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Pathogenic variants of OPA1, which encodes a dynamin GTPase involved in mitochondrial fusion, are responsible for a spectrum of neurological disorders sharing optic nerve atrophy and visual impairment. To gain insight on OPA1 neuronal specificity, we performed targeted metabolomics on rat cortical neurons with OPA1 expression inhibited by RNA interference. Of the 103 metabolites accurately measured, univariate analysis including the Benjamini-Hochberg correction revealed 6 significantly different metabolites in OPA1 down-regulated neurons, with aspartate being the most significant (p 2cum = 0.65) and a low risk of over-fitting (permQ2 = −0.16, CV-ANOVA p-value 0.036). Amongst the 46 metabolites contributing the most to the metabolic signature were aspartate, glutamate and threonine, which all decreased in OPA1 down-regulated neurons, and lysine, 4 sphingomyelins, 4 lysophosphatidylcholines and 32 phosphatidylcholines which were increased. The phospholipid signature may reflect intracellular membrane remodeling due to loss of mitochondrial fusion and/or lipid droplet accumulation. Aspartate and glutamate deficiency, also found in the plasma of OPA1 patients, is likely the consequence of respiratory chain deficiency, whereas the glutamate decrease could contribute to the synaptic dysfunction that we previously identified in this model.

Mitochondrial dynamics control the organelle's morphology, with fusion leading to the formation of elongated tubules and fission leading to isolated puncta, as well as mitochondrial functions. Recent reports have shown that disruptions of mitochondrial dynamics contribute to neurodegenerative diseases. Mutations of the inner membrane GTPase OPA1 are responsible for type 1 dominant optic atrophy, by mechanisms not fully understood. We show here that in rodent cortical primary neurons, downregulation of the OPA1 protein leads to fragmented mitochondria that become less abundant along the dendrites. Furthermore, this inhibition results in reduced expression of mitochondrial respiratory complexes as well as mitochondrial DNA, decreased mitochondrial membrane potential, and diminished reactive oxygen species levels. The onset of synaptogenesis was markedly impaired through reductions in pre- and postsynaptic structural protein expression and synapse numbers without first affecting the dendritic arborization. With longer time in culture, OPA1 extinction led to a major restriction of dendritic growth, together with reduction of synaptic proteins. Furthermore, in maturing neurons we observed a transitory increase in mitochondrial filament length, associated with marked changes in the expression levels of OPA1, which occurred at the onset of synaptogenesis simultaneously with transitory increase in reactive oxygen species levels and NRF2/NFE2L2 nuclear translocation. This observation suggests that mitochondrial hyperfilamentation acts upstream of a reactive oxygen species-dependent NRF2 transcriptional activity, possibly impacting neuronal maturation, such a process being impaired by insufficient amount of OPA1. Our findings suggest a new role for OPA1 in synaptic maturation and dendritic growth through maintenance of proper mitochondrial oxidative metabolism and distribution, highlighting the role of mitochondrial dynamics in neuronal functioning and providing insights into dominant optic atrophy pathogenesis, as OPA1 loss affecting neuronal maturation could lead to early synaptic dysfunction.

The studies addressing the molecular mechanisms governing mitochondrial fusion and fission have brought to light a small group of dynamin-like GTPases (Guanosine-Triphosphate hydrolase) as central regulators of mitochondrial morphology and cristae remodeling, apoptosis, calcium signaling, and metabolism. One of them is the mammalian OPA1 (Optic atrophy 1) protein, which resides inside the mitochondrion anchored to the inner membrane and, in a cleaved form, is associated to oligomeric complexes, in the intermembrane space of the organelle. Here, we review the studies that have made OPA1 emerge as the best understood regulator of mitochondrial inner membrane fusion and cristae remodeling. Further, we re-examine the findings behind the recent claim that OPA1 mediates adrenergic control of lipolysis by functioning as a cytosolic A-kinase anchoring protein (AKAP), on the hemimembrane that envelops the lipid droplet. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.

In chronic neurodegenerative syndromes, neurons progressively die through a generalized retraction pattern triggering retrograde axonal degeneration toward the cell bodies, which molecular mechanisms remain elusive. Recent observations suggest that direct activation of pro-apoptotic signaling in axons triggers local degenerative events associated with early alteration of axonal mitochondrial dynamics. This raises the question of the role of mitochondrial dynamics on both axonal vulnerability stress and their implication in the spreading of damages toward unchallenged parts of the neuron. Here, using microfluidic chambers, we assessed the consequences of interfering with OPA1 and DRP1 proteins on axonal degeneration induced by local application of rotenone. We found that pharmacological inhibition of mitochondrial fission prevented axonal damage induced by rotenone, in low glucose conditions. While alteration of mitochondrial dynamics per se did not lead to spontaneous axonal degeneration, it dramatically enhanced axonal vulnerability to rotenone, which had no effect in normal glucose conditions, and promoted retrograde spreading of axonal degeneration toward the cell body. Altogether, our results suggest a mitochondrial priming effect in axons as a key process of axonal degeneration. In the context of neurodegenerative diseases, like Parkinson's and Alzheimer's, mitochondria fragmentation could hasten neuronal death and initiate spatial dispersion of locally induced degenerative events.

Dominant optic atrophy (DOA) is because of mutations in the mitochondrial protein OPA1. The disease principally affects retinal ganglion cells, whose axons degenerate leading to vision impairments, and sometimes other neuronal phenotypes. The exact mechanisms underlying DOA pathogenesis are not known. We previously demonstrated that the main role of OPA1, as a mitochondrial fusogenic and anti-apoptotic protein, are inhibited by interaction with the stress inducible pro-apoptotic BNIP3 protein. Because BNIP3 was recently reported to participate in autophagy and mitophagy, we tested the involvement of these processes in DOA pathogenesis. Using an in vitro neuronal model of DOA, we identified a BNIP3 down-regulation that reduced autophagy and mitophagy. Restoring BNIP3 had a biphasic effect, first rescuing autophagy and mitophagy levels but later leading to cell death. Similarly, in an in vivo mouse model of DOA, we showed that BNIP3 levels are decreased in young adult mice and increase to normal levels upon aging, paralleling disease progression. Altogether, our results indicate that the relationship between OPA1 and BNIP3 may have important bearings on DOA pathogenesis.

Mitochondrial morphology varies according to cell type and cellular context from an interconnected filamentous network to isolated dots. This morphological plasticity depends on mitochondrial dynamics, a balance between antagonistic forces of fission and fusion. DRP1 and FIS1 control mitochondrial outer membrane fission and Mitofusins its fusion. This review focuses on OPA1, one of the few known actors of inner membrane dynamics, whose mutations provoke an optic neuropathy. Since its first identification in 2000 the characterization of the functions of OPA1 has made rapid progress thus providing numerous clues to unravel the pathogenetic mechanisms of ADOA-1.

Mitochondria continually fuse and divide to dynamically adapt to changes in metabolism and stress. Mitochondrial dynamics are also required for mitochondrial DNA (mtDNA) integrity; however, the underlying reason is not known. In this study, we examined the link between mitochondrial fusion and mtDNA maintenance in Schizosaccharomyces pombe, which cannot survive without mtDNA, by screening for suppressors of the lethality induced by loss of the dynamin-related large GTPase Msp1p. Our findings reveal that inactivation of Msp1p induces a ROS-dependent nuclear mutator phenotype that affects mitochondrial fission genes involved in suppressing mitochondrial fragmentation and mtDNA depletion. This indicates that mitochondrial fusion is crucial for maintaining the integrity of both mitochondrial and nuclear genetic information. Furthermore, our study suggests that the primary roles of Msp1p are to organize mitochondrial membranes, thus making them competent for fusion, and maintain the integrity of mtDNA.

In most eucaryote cells, release of apoptotic proteins from mitochondria involves fission of the mitochondrial network and drastic remodelling of the cristae structures. The intramitochondrial dynamin OPA1, as a potential central actor of these processes, exists as eight isoforms resulting from the alternate splicing combinations of exons (Ex) 4, 4b and 5b, which functions remain undetermined. Here, we show that Ex4 that is conserved throughout evolution confers functions to OPA1 involved in the maintenance of the DeltaPsi(m) and in the fusion of the mitochondrial network. Conversely, Ex4b and Ex5b, which are vertebrate specific, define a function involved in cytochrome c release, an apoptotic process also restricted to vertebrates. The drastic changes of OPA1 variant abundance in different organs suggest that nuclear splicing can control mitochondrial dynamic fate and susceptibility to apoptosis and pathologies.

The mitochondria are dynamic organelles that constantly fuse and divide. An equilibrium between fusion and fission controls the morphology of the mitochondria, which appear as dots or elongated tubules depending the prevailing force. Characterization of the components of the fission and fusion machineries has progressed considerably, and the emerging question now is what role mitochondrial dynamics play in mitochondrial and cellular functions. Its importance has been highlighted by the discovery that two human diseases are caused by mutations in the two mitochondrial pro-fusion genes, MFN2 and OPA1. This review will focus on data concerning the function of OPA1, mutations in which cause optic atrophy, with respect to the underlying pathophysiological processes.

OPA1 encodes a large GTPase related to dynamins, anchored to the mitochondrial cristae inner membrane, facing the intermembrane space. OPA1 haplo-insufficiency is responsible for the most common form of autosomal dominant optic atrophy (ADOA, MIM165500), a neuropathy resulting from degeneration of the retinal ganglion cells and optic nerve atrophy. Here we show that down-regulation of OPA1 in HeLa cells using specific small interfering RNA (siRNA) leads to fragmentation of the mitochondrial network concomitantly to the dissipation of the mitochondrial membrane potential and to a drastic disorganization of the cristae. These events are followed by cytochrome c release and caspase-dependent apoptotic nuclear events. Similarly, in NIH-OVCAR-3 cells, the OPA1 siRNA induces mitochondrial fragmentation and apoptosis, the latter being inhibited by Bcl2 overexpression. These results suggest that OPA1 is a major organizer of the mitochondrial inner membrane from which the maintenance of the cristae integrity depends. As loss of OPA1 commits cells to apoptosis without any other stimulus, we propose that OPA1 is involved in the cytochrome c sequestration and might be a target for mitochondrial apoptotic effectors. Our results also suggest that abnormal apoptosis is a possible pathophysiological process leading to the retinal ganglion cells degeneration in ADOA patients.

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy occurring in 1 in 50,000 individuals that features progressive loss in visual acuity leading, in many cases, to legal blindness. Phenotypic variations and loss of retinal ganglion cells, as found in Leber hereditary optic neuropathy (LHON), have suggested possible mitochondrial impairment. The OPA1 gene has been localized to 3q28-q29 (refs 13-19). We describe here a nuclear gene, OPA1, that maps within the candidate region and encodes a dynamin-related protein localized to mitochondria. We found four different OPA1 mutations, including frameshift and missense mutations, to segregate with the disease, demonstrating a role for mitochondria in retinal ganglion cell pathophysiology.

Members of the dynamin-related proteins family have been identified in a wide range of organisms, however their precise functions remain elusive. We have identified a new member of that GTPase family in the fission yeastSchizosaccharomyces pombe.We show thatMsp1+is an essential nuclear gene encoding a 101 kDa protein whose closest homologue is theS. cerevisiaeMGM1 gene product. We also report that msp1 conditional loss of function affects the maintenance of mitochondrial DNA and leads to growth arrest associated with respiratory deficiency.

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

Opa1 modulates mitochondrial fusion, cristae structure and apoptosis. The relationships between these functions and autosomal dominant optic atrophy, caused by mutations in Opa1, are poorly defined. We show that Bnip3 interacts with Opa1, leading to mitochondrial fragmentation and apoptosis. Fission is due to inhibition of Opa1-mediated fusion and is counteracted by Opa1 in an Mfn1-dependent manner. Bnip3-Opa1 interaction is necessary to trigger Opa1 complex disruption in a Bax- and/or Bak-dependent manner, ultimately leading to apoptosis. Our results uncover a direct link between Opa1 on the inner mitochondrial membrane and the apoptotic machinery on the outer membrane that modulates fusion and cristae structure by separate mechanisms. These findings might help to unravel optic atrophy aetiology as retinal ganglion cells are particularly prone to hypoxia, an inductor of Bnip3 expression.

International audience; OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA), a blinding disease that affects specifically the retinal ganglion cells (RGCs), which function consists in connecting the neuro-retina to the brain. OPA1 encodes an intra-mitochondrial dynamin, involved in inner membrane structures and ubiquitously expressed, raising the critical question of the origin of the disease pathophysiology. Here, we review the fundamental knowledge on OPA1 functions and regulations, highlighting their involvements in mitochondrial respiration, membrane dynamic and apoptosis. In light of these functions, we then describe the remarkable RGC mitochondrial network physiology and analyse data collected from animal models expressing OPA1 mutations. If, to date RGC mitochondria does not present any peculiarity at the molecular level, they represent possible targets of numerous assaults, like light, pressure, oxidative stress and energetic impairment, which jeopardize their function and survival, as observed in OPA1 mouse models. Although fascinating fields of investigation are still to be addressed on OPA1 functions and on DOA pathophysiology, we have reached a conspicuous state of knowledge with pertinent cell and animal models, from which therapeutic trials can be initiated and deeply evaluated.

To characterize the molecular links between type-1 autosomal dominant optic atrophy (ADOA) and OPA1 dysfunctions, the effects of pathogenic alleles of this dynamin on mitochondrial morphology and apoptosis were analyzed, either in fibroblasts from affected individuals, or in HeLa cells transfected with similar mutants. The alleles were missense substitutions in the GTPase domain (OPA1(G300E) and OPA1(R290Q)) or deletion of the GTPase effector domain (OPA1(Delta58)). Fragmentation of mitochondria and apoptosis increased in OPA1(R290Q) fibroblasts and in OPA1(G300E) transfected HeLa cells. OPA1(Delta58) did not influence mitochondrial morphology, but increased the sensitivity to staurosporine of fibroblasts. In these cells, the amount of OPA1 protein was half of that in control fibroblasts. We conclude that GTPase mutants exert a dominant negative effect by competing with wild-type alleles to integrate into fusion-competent complexes, whereas C-terminal truncated alleles act by haplo-insufficiency. We present a model where antagonistic fusion and fission forces maintain the mitochondrial network, within morphological limits that are compatible with cellular functions. In the retinal ganglion cells (RGCs) of patients suffering from type-1 ADOA, OPA1-driven fusion cannot adequately oppose fission, thereby rendering them more sensitive to apoptotic stimuli and eventually leading to optic nerve degeneration.

PURPOSE. Mutations in the mitochondrial dynamin-related GTPase OPA1 cause autosomal dominant optic atrophy (ADOA), but the pathophysiology of this disease is unknown. As a first step in functional studies, this study was conducted to evaluate the expression of Opal in whole retina and in isolated retinal ganglion cells (RGCs) and to test the effects of Opal downregulation in cultured RGCs. METHODS. Opal mRNA isoforms from total retina and from RGCs freshly isolated by immunopanning were determined by RT-PCR. Protein expression was examined by immunohistochemistry and Western blot with antibodies against Opal and cytochrome c, and the mitochondrial network was visualized with a mitochondrial marker. Short interfering (si)RNA targeting OPA1 mRNAs were transfected to cultured RGCs and mitochondrial network phenotypes were followed for 15 days, in comparison with those of cerebellar granule cells (CGCs). RESULTS. Opal expression did not predominate in rat postnatal RGCs as found by immunohistochemistry and Western blot analysis. The pattern of mRNA isoforms was similar in whole retina and RGCs. After a few days in culture, isolated RGCs showed line mitochondrial punctiform structures in the soma and neurites that colocalized with cytochrome c and Opal. Opal knockdown in RGCs induced mitochondrial network aggregation at a higher rate than in CGCs. CONCLUSIONS. Results suggest that the level of expression and the mRNA isoforms do not underlie the vulnerability of RGCs to OPA1 mutations. However, aggregation of the mitochondrial network induced by the downregulation of Opal appears more frequent in RGCs than in control CGCs.

The heterozygous R445H mutation in OPA1 was found in five patients with optic atrophy and deafness. Audiometry suggested that the sensorineural deafness resulted from auditory neuropathy. Skin fibroblasts showed hyperfragmentation of the mitochondrial network, decreased mitochondrial membrane potential, and adenosine triphosphate synthesis defect. In addition, OPA1 was found to be widely expressed in the sensory and neural cochlear cells of the guinea pig. Thus, optic atrophy and deafness may be related to energy defects due to a fragmented mitochondrial network.

Mitochondrial morphology is controlled by large GTPases, such as Msp1p, whose action on mitochondrial membranes is not yet understood. The sub-mitochondrial localization of Msp1p, the subject of ongoing controversies, was found to be within the intermembrane space. Overexpression of Msp1p led to aggregation of the mitochondrial network, while its downregulation resulted in fragmentation of this network. Mutations affecting the integrity of the Msp1p GTPase function had a dominant phenotype and induced mitochondrial fragmentation followed by mitochondrial DNA loss and cell death. These effects were not observed in cells deleted for Dnm1p, an actor in mitochondrial fission, suggesting that Msp1p is involved in the fusion of mitochondria.

Dominant optic atrophy (DOA) is the most common form of inherited optic neuropathy. Although heterogeneous, a major locus has been mapped to chromosome 3q28 and the responsible gene, OPA1, was recently identified. OPA1 is a mitochondrial dynamin-related GTPase implicated in the formation and maintenance of the mitochondrial network. To date, 62 mutations have been identified in a total of 201 DOA patients. Most of them (90%) are distributed from exons 8 to 28 with a majority in the GTPase domain (54%). None were found in the alternatively spliced exons 4, 4b, and 5b. Half of them are truncative mutations (50%) with a frequent recurrent allele, c.2708delTTAG. Most missense mutations (81%) cluster within the putative GTPase domain. Various pathogenic mechanisms may play a role in OPA1 DOA. Truncative mutations in the N-terminal region and perhaps missense mutations in the GTPase domain lead to a loss of function of the encoded protein and haplotype insufficiency. However, there is a cluster of truncation mutations in the in C-terminus, a putative dimerization domain, that could act through a dominant negative effect. The findings that OPA1-type DOA, as Leber optic neuropathy, is caused by the impairment of a mitochondrial protein address the question of the vulnerability of the retinal ganglion cell in response to mitochondrial defects.

The nim1/cdr1 protein kinase is required for an efficient adaptation of cell cycle parameters to changes in nutritional conditions. We have isolated msp1, a new fission yeast member of the dynamin-related large GTPase family, in a two-hybrid screen designed to identify proteins interacting with the nim1 kinase. Msp1 has been shown to be essential for the maintenance of mtDNA and hence for the inheritance of functional mitochondria. We present evidence indicating that nim1 and msp1 proteins physically interact both in vitro and in vivo in fission yeast. These interactions occur through the amino-terminal catalytic domain of nim1 and the carboxy-terminal putative regulatory domain of msp1. These results provide new evidence for the existence of a connection between mitochondrial function and the cell cycle machinery.

Mutations in the OPA1 gene are associated with autosomal dominant optic atrophy. OPA1 encodes a dynamin-related protein orthologous to Msp1 of Schizosaccharomyces pombe and Mgm1p of Saccharomyces cerevisiae, both involved in mitochondrial morphology and genome maintenance. We present immuno-fluorescence and biochemical evidences showing that OPA1 resides in the mitochondria where it is imported through its highly basic amino-terminal extension. Proteolysis experiments indicate that OPA1 is present in the inter-membrane space and electron microscopy further localizes it close to the cristae. The strong association of OPA1 with membranes suggests its anchoring to the inner membrane.

We recently identified Msp1p, a fission yeast Schizosaccharomyces pombe dynamin-related protein, which is essential for the maintenance of mitochondrial DNA. The Msp1p sequence displays typical features of a mitochondrial protein. Here we report in vitro and in vivo data that validate that prediction. We demonstrate that the targeting sequence of Msp1p is processed by recombinant mitochondrial processing peptidase and that Msp1p is imported into S. pombe mitochondria in vitro in the presence of cellular extracts. We show that the first 109 residues of Msp1p encompass a functional peptide signal that is sufficient to direct chimera to mitochondria. Immunofluorescence studies indicate that Msp1p staining colocalises with a mitochondrial marker and electron microscopy shows that the protein is located inside the mitochondria. Mitochondrial enrichment and fractionation further confirm that localisation and show that Msp1p is anchored to the matrix side of the mitochondrial inner membrane. Finally, we report that overexpression of the Msp1 protein results in gross alteration of the mitochondrial structure and function. All together our results suggest that Msp1p is an essential component for mitochondrial maintenance.