Glaucoma etiology often includes retinal ganglion cell (RGC) death associated with elevated intraocular pressure (IOP). However, even when IOP is managed well, disease can progress. It is thus important to develop therapeutic approaches that directly protect RGCs in an IOP-independent manner. Compromised nicotinamide adenine dinucleotide (NAD⁺) metabolism occurs in neurodegenerative diseases, including models of glaucoma. Here we report testing the protective effects of prophylactically systemically administered nicotinamide riboside (NR), a NAD⁺ precursor, in a mouse model of acute RGC damage (optic nerve crush (ONC)), and in a chronic model of RGC degeneration (ocular hypertension induced by intracameral injection of microbeads). For both models, treatment enhanced RGC survival, assessed by counting cells in retinal flatmounts immunostained for Brn3a+. In the ONC model, treatment preserved RGC function, as assessed by pattern electroretinogram, and suppressed retinal inflammation, as assessed by immunofluorescence staining of retinal fixed sections for glial fibrillary acidic protein (GFAP). This is the first study to demonstrate that systemic treatment with NR is protective in acute and chronic models of RGC damage. The protection is significant and, considering that NR is highly bioavailable in and well-tolerated by humans, may support the proposition of prospective human subject studies.

The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control and believed that this process involves interactions from the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE. Thereby, the aim of this study was to determine whether the clock in the retina or RPE controls the diurnal phagocytic peak and whether disruption of the circadian clock in the RPE would affect cellular function and the viability during aging. To that, we generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then, using electroretinography, spectral domain-optical coherence tomography, and optomotor response of visual function we determined the effect of Bmal1 removal in young (6 months) and old (18 months) mice. RPE morphology and lipofuscin accumulation was determined in young and old mice. Our data shows that the clock in the RPE, rather than the retina clock, controls the diurnal phagocytic peak. Surprisingly, absence of a functional RPE clock and phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the circadian clock in the RPE controls the daily peak of phagocytic activity. However, the absence of the clock in the RPE does not result in deterioration of photoreceptors or the RPE during aging.

The Per2^luc mouse model developed by Takahashi laboratory is one of the most powerful models to study circadian rhythms in real time. In this study, we report that photoreceptors degenerate in male Per2^luc mice during aging. Young (2.5- to 5-month-old) and aged (11- to 13.5-month-old) homozygous male Per2^luc mice and C57BL/6J mice were used for this study. Retina structure and function were investigated via spectral domain optical coherence tomography (SD-OCT), fundus imaging, and electroretinography (ERG). Zonula occludens-1 (ZO-1) immunofluorescence was used to analyze the retinal pigment epithelium (RPE) morphology. Fundus examination revealed no difference between young Per2^luc and wild-type (WT) mice. However, the fundus of aged Per2^luc mice showed white deposits, suggestive of age-related drusen-like formation or microglia, which were absent in age-matched WT mice. No differences in retinal structure and function were observed between young Per2^luc and WT mice. However, with age, Per2^luc mice showed a significant reduction in total retinal thickness with respect to C57BL/6J mice. The reduction was mostly confined to the photoreceptor layer. Consistent with these results, we observed a significant decrease in the amplitude of a- and b-waves of the ERG in aged Per2^luc mice. Analysis of the RPE morphology revealed that in aged Per2^luc mice there was an increase in compactness and eccentricity with a decrease in solidity with respect to the values observed in WT, pointing toward signs of aging in the RPE of Per2^luc mice. Our data demonstrate that homozygous Per2^luc mice show photoreceptor degeneration during aging and a premature aging of the RPE.

5.1 Purpose: Pigment epithelium-derived factor (PEDF) is a neurotrophic glycoprotein secreted by the retinal pigment epithelium (RPE) that supports retinal photoreceptor health. Deficits in PEDF are associated with increased inflammation and retinal degeneration in aging and diabetic retinopathy. We hypothesized that light-induced stress in C57BL/6J mice deficient in PEDF would lead to increased retinal neuronal and RPE defects, impaired expression of neurotrophic factor Insulin-like growth factor 1 (IGF-1), and overactivation of Galectin-3-mediated inflammatory signaling. Methods: C57BL/6J mice expressing the RPE65 M450/M450 allele were crossed to PEDF ^KO/KO and wildtype (PEDF ^+/+ ) littermates. Mice were exposed to 50,000 lux light for 5 hours to initiate acute damage. Changes in visual function outcomes were tracked via electroretinogram (ERG), confocal scanning laser ophthalmoscopy(cSLO), and spectral domain optical coherence tomography (SD-OCT) on days 3, 5, and 7 post-light exposure. Gene and protein expression of Galectin-3 were measured by digital drop PCR (ddPCR) and western blot. To further investigate the role of galectin-3 on visual outcomes and PEDF expression after damage, we also used a small-molecule inhibitor to reduce its activity. Results: Following light damage, PEDF ^KO/KO mice showed more severe retinal thinning, impaired visual function (reduced a-, b-, and c-wave amplitudes), and increased Galectin-3 expressing immune cell infiltration compared to PEDF ^+/+ . PEDF ^KO/KO mice had suppressed damage-associated increases in IGF-1 expression. Additionally, baseline Galectin-3 mRNA and protein expression were reduced in PEDF ^KO/KO mice compared to PEDF ^+/+ . However, after light damage, Galectin-3 expression decreases in PEDF ^+/+ mice but increases in PEDF KO/KO mice without reaching PEDF ^+/+ levels. Galectin-3 inhibition worsens retinal degeneration, reduces PEDF expression in PEDF ^+/+ mice, and mimics the effects seen in PEDF knockouts. Conclusions: Loss of PEDF alone does not elicit functional defects in C57BL/6J mice. However, under light stress, PEDF deficiency significantly increases severe retinal degeneration, visual deficits, Galectin-3 expression, and suppression of IGF-1 than PEDF ^+/+ . PEDF deficiency reduced baseline expression of Galectin-3, and pharmacological inhibition of Galectin-3 worsens outcomes and suppresses PEDF expression in PEDF ^+/+, suggesting a novel co-regulatory relationship between the two proteins in mitigating light-induced retinal damage.

Purpose: The aim of this study was to investigate the protective effects of systemically administered tauroursodeoxycholic acid (TUDCA) in an optic nerve crush (ONC) mouse model of retinal ganglion cell (RGC) death. Methods: C57BL/6J mice were injected intraperitoneally (i.p.) three times per week with TUDCA (500 mg/kg) for two weeks, after which unilateral ONC was performed. A control cohort was identically treated with a drug vehicle (phosphate buffered saline; PBS). A separate cohort did not undergo any injections or surgeries (this was termed the “Naïve” group). Pattern electroretinography (PERG) was recorded 3 days after ONC. Retinas were harvested for whole-mount immunofluorescence staining with an antibody against RGC marker Brn3a and imaged by fluorescent confocal microscopy. Apoptotic cells in the ganglion cell layer (GCL) were detected by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) performed on fixed retina sections. Glial fibrillary acidic protein (GFAP) immunostaining on fixed retina sections was conducted to detect the activation of Müller cells. Total RNA was extracted from retinas and expression of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-10 was determined by digital droplet PCR (ddPCR). Results: TUDCA treatment preserved visual function as assessed by PERG. P1 and N2 amplitudes from the PBS-treated ONC group were significantly diminished compared to those of the Naïve group (p < 0.001). TUDCA treatment prevented this diminution. The amplitudes of P1 and N2 in the TUDCA-treated ONC group were statistically indistinguishable from those of the Naïve group and were higher than the PBS-treated ONC group (TUDCA+ONC vs. PBS+ONC, P1: 6.99 ± 0.89 µV vs. 3.60 ± 0.69 µV, p < 0.01; N2: −9.30 (IQR: −13.43–−6.44) µV vs. −4.47 (IQR: −10.26–−2.17) µV). TUDCA treatment preserved RGCs. The ONC-vehicle-only group had 25% fewer RGCs (Brn3a-positive cells) than Naïve eyes (p < 0.0001). TUDCA treatment nearly completely prevented this loss, preserving all but 7.7% of the RGCs, and the number of RGCs in the TUDCA-treated ONC group was significantly higher than in the PBS-treated ONC group (TUDCA+ONC vs. PBS+ONC, 1738.00 ± 14.43 cells per field vs. 1454.00 ± 6.55 cells per field, p < 0.0001). The number of TUNEL-positive cells in the GCL (Naïve vs. PBS+ONC group: 1.00 (IQR: 0.00–2.00) % vs. 37.00 (IQR: 8.50–48.50) %, p < 0.05) and GFAP-positive fibers transversing retina sections (Naïve vs. PBS+ONC group: 33.00 ± 1.15 vs. 185.70 ± 42.37 fibers/retina, p < 0.05), and the expression of IL-6, TNF-α were significantly greater in the PBS-treated ONC group compared to that of the Naïve group (Naïve vs. PBS+ONC group, IL-6: 0.07 (IQR: 0.06–0.31) vs. 0.99 (IQR: 0.56–1.47), p < 0.05, TNF-α: 0.19 ± 0.069 vs. 1.39 ± 0.23; p < 0.01), an increase not observed with TUDCA treatment. Conclusions: Systemic TUDCA treatment significantly preserved RGC function and survival in the mouse ONC model of RGC damage. TUDCA treatment prevented RGC apoptosis, Müller glial cell activation, and retinal expression of several inflammatory cytokines. These data suggest that TUDCA is a promising therapeutic candidate for preserving RGC numbers and function.

The retinal pigment epithelium (RPE), a monolayer of pigmented cells, is critical for visual function through its interaction with the neural retina. In healthy eyes, RPE cells exhibit a uniform hexagonal arrangement, but under stress or disease, such as age-related macular degeneration (AMD), dysmorphic traits like cell enlargement and apparent multinucleation emerge. Multinucleation has been hypothesized to result from cellular fusion, a compensatory mechanism to maintain cell-to-cell contact and barrier function, as well as conserve resources in unhealthy tissue. However, traditional two-dimensional (2D) imaging using apical border markers alone may misrepresent multinucleation due to the lack of lateral markers. We present high-resolution confocal images enabling three-dimensional (3D) visualization of apical (ZO-1) and lateral (α-catenin) markers alongside nuclei. In two RPE damage models, we find that seemingly multinucleated cells are often single cells with displaced neighboring nuclei and lateral membranes. This emphasizes the need for 3D analyses to avoid misidentifying multinucleation and underlying fusion mechanisms. Lastly, images from the NaIO₃ oxidative damage model reveal variability in RPE damage, with elongated, dysmorphic cells showing increased ZsGreen reporter protein expression driven by EMT-linked CAG promoter activity, while more regular RPE cells displayed somewhat reduced green signal more typical of epithelial phenotypes.