OPA1 mutations and mitochondrial DNA damage: keeping the magic circle in shape (original) (raw)

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Unit of Molecular Neurogenetics, Foundation ‘Istituto Neurologico Carlo Besta’, Via Temolo 4 – 20126 Milano, Italy

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01 February 2008

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Structural instability of mitochondrial DNA (mtDNA), consisting either of large-scale rearrangements, tissue-specific depletion or both, is a major cause of mitochondrial dysfunction and disease in humans (Zeviani and Di Donato, 2004). Almost 20 years have elapsed since the discovery that single, large-scale deletions (Holt et al., 1988) across short direct repeats (Mita et al., 1990) occur and become clonally expanded (Mita et al., 1989) in the mtDNA of patients with the sporadic form of Kearns–Sayre syndrome (Zeviani et al., 1988) or its milder variant, progressive external ophthalmoplegia (PEO; Moraes et al., 1989). Shortly thereafter, the accumulation of multiple mtDNA-deleted species was observed in families with recurrent cases of PEO transmitted as an autosomal dominant trait (Zeviani et al., 1989). Mendelian inheritance of mtDNA mutations appeared at first to be a contradiction, since this genome is transmitted in a strictly maternal fashion; but this was explained as the consequence of a dominant mutation in a nuclear gene affecting the structural integrity of mtDNA (Zeviani et al., 1990). This hypothesis proved to be correct, as it led to the discovery of at least four genes responsible for familial PEO associated with multiple mtDNA deletions. These genes encode ANT1, the muscle-specific isoform of the mitochondrial adenine nucleotide translocator (Kaukonen et al., 2000); Twinkle, a mtDNA helicase (Spelbrink et al., 2001); and pol γA (van Goethem et al., 2001) and B (Longley et al., 2006); the subunits of the mtDNA-specific polymerase holoenzyme, pol γ. Mutations in pol γA, the larger catalytic subunit of the pol γ holoenzyme, are indeed responsible for a much wider spectrum of clinical presentations (Horvath et al., 2006), including not only dominant but also recessive PEO (Lamantea et al., 2002), juvenile-onset spino-cerebellar ataxia and epilepsy (Tzoulis et al., 2006) and infantile hepatopathic poliodystrophy (Alpers–Huttenlocher syndrome, AHS; Nguyen et al., 2005). Rather than multiple mtDNA deletions, AHS and its juvenile variant are hallmarked by mtDNA depletion in affected tissues (Ferrari et al., 2005). Other forms of mtDNA depletion are associated with mutations in a number of nuclear genes, most of which encode enzymes regulating the mitochondrial supply of deoxynucleotides (Spinazzola and Zeviani, 2007). A case apart is MNGIE, or myo-neuro-gastro-intestinal encephalomyopathy, a disease resulting from combined mtDNA depletion, point mutations and multiple deletions (Nishigaki et al., 2003). Lack of thymidine phosphorylase, a key enzyme in pyrimidine catabolism, is the cause of MNGIE (Nishino et al., 1999).

The complexity of this intricate genetic, biochemical and clinical scenario is further enriched by the contribution of papers that appear in this issue of Brain (Amati-Bonneau et al., 2008; Hudson et al., 2008). Centre stage this time is an unexpected villain. Amati-Bonneau et al. and Hudson et al. both show that some dominant missense mutations in OPA1, the optic atrophy 1 gene, cause the accumulation of multiple mtDNA deletions in skeletal muscle. This finding, which makes OPA1 the fifth gene associated with ‘mtDNA breakage syndromes’, is indeed striking for several reasons.

As the name suggests, OPA1 is linked to non-syndromic autosomal dominant optic atrophy (ADOA) (Alexander et al., 2000; Delettre et al., 2000), a condition characterized by slowly progressive visual loss starting in childhood, first described by the Danish ophthalmologist Poul Kjer in 1959 (Kjer et al., 1959). However, the syndrome reported here is more complex, consisting of a combination of ADOA with PEO, peripheral neuropathy, ataxia and deafness. Most of these patients have ragged-red and cytochrome-c-oxidase negative muscle fibres, with paracrystalline inclusions filling abnormally shaped mitochondria. These clinical and morphological features are typical of mitochondrial encephalomyopathies associated with multiple mtDNA deletions, suggesting that it is the instability of mtDNA in critical tissues to act as a common mechanism in all forms of autosomal-dominant PEO. However, the presence of early-onset optic atrophy, which is rare if not exceptional in other autosomal-dominant PEO syndromes but invariant in OPA1 mutations, indicates that a second disease mechanism is likely to be at work here, directly linked to OPA1 dysfunction. ‘OPA1-plus’ syndrome is the term proposed by Amati-Bonneau et al. for this new clinical entity. The molecular details of the two distinct pathomechanisms, which seem to act independently on different target tissues, are presently unknown.

Another matter of interest, and surprise, is that—in contrast with other PEO-associated proteins—OPA1 is not directly involved in mtDNA replication, at least as far as we know at present. OPA1 is in fact a dynamin-like GTPase located in the inner mitochondrial membrane. It works as a nucleotide-propelled mechano-enzyme, which participates in organellar fusion and remodelling of mitochondrial cristae (Duvezin-Caubet et al., 2006). In addition, OPA1 functions as a gatekeeper of the cristae junctions, thus controlling the storage and release of cytochrome c. The latter mechanism makes OPA1 an important player in the regulation of mitochondrion-driven apoptosis (Olichon et al., 2003; Lee et al., 2004) Such a conclusion is further supported by a third interesting paper published in this issue of Brain, which not only confirms the involvement of OPA1 in apoptosis but also provides some evidence of a direct physical interaction between OPA1 and several complexes of the mitochondrial respiratory chain (Zanna, Ghelli et al., 2008). Given the essential role of this protein in fundamental homoeostatic control mechanisms of the cell, it is no wonder that ablation of OPA1 is embryonically lethal in recombinant mice (Davies et al., 2007). Also, in the budding yeast Saccharomyces cerevisiae, the absence of Mgm1, the OPA1 orthologue, determines profound distortion of cristae shape, lack of growth on aerobic substrates and the formation of ‘petite’ colonies characterized by mtDNA loss (ρ0) or rearrangements (ρ-; Sesaki et al., 2003). Functional diversity reflects structural complexity: at least eight OPA1 isoforms are produced in humans by differential splicing of a single gene, but the specific roles of these numerous variants are not known either functionally or mechanistically (Duvezin-Caubet et al., 2007). In humans, most ADOA-associated OPA1 mutations cause a heterozygous frameshift or introduce a stop codon, suggesting that haplo-insufficiency is the principal genetic mechanism in non-syndromic visual loss (Cohn et al., 2007). By contrast, the defects in ‘OPA1-plus’, linked to accumulation of multiple mtDNA deletions, are all heterozygous missense mutations. This suggests that it is an additional ‘toxic’ effect, exerted by an abnormal OPA1 species, rather than the simple decrease of protein content, that causes mtDNA damage.

What then could this effect be? One possibility is suggested by the observation that four of the five OPA1-plus mutations are clustered in, or in close proximity to, the catalytic GTPase site of the protein. According to Amati-Bonneau et al., impaired, or rather increased, GTP hydrolysis by mutant OPA1 variants could affect the supply of nucleotides, which constitute the ‘building blocks’ of mtDNA biosynthesis. Imbalance of nucleotide pools are believed to determine the stalling of the mtDNA replisome, which could in turn trigger recombinogenic phenomena, such as single-strand branch migration and strand reinvasion, ultimately leading to the generation of large-scale rearrangements (Zeviani et al., 1996; Hirano et al., 2001). An objection to this hypothesis comes from the consideration that the GTPase domain of OPA1 seems to project into, and exploit, the GTP pool of the mitochondrial intermembrane space, rather than that of the mitochondrial inner compartment, where the mtDNA resides and is replicated (Olichon et al., 2002).

A second possibility is that OPA1 establishes physical contact with mtDNA, as part of mitochondrial nucleoids. These are nucleo-protein complexes that anchor the circular mtDNA to the matrix side of the inner membrane, and constitute the segregation units of the mitochondrial genome (Wang and Bogenhagen, 2006). Although the nucleoid protein set is still vaguely and incompletely characterized, nucleoid proteins are likely to play an important role not only in controlling mtDNA replication and transcription, but also in preserving its integrity from potential damage caused by oxygen radicals and other insults (Bogenhagen et al., 2007).

A third hypothesis portrays OPA1 as a major player in the biogenesis of mitochondria. Amati-Bonneau et al. show that, in conditions of strictly aerobic metabolism, OPA1-plus mutant cells display fragmentation of the mitochondrial network and dispersion of the organelles. This effect can have profound consequences on mtDNA maintenance and repair. For instance, the segregation of single nucleoids in physically and functionally separate organelles could impede spontaneously rearranged mitochondrial genomes from being functionally complemented and structurally repaired by homologous recombination with intact genomes. This could in turn determine the formation of severely dysfunctional mitochondria, then inducing proliferation by homoeostatic signals that regulate mitochondrial biogenesis. As a consequence, a vicious cycle would ensue, promoting the clonal amplification of defective genomes, accumulation of mtDNA deleted species, more mitochondrial damage and, ultimately, the onset of clinical symptoms (Moslemi et al., 1996).

These hypotheses are not mutually exclusive and each can be tested experimentally in mutant human cells or in recombinant OPA1 mouse models. Other possibilities can of course be envisaged, and more will certainly emerge with more detailed knowledge of the functions of the gene.

That mitochondrial proteins can serve more than one function, and be associated with polymorphic clinical phenotypes is nicely illustrated by yet another paper published in this issue of Brain. Votruba et al. (in press) describe the clinical effects of a murine mutation in OPA3, another mitochondrial protein associated with autosomal dominant optic atrophy, cataract and 3-methyl glutaconic aciduria in humans. Heterozygous mutant mice are normal, but homozygous individuals develop a multisystemic syndrome including, besides visual loss, dilated cardiomyopathy, extrapyramidal dysfunction, neuro-muscular impairment and reduced lifespan.

A relevant working hypothesis that proceeds from the results reported here is that additional factors controlling organelle dynamics, for instance mitofusins 1 and 2 (Detmer et al., 2007), also affect the integrity of mtDNA, thus serving as candidates for an expanding list of disorders arising from breakage of the ‘magic circle’.

Finally, the utmost fascinating lesson that this story tells us is perhaps a methodological one. As William Harvey wrote in 1657 AD, just a few days before he died: ‘Nature is nowhere accustomed more openly to display her secret mysteries than in cases where she shows traces of her workings apart from the beaten path’ (Harvey, 1657). Once again, astute clinical observations have been ushered into an unexpected scenario, offering new interpretations not only for the explanation of intriguing disease phenotypes, but also for the understanding of fundamental biological phenomena. In the spotlight this time is the connection between mitochondrial biogenesis and maintenance of the mitochondrial genome. The polymorphic clinical and molecular effects are revealed by rare, but exceptionally informative patients who, once again, have served to illuminate an unusual problem in the best traditions of clinical science.

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