Pathogenesis Of Leber's Hereditary Optic Neuropathy Research Articles

Assocation Between Leber's Hereditary Optic Neuropathy and MT-ND1 3460G>A Mutation-Induced Alterations in Mitochondrial Function, Apoptosis, and Mitophagy.

PurposeTo investigate the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-linked MT-ND1 3460G>A mutation.MethodsCybrid cell models were generated by fusing mitochondrial DNA-less ρ0 cells with enucleated cells from a patient carrying the m.3460G>A mutation and a control subject. The impact of m.3460G>A mutations on oxidative phosphorylation was evaluated using Blue Native gel electrophoresis, and measurements of oxygen consumption were made with an extracellular flux analyzer. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Assays for apoptosis and mitophagy were undertaken via immunofluorescence analysis.ResultsNineteen Chinese Han pedigrees bearing the m.3460G>A mutation exhibited variable penetrance and expression of LHON. The m.3460G>A mutation altered the structure and function of MT-ND1, as evidenced by reduced MT-ND1 levels in mutant cybrids bearing the mutation. The instability of mutated MT-ND1 manifested as defects in the assembly and activity of complex I, respiratory deficiency, diminished mitochondrial adenosine triphosphate production, and decreased membrane potential, in addition to increased production of mitochondrial ROS in the mutant cybrids carrying the m.3460G>A mutation. The m.3460G>A mutation mediated apoptosis, as evidenced by the elevated release of cytochrome c into the cytosol and increasing levels of the apoptotic-associated proteins BAK, BAX, and PARP, as well as cleaved caspases 3, 7, and 9, in the mutant cybrids. The cybrids bearing the m.3460G>A mutation exhibited reduced levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PTEN-induced kinase 1/parkin-dependent mitophagy.ConclusionsOur findings highlight the critical role of m.3460G>A mutation in the pathogenesis of LHON, manifested by mitochondrial dysfunction and alterations in apoptosis and mitophagy.

Two detrimental mutations in cattle mitogenome indicate the presence of Leber's hereditary optic neuropathy

While mitochondriopathies, mitochondrial diseases, caused by mutations in mitochondrial DNA (mtDNA), are well documented in humans, single pathogenic mtDNA mutation or disorders are unknown in livestock populations. In a survey of 799 complete cattle mtDNAs belonging to more than 120 breeds two mutations, one in ND1 (C4171T) and the other in ND4L (T10663C) gene were identified, that are confirmed to be pathogenic in humans causing Leber's hereditary optic neuropathy (LHON). In one Cika cow with T10663C mutation, which was in humans reported to cause an acute onset of visual loss or/and many other LHON associated clinical manifestations, an exophthalmia of the right eye that might fit to the pathogenesis of LHON was observed. This work supports the existence of potentially detrimental mtDNA mutations in cattle, while aetiology and pathogenesis need to be further documented.

Mitophagy activation repairs Leber's hereditary optic neuropathy-associated mitochondrial dysfunction and improves cell survival.

Leber's hereditary optic neuropathy (LHON) is a classical mitochondrial disease caused by mutations in the mitochondrial DNA encoding complex I subunits. Oxidative stress associated with complex I defect has been implicated in developing LHON phenotype such as retinal ganglion cell (RGC) death and loss of vision. However, the mechanism of LHON pathogenesis is still not very clear and thus no effective therapies are available to date. Using cybrid models for LHON, we show that autophagy is significantly compromised in cells carrying LHON-specific mtDNA mutations, which results in reduced clearance of dysfunctional mitochondria contributing to cell death. We further show that pharmacological activation of autophagy selectively clears the damaged mitochondria and thus repairs mitochondrial defects and improves overall cell survival in LHON cell models. Our results suggest that compromised autophagy is the missing link from oxidative stress to LHON pathogenesis. Activation of mitophagy ameliorates mitochondrial defects and exerts a protective role by improving cell survival in cells carrying LHON mutations that could be utilized as a potential therapeutic target for LHON treatment.

Identification of mitochondrial DNA ND1 T3866C mutation in three ethnic Han Chinese families affected with Leber's hereditary optic neuropathy

To report on the clinical, genetic and molecular characteristics of three ethnic Han Chinese families affected with Leber's hereditary optic neuropathy (LHON). The three families were all diagnosed with LHON. Ophthalmologic examinations were conducted on the probands . The ND1, ND4 and ND6 genes of the mitochondrial DNA (mtDNA) were amplified with PCR respectively for the screening of three primary mutations G3460A, G11778A and T14484C. The entire mtDNA of the probands were also amplified by PCR. Analysis of mtDNA in the three pedigrees has failed to find the presence of the three LHON associated mutations but presence of a homoplastic ND1 T3866C mutation in all probands and their matrilineal relatives . The probands had different levels of visual impairment. The penetrance in the three families has been calculated as 12.5%, 11.1% and 33.3%, respectively. The T3866C mutation has resulted in replacement of isoleucine at position 187 with theronine. The isoleucine at position 187 is located at one of the transmembrane domains of ND1 polypeptide. Above results have suggested that the ND1 T3866C mutation might have been involved in the pathogenesis of LHON in the three Chinese families studied.

Study on three common mitochondrial DNA mutations in Leber's hereditary optic neuropathy

To screen for genetic mutations in 35 patients with Leber's hereditary optic neuropathy (LHON). Polymerase chain reaction and DNA sequencing were used to screen for the presence of mitochondrial DNA mutations. The total detection rate of top 3 common LHON mutations were 20.0%, which included 6 cases of ND4 11778 G to A, 1 case of ND1 3460 G to A. No ND6 14484 T to C mutation was detected. A ND4 G11719A synonymous mutation was found in all patients. In addition, 21 other mutations were discovered among 23 patients, among which 13 had a single mutation, 8 had a second mutations, and 2 had a third mutation. Among the 21 mutations, ND4 11778 G to A had a frequency of 28.6%(6/21). ND1 3552 T to A, ND6 14470 T to C, ND4 11794 T to C, ND1 3497 C to T and 3644 T to C respectively had a frequency of 19.0% (4/21), 19.0%(4/21), 14.3%(3/21), 9.5%(2/21) and 9.5%(2/21). Among the 3 patients who harbored a ND4 11794 T to C mutation, 2 were heteroplasmic and one was homoplasmic in nature. The ND4 11778 G to A mutation is common in the Top "3" primary mutations of patients with LHON. Candidate LHON mutation ND1 3552 T to A or ND1 3644 T to C resulted in LHON pathogenesis as single or synergistic effect. The visual impairment at onset of the disease with candidate mutation were better than the eyes with the ND4 11778 G to A mutation.

Genome-wide expression profile of LHON patients with the 11778 mutation

AimTo obtain a whole genome-expression profile in Leber hereditary optic neuropathy (LHON), patients with the 11 778 mitochondrial DNA mutation.MethodsRNA was extracted from leucocytes and cDNA reverse-transcribed and hybridised to...

Mitochondrial DNA Haplogroups M7b1′2 and M8a Affect Clinical Expression of Leber Hereditary Optic Neuropathy in Chinese Families with the m.11778G→A Mutation

Leber hereditary optic neuropathy (LHON) is the most extensively studied mitochondrial disease, with the majority of the cases being caused by one of three primary mitochondrial DNA (mtDNA) mutations. Incomplete disease penetrance and gender bias are two features of LHON and indicate involvement of additional genetic or environmental factors in the pathogenesis of the disorder. Haplogroups J, K, and H have been shown to influence the clinical expression of LHON in subjects harboring primary mutations in European families. However, whether mtDNA haplogroups would affect the penetrance of LHON in East Asian families has not been evaluated yet. By studying the penetrance of LHON in 1859 individuals from 182 Chinese families (including one from Cambodia) with the m.11778G-->A mutation, we found that haplogroup M7b1'2 significantly increases the risk of visual loss, whereas M8a has a protective effect. Analyses of the complete mtDNA sequences from LHON families with m.11778G-->A narrow the association of disease expression to m.12811T-->C (Y159H) in the NADH dehydrogenase 5 gene (MT-ND5) in haplogroup M7b1'2 and suggest that the specific combination of amino acid changes (A20T-T53I) in the ATP synthase 6 protein (MT-ATP6) caused by m.8584G-->A and m.8684C-->T might account for the beneficial background effect of M8a. Protein secondary-structure prediction for the MT-ATP6 with the two M8a-specific amino acid changes further supported our inferences. These findings will assist in further understanding the pathogenesis of LHON and guide future genetic counseling in East Asian patients with m.11778G-->A.

Oxidative Stress in Chinese Patients with Leber's Hereditary Optic Neuropathy

The cellular mechanisms of Leber's hereditary optic neuropathy (LHON) are poorly understood and there is little information on the onset of blindness and neurological degeneration. Here we define the relationship between oxidative stress and LHON pathogenicity at the cellular level. Venous blood was obtained from 14 patients with LHON, 21 asymptomatic maternal relatives and 30 normal individuals (controls). The level of free radicals in blood was assessed as luminol luminescence immediately and at 10 min after addition of phytohaemagglutinin. In LHON patients and their asymptomatic relatives, free radicals increased significantly immediately after adding phytohaemagglutinin compared with baseline and normal controls. After 10 min, however, there were no significant differences between and within the groups. These results suggest that the antioxidant capacity is reduced in the blood of patients with LHON and in asymptomatic relatives, and that oxidative stress plays a significant role in the pathogenesis of LHON.

Leber hereditary optic neuropathy mutations in the ND6 subunit of mitochondrial complex I affect ubiquinone reduction kinetics in a bacterial model of the enzyme

LHON (Leber hereditary optic neuropathy) is a maternally inherited disease that leads to sudden loss of central vision at a young age. There are three common primary LHON mutations, occurring at positions 3460, 11778 and 14484 in the human mtDNA (mitochondrial DNA), leading to amino acid substitutions in mitochondrial complex I subunits ND1, ND4 and ND6 respectively. We have now examined the effects of ND6 mutations on the function of complex I using the homologous NuoJ subunit of Escherichia coli NDH-1 (NADH:quinone oxidoreductase) as a model system. The assembly level of the NDH-1 mutants was assessed using electron transfer from deamino-NADH to the 'shortcut' electron acceptor HAR (hexammine ruthenium), whereas ubiquinone reductase activity was determined using DB (decylubiquinone) as a substrate. Mutant growth in minimal medium with malate as the main carbon source was used for initial screening of the efficiency of energy conservation by NDH-1. The results indicated that NuoJ-M64V, the equivalent of the common LHON mutation in ND6, had a mild effect on E. coli NDH-1 activity, while nearby mutations, particularly NuoJ-Y59F, NuoJ-V65G and NuoJ-M72V, severely impaired the DB reduction rate and cell growth on malate. NuoJ-Met64 and NuoJ-Met72 position mutants lowered the affinity of NDH-1 for DB and explicit C-type inhibitors, whereas NuoJ-Y59C displayed substrate inhibition by oxidized DB. The results are compatible with the notion that the ND6 subunit delineates the binding cavity of ubiquinone substrate, but does not directly take part in the catalytic reaction. How these changes in the enzyme's catalytic properties contribute to LHON pathogenesis is discussed.

Leber's hereditary optic neuropathy: A multifactorial disease

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease characterized by acute or subacute visual loss predominantly affecting young men. The majority of LHON cases are caused by one of the three primary mitochondrial DNA (mtDNA) mutations: G3460A/ND1, G11778A/ND4, or T14484C/ND6. Although the primary etiological factor of LHON is a mtDNA mutation, the presence of a primary mtDNA mutation does not necessarily lead to visual loss. The pathogenesis of LHON remains unclear. The marked incomplete penetrance and gender bias indicate that additional genetic (nuclear or mitochondrial) and epigenetic factors may also be involved. Deficiency in respiratory chain function and reactive oxygen species (ROS) are believed to play a pivotal role in the pathophysiology of the disease.

Severe Impairment of Complex I–Driven Adenosine Triphosphate Synthesis in Leber Hereditary Optic Neuropathy Cybrids

Leber hereditary optic neuropathy (LHON) is a maternally inherited form of central vision loss associated with mitochondrial DNA point mutations that affect the ND subunits of complex I. To elucidate the bioenergetic consequences of complex I dysfunction in LHON. The biochemical phenotypes of LHON mutations have been investigated using the transmitochondrial cytoplasmic hybrid (cybrid) cell model derived from the osteocarcoma parental cell line 143B.TK-. Research laboratories at neuroscience and biochemistry departments at the University of Bologna, Scientific Institute "E. Medea," and University of College Medical School. Fibroblast cell lines were obtained from patients affected with LHON, as defined by the presence of 1 pathogenic mutation, and from healthy volunteers as controls to construct cybrid cell lines. Complex I (glutamate-malate)- and complex II (succinate)-dependent adenosine triphosphate (ATP) synthesis, their respective respiratory rates, and total cellular ATP content were investigated using digitonin permeabilized cybrid cells. Multiple cybrid cell lines were constructed, introducing into osteosarcoma-derived rho(0) cells either wild-type or LHON mutant mitochondria carrying each of the 3 common mutations at positions 11778/ND4, 3460/ND1, and 14484/ND6. All 3 LHON mutations impaired ATP synthesis and the respiratory control ratio driven by complex I substrates. In contrast, succinate-driven ATP synthesis, respiration rates, and respiratory control ratios were not affected. However, the defective ATP synthesis with complex I substrates did not result in reduced ATP cellular content, indicating a compensatory mechanism. The LHON pathogenic mutations profoundly impair complex I-dependent synthesis of ATP, providing a common biochemical feature that may play a major role in LHON pathogenesis. Stratification of the results by mutation suggests that the 11778/ND4 mutation may induce an uncoupling of cybrid respiration, whereas the other 2 mutations impair the oxygen consumption rate.

Increased 8-Hydroxy-2′-Deoxyguanosine in Leukocyte DNA in Leber’s Hereditary Optic Neuropathy

This study was conducted to test the hypothesis that oxidative stress is involved in the pathogenesis of Leber's hereditary optic neuropathy (LHON). The level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidized DNA base common in cells undergoing oxidative stress, was measured in leukocyte DNA from patients with LHON and normal control subjects. The 8-OHdG and deoxyguanosine (dG) content in leukocyte DNA from 25 patients with LHON with an 11778 mitochondrial (mt)DNA mutation, 14 asymptomatic maternal relatives, and 27 unrelated normal control subjects were measured using high-performance liquid chromatography and electrochemical detection methods. The mean 8-OHdG/10(5) dG ratio from leukocyte DNA was 1.34 +/- 0.99 in patients with LHON, 1.00 +/- 0.91 in their asymptomatic maternal relatives, and 0.31 +/- 0.20 in normal control subjects, respectively. There was a statistically significant difference in the mean 8-OHdG/10(5) dG ratio between patients with LHON and normal control subjects and between asymptomatic maternal relatives and normal control subjects. The difference between patients with LHON and asymptomatic maternal relatives did not reach statistical significance. Patients with LHON with an 11778 mtDNA mutation had higher oxidative DNA damage. Oxidative stress has a key role in the pathogenesis of LHON.

Optic neuropathy induced by reductions in mitochondrial superoxide dismutase.

Reactive oxygen species (ROS) are suspected to play a pivotal role in the pathogenesis of Leber hereditary optic neuropathy (LHON), caused by mutated complex I subunit genes. It seems surprising that optic neuropathy has not been described in animals with a knockout of genes encoding critical anti-ROS defenses. If ROS have a role in the optic nerve injury of LHON, then increasing mitochondrial levels of ROS should induce optic neuropathy. To develop an animal model system for study of oxidative injury to the optic nerve, mitochondrial defenses were decreased against ROS by designing hammerhead ribozymes to degrade SOD2 mRNA. Several potential ribozymes were analyzed in vitro. The one with the best kinetic characteristics was cloned into a recombinant adeno-associated virus (rAAV) vector for delivery and testing in cells and animals. The effects of the AAV-expressing ribozyme on murine cell growth, SOD2 mRNA and protein, cellular ROS levels, and apoptosis were evaluated by RNase protection assay, immunoblot analysis, and ROS- and apoptosis-activated fluorescent probes. The rAAV-ribozyme was then injected into the eyes of DBA/1J mice, and the effect on the optic nerve was evaluated by ocular histopathologic examination. The AAV-expressing ribozyme decreased SOD2 mRNA and protein levels by as much as 85%, increased cellular superoxide, reduced mitochondrial membrane potential, and culminated in the death of infected cell lines by apoptosis without significantly altering complex I and III activity, somewhat spared in the most common LHON mutation (G11778A), although adenosine triphosphate (ATP) synthesis is markedly reduced. When inoculated into the eyes of mice, the AAV-expressing ribozyme led to loss of axons and myelin in the optic nerve and ganglion cells in the retina, the hallmarks of optic nerves examined at autopsy of patients with LHON. The striking similarity of the optic neuropathy to the histopathology of LHON is powerful evidence supporting ROS as a key factor in the pathogenesis of LHON.

LHON and other optic nerve atrophies: the mitochondrial connection.

The clinical, biochemical and genetic features of Leber's hereditary optic neuropathy (LHON) are reviewed. The etiology of LHON is complex, but the primary risk factor is a mutation in one of the seven mitochondrial genes that encode subunits of respiratory chain complex I. The pathogenesis of LHON is not yet understood, but one plausible model is that increased or altered mitochondrial ROS production renders the retinal ganglion cells vulnerable to apoptotic cell death. In addition to LHON, there are a large number of other optic nerve degenerative disorders including autosomal dominant optic atrophy, the toxic/nutritional optic neuropathies and glaucoma. A review of the recent scientific literature suggests that these disorders also involve mitochondrial dysfunction or altered mitochondrial signaling pathways in their pathogenesis. This mitochondrial link provides new avenues of experimental investigation to these major causes of loss of vision.

Secondary mutations of mitochondrial DNA in Japanese patients with Leber's hereditary optic neuropathy

Purpose: Based on studies on the pathogenesis of Leber's hereditary optic neuropathy (LHON), mitochondrial DNA (mtDNA) mutations have been divided into two types: primary and secondary. Primary mutations at nucleotide positions (nt) 11778, 3460, and 14484 can each cause LHON. Secondary mutations may be simultaneously found in LHON patients with a primary mutation, may occur at higher frequency in LHON patients than in normal controls, and may play an additional role in the pathogenesis of LHON. We examined the frequencies of secondary mutations of mtDNA at nt3394, 7444, 9438, 9804, 13708, and 15257 in 19 Japanese patients with LHON associated with primary mutations and 108 normal controls. Methods: Mutations were determined by restriction enzyme analysis or DNA sequencing using polymerase chain reaction (PCR) products. Results: One patient with an nt11778 mutation also had an nt13708 mutation. Another patient with an nt3460 mutation also had an nt7444 mutation. During DNA sequencing of the PCR fragment harboring nt3394, three novel mutations in the ND1 gene (nt3316, 3496, and 3497 mutations) were found in three patients with an nt11778 mutation. The frequency of these mutations in 108 control subjects was studied further: one (0.9%) had an nt3394 mutation, none (0%) had an nt9804 mutation, one (0.9%) had an nt13708 muation, two (1.9%) had nt3316 mutations, one (0.9%) had an nt3496 mutation, and two (1.9%) had nt3497 mutations. Conclusion: It is unlikely that the frequencies of secondary mutations in Japanese patients with LHON are higher than those in normal Japanese controls. It is possible that the mutations at nt3316, 3496, and 3497 are secondary mutations of LHON.

Energy Charge Is Not Decreased in Lymphocytes of Patients With Leber??s Hereditary Optic Neuropathy With the 11778 Mutation

A defect in mitochondrial energy conservation is strongly suggested to be involved in the pathogenesis of Leber's hereditary optic neuropathy (LHON). The authors therefore compared the energy charge in lymphocytes among patients with LHON, their asymptomatic maternal lineages, and normal control subjects. Blood samples were obtained from 7 patients, 10 asymptomatic maternal relatives, and 16 normal subjects. Molecular analysis confirmed that all had the homoplasmic 11,778 point mutation in the mtDNA of their blood cells. The concentrations of adenosine triphosphate (ATP), diphosphate (ADP), and monophosphate (AMP) were determined by high-performance liquid chromatography. The energy charge was calculated as (ATP + 1/2 ADP)/(ATP + ADP + AMP). The mean energy charges of lymphocytes were 0.871 +/- 0.049 in patients with LHON, 0.884 +/- 0.061 in their asymptomatic maternal relatives, and 0.885 +/- 0.061 in normal controls, respectively. No statistically significant difference was found among the three groups. Although the study did not find the anticipated change in energy charge in peripheral blood cells, this neither confirms nor rejects the notion that a defect in the mitochondrial oxidative phosphorylation system is involved in the pathogenesis of LHON.

X-inactivation patterns in female Leber's hereditary optic neuropathy patients do not support a strong X-linked determinant.

Leber's hereditary optic neuropathy (LHON) accounts for about 3% of the cases of blindness in young adult males. The underlying mitochondrial pathogenesis of LHON has been well studied, with specific mitochondrial DNA (mtDNA) mutations of structural genes described and well characterized. However, enigmatic aspects of the disease are not explained by mutation data, such as the higher proportion of affected males, the later onset of the disease in females, and the presence of unaffected individuals with a high proportion of mutant mtDNA. A hypothesis which has been put forward to explain the unusual disease expression is a dual model of mtDNA and X-linked nuclear gene inheritance. If a nuclear X-linked modifier gene influences the expression of the mitochondrial-linked mutant gene then the affected females should be either homozygous for the nuclear determinant, or if heterozygous, lyonization should favor the mutant X. In order to determine if an X-linked gene predisposes to LHON phenotype we studied X-inactivation patterns in 35 females with known mtDNA mutations from 10 LHON pedigrees. Our results do not support a strong X-linked determinant in LHON cause: 2 of the 10 (20%) manifesting carriers showed skewing of X-inactivation, as did 3 of the 25 (12%) nonmanifesting carriers.

Antibodies to human optic nerve in Leber's hereditary optic neuropathy

Mutations of mitochondrial DNA have now been identified in the majority of patients with Leber's hereditary optic neuropathy (LHON). However, these mutations do not explain all the clinical features of LHON, and other pathogenetic factors are likely to be operating. We have analysed serum from 69 LHON patients and their relatives, 58 controls and 14 patients with ischemic or compressive optic neuropathy. A significant proportion of LHON patients had circulating antibodies to tubulin protein. This finding supports the theory that autoimmunity may play some role in the pathogenesis of LHON.