Genetic variants affecting NQO1 protein levels impact the efficacy of idebenone treatment in Leber hereditary optic neuropathy.

Idebenone, the only approved treatment for Leber hereditary optic neuropathy (LHON), promotes recovery of visual function in up to 50% of patients, but we can neither predict nor understand the non-responders. Idebenone is reduced by the cytosolic NAD(P)H oxidoreductase I (NQO1) and directly shuttles electrons to respiratory complex III, bypassing complex I affected in LHON. We show here that two polymorphic variants drastically reduce NQO1 protein levels when homozygous or compound heterozygous. This hampers idebenone reduction. In its oxidized form, idebenone inhibits complex I, decreasing respiratory function in cells. By retrospectively analyzing a large cohort of idebenone-treated LHON patients, classified by their response to therapy, we show that patients with homozygous or compound heterozygous NQO1 variants have the poorest therapy response, particularly if carrying the m.3460G>A/MT-ND1 LHON mutation. These results suggest consideration of patient NQO1 genotype and mitochondrial DNA mutation in the context of idebenone therapy.

Pathological mitophagy disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON), a disease associated with a mitochondrial DNA mutation, is characterized by blindness due to degeneration of retinal ganglion cells (RGCs) and their axons, which form the optic nerve. We show that a sustained pathological autophagy and compartment-specific mitophagy activity affects LHON patient-derived cells and cybrids, as well as induced pluripotent-stem-cell-derived neurons. This is variably counterbalanced by compensatory mitobiogenesis. The aberrant quality control disrupts mitochondrial homeostasis as reflected by defective bioenergetics and excessive reactive oxygen species production, a stress phenotype that ultimately challenges cell viability by increasing the rate of apoptosis. We counteract this pathological mechanism by using autophagy regulators (clozapine and chloroquine) and redox modulators (idebenone), as well as genetically activating mitochondrial biogenesis (PGC1-α overexpression). This study substantially advances our understanding of LHON pathophysiology, providing an integrated paradigm for pathogenesis of mitochondrial diseases and druggable targets for therapy.

Human RGR Gene and Associated Features of Age-Related Macular Degeneration in Models of Retina-Choriocapillaris Atrophy.

Age-related macular degeneration (AMD) is a progressive eye disease and the most common cause of blindness among the elderly. AMD is characterized by early atrophy of the choriocapillaris and retinal pigment epithelium (RPE). Although AMD is a multifactorial disease with many environmental and genetic risk factors, a hallmark of the disease is the origination of extracellular deposits, or drusen, between the RPE and Bruch membrane. Human retinal G-protein-coupled receptor (RGR) gene generates an exon-skipping splice variant of RGR-opsin (RGR-d; NP_001012740) that is a persistent component of small and large drusen. Herein, the findings show that abnormal RGR proteins, including RGR-d, are pathogenic in an animal retina with degeneration of the choriocapillaris, RPE, and photoreceptors. A frameshift truncating mutation resulted in severe retinal degeneration with a continuous band of basal deposits along the Bruch membrane. RGR-d produced less severe disease with choriocapillaris and RPE atrophy, including focal accumulation of abnormal RGR-d protein at the basal boundary of the RPE. Degeneration of the choriocapillaris was marked by a decrease in endothelial CD31 protein and choriocapillaris breakdown at the ultrastructural level. Fundus lesions with patchy depigmentation were characteristic of old RGR-d mice. RGR-d was mislocalized in cultured cells and caused a strong cell growth defect. These results uphold the notion of a potential hidden link between AMD and a high-frequency RGR allele.

Inhibition of autophagy curtails visual loss in a model of autosomal dominant optic atrophy.

In autosomal dominant optic atrophy (ADOA), caused by mutations in the mitochondrial cristae biogenesis and fusion protein optic atrophy 1 (Opa1), retinal ganglion cell (RGC) dysfunction and visual loss occur by unknown mechanisms. Here, we show a role for autophagy in ADOA pathogenesis. In RGCs expressing mutated Opa1, active 5' AMP-activated protein kinase (AMPK) and its autophagy effector ULK1 accumulate at axonal hillocks. This AMPK activation triggers localized hillock autophagosome accumulation and mitophagy, ultimately resulting in reduced axonal mitochondrial content that is restored by genetic inhibition of AMPK and autophagy. In C. elegans, deletion of AMPK or of key autophagy and mitophagy genes normalizes the axonal mitochondrial content that is reduced upon mitochondrial dysfunction. In conditional, RGC specific Opa1-deficient mice, depletion of the essential autophagy gene Atg7 normalizes the excess autophagy and corrects the visual defects caused by Opa1 ablation. Thus, our data identify AMPK and autophagy as targetable components of ADOA pathogenesis.

Correction: Retinal nerve fiber layer thickness predicts CSF amyloid/tau before cognitive decline.

[This corrects the article DOI: 10.1371/journal.pone.0232785.].

Retinal nerve fiber layer thickness predicts CSF amyloid/tau before cognitive decline.

BACKGROUND: Alzheimer's disease (AD) pathology precedes symptoms and its detection can identify at-risk individuals who may benefit from early treatment. Since the retinal nerve fiber layer (RNFL) is depleted in established AD, we tested whether its thickness can predict whether cognitively healthy (CH) individuals have a normal or pathological cerebrospinal fluid (CSF) Aß42 (A) and tau (T) ratio. METHODS: As part of an ongoing longitudinal study, we enrolled CH individuals, excluding those with cognitive impairment and significant ocular pathology. We classified the CH group into two sub-groups, normal (CH-NAT, n = 16) or pathological (CH-PAT, n = 27), using a logistic regression model from the CSF AT ratio that identified >85% of patients with a clinically probable AD diagnosis. Spectral-domain optical coherence tomography (OCT) was acquired for RNFL, ganglion cell-inner plexiform layer (GC-IPL), and macular thickness. Group differences were tested using mixed model repeated measures and a classification model derived using multiple logistic regression. RESULTS: Mean age (± standard deviation) in the CH-PAT group (n = 27; 75.2 ± 8.4 years) was similar (p = 0.50) to the CH-NAT group (n = 16; 74.1 ± 7.9 years). Mean RNFL (standard error) was thinner in the CH-PAT group by 9.8 (2.7) µm; p < 0.001. RNFL thickness classified CH-NAT vs. CH-PAT with 87% sensitivity and 56.3% specificity. CONCLUSIONS: Our retinal data predict which individuals have CSF biomarkers of AD pathology before cognitive deficits are detectable with 87% sensitivity. Such results from easy-to-acquire, objective and non-invasive measurements of the RNFL merit further study of OCT technology to monitor or screen for early AD pathology.

Choroidal thickness and the retinal ganglion cell complex in chronic Leber's hereditary optic neuropathy: a prospective study using swept-source optical coherence tomography.

BACKGROUND/OBJECTIVES: Choroidal thinning has been suggested in Leber's hereditary optic neuropathy (LHON). No study has been conducted of the choroid in relation to the retinal ganglion cell-inner plexiform layer (RGC-IPL). We sought to measure choroidal thickness in chronic LHON and to correlate thickness changes with the RGC-IPL. SUBJECTS/METHODS: Chronic LHON, 11778 mitochondrial DNA (mtDNA) mutation, patients (26 eyes; mean age: 35.1 ± 16.1 years) were prospectively recruited at Doheny Eye Center, University of California Los Angeles from March 2016 to July 2017. Age-matched healthy controls (27 eyes; mean age: 32.4 ± 11.1 years) were enroled for comparison. Swept-source optical coherence tomography (SS-OCT) imaging was performed in chronic LHON patients and compared with age-matched healthy controls. RESULTS: The macular choroid was significantly thinner in chronic LHON (250.5 ± 62.2 µm) compared with controls (313.9 ± 60.2 µm; p < 0.0001). The peripapillary choroid was also significantly thinner in chronic LHON (135.7 ± 51.4 µm) compared with controls (183.0 ± 61.8 µm, p < 0.001). Choroidal thickness strongly correlated with retinal nerve fibre layer (RNFL) thickness in both the macular (R2 = 0.72; 95% CI, 0.57-0.84) and peripapillary regions (R2 = 0.53; 95% CI, 0.31-0.70). Choroidal thickness was also significantly correlated with macular RGC-IPL thickness (R2 = 0.51; 95% CI, 0.26-0.73). CONCLUSIONS: Choroidal thinning in chronic LHON correlated strongly with both RNFL and RGC-IPL thicknesses. These findings may suggest a pathophysiological mechanism involving vascular pathology of the choroid in relation to the retinal ganglion cell complex in LHON.

The retinal choroid as an oculovascular biomarker for Alzheimer's dementia: A histopathological study in severe disease.

INTRODUCTION: Previous in vivo optical coherence tomography studies have proposed the retinal choroid as a potential oculovascular biomarker for Alzheimer's disease (AD). However, the clinical use of the choroid as a purported surrogate marker remains poorly understood. We pursued a histopathological approach to assess choroidal thickness and vascular morphology in severe disease. METHODS: Human postmortem tissues from 8 patients with AD (mean age: 80.1 ± 12.7 years) and from 11 age-matched controls (mean age: 78.4 ± 16.57 years) were analyzed. Thickness, area, and vascularity of the retinal choroid and its sublayers were measured from the nasal and temporal quadrants of the superior retina. RESULTS: Nasally, the choroid was thinner in the patients with AD than in the controls (22% thickness reduction; P < .001), but to our surprise, the choroid was thicker in the patients with AD than in the controls (~60% increase; P < .03) within the macula, temporally. The choroidal area was also significantly greater in the patients with AD than in the controls (~60% increase; P < .03). Choroidal thickening in AD was strongly correlated with the stromal vessel number (R2 = 0.96, P < .001). DISCUSSION: We found significant differences in the retinal choroid by layer and by region, nasally and temporally with respect to the optic nerve. Intriguingly, the choroid was markedly thicker in the central macular region and was strongly associated with vessel number in the stromal vascular layer. These quantified histological findings in severe disease expand our understanding of vascular pathology in AD and suggest vascularity as a potential biomarker supplementary to thickness when evaluating the retinal choroid in AD.

The Retina in Alzheimer's Disease: Histomorphometric Analysis of an Ophthalmologic Biomarker.

Purpose: To provide a histopathologic, morphometric analysis of the retina in Alzheimer's disease (AD). Methods: Human postmortem retinas from eight patients with AD (mean age: 80 ± 12.7 years) and from 11 age-matched controls (mean age: 78 ± 16.57 years) were analyzed. The retinas were sampled from the superior quadrant on both the temporal and nasal sides with respect to the optic nerve. Thickness of the inner and outer layers involving the retinal nerve fiber layer (RNFL), retinal ganglion cell layer (RGCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) were measured and compared between controls and AD. A total of 16 measurements of retinal thickness were acquired for each layer. Results: RNFL thinning supero-temporally was significant closest to the optic nerve (∼35% thickness reduction; P < 0.001). Supero-nasally, RNFL was thinner throughout all points (∼40% reduction; P < 0.001). Supero-temporally, RGCL thinning was pronounced toward the macula (∼35% thickness reduction; P < 0.001). Supero-nasally, RGCL showed uniform thinning throughout (∼35% reduction; P < 0.001). IPL thinning supero-temporally was statistically significant in the macula (∼15% reduction; P < 0.01). Supero-nasal IPL featured uniform thinning throughout (∼25% reduction; P < 0.001). Supero-temporally, INL and ONL thinning were pronounced toward the macula (∼25% reduction; P < 0.01). Supero-nasally, INL and ONL were thinner throughout (∼25% reduction; P < 0.01). Conclusions: Our study revealed marked thinning in both the inner and outer layers of the retina. These quantified histopathologic findings provide a more comprehensive understanding of the retina in AD than previously reported.

Optic neuropathies: the tip of the neurodegeneration iceberg.

The optic nerve and the cells that give origin to its 1.2 million axons, the retinal ganglion cells (RGCs), are particularly vulnerable to neurodegeneration related to mitochondrial dysfunction. Optic neuropathies may range from non-syndromic genetic entities, to rare syndromic multisystem diseases with optic atrophy such as mitochondrial encephalomyopathies, to age-related neurodegenerative diseases such as Alzheimer's and Parkinson's disease where optic nerve involvement has, until recently, been a relatively overlooked feature. New tools are available to thoroughly investigate optic nerve function, allowing unparalleled access to this part of the central nervous system. Understanding the molecular pathophysiology of RGC neurodegeneration and optic atrophy, is key to broadly understanding the pathogenesis of neurodegenerative disorders, for monitoring their progression in describing the natural history, and ultimately as outcome measures to evaluate therapies. In this review, the different layers, from molecular to anatomical, that may contribute to RGC neurodegeneration and optic atrophy are tackled in an integrated way, considering all relevant players. These include RGC dendrites, cell bodies and axons, the unmyelinated retinal nerve fiber layer and the myelinated post-laminar axons, as well as olygodendrocytes and astrocytes, looked for unconventional functions. Dysfunctional mitochondrial dynamics, transport, homeostatic control of mitobiogenesis and mitophagic removal, as well as specific propensity to apoptosis may target differently cell types and anatomical settings. Ultimately, we can envisage new investigative approaches and therapeutic options that will speed the early diagnosis of neurodegenerative diseases and their cure.

Retinal Ganglion Cells and Circadian Rhythms in Alzheimer's Disease, Parkinson's Disease, and Beyond.

There is increasing awareness on the role played by circadian rhythm abnormalities in neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). The characterization of the circadian dysfunction parallels the mounting evidence that the hallmarks of neurodegeneration also affect the retina and frequently lead to loss of retinal ganglion cells (RGCs) and to different degrees of optic neuropathy. In the RGC population, there is the subgroup of cells intrinsically photosensitive and expressing the photopigment melanopsin [melanopsin-containing retinal ganglion cells (mRGCs)], which are now well known to drive the entrainment of circadian rhythms to the light-dark cycles. Thus, the correlation between the pathological changes affecting the retina and mRGCs with the circadian imbalance in these neurodegenerative diseases is now clearly emerging, pointing to the possibility that these patients might be amenable to and benefit from light therapy. Currently, this connection is better established for AD and PD, but the same scenario may apply to other neurodegenerative disorders, such as Huntington's disease. This review highlights similarities and differences in the retinal/circadian rhythm axis in these neurodegenerative diseases posing a working frame for future studies.

Melanopsin-expressing retinal ganglion cells are resistant to cell injury, but not always.

Melanopsin retinal ganglion cells (mRGCs) are intrinsically photosensitive RGCs deputed to non-image forming functions of the eye such as synchronization of circadian rhythms to light-dark cycle. These cells are characterized by unique electrophysiological, anatomical and biochemical properties and are usually more resistant than conventional RGCs to different insults, such as axotomy and different paradigms of stress. We also demonstrated that these cells are relatively spared compared to conventional RGCs in mitochondrial optic neuropathies (Leber's hereditary optic neuropathy and Dominant Optic Atrophy). However, these cells are affected in other neurodegenerative conditions, such as glaucoma and Alzheimer's disease. We here review the current evidences that may underlie this dichotomy. We also present our unpublished data on cell experiments demonstrating that melanopsin itself does not explain the robustness of these cells and some preliminary data on immunohistochemical assessment of mitochondria in mRGCs.

Subchoroidal Release of VEGF and bFGF Produces Choroidal Neovascularization in Rabbit.

PURPOSE: Intravitreal vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) produced florid retinal neovascularization and hemorrhage in the rabbit. This study seeks to determine whether sustained subchoroidal release of both VEGF and bFGF can induce robust choroidal neovascularization (CNV) in the rabbit. METHODS: Subchoroidal implantation through the sclera of polymeric pellets containing both 15 µg VEGF and 15 µg bFGF was performed on adult pigmented male Dutch belted rabbits (N = 6) and NZW albinos (N = 8). As negative controls, blank pellets with no growth factors were implanted in both Dutch belted rabbits (N = 6) and NZW albino rabbits (N = 4). Development of CNV was documented weekly over a 4-week period with indirect ophthalmoscopy, color fundus photography, and fluorescein angiography. Eyes were enucleated and prepared for histologic and immunohistochemical analyses at the end of the study. Amounts of VEGF and bFGF that were released in vitro from the pellets were measured by ELISA. RESULTS: In all eyes with subchoroidal implants containing both VEGF and bFGF, strong fluorescein leakage was observed at 2, 3, and 4 weeks (P < 0.005); no leakage was seen initially in week 1. Negative control groups with blank implants showed no fluorescein leakage throughout the 4-week study period. Histologic analysis confirmed the presence of experimental CNV. New subretinal blood vessel growth occurred in all eyes with VEGF/bFGF implants. Negative control eyes with blank implants showed no vascular changes. In vitro sustained release of both VEGF and bFGF was confirmed by ELISA. CONCLUSION: Sustained subchoroidal release of both VEGF and bFGF produced experimental CNV rapidly in the rabbit. Understanding how these growth factors induce CNV may suggest novel therapeutic strategies in the large rabbit eye.

Melanopsin retinal ganglion cell loss in Alzheimer disease.

OBJECTIVE: Melanopsin retinal ganglion cells (mRGCs) are photoreceptors driving circadian photoentrainment, and circadian dysfunction characterizes Alzheimer disease (AD). We investigated mRGCs in AD, hypothesizing that they contribute to circadian dysfunction. METHODS: We assessed retinal nerve fiber layer (RNFL) thickness by optical coherence tomography (OCT) in 21 mild-moderate AD patients, and in a subgroup of 16 we evaluated rest-activity circadian rhythm by actigraphy. We studied postmortem mRGCs by immunohistochemistry in retinas, and axons in optic nerve cross-sections of 14 neuropathologically confirmed AD patients. We coimmunostained for retinal amyloid ß (Aß) deposition and melanopsin to locate mRGCs. All AD cohorts were compared with age-matched controls. RESULTS: We demonstrated an age-related optic neuropathy in AD by OCT, with a significant reduction of RNFL thickness (p = 0.038), more evident in the superior quadrant (p = 0.006). Axonal loss was confirmed in postmortem AD optic nerves. Abnormal circadian function characterized only a subgroup of AD patients. Sleep efficiency was significantly reduced in AD patients (p = 0.001). We also found a significant loss of mRGCs in postmortem AD retinal specimens (p = 0.003) across all ages and abnormal mRGC dendritic morphology and size (p = 0.003). In flat-mounted AD retinas, Aß accumulation was remarkably evident inside and around mRGCs. INTERPRETATION: We show variable degrees of rest-activity circadian dysfunction in AD patients. We also demonstrate age-related loss of optic nerve axons and specifically mRGC loss and pathology in postmortem AD retinal specimens, associated with Aß deposition. These results all support the concept that mRGC degeneration is a contributor to circadian rhythm dysfunction in AD.

Proteomic Analysis of Embryonic and Young Human Vitreous.

PURPOSE: The proteomic profile of vitreous from second-trimester human embryos and young adults was characterized using mass spectrometry and analyzed for changes in protein levels that may relate to structural changes occurring during this time. This vitreous proteome was compared to previous reports to confirm proteins already identified and reveal novel ones. METHODS: Vitreous from 17 human embryos aged 14 to 20 weeks gestation (WG) and from a 12-, a 14-, a 15-, and a 28-year-old was individually analyzed using tandem mass spectrometry-based proteomics. Peptide spectral count associations with embryonic age were assessed using a general linear model of fold changes and Spearman's rank correlation. Differences between embryonic and young adult vitreous proteomes were also compared. Immunohistochemistry was used to evaluate three proteins in five additional fetal (10-18 WG) human eyes. RESULTS: There were 1217 proteins identified in fetal and young adult human vitreous, 206 after quantile normalization and variance filtering. In embryos, the peptide counts of 37 proteins changed significantly from 14 to 20 WG: 75.7% increased, 24.3% decreased. Immunohistochemistry confirmed the absence of clusterin and cadherin in 10 and 14 WG eyes and their presence at 18 WG. Comparing embryonic to young adult vitreous, 47 proteins were significantly higher or lower. A total of 768 proteins not previously identified in the literature are presented. CONCLUSIONS: Proteins previously unreported in the human vitreous were identified. The human vitreous proteome undergoes significant changes during embryogenesis and young adulthood. A number of protein levels change considerably during the second trimester, with the majority decreasing.

Phenotypic and functional characterization of Bst+/- mouse retina.

The belly spot and tail (Bst(+/-)) mouse phenotype is caused by mutations of the ribosomal protein L24 (Rpl24). Among various phenotypes in Bst(+/-) mice, the most interesting are its retinal abnormalities, consisting of delayed closure of choroid fissures, decreased ganglion cells and subretinal vascularization. We further characterized the Bst(+/-) mouse and investigated the underlying molecular mechanisms to assess the feasibility of using this strain as a model for stem cell therapy of retinal degenerative diseases due to retinal ganglion cell (RGC) loss. We found that, although RGCs are significantly reduced in retinal ganglion cell layer in Bst(+/-) mouse, melanopsin(+) RGCs, also called ipRGCs, appear to be unchanged. Pupillary light reflex was completely absent in Bst(+/-) mice but they had a normal circadian rhythm. In order to examine the pathological abnormalities in Bst(+/-) mice, we performed electron microscopy in RGC and found that mitochondria morphology was deformed, having irregular borders and lacking cristae. The complex activities of the mitochondrial electron transport chain were significantly decreased. Finally, for subretinal vascularization, we also found that angiogenesis is delayed in Bst(+/-) associated with delayed hyaloid regression. Characterization of Bst(+/-) retina suggests that the Bst(+/-) mouse strain could be a useful murine model. It might be used to explore further the pathogenesis and strategy of treatment of retinal degenerative diseases by employing stem cell technology.

Inner retinal optic neuropathy: vitreomacular surgery-associated disruption of the inner retina.

PURPOSE: Macular pucker (MP) and macular hole (MH) are vitreomaculopathies treated by vitrectomy and membrane peel. The complication of postoperative central scotoma can be associated with significant reduction in visual acuity (VA). We seek to determine whether retinal nerve fiber layer (RNFL) disruption is the pathophysiologic basis of this defect. Mitigating clinical circumstances also were sought. METHODS: Eleven eyes from 10 pseudophakic patients who had undergone vitrectomy with peeling for either MH or MP were studied with clinical measures, including optical coherence tomography (OCT). Membrane specimens were evaluated by immunohistochemistry for neurofilament, a marker for the inner retina. Ten eyes from 10 pseudophakic patients who underwent repeat surgery for persistent or recurrent pathology were evaluated to determine the relationship between the timing of reoperation and clinical outcome. RESULTS: Cases with a postoperative central scotoma (N=4) had worse VA (~20/600) compared to those without (N=7, ~20/30, P=0.01). Eyes with a central scotoma had significantly reduced RNFL thickness in the temporal quadrant (53.67 vs. 72.33 µm, P=0.05) by OCT. A central scotoma was associated with more disruption of the inner retina on immunohistochemistry (P=0.03). In patients with persistent or recurrent pathology, waiting six months before reoperation resulted in better functional outcomes (P=0.03). CONCLUSIONS: Central scotomata and poor VA were associated with disruption of the RNFL during membrane peeling. Affected patients have RNFL thinning and signs of optic neuropathy, for which we propose the term inner retinal optic neuropathy (IRON). In patients requiring reoperation, waiting six months between surgeries may reduce the risk of IRON.