Inducible RPE-specific GPX4 knockout causes oxidative stress and retinal degeneration with features of age-related macular degeneration.

Age-related macular degeneration (AMD) is one of the leading causes of vision loss in the elderly. This disease involves oxidative stress burden in the retina leading to death of retinal pigment epithelial (RPE) cells and photoreceptors. The retina is susceptible to oxidative stress, in part due to high metabolic activity and high concentration of polyunsaturated fatty acids that undergo lipid peroxidation chain reactions. Antioxidant enzymes exist in the retina to combat this stress, including glutathione peroxidase 4 (GPX4). GPX4 specifically reduces oxidized lipids, protecting against lipid peroxidation-induced oxidative stress, which is noted in dry AMD. We hypothesize that Gpx4 knockout within the RPE will result in an environment of chronic oxidative stress yielding degeneration akin to AMD. C57BL/6J mice with a floxed Gpx4 gene were mated with Rpe65Cre/ER mice. Offspring containing Rpe65Cre ± alleles and either Gpx4 WT or Gpx4 fl/fl alleles were administered tamoxifen to induce Gpx4 knockout in Gpx4 fl/fl mice. At sequential timepoints, retinal phenotypes were assessed via in vivo imaging utilizing confocal scanning laser ophthalmoscopy and optical coherence tomography (OCT), and visual function was probed by electroretinography. Retinas were studied post-mortem by immunohistochemical analyses, electron microscopy, plastic sectioning, and quantitative polymerase chain reaction and Western analyses. The RPE-specific Gpx4 knockout model was validated via Western analysis indicating diminished GPX4 protein only within the RPE and not the neural retina. Following Gpx4 knockout, RPE cells became dysfunctional and died, with significant cell loss occurring 2 weeks post-knockout. Progressive thinning of the photoreceptor layer followed RPE degeneration and was accompanied by loss of visual function. OCT and light microscopy showed hyperreflective foci and enlarged, pigmented cells in and above the RPE layer. Electron microscopy revealed decreased mitochondrial cristae and loss of basal and apical RPE ultrastructure. Finally, there was increased carboxyethylpyrrole staining, indicating oxidation of docosahexaenoic acid, and increased levels of mRNAs encoding oxidative stress-associated genes in the RPE and photoreceptors. Overall, we show that RPE-localized GPX4 is necessary for the health of the RPE and outer retina, and that knockout recapitulates phenotypes of dry AMD.

Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration.

This study characterizes a fluorescent Slc17a6 -tdTomato neuronal reporter mouse line offering strong labeling in axons throughout the optic nerve, dendrites and soma in 99% of retinal ganglion cells (RGCs). The model facilitates neuronal assessment ex vivo with wholemounts quantified to show neurodegeneration following optic nerve crush or elevated IOP as related to glaucoma, in vitro with robust Ca 2+ responses to P2X7 receptor stimulation in neuronal cultures, and in vivo using a confocal scanning laser ophthalmoscope (cSLO). While the tdTomato signal showed strong overlap with RGC markers, BRN3A and RBPMS, there was no cross-labeling of displaced amacrine cells in the ganglion cell layer. Controls indicated no impact of Slc17a6 -tdTomato expression on light-dependent neuronal function, as determined with a microelectrode array (MEA), or on structure, as measured with optical coherence tomography (OCT). In summary, this novel neuronal reporter mouse model offers an effective means to increase the efficiency for real-time, specific visualization of retinal ganglion cells. It holds substantial promise for enhancing our understanding of RGC pathology in glaucoma and other diseases of the optic nerve, and could facilitate the screening of targeted therapeutic interventions for neurodegeneration.

Tamoxifen protects photoreceptors in the sodium iodate model.

Because the selective estrogen receptor modulator tamoxifen was shown to be retina-protective in the light damage and rd10 models of retinal degeneration, the purpose of this study was to test whether tamoxifen is retina-protective in a model where retinal pigment epithelium (RPE) toxicity appears to be the primary insult: the sodium iodate (NaIO3) model. C57Bl/6J mice were given oral tamoxifen (in the diet) or the same diet lacking tamoxifen, then given an intraperitoneal injection of NaIO3 at 25 mg/kg. The mice were imaged a week later using optical coherence tomography (OCT). ImageJ with a custom macro was utilized to measure retinal thicknesses in OCT images. Electroretinography (ERG) was used to measure retinal function one week post-injection. After euthanasia, quantitative real-time PCR (qRT-PCR) was performed. Tamoxifen administration partially protected photoreceptors. There was less photoreceptor layer thinning in OCT images of tamoxifen-treated mice. qRT-PCR revealed, in the tamoxifen-treated group, less upregulation of antioxidant and complement factor 3 mRNAs, and less reduction in the rhodopsin and short-wave cone opsin mRNAs. Furthermore, ERG results demonstrated preservation of photoreceptor function for the tamoxifen-treated group. Cone function was better protected than rods. These results indicate that tamoxifen provided structural and functional protection to photoreceptors against NaIO3. RPE cells were not protected. These neuroprotective effects suggest that estrogen-receptor modulation may be retina-protective. The fact that cones are particularly protected is intriguing given their importance for human visual function and their survival until the late stages of retinitis pigmentosa. Further investigation of this protective pathway could lead to new photoreceptor-protective therapeutics.

Microsomal triglyceride transfer protein is necessary to maintain lipid homeostasis and retinal function.

Lipid processing by the retinal pigment epithelium (RPE) is necessary to maintain retinal health and function. Dysregulation of retinal lipid homeostasis due to normal aging or age-related disease triggers lipid accumulation within the RPE, on Bruch's membrane (BrM), and in the subretinal space. In its role as a hub for lipid trafficking into and out of the neural retina, the RPE packages a significant amount of lipid into lipid droplets for storage and into apolipoprotein B (APOB)-containing lipoproteins (Blps) for export. Microsomal triglyceride transfer protein (MTP), encoded by the MTTP gene, is essential for Blp assembly. Herein we test the hypothesis that MTP expression in the RPE is essential to maintain lipid balance and retinal function using the newly generated RPEΔMttp mouse model. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic depletion of Mttp from the RPE results in intracellular lipid accumulation, increased photoreceptor-associated cholesterol deposits, and photoreceptor cell death, and loss of rod but not cone function. RPE-specific reduction in Mttp had no significant effect on plasma lipids and lipoproteins. While APOB was decreased in the RPE, most ocular retinoids remained unchanged, with the exception of the storage form of retinoid, retinyl ester. Thus suggesting that RPE MTP is critical for Blp synthesis and assembly but is not directly involved in plasma lipoprotein metabolism. These studies demonstrate that RPE-specific MTP expression is necessary to establish and maintain retinal lipid homeostasis and visual function.

Optimizing the sodium iodate model: Effects of dose, gender, and age.

Sodium iodate (NaIO3) is a commonly used model for age-related macular degeneration (AMD), but its rapid and severe induction of retinal pigment epithelial (RPE) and photoreceptor degeneration can lead to the premature dismissal of potentially effective therapeutics. Additionally, little is known about how sex and age affect the retinal response to NaIO3. This study aims to establish a less severe yet reproducible regimen by testing low doses of NaIO3 while considering age- and sex-related effects, enabling a broader range of therapeutic evaluations. In this study, young (3-5 months) and old (18-24 months) male and female C57Bl/6J mice were given an intraperitoneal (IP) injection of 15, 20, or 25 mg/kg NaIO3. Damage assessment one week post-injection included in vivo imaging, histological examination, and qRT-PCR analysis. The results revealed that young mice showed no damage at 15 mg/kg IP NaIO3, with varying degrees of damage observed at 20 mg/kg. At 25 mg/kg, most young mice displayed widespread retinal damage, with females exhibiting less retinal thinning than males. In contrast, older mice at 20 and 25 mg/kg displayed a more patchy degeneration pattern, outer retinal undulations, and greater variability in degeneration than the young mice. The most effective model for minimizing damage while maintaining consistency utilizes young female mice injected with 25 mg/kg NaIO3. The observed sex- and age-related differences underscore the importance of considering these variables in research, aligning with the National Institutes of Health's guidance. While the model does not fully replicate the complexity of AMD, these findings enhance its utility as a valuable tool for testing RPE/photoreceptor protective or replacement therapies.

Microsomal triglyceride transfer protein is necessary to maintain lipid homeostasis and retinal function.

Lipid processing by the retinal pigment epithelium (RPE) is necessary to maintain retinal health and function. Dysregulation of retinal lipid homeostasis due to normal aging or to age-related disease triggers lipid accumulation within the RPE, on Bruch's membrane (BrM), and in the subretinal space. In its role as a hub for lipid trafficking into and out of the neural retina, the RPE packages a significant amount of lipid into lipid droplets for storage and into apolipoprotein B (apoB)-containing lipoproteins (Blps) for export. Microsomal triglyceride transfer protein (MTP), encoded by the MTTP gene, is essential for Blp assembly. Herein we test the hypothesis that MTP expression in the RPE is essential to maintain lipid balance and retinal function using the newly generated RPEΔMttp mouse model. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Mttp from the RPE results in intracellular lipid accumulation, increased photoreceptor -associated cholesterol deposits and photoreceptor cell death, and loss of rod but not cone function. RPE-specific ablation of Mttp had no significant effect on plasma lipids and lipoproteins. While, apoB was decreased in the RPE, ocular retinoid concentrations remained unchanged. Thus suggesting that RPE MTP is critical for Blp synthesis and assembly but not directly involved in ocular retinoid and plasma lipoprotein metabolism. These studies demonstrate that RPE-specific MTP expression is necessary to establish and maintain retinal lipid homeostasis and visual function.

A comparison of optophysiological biomarkers of photoreceptor stress and phototoxicity in BALB/cJ, B6 (Cg)-Tyrc-2J/J, and C57Bl/6J mouse strains.

Ophthalmic imaging instruments, including the confocal scanning laser ophthalmoscope and spectral-domain optical coherence tomography system, originally intended for revealing ocular microstructures in the human eye, have been deployed by vision researchers to evaluate the eyes of numerous small and large animal species for more than two decades. In this study, we have used these two instruments to obtain imaging data sequentially from the retinas of three prominent, widely used experimental mouse models to document changes induced by two contrasting vivarium lighting conditions. Mice studied include albino BALB/cJ and B6(Cg)-Tyrc-2J/J and pigmented C57Bl/6J. Mice were reared under dim light conditions until ~8 weeks of age where they underwent baseline imaging. Following, mice were returned to the dim vivarium or relocated to the top rack cage position in a standard vivarium. Mice were then followed for several months by ocular imaging to catalog the retinal dynamics as a function of long-term dim vs. elevated, standard vivarium lighting exposure levels. Upon exposure to elevated light levels, B6(Cg)-Tyrc-2J/J underwent similar changes as BALB/cJ in regard to photoreceptor outer segment shortening, photoreceptor layer proximal aspect hyperreflective changes, and the development of retinal infoldings and autofluorescent sub-retinal inflammatory monocyte infiltrate. Noteworthy, however, is that infoldings and infiltrate occurred at a slower rate of progression in B6(Cg)-Tyrc-2J/J vs. BALB/cJ. The photoreceptor outer nuclear layer thickness of BALB/cJ degenerated steadily following elevated light onset. In contrast, B6(Cg)-Tyrc-2J/J degeneration was unremarkable for many weeks before experiencing a noticeable change in the rate of degeneration that was concomitant with a plateau and decreasing trend in number of retinal infoldings and monocyte infiltrate. Pathological changes in C57Bl/6J mice were unremarkable for all imaging biomarkers assessed with exception to autofluorescent sub-retinal inflammatory monocyte infiltrate, which showed significant accumulation in dim vs. elevated light exposed mice following ~1 year of observation. These data were evaluated using Spearman's correlation and Predictive Power Score matrices to determine the best imaging optophysiological biomarkers for indicating vivarium light stress and light-induced photoreceptor degeneration. This study suggests that changes in proximal aspect hyperreflectivity, outer segment shortening, retinal infoldings and autofluorescent sub-retinal inflammatory monocyte infiltrate are excellent indicators of light stress and light-induced degeneration in albino B6(Cg)-Tyrc-2J/J and BALB/cJ mouse strains.