Autophagy controls the pathogenicity of OPA1 mutations in dominant optic atrophy

Optic Atrophy 1 (OPA1) gene mutations cause diseases ranging from isolated dominant optic atrophy (DOA) to various multisystemic disorders. OPA1, a large GTPase belonging to the dynamin family, is involved in mitochondrial network dynamics. The majority of OPA1 mutations encodes truncated forms of the protein and causes DOA through haploinsufficiency, whereas missense OPA1 mutations are predicted to cause disease through deleterious dominant‐negative mechanisms. We used 3D imaging and biochemical analysis to explore autophagy and mitophagy in fibroblasts from seven patients harbouring OPA1 mutations. We report new genotype–phenotype correlations between various types of OPA1 mutation and mitophagy. Fibroblasts bearing dominant‐negative OPA1 mutations showed increased autophagy and mitophagy in response to uncoupled oxidative phosphorylation. In contrast, OPA1 haploinsufficiency was correlated with a substantial reduction in mitochondrial turnover and autophagy, unless subjected to experimental mitochondrial injury. Our results indicate distinct alterations of mitochondrial physiology and turnover in cells with OPA1 mutations, suggesting that the level and profile of OPA1 may regulate the rate of mitophagy.     

Sustained intracellular calcium rise mediates neuronal mitophagy in models of autosomal dominant optic atrophy

Mitochondrial dysfunction and mitophagy are often hallmarks of neurodegenerative diseases such as autosomal dominant optic atrophy (ADOA) caused by mutations in the key mitochondrial dynamics protein optic atrophy 1 (Opa1). However, the second messengers linking mitochondrial dysfunction to initiation of mitophagy remain poorly characterized. Here, we show in mammalian and nematode neurons that Opa1 mutations trigger Ca²⁺-dependent mitophagy. Deletion or expression of mutated Opa1 in mouse retinal ganglion cells and Caenorhabditis elegans motor neurons lead to mitochondrial dysfunction, increased cytosolic Ca²⁺ levels, and decreased axonal mitochondrial density. Chelation of Ca²⁺ restores mitochondrial density in neuronal processes, neuronal function, and viability. Mechanistically, sustained Ca²⁺ levels activate calcineurin and AMPK, placed in the same genetic pathway regulating axonal mitochondrial density. Our data reveal that mitophagy in ADOA depends on Ca²⁺-calcineurin-AMPK signaling cascade.Subject terms: Cell biology, Neurological disorders     

Mitochondrial disorder with OPA1 mutation lacking optic atrophy

OPA1 is highly expressed in retina and optic nerve. OPA1 mutations were first identified in patients with non-syndromic autosomal dominant optic atrophy. Recently, OPA1 mutations were detected in a multisystemic disorder which has optic atrophy as the core clinical feature and multiple mitochondrial DNA (mtDNA) deletions in muscle. We report a patient with a multisystemic disorder and multiple muscle mtDNA deletions, carrying an in-frame deletion in OPA1 in the absence of optic atrophy. This patient provides evidence that optic atrophy is not the main clinical manifestation of OPA1-related disorders. OPA1 analysis should be considered in mitochondrial disorders despite the lack of optic atrophy.     

The molecular mechanisms of OPA1-mediated optic atrophy in Drosophila model and prospects for antioxidant treatment

Mutations in optic atrophy 1 (OPA1), a nuclear gene encoding a mitochondrial protein, is the most common cause for autosomal dominant optic atrophy (DOA). The condition is characterized by gradual loss of vision, color vision defects, and temporal optic pallor. To understand the molecular mechanism by which OPA1 mutations cause optic atrophy and to facilitate the development of an effective therapeutic agent for optic atrophies, we analyzed phenotypes in the developing and adult Drosophila eyes produced by mutant dOpa1 (CG8479), a Drosophila ortholog of human OPA1. Heterozygous mutation of dOpa1 by a P-element or transposon insertions causes no discernable eye phenotype, whereas the homozygous mutation results in embryonic lethality. Using powerful Drosophila genetic techniques, we created eye-specific somatic clones. The somatic homozygous mutation of dOpa1 in the eyes caused rough (mispatterning) and glossy (decreased lens and pigment deposition) eye phenotypes in adult flies; this phenotype was reversible by precise excision of the inserted P-element. Furthermore, we show the rough eye phenotype is caused by the loss of hexagonal lattice cells in developing eyes, suggesting an increase in lattice cell apoptosis. In adult flies, the dOpa1 mutation caused an increase in reactive oxygen species (ROS) production as well as mitochondrial fragmentation associated with loss and damage of the cone and pigment cells. We show that superoxide dismutase 1 (SOD1), Vitamin E, and genetically overexpressed human SOD1 (hSOD1) is able to reverse the glossy eye phenotype of dOPA1 mutant large clones, further suggesting that ROS play an important role in cone and pigment cell death. Our results show dOpa1 mutations cause cell loss by two distinct pathogenic pathways. This study provides novel insights into the pathogenesis of optic atrophy and demonstrates the promise of antioxidants as therapeutic agents for this condition.     

Mitochondrial oxidative phosphorylation is defective in the long-lived mutant clk-1

The long-lived mutant of Caenorhabditis elegans, clk-1, is unable to synthesize ubiquinone, CoQ(9). Instead, the mutant accumulates demethoxyubiquinone(9) and small amounts of rhodoquinone(9) as well as dietary CoQ(8). We found a profound defect in oxidative phosphorylation, a test of integrated mitochondrial function, in clk-1 mitochondria fueled by NADH-linked electron donors, i.e. complex I-dependent substrates. Electron transfer from complex I to complex III, which requires quinones, is severely depressed, whereas the individual complexes are fully active. In contrast, oxidative phosphorylation initiated through complex II, which also requires quinones, is completely normal. Here we show that complexes I and II differ in their ability to use the quinone pool in clk-1. This is the first direct demonstration of a differential interaction of complex I and complex II with the endogenous quinone pool. This study uses the combined power of molecular genetics and biochemistry to highlight the role of quinones in mitochondrial function and aging.     

Mutations in TOPORS cause autosomal dominant retinitis pigmentosa with perivascular retinal pigment epithelium atrophy

We report mutations in the gene for topoisomerase I-binding RS protein (TOPORS) in patients with autosomal dominant retinitis pigmentosa (adRP) linked to chromosome 9p21.1 (locus RP31). A positional-cloning approach, together with the use of bioinformatics, identified TOPORS (comprising three exons and encoding a protein of 1,045 aa) as the gene responsible for adRP. Mutations that include an insertion and a deletion have been identified in two adRP-affected families--one French Canadian and one German family, respectively. Interestingly, a distinct phenotype is noted at the earlier stages of the disease, with an unusual perivascular cuff of retinal pigment epithelium atrophy, which was found surrounding the superior and inferior arcades in the retina. TOPORS is a RING domain-containing E3 ubiquitin ligase and localizes in the nucleus in speckled loci that are associated with promyelocytic leukemia bodies. The ubiquitous nature of TOPORS expression and a lack of mutant protein in patients are highly suggestive of haploinsufficiency, rather than a dominant negative effect, as the molecular mechanism of the disease and make rescue of the clinical phenotype amenable to somatic gene therapy.     

Novel mutations of the PKD1 gene in Korean patients with autosomal dominant polycystic kidney disease

The gene for the most common form of autosomal dominant polycystic kidney disease (ADPKD), PKD1, has recently been characterized and shown to encode an integral membrane protein, polycystin-1, which is involved in cell-cell and cell-matrix interactions. Until now, approximately 30 mutations of the 3' single copy region of the PKD1 gene have been reported in European and American populations. However, there is no report of mutations in Asian populations. Using the polymerase chain reaction and single-strand conformation polymorphism (SSCP) analysis, 91 Korean patients with ADPKD were screened for mutation in the 3' single copy region of the PKD1 gene. As a result, we have identified and characterized six mutations: three frameshift mutations (11548del8bp, 11674insG and 12722delT), a nonsense mutation (Q4010X), and two missense mutations (R3752W and D3814N). Five mutations except for Q4010X are reported here for the first time. Our findings also indicate that many different mutations are likely to be responsible for ADPKD in the Korean population. The detection of additional disease-causing PKD1 mutations will help in identifying the location of the important functional regions of polycystin-1 and help us to better understand the pathophysiology of ADPKD.     

Refinement of the dominant optic atrophy locus (OPA1) to a 1.4-cM interval on chromosome 3q28-3q29, within a 3-Mb YAC contig

Dominant optic atrophy, type Kjer, is an autosomal dominant eye disease that is characterized by progressive optic atrophy with onset in early childhood, decrease of visual acuity, colour vision defects and centrocecal scotoma. By examination of 5 Danish families and the use of polymorphic markers, we have refined the localization of the OPA1 locus and assigned it to a 1.4-cM interval on chromosome 3q28-3q29, between markers D3S3669 and D3S3562. This localizes the gene on a 3-Mb YAC contig covering the disease locus. We have also located a possible candidate gene HRY to this contig. Received: 1 April 1996 / Revised: 8 August 1996     

Mutation of the PAX6 gene in patients with autosomal dominant keratitis.

Autosomal dominant keratitis (ADK) is an eye disorder chiefly characterized by corneal opacification and vascularization and by foveal hypoplasia. Aniridia (shown recently to result from mutations in the PAX6 gene) has overlapping clinical findings and a similar pattern of inheritance with ADK. On the basis of these similarities, we used a candidate-gene approach to investigate whether mutations in the PAX6 gene also result in ADK. Significant linkage was found between two polymorphic loci in the PAX6 region and ADK in a family with 15 affected members in four generations (peak LOD score = 4.45; theta = .00 with D11S914), consistent with PAX6 mutations being responsible for ADK. SSCP analysis and direct sequencing revealed a mutation in the PAX6 exon 11 splice-acceptor site. The predicted consequent incorrect splicing results in truncation of the PAX6 proline-serine-threonine activation domain. The SeyNeu mouse results from a mutation in the Pax-6 exon 10 splice-donor site that produces a PAX6 protein truncated from the same point as occurs in our family with ADK. Therefore, the SeyNeu mouse is an excellent animal model of ADK. The finding that mutations in PAX6 underlie ADK, along with a recent report that mutations in PAX6 also underlie Peters anomaly, implicates PAX6 broadly in human anterior segment malformations.     

Mitochondrial cristae-remodeling protein OPA1 in POMC neurons couples Ca2+ homeostasis with adipose tissue lipolysis

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Mitochondrial defects and oxidative damage in patients with peripheral arterial disease

Abnormal mitochondrial function is present in patients with peripheral arterial disease and may contribute to its clinical manifestations. However, the specific biochemical mitochondrial defects and their association with increased oxidative stress have not been fully characterized. Gastrocnemius muscle was obtained from peripheral arterial disease patients (n = 25) and age-matched controls (n = 16) and mitochondrial parameters were measured. Complexes I through IV of the electron transport chain were individually evaluated to assess for isolated defects. Muscle was also evaluated for protein and lipid oxidative changes by measuring the levels of protein carbonyls, lipid hydroperoxides, and total 4-hydroxy-2-nonenal binding and for the activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. Mitochondrial electron transport chain complexes I, III, and IV in arterial disease patients demonstrated significant reductions in enzymatic activities and mitochondrial respiration compared to controls. Oxidative stress biomarker analysis demonstrated significantly increased levels of protein carbonyls, lipid hydroperoxides, and 4-hydroxy-2-nonenal compared to control muscle. Antioxidant enzyme activities were altered, with a significant decrease in superoxide dismutase activity and significant increases in catalase and glutathione peroxidase. Peripheral arterial disease is associated with abnormal mitochondrial function and evidence of significant oxidative stress.     

Mitochondrial myopathy with loosely coupled oxidative phosphorylation in a case of Zellweger syndrome. A cytochemical-ultrastructural study

A newborn female, the second child of consanguineous parents, exhibited general muscle hypotonia, apathy, hepatomegaly and failure to thrive from birth and signs of craniofacial dysmorphia were present. Pipecolic and trihydroxicoprostanoic acid were excreted in the urine and serum transferrin, ferritin and iron were markedly elevated. At the age of 7 weeks the baby died of respiratory insufficiency. Besides malformations of the brain, renal cysts, liver damage with hypoplastic intrahepatic bile ducts and cholestasis, increased storage of iron and cytochemically proven deficiency of peroxisomes in liver and kidney, morphological studied provided evidence of a mitochondrial myopathy in striated muscle with the accumulation of enlarged bizarre mitochondria, showing only minor structural abnormalities. No defects of NADH-reductase, succinate-dehydrogenase or cytochrome-c-oxidase were demonstrated histochemically. Cytochemical-ultrastructural investigation of mitochondrial ATPase revealed activation of the ATP-synthesising enzyme even before the addition of an uncoupler, this indicating loosely coupled oxidative phosphorylation. In addition a high rate of subcellular autophagy with segregation of mitochondria and focal loss of fibrils was present. Muscle damage in Zellweger syndrome appears to be the consequence of complex, interacting metabolic processes. The mitochondrial myopathy thereby induced allows a better understanding of general muscle hypotonia, one of the leading symptoms of this disorder.     

Mitochondrial DNA mutation m.3635G>A may be associated with Leber hereditary optic neuropathy in Chinese

Leber hereditary optic neuropathy (LHON) was the first disease to be linked to the presence of a mitochondrial DNA (mtDNA) mutation. Nowadays over 95% of LHON cases are known to be caused by one of three primary mutations (m.11778G>A, m.14484T>C, and m.3460G>A). Reports for other (rare) primary mutations in LHON patients are not infrequent. Among those is the mutation m.3635G>A in the MT-ND1 gene which was reported to be pathogenic in a Russian LHON family. In this study, we report on a Chinese family with clinical features of LHON but without any of the three well-known primary mutations. Analysis of the complete mitochondrial genome in the proband revealed the presence of m.3635G>A and m.6228C>T, along with a full array of other variants that suggest the haplogroup M7b1. Evolutionary analysis indicates that site 3635, but not 6228, is highly conserved in vertebrates. Protein secondary-structure modeling for the MT-ND1 protein harboring amino acid change S110N indicates that mutant m.3635G>A decreases the protein hydrophobicity. Our current observations provide further support for a pathogenic role of m.3635G>A in patients with LHON.     

Yeast NDI1 improves oxidative phosphorylation capacity and increases protection against oxidative stress and cell death in cells carrying a Leber's hereditary optic neuropathy mutation

G11778A in the subunit ND4 gene of NADH dehydrogenase complex is the most common primary mutation found in Leber's hereditary optic neuropathy (LHON) patients. The NDI1 gene, which encodes the internal NADH-quinone oxidoreductase in Saccharomyces cerevisiae, was introduced into the nuclear genome of a mitochondrial defective human cell line, Le1.3.1, carrying the G11778A mutation. In transformant cell lines, LeNDI1-1 and -2, total and complex I-dependent respiration were fully restored and largely resistant to complex I inhibitor, rotenone, indicating a dominant role of NDI1 in the transfer of electrons in the host cells. Whereas the original mutant Le1.3.1 cell grows poorly in medium containing galactose, the transformants have a fully restored growth capacity in galactose medium, although the ATP production was not totally recovered. Furthermore, the increased oxidative stress in the cells carrying the G11778A mutation was alleviated in transformants, demonstrated by a decreased reactive oxygen species (ROS) level. Finally, transformants were also shown to be desensitized to induction to apoptosis and also exhibit greater resistance to paraquat-induced cell death. It is concluded that the yeast NDI1 enzyme can improve the oxidative phosphorylation capacity in cells carrying the G11778A mutation and protect the cells from oxidative stress and cell death.     

Elevated levels of arylsulfatase C activity in cultured skin fibroblasts of patients with autosomal dominant ichthyosis vulgaris

Summary Assays of arylsulfatase C and steroidsulfatase activities in cultured skin fibroblasts from 20 persons with autosomal dominant ichthyosis and 8 controls showed significantly elecated arylsulfatase C activities in cells from patients with autosomal dominant ichthyosis in comparison with those in control cells. This difference was not detected for steroidsulfatase activity. In leukocytes, activities of arylsulfatase C and of steroidsulfatase were similar for patients and controls.