A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy.

Purpose: To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. Methods: Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. Results: Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. Conclusions: The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models.

PBN (Phenyl-N-Tert-Butylnitrone)-Derivatives Are Effective in Slowing the Visual Cycle and Rhodopsin Regeneration and in Protecting the Retina from Light-Induced Damage.

A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial (RPE) cells in eyes affected by Stargardt's disease, age-related macular degeneration (AMD), and other retinal degenerations. A novel therapeutic approach for treating such degenerations involves slowing down the visual cycle, which could reduce the amount of A2E in the RPE. This can be accomplished by inhibiting RPE65, which produces 11-cis-retinol from all-trans-retinyl esters. We recently showed that phenyl-N-tert-butylnitrone (PBN) inhibits RPE65 enzyme activity in RPE cells. In this study we show that like PBN, certain PBN-derivatives (PBNDs) such as 4-F-PBN, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN can inhibit RPE65 and synthesis of 11-cis-retinol in in vitro assays using bovine RPE microsomes. We further demonstrate that systemic (intraperitoneal, IP) administration of these PBNDs protect the rat retina from light damage. Electroretinography (ERG) and histological analysis showed that rats treated with PBNDs retained ~90% of their photoreceptor cells compared to a complete loss of function and 90% loss of photoreceptors in the central retina in rats treated with vehicle/control injections. Topically applied PBN and PBNDs also significantly slowed the rate of the visual cycle in mouse and baboon eyes. One hour dark adaptation resulted in 75-80% recovery of bleachable rhodopsin in control/vehicle treated mice. Eye drops of 5% 4-CH3-PBN were most effective, inhibiting the regeneration of bleachable rhodopsin significantly (60% compared to vehicle control). In addition, a 10% concentration of PBN and 5% concentration of 4-CH3-PBN in baboon eyes inhibited the visual cycle by 60% and by 30%, respectively. We have identified a group of PBN related nitrones that can reach the target tissue (RPE) by systemic and topical application and slow the rate of rhodopsin regeneration and therefore the visual cycle in mouse and baboon eyes. PBNDs can also protect the rat retina from light damage. There is potential in developing these compounds as preventative therapeutics for the treatment of human retinal degenerations in which the accumulation of lipofuscin may be pathogenic.

Leukemia inhibitory factor coordinates the down-regulation of the visual cycle in the retina and retinal-pigmented epithelium.

Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-cis-retinal chromophore to photoreceptors in vivo. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-cis-retinal.

Alpha-phenyl-N-tert-butylnitrone (PBN) prevents light-induced degeneration of the retina by inhibiting RPE65 protein isomerohydrolase activity.

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 µm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.

Factors that determine Ca2+ sensitivity of photoreceptor guanylyl cyclase. Kinetic analysis of the interaction between the Ca2+-bound and the Ca2+-free guanylyl cyclase activating proteins (GCAPs) and recombinant photoreceptor guanylyl cyclase 1 (RetGC-1).

We explored the possibility that, in the regulation of an effector enzyme by a Ca(2+)-sensor protein, the actual Ca(2+) sensitivity of the effector enzyme can be determined not only by the affinity of the Ca(2+)-sensor protein for Ca(2+) but also by the relative affinities of its Ca(2+)-bound versus Ca(2+)-free form for the effector enzyme. As a model, we used Ca(2+)-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg(838)Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca(2+) sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca(2+) range. We find that at physiological concentrations of Mg(2+) this mutation increases the free Ca(2+) concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 microM. Similar to rod outer segment cyclase, Ca(2+) sensitivity of recombinant RetGC-1 is strongly affected by Mg(2+), but the shift in Ca(2+) sensitivity for the R838S mutant relative to the wild type is Mg(2+)-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca(2+)-bound and Ca(2+)-free GCAP-1 and found that the net shift in Ca(2+) sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca(2+)-free versus Ca(2+)-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca(2+)/GCAP is determined by both the affinity of GCAP for Ca(2+) and relative affinities of the effector enzyme for the Ca(2+)-free versus Ca(2+)-loaded GCAP.