Drosophila model to clarify the pathological significance of OPA1 in autosomal dominant optic atrophy.

Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase (OPA1) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 (hOPA1) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1. This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies.

Creation of an Isogenic Human iPSC-Based RGC Model of Dominant Optic Atrophy Harboring the Pathogenic Variant c.1861C>T (p.Gln621Ter) in the OPA1 Gene.

Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action.

Genetic diversity of 1,845 rhesus macaques improves genetic variation interpretation and identifies disease models.

Understanding and treating human diseases require valid animal models. Leveraging the genetic diversity in rhesus macaque populations across eight primate centers in the United States, we conduct targeted-sequencing on 1845 individuals for 374 genes linked to inherited human retinal and neurodevelopmental diseases. We identify over 47,000 single nucleotide variants, a substantial proportion of which are shared with human populations. By combining rhesus and human allele frequencies with established variant prediction methods, we develop a machine learning-based score that outperforms established methods in predicting missense variant pathogenicity. Remarkably, we find a marked number of loss-of-function variants and putative deleterious variants, which may lead to the development of rhesus disease models. Through phenotyping of macaques carrying a pathogenic OPA1:p.A8S variant, we identify a genetic model of autosomal dominant optic atrophy. Finally, we present a public website housing variant and genotype data from over two thousand rhesus macaques.

Identifying therapeutic compounds for autosomal dominant optic atrophy (ADOA) through screening in the nematode C. elegans.

Autosomal Dominant Optic Atrophy (ADOA) is a rare neurodegenerative condition, characterized by the bilateral loss of vision due to the degeneration of retinal ganglion cells. Its primary cause is linked to mutations in OPA1 gene, which ultimately affect mitochondrial structure and function. The current lack of successful treatments for ADOA emphasizes the need to investigate the mechanisms driving disease pathogenesis and exploit the potential of animal models for preclinical trials. Among such models, Caenorhabditis elegans stands out as a powerful tool, due its simplicity, its genetic tractability, and its relevance to human biology. Despite the lack of a visual system, the presence of mutated OPA1 in the nematode recapitulates ADOA pathology, by stimulating key pathogenic features of the human condition that can be studied in a fast and relatively non-laborious manner. Here, we provide a detailed guide on how to assess the therapeutic efficacy of chemical compounds, in either small or large scale, by evaluating three crucial phenotypes of humanized ADOA model nematodes, that express pathogenic human OPA1 in their GABAergic motor neurons: axonal mitochondria number, neuronal cell death and defecation cycle time. The described methods can deepen our understanding of ADOA pathogenesis and offer a practical framework for developing novel treatment schemes, providing hope for improved therapeutic outcomes and a better quality of life for individuals affected by this currently incurable condition.

Targeting DRP1 with Mdivi-1 to correct mitochondrial abnormalities in ADOA+ syndrome.

Autosomal dominant optic atrophy plus (ADOA+) is characterized by primary optic nerve atrophy accompanied by a spectrum of degenerative neurological symptoms. Despite ongoing research, no effective treatments are currently available for this condition. Our study provided evidence for the pathogenicity of an unreported c.1780T>C variant in the OPA1 gene through patient-derived skin fibroblasts and an engineered HEK293T cell line with OPA1 downregulation. We demonstrate that OPA1 insufficiency promoted mitochondrial fragmentation and increased DRP1 expression, disrupting mitochondrial dynamics. Consequently, this disruption enhanced mitophagy and caused mitochondrial dysfunction, contributing to the ADOA+ phenotype. Notably, the Drp1 inhibitor, mitochondrial division inhibitor-1 (Mdivi-1), effectively mitigated the adverse effects of OPA1 impairment. These effects included reduced Drp1 phosphorylation, decreased mitochondrial fragmentation, and balanced mitophagy. Thus, we propose that intervening in DRP1 with Mdivi-1 could correct mitochondrial abnormalities, offering a promising therapeutic approach for managing ADOA+.

[Chinese expert consensus on the clinical diagnosis and treatment of autosomal dominant optic atrophy (2024)].

Autosomal dominant optic atrophy (ADOA) primarily affects retinal ganglion cells and their axons, resulting in varying degrees of central vision loss from childhood. Due to the rarity of ADOA in clinical practice, Chinese ophthalmologists currently lack sufficient understanding of the disease and experience non-standardized diagnostic procedures and high clinical and genetic misdiagnosis rates. To address these issues, the Ophthalmology Group of China Alliance for Rare Diseases/Beijing Society of Rare Disease Clinical Care and Accessibility and the Neuro-ophthalmology Group of Ophthalmology Branch of Chinese Medical Association have established an expert panel to form consensus opinions based on extensive discussions. This consensus would enhance the knowledge and diagnostic capabilities of Chinese clinicians regarding ADOA and promote awareness of related treatment principles and genetic counseling.

Renal coloboma syndrome/dominant optic atrophy with severe retinal atrophy and de novo digenic mutations in PAX2 and OPA1.

Renal coloboma syndrome (RCS) and dominant optic atrophy are mainly caused by heterozygous mutations in PAX2 and OPA1, respectively. We describe a patient with digenic mutations in PAX2 and OPA1. A female infant was born without perinatal abnormalities. Magnetic resonance imaging at 4 months of age showed bilateral microphthalmia and optic nerve hypoplasia. Appropriate body size was present at 2 years of age, and mental development was favorable. Color fundus photography revealed severe retinal atrophy in both eyes. Electroretinography showed slight responses in the right eye, but no responses in the left eye, suggesting a high risk of blindness. Urinalysis results were normal, creatinine-based estimated glomerular filtration rate was 63.5 mL/min/1.73 m2, and ultrasonography showed bilateral hypoplastic kidneys. Whole exome sequencing revealed de novo frameshift mutations in PAX2 and OPA1. Both variants were classified as pathogenic (PVS1, PS2, PM2) based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Genetic testing for ocular diseases should be considered for patients with suspected RCS and a high risk of total blindness.

Inherited Optic Neuropathies: Real-World Experience in the Paediatric Neuro-Ophthalmology Clinic.

Inherited optic neuropathies affect around 1 in 10,000 people in England; in these conditions, vision is lost as retinal ganglion cells lose function or die (usually due to pathological variants in genes concerned with mitochondrial function). Emerging gene therapies for these conditions have emphasised the importance of early and expedient molecular diagnoses, particularly in the paediatric population. Here, we report our real-world clinical experience of such a population, exploring which children presented with the condition, how they were investigated and the time taken for a molecular diagnosis to be reached. A retrospective case-note review of paediatric inherited optic neuropathy patients (0-16 years) in the tertiary neuro-ophthalmology service at Moorfields Eye Hospital between 2016 and 2020 identified 19 patients. Their mean age was 9.3 ± 4.6 (mean ± SD) years at presentation; 68% were male, and 32% were female; and 26% had comorbidities, with diversity of ethnicity. Most patients had undergone genetic testing (95% (n = 18)), of whom 43% (n = 8) received a molecular diagnosis. On average, this took 54.8 ± 19.5 weeks from presentation. A cerebral MRI was performed in 70% (n = 14) and blood testing in 75% (n = 15) of patients as part of their workup. Continual improvement in the investigative pathways for inherited optic neuropathies will be paramount as novel therapeutics become available.

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.

AFG3L2 and ACO2-Linked Dominant Optic Atrophy: Genotype-Phenotype Characterization Compared to OPA1 Patients.

PURPOSE: Heterozygous mutations in the AFG3L2 gene (encoding a mitochondrial protease indirectly reflecting on OPA1 cleavage) and ACO2 gene (encoding the mitochondrial enzyme aconitase) are associated with isolated forms of Dominant Optic Atrophy (DOA). We aimed at describing their neuro-ophthalmological phenotype as compared with classic OPA1-related DOA. DESIGN: Cross-sectional study. METHODS: The following neuro-ophthalmological parameters were collected: logMAR visual acuity (VA), color vision, mean deviation and foveal threshold at visual fields, average and sectorial retinal nerve fiber layer (RNFL), and ganglion cell layer (GCL) thickness on optical coherence tomography. ACO2 and AFG3L2 patients were compared with an age- and sex-matched group of OPA1 patients with a 1:2 ratio. All eyes were analyzed using a clustered Wilcoxon rank sum test with the Rosner-Glynn-Lee method. RESULTS: A total of 44 eyes from 23 ACO2 patients and 26 eyes from 13 AFG3L2 patients were compared with 143 eyes from 72 OPA1 patients. All cases presented with bilateral temporal-predominant optic atrophy with various degree of visual impairment. Comparison between AFG3L2 and OPA1 failed to reveal any significant difference. ACO2 patients compared to both AFG3L2 and OPA1 presented overall higher values of nasal RNFL thickness (P = .029, P = .023), average thickness (P = .012, P = .0007), and sectorial GCL thickness. These results were confirmed also comparing separately affected and subclinical patients. CONCLUSIONS: Clinically, DOA remains a fairly homogeneous entity despite the growing genetic heterogeneity. ACO2 seems to be associated with an overall better preservation of retinal ganglion cells, probably depending on the different pathogenic mechanism involving mtDNA maintenance, as opposed to AFG3L2, which is involved in OPA1 processing and is virtually indistinguishable from classic OPA1-DOA.

Sectorial Ganglion Cell Complex Thickness as Biomarker of Vision Outcome in Patients With Dominant Optic Atrophy.

Purpose: Dominant optic atrophy (DOA) is an inherited condition caused by autosomal dominant mutations involving the OPA-1 gene. The aim of this study was to assess the relationship between macular ganglion cell and inner plexiform layer (GC-IPL) thickness obtained from structural optical coherence tomography (OCT) and visual outcomes in DOA patients. Methods: The study recruited 33 patients with confirmed OPA-1 heterozygous mutation and DOA. OCT scans were conducted to measure the GC-IPL thickness. The average and sectorial Early Treatment Diabetic Retinopathy Study (ETDRS) charts (six-sector macular analysis to enhance the topographical analysis) centered on the fovea were considered. Several regression analyses were carried out to investigate the associations between OCT metrics and final best-corrected visual acuity (BCVA) as the dependent variable. Results: The mean BCVA was 0.43 ± 0.37 logMAR, and the average macular GC-IPL thickness was 43.65 ± 12.56 µm. All of the GC-IPL sectors were significantly reduced and correlated with BCVA. The univariate linear regression and the multivariate stepwise regression modeling showed that the strongest association with final BCVA was observed with the internal superior GC-IPL thickness. Dividing patients based on BCVA, we found a specific pattern. Specifically, in patients with BCVA ≤ 0.3 logMAR, the external superior and inferior sectors together with the internal superior were more significant; whereas, for BCVA > 0.3 logMAR, the external superior sector and internal superior sector were more significant. Conclusions: The study identified OCT biomarkers associated with visual outcomes in DOA patients. Moreover, we assessed a specific OCT biomarker for DOA progression, ranging from patients in the early stages of disease with more preserved GC-IPL sectorial thickness to advanced stages with severe thinning.

OPA1 mutation affects autophagy and triggers senescence in autosomal dominant optic atrophy plus fibroblasts.

In several cases of mitochondrial diseases, the underlying genetic and bioenergetic causes of reduced oxidative phosphorylation (OxPhos) in mitochondrial dysfunction are well understood. However, there is still limited knowledge about the specific cellular outcomes and factors involved for each gene and mutation, which contributes to the lack of effective treatments for these disorders. This study focused on fibroblasts from a patient with Autosomal Dominant Optic Atrophy (ADOA) plus syndrome harboring a mutation in the Optic Atrophy 1 (OPA1) gene. By combining functional and transcriptomic approaches, we investigated the mitochondrial function and identified cellular phenotypes associated with the disease. Our findings revealed that fibroblasts with the OPA1 mutation exhibited a disrupted mitochondrial network and function, leading to altered mitochondrial dynamics and reduced autophagic response. Additionally, we observed a premature senescence phenotype in these cells, suggesting a previously unexplored role of the OPA1 gene in inducing senescence in ADOA plus patients. This study provides novel insights into the mechanisms underlying mitochondrial dysfunction in ADOA plus and highlights the potential importance of senescence in disease progression.

Human retinal organoids with an OPA1 mutation are defective in retinal ganglion cell differentiation and function.

Autosomal dominant optic atrophy (ADOA), mostly caused by heterozygous OPA1 mutations and characterized by retinal ganglion cell (RGC) loss and optic nerve degeneration, is one of the most common types of inherited optic neuropathies. Previous work using a two-dimensional (2D) differentiation model of induced pluripotent stem cells (iPSCs) has investigated ADOA pathogenesis but failed to agree on the effect of OPA1 mutations on RGC differentiation. Here, we use 3D retinal organoids capable of mimicking in vivo retinal development to resolve the issue. We generated isogenic iPSCs carrying the hotspot OPA1 c.2708_2711delTTAG mutation and found that the mutant variant caused defective initial and terminal differentiation and abnormal electrophysiological properties of organoid-derived RGCs. Moreover, this variant inhibits progenitor proliferation and results in mitochondrial dysfunction. These data demonstrate that retinal organoids coupled with gene editing serve as a powerful tool to definitively identify disease-related phenotypes and provide valuable resources to further investigate ADOA pathogenesis and screen for ADOA therapeutics.

OPA1 deficiency impairs oxidative metabolism in cycling cells, underlining a translational approach for degenerative diseases.

Dominant optic atrophy is an optic neuropathy with varying clinical symptoms and progression. A severe disorder is associated with certain OPA1 mutations and includes additional symptoms for >20% of patients. This underscores the consequences of OPA1 mutations in different cellular populations, not only retinal ganglionic cells. We assessed the effects of OPA1 loss of function on oxidative metabolism and antioxidant defences using an RNA-silencing strategy in a human epithelial cell line. We observed a decrease in the mitochondrial respiratory chain complexes, associated with a reduction in aconitase activity related to an increase in reactive oxygen species (ROS) production. In response, the NRF2 (also known as NFE2L2) transcription factor was translocated into the nucleus and upregulated SOD1 and GSTP1. This study highlights the effects of OPA1 deficiency on oxidative metabolism in replicative cells, as already shown in neurons. It underlines a translational process to use cycling cells to circumvent and describe oxidative metabolism. Moreover, it paves the way to predict the evolution of dominant optic atrophy using mathematical models that consider mitochondrial ROS production and their detoxifying pathways.

Visual Function and Inner Retinal Structure in Relation to Birth Factors in Autosomal Dominant Optic Atrophy.

Purpose: The extreme variation in expressivity of autosomal dominant optic atrophy (ADOA) is unexplained. It is present from early childhood, why there is reason to search for pre- and perinatal risk factors for poor vision in ADOA. The process of ganglion cell pruning in the fetus is of interest because mitochondria are involved in apoptosis. We hypothesized that suboptimal mitochondrial function makes the developing retina and optic nerve vulnerable to fetal stress in ADOA. We have examined visual function and inner retinal layer structure in relation to birth parameters in ADOA. Methods: The study included 142 participants with OPA1 ADOA, 62 unaffected first-degree relatives, and 90 unrelated control subjects. Outcome measures included best-corrected visual acuity, microperimetric sensitivity, nerve fiber layer (NFL) volume, and ganglion cell layer (GCL) volume. Descriptive parameters included birth weight, maternal age at birth, birth complications, and gestational age. Analysis was made using mixed modeling. Results: The analysis showed a significant positive association between microperimetric sensitivity and longer gestational age in ADOA (0.5 dB/week, P = 0.017). Interaction analysis showed a significant different association between microperimetric sensitivity and gestational age between participants with ADOA and the control groups (P = 0.007) and a significant difference in association between NFL volume and birth weight (P = 0.04) and gestational age (P = 0.02) between variant types. Conclusions: The study suggests that gestational age and birth weight may affect the expressivity of ADOA. The results support that prospectively collected pre- and perinatal data should be included in future studies of the natural history of ADOA.

OPA1 disease-causing mutants have domain-specific effects on mitochondrial ultrastructure and fusion.

Inner mitochondrial membrane fusion and cristae shape depend on optic atrophy protein 1, OPA1. Mutations in OPA1 lead to autosomal dominant optic atrophy (ADOA), an important cause of inherited blindness. The Guanosin Triphosphatase (GTPase) and GTPase effector domains (GEDs) of OPA1 are essential for mitochondrial fusion; yet, their specific roles remain elusive. Intriguingly, patients carrying OPA1 GTPase mutations have a higher risk of developing more severe multisystemic symptoms in addition to optic atrophy, suggesting pathogenic contributions for the GTPase and GED domains, respectively. We studied OPA1 GTPase and GED mutations to understand their domain-specific contribution to protein function by analyzing patient-derived cells and gain-of-function paradigms. Mitochondria from OPA1 GTPase (c.870+5G>A and c.889C>T) and GED (c.2713C>T and c.2818+5G>A) mutants display distinct aberrant cristae ultrastructure. While all OPA1 mutants inhibited mitochondrial fusion, some GTPase mutants resulted in elongated mitochondria, suggesting fission inhibition. We show that the GED is dispensable for fusion and OPA1 oligomer formation but necessary for GTPase activity. Finally, splicing defect mutants displayed a posttranslational haploinsufficiency-like phenotype but retained domain-specific dysfunctions. Thus, OPA1 domain-specific mutants result in distinct impairments in mitochondrial dynamics, providing insight into OPA1 function and its contribution to ADOA pathogenesis and severity.

Mitochondrial optic neuropathies.

Mitochondrial optic neuropathies have a leading role in the field of mitochondrial medicine ever since 1988, when the first mutation in mitochondrial DNA was associated with Leber's hereditary optic neuropathy (LHON). Autosomal dominant optic atrophy (DOA) was subsequently associated in 2000 with mutations in the nuclear DNA affecting the OPA1 gene. LHON and DOA are both characterized by selective neurodegeneration of retinal ganglion cells (RGCs) triggered by mitochondrial dysfunction. This is centered on respiratory complex I impairment in LHON and defective mitochondrial dynamics in OPA1-related DOA, leading to distinct clinical phenotypes. LHON is a subacute, rapid, severe loss of central vision involving both eyes within weeks or months, with age of onset between 15 and 35 years old. DOA is a more slowly progressive optic neuropathy, usually apparent in early childhood. LHON is characterized by marked incomplete penetrance and a clear male predilection. The introduction of next-generation sequencing has greatly expanded the genetic causes for other rare forms of mitochondrial optic neuropathies, including recessive and X-linked, further emphasizing the exquisite sensitivity of RGCs to compromised mitochondrial function. All forms of mitochondrial optic neuropathies, including LHON and DOA, can manifest either as pure optic atrophy or as a more severe multisystemic syndrome. Mitochondrial optic neuropathies are currently at the forefront of a number of therapeutic programs, including gene therapy, with idebenone being the only approved drug for a mitochondrial disorder.

The effects of nuclear DNA mutations on mitochondrial function.

ABSTRACT: The multiple functions of mitochondria, including adenosine triphosphate synthesis, are controlled by the coordination of both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) genomes. Mitochondrial disorders manifest because of impairment of energy metabolism. This article focuses on mutations in two nuclear genes and their effect on mitochondrial function. Mutations in the polymerase gamma, or POLG, gene are associated with multisystemic disease processes, including Alpers Syndrome, a severe childhood-onset syndrome. Mutations in the OPA1 gene are associated with autosomal dominant optic atrophy and other neurologic, musculoskeletal, and ophthalmologic symptoms. When assessing for disorders affecting energy metabolism, sequencing of both the mtDNA genome and the nDNA whole exome sequencing is necessary.