The human OPA1delTTAG mutation induces adult onset and progressive auditory neuropathy in mice.

Dominant optic atrophy (DOA) is one of the most prevalent forms of hereditary optic neuropathies and is mainly caused by heterozygous variants in OPA1, encoding a mitochondrial dynamin-related large GTPase. The clinical spectrum of DOA has been extended to a wide variety of syndromic presentations, called DOAplus, including deafness as the main secondary symptom associated to vision impairment. To date, the pathophysiological mechanisms underlying the deafness in DOA remain unknown. To gain insights into the process leading to hearing impairment, we have analyzed the Opa1delTTAG mouse model that recapitulates the DOAplus syndrome through complementary approaches combining morpho-physiology, biochemistry, and cellular and molecular biology. We found that Opa1delTTAG mutation leads an adult-onset progressive auditory neuropathy in mice, as attested by the auditory brainstem response threshold shift over time. However, the mutant mice harbored larger otoacoustic emissions in comparison to wild-type littermates, whereas the endocochlear potential, which is a proxy for the functional state of the stria vascularis, was comparable between both genotypes. Ultrastructural examination of the mutant mice revealed a selective loss of sensory inner hair cells, together with a progressive degeneration of the axons and myelin sheaths of the afferent terminals of the spiral ganglion neurons, supporting an auditory neuropathy spectrum disorder (ANSD). Molecular assessment of cochlea demonstrated a reduction of Opa1 mRNA level by greater than 40%, supporting haploinsufficiency as the disease mechanism. In addition, we evidenced an early increase in Sirtuin 3 level and in Beclin1 activity, and subsequently an age-related mtDNA depletion, increased oxidative stress, mitophagy as well as an impaired autophagic flux. Together, these results support a novel role for OPA1 in the maintenance of inner hair cells and auditory neural structures, addressing new challenges for the exploration and treatment of OPA1-linked ANSD in patients.

SPACR Encoded by IMPG1 Is Essential for Photoreceptor Survival by Interplaying between the Interphotoreceptor Matrix and the Retinal Pigment Epithelium.

Several pathogenic variants have been reported in the IMPG1 gene associated with the inherited retinal disorders vitelliform macular dystrophy (VMD) and retinitis pigmentosa (RP). IMPG1 and its paralog IMPG2 encode for two proteoglycans, SPACR and SPACRCAN, respectively, which are the main components of the interphotoreceptor matrix (IPM), the extracellular matrix surrounding the photoreceptor cells. To determine the role of SPACR in the pathological mechanisms leading to RP and VMD, we generated a knockout mouse model lacking Impg1, the mouse ortholog. Impg1-deficient mice show abnormal accumulation of autofluorescent deposits visible by fundus imaging and spectral-domain optical coherence tomography (SD-OCT) and attenuated electroretinogram responses from 9 months of age. Furthermore, SD-OCT of Impg1-/- mice shows a degeneration of the photoreceptor layer, and transmission electron microscopy shows a disruption of the IPM and the retinal pigment epithelial cells. The decrease in the concentration of the chromophore 11-cis-retinal supports this loss of photoreceptors. In conclusion, our results demonstrate the essential role of SPACR in maintaining photoreceptors. Impg1-/- mice provide a novel model for mechanistic investigations and the development of therapies for VMD and RP caused by IMPG1 pathogenic variants.

Protective role of the mitochondrial fusion protein OPA1 in hypertension.

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.

Optic neuropathy linked to ACAD9 pathogenic variants: A potentially riboflavin-responsive disorder?

Mitochondrial complex I (CI) deficiencies (OMIM 252010) are the commonest inherited mitochondrial disorders in children. Acyl-CoA dehydrogenase 9 (ACAD9) is a flavoenzyme involved chiefly in CI assembly and possibly in fatty acid oxidation. Biallelic pathogenic variants result in CI dysfunction, with a phenotype ranging from early onset and sometimes fatal mitochondrial encephalopathy with lactic acidosis to late-onset exercise intolerance. Cardiomyopathy is often associated. We report a patient with childhood-onset optic and peripheral neuropathy without cardiac involvement, related to CI deficiency. Genetic analysis revealed compound heterozygous pathogenic variants in ACAD9, expanding the clinical spectrum associated to ACAD9 mutations. Importantly, riboflavin treatment (15 mg/kg/day) improved long-distance visual acuity and demonstrated significant rescue of CI activity in vitro.

Dominant mutations in mtDNA maintenance gene SSBP1 cause optic atrophy and foveopathy.

Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDSs), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in single-strand binding protein 1 (SSBP1) in 5 unrelated families, leading to the R38Q and R107Q amino acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a revised SSBP1 crystal structure. Structural analysis suggested that both mutations affect dimer interactions and presumably distort the DNA-binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication, and induces mtDNA depletion. Our study showing that mutations in SSBP1 cause a form of dominant optic atrophy frequently accompanied with foveopathy brings insights into mtDNA maintenance disorders.

Hereditary spastic paraplegia and prominent sensorial involvement: think MAG mutations!

Homozygous mutations in MAG, encoding the myelin-associated glycoprotein, a transmembrane component of the myelin sheath, have been associated with SPG 75 recessive spastic paraplegia. Here, we report the first patient with two compound heterozygous novel MAG mutations (p.A151V and p.S373R) and early developmental delay with a progressive complex phenotype characterized by spastic paraplegia, peripheral sensorimotor neuropathy, intellectual disability, and sensorial dysfunctions with severe optic atrophy and hearing involvement. Brain imaging showed progressive global cerebellar atrophy. We propose that complex hereditary spastic paraplegia, with axonal and demyelinating polyneuropathy, sensorial impairment and intellectual disability might suggest MAG mutations.

OPA1 gene therapy prevents retinal ganglion cell loss in a Dominant Optic Atrophy mouse model.

Dominant optic atrophy (DOA) is a rare progressive and irreversible blinding disease which is one of the most frequent forms of hereditary optic neuropathy. DOA is mainly caused by dominant mutation in the OPA1 gene encoding a large mitochondrial GTPase with crucial roles in membrane dynamics and cell survival. Hereditary optic neuropathies are commonly characterized by the degeneration of retinal ganglion cells, leading to the optic nerve atrophy and the progressive loss of visual acuity. Up to now, despite increasing advances in the understanding of the pathological mechanisms, DOA remains intractable. Here, we tested the efficiency of gene therapy on a genetically-modified mouse model reproducing DOA vision loss. We performed intravitreal injections of an Adeno-Associated Virus carrying the human OPA1 cDNA under the control of the cytomegalovirus promotor. Our results provide the first evidence that gene therapy is efficient on a mouse model of DOA as the wild-type OPA1 expression is able to alleviate the OPA1-induced retinal ganglion cell degeneration, the hallmark of the disease. These results displayed encouraging effects of gene therapy for Dominant Optic Atrophy, fostering future investigations aiming at clinical trials in patients.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo-rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Targeted Metabolomics Reveals Early Dominant Optic Atrophy Signature in Optic Nerves of Opa1delTTAG/+ Mice.

Purpose: Dominant optic atrophy (MIM No. 165500) is a blinding condition related to mutations in OPA1, a gene encoding a large GTPase involved in mitochondrial inner membrane dynamics. Although several mouse models mimicking the disease have been developed, the pathophysiological mechanisms responsible for retinal ganglion cell degeneration remain poorly understood. Methods: Using a targeted metabolomic approach, we measured the concentrations of 188 metabolites in nine tissues, that is, brain, three types of skeletal muscle, heart, liver, retina, optic nerve, and plasma in symptomatic 11-month-old Opa1delTTAG/+ mice. Results: Significant metabolic signatures were found only in the optic nerve and plasma of female mice. The optic nerve signature was characterized by altered concentrations of phospholipids, amino acids, acylcarnitines, and carnosine, whereas the plasma signature showed decreased concentrations of amino acids and sarcosine associated with increased concentrations of several phospholipids. In contrast, the investigation of 3-month-old presymptomatic Opa1delTTAG/+ mice showed no specific plasma signature but revealed a significant optic nerve signature in both sexes, although with a sex effect. The Opa1delTTAG/+ versus wild-type optic nerve signature was characterized by the decreased concentrations of 10 sphingomyelins and 10 lysophosphatidylcholines, suggestive of myelin sheath alteration, and by alteration in the concentrations of metabolites involved in neuroprotection, such as dimethylarginine, carnitine, spermine, spermidine, carnosine, and glutamate, suggesting a concomitant axonal metabolic dysfunction. Conclusions: Our comprehensive metabolomic investigations revealed in symptomatic as well as in presymptomatic Opa1delTTAG/+ mice, a specific sensitiveness of the optic nerve to Opa1 insufficiency, opening new routes for protective therapeutic strategies.

Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model.

BACKGROUND: Recent data suggests the involvement of mitochondrial dynamics in cardiac ischemia/reperfusion (I/R) injuries. Whilst excessive mitochondrial fission has been described as detrimental, the role of fusion proteins in this context remains uncertain. OBJECTIVES: To investigate whether Opa1 (protein involved in mitochondrial inner-membrane fusion) deficiency affects I/R injuries. METHODS AND RESULTS: We examined mice exhibiting Opa1delTTAG mutations (Opa1+/-), showing 70% Opa1 protein expression in the myocardium as compared to their wild-type (WT) littermates. Cardiac left-ventricular systolic function assessed by means of echocardiography was observed to be similar in 3-month-old WT and Opa1+/- mice. After subjection to I/R, infarct size was significantly greater in Opa1+/- than in WTs both in vivo (43.2±4.1% vs. 28.4±3.5%, respectively; p<0.01) and ex vivo (71.1±3.2% vs. 59.6±8.5%, respectively; p<0.05). No difference was observed in the expression of other main fission/fusion protein, oxidative phosphorylation, apoptotic markers, or mitochondrial permeability transition pore (mPTP) function. Analysis of calcium transients in isolated ventricular cardiomyocytes demonstrated a lower sarcoplasmic reticulum Ca2+ uptake, whereas cytosolic Ca2+ removal from the Na+/Ca2+ exchanger (NCX) was increased, whilst SERCA2a, phospholamban, and NCX protein expression levels were unaffected in Opa1+/- compared to WT mice. Simultaneous whole-cell patch-clamp recordings of mitochondrial Ca2+ movements and ventricular action potential (AP) showed impairment of dynamic mitochondrial Ca2+ uptake and a marked increase in the AP late repolarization phase in conjunction with greater occurrence of arrhythmia in Opa1+/- mice. CONCLUSION: Opa1 deficiency was associated with increased sensitivity to I/R, imbalance in dynamic mitochondrial Ca2+ uptake, and subsequent increase in NCX activity.

Increased steroidogenesis promotes early-onset and severe vision loss in females with OPA1 dominant optic atrophy.

OPA1 mutations are responsible for autosomal dominant optic atrophy (ADOA), a progressive blinding disease characterized by retinal ganglion cell (RGC) degeneration and large phenotypic variations, the underlying mechanisms of which are poorly understood. OPA1 encodes a mitochondrial protein with essential biological functions, its main roles residing in the control of mitochondrial membrane dynamics as a pro-fusion protein and prevention of apoptosis. Considering recent findings showing the importance of the mitochondrial fusion process and the involvement of OPA1 in controlling steroidogenesis, we tested the hypothesis of deregulated steroid production in retina due to a disease-causing OPA1 mutation and its contribution to the visual phenotypic variations. Using the mouse model carrying the human recurrent OPA1 mutation, we disclosed that Opa1 haploinsufficiency leads to very high circulating levels of steroid precursor pregnenolone in females, causing an early-onset vision loss, abolished by ovariectomy. In addition, steroid production in retina is also increased which, in conjunction with high circulating levels, impairs estrogen receptor expression and mitochondrial respiratory complex IV activity, promoting RGC apoptosis in females. We further demonstrate the involvement of Muller glial cells as increased pregnenolone production in female cells is noxious and compromises their role in supporting RGC survival. In parallel, we analyzed ophthalmological data of a multicentre OPA1 patient cohort and found that women undergo more severe visual loss at adolescence and greater progressive thinning of the retinal nerve fibres than males. Thus, we disclosed a gender-dependent effect on ADOA severity, involving for the first time steroids and Müller glial cells, responsible for RGC degeneration.

OPA1-related disorders: Diversity of clinical expression, modes of inheritance and pathophysiology.

Mutations in the Optic Atrophy 1 gene (OPA1) were first identified in 2000 as the main cause of Dominant Optic Atrophy, a disease specifically affecting the retinal ganglion cells and the optic nerve. Since then, an increasing number of symptoms involving the central, peripheral and autonomous nervous systems, with considerable variations of age of onset and severity, have been reported in OPA1 patients. This variety of phenotypes is attributed to differences in the effects of OPA1 mutations, to the mode of inheritance, which may be mono- or bi-allelic, and eventually to somatic mitochondrial DNA mutations. The diversity of the pathophysiological mechanisms involved in OPA1-related disorders is linked to the crucial role played by OPA1 in the maintenance of mitochondrial structure, genome and function. The neurological expression of these disorders highlights the importance of mitochondrial dynamics in neuronal processes such as dendritogenesis, axonal transport, and neuronal survival. Thus, OPA1-related disorders may serve as a paradigm in the wider context of neurodegenerative syndromes, particularly for the development of novel therapeutic strategies against these diseases.

In vivo time-lapse imaging of mitochondria in healthy and diseased peripheral myelin sheath.

The myelin sheath that covers a large amount of neurons is critical for their homeostasis, and myelinating glia mitochondria have recently been shown to be essential for neuron survival. However morphological and physiological properties of these organelles remain elusive. Here we report a method to analyze mitochondrial dynamics and morphology in myelinating Schwann cells of living mice using viral transduction and time-lapse multiphoton microscopy. We describe the distribution, shape, size and dynamics of mitochondria in live cells. We also report mitochondrial alterations in Opa1(delTTAG) mutant mice cells at presymptomatic stages, suggesting that mitochondrial defects in myelin contribute to OPA1 related neuropathy and represent a biomarker for the disease.

Neuroradiological findings expand the phenotype of OPA1-related mitochondrial dysfunction.

OBJECTIVE: OPA1 mutations are responsible for more than half of autosomal dominant optic atrophy (ADOA), a blinding disease affecting the retinal ganglion neurons. In most patients the clinical presentation is restricted to the optic nerve degeneration, albeit in 20% of them, additional neuro-sensorial symptoms might be associated to the loss of vision, as frequently encountered in mitochondrial diseases. This study describes clinical and neuroradiological features of OPA1 patients. METHODS: Twenty two patients from 17 families with decreased visual acuity related to optic atrophy and carrying an OPA1 mutation were enrolled. Patients underwent neuro-ophthalmological examinations. Brain magnetic resonance imaging (T1, T2 and flair sequences) was performed on a 1.5-Tesla MR Unit. Twenty patients underwent 2-D proton spectroscopic imaging. RESULTS: Brain imaging disclosed abnormalities in 12 patients. Cerebellar atrophy mainly involving the vermis was observed in almost a quarter of the patients; other abnormalities included unspecific white matter hypersignal, hemispheric cortical atrophy, and lactate peak. Neurological examination disclosed one patient with a transient right hand motor deficit and ENT examination revealed hearing impairment in 6 patients. Patients with abnormal MRI were characterized by: (i) an older age (ii) more severe visual impairment with chronic visual acuity deterioration, and (iii) more frequent associated deafness. CONCLUSIONS: Our results demonstrate that brain imaging abnormalities are common in OPA1 patients, even in those with normal neurological examination. Lactate peak, cerebellar and cortical atrophies are consistent with the mitochondrial dysfunction related to OPA1 mutations and might result from widespread neuronal degeneration.

Why mitochondria must fuse to maintain their genome integrity.

SIGNIFICANCE: The maintenance of mitochondrial genome integrity is a major challenge for cells to sustain energy production by respiration. RECENT ADVANCES: Recently, mitochondrial membrane dynamics emerged as a key process contributing to prevent mitochondrial DNA (mtDNA) alterations. Indeed, both fundamental and clinical data suggest that disruption of mitochondrial fusion, related to mutations in the OPA1, MFN2, PINK1, and PARK2 genes, leads to the accumulation of mutations in the mitochondrial genome. CRITICAL ISSUES: We discuss here the possibility that mitochondrial fusion acts as a direct mechanism to prevent the generation of altered mtDNA and to eliminate mutated deleterious genomes either by trans-complementation or by mitophagy. FUTURE DIRECTIONS: Finally, we conclude this review with a short evolutionary comparison between the mechanisms involved in mitochondrial and bacterial modes of genome distribution and plasticity, highlighting possible common conserved processes required for the maintenance of their genome integrity, which should inspire our future investigations.

The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse.

Dominant optic atrophy is a rare inherited optic nerve degeneration caused by mutations in the mitochondrial fusion gene OPA1. Recently, the clinical spectrum of dominant optic atrophy has been extended to frequent syndromic forms, exhibiting various degrees of neurological and muscle impairments frequently found in mitochondrial diseases. Although characterized by a specific loss of retinal ganglion cells, the pathophysiology of dominant optic atrophy is still poorly understood. We generated an Opa1 mouse model carrying the recurrent Opa1(delTTAG) mutation, which is found in 30% of all patients with dominant optic atrophy. We show that this mouse displays a multi-systemic poly-degenerative phenotype, with a presentation associating signs of visual failure, deafness, encephalomyopathy, peripheral neuropathy, ataxia and cardiomyopathy. Moreover, we found premature age-related axonal and myelin degenerations, increased autophagy and mitophagy and mitochondrial supercomplex instability preceding degeneration and cell death. Thus, these results support the concept that Opa1 protects against neuronal degeneration and opens new perspectives for the exploration and the treatment of mitochondrial diseases.

OPA1 links human mitochondrial genome maintenance to mtDNA replication and distribution.

Eukaryotic cells harbor a small multiploid mitochondrial genome, organized in nucleoids spread within the mitochondrial network. Maintenance and distribution of mitochondrial DNA (mtDNA) are essential for energy metabolism, mitochondrial lineage in primordial germ cells, and to prevent mtDNA instability, which leads to many debilitating human diseases. Mounting evidence suggests that the actors of the mitochondrial network dynamics, among which is the intramitochondrial dynamin OPA1, might be involved in these processes. Here, using siRNAs specific to OPA1 alternate spliced exons, we evidenced that silencing of the OPA1 variants including exon 4b leads to mtDNA depletion, secondary to inhibition of mtDNA replication, and to marked alteration of mtDNA distribution in nucleoid and nucleoid distribution throughout the mitochondrial network. We demonstrate that a small hydrophobic 10-kDa peptide generated by cleavage of the OPA1-exon4b isoform is responsible for this process and show that this peptide is embedded in the inner membrane and colocalizes and coimmunoprecipitates with nucleoid components. We propose a novel synthetic model in which a peptide, including two trans-membrane domains derived from the N terminus of the OPA1-exon4b isoform in vertebrates or from its ortholog in lower eukaryotes, might contribute to nucleoid attachment to the inner mitochondrial membrane and promotes mtDNA replication and distribution. Thus, this study places OPA1 as a direct actor in the maintenance of mitochondrial genome integrity.

[Mitochondrial genome instability and associated diseases]. / Instabilité du génome mitochondrial et pathologies associées.

Some cases of mitochondrial diseases are due to mitochondrial DNA instability: multiple deletions or depletions. These anomalies are responsible for a mitochondrial respiratory chain impairment leading to various clinical involvements ranging from mild features with multiple mtDNA deletions to severe organ failure and premature death caused by mtDNA depletions. Both, deletions and depletions share an important and common feature between these two specificities: indeed, both are expressed in a tissue-specific manner.