Sem10Biol-Mitocondriopatias

1

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.

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 appre-ciation 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 intramito-chondrial 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 conse-quences for mitochondrial function and cell physio-logy? 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 mito-chondrial population, including its respiratory capacity. Moreover, mitochondrial dynamics has key roles in mammalian development, several neurodegenerative diseases and apoptosis.

Mitochondria as dynamic organellesBy 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 distribu-tions. 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 balanced4 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 inter-connected5–8 (FIG. 2). Conversely, cells with a low fusion-to-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 dur-ing development. For example, during Drosophila melanogaster spermatogenesis, many mitochondria synchronously fuse to form the Nebenkern structure, which is required for sperm motility9.

Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.Correspondence to D.C.C. e-mail: [email protected]:10.1038/nrm2275Published online 10 October 2007

CristaeInvaginations of the mitochondrial inner membrane.

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

Functions and dysfunctions of mitochondrial dynamicsScott 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.

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© 2007 Nature Publishing Group

mailto:[email protected]

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AnterogradeThe direction from the cell body towards the periphery.

RetrogradeThe direction from peripheral regions towards the cell body.

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

Dynamic subcellular distribution. Mitochondrial trans-port 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 mam-malian 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 par-ticularly 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 continu-ous; 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

lines of evidence from neuronal studies suggest that mito-chondrial 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 acti-vation, further arguing that the recruitment of mitochon-dria 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 mito-chondria with high membrane potential preferentially migrate in the anterograde direction, whereas mitochon-dria 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 mem-brane 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 mito-chondria placed in low ADP conditions have limited respiration and have an ‘orthodox’ morphology, charac-terized 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

Box 1 | What happens to mitochondrial components after fusion?

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 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.

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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4803&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum



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

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

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.

Mediators of fusion and fissionMolecular analysis of mitochondrial morphology began with the discovery in 1997 of the D. melanogaster fusion factor fuzzy onions (FZo), a mitochondrial outer mem-brane GTPase that is required for the fusion of mito-chondria during spermatogenesis9. FZo is the founding

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 compre-hensive 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 mem-branes24,26. 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 trans-port 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 morphol-ogy screens in yeast identified members of the ergosterol synthesis pathway as being required for normal mito-chondrial morphology35,36. Recently, mitochondrial phospholipase D has been identified as a protein that is important for mitochondrial fusion37. This mitochon-drial outer membrane enzyme hydrolyses cardiolipin to generate phosphatidic acid. Interestingly, ergosterol has been associated with yeast vacuole fusion38, and phos-phatidic 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.

Figure 1 | mitochondria as dynamic organelles. a | Mitochondrial fusion and fission control mitochondrial number and size. With fusion, 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.

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Mitochondrial membrane potentialThe electrochemical gradient that exists across the mitochondrial inner membrane.

ErgosterolA 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 proteinA 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.

β-barrel proteinA 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.

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

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 fis-sion 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 suffi-cient 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.

Other regulators of dynamicsMitochondrial fusion and fission activities are probably coordinated with cellular physiology. In yeast, the steady-state 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 abil-ity 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.

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 mitochon-drial 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 mito-chondrial 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 mitochon-dria 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+-bind-ing eF‑hand domains59. In yeast, disruption of the Miro orthologue Gem1 results in abnormalities in mitochon-drial 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, mitochon-dria 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.

Figure 2 | mitochondrial fusion and fission regulate morphology. Mitochondrial length, size and connectivity are determined by the 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.

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

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

Mitochondrial F1F0 ATP synthaseA 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.

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

Mitochondrial F1FoATP 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 elec-tron 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

mitofilin in mammalian cells causes dramatic abnorm-alities 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.

Biological functions of mitochondrial dynamicsWhy do mitochondria continually fuse and divide? Recent studies show that these processes have impor-tant 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 sub-cellular locations. This function is especially important in highly polarized cells, such as neurons. Finally, mito-chondrial 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 func-tional mitochondrial population in the cell. Fibroblasts that lack both MFn1 and MFn2 have reduced respira-tory 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 path-way 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 phos-phorylation. 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

Figure 3 | mitochondrial fusion. a | Mitochondrial fusion consists of outer membrane (OM) fusion followed by inner membrane (IM) fusion. Normally these events occur 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.

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ChemotaxisThe directed movement of cells in response to a chemical stimulus.

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 fusion- deficient cells still maintain significant numbers of mtDnA nucleoids; however, due to ongoing mitochon-drial fission, these nucleoids are encased by a small mitochondrial mass, and therefore the functional mito-chondrial 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 sub-strates, 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 dominant-negative DRP1 allele has been reported. This patient died at ~1 month of age and had a wide range of abnorm-alities, including reduced head growth, increased lactic acid and optic atrophy. Fibroblasts from this patient showed elongated mitochondria and peroxisomes77. 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 depend-ence of neurons probably stems from their high energy demands and the special importance of proper mito-chondrial 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 mito-chondrial 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 ATP-dependent process), and the defects in synaptic transmis-sion 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 mito-chondrial 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 mitochon-dria and leads to an increased spine number13. In the cerebellum, the distribution of mitochondria in the den-dritic 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 chemotaxis79. 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

Figure 4 | mitochondrial fission. a | In yeast, mitochondrial fission is mediated by the dynamin-related protein Dnm1. Cytoplasmic Dnm1 localizes to the mitochondrial outer 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.

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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.

HaploinsufficiencyA 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 inter-membrane 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, disrup-tion 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

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 sys-tem88. 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 release64,90. 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 diseaseSeveral 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 symp-toms of ADoA remains to be clarified. non-neuronal cells from patients with ADoA can have aggregated, frag-mented 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 defi-cient for oPA1 have fragmented mitochondria, defects in respiration, aberrant cristae structure and increased susceptibility to apoptosis28,31,62,98.

Figure 5 | mitochondrial dynamics protects mitochondrial function. a | In wild-type 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.

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Apoptoticstimuli

Cristae junctions

Cytochrome c


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

Mouse models of ADoA that contain oPA1 muta-tions 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 con-sistent with an essential requirement for mitochondrial fusion during embryonic development6.

MFN2 and Charcot-Marie-Tooth 2A. Charcot-Marie-Tooth (CMT) disease, one of the most common heredi-tary 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 muta-tions or short, in-frame deletions100. Most mutations cluster in or near the GTPase domain, but some also

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 mitochon-dria 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 con-cerning 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, there-fore, 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 obser-vations). 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 cere-bellar ataxia72. Purkinje cells are the sole efferent neurons of the cerebellum, and they have exquisitely formed den-dritic 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 mito-chondria that fail to distribute effectively along dendritic

Figure 6 | mitochondrial dynamics during apoptosis. At an early stage of apoptosis, 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.

Table 1 | Disorders associated with mitochondrial perturbations

Disease mitochondrial function

gene Description

CMT2A Fusion MFN2 Autosomal dominant peripheral neuropathy

ADOA Fusion OPA1 Autosomal dominant optic atrophy (ADOA)

CMT4A Fission? GDAP1 Autosomal recessive peripheral neuropathy

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

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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 dyn-amics. 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.

PerspectivesThe 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 oppos-ing 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 func-tion. 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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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).

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

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneBAK | 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 | Ugo1OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMCMT2A | CMT4A

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

all links are active in the online pDf

R E V I E W S



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
























http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4976&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gen




http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=OMIM&dopt=DocSum&list_uids=609260%2C118210

http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=214400

http://www.its.caltech.edu/~chanlab

151salud pública de méxico / vol.43, no.2, marzo-abril de 2001

Enfermedades del mtDNA ARTÍCULO DE REVISIÓN

Enfermedades genéticasdel 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.Genetic diseasesof the mitochondrial DNA.Salud Publica Mex 2001;43:151-161.The English version of this paperis available at: http://www.insp.mx/salud/index.html

AbstractMitochondrial diseases are a group of disorders producedby defects in the oxidative phosphorylation system (Ox-phos system), the final pathway of the mitochondrial ener-getic metabolism, resulting in a deficiency of the biosynthesisof ATP. Part of the polypeptide subunits involved in theOxphos system are codified by the mitochondrial DNA.In the last years, mutations in this genetic system have beendescribed and associated to well defined clinical syndromes.The clinical features of these disorders are very heteroge-neous affecting, in most cases, to different organs and tis-sues and their correct diagnosis require precise clinical,morphological, biochemical and genetic data. The peculiargenetic characteristics of the mitochondrial DNA (mater-nal inheritance, polyplasmia and mitotic segregation) giveto these disorders very distinctive properties. The Englishversion of this paper is available at: http://www.insp.mx/salud/index.html

Key words: DNA, mitochondrial; mitochondrial diseases;Spain

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 estádisponible en: http://www.insp.mx/salud/index.html

ResumenLas enfermedades mitocondriales son un grupo de tras-tornos que están producidos por un fallo en el sistema defosforilación oxidativa (sistema Oxphos), la ruta final delmetabolismo energético mitocondrial, con la consiguientedeficiencia en la biosíntesis del trifosfato de adenosina (ATP,por sus siglas en inglés). Parte de los polipéptidos quecomponen este sistema están codificados en el ácido deso-xirribonucleico (DNA) mitocondrial y, en los últimos años,se han descrito mutaciones que se han asociado con sín-dromes clínicos bien definidos. Las características genéti-cas del DNA mitocondrial, herencia materna, poliplasmia ysegregación mitótica, confieren a estas enfermedades pro-piedades muy particulares. Las manifestaciones clínicas deestas enfermedades son muy heterogéneas y afectan a dis-tintos órganos y tejidos por lo que su correcto diagnósticoimplica la obtención de datos clínicos, morfológicos, bio-químicos y genéticos. El texto completo en inglés de esteartículo está disponible en: http://www.insp.mx/salud/index.html

Palabras clave: ADN mitocóndrico; enfermedades mito-condriales; España

Este trabajo ha sido subvencionado por la Dirección General de Enseñanza Superior e Investigación Científica (PB97-1019), el Fondo de InvestigacionesSanitarias (FIS 98-0049-01), la Diputación General de Aragón (P24/97) de España, así como por el Consejo Nacional de Ciencia y Tecnología (Conacyt) deMéxico.

(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 2000Solicitud 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: [email protected]

L as mitocondrias son organelos subcelulares que seencuentran en el citoplasma de las células euca-

riotas, cuya función principal es la producción de laenergía celular en forma de trifosfato de adenosina

(ATP, por sus siglas en inglés). Una de las particulari-dades de estos organelos es la de poseer un sistemagenético propio con toda la maquinaria necesariapara su expresión, es decir, para replicar, transcribir y

ARTÍCULO DE REVISIÓN

152 salud pública de méxico / vol.43, no.2, marzo-abril de 2001

Solano A y col.

traducir la información genética que contiene. El áci-do desoxirribonucleico mitocondrial (mtDNA, por sussiglas en inglés) humano es una molécula circularcompuesta por 16 569 pares de bases1 que contieneinformación para 37 genes: dos ácidos ribonucleicosribosómicos (rRNA), componentes de los ribosomasespecíficos mitocondriales, 22 de transferencia (tRNA),que son capaces de leer todo el código genético, y 13polipéptidos que forman parte de cuatro de los cincocomplejos multienzimáticos del sistema de fosfo-rilación oxidativa (sistema Oxphos), etapa terminal dela ruta de producción de ATP. Estos péptidos corres-ponden a siete subunidades (ND1, 2, 3, 4, 4L, 5, 6) deldinucleótido de nicotinamida y adenina reducido(NADH): ubiquinona óxido-reductasa (complejo I);una subunidad (cyt b) de la ubiquinol: citocromo cóxido-reductasa (complejo III); tres subunidades (COI, II, III) de la citocromo c oxidasa (complejo IV), ydos subunidades de la ATP sintetasa (complejo V)2

(figura 1). El resto de los polipéptidos componentesde estos complejos, así como el complejo II completo,están codificados en el DNA nuclear. La biogénesis deeste sistema constituye un caso único en la célula yaque para su formación se requiere la expresión coordi-nada de los dos sistemas genéticos.

Los caracteres moleculares básicos y peculiaresdel sistema genético mitocondrial se descubrieron alinicio de los años ochenta,1,3-6 y en 1988 se encontra-ron las primeras mutaciones asociadas a enferme-dades.7-9 Desde entonces, el número de mutacionesen el mtDNA y de enfermedades asociadas ha creci-do de modo espectacular y ha generado lo que hoy sepodría llamar como una “medicina mitocondrial”.10,11

Se designa con el nombre de enfermedades mi-tocondriales a un grupo de trastornos cuya caracte-rística común es un defecto en la producción de ATP.Sin embargo, frecuentemente este término se aplica atrastornos producidos por daños en el sistema Ox-phos, debido a que durante muchos años sólo sehabían encontrado mutaciones en el mtDNA relacio-nados con los mismos. Hoy en día, se han comenzadoa identificar genes nucleares codificantes de proteí-nas de los complejos del sistema Oxphos o responsa-bles de su ensamblaje. En este trabajo nos limitaremosa describir las enfermedades debidas a daños en el sis-tema genético mitocondrial por ser las más conocidasy por presentar un modo de herencia muy particular.

Caracteres específicos de la genéticamitocondrial

El tipo de herencia del sistema genético mitocondrial,su localización en un organelo citoplasmático, la dis-posición contínua de los genes sin nucleótidos inter-medios ni intrones y la poliplasmia (alto número decopias en cada célula) proporcionan caracteres gené-ticos que los diferencian claramente de los del DNAnuclear. Cada célula contiene entre unas 1 000 y 10 000copias de mtDNA dependiendo del tejido, pasando porunos cuantos cientos en los espermatozoides y hastaunas 100 000 en el oocito. Cada mitocondria contieneentre 2 y 10 moléculas.

Herencia materna. El mtDNA se hereda por vía mater-na con un patrón vertical no mendeliano. La madretrasmite su genoma mitocondrial a todos sus hijos, perosolamente las hijas lo pasarán a todos los miembrosde la siguiente generación y así sucesivamente. Estose debe al elevado número de moléculas de mtDNAque existe en los óvulos (entre 100 000 y 200 000 co-pias) en comparación con unos pocos cientos que hayen los espermatozoides. Además, las mitocondrias

FIGURA 1. MAPA GENÉTICO DEL DNA MITOCONDRIAL

HUMANO. SE REPRESENTA LAS DOS HEBRAS DEL DNA CON

LOS GENES QUE CODIFICAN: RRNA (12S Y 16S), TRNA,SEÑALADOS CON LA ABREVIATURA DEL AMINOÁCIDO QUE

TRANSPORTAN, Y SECUENCIAS CODIFICADORAS DE PROTEÍ-NAS (CO: SUBUNIDADES CITOCROMO C OXIDASA; CYT B:CITOCROMO B Y ND: SUBUNIDADES DE NADH DESHIDRO-GENASA). H1, H2 Y L INDICAN LOS LUGARES DE INICIACIÓN

DE LA TRANSCRIPCIÓN DE LAS HEBRAS PESADA Y LIGERA,RESPECTIVAMENTE. OH Y OL SIMBOLIZAN LOS ORÍGENES DE

REPLICACIÓN DE LA CADENA PESADA Y LIGERA

Gln

Ala AsnCysTyr

Ser

ProGlu

ND 6

CadenaLigera

F-met LeuSerHis

•

••••

•

••

•

•

•

••

•

•

•••

••

lle

ND 1

rARN 16S

ValrARN 12S

OH

Leu

•

Phe BUCLE-DThr

Cyt. b• Cadenapesada

ND 5

ND 4

ND 4L

ND 3

Arg

Gly

CO IIIATPasa 6

ATPasa 8

LysCO IIAsp

CO I

>

>OL

Trp

ND 2



que puedan entrar en el óvulo fecundado se eliminanpor un proceso activo.12

Segregación mitótica. El fenotipo de una línea celularpuede variar durante la división celular debido a quelas mitocondrias se distribuyen al azar entre las célu-las hijas por lo que si en una célula coexisten dos po-blaciones de mtDNA, una normal y otra mutada(heteroplasmia), a lo largo de las divisiones se podránoriginar tres genotipos diferentes: homoplásmico parael DNA mitocondrial normal, homoplásmico para elDNA mutado y heteroplásmico. Por tanto, el fenotipode una célula con heteroplasmia dependerá del por-centaje de DNA mutado que contenga. Si el númerode moléculas de mtDNA dañado es relativamentebajo se produce una complementación con las molécu-las de DNA normal y no se manifestará el defectogenético. Cuando el DNA mutado sobrepasa un um-bral determinado se manifestará un fenotipo patogé-nico (efecto umbral), es decir, si la producción de ATPllega a estar por debajo de los mínimos necesarios parael funcionamiento de los tejidos, debido a la produc-ción defectuosa de proteínas codificadas en el mtDNA,se produce la aparición de la enfermedad. El númerode moléculas de DNA es diferente en cada órgano ytejido según la cantidad de energía requerida para sufuncionamiento. Por ello, los tejidos que preferente-mente se afectan son la visión, el sistema nervioso cen-tral, músculo esquelético, corazón, islotes pancreáticos,riñón e hígado.Alta velocidad de mutación. El mtDNA presenta unatasa de mutación espontánea 10 veces superior a ladel DNA nuclear. Este fenómeno puede estar causadoporque en la mitocondria se producen continuamenteradicales de oxígeno, como consecuencia de la oxi-dación final de los compuestos carbonados, quepueden dañar a un DNA que no está protegido porproteínas. Debido a este hecho, la variación de se-cuencias entre individuos de una misma especie esmuy grande, hasta unos 70 nucleótidos,13 y en un mis-mo individuo se estará generando, a lo largo de lavida, una pequeña heterogeneidad en el mtDNA. Deeste modo, se ha llegado a proponer que la disminu-ción en la capacidad respiratoria de los tejidos quetiene lugar en el envejecimiento pueda ser debida auna acumulación de este daño mitocondrial.14 Estateoría tiene su primera evidencia en un trabajo delgrupo de Attardi, que documenta que las mitocon-drias se deterioran con la edad como consecuenciade la acumulación de mutaciones.15 Las variaciones desecuencia existentes entre diferentes individuos hanresultado muy útiles para estudios antropológicos, et-nológicos y forenses, y es la base de la hipótesis de

que el hombre desciende de una mujer que vivió enAfrica hace unos 250 000 años (“Eva mitocondrial”).16

Enfermedades genéticas del DNAmitocondrial

Las enfermedades originadas por daños en el genomamitocondrial tienen en común el estar producidas poruna deficiencia en la biosíntesis de ATP, ya que toda lainformación que contiene este DNA está dirigida ala síntesis de proteínas componentes del sistemaOxphos. Las manifestaciones de estas enfermedadesson muy variadas y pueden afectar a todos los órga-nos y tejidos, ya que la síntesis de ATP se produce entodos ellos y a cualquier edad. Estas pueden presentaruna serie de aspectos clínicos, morfológicos y bioquí-micos muy concretos que dan lugar a síndromes biencaracterizados pero, en la mayor parte de los casos,principalmente en edad pediátrica, los síntomas sonmuy poco informativos y es sólo la presencia deanormalidades neurológicas, a veces acompañadasde aumento de ácido láctico y de otros síntomas clíni-cos secundarios que afectan a diversos órganos, lo queda alguna orientación en el diagnóstico de una en-fermedad mitocondrial.17 Entre las manifestacionesclínicas más comunes se encuentran una o varias delas siguientes: desórdenes motores, accidentes cere-brovasculares, convulsiones, demencia, intolerancia alejercicio, ptosis, oftalmoplejia, retinopatía pigmenta-ria, atrofia óptica, ceguera, sordera, cardiomiopatía,disfunciones hepáticas y pancreáticas, diabetes, defec-tos de crecimiento, anemia sideroblástica, pseudoobstrucción intestinal, nefropatías, acidosis metabó-lica y otras más secundarias.

La presencia de uno o más de estos síntomas re-quiere a continuación de un estudio morfológico, his-toquímico y bioquímico para asegurar la naturalezade estas enfermedades. Así, con mucha frecuencia seencuentran: fibras rojo-rasgadas (acumulación de mi-tocondrias anormales en tamaño y número) en biop-sias musculares teñidas con tricromo de Gomori yfibras no reactivas a la tinción histoquímica de lacitocromo c oxidasa; defectos en uno o varios com-plejos de la cadena respiratoria; y desarreglos me-tabólicos con elevación de lactato, piruvato o unaaminoaciduria generalizada causados por una dis-función de la cadena respiratoria que conlleva un au-mento de equivalentes reductores en la mitocondriay citoplasma, y una alteración del funcionamiento delciclo de Krebs debido al exceso de NADH, lo que pro-voca una acumulación de piruvato y su posteriorconversión a lactato que difunde a la sangre. Sin em-



Solano A y col.

bargo, la ausencia de algunos de estos caracteres nodebe descartar la posibilidad de enfermedad mito-condrial, especialmente en pacientes en edad pediá-trica. Además, los estudios familiares pueden serdecisivos si se comprueba la existencia de herenciamaterna de la enfermedad. El estudio genético del pa-ciente y familiares relacionados por vía maternapueden asegurar finalmente que nos encontramosante este tipo de trastornos. De hecho, hoy en día, eldesarrollo y rapidez de las técnicas de genética mo-lecular permiten, en ocasiones, una confirmación dela enfemedad antes de haber realizado muchas de laspruebas anteriormente citadas. La complejidad deldiagnóstico de estas enfermedades hace preciso quelos pacientes tengan que acudir a centros muy espe-cializados donde se pueda llevar a cabo evaluacionesclínicas, metabólicas, patológicas, bioquímicas y ge-néticas, y a que en su diagnóstico estén implicadosespecialistas de muy diverso origen.

Desde que en 1988 se describieran las primerasenfermedades causadas por daños en el mtDNA,7-9 sehan encontrado más de 150 mutaciones (más 100 dele-ciones y unas 50 mutaciones puntuales) asociadas aenfermedades humanas. El interés por su estudio hacrecido enormemente debido al gran aumento de pa-cientes diagnosticados con estos trastornos y a que sepresentan desde en recién nacidos hasta en adultosde todas las edades. Además, muchas de estas mu-taciones se trasmiten por línea materna, como se haindicado anteriormente, lo que hace que el diagnós-tico en un individuo pueda tener implicaciones enmuchas generaciones de una familia.

A pesar de la importancia que las enfermedadesmitocondriales tienen últimamente y de ser respon-sables de una considerable morbilidad, hasta ahorano se han realizado estudios exhaustivos sobre suprevalencia en la población general. Las razones sonmúltiples:18 complejidad de las manifestaciones clí-nicas, necesidad de biopsias musculares para sudiagnóstico (no siempre se pueden detectar las mu-taciones en muestras de sangre); necesidad de se-cuenciar todo el genoma mitocondrial para poderlocalizar mutaciones no detectadas hasta ahora, pro-blemas éticos para realizar análisis genéticos presin-tomáticos en niños, diagnóstico erróneo de muchospacientes al no ser atendidos en centros especializa-dos, etcétera. Sin embargo, a pesar de todas estas difi-cultades, el grupo del doctor Turnbull, en Newcastle,Reino Unido, ha publicado muy recientemente losprimeros datos epidemiológicos de las enfermeda-des del mtDNA, centrados en la población blanca deEuropa del Norte residente en el noreste de Inglate-rra.18 Así, ha mostrado que los defectos en el mtDNA

son la causa de enfermedad en 6.57 de cada 100 000individuos de la población adulta trabajadora y que7.59 por cada 100 000 adultos y niños no afectadoscorren el riesgo de desarrollar una de estas enfer-medades. En total, 12.48 por 100 000 individuos (1 decada 8 000) tienen o presentan un riesgo de padeceruna enfermedad causada por daños en el mtDNA.Estos datos representan un mínimo de prevalenciaporque, muy probablemente, el número de pacientesque han quedado sin diagnosticar es elevado por ha-ber sido atendidos por médicos de asistencia prima-ria, y no en clínicas neurológicas, y que hayan podidopasar desapercibidos por presentar solamente algu-nos de los síntomas acompañantes de estas enfer-medades como diabetes o ptosis.

Los datos obtenidos por el grupo de Newcastlehan permitido comprobar que la prevalencia de lasenfermedades debidas a daños en el mtDNA, consi-deradas en su conjunto, es equivalente a la de otrasenfermedades neurológicas como la enfermedad deHuntington y la esclerosis amiotrófica lateral (6.4 y6.2 por cada 100 000 individuos, respectivamente),y superior a la de otras enfermedades neuromus-culares hereditarias como la distrofia de Duchenne(3.2 por cada 100 000 individuos).18

La experiencia de nuestro servicio de diagnós-tico en la Universidad de Zaragoza es que un 16% delos pacientes remitidos para el estudio genético pre-sentan una deleción o mutación puntual.19,20,* No he-mos realizado ningún estudio de lo que este númerorepresenta entre la población general española; pero,seguro que tanto en el estudio realizado en Inglaterracomo en nuestro laboratorio, el número de pacientesserá muy superior cuando se secuencie el mtDNAde todos los posibles implicados y se detecten nue-vas mutaciones. Estos números, junto al hecho deque no exista una terapia eficaz, y que, aunque algu-nas de estas enfermedades puedan mejorar o estabi-lizarse a lo largo de su curso, ilustran la importanciaque tienen en relación con la salud pública, particu-larmente en cuanto a su atención y consejo genético,pues la mayoría tiene un desenlace fatal.

La heterogeneidad de las manifestaciones clíni-cas, morfológicas y bioquímicas de las enfermedadesdel mtDNA, hace que su clasificación se base muyfrecuentemente en las características genéticas delas mutaciones, a pesar de que, en algunos casos, unamisma mutación pueda dar lugar a fenotipos clíni-cos muy diversos. Así, las enfermedades del mtDNA

* Solano A. Enfermedades del mtDNA (tesis doctoral). Zaragoza:Universidad de Zaragoza. En realización, 2000.



se pueden dividir en tres grandes grupos según esténasociadas a mutaciones puntuales, a reorganizacioneso a disminución de número de copias del mtDNA. Enla severidad de la manifestación de la enfermedad in-tervienen varios factores: la naturaleza de la muta-ción, el grado de heteroplasmia, los requerimientosenergéticos del tejido y la capacidad del tejido paracompensar el daño celular. A continuación se presentaun resumen de las enfermedades más comunes aso-ciadas a estos tipos de mutaciones.

Enfermedades asociadas a mutaciones puntualesen el mtDNA

Dado el alto índice de mutación del mtDNA, como seha indicado anteriormente, es posible encontrar ungran número de mutaciones puntuales. Sin embargo,la mayoría son mutaciones silenciosas que no causanningún tipo de defecto. Las mutaciones patológicas sepueden encontrar tanto en los genes de tRNA, derRNA, como en los codificantes de proteínas, y res-ponden siempre a un tipo de herencia materna.

Neuropatía óptica hereditaria de Leber. La neuropatía óp-tica hereditaria de Leber (LHON) se caracteriza por lapérdida bilateral de la visión central, originada poratrofia del nervio óptico. Aparece en la segunda o ter-cera década de la vida y afecta a más hombres que amujeres. Aunque normalmente sólo la visión estáafectada, hay casos en los que también aparecen tras-tornos en la conducción cardiaca, neuropatía perifé-rica y ataxia cerebelar.

Esta fue la primera enfermedad humana de he-rencia materna que se asoció a una mutación en elmtDNA. Después se llegó a asociar hasta con 16 mu-taciones puntuales (cuadro I), localizadas todas ellasen genes codificantes de proteínas, y que se clasifica-ron en primarias, secundarias o intermedias según surelación con la aparición de la enfermedad. Sin em-bargo, últimamente sólo tres, G3 460A, G11 778A yT14 484C, están consideradas como primarias o pa-togénicas verdaderas, siendo la G11 778A la respon-sable en 50% de los casos y la que provoca la formamás severa de la enfermedad. Las tres se encuentranen genes que codifican algún polipéptido del com-plejo I del sistema Oxphos. La detección de estas mu-taciones se suele hacer en células sanguíneas donde seencuentran tanto en forma homo como heteroplás-mica. El resto de las mutaciones se consideran comosecundarias, suelen acompañar a las anteriores enforma homoplásmica y se desconoce su relación di-recta con la enfermedad. Entre estas últimas vale lapena mencionar que la mutación G15257A, consi-

derada como intermedia por algunos autores, se haencontrado en varias familias analizadas en nuestrolaboratorio por lo que pensamos que puede contribuirde forma decisiva a la aparición de la enfermedad.

La prevalencia de la enfermedad en hombres hasugerido la influencia de un gen nuclear, y aunquese ha descrito un ligamiento de la enfermedad conel locus (DXS7) situado en el cromosoma X en fami-lias finlandesas,21 no se ha podido confirmar en fa-milias de otro origen.Síndrome de neuropatía, ataxia y retinopatía pigmenta-ria. Este síndrome está caracterizado por debilidadmuscular neurogénica, ataxia y retinitis pigmentosa.Suele ir acompañado de demencia, convulsiones yneuropatía sensorial axonal, presenta una herenciamaterna 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 for-ma heteroplásmica y en todos los tejidos estudiados:leucocitos, fibroblastos, músculo, riñón y cerebro.Existe una alta correlación entre la proporción delDNA mutado y la severidad de la enfermedad.Síndrome de Leigh de herencia materna. El síndrome deLeigh de herencia materna (MILS) es una enferme-dad muy heterogénea que se puede presentar aso-ciada a diferentes tipos de herencia, autosómicarecesiva, ligada al cromosoma X o materna (mitocon-drial) según el gen que esté dañado. Es una enfer-medad devastadora que se caracteriza por trastornosdegenerativos multisistémicos que aparecen en elprimer año de vida, disfunciones del tallo cerebral yde los ganglios basales, desmielinización, regresiónpsicomotora, retraso en el desarrollo, ataxia, convul-siones, neuropatía periférica. El diagnóstico se confir-ma por la presencia de lesiones necróticas cerebralesfocales en el tálamo, tallo cerebral y núcleo dentado.La forma de la enfermedad, que se hereda por víamaterna, está producida por la mutación en el gen dela subunidad 6 de la ATPasa, T8993G, la misma queproduce el síndrome de neuropatía, ataxia y retino-patía pigmentaria, pero con un porcentaje de la mu-tación superior a 90%. Otras formas menos severasde esta enfermedad se han asociado con un cambioT→C* en la misma posición del mtDNA.Síndrome de epilepsia mioclónica con fibras rojo-rasgadas(MERRF). Este síndrome de herencia materna, está ca-racterizado por epilepsia mioclónica, convulsionesgeneralizadas y miopatía con presencia de fibras rojo-rasgadas. Otros síntomas clínicos que pueden acompa-ñar a los anteriores son demencia, sordera, neuropatía,

* Timina por citosina.



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atrofia óptica, fallo respiratorio y cardiomiopatía. Apa-rece tanto en la infancia como en edad adulta y es decurso progresivo. Está asociado a la presencia de mu-taciones en el gen del mtDNA para el tRNALys. En lamayoría de los casos (80%-90%) se debe a una mutaciónA8344G, pero también se han encontrado otras mi-

noritarias como T8356C (cuadro I), todas en formaheteroplásmica. El porcentaje de heteroplasmia nece-sario para la afectación varía entre individuos jóve-nes (95%) e individuos por encima de los 60-70 años(60%) del DNA mutado.13 La presencia de estas muta-ciones en tRNA daña la síntesis de proteínas.

LHONMutaciones primarias G3460A ND1 22,23

G11778A ND4 7

T14484C ND6 24

Mutaciones intermedias G5244A ND2 25

G15257A Citocromo b 25

Mutaciones secundarias T3394C ND1 24

T4160C ND1 26

T4216C ND1 27

A4917G ND2 27

G7444A CO I 28

T9101C ATPasa 6 29

G9438A CO III 30

G9804A CO III 30

G13708A ND5 27

G13730A ND5 31

G14459A ND6 32

G15812A cyt b 25

NARP T8993G ATPasa 6 33

Leigh (MILS) T8993G ATPasa 6 34,35

T8993C ATPasa 6 36

MELAS A3243G tRNALeu(uur) 37

C3256T tRNALeu(uur) 38

T3271C tRNALeu(uur) 39

T3291C tRNALeu(uur) 40

T9957C COIII 41

MERRF A8344G tRNALys 42

T8356C tRNALys 43

Diabetes y sordera A3243G tRNALeu(uur) 44

Cardiomiopatía (MICM) A3260G tRNALeu(uur) 45


A4269G tRNAIle 47

A4300G tRNAIle 48

A4317G tRNAIle 49

C4320T tRNAIle 50

G8363A tRNALys 51

T9997C tRNAGly 52

Cuadro IMUTACIONES EN EL MTDNA Y ENFERMEDADES ASOCIADAS

Enfermedad Mutación Gen Mutación Genen el mtDNA afectado Referencias Enfermedad en el mtDNA afectado Referencias

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

Miopatía mitocondrial T3250C tRNALeu(uur) 53

A3302G tRNALeu(uur) 54

C15990T tRNAPro 55

Sordera inducida poraminoglicósidos A1555G 12S rRNA 56

Sordera sensoneural T7445C tRNASer(ucn) 57

Anemia sideroblástica G12301A tRNALeu(cun) 58

Lipomatosis múltiplesimétrica A8344G tRNALys 59

CPEO Deleción única 60


A5692G tRNAAsn 62

G5703A tRNAAsn 63


Intolerancia al ejercicio G15084A Citocromo b 64




delecion de 24pb Citocromo b 64

LIMM A15923G tRNAThr 65

Muerte súbita A3251G tRNALeu(uur) 66

Necrosis bilateraldel estriado T9176C ATPase 6 67

T8851C ATPase 6 68

Multisistémicas A3251G tRNALeu(uur) 66



Corea y demencia G5549A tRNATrp 70

LHON y distonía G14459A ND6 32

Diabetes y miopatía T14709C ND6 71

Pearson Deleción única 72

Kearns-Sayre Deleción única 73



Síndrome de encefalomiopatía mitocondrial con acido-sis láctica y episodios de accidentes cerebro-vasculares(MELAS). Se trata de una encefalomiopatía mitocon-drial, de herencia materna, caracterizada por accidentescerebrovasculares producidos a edad temprana queprovocan una disfunción cerebral subaguda y cam-bios en la estructura cerebral, y por acidosis láctica.Estos caracteres suelen ir acompañados de convul-siones generalizadas, dolor de cabeza, sordera, demen-cia y, a veces, presenta fibras rojo-rasgadas.

Esta enfermedad ha sido asociada fundamental-mente con mutaciones en el gen del tRNALeu(UUR) delmtDNA. La mayor parte de los casos (80%) está asocia-da a la mutación A3.243G, pero también se han encon-trado otras con menor incidencia y alguna en genescodificantes de proteínas (cuadro I), todas en formaheteroplásmica. Al igual que en la epilepsia mioclóni-ca, las mutaciones en el tRNA dañan la síntesis de pro-teínas mitocondriales.Diabetes de herencia materna con sordera. Además de losdos tipos clásicos de diabetes dependiente y no de-pendiente de insulina (tipo 1 y 2, respectivamente), seha descrito recientemente un nuevo tipo de diabetesasociada a sordera, que no encuadra dentro de la cla-sificación de la Organización Mundial de la Salud.Esta diabetes, de herencia materna, está producidapor 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 apro-ximadamente de un 1.5% de la población diabéticatotal.74 Por otra parte, la diabetes es una de las enferme-dades que se han descrito asociadas a otros síndromesmitocondriales como la encefalomiopatía mitocon-drial, oftalmoplejia progresiva externa crónica, Kearns-Sayre, Pearson y diabetes insípida, diabetes mellitus,atrofia óptica y sordera (DIDMOAD).

Otras enfermedades del mtDNA asociadas a mutacionespuntuales

Además de las enfermedades descritas anteriormente,hay otras muchas que se han asociado a otras muta-ciones puntuales (cuadro I). Entre ellas, se pueden citarlas cardiomiopatías de herencia materna relacionadasfundamentalmente con mutaciones en el tRNAIle: lasordera inducida por aminoglicósidos que está pro-ducida por una mutación en el rRNA 12S (A1555G), yotros tipos de sordera sindrómica o no sindrómica deherencia materna; LHON y distonía; miopatías de he-rencia materna unidas a mutaciones en tRNALeu,tRNAPro, tRNAAsn, tRNATyr; oftalmoplejia progresivaexterna crónica; anemia sideroblástica; deficienciafatal de la cadena respiratoria infantil; lipomatosis si-

métrica múltiple asociada a la mutación A8.344G delgen del tRNALys (descrita en nuestro laboratorio) y,recientemente, se ha relacionado la intolerancia alejercicio, como entidad propia, a mutaciones pun-tuales en el gen del citocromo b. Así, se han descritomutaciones en este gen que crean un codón de termi-nación, que cambian un aminoácido o, incluso, unadeleción de 24 pares de bases. En el cuadro I se citanéstos y otros síndromes que se han asociado a muta-ciones puntuales y, sin ninguna duda, el espectro defenotipos relacionados con mutaciones en el mtDNAaumentará más en un futuro. Asimismo, cabe mencio-nar que alguna de las mutaciones, como la A3.243G,puede estar relacionada con muy diversos fenotiposclínicos como sindromes de encefalopatía mitocon-drial con acidosis láctica y episodios de accidentescerebrovasculares de epilepsia mioclónica con fibrasrojo-rasgadas y solapados, cardiomiopatías, CPEO,etcétera. Actualmente, se estudia la posible implica-ción del mtDNA en enfermedades neurodegenerati-vas como Parkinson y Alzheimer.

Enfermedades asociadas a reorganizaciones en el DNAmitocondrial

Además de las mutaciones puntuales, el mtDNA pue-de sufrir otro tipo de daños como son la pérdida departe del mismo (deleciones) o la adición de un nuevofragmento del DNA (duplicaciones), que, como en loscasos anteriores, afectan a la biogénesis del sistemaOxphos y, por tanto, a la síntesis de ATP. En la actua-lidad hay descritos más de 100 tipos de deleciones ysólo unos cuantos casos de inserciones. Este tipo demutaciones suelen ser espontáneas, probablementecausadas por daños en genes nucleares que controlanla replicación del mtDNA, aunque hay descritos casosde herencia materna.75 Se presentan siempre en formaheteroplásmica, ya que la homoplasmia sería incom-patible con la vida, y se sabe que la gravedad de loscasos aumenta con la edad debido a la ventaja replica-tiva de estas moléculas de DNA más pequeñas en re-lación con la de tamaño normal.

Los tres tipos de síndromes más comunes en losque se presentan deleciones son los de Pearson, oftal-moplejia progresiva externa crónica y Kearns-Sayre.

Síndrome de médula ósea-páncreas de Pearson. Es una en-fermedad que aparece en los primeros años de vida yque afecta a la hematopoyesis y a la función pancreá-tica exocrina. Las características clínicas más comunesson anemia sideroblástica con vacuolización de pre-cursores de la médula ósea que se manifiesta con unaanemia macrocítica, trombocitopenia y neutropenia.



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Los niños afectados suelen morir antes de los tresaños de edad y los que sobreviven suelen desarrollarposteriormente el fenotipo de Kearns-Sayre, que ve-remos más adelante. Estos pacientes presentan dele-ciones grandes únicas del mtDNA, en general sonesporádicas aunque se ha descrito algún caso de he-rencia materna.Oftalmoplejia progresiva externa crónica. Esta enferme-dad está caracterizada por oftalmoplejia, ptosis bila-teral de los párpados y miopatía. Suele ir acompañadatambién de intolerancia al ejercicio y debilidad mus-cular. En el músculo se encuentran fibras rojo-rasga-das COX negativas. En general, es una enfermedadbenigna que suele aparecer en la adolescencia o enadultos jóvenes. Aparece de forma esporádica sin his-toria familiar. Se ha asociado fundamentalmente adeleciones grandes y únicas en el mtDNA (ver másadelante). Asimismo, se han encontrado otras for-mas de CPEO con mutaciones puntuales de herenciamaterna (cuadro I) o con deleciones múltiples de he-rencia autosómica recesiva o dominante.Síndrome de Kearns-Sayre. Este síndrome es, por otraparte, una enfermedad multisistémica progresivacaracterizada clinicamente por CPEO, retinopatía pig-mentaria atípica, ataxia, miopatía mitocondrial, blo-queo de la conducción cardiaca, elevados niveles deproteína CSF (fluido cerebro espinal, por sus siglas eninglés), sordera y demencia. Aparece antes de los 20años de edad.

Estas tres enfermedades están causadas por dele-ciones (de 2 a 9 kb) en el mtDNA que suelen aparecerde forma espontánea. En general, la deleción es única,pero también se han descrito casos de deleciones múl-tiples. La gravedad de la enfermedad depende del por-centaje de DNA mutado en el individuo. En generalestán localizadas en el arco grande comprendido entrelos orígenes de replicación del DNA y mantienen siem-pre las secuencias requeridas para la replicación delDNA y los promotores de la transcripción. Entre to-das las deleciones conocidas, hay una que aparece conmás frecuencia (hasta en 50%), la llamada delecióncomún, que elimina un tramo de DNA de 4 977 paresde bases (entre los nucleótidos 8 483 a 13 460), quecomprende los genes localizados entre la subunidad8 de la ATPasa y ND5 (figura 1). No existe una clararelación entre el fenotipo y el tipo, tamaño o porcen-taje del DNA delecionado ya que la misma deleciónpuede dar lugar a varios fenotipos diferentes. La ma-yor parte de las deleciones encontradas están flan-queadas por repeticiones directas de longitud variable(3-13 nt). Este hecho sugiere que la delección se pro-duce por errores sucedidos en el proceso de replica-

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, portanto, que sean dependientes de complementacióncon moléculas de mtDNA normales en la misma mi-tocondria. El umbral se suele alcanzar cuando el por-centaje de moléculas delecionadas supera 60%.

Existen otras enfermedades como una diabetescon sordera y atrofia óptica; miopatías en general; elsíndrome de encefalomiopatía mitocondrial neuro-gastrointestinal; el de diabetes mellitus, diabetes in-sípida, atrofia óptica y sordera, etcétera, que estánasociadas a la presencia de deleciones en el mtDNA.

Como se ha mencionado anteriormente, entre lasreorganizaciones del mtDNA se pueden encontrarduplicaciones en pacientes con defectos en el sistemaOxphos. Estas pueden ser también esporádicas o deherencia materna. Se han encontrado en pacientesde Kearns-Sayre, Pearson, diabetes mellitus, tubu-lopatía renal y miopatía mitocondrial e incluso en in-dividuos normales. El mecanismo por el cual puedencausar la patogenicidad no está nada claro todavía.

Enfermedades asociadas a depleciones de DNAmitocondrial

El tercer tipo de daños en el genoma mitocondrial quepuede causar enfermedades no se debe a mutacionespropiamente dichas sino a una disminución de los ni-veles del mtDNA. El espectro clínico que produce ladepleción es muy variado. Los casos descritos hastaahora afectan fundamentalmente a niños con combi-naciones variables de miopatía, nefropatía o hepa-topatía, miopatía infantil fatal por fallo respiratorio yalgún otro con implicación multisistémica. La deple-ción puede estar producida por mutaciones en genesnucleares que controlan el número de copias delmtDNA. Es, por tanto, un trastorno de herencia men-deliana que afecta a la coordinación núcleo-mito-condria, y que parece ser autosómico recesivo.

Regreso a la genética mendeliana

Debido al doble origen genético nuclear y mitocon-drial del sistema Oxphos las enfermedades genéti-cas mitocondriales pueden estar originadas, ademásde por mutaciones en genes del mtDNA con herenciamaterna, como ya hemos visto, por mutaciones engenes nucleares que codifican proteínas mitocon-driales, por mutaciones que afecten al procesamientopostraduccional, al importe de proteínas por la mito-condria y al ensamblaje de los complejos, y por mu-taciones que afecten al control nuclear del genoma



mitocondrial, todas ellas con un tipo de herencia men-deliana.

El primer caso ha sido hasta ahora el más estudia-do por estar el genoma mitocondrial completamentesecuenciado y, por tanto, por la facilidad de encontrarmutaciones que afecten a los genes que codifica. Sinembargo, la mayor parte de los genes que compo-nen el sistema Oxphos es de origen nuclear y cabeesperar que la mayoría de las enfermedades causadaspor la deficiencia de este sistema sean debidas a mu-taciones en el DNA nuclear. Así, por ejemplo, a pesarde que una de las causas del síndrome de Leigh seauna mutación en el mtDNA de herencia materna, sesabe que se transmite con más frecuencia por herenciaautosómica recesiva, y se han encontrado e identifi-cado mutaciones en genes de subunidades del com-plejo I, codificadas por el DNA nuclear76,77 y en elcomplejo II,78 codificado enteramente en el núcleo.Asimismo, se han localizado mutaciones en un gennuclear (SURF 1) que codifica una proteína que, aunqueno forma parte del complejo IV, es necesaria para suensamblaje.79,80

Además, en la mitocondria existen muchas otrasrutas metabólicas en las que no participa para nada elmtDNA y cuya deficiencia puede causar encefalomio-patías mitocondriales. Por todo ello, la genética men-deliana de las enfermedades mitocondriales estátodavía prácticamente por descubrir y nos proporcio-nará mucha información sobre estos trastornos.

Referencias

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