Caractérisation de Msp1, un acteur de la dynamique mitochondriale, chez la levure à fission

Notre équipe s'intéresse à la dynamique mitochondriale, qui contrôle la forme des mitochondries et régule leurs principales fonctions, et dont l'altération provoque des pathologies neurodégénératives. Ce processus correspond à l'établissement d'un équilibre dynamique entre des forces de fusion et de fission des mitochondries. Quand la force de fission est prépondérante les mitochondries apparaissent sous formes de dots isolés les uns des autres. À l'inverse si la force de fusion prédomine les mitochondries apparaissent sous la forme d'un réseau de filaments plus ou moins longs et interconnectés. Récemment, de nombreuses protéines qui contrôlent la dynamique mitochondriale ont été identifiées. Dans notre équipe il a été isolé puis caractérisé l'une d'entre elle, Msp1 chez la levure à fission et OPA1 son homologue humain. Msp1/OPA1 contrôle la fusion des mitochondries et la mort des cellules. Les mutations de OPA1 provoquent une atrophie optique dominante de type 1, qui touche le nerf optique et peut conduire à la cécité. Le sujet de thèse qui m'a été confié concerne l'isolement et la caractérisation de partenaires génétiques de Msp1. Pour cela un préalable était requis : disposer d'un mutant thermosensible de la dynamine. Au cours de la première moitié de ma thèse, j'ai construit un mutant thermosensible de Msp1 porteur d'une mutation du domaine GTPase (P300S) par recombinaison homologue. J'ai ensuite caractérisé ce mutant et recherché des conditions de létalité induites par l’inactivation de Msp1. J'ai montré que cette souche est affectée dans la morphologie de ses mitochondries, sa respiration et présente une sensibilité accrue au stress oxydatif. J'ai également montré que cette souche est incapable de proliférer en milieu galactose ce qui m'a permis de mettre au point un crible génétique visant à isoler des suppresseurs multicopies de la létalité induite par l'inactivation de Msp1. En parallèle à ces travaux, j'ai contribué à l'étude des mécanismes de protéolyse de Msp1p. Msp1p existe chez la levure sous deux isoformes : une forme longue (l-msp1) issue du clivage par la peptidase mitochondriale lors de l'import de la dynamine et une forme courte (s-msp1p) dont la formation était inconnue. A l'aide de mutants de différents protéases mitochondriales que nous avons construits, nous avons montré que la protéase Rhomboïde 1 est impliquée dans la formation de s-msp1p, et que la m-AAA protéase, Paraplégine, contrôle la stabilité de la forme l-msp1p. Nous avons également utilisé ces mutants pour caractériser les mécanismes de protéolyse des huit variants d'épissage d'OPA1, que nous avons surexprimés chez ces levures. Pour la plupart des variants d'épissage, la m-AAA protéase, contrairement aux protéases Yme1p et Rhomboïde 1, est impliquée dans le clivage de la dynamine, permettant de revisiter certains résultats contradictoires parus dans la littérature. Mes travaux de thèse pourraient déboucher sur une meilleure compréhension du rôle de Msp1 et permettre d'identifier de nouveaux acteurs et régulateurs de la dynamique mitochondriale qui pourraient constituer de nouveaux gènes candidats pour des pathologies mitochondriales.Mitochondrial dynamics is a regulated interplay between antagonistic fission and fusion forces acting on mitochondrial membranes. This process determines mitochondrial morphology; when fission or fusion predominates, mitochondria appear as isolated dots or as a filamentous and interconnected network, respectively. Proteins that control the morphology of mitochondria have been identified. Our team has isolated and characterized Msp1p in the fission yeast Schizosaccharomyces pombe, and its human orthologue OPA1, which control mitochondrial fusion. Furthermore we have found that mutations in the OPA1 gene are associated with the most frequent form of autosomal dominant optic atrophy that features a progressive loss of retinal ganglion cells, often leading to blindness. Understanding the function of the dynamin Msp1p requires identification of its partners, which was part of my thesis. To perform a genetic screen for msp1+ interactors, I generated a thermosensitive (ts) mutant of msp1+ (Msp1P300S) by homology to a ts substitution described at a position conserved in other dynamins. The mutation was integrated at the msp1+ locus by homologous recombination. I then characterized this mutant to search for conditions of lethality. At restrictive temperature, the mutant had fragmented mitochondria when grown on respiratory, glucose-containing medium, and further showed altered respiration and increased ROS production on non-fermentable ethanol/glycerol-containing medium. Perturbation of colony pigmentation in ade6 background, which relates to mitochondrial dysfunctions, and lethality on non-fermentable galactose-containing media, were also observed. As a perspective, we plan to take advantage of this latter property to screen a cDNA library for the presence of multicopy suppressors of thermolethality. In addition to this work, we have studied the mechanisms of maturation of Msp1p, which is processed into a long and a short isoform (l- and s-Msp1p) during its biogenesis. Using mutants of various mitochondrial proteases, we demonstrated that the two isoforms of Msp1p are independently formed from the same precursor and that generation of s-Msp1p implicates Rhomboid 1. Furthermore, we showed that the m-AAA protease Paraplegin might control the stability of l-Msp1p. These proteases mutants were also used to study the proteolysis of the eight OPA1 splicing variants heterologously expressed in S. pombe. Most of these variants were only processed by Paraplegin, a finding that could allow to precise contradictory results obtained in mammals where Yme1p and Rhomboid 1 were implicated in the maturation of OPA1. Together, my thesis works will allow to better understand the mode of action of Msp1 in mitochondrial functions and to identify new actors and regulators of mitochondrial dynamics possibly implicated in orphan mitochondrial pathologies

Genetic background modulates phenotypic expressivity in OPA1 mutated mice, relevance to DOA pathogenesis

Dominant optic atrophy (DOA) is mainly caused by OPA1 mutations and is characterized by the degeneration of retinal ganglion cells (RGCs), whose axons form the optic nerve. The penetrance of DOA is incomplete and the disease is marked by highly variable expressivity, ranging from asymptomatic patients to some who are totally blind or who suffer from multisystemic effects. No clear genotype–phenotype correlation has been established to date. Taken together, these observations point toward the existence of modifying genetic and/or environmental factors that modulate disease severity. Here, we investigated the influence of genetic background on DOA expressivity by switching the previously described DOA mouse model bearing the c.1065 + 5G → A Opa1 mutation from mixed C3H; C57BL/6 J to a pure C57BL/6 J background. We no longer observed retinal and optic nerve abnormalities; the findings indicated no degeneration, but rather a sex-dependent negative effect on RGC connectivity. This highlights the fact that RGC synaptic alteration might precede neuronal death, as has been proposed in other neurodegenerative diseases, providing new clinical considerations for early diagnosis as well as a new therapeutic window for DOA. Furthermore, our results demonstrate the importance of secondary genetic factors in the variability of DOA expressivity and offer a model for screening for aggravating environmental and genetic factors

PGC1-α mRNA expression cross talk with tumor volume growth and total antioxidant capacity in breast cancer model mice: following discontinuous aerobic exercise and vitamin D intake

The modifications of PGC-1 induce the change of the carcinogenesis and tumor growth and lead to increased antioxidant enzymes. The present study aimed to determine the cross talk between PGC1-α mRNA expression, tumor volume growth, and total antioxidant capacity in breast cancer model mice, followed by discontinuous aerobic exercise and vitamin D. In the present study, 40 female NMRI mice were randomly assigned into five equal groups (n=8): healthy control group (H.C), cancer control group (Ca.C), cancer with the vitamin D group (U.Ca.VD), cancer exercise training group (Ca. Ex), and cancer exercise training with the vitamin D group (Ca.Ex.VD). As the results indicate, the bodyweight of cancer groups (p=0.041, F=3.61) and the tumor growth rate significantly reduced compared to the H.C group. The results indicated that the PGC-1α mRNA expression and TAC (p=0.013, F=5.16) change significantly different between the study groups. Besides, based on the results, a significant positive correlation was observed between PGC1-α and tumor volume growth among the groups, whereas a negative relationship exists between PGC1-α and TAC and among TAC and tumor volume growth only in the Ca. Ex.VD group. The correlation between the variables confirms using vitamin D treatment with the implementation of discontinuous aerobic exercise, as a synergistic effect, improves the total antioxidant capacity and is effective in controlling tumor growth. We recommend that further studies be done on exercise training along with supplementation intake synergistic

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

A yeast-based screening assay identifies repurposed drugs that suppress mitochondrial fusion and mtDNA maintenance defects

Mitochondria continually move, fuse and divide, and these dynamics are essential for the proper function of the organelles. Indeed, the dynamic balance of fusion and fission of mitochondria determines their morphology and allows their immediate adaptation to energetic needs as well as preserving their integrity. As a consequence, mitochondrial fusion and fission dynamics and the proteins that control these processes, which are conserved from yeast to human, are essential, and their disturbances are associated with severe human disorders, including neurodegenerative diseases. For example, mutations in OPA1, which encodes a conserved factor essential for mitochondrial fusion, lead to optic atrophy 1, a neurodegeneration that affects the optic nerve, eventually leading to blindness. Here, by screening a collection of ∼1600 repurposed drugs on a fission yeast model, we identified five compounds able to efficiently prevent the lethality associated with the loss of Msp1p, the fission yeast ortholog of OPA1. One compound, hexestrol, was able to rescue both the mitochondrial fragmentation and mitochondrial DNA (mtDNA) depletion induced by the loss of Msp1p, whereas the second, clomifene, only suppressed the mtDNA defect. Yeast has already been successfully used to identify candidate drugs to treat inherited mitochondrial diseases; this work may therefore provide useful leads for the treatment of optic atrophies such as optic atrophy 1 or Leber hereditary optic neuropathy