Nicotinamide-Rich Diet in DBA/2J Mice Preserves Retinal Ganglion Cell Metabolic Function as Assessed by PERG Adaptation to Flicker.

Flickering light increases metabolic demand in the inner retina. Flicker may exacerbate defective mitochondrial function in glaucoma, which will be reflected in the pattern electroretinogram (PERG), a sensitive test of retinal ganglion cell (RGC) function. We tested whether flicker altered the PERG of DBA/2J (D2) glaucomatous mice and whether vitamin B3-rich diet contributed to the flicker effect. D2 mice fed with either standard chow (control, n = 10) or chow/water enriched with nicotinamide (NAM, 2000 mg/kg per day) (treated, n = 10) were monitored from 3 to 12 months. The PERG was recorded with superimposed flicker (F-PERG) at either 101 Hz (baseline) or 11 Hz (test), and baseline-test amplitude difference (adaptation) evaluated. At endpoint, flat-mounted retinas were immunostained (RBPMS and mito-tracker). F-PERG adaptation was 41% in 3-month-old D2 and decreased with age more in control D2 than in NAM-fed D2 (GEE, p < 0.01). At the endpoint, F-PERG adaptation was 0% in control D2 and 17.5% in NAM-fed D2, together with higher RGC density (2.4×), larger RGC soma size (2×), and greater intensity of mitochondrial staining (3.75×). F-PERG adaptation may provide a non-invasive tool to assess RGC autoregulation in response to increased metabolic demand and test the effect of dietary/pharmacological treatments on optic nerve disorders.

Gene Therapy for Leber Hereditary Optic Neuropathy: Initial Results.

PURPOSE: Leber hereditary optic neuropathy (LHON) is a disorder characterized by severe and rapidly progressive visual loss when caused by a mutation in the mitochondrial gene encoding NADH:ubiquinone oxidoreductase subunit 4 (ND4). We have initiated a gene therapy trial to determine the safety and tolerability of escalated doses of an adeno-associated virus vector (AAV) expressing a normal ND4 complementary DNA in patients with a G to A mutation at nucleotide 11778 of the mitochondrial genome. DESIGN: In this prospective open-label trial (NCT02161380), the study drug (self-complementary AAV [scAAV]2(Y444,500,730F)-P1ND4v2) was intravitreally injected unilaterally into the eyes of 5 blind participants with G11778A LHON. Four participants with visual loss for more than 12 months were treated. The fifth participant had visual loss for less than 12 months. The first 3 participants were treated at the low dose of vector (5 × 10(9) vg), and the fourth participant was treated at the medium dose (2.46 × 10(10) vg). The fifth participant with visual loss for less than 12 months received the low dose. Treated participants were followed for 90 to 180 days and underwent ocular and systemic safety assessments along with visual structure and function examinations. PARTICIPANTS: Five legally blind patients with G11778A LHON. MAIN OUTCOME MEASURES: Loss of visual acuity. RESULTS: Visual acuity as measured by the Early Treatment Diabetic Retinopathy Study (ETDRS) eye chart remained unchanged from baseline to 3 months in the first 3 participants. For 2 participants with 90-day follow-up, acuity increased from hand movements to 7 letters in 1 and by 15 letters in 1, representing an improvement equivalent to 3 lines. No one lost vision, and no serious adverse events were observed. Minor adverse events included a transient increase of intraocular pressure (IOP), exposure keratitis, subconjunctival hemorrhage, a sore throat, and a transient increase in neutralizing antibodies (NAbs) against AAV2 in 1 participant. All blood samples were negative for vector DNA. CONCLUSIONS: No serious safety problems were observed in the first 5 participants enrolled in this phase I trial of virus-based gene transfer in this mitochondrial disorder. Additional study follow-up of these and additional participants planned for the next 4 years is needed to confirm these preliminary observations.

Mutant NADH dehydrogenase subunit 4 gene delivery to mitochondria by targeting sequence-modified adeno-associated virus induces visual loss and optic atrophy in mice.

PURPOSE: Although mutated G11778A NADH ubiquinone oxidoreductase subunit 4 (ND4) mitochondrial DNA (mtDNA) is firmly linked to the blindness of Leber hereditary optic neuropathy (LHON), a bona fide animal model system with mutated mtDNA complex I subunits that would enable probing the pathogenesis of optic neuropathy and testing potential avenues for therapy has yet to be developed. METHODS: The mutant human ND4 gene with a guanine to adenine transition at position 11778 with an attached FLAG epitope under control of the mitochondrial heavy strand promoter (HSP) was inserted into a modified self-complementary (sc) adeno-associated virus (AAV) backbone. The HSP-ND4FLAG was directed toward the mitochondria by adding the 23 amino acid cytochrome oxidase subunit 8 (COX8) presequence fused in frame to the N-terminus of green fluorescent protein (GFP) into the AAV2 capsid open reading frame. The packaged scAAV-HSP mutant ND4 was injected into the vitreous cavity of normal mice (OD). Contralateral eyes received scAAV-GFP (OS). Translocation and integration of mutant human ND4 in mouse mitochondria were assessed with PCR, reverse transcription-polymerase chain reaction (RT-PCR), sequencing, immunoblotting, and immunohistochemistry. Visual function was monitored with serial pattern electroretinography (PERG) and in vivo structure with spectral domain optical coherence tomography (OCT). Animals were euthanized at 1 year and processed for light and transmission electron microscopy. RESULTS: The PCR products of the mitochondrial and nuclear DNA extracted from infected retinas and optic nerves gave the expected 500 base pair bands. RT-PCR confirmed transcription of the mutant human ND4 DNA in mice. DNA sequencing confirmed that the PCR and RT-PCR products were mutant human ND4 (OD only). Immunoblotting revealed the expression of mutant ND4FLAG (OD only). Pattern electroretinograms showed a significant decrement in retinal ganglion cell function OD relative to OS at 1 month and 6 months after AAV injections. Spectral domain optical coherence tomography showed optic disc edema starting at 1 month post injection followed by optic nerve head atrophy with marked thinning of the inner retina at 1 year. Histopathology of optic nerve cross sections revealed reductions in the optic nerve diameters of OD versus OS where transmission electron microscopy revealed significant loss of optic nerve axons in mutant ND4 injected eyes where some remaining axons were still in various stages of irreversible degeneration with electron dense aggregation. Electron lucent mitochondria accumulated in swollen axons where fusion of mitochondria was also evident. CONCLUSIONS: Due to the UGA codon at amino acid 16, mutant G11778A ND4 was translated only in the mitochondria where its expression led to significant loss of visual function, loss of retinal ganglion cells, and optic nerve degeneration recapitulating the hallmarks of human LHON.

Induction of rapid and highly efficient expression of the human ND4 complex I subunit in the mouse visual system by self-complementary adeno-associated virus.

OBJECTIVE: To demonstrate the high efficiency and rapidity of allotopic expression of a normal human ND4 subunit of complex I in the vertebrate retina using a self-complementary adeno-associated virus (scAAV) vector for ocular gene delivery to treat acute visual loss in Leber hereditary optic neuropathy (LHON). METHODS: The nuclear-encoded human ND4 subunit fused to the P1 isoform of subunit C of adenosine triphosphate synthase (ATPc) mitochondrial targeting sequence and FLAG epitope was packaged in scAAV2 capsids or single-stranded (ss) AAV2 capsids. These constructs were injected into the vitreous cavities of mice. The contralateral eyes were injected with scAAV-green fluorescent protein (GFP). One week later, pattern electroretinograms and gene expression of the human ND4 subunit and GFP were evaluated. Quantitative analysis of ND4FLAG-injected eyes was assessed relative to Thy1.2-labeled retinal ganglion cells (RGCs). RESULTS: Pattern electroretinogram amplitudes remained normal in eyes inoculated with scAAV-ND4FLAG, ssAAV-ND4FLAG, and GFP. Confocal microscopy revealed the typical perinuclear mitochondrial expression of scAAV-ND4FLAG in almost the entire retinal flat mount. In contrast, scAAV-GFP expression was cytoplasmic and nuclear. Relative to Thy1.2-positive RGCs, quantification of scAAV-ND4FLAG-positive RGCs was 91% and that of ssAAV-ND4FLAG-positive RGCs was 51%. CONCLUSION: Treatment of acute visual loss due to LHON may be possible with a normal human ND4 subunit gene of complex I, mutated in most cases of LHON, when delivered by an scAAV vector. Clinical Relevance Unlike most retinal degenerations that result in slowly progressive loss of vision over many years, LHON due to mutated mitochondrial DNA results in apoplectic, bilateral severe and usually irreversible visual loss. For rescue of acute visual loss in LHON, a highly efficient and rapid gene expression system is required.

Reproducibility of pattern electroretinogram in glaucoma patients with a range of severity of disease with the new glaucoma paradigm.

PURPOSE: To determine the reproducibility of the pattern electroretinogram with the new Pattern Electroretinogram for Glaucoma (PERGLA) recording paradigm in glaucoma patients with a range of severity. DESIGN: Experimental study. PARTICIPANTS: Fifty-three glaucoma patients were recruited for the study (mean age +/- standard deviation [SD], 69+/-11 years). Their mean deviation (MD) global indices on static automatic perimetry ranged from 2.16 to -31.36 decibels (mean MD, -9.05). INTERVENTION: All patients had pattern electroretinogram recordings done 5 times by the same operator, on 5 different days with the standardized PERGLA paradigm. MAIN OUTCOME MEASURES: Pattern electroretinogram amplitude (microvolts), phase (pi radians), response variability (coefficient of variation [CV] = SD/mean x 100) of amplitude and phase of 2 partial averages that build up the pattern electroretinogram waveform, interocular asymmetry in amplitude and phase (in terms of the CV generated by the pattern electroretinogram software), signal-to-noise (S/N) ratio, SDs, CV, and intraclass correlation coefficient (ICC). All analyses were done on one eye of each subject, except when interocular asymmetry was studied. RESULTS: The CVs of intrasession variabilities in amplitude and phase were 12.08% and 2.20%, respectively, and those of intersession variabilities were 20.82% and 4.17%. The pattern electroretinogram produced intersession ICCs in amplitude and phase of 0.791 and 0.765, respectively. These ICCs were significantly higher than the ICCs for pattern electroretinogram interocular asymmetry in amplitude and phase (0.659 [P<0.05] and 0.571 [P<0.05], respectively). On average, the pattern electroretinogram S/N ratio in glaucomatous patients was about 5:1. CONCLUSIONS: The reproducibility of PERGLA in glaucomatous patients is sufficiently good for it to be considered a useful complementary clinical tool. Being more reproducible, direct measures of amplitude and phase should be more useful in monitoring progression than interocular asymmetry comparisons.

Pattern electroretinogram abnormality and glaucoma.

PURPOSE: To determine the existence of retinal ganglion cell dysfunction and associated risk factors in glaucoma suspects with increased optic disc cupping and normal visual field. DESIGN: Cross-sectional, observational study. PARTICIPANTS: Two hundred glaucoma suspect (GS) patients were identified based on optic disc abnormalities (vertical cup-to-disc ratios [C/D]>0.5; vertical C/D asymmetry >or= 0.2; disc hemorrhages; notching) in association with known glaucoma risk factors (positive family history, African American descent, increased intraocular pressure [IOP]), but normal visual fields. Forty-two patients had early manifest glaucoma (EMG). Sixteen normal black subjects were added to update previous pattern electroretinogram (PERG) normative data and to establish a normal control (NC) group with a racial breakdown comparable with that of the study groups. METHODS: Pattern electroretinograms were recorded simultaneously from both eyes using skin electrodes and automated analysis; visual fields were monitored with standard white-on-white automated perimetry (SAP) central 24-2 program; vertical C/D was evaluated by an independent reader from stereo disc photographs; and univariate and multivariate statistical analysis between PERG and other outcome measures was evaluated. MAIN OUTCOME MEASURES: Pattern electroretinogram amplitude (microV), phase (pi rad), and interocular asymmetry in amplitude and phase (%); and SAP mean deviation (MD; decibels), vertical C/D, age (years), IOP (mmHg), and race (black vs. nonblack). RESULTS: The PERG results were abnormal in at least 1 of the outcome measures in 52% of GS patients and 69% of EMG patients. The PERG amplitude was correlated weakly with both MD (P<0.01) and vertical C/D (P = 0.05). The correlation between PERG amplitude and MD and C/D was stronger (P<0.001) for interocular differences rather than absolute measures. Interocular PERG amplitude asymmetry increased with severity of disease (EMG>GS>NC; P<0.01). The PERG amplitude decline with age was steeper in patients with a more negative MD (P<0.01) and in patients with a more negative MD and a larger vertical C/D (P = 0.06). Black race (but not family history) was associated with lower PERG amplitude (P = 0.005) in GS and EMG patients, but not in normal controls (P = 0.44). CONCLUSIONS: The correlation between PERG abnormality and known risk factors for glaucoma indicates that PERG has a predictive potential for the development or progression of the disease, or both.