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• W637966712 abstract "The mitochondrion is an essential cytoplasmic organelle that provides most of the energynecessary for eukaryotic cell physiology. Mitochondrial structure and functions aremaintained by proteins of both mitochondrial and nuclear origin. These organelles areorganized in an extended network that dynamically fuses and divides. Mitochondrialmorphology results from the equilibrium between fusion and fission processes, controlled bya family of “mitochondria-shaping” proteins. It is becoming clear that defects inmitochondrial dynamics can impair mitochondrial respiration, morphology and motility,leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders invivo in humans.Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal DominantOptic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion celldegeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved inmitochondrial network dynamics, cytochrome c storage and apoptosis. This protein isanchored or associated on the inner mitochondrial membrane facing the intermembrane space.Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5bhave been described (Delettre et al., 2001). These variants greatly vary among diverse organsand the presence of specific isoforms has been associated with various mitochondrialfunctions. The different spliced exons encode domains included in the amino-terminal regionand contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown thatexon 4, that is conserved throughout evolution, confers functions to OPA1 involved inmaintenance of the mitochondrial membrane potential and in the fusion of the network.Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation ofcytochrome c release from mitochondria, and activation of apoptosis, a process restricted tovertebrates (Olichon et al., 2007).While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recentlythat OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 causeaccumulation of multiple deletions in skeletal muscle. The syndrome associated to thesemutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy,progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati-Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA“breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009).In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b areimportant for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane.Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimatelyassociated to the maintenance of the mitochondrial genome, as their silencing leads tomtDNA depletion. The mechanism leading to mtDNA loss is associated with replicationinhibition in cells where exon 4b containing isoforms were down-regulated. Furthermoresilencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoidsdistribution in the mitochondrial network.In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptideembedded in the inner membrane by a second transmembrane domain, that seems to becrucial for mitochondrial genome maintenance and does correspond to the secondtransmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is alsomandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesiswe show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA andspecifically interacts with two major components of the mitochondrial nucleoids: thepolymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1-exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, aprocess that is required for the initiation of mtDNA replication and for the distribution ofnucleoids along the network.These data provide new crucial insights in understanding the mechanism involved inmaintenance of mtDNA integrity, because they clearly demonstrate that, besides genesimplicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involvedin the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play alsoan important role. Noticeably, the effect on mtDNA is different depending on the specificOPA1 isoforms down-regulated, suggesting the involvement of two different combinedmechanisms.Over two hundred OPA1 mutations, spread throughout the coding region of the gene, havebeen described to date, including substitutions, deletions or insertions. Some mutations arepredicted to generate a truncated protein inducing haploinsufficiency, whereas the missensenucleotide substitutions result in aminoacidic changes which affect conserved positions of theOPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOApatients are poorly understood. Phosphorus MR spectroscopy in patients with thec.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi etal., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our groupthat OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidativephosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008).In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derivedfrom DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency(DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), andcompared with control fibroblasts. Although both types of DOA fibroblasts maintained asimilar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize theoxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis throughcomplex I, measured in digitonin-permeabilized cells, was significantly reduced in cells withOPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missensesubstitutions.Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblastlines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>Cmutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizingresponse to oligomycin in DOA-H cell lines only. This finding clearly supports thehypothesis that these mutations cause a significant alteration in the respiratory chain function,which can be unmasked only when the operation of the ATP synthase is prevented.Noticeably, oligomycin-induced depolarization in these cells was almost completelyprevented by preincubation with cyclosporin A, a well known inhibitor of the permeabilitytransition pore (PTP). This results is very important because it suggests for the first time thatthe voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issuehas not yet been addressed in the present study, several are the mechanisms that have beenproposed to lead to PTP deregulation, including in particular increased reactive oxygenspecies production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTPopening is currently under investigation. Identification of the mechanisms leading to alteredthreshold for PTP regulation will help our understanding of the pathophysiology of DOA, butalso provide a strategy for therapeutic intervention." @default.
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• W637966712 date "2010-04-20" @default.
• W637966712 modified "2023-09-23" @default.
• W637966712 title "Studies of OPA1 pathogenic mechanisms in DominantOptic Atrophy and novel protein function inmitochondrial DNA stability" @default.
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