OPA1 is a GTPase that controls mitochondrial fusion, cristae integrity, and mtDNA maintenance. In humans, eight isoforms are expressed as combinations of long and short forms, but it is unclear whether OPA1 functions are associated with specific isoforms and/or domains. To address this, we expressed each of the eight isoforms or different constructs of isoform 1 in Opa1 MEFs. We observed that any isoform could restore cristae structure, mtDNA abundance, and energetic efficiency independently of mitochondrial network morphology. Long forms supported mitochondrial fusion; short forms were better able to restore energetic efficiency. The complete rescue of mitochondrial network morphology required a balance of long and short forms of at least two isoforms, as shown by combinatorial isoform silencing and co-expression experiments. Thus, multiple OPA1 isoforms are required for mitochondrial dynamics, while any single isoform can support all other functions. These findings will be useful in designing gene therapies for patients with OPA1 haploinsufficiency.
ObjectiveMounting evidence links neurodegenerative disorders such as Parkinson disease and Alzheimer disease with mitochondrial dysfunction, and recent emphasis has focused on mitochondrial dynamics and quality control. Mitochondrial dynamics and mtDNA maintenance is another link recently emerged, implicating mutations in the mitochondrial fusion genes OPA1 and MFN2 in the pathogenesis of multisystem syndromes characterized by neurodegeneration and accumulation of mtDNA multiple deletions in postmitotic tissues. Here, we report 2 Italian families affected by dominant chronic progressive external ophthalmoplegia (CPEO) complicated by parkinsonism and dementia.MethodsPatients were extensively studied by optical coherence tomography (OCT) to assess retinal nerve fibers, and underwent muscle and brain magnetic resonance spectroscopy (MRS), and muscle biopsy and fibroblasts were analyzed. Candidate genes were sequenced, and mtDNA was analyzed for rearrangements.ResultsAffected individuals displayed a slowly progressive syndrome characterized by CPEO, mitochondrial myopathy, sensorineural deafness, peripheral neuropathy, parkinsonism, and/or cognitive impairment, in most cases without visual complains, but with subclinical loss of retinal nerve fibers at OCT. Muscle biopsies showed cytochrome c oxidase‐negative fibers and mtDNA multiple deletions, and MRS displayed defective oxidative metabolism in muscle and brain. We found 2 heterozygous OPA1 missense mutations affecting highly conserved amino acid positions (p.G488R, p.A495V) in the guanosine triphosphatase domain, each segregating with affected individuals. Fibroblast studies showed a reduced amount of OPA1 protein with normal mRNA expression, fragmented mitochondria, impaired bioenergetics, increased autophagy and mitophagy.InterpretationThe association of CPEO and parkinsonism/dementia with subclinical optic neuropathy widens the phenotypic spectrum of OPA1 mutations, highlighting the association of defective mitochondrial dynamics, mtDNA multiple deletions, and altered mitophagy with parkinsonism. Ann Neurol 2015;78:21–38
OPA1 is a dynamin-related GTPase that controls mitochondrial dynamics, cristae integrity, energetics and mtDNA maintenance. The exceptional complexity of this protein is determined by the presence, in humans, of eight different isoforms that, in turn, are proteolytically cleaved into combinations of membrane-anchored long forms and soluble short forms. Recent advances highlight how each OPA1 isoform is able to fulfill "essential" mitochondrial functions, whereas only some variants carry out "specialized" features. Long forms determine fusion, long or short forms alone build cristae, whereas long and short forms together tune mitochondrial morphology. These findings offer novel challenging therapeutic potential to gene therapy.
Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.
Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that modulate LHON penetrance must take into account also the exposure to environmental triggers such as tobacco smoke.
Mitochondrial diseases are highly heterogeneous metabolic disorders caused by genetic alterations in the mitochondrial DNA (mtDNA) or in the nuclear genome. In this study, we investigated a panel of blood biomarkers in a cohort of 123 mitochondrial patients, with prominent neurological and muscular manifestations. These biomarkers included creatine, fibroblast growth factor 21 (FGF21) and growth/differentiation factor 15 (GDF-15), and the novel cell free circulating-mtDNA (ccf-mtDNA). All biomarkers were significantly increased in the patient group. After stratification by the specific phenotypes, ccf-mtDNA was significantly increased in the Mitochondrial Encephalomyopathy Lactic Acidosis Stroke-like episodes syndrome (MELAS) group, and FGF21 and GDF-15 were significantly elevated in patients with MELAS and Myoclonic Epilepsy Ragged Red Fibers syndrome. On the contrary, in our cohort, creatine was not associated to a specific clinical phenotype. Longitudinal assessment in four MELAS patients showed increased levels of ccf-mtDNA in relation to acute events (stroke-like episodes/status epilepticus) or progression of neurodegeneration. Our results confirm the association of FGF21 and GDF-15 with mitochondrial translation defects due to tRNA mutations. Most notably, the novel ccf-mtDNA was strongly associated with MELAS and may be used for monitoring the disease course or to evaluate the efficacy of therapies, especially in the acute phase. Key messages • FGF21/GDF15 efficiently identifies mitochondrial diseases due to mutations in tRNA genes. • The novel ccf-mtDNA is associated with MELAS and increases during acute events. • Creatine only discriminates severe mitochondrial patients. • FGF21, GDF-15, and ccf-mtDNA are possibly useful for monitoring therapy efficacy.
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