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Purpose: Retinal ganglion cells (RGCs) are susceptible to mitochondrial deficits and also the major cell type affected in patients with mutations in the OPA1 gene in autosomal dominant optic atrophy (ADOA). Here, we characterized mitochondria in RGCs in vitro from a heterozygous B6; C3-Opa1Q285STOP (Opa1+/-) mouse model to investigate mitochondrial changes underlying the pathology in ADOA., Methods: Mouse RGCs were purified from wild-type and Opa1+/- mouse retina by two-step immunopanning. The mitochondria in neurites of RGCs were labeled with MitoTracker Red for structure and motility measurement by time-lapse imaging. Mitochondrial bioenergetics were determined by the real-time measurement of oxygen consumption rate using a Seahorse XFe 96 Extracellular Flux Analyzer., Results: We observed a significant decrease in mitochondrial length in Opa1+/- RGCs with a remarkably higher proportion and density of motile mitochondria along the neurites. We also observed an increased transport velocity with a higher number of contacts between mitochondria in Opa1+/- RGC neurites. The oxygen consumption assays showed a severe impairment in basal respiration, Adenosine triphosphate-linked (ATP-linked) oxygen consumption, as well as reserve respiratory capacity, in RGCs from Opa1+/- mouse retina., Conclusions: Opa1 deficiency leads to significant fragmentation of mitochondrial morphology, activation of mitochondrial motility and impaired respiratory function in RGCs from the B6; C3-Opa1Q285STOP mouse model. This highlights the significant alterations in the intricate interplay between mitochondrial morphology, motility, and energy production in RGCs with Opa1 deficiency long before the onset of clinical symptoms of the pathology.

Purpose: To assess the topographic relationship between the photopic negative response (PhNR) and retinal ganglion cell distribution in healthy individuals., Method: Data was recorded from 16 healthy participants. The amplitude of PhNRs obtained in response to focal long duration (250 ms) and brief flash (5 ms), red (660 nm) on blue (469 nm) stimuli of increasing size (5° - full field) were measured. The number of retinal ganglion cell receptive fields (RGCf) in each stimulus area was established from the literature and regression analysis used to determine the relationships between: PhNR amplitude and number of RGCfs stimulated, PhNR density and the RGCf density and response per RGCf as a function of eccentricity., Results: The overall amplitude of the PhNR increased with stimulus size and the response density declined from ∼0.1 μV/deg in the macular region to ∼0.003 μV/deg approximately 45° from the fovea. Contrary to expectations, the relationship between the PhNR and number of RGCf was nonlinear, the response from more eccentric neurons being about three times greater than those in the macular region., Conclusions: Although the amplitude of the PhNR broadly maps on to the topographic distribution of RGCf the increase in PhNR amplitude with increasing eccentricity is only partly explained by RGCf numbers. Increases in the PhNR amplitude may be due to topographic variations in the contributions from other non-RGC neurons, as well as eccentricity-related morphologic and physiologic differences in RGCs.

Purpose: Progressive retinal ganglion cell (RGC) loss is the pathological hallmark of autosomal dominant optic atrophy (DOA) caused by pathogenic OPA1 mutations. The aim of this study was to conduct an in-depth psychophysical study of the visual losses in DOA and to infer any selective vulnerability of visual pathways subserved by different RGC subtypes., Methods: We recruited 25 patients carrying pathogenic OPA1 mutations and age-matched healthy individuals. Spatial contrast sensitivity functions (SCSFs) and chromatic contrast sensitivity were quantified, the latter using the Cambridge Colour Test. In 11 patients, long (L) and short (S) wavelength-sensitive cone temporal acuities were measured as a function of target illuminance, and L-cone temporal contrast sensitivity (TCSF) as a function of temporal frequency., Results: Spatial contrast sensitivity functions were abnormal, with the loss of sensitivity increasing with spatial frequency. Further, the highest L-cone temporal acuity fell on average by 10 Hz and the TCSFs by 0.66 log10 unit. Chromatic thresholds along the protan, deutan, and tritan axes were 8, 9, and 14 times higher than normal, respectively, with losses increasing with age and S-cone temporal acuity showing the most significant age-related decline., Conclusions: Losses of midget parvocellular, parasol magnocellular, and bistratified koniocellular RGCs could account for the losses of high spatial frequency sensitivity and protan and deutan sensitivities, high temporal frequency sensitivity, and S-cone temporal and tritan sensitivities, respectively. The S-cone-related losses showed a significant deterioration with increasing patient age and could therefore prove useful biomarkers of disease progression in DOA.

Purpose: To assess the effect of autosomal dominant optic atrophy (ADOA) on ON and OFF retinal ganglion cell (RGC) function by evaluating the ON and OFF components of the photopic negative response (PhNR)., Methods: Twelve participants from six families with OPA1 ADOA and 16 age-matched controls were recruited. Electrophysiological assessment involved pattern ERGs (PERGs), focal (20°) and full-field long-duration (250 ms) flash ERGs using a red light-emitting diode flash on a rod-saturating blue background, and full-field brief (300 μs) xenon flash ERGs using a red filter over a continuous rod saturating blue background. Amplitudes and implicit times of the ERG components were analyzed and the diagnostic potential of each electrophysiological technique was determined by generating receiver operating characteristic (ROC) curves., Results: Mean amplitudes of the N95 and all PhNRs, except the full-field PhNRON, were significantly reduced in participants with ADOA (P < 0.01). Subtraction of the group-averaged focal ERG of ADOA participants from that of controls showed an equal loss in the focal PhNRON and PhNROFF components, whereas in the full-field ERG the loss in the PhNROFF was greater than that in the PhNRON component. The areas under the ROC curve (AUC) for the focal PhNRON (0.92), focal PhNROFF (0.95), and full-field PhNROFF (0.83), were not significantly different from that of the PERG N95 (0.99)., Conclusions: In patients with ADOA, the PhNRON and PhNROFF components are nearly symmetrically reduced in the long-duration ERG, suggesting that ON- and OFF-RGC pathways may be equally affected.

Purpose: To investigate the developmental and ocular expression of Opa3 in a mouse model of 3-methylglutaconicaciduria type III and the effect of mutation on protein localization and mitochondrial morphology., Methods: The B6 C3-Opa3(L122P) mouse carrying a missense mutation in exon 2 (c.365T>C; p.L122P) of Opa3, which displays features of recessive 3-methylglutaconic aciduria type III was studied. The expression of Opa3 was determined with RT-PCR, quantitative PCR, and Western blot, in embryos (embryonic day [E]8 to postnatal day [P]0) and adult tissues, and by ocular immunohistochemistry. Mitochondria were stained using a mitochondrion-selective probe in mouse embryonic fibroblasts from Opa3(-/-) mutants and imaged by electron microscopy of the retinas., Results: The splice variants Opa3a and Opa3b were expressed in the lenses and the retinas in the Opa3(-/-) mice, with the expression of the Opa3a isoform predominant. Opa3 was expressed throughout embryonic development, with high levels of expression in the developing brain, retina, optic nerve, and lens. Opa3 localized to the mitochondria, and the L122P mutant protein did not mislocalize. Neither protein localized to the peroxisome. Opa3(-/-) mice displayed disrupted mitochondrial morphology in the retina. Wild-type Opa3 protein increased as the lenses aged, despite the reduction in Opa3 mRNA occurring as a part of lens differentiation. However, mutant Opa3 mRNA was upregulated in homozygous mutant lenses, suggesting a compensatory increase in expression, which may further increase Opa3 protein levels., Conclusions: Mutant Opa3 protein retains its mitochondrial localization and induces disrupted mitochondrial morphology. Opa3 accumulates in the lens. The results may reflect a slow turnover of Opa3 protein in vivo and may be important in normal lens physiology.

Purpose: To characterize changes in corneal collagen arrangement during mouse postnatal development., Methods: Small-angle X-ray scatter patterns were gathered from the centers of 32 excised mice corneas aged between postnatal days 10 (before eye opening) and 28 (onset of sexual maturity). These were analyzed to produce measurements of the average separation distance between corneal collagen fibrils. Changes in the predominant orientation of corneal collagen and its relative distribution during the same developmental period were determined using wide-angle X-ray scatter data collected at 0.2-mm intervals over the entire cornea and limbal region of each specimen., Results: Collagen interfibrillar spacing decreased in the days leading up to eye opening (61.3 +/- 2.9 nm at day 10 to 45.5 +/- 4.5 nm at day 14), after which it remained constant. However, changes in collagen orientation and distribution occurred throughout the entire developmental period. After eye opening at day 12, collagen alignment gradually increased in the peripheral cornea and limbus. By day 28, an annulus of highly aligned collagen surrounded the cornea., Conclusions: Changes in corneal thickness before and after eye opening are not caused by widespread alterations in the collagen fibrillar array but are more likely caused by expansion and contraction of regions devoid of regularly arranged collagen. The postnatal development of a corneal annulus of collagen, thought to play a role in stabilizing the curvature of the cornea, may be triggered by visual factors.

Purpose: The majority of patients with autosomal dominant optic atrophy (DOA) harbor pathogenic OPA1 mutations and certain missense mutations, mostly within the GTPase domain, have recently been shown to cause multiple mitochondrial DNA (mtDNA) deletions in skeletal muscle. This raises the possibility that the optic neuropathy could be the result of secondary mtDNA defects accumulating within retinal ganglion cells (RGCs). To explore this hypothesis, the authors looked for evidence of mitochondrial dysfunction in a mouse model of DOA and documented the visual and neurologic progression in aging mutant mice., Methods: Visual function was assessed with a rotating optokinetic (OKN) drum at ages 13 and 18 months and neurologic phenotyping was performed using the primary SHIRPA screen at age 13 months, comparing mutant Opa1(+/)(-) mice with wild-type C57Bl/6 mice. The presence of cytochrome c oxidase (COX) deficiency and multiple mtDNA deletions was investigated in gastrocnemius muscle and eye specimens harvested from 2- and 11-month-old Opa1(+/+) and Opa1(+/)(-) mice., Results: At age 13 months, Opa1(+/)(-) mice had a statistically significant reduction in OKN responses compared to C57Bl/6 controls with both 2 degrees and 8 degrees gratings (P < 0.001). At age 18 months, the difference between the two groups was significant for the 8 degrees grating (P = 0.003) but not for the 2 degrees grating (P = 0.082). Opa1(+/)(-) mice did not exhibit any significant neuromuscular deficits and no COX deficient areas or secondary mtDNA deletions were identified in skeletal muscle or the RGC layer. There was also no evidence of significant mtDNA depletion or proliferation in skeletal muscle from Opa1(+/)(-) mice., Conclusions: COX deficiency and mtDNA abnormalities do not contribute to optic nerve dysfunction in pure DOA.

Purpose: To examine retinal ganglion cell (RGC) and axonal abnormalities in an ENU-induced mutant mouse carrying a protein-truncating nonsense mutation in OPA1. Mutations in the OPA1 gene cause autosomal dominant optic atrophy (ADOA) in which loss of RGCs followed by myelin degeneration in the optic nerve leads to progressive decrease in visual acuity., Methods: Ultrastructure of the optic nerve was examined in heterozygous mutants and wild-type littermate controls at 6, 9, and 24 months using electron microscopy. The RGC layer was examined at 6 and 24 months., Results: There was an increase in the number of autophagosomes in the RGC layer in heterozygous mutants compared with wild type at 24 months. Signs of optic nerve degeneration were seen as early as 9 months in Opa1(+/-) mice, with more severe degeneration by 24 months. By 24 months, degeneration of axons was also seen in control mice. Numbers of opaque mitochondria in the Opa1(+/-) mice increased at 6 and 24 months, possibly representing an increase in the density of cristae to fulfill the energy requirements of the axon. In addition, mitochondria with vesiculation of the inner membranes, similar to the mutant mitochondria described in a mouse model of Charcot-Marie-Tooth type 2A, were observed., Conclusions: Mutations in OPA1 cause pathologic changes to optic nerve axons that are similar to, but occur earlier than, age-related degeneration. Increased autophagy is likely to result from an increase in abnormal mitochondria and could be one mechanism contributing to RGC loss and subsequent optic atrophy seen in ADOA.

Purpose: Autosomal dominant optic atrophy (ADOA) is characterized by primary degeneration of retinal ganglion cells and atrophy of the optic nerve. The OPA1 gene encodes a 960-amino-acid protein. In the current study the temporal and spatial localization of OPA1 were examined in developing and adult murine ocular tissues and the adult human eye. Because the Bst/+ mouse has been postulated as a model of ADOA, the mOPA1 expression in the Bst/+ retina was also examined., Methods: A polyclonal antibody generated against a C-terminal peptide of OPA1 was used to assess by immunohistochemistry the expression of mOPA1 in the wild-type embryonic and postnatal mouse ocular tissues and the Bst/+ retina. Western blot analyses of total proteins from a panel of adult human tissues were used to examine the expression of human OPA1, and spatial localization was assessed by immunohistochemistry., Results: The ocular expression of mOPA1 begins at E15 in the inner retina in a location corresponding to that of the subsequently developing ganglion cell layer (GCL) and peaks between postnatal day (P)0 and P1 in the retina and the optic nerve. There is a sharp decline in mOPA1 expression after P2, but it is expressed at a basal level until at least P12 in the GCL, inner plexiform layer (IPL), and inner nuclear layer (INL) of the retina as well as in the optic nerve. In the adult Bst/+ retina, mOPA1 is strongly expressed in the GCL and IPL and weakly in the INL. In the adult human eye, OPA1 is expressed in the GCL, IPL, INL, and outer plexiform layer (OPL) of the retina and in the optic nerve, where it is observed only in the myelinated region., Conclusions: OPA1 is not restricted to the GCL of the mammalian retina, and its expression extends into the IPL, INL, and OPL. OPA1 is distinctly expressed in the myelinated region beyond the lamina cribrosa in the human optic nerve, whereas its expression is weaker in the mouse optic nerve. In the Bst/+ mouse retina, despite the structural defects, mOPA1 expression is comparable to that observed in the wild-type adult mouse retina. These observations suggest a wider role for OPA1 than previously anticipated.

Purpose: To characterize the spectrum of mutations in the OPA1 gene in a large international panel of patients with autosomal dominant optic atrophy (adOA), to improve understanding of the range of functional deficits attributable to sequence variants in this gene, and to assess any genotype-phenotype correlations., Methods: All 28 coding exons of OPA1, intron-exon splice sites, 273 bp 5' to exon 1, and two intronic regions with putative function were screened in 94 apparently unrelated white patients of European origin with adOA by single-strand conformational polymorphism (SSCP)-heteroduplex analysis and direct sequencing. Clinical data were collated, and putative mutations were tested for segregation in the respective families by SSCP analysis or direct sequencing and in 100 control chromosomes. Further characterization of selected splice site mutations was performed by RT-PCR of patient leukocyte RNA. Staining of mitochondria in leukocytes of patients and control subjects was undertaken to assess gross differences in morphology and cellular distribution., Results: Twenty different mutations were detected, of which 14 were novel disease mutations (missense, nonsense, deletion-frameshift, and splice site alterations) and six were known mutations. Mutations were found in 44 (47%) of the 94 families included in the study. Ten new polymorphisms in the OPA1 gene were also identified. Mutations occur throughout the gene, with three clusters emerging: in the mitochondrial leader, in the highly conserved guanosine triphosphate (GTP)-binding domain, and in the -COOH terminus. Examination of leukocyte mitochondria from two unrelated patients with splice site mutations in OPA1 revealed no abnormalities of morphology or cellular distribution when compared with control individuals., Conclusions: This study describes 14 novel mutations in the OPA1 gene in patients with adOA, bringing the total number so far reported to 54. It is likely that many cases of adOA are due to mutations outside the coding region of OPA1 or to large-scale rearrangements. Evaluation of the mutation spectrum indicates more than one pathophysiological mechanism for adOA. Preliminary data suggests that phenotype-genotype correlation is complex, implying a role for other genetic modifying or environmental factors. No evidence was found of pathologic changes in leukocyte mitochondria of patients with adOA.

Purpose: To compare visual and pupil afferent function in dominant optic atrophy (DOA)., Methods: Patients with DOA who belonged to families showing evidence of linkage to the locus on chromosome 3q28-qter were recruited from the Moorfields Genetic Register. Patients and healthy control subjects underwent visual and pupil perimetry using a modified automated perimeter (Octopus 1-2-3; Interzeag, Schlieren, Switzerland). Five stimulus locations were tested: fixation, and at 17 degrees eccentricity along the 45 degrees and 135 degrees meridians in all four quadrants. The visual deficit (difference in decibels between the patient's luminance threshold and that in age-matched healthy control subjects) was compared directly with the pupil deficit (difference in decibels between the stimulus intensity giving the patient's pupil response and that giving an equivalent pupil response in healthy control subjects) at each test location., Results: Visual deficits and pupil afferent deficits were found at all five locations. The visual deficits were significantly greater than the pupil deficits at the four peripheral locations (median difference = 6.3 dB, P: < 0.001). At fixation, the difference was not significant (median difference = 2.3 dB, P: = 0.407)., Conclusions: Pupil function appears less affected than visual function at four of five locations tested. This result provides evidence that the retinotectal fibers serving the pupil light reflex are less susceptible to damage from the OPA1 genetic defect than the retinogeniculate fibers serving vision.