la visión, el sistema nervioso central, músculo esquelético, corazón, islotes pancreáticos, riñón e hígado. Alta velocidad de mutación. El mtDNA presenta una tasa de mutación espontánea 10 veces superior a la del DNA nuclear. Este fenómeno puede estar causado porque en la mitocondria se producen continuamente radicales de oxígeno, como consecuencia de la oxidación final de los compuestos carbonados, que pueden dañar a un DNA que no está protegido por proteínas. Debido a este hecho, la variación de secuencias entre individuos de una misma especie es muy grande, hasta unos 70 nucleótidos,13 y en un mismo individuo se estará generando, a lo largo de la vida, una pequeña heterogeneidad en el mtDNA. De este modo, se ha llegado a proponer que la disminución en la capacidad respiratoria de los tejidos que tiene lugar en el envejecimiento pueda ser debida a una acumulación de este daño mitocondrial. 14 Esta teoría tiene su primera evidencia en un trabajo del grupo de Attardi, que documenta que las mitocondrias se deterioran con la edad como consecuencia de la acumulación de mutaciones.15 Las variaciones de secuencia existentes entre diferentes individuos han resultado muy útiles para estudios antropológicos, etnológicos y forenses, y es la base de la hipótesis de

que el hombre desciende de una mujer que vivió en Africa hace unos 250 000 años (“Eva mitocondrial”).16 Enfermedades genéticas del DNA mitocondrial Las enfermedades originadas por daños en el genoma mitocondrial tienen en común el estar producidas por una deficiencia en la biosíntesis de ATP, ya que toda la información que contiene este DNA está dirigida a la síntesis de proteínas componentes del sistema Oxphos. Las manifestaciones de estas enfermedades son muy variadas y pueden afectar a todos los órganos y tejidos, ya que la síntesis de ATP se produce en todos ellos y a cualquier edad. Estas pueden presentar una serie de aspectos clínicos, morfológicos y bioquímicos muy concretos que dan lugar a síndromes bien caracterizados pero, en la mayor parte de los casos, principalmente en edad pediátrica, los síntomas son muy poco informativos y es sólo la presencia de anormalidades neurológicas, a veces acompañadas de aumento de ácido láctico y de otros síntomas clínicos secundarios que afectan a diversos órganos, lo que da alguna orientación en el diagnóstico de una enfermedad mitocondrial.17 Entre las manifestaciones clínicas más comunes se encuentran una o varias de las siguientes: desórdenes motores, accidentes cerebrovasculares, convulsiones, demencia, intolerancia al ejercicio, ptosis, oftalmoplejia, retinopatía pigmentaria, atrofia óptica, ceguera, sordera, cardiomiopatía, disfunciones hepáticas y pancreáticas, diabetes, defectos de crecimiento, anemia sideroblástica, pseudo obstrucción intestinal, nefropatías, acidosis metabólica y otras más secundarias. La presencia de uno o más de estos síntomas requiere a continuación de un estudio morfológico, histoquímico y bioquímico para asegurar la naturaleza de estas enfermedades. Así, con mucha frecuencia se encuentran: fibras rojo-rasgadas (acumulación de mitocondrias anormales en tamaño y número) en biopsias musculares teñidas con tricromo de Gomori y fibras no reactivas a la tinción histoquímica de la citocromo c oxidasa; defectos en uno o varios complejos de la cadena respiratoria; y desarreglos metabólicos con elevación de lactato, piruvato o una aminoaciduria generalizada causados por una disfunción de la cadena respiratoria que conlleva un aumento de equivalentes reductores en la mitocondria y citoplasma, y una alteración del funcionamiento del ciclo de Krebs debido al exceso de NADH, lo que provoca una acumulación de piruvato y su posterior conversión a lactato que difunde a la sangre. Sin em-

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bargo, la ausencia de algunos de estos caracteres no debe descartar la posibilidad de enfermedad mitocondrial, especialmente en pacientes en edad pediátrica. Además, los estudios familiares pueden ser decisivos si se comprueba la existencia de herencia materna de la enfermedad. El estudio genético del paciente y familiares relacionados por vía materna pueden asegurar finalmente que nos encontramos ante este tipo de trastornos. De hecho, hoy en día, el desarrollo y rapidez de las técnicas de genética molecular permiten, en ocasiones, una confirmación de la enfemedad antes de haber realizado muchas de las pruebas anteriormente citadas. La complejidad del diagnóstico de estas enfermedades hace preciso que los pacientes tengan que acudir a centros muy especializados donde se pueda llevar a cabo evaluaciones clínicas, metabólicas, patológicas, bioquímicas y genéticas, y a que en su diagnóstico estén implicados especialistas de muy diverso origen. Desde que en 1988 se describieran las primeras enfermedades causadas por daños en el mtDNA,7-9 se han encontrado más de 150 mutaciones (más 100 deleciones y unas 50 mutaciones puntuales) asociadas a enfermedades humanas. El interés por su estudio ha crecido enormemente debido al gran aumento de pacientes diagnosticados con estos trastornos y a que se presentan desde en recién nacidos hasta en adultos de todas las edades. Además, muchas de estas mutaciones se trasmiten por línea materna, como se ha indicado anteriormente, lo que hace que el diagnóstico en un individuo pueda tener implicaciones en muchas generaciones de una familia. A pesar de la importancia que las enfermedades mitocondriales tienen últimamente y de ser responsables de una considerable morbilidad, hasta ahora no se han realizado estudios exhaustivos sobre su prevalencia en la población general. Las razones son múltiples:18 complejidad de las manifestaciones clínicas, necesidad de biopsias musculares para su diagnóstico (no siempre se pueden detectar las mutaciones en muestras de sangre); necesidad de secuenciar todo el genoma mitocondrial para poder localizar mutaciones no detectadas hasta ahora, problemas éticos para realizar análisis genéticos presintomáticos en niños, diagnóstico erróneo de muchos pacientes al no ser atendidos en centros especializados, etcétera. Sin embargo, a pesar de todas estas dificultades, el grupo del doctor Turnbull, en Newcastle, Reino Unido, ha publicado muy recientemente los primeros datos epidemiológicos de las enfermedades del mtDNA, centrados en la población blanca de Europa del Norte residente en el noreste de Inglaterra.18 Así, ha mostrado que los defectos en el mtDNA
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son la causa de enfermedad en 6.57 de cada 100 000 individuos de la población adulta trabajadora y que 7.59 por cada 100 000 adultos y niños no afectados corren el riesgo de desarrollar una de estas enfermedades. En total, 12.48 por 100 000 individuos (1 de cada 8 000) tienen o presentan un riesgo de padecer una enfermedad causada por daños en el mtDNA. Estos datos representan un mínimo de prevalencia porque, muy probablemente, el número de pacientes que han quedado sin diagnosticar es elevado por haber sido atendidos por médicos de asistencia primaria, y no en clínicas neurológicas, y que hayan podido pasar desapercibidos por presentar solamente algunos de los síntomas acompañantes de estas enfermedades como diabetes o ptosis. Los datos obtenidos por el grupo de Newcastle han permitido comprobar que la prevalencia de las enfermedades debidas a daños en el mtDNA, consideradas en su conjunto, es equivalente a la de otras enfermedades neurológicas como la enfermedad de Huntington y la esclerosis amiotrófica lateral (6.4 y 6.2 por cada 100 000 individuos, respectivamente), y superior a la de otras enfermedades neuromusculares hereditarias como la distrofia de Duchenne (3.2 por cada 100 000 individuos).18 La experiencia de nuestro servicio de diagnóstico en la Universidad de Zaragoza es que un 16% de los pacientes remitidos para el estudio genético presentan una deleción o mutación puntual.19,20,* No hemos realizado ningún estudio de lo que este número representa entre la población general española; pero, seguro que tanto en el estudio realizado en Inglaterra como en nuestro laboratorio, el número de pacientes será muy superior cuando se secuencie el mtDNA de todos los posibles implicados y se detecten nuevas mutaciones. Estos números, junto al hecho de que no exista una terapia eficaz, y que, aunque algunas de estas enfermedades puedan mejorar o estabilizarse a lo largo de su curso, ilustran la importancia que tienen en relación con la salud pública, particularmente en cuanto a su atención y consejo genético, pues la mayoría tiene un desenlace fatal. La heterogeneidad de las manifestaciones clínicas, morfológicas y bioquímicas de las enfermedades del mtDNA, hace que su clasificación se base muy frecuentemente en las características genéticas de las mutaciones, a pesar de que, en algunos casos, una misma mutación pueda dar lugar a fenotipos clínicos muy diversos. Así, las enfermedades del mtDNA

* Solano A. Enfermedades del mtDNA (tesis doctoral). Zaragoza: Universidad de Zaragoza. En realización, 2000. salud pública de méxico / vol.43, no.2, marzo-abril de 2001

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se pueden dividir en tres grandes grupos según estén asociadas a mutaciones puntuales, a reorganizaciones o a disminución de número de copias del mtDNA. En la severidad de la manifestación de la enfermedad intervienen varios factores: la naturaleza de la mutación, el grado de heteroplasmia, los requerimientos energéticos del tejido y la capacidad del tejido para compensar el daño celular. A continuación se presenta un resumen de las enfermedades más comunes asociadas a estos tipos de mutaciones. Enfermedades asociadas a mutaciones puntuales en el mtDNA Dado el alto índice de mutación del mtDNA, como se ha indicado anteriormente, es posible encontrar un gran número de mutaciones puntuales. Sin embargo, la mayoría son mutaciones silenciosas que no causan ningún tipo de defecto. Las mutaciones patológicas se pueden encontrar tanto en los genes de tRNA, de rRNA, como en los codificantes de proteínas, y responden siempre a un tipo de herencia materna. Neuropatía óptica hereditaria de Leber. La neuropatía óptica hereditaria de Leber (LHON) se caracteriza por la pérdida bilateral de la visión central, originada por atrofia del nervio óptico. Aparece en la segunda o tercera década de la vida y afecta a más hombres que a mujeres. Aunque normalmente sólo la visión está afectada, hay casos en los que también aparecen trastornos en la conducción cardiaca, neuropatía periférica y ataxia cerebelar. Esta fue la primera enfermedad humana de herencia materna que se asoció a una mutación en el mtDNA. Después se llegó a asociar hasta con 16 mutaciones puntuales (cuadro I), localizadas todas ellas en genes codificantes de proteínas, y que se clasificaron en primarias, secundarias o intermedias según su relación con la aparición de la enfermedad. Sin embargo, últimamente sólo tres, G3 460A, G11 778A y T14 484C, están consideradas como primarias o patogénicas verdaderas, siendo la G11 778A la responsable en 50% de los casos y la que provoca la forma más severa de la enfermedad. Las tres se encuentran en genes que codifican algún polipéptido del complejo I del sistema Oxphos. La detección de estas mutaciones se suele hacer en células sanguíneas donde se encuentran tanto en forma homo como heteroplásmica. El resto de las mutaciones se consideran como secundarias, suelen acompañar a las anteriores en forma homoplásmica y se desconoce su relación directa con la enfermedad. Entre estas últimas vale la pena mencionar que la mutación G15257A, consisalud pública de méxico / vol.43, no.2, marzo-abril de 2001

derada como intermedia por algunos autores, se ha encontrado en varias familias analizadas en nuestro laboratorio por lo que pensamos que puede contribuir de forma decisiva a la aparición de la enfermedad. La prevalencia de la enfermedad en hombres ha sugerido la influencia de un gen nuclear, y aunque se ha descrito un ligamiento de la enfermedad con el locus (DXS7) situado en el cromosoma X en familias finlandesas,21 no se ha podido confirmar en familias de otro origen. Síndrome de neuropatía, ataxia y retinopatía pigmentaria. Este síndrome está caracterizado por debilidad muscular neurogénica, ataxia y retinitis pigmentosa. Suele ir acompañado de demencia, convulsiones y neuropatía sensorial axonal, presenta una herencia materna y se ha asociado a una mutación puntual, T8993G, en el gen de la subunidad 6 de la ATPasa (cuadro I). La mutación aparece normalmente en forma heteroplásmica y en todos los tejidos estudiados: leucocitos, fibroblastos, músculo, riñón y cerebro. Existe una alta correlación entre la proporción del DNA mutado y la severidad de la enfermedad. Síndrome de Leigh de herencia materna. El síndrome de Leigh de herencia materna (MILS) es una enfermedad muy heterogénea que se puede presentar asociada a diferentes tipos de herencia, autosómica recesiva, ligada al cromosoma X o materna (mitocondrial) según el gen que esté dañado. Es una enfermedad devastadora que se caracteriza por trastornos degenerativos multisistémicos que aparecen en el primer año de vida, disfunciones del tallo cerebral y de los ganglios basales, desmielinización, regresión psicomotora, retraso en el desarrollo, ataxia, convulsiones, neuropatía periférica. El diagnóstico se confirma por la presencia de lesiones necróticas cerebrales focales en el tálamo, tallo cerebral y núcleo dentado. La forma de la enfermedad, que se hereda por vía materna, está producida por la mutación en el gen de la subunidad 6 de la ATPasa, T8993G, la misma que produce el síndrome de neuropatía, ataxia y retinopatía pigmentaria, pero con un porcentaje de la mutación superior a 90%. Otras formas menos severas de esta enfermedad se han asociado con un cambio T→C* en la misma posición del mtDNA. Síndrome de epilepsia mioclónica con fibras rojo-rasgadas (MERRF). Este síndrome de herencia materna, está caracterizado por epilepsia mioclónica, convulsiones generalizadas y miopatía con presencia de fibras rojorasgadas. Otros síntomas clínicos que pueden acompañar a los anteriores son demencia, sordera, neuropatía,

* Timina por citosina. 155

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atrofia óptica, fallo respiratorio y cardiomiopatía. Aparece tanto en la infancia como en edad adulta y es de curso progresivo. Está asociado a la presencia de mutaciones en el gen del mtDNA para el tRNALys. En la mayoría de los casos (80%-90%) se debe a una mutación A8344G, pero también se han encontrado otras mi-

noritarias como T8356C (cuadro I), todas en forma heteroplásmica. El porcentaje de heteroplasmia necesario para la afectación varía entre individuos jóvenes (95%) e individuos por encima de los 60-70 años (60%) del DNA mutado.13 La presencia de estas mutaciones en tRNA daña la síntesis de proteínas.

Cuadro I

MUTACIONES EN EL MTDNA Y ENFERMEDADES ASOCIADAS
Enfermedad Mutación en el mtDNA Gen afectado Referencias Enfermedad Mutación en el mtDNA Gen afectado tRNALeu(uur) tRNALeu(uur) tRNAPro 12S rRNA tRNASer(ucn) tRNALeu(cun) tRNALys tRNALeu(uur) tRNAAsn tRNAAsn tRNALeu(uur) Citocromo b Citocromo b Citocromo b Citocromo b Citocromo b tRNAThr tRNALeu(uur) ATPase 6 ATPase 6 tRNALeu(uur) tRNALeu(uur) tRNALeu(uur) tRNATrp ND6 ND6 Referencias
53 54 55

LHON Mutaciones primarias

Mutaciones intermedias Mutaciones secundarias

NARP Leigh (MILS) MELAS

MERRF Diabetes y sordera Cardiomiopatía (MICM)

G3460A G11778A T14484C G5244A G15257A T3394C T4160C T4216C A4917G G7444A T9101C G9438A G9804A G13708A G13730A G14459A G15812A T8993G T8993G T8993C A3243G C3256T T3271C T3291C T9957C A8344G T8356C A3243G A3260G C3303T A4269G A4300G A4317G C4320T G8363A T9997C

ND1 ND4 ND6 ND2 Citocromo b ND1 ND1 ND1 ND2 CO I ATPasa 6 CO III CO III ND5 ND5 ND6 cyt b ATPasa 6 ATPasa 6 ATPasa 6 tRNALeu(uur) tRNALeu(uur) tRNALeu(uur) tRNALeu(uur) COIII tRNALys tRNALys tRNALeu(uur) tRNALeu(uur) tRNALeu(uur) tRNAIle tRNAIle tRNAIle tRNAIle tRNALys tRNAGly

22,23 7 24 25 25 24 26 27 27 28 29 30 30 27 31 32 25 33 34,35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

Miopatía mitocondrial T3250C A3302G C15990T Sordera inducida por aminoglicósidos A1555G Sordera sensoneural T7445C Anemia sideroblástica G12301A Lipomatosis múltiple simétrica A8344G CPEO Deleción única A3243G A5692G G5703A C3256T Intolerancia al ejercicio G15084A G15168A G15723A G14846A delecion de 24pb LIMM A15923G Muerte súbita A3251G Necrosis bilateral del estriado T9176C T8851C Multisistémicas A3251G A3252G C3256T Corea y demencia G5549A LHON y distonía G14459A Diabetes y miopatía T14709C Pearson Deleción única Kearns-Sayre Deleción única

56 57 58

59 60 61 62 63 63 64 64 64 64 64 65 66

67 68 66 69 63 70 32 71 72 73

LIMM: miopatía mitocondrial infantil letal; LHON: neuropatía óptica hereditaria de Leber; MELAS: encefalomiopatía mitocondrial con acidosis láctica y episodios de accidentes cerebrovasculares; MERRF: epilepsia mioclónica con fibras rojo-rasgadas; MICM: Cardiomiopatía de herencia materna; MILS: síndrome de Leigh de herencia materna; PEO: oftalmoplejia progresiva externa

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Síndrome de encefalomiopatía mitocondrial con acidosis láctica y episodios de accidentes cerebro-vasculares (MELAS). Se trata de una encefalomiopatía mitocondrial, de herencia materna, caracterizada por accidentes cerebrovasculares producidos a edad temprana que provocan una disfunción cerebral subaguda y cambios en la estructura cerebral, y por acidosis láctica. Estos caracteres suelen ir acompañados de convulsiones generalizadas, dolor de cabeza, sordera, demencia y, a veces, presenta fibras rojo-rasgadas. Esta enfermedad ha sido asociada fundamentalmente con mutaciones en el gen del tRNALeu(UUR) del mtDNA. La mayor parte de los casos (80%) está asociada a la mutación A3.243G, pero también se han encontrado otras con menor incidencia y alguna en genes codificantes de proteínas (cuadro I), todas en forma heteroplásmica. Al igual que en la epilepsia mioclónica, las mutaciones en el tRNA dañan la síntesis de proteínas mitocondriales. Diabetes de herencia materna con sordera. Además de los dos tipos clásicos de diabetes dependiente y no dependiente de insulina (tipo 1 y 2, respectivamente), se ha descrito recientemente un nuevo tipo de diabetes asociada a sordera, que no encuadra dentro de la clasificación de la Organización Mundial de la Salud. Esta diabetes, de herencia materna, está producida por la mutación A3.243G en el gen del tRNALeu(UUR) (cuadro I), la misma descrita para el síndrome de (MELAS). La frecuencia de diabetes y sordera es aproximadamente de un 1.5% de la población diabética total.74 Por otra parte, la diabetes es una de las enfermedades que se han descrito asociadas a otros síndromes mitocondriales como la encefalomiopatía mitocondrial, oftalmoplejia progresiva externa crónica, KearnsSayre, Pearson y diabetes insípida, diabetes mellitus, atrofia óptica y sordera (DIDMOAD). Otras enfermedades del mtDNA asociadas a mutaciones puntuales Además de las enfermedades descritas anteriormente, hay otras muchas que se han asociado a otras mutaciones puntuales (cuadro I). Entre ellas, se pueden citar las cardiomiopatías de herencia materna relacionadas fundamentalmente con mutaciones en el tRNAIle: la sordera inducida por aminoglicósidos que está producida por una mutación en el rRNA 12S (A1555G), y otros tipos de sordera sindrómica o no sindrómica de herencia materna; LHON y distonía; miopatías de herencia materna unidas a mutaciones en tRNA Leu, tRNAPro, tRNAAsn, tRNATyr; oftalmoplejia progresiva externa crónica; anemia sideroblástica; deficiencia fatal de la cadena respiratoria infantil; lipomatosis sisalud pública de méxico / vol.43, no.2, marzo-abril de 2001

métrica múltiple asociada a la mutación A8.344G del gen del tRNALys (descrita en nuestro laboratorio) y, recientemente, se ha relacionado la intolerancia al ejercicio, como entidad propia, a mutaciones puntuales en el gen del citocromo b. Así, se han descrito mutaciones en este gen que crean un codón de terminación, que cambian un aminoácido o, incluso, una deleción de 24 pares de bases. En el cuadro I se citan éstos y otros síndromes que se han asociado a mutaciones puntuales y, sin ninguna duda, el espectro de fenotipos relacionados con mutaciones en el mtDNA aumentará más en un futuro. Asimismo, cabe mencionar que alguna de las mutaciones, como la A3.243G, puede estar relacionada con muy diversos fenotipos clínicos como sindromes de encefalopatía mitocondrial con acidosis láctica y episodios de accidentes cerebrovasculares de epilepsia mioclónica con fibras rojo-rasgadas y solapados, cardiomiopatías, CPEO, etcétera. Actualmente, se estudia la posible implicación del mtDNA en enfermedades neurodegenerativas como Parkinson y Alzheimer. Enfermedades asociadas a reorganizaciones en el DNA mitocondrial Además de las mutaciones puntuales, el mtDNA puede sufrir otro tipo de daños como son la pérdida de parte del mismo (deleciones) o la adición de un nuevo fragmento del DNA (duplicaciones), que, como en los casos anteriores, afectan a la biogénesis del sistema Oxphos y, por tanto, a la síntesis de ATP. En la actualidad hay descritos más de 100 tipos de deleciones y sólo unos cuantos casos de inserciones. Este tipo de mutaciones suelen ser espontáneas, probablemente causadas por daños en genes nucleares que controlan la replicación del mtDNA, aunque hay descritos casos de herencia materna.75 Se presentan siempre en forma heteroplásmica, ya que la homoplasmia sería incompatible con la vida, y se sabe que la gravedad de los casos aumenta con la edad debido a la ventaja replicativa de estas moléculas de DNA más pequeñas en relación con la de tamaño normal. Los tres tipos de síndromes más comunes en los que se presentan deleciones son los de Pearson, oftalmoplejia progresiva externa crónica y Kearns-Sayre. Síndrome de médula ósea-páncreas de Pearson. Es una enfermedad que aparece en los primeros años de vida y que afecta a la hematopoyesis y a la función pancreática exocrina. Las características clínicas más comunes son anemia sideroblástica con vacuolización de precursores de la médula ósea que se manifiesta con una anemia macrocítica, trombocitopenia y neutropenia.
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Los niños afectados suelen morir antes de los tres años de edad y los que sobreviven suelen desarrollar posteriormente el fenotipo de Kearns-Sayre, que veremos más adelante. Estos pacientes presentan deleciones grandes únicas del mtDNA, en general son esporádicas aunque se ha descrito algún caso de herencia materna. Oftalmoplejia progresiva externa crónica. Esta enfermedad está caracterizada por oftalmoplejia, ptosis bilateral de los párpados y miopatía. Suele ir acompañada también de intolerancia al ejercicio y debilidad muscular. En el músculo se encuentran fibras rojo-rasgadas COX negativas. En general, es una enfermedad benigna que suele aparecer en la adolescencia o en adultos jóvenes. Aparece de forma esporádica sin historia familiar. Se ha asociado fundamentalmente a deleciones grandes y únicas en el mtDNA (ver más adelante). Asimismo, se han encontrado otras formas de CPEO con mutaciones puntuales de herencia materna (cuadro I) o con deleciones múltiples de herencia autosómica recesiva o dominante. Síndrome de Kearns-Sayre. Este síndrome es, por otra parte, una enfermedad multisistémica progresiva caracterizada clinicamente por CPEO, retinopatía pigmentaria atípica, ataxia, miopatía mitocondrial, bloqueo de la conducción cardiaca, elevados niveles de proteína CSF (fluido cerebro espinal, por sus siglas en inglés), sordera y demencia. Aparece antes de los 20 años de edad. Estas tres enfermedades están causadas por deleciones (de 2 a 9 kb) en el mtDNA que suelen aparecer de forma espontánea. En general, la deleción es única, pero también se han descrito casos de deleciones múltiples. La gravedad de la enfermedad depende del porcentaje de DNA mutado en el individuo. En general están localizadas en el arco grande comprendido entre los orígenes de replicación del DNA y mantienen siempre las secuencias requeridas para la replicación del DNA y los promotores de la transcripción. Entre todas las deleciones conocidas, hay una que aparece con más frecuencia (hasta en 50%), la llamada deleción común, que elimina un tramo de DNA de 4 977 pares de bases (entre los nucleótidos 8 483 a 13 460), que comprende los genes localizados entre la subunidad 8 de la ATPasa y ND5 (figura 1). No existe una clara relación entre el fenotipo y el tipo, tamaño o porcentaje del DNA delecionado ya que la misma deleción puede dar lugar a varios fenotipos diferentes. La mayor parte de las deleciones encontradas están flanqueadas por repeticiones directas de longitud variable (3-13 nt). Este hecho sugiere que la delección se produce por errores sucedidos en el proceso de replica158

ción dependientes de la presencia de estas repeticiones. La pérdida de genes, especialmente la de los tRNA, hace que estos genomas no se puedan traducir y, por tanto, que sean dependientes de complementación con moléculas de mtDNA normales en la misma mitocondria. El umbral se suele alcanzar cuando el porcentaje de moléculas delecionadas supera 60%. Existen otras enfermedades como una diabetes con sordera y atrofia óptica; miopatías en general; el síndrome de encefalomiopatía mitocondrial neurogastrointestinal; el de diabetes mellitus, diabetes insípida, atrofia óptica y sordera, etcétera, que están asociadas a la presencia de deleciones en el mtDNA. Como se ha mencionado anteriormente, entre las reorganizaciones del mtDNA se pueden encontrar duplicaciones en pacientes con defectos en el sistema Oxphos. Estas pueden ser también esporádicas o de herencia materna. Se han encontrado en pacientes de Kearns-Sayre, Pearson, diabetes mellitus, tubulopatía renal y miopatía mitocondrial e incluso en individuos normales. El mecanismo por el cual pueden causar la patogenicidad no está nada claro todavía. Enfermedades asociadas a depleciones de DNA mitocondrial El tercer tipo de daños en el genoma mitocondrial que puede causar enfermedades no se debe a mutaciones propiamente dichas sino a una disminución de los niveles del mtDNA. El espectro clínico que produce la depleción es muy variado. Los casos descritos hasta ahora afectan fundamentalmente a niños con combinaciones variables de miopatía, nefropatía o hepatopatía, miopatía infantil fatal por fallo respiratorio y algún otro con implicación multisistémica. La depleción puede estar producida por mutaciones en genes nucleares que controlan el número de copias del mtDNA. Es, por tanto, un trastorno de herencia mendeliana que afecta a la coordinación núcleo-mitocondria, y que parece ser autosómico recesivo. Regreso a la genética mendeliana Debido al doble origen genético nuclear y mitocondrial del sistema Oxphos las enfermedades genéticas mitocondriales pueden estar originadas, además de por mutaciones en genes del mtDNA con herencia materna, como ya hemos visto, por mutaciones en genes nucleares que codifican proteínas mitocondriales, por mutaciones que afecten al procesamiento postraduccional, al importe de proteínas por la mitocondria y al ensamblaje de los complejos, y por mutaciones que afecten al control nuclear del genoma
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mitocondrial, todas ellas con un tipo de herencia mendeliana. El primer caso ha sido hasta ahora el más estudiado por estar el genoma mitocondrial completamente secuenciado y, por tanto, por la facilidad de encontrar mutaciones que afecten a los genes que codifica. Sin embargo, la mayor parte de los genes que componen el sistema Oxphos es de origen nuclear y cabe esperar que la mayoría de las enfermedades causadas por la deficiencia de este sistema sean debidas a mutaciones en el DNA nuclear. Así, por ejemplo, a pesar de que una de las causas del síndrome de Leigh sea una mutación en el mtDNA de herencia materna, se sabe que se transmite con más frecuencia por herencia autosómica recesiva, y se han encontrado e identificado mutaciones en genes de subunidades del complejo I, codificadas por el DNA nuclear76,77 y en el complejo II,78 codificado enteramente en el núcleo. Asimismo, se han localizado mutaciones en un gen nuclear (SURF 1) que codifica una proteína que, aunque no forma parte del complejo IV, es necesaria para su ensamblaje.79,80 Además, en la mitocondria existen muchas otras rutas metabólicas en las que no participa para nada el mtDNA y cuya deficiencia puede causar encefalomiopatías mitocondriales. Por todo ello, la genética mendeliana de las enfermedades mitocondriales está todavía prácticamente por descubrir y nos proporcionará mucha información sobre estos trastornos.

Referencias
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