Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1.

Mitochondrial fusion depends on the dynamin-like guanosine triphosphatase OPA1, whose activity is controlled by proteolytic cleavage. Dysfunction of mitochondria induces OPA1 processing and results in mitochondrial fragmentation, allowing the selective removal of damaged mitochondria. In this study, we demonstrate that two classes of metallopeptidases regulate OPA1 cleavage in the mitochondrial inner membrane: isoenzymes of the adenosine triphosphate (ATP)-dependent matrix AAA (ATPase associated with diverse cellular activities [m-AAA]) protease, variable assemblies of the conserved subunits paraplegin, AFG3L1 and -2, and the ATP-independent peptidase OMA1. Functionally redundant isoenzymes of the m-AAA protease ensure the balanced accumulation of long and short isoforms of OPA1 required for mitochondrial fusion. The loss of AFG3L2 in mouse tissues, down-regulation of AFG3L1 and -2 in mouse embryonic fibroblasts, or the expression of a dominant-negative AFG3L2 variant in human cells decreases the stability of long OPA1 isoforms and induces OPA1 processing by OMA1. Moreover, cleavage by OMA1 causes the accumulation of short OPA1 variants if mitochondrial DNA is depleted or mitochondrial activities are impaired. Our findings link distinct peptidases to constitutive and induced OPA1 processing and shed new light on the pathogenesis of neurodegenerative disorders associated with mutations in m-AAA protease subunits.

Genetic interaction between the m-AAA protease isoenzymes reveals novel roles in cerebellar degeneration.

The mitochondrial m-AAA protease has a crucial role in axonal development and maintenance. Human mitochondria possess two m-AAA protease isoenzymes: a hetero-oligomeric complex, composed of paraplegin and AFG3L2 (Afg3 like 2), and a homo-oligomeric AFG3L2 complex. Loss of function of paraplegin (encoded by the SPG7 gene) causes hereditary spastic paraplegia, a disease characterized by retrograde degeneration of cortical motor axons. Spg7(-/-) mice show a late-onset degeneration of long spinal and peripheral axons with accumulation of abnormal mitochondria. In contrast, Afg3l2(Emv66/Emv66) mutant mice, lacking the AFG3L2 protein, are affected by a severe neuromuscular phenotype, due to defects in motor axon development. The role of the homo-oligomeric m-AAA protease and the extent of cooperation and redundancy between the two isoenzymes in adult neurons are still unclear. Here we report an early-onset severe neurological phenotype in Spg7(-/-) Afg3l2(Emv66/+) mice, characterized by loss of balance, tremor and ataxia. Spg7(-/-) Afg3l2(Emv66/+) mice display acceleration and worsening of the axonopathy observed in paraplegin-deficient mice. In addition, they show prominent cerebellar degeneration with loss of Purkinje cells and parallel fibers, and reactive astrogliosis. Mitochondria from affected tissues are prone to lose mt-DNA and have unstable respiratory complexes. At late stages, neurons contain structural abnormal mitochondria defective in COX-SDH reaction. Our data demonstrate genetic interaction between the m-AAA isoenzymes and suggest that different neuronal populations have variable thresholds of susceptibility to reduced levels of the m-AAA protease. Moreover, they implicate impaired mitochondrial proteolysis as a novel pathway in cerebellar degeneration.

OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria.

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.

Redox cycling of adrenaline and adrenochrome catalysed by mitochondrial Complex I.

Complex I in bovine heart submitochondrial particles catalyses the NADH-supported generation of superoxide anion; adrenaline is oxidised by superoxide to adrenochrome that, on its hand, is reduced by Complex I, thus establishing a redox cycle that amplifies the superoxide production. The routes in Complex I for superoxide formation and for adrenochrome reduction appear to be different, since they have a different sensitivity to Complex I inhibitors. The results are discussed in terms of current assays for superoxide detection and of pathologies linked to catecholamine oxidation.

The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria.

AAA proteases comprise a conserved family of membrane bound ATP-dependent proteases that ensures the quality control of mitochondrial inner-membrane proteins. Inactivation of AAA proteases causes pleiotropic phenotypes in various organisms, including respiratory deficiencies, mitochondrial morphology defects, and axonal degeneration in hereditary spastic paraplegia (HSP). The molecular basis of these defects, however, remained unclear. Here, we describe a regulatory role of an AAA protease for mitochondrial protein synthesis in yeast. The mitochondrial ribosomal protein MrpL32 is processed by the m-AAA protease, allowing its association with preassembled ribosomal particles and completion of ribosome assembly in close proximity to the inner membrane. Maturation of MrpL32 and mitochondrial protein synthesis are also impaired in a HSP mouse model lacking the m-AAA protease subunit paraplegin, demonstrating functional conservation. Our findings therefore rationalize mitochondrial defects associated with m-AAA protease mutants in yeast and shed new light on the mechanism of axonal degeneration in HSP.

The mitochondrial production of reactive oxygen species in relation to aging and pathology.

Mitochondria are known to be strong producers of reactive oxygen species (ROS) and, at the same time, particularly susceptible to the oxidative damage produced by their action on lipids, proteins, and DNA. In particular, damage to mtDNA induces alterations to the polypeptides encoded by mtDNA in the respiratory complexes, with consequent decrease of electron transfer, leading to further production of ROS and thus establishing a vicious circle of oxidative stress and energetic decline. This deficiency in mitochondrial energetic capacity is considered the cause of aging and age-related degenerative diseases. Complex I would be the enzyme most affected by ROS, since it contains seven of the 13 subunits encoded by mtDNA. Accordingly, we found that complex I activity is significantly affected by aging in rat brain and liver mitochondria as well as in human platelets. Moreover, due to its rate control over aerobic respiration, such alterations are reflected on the entire oxidative phosphorylation system. We also investigated the role of mitochondrial complex I in superoxide production and found that the one-electron donor to oxygen is most probably the Fe-S cluster N2. Short chain coenzyme Q (CoQ) analogues enhance ROS formation, presumably by mediating electron transfer from N2 to oxygen, both in bovine heart SMP and in cultured HL60 cells. Nevertheless, we have accumulated much evidence of the antioxidant role of reduced CoQ(10) in several cellular systems and demonstrated the importance of DT-diaphorase and other internal cellular reductases to reduce exogenous CoQ(10) after incorporation.

The various sources of mitochondrial oxygen radicals: a minireview.

The mitochondrial respiratory chain is the main source of reactive oxygen species in the cell. The main sites of superoxide radical production are within Complexes I and III; however, Complex II, glycerol phosphate dehydrogenase and dihydroorotate dehydrogenase, are also sources of oxygen radicals. By using specific inhibitors within the complexes it is possible to obtain indications at the sites where redox components react with oxygen and in the compartments where they are released. The production of oxygen radicals is enhanced when the electron flow is lowered, and partial uncoupling is believed to be a means of preventing excess radical production.

Mitochondrial production of oxygen radical species and the role of Coenzyme Q as an antioxidant.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), which is considered as the pathogenic agent of many diseases and of aging. We have investigated the role of complex I in superoxide radical production and found by the combined use of specific inhibitors of complex I that the one-electron donor to oxygen in the complex is a redox center located prior to the sites where three different types of Coenzyme Q (CoQ) competitors bind, to be identified with an Fe-S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short-chain Coenzyme Q analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective, raising doubts on its safety as a drug. Cells counteract oxidative stress by antioxidants. CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT-diaphorase are two such systems, likewise, they are overexpressed under oxidative stress conditions.

Ubiquinol and a coenzyme Q reducing system protect platelet mitochondrial function of transfusional buffy coats from oxidative stress.

The conditions under which Coenzyme Q (CoQ) may protect platelet mitochondrial function of transfusional buffy coats from aging and from induced oxidative stress were investigated. The Pasteur effect, i.e. the enhancement of lactate production after inhibition of mitochondrial respiratory chain, was exploited as a marker of mitochondrial function as it allows to calculate the ratio of mitochondrial ATP to glycolytic ATP. Reduced CoQ10 improves platelet mitochondrial function of transfusional buffy coats and protects the cells from induced oxidative stress. Oxidized CoQ is usually less effective, despite the presence, shown for the first time in this study, of quinone reductase activities in the platelet plasma membranes. The addition of a CoQ reducing system to platelets is effective in enhancing the protection of platelet mitochondrial function from the oxidative stress. The results support on one hand a possibility of protection of mitochondrial function in aging by exogenous CoQ intake, on the other a possible application in protection of transfusional buffy coats from storage conditions and oxidative deterioration.