Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse

PURPOSE. To test the hypothesis that Nrl Ϫ/Ϫ photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS. The photoreceptor layer of fixed retinal tissue of 4-to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single-and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the awave of the ERG. RESULTS. Outer segments of Nrl Ϫ/Ϫ photoreceptors (ϳ7 m) are shorter than those of wild-type (WT) rods (ϳ25 m) and cones (ϳ15 m); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl Ϫ/Ϫ photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl -/-a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the max of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was ϳ50 ms, more than two times faster than that of rods. CONCLUSIONS. Many morphological, molecular, and electrophysiological features of the Nrl Ϫ/Ϫ photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease. (Invest Ophthalmol Vis Sci

The oral iron chelator deferiprone protects iron overload-induced retinal degeneration. Invest Ophthalmol Vis Sci 52:959–968

PURPOSE. Iron-induced oxidative stress may exacerbate agerelated macular degeneration (AMD). Ceruloplasmin/Hephaestin double-knockout (DKO) mice with age-dependent retinal iron accumulation and some features of AMD were used to test retinal protection by the oral iron chelator deferiprone (DFP). METHODS. Cultured retinal pigment epithelial (ARPE-19) cells and mice were treated with DFP. Transferrin receptor mRNA (Tfrc), an indicator of iron levels, was quantified by qPCR. In mice, retinal oxidative stress was assessed by mass spectrometry, and degeneration by histology and electroretinography. RESULTS. DFP at 60 M decreased labile iron in ARPE-19 cells, increasing Tfrc and protecting 70% of cells against a lethal dose of H 2 O 2 . DFP 1 mg/mL in drinking water increased retinal Tfrc mRNA 2.7-fold after 11 days and also increased transferrin receptor protein. In DKOs, DFP over 8 months decreased retinal iron levels to 72% of untreated mice, diminished retinal oxidative stress to 70% of the untreated level, and markedly ameliorated retinal degeneration. DFP was not retina toxic in wild-type (WT) or DKO mice, as assessed by histology and electroretinography. CONCLUSIONS. Oral DFP was not toxic to the mouse retina. It diminished retinal iron levels and oxidative stress and protected DKO mice against iron overload-induced retinal degeneration. Further testing of DFP for retinal disease involving oxidative stress is warranted. (Invest Ophthalmol Vis Sci. 2011;52:959 -968) DOI:10.1167/iovs.10-6207 I ron is crucial for optimal cellular metabolism, but is also a potent generator of oxidative stress if present in excess, especially in the form of labile ferrous iron. Inability of the body to actively excrete excess iron leads to age-dependent iron accumulation in certain tissues, including the macula. 1 Excess tissue iron generates reactive oxygen species (ROS) via the Fenton reaction, leading to oxidative damage. Free radicals and oxidative stress have been implicated in a growing number of conditions, from normal aging to cancer, diabetes, and neurodegenerative diseases, making iron overload or metabolic mishandling of iron an important target for therapeutic intervention. 2-6 Since iron catalyzes the production of the hydroxyl radical, the most damaging of the free radicals, it is likely to exacerbate oxidative damage in a tissue that is already prone to oxidative insult. Retinal pigment epithelial (RPE) cells and photoreceptors are especially vulnerable to oxidative damage due to high oxygen tension, ROS production by large numbers of mitochondria, and abundant, easily oxidized polyunsaturated fatty acids in photoreceptor membranes. 7 Indeed, several neurodegenerative disorders with iron dysregulation feature retinal degeneration. 8 These include the rare hereditary disorders aceruloplasminemia, Friedreich's ataxia, and pantothenate kinase-associated neurodegeneration. Further, traumatic siderosis causes rapid retinal degeneration. 9 Similarly, retinal degeneration in several mouse models is associated with retinal iron dysregulation. -12 Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly worldwide. Although the pathogenesis of AMD is incompletely understood, growing evidence suggests that, in addition to inflammation, complement activation, and other hereditary and environmental influences, 9 Supporting this hypothesis, patients lacking the ferroxidase ceruloplasmin (Cp) as a result of the autosomal recessive condition aceruloplasminemia, have retinal iron accumulation and early-onset macular degeneration. From th

Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration

<div><p>Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO.</p></div

Photomicrographs of plastic sections of WT and CD59aKO retinas, and quantification of ONL nuclei.

<p>Histology of mouse retinas showing thinning of the ONL (white brackets) and IS/OS in post-LD WT retinas, while CD59aKO retinas showed less thinning of the ONL and IS/OS (A). Plot of the number of nuclei per column in the ONL. In WT retinas on both the Balb/c and C57BL/6 background, there was a significant decrease of ONL thickness across all sampled retinal regions (a-d) comparing non-light- damaged (NLD) (black line in B and C) and LD (green line in B and C). However, there was a smaller reduction of ONL thickness comparing NLD (blue line in B and C) and LD CD59aKO retinas (red line in B and C). Data are expressed as means ± SD. N = 4. *<i>P</i><0.01, and significance markings refer to overall differences between groups across all four retinal regions (a-d).</p

CD59a expression in mouse retina and mRNA levels of CD59a in NSR and isolated RPE.

<p>Photomicrograph showing CD59a immunolabeling in all layers of mouse retina, RPE and choroid (B), but not in CD59aKO eyes (C). Graph of qPCR results showing that mRNA levels of CD59a in NSR were more than 60-fold higher than that in isolated RPE cells in WT eyes (**<i>P</i><0.001) (D). Data are expressed as means ± SD. N = 4. RPE, retinal pigment epithelium; OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.</p

ERG responses before and after LD.

<p>Before LD, rod-a (<i>P</i><0.05) and rod-b (<i>P</i><0.05) waves were decreased in CD59aKO Balb/c mice compared to WT (A). At day 7 after LD, however, rod-a (<i>P</i> = 0.04) and rod-b (<i>P</i> = 0.01) waves were all larger in CD59aKO compared to WT (A). Data are expressed as means ± SD. N = 4. Similarly, rod-a (<i>P</i><0.05) and rod-b (<i>P</i><0.01) wave amplitudes were smaller in CD59aKO C57BL/6J mice compared to C57BL/6J mice at baseline, and while C57BL/6J mice have diminished ERG amplitudes 10 days after LD, amplitudes in CD59aKO mice remain unchanged (B). Data are expressed as means ± SD. N = 6–7.</p