mtDNA Mutations Research Articles

Harnessing HetHydrogel: A Universal Platform to Dropletize Single-Cell Multiomics.

A universal platform is developed for dropletizing single cell plate-based multiomic assays, consisting of three main pillars: a miniaturized open Heterogeneous Hydrogel reactor (abbreviated HetHydrogel) for multi-step biochemistry, its tunable permeability that allows Tn5 tagmentation, and single cell droplet barcoding. Through optimizing the HetHydrogel manufacturing procedure, the chemical composition, and cell permeation conditions, simultaneous high-throughput mitochondrial DNA genotyping and chromatin profiling at the single-cell level are demonstrated using a mixed-species experiment. This platform offers a powerful way to investigate the genotype-phenotype relationships of various mtDNA mutations in biological processes. The HetHydrogel platform is believed to have the potential to democratize droplet technologies, upgrading a whole range of plate-based single cell assays to high throughput format.

Therapeutic benefit of idebenone in patients with Leber hereditary optic neuropathy: The LEROS nonrandomized controlled trial

Leber hereditary optic neuropathy (LHON) is a mitochondrial disease leading to rapid and severe bilateral vision loss. Idebenone has been shown to be effective in stabilizing and restoring vision in patients treated within 1 year of onset of vision loss. The open-label, international, multicenter, natural history-controlled LEROS study (ClinicalTrials.gov NCT02774005) assesses the efficacy and safety of idebenone treatment (900mg/day) in patients with LHON up to 5 years after symptom onset (N= 199) and over a treatment period of 24months, compared to an external natural history control cohort (N= 372), matched by time since symptom onset. LEROS meets its primary endpoint and confirms the long-term efficacy of idebenone in the subacute/dynamic and chronic phases; the treatment effect varies depending on disease phase and the causative mtDNA mutation. The findings of the LEROS study will help guide the clinical management of patients with LHON.

Miro1 improves the exogenous engraftment efficiency and therapeutic potential of mitochondria transfer using Wharton's jelly mesenchymal stem cells

Mitochondria are important for maintaining cellular energy metabolism and regulating cellular senescence. Mitochondrial DNA (mtDNA) encodes subunits of the OXPHOS complexes which are essential for cellular respiration and energy production. Meanwhile, mtDNA variants have been associated with the pathogenesis of neurodegenerative diseases, including MELAS, for which no effective treatment has been developed. To alleviate the pathological conditions involved in mitochondrial disorders, mitochondria transfer therapy has shown promise. Wharton's jelly mesenchymal stem cells (WJMSCs) have been identified as suitable mitochondria donors for mitochondria-defective cells, wherein mitochondrial functions can be rescued. Miro1 participates in mitochondria trafficking by anchoring mitochondria to microtubules. In this study, we identified Miro1 over-expression as a factor that could help to enhance the efficiency of mitochondrial delivery. More specifically, we reveal that Miro1 over-expressed WJMSCs significantly improved intercellular communications, cell proliferation rates, and mitochondrial membrane potential, while restoring mitochondrial bioenergetics in mitochondria-defective fibroblasts. Furthermore, Miro1 over-expressed WJMSCs decreased rates of induced apoptosis and ROS production in MELAS fibroblasts; although, Miro1 over-expression did not rescue mtDNA mutation ratios nor mitochondrial biogenesis. This study presents a potentially novel therapeutic strategy for treating mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and other diseases associated with dysfunctional mitochondria, while the pathophysiological relevance of our results should be further verified by animal models and clinical studies.

Mitochondrial DNA integrity and metabolome profile are preserved in the human induced pluripotent stem cell reference line KOLF2.1J

Quality control of human induced pluripotent stem cells (iPSCs) is critical to ensure reproducibility of research. Recently, KOLF2.1J was characterized and published as a male iPSC reference line to study neurological disorders. Emerging evidence suggests potential negative effects of mtDNA mutations, but its integrity was not analyzed in the original publication. To assess mtDNA integrity, we conducted a targeted mtDNA analysis followed by untargeted metabolomics analysis. We found that KOLF2.1J mtDNA integrity was intact at the time of publication and is still preserved in the commercially distributed cell line. In addition, the basal KOLF2.1J metabolome profile was similar to that of the two commercially available iPSC lines IMR90 and iPSC12, but clearly distinct from an in-house-generated ERCC6R683X/R683X iPSC line modeling Cockayne syndrome. Conclusively, we validate KOLF2.1J as a reference iPSC line, and encourage scientists to conduct mtDNA analysis and unbiased metabolomics whenever feasible.

Mitochondria: The master regulator of aging

Mitochondria are ATP-producing organelles in eukaryotic organisms that serve as the cell’s power plants. Besides, mitochondria are integral to regulating cellular homeostasis and metabolism as a result of their essential roles in reactive oxygen species (ROS) production, bioenergetics, catabolism and anabolism, heme and iron-sulfur biosynthesis, iron and calcium homeostasis, apoptosis and signal transduction, as well as immunity and inflammation. It is well accepted that mitochondria are evolutionarily derived from endosymbiotic alphaproteobacteria within eukaryotic cells adapted for effective energy transduction. Although most of the mitochondrial DNA (mtDNA) is thought to have been transported to the eukaryotic nucleus during evolution, mitochondria may have preserved protein-coding genes within their own DNA. Accumulating data show that a progressive decline of mitochondria regulates aging. The present review aims to outline the role of mitochondria in various aspects of aging, including unfolded protein response, generation of ROS, and the contribution of somatic mtDNA mutations as well as inflammation in aging. Moreover, we propose mitochondria-targeted nanoparticles and mitochondrial genome editing as novel tools to modify mitochondrial genome aberrations.

MtDNA deletions and aging.

Aging is the major risk factor in most of the leading causes of mortality worldwide, yet its fundamental causes mostly remain unclear. One of the clear hallmarks of aging is mitochondrial dysfunction. Mitochondria are best known for their roles in cellular energy generation, but they are also critical biosynthetic and signaling organelles. They also undergo multiple changes with organismal age, including increased genetic errors in their independent, circular genome. A key group of studies looking at mice with increased mtDNA mutations showed that premature aging phenotypes correlated with increased deletions but not point mutations. This generated an interest in mitochondrial deletions as a potential fundamental cause of aging. However, subsequent studies in different models have yielded diverse results. This review summarizes the research on mitochondrial deletions in various organisms to understand their possible roles in causing aging while identifying the key complications in quantifying deletions across all models.

ETHICALLY MANAGING LEIGH’S SYNDROME: A CLINICAL CASE STUDY

Leigh syndrome, a rare inherited neurometabolic disorder described as subacute necrotizing encephalomyelopathy, affects the central nervous system and falls within a group of mitochondrial cytopathies involving disordered oxidative phosphorylation in the affected neural cells leading to disrupted enzyme of mitochondrial aberrations are attributed to variously inherited nDNA or mtDNA mutations The present study strives to demonstrate the efficacy, and therefore the need and ethical justification of therapeutic intervention at-least in the speech-language and swallowing realm; despite poor prognosis and the high risk of early death, in such infants through a representative case study. The case “x”, a three years old girl suffered classical cognitive, neuromotor, and linguistic regression following suspected pneumonia at 18 months (probable trigger), before which she had 10-15 words vocabulary, able to recognize body parts, follow simple verbal command. The child presented severe dystonia, hypotonia, moderate dysphagia with persistent of sucking reflexes, oromotor inadequacies, absence of spoken words, absence of receptive language except to name-calling. Intensive physiotherapeutic and speech-language intervention were initiated. Orofacial exercises, dysphagia management protocols including the compensatory approaches and dietary modifications as well as direct language therapy for increase in vocabulary were taken up. After 2 months of therapy, there were significant improvements especially in swallowing rate and strength, integration of infantile reflexes and increase in linguistic receptivity, which clearly warrants continued therapy and consideration of AAC. Keywords: Neurological disorder, Speech disorder, Leigh’s syndrome, Speech therapy.

MitoTALEN reduces the mutant mtDNA load in neurons

Mutations within mitochondrial DNA (mtDNA) frequently give rise to severe encephalopathies. Given that a majority of these mtDNA defects exist in a heteroplasmic state, we harnessed the precision of mitochondrial-targeted TALEN (mitoTALEN) to selectively eliminate mutant mtDNA within the central nervous system (CNS) of a murine model harboring a heteroplasmic mutation in the mitochondrial tRNA alanine gene (m.5024C>T). This targeted approach was accomplished by the utilization of AAV-PHP.eB and a neuron-specific synapsin promoter for effective neuronal delivery and expression of mitoTALEN. We found that most CNS regions were effectively transduced and showed a significant reduction in mutant mtDNA. This reduction was accompanied by an increase in mitochondrial tRNA alanine levels, which are drastically reduced by the m.5024C>T mutation. These results showed that mitochondrial-targeted gene editing can be effective in reducing CNS mutant mtDNA in vivo, paving the way for clinical trials in patients with mitochondrial encephalopathies.

Enhanced ROS Production in Mitochondria from Prematurely Aging mtDNA Mutator Mice.

An increase in mitochondrial DNA (mtDNA) mutations and an ensuing increase in mitochondrial reactive oxygen species (ROS) production have been suggested to be a cause of the aging process ("the mitochondrial hypothesis of aging"). In agreement with this, mtDNA-mutator mice accumulate a large amount of mtDNA mutations, giving rise to defective mitochondria and an accelerated aging phenotype. However, incongruously, the rates of ROS production in mtDNA mutator mitochondria have generally earlier been reported to be lower - not higher - than in wildtype, thus apparently invalidating the "mitochondrial hypothesis of aging". We have here re-examined ROS production rates in mtDNA-mutator mice mitochondria. Using traditional conditions for measuring ROS (succinate in the absence of rotenone), we indeed found lower ROS in the mtDNA-mutator mitochondria compared to wildtype. This ROS mainly results from reverse electron flow driven by the membrane potential, but the membrane potential reached in the isolated mtDNA-mutator mitochondria was 33 mV lower than that in wildtype mitochondria, due to the feedback inhibition of succinate oxidation by oxaloacetate, and to a lower oxidative capacity in the mtDNA-mutator mice, explaining the lower ROS production. In contrast, in normal forward electron flow systems (pyruvate (or glutamate) + malate or palmitoyl-CoA + carnitine), mitochondrial ROS production was higher in the mtDNA-mutator mitochondria. Particularly, even during active oxidative phosphorylation (as would be ongoing physiologically), higher ROS rates were seen in the mtDNA-mutator mitochondria than in wildtype. Thus, when examined under physiological conditions, mitochondrial ROS production rates are indeed increased in mtDNA-mutator mitochondria. While this does not prove the validity of the mitochondrial hypothesis of aging, it may no longer be said to be negated in this respect. This paper is dedicated to the memory of Professor Vladimir P. Skulachev.

Mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade response in melanoma

The mitochondrial genome (mtDNA) encodes essential machinery for oxidative phosphorylation and metabolic homeostasis. Tumor mtDNA is among the most somatically mutated regions of the cancer genome, but whether these mutations impact tumor biology is debated. We engineered truncating mutations of the mtDNA-encoded complex I gene, Mt-Nd5, into several murine models of melanoma. These mutations promoted a Warburg-like metabolic shift that reshaped tumor microenvironments in both mice and humans, consistently eliciting an anti-tumor immune response characterized by loss of resident neutrophils. Tumors bearing mtDNA mutations were sensitized to checkpoint blockade in a neutrophil-dependent manner, with induction of redox imbalance being sufficient to induce this effect in mtDNA wild-type tumors. Patient lesions bearing >50% mtDNA mutation heteroplasmy demonstrated a response rate to checkpoint blockade that was improved by ~2.5-fold over mtDNA wild-type cancer. These data nominate mtDNA mutations as functional regulators of cancer metabolism and tumor biology, with potential for therapeutic exploitation and treatment stratification.

Forward Genetic Screens Identify Mechanisms of Resistance to Small-Molecule Lactate Dehydrogenase Inhibitors.

Altered metabolism is a hallmark of cancer; however, it has been difficult to specifically target metabolism in cancer for therapeutic benefit. Cancers with genetically defined defects in metabolic enzymes constitute a subset of cancers where targeting metabolism is potentially accessible. Hürthle cell carcinoma of the thyroid (HTC) tumors frequently harbor deleterious mitochondrial DNA (mtDNA) mutations in subunits of complex I of the mitochondrial electron transport chain (ETC). Previous work has shown that HTC models with deleterious mtDNA mutations exhibit mitochondrial ETC defects that expose lactate dehydrogenase (LDH) as a therapeutic vulnerability. Here, we performed forward genetic screens to identify mechanisms of resistance to small-molecule LDH inhibitors. We identified two distinct mechanisms of resistance: upregulation of an LDH isoform and a compound-specific resistance mutation. Using these tools, we demonstrate that the anticancer activity of LDH inhibitors in cell line and xenograft models of complex I mutant HTC is through on-target LDH inhibition.

Epigenetic regulation of the nuclear genome associated with mitochondrial dysfunction in Leber’s hereditary optic neuropathy (LHON)

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial hereditary disease in which visual loss affects complex 1 activity of the electron transport chain of mitochondria. It first manifests as painless dulling or blurry in one or even both eyes, and as it develops, sharpness and color perception are lost. In addition to primary mitochondrial DNA (mtDNA) mutations, there are also other environmental and epigenetic factors involved in the pathogenesis of LHON. One of the most common locations for deadly pathogenic mutations in humans is the human complex I accessory NDUFS4 subunit gene. The iron-sulfur clusters of the electron input domain were distorted in the absence of NDUFS4, which reduced complex I function and elevated the production of reactive oxygen species. Therefore, here, we studied the epigenetic alterations of NDUFS4 by focusing on histone activation and repressive markers. We isolated peripheral blood mononuclear cells (PBMCs) from LHON patients and healthy individuals and examined epigenetic modifications in ND4 mutant cells and control cells. Chromatin immunoprecipitation-qRT PCR (ChIP-qRT PCR) assays were performed to investigate the modifications of histones. In comparison to their controls, both LHON patients and ND4 mutant cells exhibited a significant enrichment in activation and repressive markers. This finding indicates that these modifications might mitigate the impact of LHON mutations on complex 1 and aid in elucidating the mechanism underlying the progression of LHON disease.

Friend turned foe: selfish behavior of a spontaneously arising mitochondrial deletion in an experimentally evolved Caenorhabditis elegans population.

Selfish mitochondrial DNA (mtDNA) mutations are variants that can proliferate within cells and enjoy a replication or transmission bias without fitness benefits for the host. mtDNA deletions in Caenorhabditis elegans can reach high heteroplasmic frequencies despite significantly reducing fitness, illustrating how new mtDNA variants can give rise to genetic conflict between different levels of selection and between the nuclear and mitochondrial genomes. During a mutation accumulation experiment in C. elegans, a 1,034-bp deletion originated spontaneously and reached an 81.7% frequency within an experimental evolution line. This heteroplasmic mtDNA deletion, designated as meuDf1, eliminated portions of 2 protein-coding genes (coxIII and nd4) and tRNA-thr in entirety. mtDNA copy number in meuDf1 heteroplasmic individuals was 35% higher than in individuals with wild-type mitochondria. After backcrossing into a common genetic background, the meuDf1 mitotype was associated with reduction in several fitness traits and independent competition experiments found a 40% reduction in composite fitness. Experiments that relaxed individual selection by single individual bottlenecks demonstrated that the deletion-bearing mtDNA possessed a strong transmission bias, thereby qualifying it as a novel selfish mitotype.

NAD+ dependent UPRmt activation underlies intestinal aging caused by mitochondrial DNA mutations

Aging in mammals is accompanied by an imbalance of intestinal homeostasis and accumulation of mitochondrial DNA (mtDNA) mutations. However, little is known about how accumulated mtDNA mutations modulate intestinal homeostasis. We observe the accumulation of mtDNA mutations in the small intestine of aged male mice, suggesting an association with physiological intestinal aging. Using polymerase gamma (POLG) mutator mice and wild-type mice, we generate male mice with progressive mtDNA mutation burdens. Investigation utilizing organoid technology and in vivo intestinal stem cell labeling reveals decreased colony formation efficiency of intestinal crypts and LGR5-expressing intestinal stem cells in response to a threshold mtDNA mutation burden. Mechanistically, increased mtDNA mutation burden exacerbates the aging phenotype of the small intestine through ATF5 dependent mitochondrial unfolded protein response (UPRmt) activation. This aging phenotype is reversed by supplementation with the NAD+ precursor, NMN. Thus, we uncover a NAD+ dependent UPRmt triggered by mtDNA mutations that regulates the intestinal aging.

Huntington's disease affects mitochondrial network dynamics predisposing to pathogenic mitochondrial DNA mutations.

Huntington's disease (HD) predominantly affects the brain, causing a mixed movement disorder, cognitive decline and behavioural abnormalities. It also causes a peripheral phenotype involving skeletal muscle. Mitochondrial dysfunction has been reported in tissues of HD models, including skeletal muscle, and lymphoblast and fibroblast cultures from patients with HD. Mutant huntingtin protein (mutHTT) expression can impair mitochondrial quality control and accelerate mitochondrial ageing. Here, we obtained fresh human skeletal muscle, a post-mitotic tissue expressing the mutated HTT allele at physiological levels since birth, and primary cell lines from HTT CAG repeat expansion mutation carriers and matched healthy volunteers to examine whether such a mitochondrial phenotype exists in human HD. Using ultra-deep mitochondrial DNA (mtDNA) sequencing, we showed an accumulation of mtDNA mutations affecting oxidative phosphorylation. Tissue proteomics indicated impairments in mtDNA maintenance with increased mitochondrial biogenesis of less efficient oxidative phosphorylation (lower complex I and IV activity). In full-length mutHTT expressing primary human cell lines, fission-inducing mitochondrial stress resulted in normal mitophagy. In contrast, expression of high levels of N-terminal mutHTT fragments promoted mitochondrial fission and resulted in slower, less dynamic mitophagy. Expression of high levels of mutHTT fragments due to somatic nuclear HTT CAG instability can thus affect mitochondrial network dynamics and mitophagy, leading to pathogenic mtDNA mutations. We show that life-long expression of mutant HTT causes a mitochondrial phenotype indicative of mtDNA instability in fresh post-mitotic human skeletal muscle. Thus, genomic instability may not be limited to nuclear DNA, where it results in somatic expansion of the HTT CAG repeat length in particularly vulnerable cells such as striatal neurons. In addition to efforts targeting the causative mutation, promoting mitochondrial health may be a complementary strategy in treating diseases with DNA instability such as HD.

Comparison of capture-based mtDNA sequencing performance between MGI and illumina sequencing platforms in various sample types

BackgroundMitochondrial genome abnormalities can lead to mitochondrial dysfunction, which in turn affects cellular biology and is closely associated with the development of various diseases. The demand for mitochondrial DNA (mtDNA) sequencing has been increasing, and Illumina and MGI are two commonly used sequencing platforms for capture-based mtDNA sequencing. However, there is currently no systematic comparison of mtDNA sequencing performance between these two platforms. To address this gap, we compared the performance of capture-based mtDNA sequencing between Illumina's NovaSeq 6000 and MGI's DNBSEQ-T7 using tissue, peripheral blood mononuclear cell (PBMC), formalin-fixed paraffin-embedded (FFPE) tissue, plasma, and urine samples.ResultsOur analysis indicated a high degree of consistency between the two platforms in terms of sequencing quality, GC content, and coverage. In terms of data output, DNBSEQ-T7 showed higher rates of clean data and duplication compared to NovaSeq 6000. Conversely, the amount of mtDNA data obtained by per gigabyte sequencing data was significantly lower in DNBSEQ-T7 compared to NovaSeq 6000. In terms of detection mtDNA copy number, both platforms exhibited good consistency in all sample types. When it comes to detection of mtDNA mutations in tissue, FFPE, and PBMC samples, the two platforms also showed good consistency. However, when detecting mtDNA mutations in plasma and urine samples, significant differenceof themutation number detected was observed between the two platforms. For mtDNA sequencing of plasma and urine samples, a wider range of DNA fragment size distribution was found in NovaSeq 6000 when compared to DNBSEQ-T7. Additionally, two platforms exhibited different characteristics of mtDNA fragment end preference.ConclusionsIn summary, the two platforms generally showed good consistency in capture-based mtDNA sequencing. However, it is necessary to consider the data preferences generated by two sequencing platforms when plasma and urine samples were analyzed.

Mutational profiling of mitochondrial DNA reveals an epithelial ovarian cancer-specific evolutionary pattern contributing to high oxidative metabolism.

Epithelial ovarian cancer (EOC) heavily relies on oxidative phosphorylation (OXPHOS) and exhibits distinct mitochondrial metabolic reprogramming. Up to now, the evolutionary pattern of somatic mitochondrial DNA (mtDNA) mutations in EOC tissues and their potential roles in metabolic remodelling have not been systematically elucidated. Based on a large somatic mtDNA mutation dataset from private and public EOC cohorts (239 and 118 patients, respectively), we most comprehensively characterised the EOC-specific evolutionary pattern of mtDNA mutations and investigated its biological implication. Mutational profiling revealed that the mitochondrial genome of EOC tissues was highly unstable compared with non-cancerous ovary tissues. Furthermore, our data indicated the delayed heteroplasmy accumulation of mtDNA control region (mtCTR) mutations and near-complete absence of mtCTR non-hypervariable segment (non-HVS) mutations in EOC tissues, which is consistent with stringent negative selection against mtCTR mutation. Additionally, we observed a bidirectional and region-specific evolutionary pattern of mtDNA coding region mutations, manifested as significant negative selection against mutations in complex V (ATP6/ATP8) and tRNA loop regions, and potential positive selection on mutations in complex III (MT-CYB). Meanwhile, EOC tissues showed higher mitochondrial biogenesis compared with non-cancerous ovary tissues. Further analysis revealed the significant association between mtDNA mutations and both mitochondrial biogenesis and overall survival of EOC patients. Our study presents a comprehensive delineation of EOC-specific evolutionary patterns of mtDNA mutations that aligned well with the specific mitochondrial metabolic remodelling, conferring novel insights into the functional roles of mtDNA mutations in EOC tumourigenesis and progression.

Maternally Inherited Essential Hypertension May Be Associated with the Mutations in Mitochondrial tRNAGlu Gene.

Mitochondrial DNA (mtDNA) mutations are associated with essential hypertension (EH), but the molecular mechanism remains largely unknown. The aim of this study is to explore the association between mtDNA mutations and EH. Two maternally inherited families with EH are underwent clinical, genetic and biochemical assessments. mtDNA mutations are screened by PCR-Sanger sequencing and phylogenetic, and bioinformatics analyses are performed to evaluate the pathogenicity of mtDNA mutations. We also generate cytoplasmic hybrid (cybrid) cell lines to analysis mitochondrial functions. Matrilineal relatives exhibit variable degree of clinical phenotypes. Molecular analysis reveals the presence of m.A14693G and m.A14696G mutations in two pedigrees. Notably, the m.A14693G mutation occurs at position 54 in the TψC loop of tRNAGlu, a position which is critical for post-transcriptionally modification of tRNAGlu. While the m.A14696G mutation creates a novel base-pairing (51C-64G). Bioinformatic analysis shows that these mutations alter tRNAGlu secondary structure. Additionally, patients with tRNAGlu mutations exhibit markedly decreased in mtDNA copy number, mitochondrial membrane potential (MMP) and ATP, whereas the levels of reactive oxygen species (ROS) increase significantly. The m.A14696G and m.A14693G mutations lead to failure in tRNAGlu metabolism and cause mitochondrial dysfunction that is responsible for EH.

Long‐term efficacy of idebenone in patients with Leber hereditary optic neuropathy in the LEROS study: Analysing change in visual acuity according to causative mutation and disease phase

Aims/Purpose: Leber hereditary optic neuropathy (LHON) is a mitochondrial disease resulting in severe vision loss. The causative mitochondrial DNA (mtDNA) mutation impacts disease progression and prognosis. In LEROS, a Phase IV, open‐label interventional study (Clinicaltrials.gov NCT02774005), visual acuity (VA) outcomes following 24 months of idebenone treatment (IDE) were compared to those of an external Natural History (NH) cohort. Here, we compare VA change from baseline to Month 24 according to primary mtDNA mutation and disease stage.Methods: Eyes of patients with LHON and either a m.G11778A, m.T14484C or m.G3460A, mtDNA mutation were stratified by time since onset: subacute/dynamic (≤1 year) and chronic (>1 but ≤5 years). Data from 181 patients were compared to retrospective data from the NH cohort (N = 372), matched by time since symptom onset at baseline. We compare the difference in least squares‐mean VA change, or delta VA, from baseline to Month 24 in IDE versus NH eyes (a negative value favours IDE).Results: In treated subacute/dynamic m.G11778A eyes (n = 60 IDE), delta VA was −0.32 logMAR (p = 0.002) versus the NH cohort (n = 47). In chronic m.G11778A eyes, delta VA was −0.11 logMAR (n = 82 IDE vs n = 51 NH; p = 0.043). In subacute/dynamic m.T14484C eyes, delta VA was +0.20 logMAR (n = 35 IDE vs n = 10 NH; p = 0.285). In chronic m.T14484C eyes, delta VA was −0.52 logMAR (n = 11 IDE vs n = 18 NH; p < 0.001). In subacute/dynamic m.G3460A eyes, delta VA was +0.53 logMAR (n = 26 IDE vs n = 18 NH; p = 0.001). In chronic m.G3460A eyes, delta VA was −0.13 logMAR (n = 23 IDE vs n = 24 NH; p = 0.162).Conclusions: Idebenone improved VA in a large proportion of eyes; this benefit manifested to varying degrees depending on disease stage and mtDNA mutation. m.G11778A eyes saw a consistent benefit regardless of disease stage. m.T14484C and m.G3460A results should be interpreted with caution in some cases due to the relatively low number of eyes; further study is warranted.

Molecular mechanisms that cause cell death in LHON

Mitochondrial impairments, caused by mtDNA mutations, lead to RGC death and optic atrophy causing blindness in LHON. It was once thought that this was a disease of impaired bioenergetics. More recent thinking, considering the tempo of damage, studies in cybrids and a faithful mouse genetic model of LHON, has led to the realization that the problem is more that of the pathological generation of Reactive Oxygen Species (ROS). For example, synaptosome analysis from the brains of LHON mice show normal levels of ATP, but the blockage of Complex I activity was associated with very high levels of ROS. We hypothesized that the mtDNA mutation, specifically for 3460 in ND1, changed the atomic structures around COQ10 such that ROS were generated. In order to test that hypothesis, we conducted a series of experiments with statistical ensembles, built with Monte Carlo and Boltzmann sampling for simulations in computational chemistry. We began with the single amino acid mutation in ND1 of Complex I that switches hydrophobic alanine to hydrophilic threonine, at the entry of the Coenzyme Q10 binding channel. We explicated the mechanistic consequences of this mutation, using the tools of Molecular Dynamics and Free Energy Perturbation simulations. We showed that the mutant remained capable of binding Coenzyme Q10. Indeed, the mutated channel was even more proficient than the wild type in reducing ubiquinone to ubiquinol due to the closer average proximity of the Coenzyme Q10 headgroup to the chain of iron–sulfur clusters, from which it received electrons via quantum mechanical tunnelling. However, the mutation kinetically hindered the exit of the reduced (ubiquinol) form of Q10 from the ND1 channel, slowing the passage of its headgroup due to unfavourable electrostatics. This effect lead to overproduction of ROS via re‐oxidation of Coenzyme Q10 as electrons were sent back up the Iron–sulfur cluster chain, where the ROS production occurs. Another line of studies has shown that ROS lead to the death of RGCs affecting the smallest axons first. In those areas of the optic nerve in which larger axons are preponderate, the ROS are absorbed by the larger axons and little more happens. However, there is a dearth of large axons inferior temporally where the papillomacular bundle enters the optic nerve. Then, the ROS begins a reiterative and vicious cycle of cell death and the production of more ROS. This explains both the very peculiar spatial and temporal features of the optic neuropathy in LHON.