Revival of light signalling in the postmortem mouse and human retina.

Death is defined as the irreversible cessation of circulatory, respiratory or brain activity. Many peripheral human organs can be transplanted from deceased donors using protocols to optimize viability. However, tissues from the central nervous system rapidly lose viability after circulation ceases1,2, impeding their potential for transplantation. The time course and mechanisms causing neuronal death and the potential for revival remain poorly defined. Here, using the retina as a model of the central nervous system, we systemically examine the kinetics of death and neuronal revival. We demonstrate the swift decline of neuronal signalling and identify conditions for reviving synchronous in vivo-like trans-synaptic transmission in postmortem mouse and human retina. We measure light-evoked responses in human macular photoreceptors in eyes removed up to 5 h after death and identify modifiable factors that drive reversible and irreversible loss of light signalling after death. Finally, we quantify the rate-limiting deactivation reaction of phototransduction, a model G protein signalling cascade, in peripheral and macular human and macaque retina. Our approach will have broad applications and impact by enabling transformative studies in the human central nervous system, raising questions about the irreversibility of neuronal cell death, and providing new avenues for visual rehabilitation.

Constitutive and Regulated Shedding of Soluble FGF Receptors Releases Biologically Active Inhibitors of FGF-2.

The identification of soluble fibroblast growth factor (FGF) receptors in blood and the extracellular matrix has led to the prediction that these proteins modulate the diverse biological activities of the FGF family of ligands in vivo. A recent structural characterization of the soluble FGF receptors revealed that they are primarily generated by proteolytic cleavage of the FGFR-1 ectodomain. Efforts to examine their biological properties are now focused on understanding the functional consequences of FGFR-1 ectodomain shedding and how the shedding event is regulated. We have purified an FGFR-1 ectodomain that is constitutively cleaved from the full-length FGFR-1(IIIc) receptor and released into conditioned media. This shed receptor binds FGF-2; inhibits FGF-2-induced cellular proliferation; and competes with high affinity, cell surface FGF receptors for ligand binding. FGFR-1 ectodomain shedding downregulates the number of high affinity receptors from the cell surface. The shedding mechanism is regulated by ligand binding and by activators of PKC, and the two signaling pathways appear to be independent of each other. Deletions and substitutions at the proposed cleavage site of FGFR-1 do not prevent ectodomain shedding. Broad spectrum inhibitors of matrix metalloproteases decrease FGFR-1 ectodomain shedding, suggesting that the enzyme responsible for constitutive, ligand-activated, and protein kinase C-activated shedding is a matrix metalloprotease. In summary, shedding of the FGFR-1 ectodomain is a highly regulated event, sharing many features with a common system that governs the release of diverse membrane proteins from the cell surface. Most importantly, the FGFR ectodomains are biologically active after shedding and are capable of functioning as inhibitors of FGF-2.

Functional diversity of human intrinsically photosensitive retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are a subset of cells that participate in image-forming and non-image-forming visual responses. Although both functional and morphological subtypes of ipRGCs have been described in rodents, parallel functional subtypes have not been identified in primate or human retinas. In this study, we used a human organ donor preparation method to measure human ipRGCs' photoresponses. We discovered three functional ipRGC subtypes with distinct sensitivities and responses to light. The response of one ipRGC subtype appeared to depend on exogenous chromophore supply, and this response is conserved in both human and mouse retinas. Rods and cones also provided input to ipRGCs; however, each subtype integrated outer retina light signals in a distinct fashion.

Bis(monoacylglycero)phosphate lipids in the retinal pigment epithelium implicate lysosomal/endosomal dysfunction in a model of Stargardt disease and human retinas.

Stargardt disease is a juvenile onset retinal degeneration, associated with elevated levels of lipofuscin and its bis-retinoid components, such as N-retinylidene-N-retinylethanolamine (A2E). However, the pathogenesis of Stargardt is still poorly understood and targeted treatments are not available. Utilizing high spatial and high mass resolution matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS), we determined alterations of lipid profiles specifically localized to the retinal pigment epithelium (RPE) in Abca4 -/- Stargardt model mice compared to their relevant background strain. Extensive analysis by LC-MS/MS in both positive and negative ion mode was required to accurately confirm the identity of one highly expressed lipid class, bis(monoacylgylercoro)phosphate (BMP) lipids, and to distinguish them from isobaric species. The same BMP lipids were also detected in the RPE of healthy human retina. BMP lipids have been previously associated with the endosomal/lysosomal storage diseases Niemann-Pick and neuronal ceroid lipofuscinosis and have been reported to regulate cholesterol levels in endosomes. These results suggest that perturbations in lipid metabolism associated with late endosomal/lysosomal dysfunction may play a role in the pathogenesis of Stargardt disease and is evidenced in human retinas.

Biochemical Measurements of Free Opsin in Macular Degeneration Eyes: Examining the 11-CIS Retinal Deficiency Hypothesis of Delayed Dark Adaptation (An American Ophthalmological Society Thesis).

PURPOSE: To test the hypothesis that delayed dark adaptation in patients with macular degeneration is due to an excess of free unliganded opsin (apo-opsin) and a deficiency of the visual chromophore, 11-cis retinal, in rod outer segments. METHODS: A total of 50 human autopsy eyes were harvested from donors with and without macular degeneration within 2-24 hrs. postmortem. Protocols were developed which permitted dark adaptation of normal human eyes after death and enucleation. Biochemical methods of purifying rod outer segments were optimized and the concentration of rhodopsin and apo-opsin was measured with UV-visible scanning spectroscopy. The presence of apo-opsin was calculated by measuring the difference in the rhodopsin absorption spectra before and after the addition of 11-cis retinal. RESULTS: A total of 20 normal eyes and 16 eyes from donors with early, intermediate and advanced stages of macular degeneration were included in the final analysis. Dark adaptation was achieved by harvesting whole globes in low light, transferring into dark (light-proof) canisters and dissecting the globes using infrared light and image converters for visualization. Apo-opsin was readily detected in positive controls after the addition of 11-cis retinal. Normal autopsy eyes showed no evidence of apo-opsin. Eyes with macular degeneration also showed no evidence of apo-opsin, regardless of the severity of disease. CONCLUSIONS: Methods have been developed to study dark adaptation in human autopsy eyes. Eyes with age-related macular degeneration do not show a deficiency of 11-cis retinal or an excess of apo-opsin within rod outer segments.

Determination of N-retinylidene-N-retinylethanolamine (A2E) levels in central and peripheral areas of human retinal pigment epithelium.

The bis-retinoid N-retinylidene-N-retinylethanolamine (A2E) is one of the major components of lipofuscin, a fluorescent material that accumulates with age in the lysosomes of the retinal pigment epithelium (RPE) of the human eye. Lipofuscin, as well as A2E, exhibit a range of cytotoxic properties, which are thought to contribute to the pathogenesis of degenerative diseases of the retina such as Age-related Macular Degeneration. Consistent with such a pathogenic role, high levels of lipofuscin fluorescence are found in the central area of the human RPE, and decline toward the periphery. Recent reports have however suggested a surprising incongruence between the distributions of lipofuscin and A2E in the human RPE, with A2E levels being lowest in the central area and increasing toward the periphery. To appraise such a possibility, we have quantified the levels of A2E in the central and peripheral RPE areas of 10 eyes from 6 human donors (ages 75-91 years) with HPLC and UV/VIS spectroscopy. The levels of A2E in the central area were on average 3-6 times lower than in peripheral areas of the same eye. Furthermore, continuous accumulation of selected ions (CASI) imaging mass spectrometry showed the presence of A2E in the central RPE, and at lower intensities than in the periphery. We have therefore corroborated that in human RPE the levels of A2E are lower in the central area compared to the periphery. We conclude that the levels of A2E cannot by themselves provide an explanation for the higher lipofuscin fluorescence found in the central area of the human RPE.

Lack of correlation between the spatial distribution of A2E and lipofuscin fluorescence in the human retinal pigment epithelium.

PURPOSE: The accumulation of lipofuscin in the RPE is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of our study was to correlate the distribution of lipofuscin and A2E across the human RPE. METHODS: Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on flat-mounted RPE tissue sections and retinal cross-sections. RESULTS: Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased toward the central region. High-resolution MALDI-IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels. CONCLUSIONS: This report to our knowledge is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging.

Oral proton pump inhibitors disrupt horizontal cell-cone feedback and enhance visual hallucinations in macular degeneration patients.

PURPOSE: Visual hallucinations (VHs) occur in macular degeneration patients with poor vision but normal cognitive function. The underlying mechanisms are poorly understood. We report the identification of pharmaceutical agents that enhance VH and use these agents to examine the contribution of retinal neurons to this syndrome. METHODS: We detail clinical observations on VH in five macular degeneration patients treated with proton pump inhibitors having the core structure, 2-pyridyl-methylsulfinyl-benzimidazole. We tested possible retinal mechanisms using paired whole cell recordings to examine effects of these compounds on feedback interactions between horizontal cells and cones in amphibian retina. RESULTS: Five patients with advanced wet macular degeneration described patterned VHs that were induced or enhanced by oral proton pump inhibitors. The abnormal images increased with light, disappeared in the dark, and originated in the retina, based on ophthalmodynamometry. Simultaneous paired whole cell recordings from amphibian cones and horizontal cells showed that 2-pyridyl-methylsulfinyl-benzimidazoles blocked the negative shift in voltage dependence and increase in amplitude of the calcium current (ICa) in cones that is induced by changes in horizontal cell membrane potential. These effects disrupt the negative feedback from horizontal cells to cones that is important for the formation of center-surround receptive fields in bipolar and ganglion cells, and thus for normal spatial and chromatic perception. CONCLUSIONS: Our study suggests that changes in the output of retinal neurons caused by disturbances in outer retinal feedback mechanisms can enhance patterned visual hallucinations.

Mass spectrometry provides accurate and sensitive quantitation of A2E.

Orange autofluorescence from lipofuscin in the lysosomes of the retinal pigment epithelium (RPE) is a hallmark of aging in the eye. One of the major components of lipofuscin is A2E, the levels of which increase with age and in pathologic conditions, such as Stargardt disease or age-related macular degeneration. In vitro studies have suggested that A2E is highly phototoxic and, more specifically, that A2E and its oxidized derivatives contribute to RPE damage and subsequent photoreceptor cell death. To date, absorption spectroscopy has been the primary method to identify and quantitate A2E. Here, a new mass spectrometric method was developed for the specific detection of low levels of A2E and compared to a traditional method of analysis. The new mass spectrometric method allows the detection and quantitation of approximately 10,000-fold less A2E than absorption spectroscopy and the detection and quantitation of low levels of oxidized A2E, with localization of the oxidation sites. This study suggests that identification and quantitation of A2E from tissue extracts by chromatographic absorption spectroscopy overestimates the amount of A2E. This mass spectrometric approach makes it possible to detect low levels of A2E and its oxidized metabolites with greater accuracy than traditional methods, thereby facilitating a more exact analysis of bis-retinoids in animal models of inherited retinal degeneration as well as in normal and diseased human eyes.

Recent research on polyphenolics in vision and eye health.

A long-standing yet controversial bioactivity attributed to polyphenols is their beneficial effects in vision. Although anecdotal case reports and in vitro research studies provide evidence for the visual benefits of anthocyanin-rich berries, rigorous clinical evidence of their benefits is still lacking. Recent in vitro studies demonstrate that anthocyanins and other flavonoids interact directly with rhodopsin and modulate visual pigment function. Additional in vitro studies show flavonoids protect a variety of retinal cell types from oxidative stress-induced cell death, a neuroprotective property of significance because the retina has the highest metabolic rate of any tissue and is particularly vulnerable to oxidative injury. However, more information is needed on the bioactivity of in vivo conjugates and the accumulation of flavonoids in ocular tissues. The direct and indirect costs of age-related vision impairment provide a powerful incentive to explore the potential for improved vision health through the intake of dietary polyphenolics.

The flavonoid, eriodictyol, induces long-term protection in ARPE-19 cells through its effects on Nrf2 activation and phase 2 gene expression.

PURPOSE: Eriodictyol, a flavonoid found in citrus fruits, is among the most potent compounds reported to protect human RPE cells from oxidative stress-induced cell death. The present study sought to determine whether eriodictyol-induced phase 2 protein expression further enhances the resistance of human ARPE-19 cells to oxidative stress. METHODS: The ability of eriodictyol to activate Nrf2 and to induce the phase 2 proteins heme-oxygenase (HO)-1 and NAD(P)H:quinone oxidoreductase (NQO)-1, and the cellular antioxidant glutathione (GSH) were analyzed. Cytoprotection assays in ARPE-19 cells that were overexpressing HO-1 or NQO-1 were performed, cell survival after short-term and long-term eriodictyol treatment was compared, and the mechanism of protection using a dominant negative Nrf2 and shRNA specific for HO-1 was tested. RESULTS: Eriodictyol induced the nuclear translocation of Nrf2, enhanced the expression of HO-1 and NQO-1, and increased the levels of intracellular glutathione. ARPE-19 cells that overexpress HO-1 or NQO-1 were more resistant to oxidative stress-induced cell death than control cells. Eriodictyol induced long-term protection significantly greater than its short-term protection. This effect was correlated temporally with the activation of Nrf2 and the induction of phase 2 enzymes and could be blocked with the use of a dominant negative Nrf2 and shRNA specific to HO-1. CONCLUSIONS: These findings indicate that the greatest benefit from eriodictyol may be its ability to regulate gene expression and enhance multiple cellular defenses to oxidative injury.

Flavonoids protect retinal ganglion cells from ischemia in vitro.

Retinal ischemia is a common cause of visual impairment and blindness. However, despite the significant advances that have been made in understanding the pathophysiology of retinal ischemia, effective treatments are still lacking. The goal of these studies was to use an in vitro model to identify molecules that could be neuroprotective for retinal ganglion cells exposed to ischemia. Ischemia was induced in the rat retinal ganglion cell line, RGC-5, using iodoacetic acid (IAA). Brief treatment with IAA resulted in RGC-5 cell death within 24 h by a non-apoptotic mechanism. Similar to ischemia in vivo, IAA treatment caused a rapid loss of ATP to approximately 50% of control levels. In contrast, changes in markers of oxidative stress occurred more slowly and included an increase in reactive oxygen species and a decrease in glutathione. Specific flavonoids were able to prevent the cell death caused by IAA treatment. Some of the flavonoids also prevented the loss of ATP as well as the changes in markers of oxidative stress. In contrast, classical antioxidants had only a very modest effect on IAA-induced cell death. These results suggest that specific flavonoids may be useful in preventing ischemia-induced retinal ganglion cell death in vivo.

Flavonoids protect human retinal pigment epithelial cells from oxidative-stress-induced death.

PURPOSE: To determine whether specific dietary and synthetic flavonoids can protect human retinal pigment epithelial (RPE) cells from oxidative-stress-induced death. METHODS: The efficacy and potency were determined of a variety of dietary and synthetic flavonoids on the survival of human ARPE-19 cells and primary human RPE cells treated with either hydrogen peroxide (H2O2) or t-butyl hydroperoxide (t-BOOH). We determined the effective concentrations (EC50s) and the toxicities (LD50s) of the flavonoids after 24 hours, by using the MTT assay. The efficacy of vitamins E and C on RPE cell survival were compared under identical conditions. The ability of specific flavonoids to protect RPE cells from cell death was determined at various time intervals after the cells were exposed to oxidative stress. The ability of flavonoids to block the accumulation of intracellular reactive oxygen species was examined with dichlorofluorescein (DCF) fluorescence. Finally, the ability of flavonoids to induce phase-2 detoxifying enzymes was tested by immunoblot analysis for the transcription factor Nrf2 and the phase-2 gene product heme-oxygenase 1. RESULTS: Specific flavonoids protected human RPE cells from oxidative-stress-induced death with efficacies between 80% and 100% and potencies in the high-nanomolar and low-micromolar range. The toxicities of most of the effective flavonoids were low. The effective flavonoids included the dietary flavonoids fisetin, luteolin, quercetin, eriodictyol, baicalein, galangin and EGCG, and the synthetic flavonoids, 3,6-dihydroxy flavonol and 3,7 dihydroxy flavonol. Several flavonoids can protect RPE cells even when they are added after the cells have been exposed to oxidative stress. The flavonoids acted through an intracellular route to block the accumulation of reactive oxygen species. Many of these flavonoids induced the expression of Nrf2 and the phase-2 gene product heme-oxygenase 1 in human RPE cells. CONCLUSIONS: The results identify a select group of flavonoids that protect RPE cells from oxidative-stress-induced death with a high degree of potency and low toxicity. Many of these flavonoids also induce the expression of phase-2 detoxification proteins which could function to provide additional protection against oxidative stress. This select group of flavonoids and the foods that contain high levels of these compounds may have some clinical benefit for patients with retinal diseases associated with oxidative stress.

Flavonoids protect retinal ganglion cells from oxidative stress-induced death.

PURPOSE: Mounting evidence suggests that oxidative stress contributes to the pathogenesis of many ocular diseases, including glaucoma and diabetic retinopathy. No treatments are available to prevent the neuronal degeneration that occurs in these disorders. The purpose of this study was to determine whether flavonoids, natural products that are abundant in fruits and vegetables, can protect retinal ganglion cells from oxidative stress-induced death. METHODS: The ability of flavonoids to protect an immortalized retinal ganglion cell line, RGC-5 cells, was tested using three model systems of oxidative stress-induced cell death: glutathione (GSH) depletion, t-butyl peroxide (t-BOOH) treatment, and hydrogen peroxide (H2O2) treatment. RESULTS: GSH depletion causes retinal ganglion cell death by a pathway involving the production of endogenous reactive oxygen species (ROS), whereas the other two treatments use exogenous sources of ROS. It was found that specific flavonoids could protect retinal ganglion cells from cell death initiated by all three of the inducers of oxidative stress with high levels of potency and low toxicity. In the case of GSH depletion, different flavonoids could be shown to act at distinct steps in the cell death pathway. Several of the protective flavonoids also induced the synthesis of the transcription factor NF-E2-related factor 2 and phase 2 antioxidant enzymes such as heme oxygenase 1, markers for the activation of the antioxidant response element. CONCLUSIONS: These results suggest that certain flavonoids can function as potent and effective neuroprotective agents for retinal ganglion cells.

The molecular basis of oxidative stress-induced cell death in an immortalized retinal ganglion cell line.

PURPOSE: To characterize the molecular basis of oxidative stress-induced death, a process that has been implicated in several chronic eye diseases, in RGC-5 cells, an immortalized retinal ganglion cell (RGC) line. METHODS: The responses of RGC-5 cells to oxidative stress induced by three different treatments--glutathione depletion, tert-butyl peroxide addition, and hydrogen peroxide addition--were examined and compared. The level of cell death was monitored with the MTT assay. The effects of glutathione depletion on the intracellular levels of glutathione, reactive oxygen species, and calcium were determined. The type of cell death was assessed with assays for DNA fragmentation and caspase activation. Compounds that were shown to be protective of central nervous system-derived nerve cells exposed to oxidative stress were tested to see whether they could also protect the RGC-5 cells. In addition, several compounds that have been found to be protective in primary cultures of RGCs or in animal models of retinal dysfunction were tested against each of the inducers of oxidative stress. RESULTS: The cell death triggered by all three inducers of oxidative stress shared several features, suggesting that there is a final common pathway of oxidative stress-induced death in the RGCs. In addition, several compounds were identified that protected RGCs from multiple forms of oxidative stress. CONCLUSIONS: The RGC-5 line is an excellent model for studying mechanisms of RGC death in response to oxidative stress and for the identification of neuroprotective compounds.