What OPA1 models teach us about potential therapy

PurposeThe majority of patients with autosomal dominant optic atrophy (DOA) harbour pathogenic OPA1 mutations. Although DOA is characterised by the preferential loss of retinal ganglion cells (RGC), about 20% of patients with OPA1 mutations will develop a more severe disease variant (DOA+), with additional neuromuscular features. In this prospective, observational case series, optical coherence tomography (OCT) was used to define the pattern of retinal nerve fibre layer (RNFL) loss in patients with both the pure and syndromal forms of DOA.MethodsForty patients with a molecular diagnosis of DOA due to OPA1 mutations were prospectively recruited from our neuro-ophthalmology clinic: 26 patients with isolated optic atrophy, and 14 patients manifesting DOA+ features. Peripapillary RNFL thickness was measured with the Fast RNFL (3.4) acquisition protocol on a Stratus OCT™.ResultsThere was a statistically significant reduction in average RNFL thickness in the OPA1 group compared with normal controls (P < 0.0001). The percentage decrease was greatest in the temporal quadrant (59.0%), followed by the inferior (49.6%), superior (41.8%), and nasal (25.9%) quadrants. Patients with DOA+ features had worse visual outcomes compared with patients with pure DOA, and except in the temporal quadrant, RNFL measurements were significantly thinner for the DOA+ group. There was an inverse correlation between average RNFL thickness and LogMAR visual acuity (P < 0.0001).ConclusionsRGC loss in DOA is characterised by severe involvement of the temporal papillomacular bundle, with relative sparing of the nasal fibres. RNFL thinning is more pronounced in patients with DOA+ phenotypes.

Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Fort Lauderdale, Florida, May 5-10, 2002.

Chapter 3 - Mitochondrial optic neuropathies

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.

Hereditary optic neuropathies (HONs) such as dominant optic atrophy (DOA) and Leber Hereditary Optic Neuropathy (LHON) are mitochondrial diseases characterized by a degenerative loss of retinal ganglion cells (RGCs) and are a cause of blindness worldwide. To date, there are only limited disease-modifying treatments for these disorders. The discovery of induced pluripotent stem cell (iPSC) technology has opened several promising opportunities in the field of HON research and the search for therapeutic approaches. This systematic review is focused on the two most frequent HONs (LHON and DOA) and on the recent studies related to the application of human iPSC technology in combination with biomaterials technology for their potential use in the development of RGC replacement therapies with the final aim of the improvement or even the restoration of the vision of HON patients. To this purpose, the combination of natural and synthetic biomaterials modified with peptides, neurotrophic factors, and other low- to medium-molecular weight compounds, mimicking the ocular extracellular matrices, with human iPSC or iPSC-derived cell retinal progenitors holds enormous potential to be exploited in the near future for the generation of transplantable RGC populations.

Inherited optic neuropathies (IONs) cause progressive irreversible visual loss in children and young adults. There are limited disease-modifying treatments, and most patients progress to become severely visually impaired, fulfilling the legal criteria for blind registration. The seminal discovery of the technique for reprogramming somatic nondividing cells into induced pluripotent stem cells (iPSCs) has opened several exciting opportunities in the field of ION research and treatment. A systematic review of the literature was conducted with PubMed using the following search terms: autosomal dominant optic atrophy, ADOA, dominant optic atrophy, DOA, Leber hereditary optic neuropathy, LHON, optic atrophy, induced pluripotent stem cell, iPSC, iPSC derived, iPS, stem cell, retinal ganglion cell, and RGC. Clinical trials were identified on the ClinicalTrials.gov website. This review article is focused on disease modeling and the therapeutic strategies being explored with iPSC technologies for the 2 most common IONs, namely, dominant optic atrophy and Leber hereditary optic neuropathy. The rationale and translational advances for cell-based and gene-based therapies are explored, as well as opportunities for neuroprotection and drug screening. iPSCs offer an elegant, patient-focused solution to the investigation of the genetic defects and disease mechanisms underpinning IONs. Furthermore, this group of disorders is uniquely amenable to both the disease modeling capability and the therapeutic potential that iPSCs offer. This fast-moving area will remain at the forefront of both basic and translational ION research in the coming years, with the potential to accelerate the development of effective therapies for patients affected with these blinding diseases.

Autosomal dominant optic atrophy (DOA) is the most common inherited optic neuropathy in the United Kingdom. DOA has an insidious onset in early childhood, typically presenting with bilateral, central visual loss caused by the preferential loss of retinal ganglion cells. 60%–70% of genetically confirmed DOA cases are associated with variants in OPA1, a ubiquitously expressed GTPase that regulates mitochondrial homeostasis through coordination of inner membrane fusion, maintenance of cristae structure, and regulation of bioenergetic output. Whether genetic correction of OPA1 pathogenic variants can alleviate disease-associated phenotypes remains unknown. Here, we demonstrate generation of patient-derived OPA1 c.1334G>A: p.R445H mutant induced pluripotent stem cells (iPSCs), followed by correction of OPA1 through CRISPR-Cas9-guided homology-directed repair (HDR) and evaluate the effect of OPA1 correction on mitochondrial homeostasis. CRISPR-Cas9 gene editing demonstrated an efficient method of OPA1 correction, with successful gene correction in 57% of isolated iPSCs. Correction of OPA1 restored mitochondrial homeostasis, re-establishing the mitochondrial network and basal respiration and ATP production levels. In addition, correction of OPA1 re-established the levels of wild-type (WT) mitochondrial DNA (mtDNA) and reduced susceptibility to apoptotic stimuli. These data demonstrate that nuclear gene correction can restore mitochondrial homeostasis and improve mtDNA integrity in DOA patient-derived cells carrying an OPA1 variant.

Preferential degeneration of retinal ganglion cells (RGCs) is a defining feature of the inherited optic neuropathies (IONs), a group of monogenic eye diseases predominately comprising Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (DOA). Their pathogenesis is characterised by mitochondrial dysfunction, which causes loss of RGCs leading to irreversible vision loss. Although currently incurable, there are several emerging therapeutic avenues encompassing gene therapies, precision medicine strategies and neuroprotection. These are underscored by recent technological advances such as next-generation sequencing and improved disease modelling. In this review, we discuss these advances and the impact these will have on future diagnostic and treatment capabilities. We first focus on the clinical presentation and pathogenic mechanisms of LHON and DOA, followed by a discussion of emerging technology to facilitate diagnosis and treatment. We highlight the current unmet clinical demand of IONs, and the promise of current and future research developments.

Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, characterised by the selective loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a dynamin-related GTPase protein. OPA1 plays a key role in the maintenance of the mitochondrial network, mitochondrial DNA integrity and bioenergetic function. However, our current understanding of how OPA1 dysfunction contributes to vision loss in DOA patients has been limited by access to patient-derived RGCs. Here, we used induced pluripotent stem cell (iPSC)-RGCs to study how OPA1 dysfunction affects cellular homeostasis in human RGCs. iPSCs derived from a DOA+ patient with the OPA1 R445H variant and isogenic CRISPR-Cas9-corrected iPSCs were differentiated to iPSC-RGCs. Defects in mitochondrial networks and increased levels of reactive oxygen species were observed in iPSC-RGCs carrying OPA1 R445H. Ultrastructural analyses also revealed changes in mitochondrial shape and cristae structure, with decreased endoplasmic reticulum (ER): mitochondrial contact length in DOA iPSC-RGCs. Mitochondrial membrane potential was reduced and its maintenance was also impaired following inhibition of the F1Fo-ATP synthase with oligomycin, suggesting that mitochondrial membrane potential is maintained in DOA iPSC-RGCs through reversal of the ATP synthase and ATP hydrolysis. These impairments in mitochondrial structure and function were associated with defects in cytosolic calcium buffering following ER calcium release and store-operated calcium entry, and following stimulation with the excitatory neurotransmitter glutamate. In response to mitochondrial calcium overload, DOA iPSC-RGCs exhibited increased sensitivity to opening of the mitochondrial permeability transition pore. These data reveal novel aspects of DOA pathogenesis in R445H patient-derived RGCs. The findings suggest a mechanism in which primary defects in mitochondrial network dynamics disrupt core mitochondrial functions, including bioenergetics, calcium homeostasis, and opening of the permeability transition pore, which may contribute to vision loss in DOA patients.

Leber hereditary optic neuropathy and autosomal dominant optic atrophy are the two most common inherited optic neuropathies. The latter has been associated with mutations in the OPA1 and OPA3 genes. To date, only six families with OPA3-associated dominant optic atrophy have been reported. In order to identify additional families, we performed Sanger sequencing of the OPA3 gene in 75 unrelated optic neuropathy patients. Affected individuals from two families were found to harbour the c.313C > G, p.(Gln105Glu) change in heterozygous state; this genetic defect has been previously reported in four dominant optic atrophy families. Intra- and interfamilial variability in age of onset and presenting symptoms was observed. Although dominant OPA3 mutations are typically associated with optic atrophy and cataracts, the former can be observed in isolation; we report a case with no lens opacities at age 38. Conversely, it is important to consider OPA3-related disease in individuals with bilateral infantile-onset cataracts and to assess optic nerve health in those whose vision fail to improve following lens surgery. The papillomacular bundle is primarily affected and vision is typically worse than 20/40. Notably, we describe one subject who retained normal acuities into the fifth decade of life. The condition can be associated with extraocular clinical features: two affected individuals in the present study had sensorineural hearing loss. The clinical heterogeneity observed in the individuals reported here (all having the same genetic defect in OPA3) suggests that the molecular pathology of the disorder is likely to be complex.

Dominant optic atrophy: Culprit mitochondria in the optic nerve.

Hereditary optic neuropathies are diseases affecting the optic nerve. The most common are mitochondrial hereditary optic neuropathies, i.e., the maternally inherited Leber’s hereditary optic neuropathy (LHON) and dominant optic atrophy (DOA). They both share a mitochondrial pathogenesis that leads to the selective loss of retinal ganglion cells and axons, in particular of the papillo-macular bundle. Typically, LHON is characterized by an acute/subacute loss of central vision associated with impairment of color vision and swelling of retinal nerve fibers followed by optic atrophy. DOA, instead, is characterized by a childhood-onset and slowly progressive loss of central vision, worsening over the years, leading to optic atrophy. The diagnostic workup includes neuro-ophthalmologic evaluation and genetic testing of the three most common mitochondrial DNA mutations affecting complex I (11778/ND4, 3460/ND1, and 14484/ND6) for LHON and sequencing of the nuclear gene OPA1 for DOA. Therapeutic strategies are still limited including agents that bypass the complex I defect and exert an antioxidant effect (idebenone). Further strategies are aimed at stimulating compensatory mitochondrial biogenesis. Gene therapy is also a promising avenue that still needs to be validated.

SummaryThe most frequent mitochondrial eye diseases are Leber's Hereditary Optic Neuropathy (LHON) and Dominant Optic Atrophy (DOA). They are both characterized by optic atrophy due to retinal ganglion cell loss. Extra‐ocular features have been described in both diseases. In particular, neurological involvement has been reported even though, for some of the neurological features in LHON, the possibility of a chance association is still debated.In LHON the main neurological features are: 1) Multiple Sclerosis “like” phenotype 2) Movement disorders including dystonia, parkinsonism and myoclonus 3) Peripheral neuropathy 5) Migraine 6) Hearing loss 6) “LHON‐MELAS‐Leigh” overlap.In DOA, the “plus” subtype is a well‐recognized entity characterized by the occurrence, over the decades, of optic atrophy and sensorineural deafness, polyneuropathy and myopathy with chronic external ophthalmoplegia, and in some families Parkinsonism and dementia. DOA “plus” is typically associated with missense mutations affecting the OPA1 GTPase domain, and skeletal muscle is remarkable for COX deficient fibers and mtDNA multiple deletions. More recently, a Multiple Sclerosis “like” phenotype has been described also in DOA.

Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mitochondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy. Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mitochondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under different retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in cortical neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochondrial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion cell death.

Dominant optic atrophy (DOA) is the most commonly inherited optic neuropathy. The majority of DOA is caused by mutations in the OPA1 gene, which encodes a dynamin-related GTPase located to the mitochondrion. OPA1 has been shown to regulate mitochondrial dynamics and promote fusion. Within the mitochondrion, proteolytically processed OPA1 proteins form complexes to maintain membrane integrity and the respiratory chain complexity. Although OPA1 is broadly expressed, human OPA1 mutations predominantly affect retinal ganglion cells (RGCs) that are responsible for transmitting visual information from the retina to the brain. Due to the scarcity of human RGCs, DOA has not been studied in depth using the disease affected neurons. To enable studies of DOA using stem-cell-derived human RGCs, we performed CRISPR-Cas9 gene editing to generate OPA1 mutant pluripotent stem cell (PSC) lines with corresponding isogenic controls. CRISPR-Cas9 gene editing yielded both OPA1 homozygous and heterozygous mutant ESC lines from a parental control ESC line. In addition, CRISPR-mediated homology-directed repair (HDR) successfully corrected the OPA1 mutation in a DOA patient's iPSCs. In comparison to the isogenic controls, the heterozygous mutant PSCs expressed the same OPA1 protein isoforms but at reduced levels; whereas the homozygous mutant PSCs showed a loss of OPA1 protein and altered mitochondrial morphology. Furthermore, OPA1 mutant PSCs exhibited reduced rates of oxygen consumption and ATP production associated with mitochondria. These isogenic PSC lines will be valuable tools for establishing OPA1-DOA disease models in vitro and developing treatments for mitochondrial deficiency associated neurodegeneration.