Relations structure/fonction de la dynamine mitochondriale Msp1 : étude de ses relations avec la membrane interne et de ses activités biochimiques

La dynamique mitochondriale est définie comme un équilibre entre des forces antagonistes de fusion et de fission des membranes qui modulent la morphologie des mitochondries et jouent un rôle majeur dans la régulation des fonctions de l'organelle. Notre équipe a identifié l'un des acteurs de cette dynamique, une GTPase de la famille des dynamines appelée Msp1 chez la levure S. pombe et OPA1 chez l'homme. Le dysfonctionnement d'OPA1 est responsable de l'atrophie optique dominante de type 1. Cette dynamine agit sur la fusion des mitochondries, la structure des crêtes et le maintien de l'ADN mitochondrial (ADNmt). Elle module aussi la respiration et la synthèse d'ATP, avec des conséquences sur la viabilité cellulaire et sur l'apoptose chez les eucaryotes supérieurs. Au vue de cette pléiotropie, notre hypothèse est que la dynamine OPA1/Msp1 intervient dans l'organisation de la membrane interne des mitochondries. Mon travail de thèse a été initié pour définir les activités biochimiques de Msp1 à l'origine de ses effets biologiques, et les bases structurales sous-jacentes. Le rôle de deux domaines hydrophobes (TM1 et TM2) dans l'association aux membranes et les fonctions de Msp1 a été analysé in vivo dans la levure S. pombe. Les propriétés biochimiques de Msp1 dans l'organisation des membranes ont été étudiées in vitro après purification et reconstitution dans des liposomes de composition phospholipidique similaire à celle de la membrane interne. Nos études in vivo ont montré que TM1 et TM2, et un troisième site d'interaction membranaire (SIM3), ont des rôles distincts dans l'association de Msp1 aux membranes, et dans la fusion des mitochondries et le maintien de l'ADNmt. Nous suggérons que l'organisation locale de la membrane interne est modulée par les proportions relatives des sites (TM1 + TM2) / SIM3, reflet du rapport entre une forme longue, l-Msp1, et une forme courte, s-Msp1, qui co-existent naturellement chez la levure et diffèrent par la présence de TM1 et TM2 dans l-Msp1 uniquement, SIM3 étant commun aux deux isoformes. Dans notre système in vitro, l'association de l-Msp1 et s-Msp1 aux liposomes et leur topologie reproduisent la situation observée dans les mitochondries. Nous avons montré que s-Msp1 a une activité GTPase caractéristique des dynamines avec une faible affinité apparente pour le GTP et une forte activité catalytique et qu'un motif coiled-coil C-terminal est nécessaire au développement de cette activité. Grâce à ce système, nous mettons en évidence pour la première fois que Msp1 favorise l'hémifusion des liposomes et a donc un rôle direct dans la fusion membranaire. L'hémifusion est induite par les deux isoformes l Msp1 et s-Msp1 et est indépendante de l'activité GTPase et de l'oligomérisation de la dynamine. SIM3, qui est commun à l-Msp1 et s-Msp1, pourrait donc contenir les éléments structuraux requis pour l'hémifusion. L'ensemble de ce travail nous permet de proposer un modèle dans lequel Msp1 agirait comme organisateur de la membrane et comme commutateur moléculaire. A chacun de ses sites d'action, la dynamine entrerait dans des complexes multi-protéiques distincts dont la composition dépendrait des proportions relatives de chacune des isoformes, donc du rapport (TM1+TM2) / SIM3. La modification de ce rapport par l'hydrolyse du GTP modulerait l'organisation locale de la membrane et donc la composition et l'activité de ces complexes.Mitochondrial dynamics is defined as an equilibrium between antagonistic fusion and fission forces that control mitochondrial morphology and play a major role in the regulation of mitochondrial functions. Our group identified one actor of mitochondrial dynamics, a GTPase of the dynamins' family called Msp1 in the S.pombe yeast and OPA1 in humans. OPA1 dysfunction leads to type 1 dominant optic atrophy. This dynamin acts in mitochondrial fusion and is involved in the maintenance of cristae structure and of mitochondrial DNA (mtDNA). It also controls respiration and ATP synthesis with impacts on cellular viability and apoptosis in higher eukaryotes. Given this large spectrum of action, our hypothesis is that Msp1/OPA1 acts on the organization of the mitochondrial inner membrane. My PhD thesis was initiated to define the biochemical activities of Msp1 responsible for its biological effects, and the underlying structural basis. The role of two hydrophobic domains (TM1 and TM2) in Msp1 membrane association and functions was analysed in vivo in S. pombe cells. The biochemical properties of Msp1 in membranes organization were studied in vitro after purification and reconstitution into liposomes of phospholipd composition similar to that of the mitochondrial inner membrane. Our in vivo studies showed that TM1 and TM2, and a third site of membrane interaction (SIM3), play distinct roles in Msp1 membrane association and in mitochondrial fusion and mtDNA maintenance. We suggest that the local organisation of the inner membrane varies with the relative proportions of the domains (TM1+TM2)/SIM3 reflecting the ratio between a long, l-Msp1, and a short, s-Msp1, isoform that naturally occur in S. pombe and differ by the presence of TM1 and TM2 in l-Msp1 only, SIM 3 being common to both. In our in vitro system, the association and topology of l-Msp1 and s-Msp1 onto liposomes are similar to that observed in mitochondria. We showed that s-Msp1 has a GTPase activity similar to that of dynamins, with a low affinity for GTP and a high catalytic activity, and that a C-terminal coiled-coil motif is essential for this activity. With this system we show for the first time that Msp1 induces hemifusion of liposomes, indicating a direct role in membrane fusion. Hemifusion was induced by both l-Msp1 and s-Msp1 isoforms and was independent from GTPase activity and from self-assembly. SIM3, which is common to l-Msp1 and s-Msp1, may thus contain the structural requirements for hemifusion. Altogether, this work allows us to propose a model in which Msp1 could act as a membrane organizer and as a molecular switch. At each of its sites of action, the dynamin could enter in distinct multi-protein complexes, the composition of which could depend on the relative proportions of each Msp1 isoforms, i.e. of the (TM1+TM2)/SIM3 ratio. Modification of this ratio by GTP hydrolysis would modulate local membrane organisation, and hence the composition and activity of these complexes

Processing of the dynamin Msp1p in S. pombe reveals an evolutionary switch between its orthologs Mgm1p in S. cerevisiae and OPA1 in mammals

AbstractMitochondrial fusion depends on the evolutionary conserved dynamin, OPA1/Mgm1p/Msp1p, whose activity is controlled by proteolytic processing. Since processing diverges between Mgm1p (Saccharomyces cerevisiae) and OPA1 (mammals), we explored this process in another model, Msp1p in Schizosaccharomyces pombe. Generation of the short isoform of Msp1p neither results from the maturation of the long isoform nor correlates with mitochondrial ATP levels. Msp1p is processed by rhomboid and a protease of the matrix ATPase associated with various cellular activities (m-AAA) family. The former is involved in the generation of short Msp1p and the latter in the stability of long Msp1p. These results reveal that Msp1p processing may represent an evolutionary switch between Mgm1p and OPA1

The m.13051G>A mitochondrial DNA mutation results in variable neurology and activated mitophagy.

Maternally inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber hereditary optic neuropathy (LHON) in -1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects1 and there is no proven prophylactic treatment

Dysregulated mitophagy and mitochondrial organization in optic atrophy due to OPA1 mutations

Objective: To investigate mitophagy in 5 patients with severe dominantly inherited optic atrophy (DOA), caused by depletion of OPA1 (a protein that is essential for mitochondrial fusion), compared with healthy controls. Methods: Patients with severe DOA (DOA plus) had peripheral neuropathy, cognitive regression, and epilepsy in addition to loss of vision. We quantified mitophagy in dermal fibroblasts, using 2 high throughput imaging systems, by visualizing colocalization of mitochondrial fragments with engulfing autophagosomes. Results: Fibroblasts from 3 biallelic OPA1(2/2) patients with severe DOA had increased mitochondrial fragmentation and mitochondrial DNA (mtDNA)–depleted cells due to decreased levels of OPA1 protein. Similarly, in siRNA-treated control fibroblasts, profound OPA1 knockdown caused mitochondrial fragmentation, loss of mtDNA, impaired mitochondrial function, and mitochondrial mislocalization. Compared to controls, basal mitophagy (abundance of autophagosomes colocalizing with mitochondria) was increased in (1) biallelic patients, (2) monoallelic patients with DOA plus, and (3) OPA1 siRNA–treated control cultures. Mitophagic flux was also increased. Genetic knockdown of the mitophagy protein ATG7 confirmed this by eliminating differences between patient and control fibroblasts. Conclusions: We demonstrated increased mitophagy and excessive mitochondrial fragmentation in primary human cultures associated with DOA plus due to biallelic OPA1 mutations. We previously found that increased mitophagy (mitochondrial recycling) was associated with visual loss in another mitochondrial optic neuropathy, Leber hereditary optic neuropathy (LHON). Combined with our LHON findings, this implicates excessive mitochondrial fragmentation, dysregulated mitophagy, and impaired response to energetic stress in the pathogenesis of mitochondrial optic neuropathies, potentially linked with mitochondrial mislocalization and mtDNA depletion

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts

Disability, fatigue, pain and their associates in early diffuse cutaneous systemic sclerosis: the European Scleroderma Observational Study

Objectives; Our aim was to describe the burden of early dcSSc in terms of disability, fatigue and pain in the European Scleroderma Observational Study cohort, and to explore associated clinical features. Methods; Patients completed questionnaires at study entry, 12 and 24 months, including the HAQ disability index (HAQ-DI), the Cochin Hand Function Scale (CHFS), the Functional Assessment of Chronic Illness Therapy-fatigue and the Short Form 36 (SF36). Associates examined included the modified Rodnan skin score (mRSS), current digital ulcers and internal organ involvement. Correlations between 12-month changes were also examined. Results; The 326 patients recruited (median disease duration 11.9 months) displayed high levels of disability [mean (S.D.) HAQ-DI 1.1 (0.83)], with ‘grip’ and ‘activity’ being most affected. Of the 18 activities assessed in the CHFS, those involving fine finger movements were most affected. High HAQ-DI and CHFS scores were both associated with high mRSS (ρ = 0.34, P < 0.0001 and ρ = 0.35, P < 0.0001, respectively). HAQ-DI was higher in patients with digital ulcers (P = 0.004), pulmonary fibrosis (P = 0.005), cardiac (P = 0.005) and muscle involvement (P = 0.002). As anticipated, HAQ-DI, CHFS, the Functional Assessment of Chronic Illness Therapy and SF36 scores were all highly correlated, in particular the HAQ-DI with the CHFS (ρ = 0.84, P < 0.0001). Worsening HAQ-DI over 12 months was strongly associated with increasing mRSS (ρ = 0.40, P < 0.0001), decreasing hand function (ρ = 0.57, P < 0.0001) and increasing fatigue (ρ = −0.53, P < 0.0001). Conclusion; The European Scleroderma Observational Study highlights the burden of disability in early dcSSc, with high levels of disability and fatigue, associating with the degree of skin thickening (mRSS). Impaired hand function is a major contributor to overall disability

Treatment outcome in early diffuse cutaneous systemic sclerosis: the European Scleroderma Observational Study (ESOS).

OBJECTIVES: The rarity of early diffuse cutaneous systemic sclerosis (dcSSc) makes randomised controlled trials very difficult. We aimed to use an observational approach to compare effectiveness of currently used treatment approaches. METHODS: This was a prospective, observational cohort study of early dcSSc (within three years of onset of skin thickening). Clinicians selected one of four protocols for each patient: methotrexate, mycophenolate mofetil (MMF), cyclophosphamide or 'no immunosuppressant'. Patients were assessed three-monthly for up to 24 months. The primary outcome was the change in modified Rodnan skin score (mRSS). Confounding by indication at baseline was accounted for using inverse probability of treatment (IPT) weights. As a secondary outcome, an IPT-weighted Cox model was used to test for differences in survival. RESULTS: Of 326 patients recruited from 50 centres, 65 were prescribed methotrexate, 118 MMF, 87 cyclophosphamide and 56 no immunosuppressant. 276 (84.7%) patients completed 12 and 234 (71.7%) 24 months follow-up (or reached last visit date). There were statistically significant reductions in mRSS at 12 months in all groups: -4.0 (-5.2 to -2.7) units for methotrexate, -4.1 (-5.3 to -2.9) for MMF, -3.3 (-4.9 to -1.7) for cyclophosphamide and -2.2 (-4.0 to -0.3) for no immunosuppressant (p value for between-group differences=0.346). There were no statistically significant differences in survival between protocols before (p=0.389) or after weighting (p=0.440), but survival was poorest in the no immunosuppressant group (84.0%) at 24 months. CONCLUSIONS: These findings may support using immunosuppressants for early dcSSc but suggest that overall benefit is modest over 12 months and that better treatments are needed. TRIAL REGISTRATION NUMBER: NCT02339441

Mitophagy plays a central role in mitochondrial ageing

The mechanisms underlying ageing have been discussed for decades, and advances in molecular and cell biology of the last three decades have accelerated research in this area. Over this period, it has become clear that mitochondrial function, which plays a major role in many cellular pathways from ATP production to nuclear gene expression and epigenetics alterations, declines with age. The emerging concepts suggest novel mechanisms, involving mtDNA quality, mitochondrial dynamics or mitochondrial quality control. In this review, we discuss the impact of mitochondria in the ageing process, the role of mitochondria in reactive oxygen species production, in nuclear gene expression, the accumulation of mtDNA damage and the importance of mitochondrial dynamics and recycling. Declining mitophagy (mitochondrial quality control) may be an important component of human ageing

Mitophagy plays a central role in mitochondrial ageing

The mechanisms underlying ageing have been discussed for decades, and advances in molecular and cell biology of the last three decades have accelerated research in this area. Over this period, it has become clear that mitochondrial function, which plays a major role in many cellular pathways from ATP production to nuclear gene expression and epigenetics alterations, declines with age. The emerging concepts suggest novel mechanisms, involving mtDNA quality, mitochondrial dynamics or mitochondrial quality control. In this review, we discuss the impact of mitochondria in the ageing process, the role of mitochondria in reactive oxygen species production, in nuclear gene expression, the accumulation of mtDNA damage and the importance of mitochondrial dynamics and recycling. Declining mitophagy (mitochondrial quality control) may be an important component of human ageing