Prominin 1 is crucial for the early development of photoreceptor outer segments.

Prominin 1 (PROM1) is a pentaspan transmembrane glycoprotein localized on the nascent photoreceptor discs. Mutations in PROM1 are linked to various retinal diseases. In this study, we assessed the role of PROM1 in photoreceptor biology and physiology using the PROM1 knockout murine model (rd19). Our study found that PROM1 is essential for vision and photoreceptor development. We found an early reduction in photoreceptor response beginning at post-natal day 12 (P12) before eye opening in the absence of PROM1 with no apparent loss in photoreceptor cells. However, at this stage, we observed an increased glial cell activation, indicative of cell damage. Contrary to our expectations, dark rearing did not mitigate photoreceptor degeneration or vision loss in PROM1 knockout mice. In addition to physiological defects seen in PROM1 knockout mice, ultrastructural analysis revealed malformed outer segments characterized by whorl-like continuous membranes instead of stacked disks. In parallel to the reduced rod response at P12, proteomics revealed a significant reduction in the levels of protocadherin, a known interactor of PROM1, and rod photoreceptor outer segment proteins, including rhodopsin. Overall, our results underscore the indispensable role of PROM1 in photoreceptor development and maintenance of healthy vision.

Chirped flicker optoretinography for in vivo characterization of human photoreceptors' frequency response to light.

Flicker electroretinography (ERG) has served as a valuable noninvasive objective tool for investigating retinal physiological function through the measurement of electrical signals originating from retinal neurons in response to temporally modulated light stimulation. Deficits in the response at certain frequencies can be used as effective biomarkers of cone-pathway dysfunction. In this Letter, we present the progress we made on its optical counterpart-photopic flicker optoretinography (f-ORG). Specifically, we focus on the measurement of the response of light-adapted retinal photoreceptors to a flicker stimulus with chirped frequency modulation. In contrast to measurements performed at discrete frequencies, this technique enables a significantly accelerated characterization of photoreceptor outer segment optical path length modulation amplitudes in the nanometer range as a function of stimulus frequency, enabling the acquisition of the characteristic frequency response in less than 2 sec.

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.

The Logistical Backbone of Photoreceptor Cell Function: Complementary Mechanisms of Dietary Vitamin A Receptors and Rhodopsin Transporters.

In this review, we outline our current understanding of the mechanisms involved in the absorption, storage, and transport of dietary vitamin A to the eye, and the trafficking of rhodopsin protein to the photoreceptor outer segments, which encompasses the logistical backbone required for photoreceptor cell function. Two key mechanisms of this process are emphasized in this manuscript: ocular and systemic vitamin A membrane transporters, and rhodopsin transporters. Understanding the complementary mechanisms responsible for the generation and proper transport of the retinylidene protein to the photoreceptor outer segment will eventually shed light on the importance of genes encoded by these proteins, and their relationship on normal visual function and in the pathophysiology of retinal degenerative diseases.

Sphingolipid biosynthetic inhibitor L-Cycloserine prevents oxidative-stress-mediated death in an in vitro model of photoreceptor-derived 661W cells.

Oxidative stress plays a pivotal role in the pathogenesis of several neurodegenerative diseases. Retinal degeneration causes irreversible death of photoreceptor cells, ultimately leading to vision loss. Under oxidative stress, the synthesis of bioactive sphingolipid ceramide increases, triggering apoptosis in photoreceptor cells and leading to their death. This study investigates the effect of L-Cycloserine, a small molecule inhibitor of ceramide biosynthesis, on sphingolipid metabolism and the protection of photoreceptor-derived 661W cells from oxidative stress. The results demonstrate that treatment with L-Cycloserine, an inhibitor of Serine palmitoyl transferase (SPT), markedly decreases bioactive ceramide and associated sphingolipids in 661W cells. A nontoxic dose of L-Cycloserine can provide substantial protection of 661W cells against H2O2-induced oxidative stress by reversing the increase in ceramide level observed under oxidative stress conditions. Analysis of various antioxidant, apoptotic and sphingolipid pathway genes and proteins also confirms the ability of L-Cycloserine to modulate these pathways. Our findings elucidate the generation of sphingolipid mediators of cell death in retinal cells under oxidative stress and the potential of L-Cycloserine as a therapeutic candidate for targeting ceramide-induced degenerative diseases by inhibiting SPT. The promising therapeutic prospect identified in our findings lays the groundwork for further validation in in-vivo and preclinical models of retinal degeneration.

Visual afferents from an eye in the terrestrial slug Limax valentianus.

Terrestrial gastropods have a lens-bearing eye on the tip of their tentacles. There are two morphologically distinct photoreceptors, called Type-I and Type-II photoreceptors, in the retina. Type-I photoreceptors are equipped with highly developed photoreceptive microvilli in their outer rhabdomeric segment, whereas Type-II photoreceptors have short and fewer microvilli. Although both types of photoreceptors send afferent projections directly to the brain, their destinations in the brain, called optic neuropiles, have not been sufficiently investigated. Our recent studies revealed that there are commissural fibers in the cerebral ganglia that transmit photic information acquired by bilateral eyes. Moreover, some of the retinal photoreceptors are connected by gap junctions to the photosensitive brain neurons, suggesting the functional interaction of the photic information between the eye and brain photoreceptors, as well as between bilateral eyes. However, it has not been clarified which type of retinal photoreceptors send commissural projections to the contralateral hemiganglion nor interact with the brain photoreceptors. In the present study, we demonstrated by molecular histological analyses and tracer injections that (1) Type-I and Type-II photoreceptors send glutamatergic afferent projections to the medial and lateral lobes of the ipsilateral optic neuropile, respectively, (2) direct synaptic interaction between bilateral optic nerves occurs in the medial lobe of the optic neuropile, and (3) brain photosensory neurons form gap junctions with the medial lobe of the contralateral optic neuropile. These results reveal an ordered pattern of afferent projections from the retina and provide insight into the different functional roles of retinal photoreceptors.

Transplanted human photoreceptors transfer cytoplasmic material but not to the recipient mouse retina.

BACKGROUND: The discovery of material transfer between transplanted and host mouse photoreceptors has expanded the possibilities for utilizing transplanted photoreceptors as potential vehicles for delivering therapeutic cargo. However, previous research has not directly explored the capacity for human photoreceptors to engage in material transfer, as human photoreceptor transplantation has primarily been investigated in rodent models of late-stage retinal disease, which lack host photoreceptors. METHODS: In this study, we transplanted human stem-cell derived photoreceptors purified from human retinal organoids at different ontological ages (weeks 10, 14, or 20) into mouse models with intact photoreceptors and assessed transfer of human proteins and organelles to mouse photoreceptors. RESULTS: Unexpectedly, regardless of donor age or mouse recipient background, human photoreceptors did not transfer material in the mouse retina, though a rare subset of donor cells (< 5%) integrated into the mouse photoreceptor cell layer. To investigate the possibility that a species barrier impeded transfer, we used a flow cytometric assay to examine material transfer in vitro. Interestingly, dissociated human photoreceptors transferred fluorescent protein with each other in vitro, yet no transfer was detected in co-cultures of human and mouse photoreceptors, suggesting that material transfer is species specific. CONCLUSIONS: While xenograft models are not a tractable system to study material transfer of human photoreceptors, these findings demonstrate that human retinal organoid-derived photoreceptors are competent donors for material transfer and thus may be useful to treat retinal degenerative disease.

Drp1-dependent mitochondrial fragmentation mediates photoreceptor abnormalities in type 1 diabetic retina.

Recent studies have highlighted that retinal neurodegeneration precedes microvascular changes in diabetic retinopathy (DR), but the specific mechanisms remain unclear. Given the pivotal role of dysfunctional mitochondria and oxidative stress in early DR, our objective was to observe mitochondria-related alterations in the neural retina of type one diabetic mellitus mice with no evidence of DR (T1DM-NDR). We aimed to identify the key mitochondrial-related proteins contributing to mitochondrial injury. Our study revealed that T1DM-NDR mice exhibited outer retina thinning, including the ellipsoid zone, inner segment, and outer segment. Additionally, there was an impaired amplitude of the b-wave in electroretinogram (ERG) and a disorganized arrangement of the photoreceptor layer. In both the retina of DM mice and high glucose (HG)-treated 661w cells, mitochondria appeared swollen and fragmented, with disrupted cristae, disorganized or shortened branches in the mitochondrial network, and decreased mitochondrial membrane potential. Among the mitochondrial-related proteins, dynamin-related protein 1 (Drp1) was upregulated, and the ratio of phosphorylated Drp1 protein at serine 616 (S616) and serine 637 (S637) sites significantly increased in the retina of DM mice. The administration of Mdivi-1 ameliorated high-glucose-induced dysfunctional mitochondria, thereby protecting T1DM-NDR mice retina from morphological and functional injuries. Our findings suggest that hyperglycemia promotes Drp1-mediated mitochondrial dysfunction, which may be a significant factor in the development of DR. The inhibition of high-glucose-induced mitochondrial fission emerges as a potential and innovative intervention strategy for preventing DR.

Photoreceptor deficits appear at eye opening in Rs1 mutant mouse models of X-linked retinoschisis.

X-linked retinoschisis (XLRS) is an early onset degenerative retinal disease characterized by cystic lesions in the middle layers of the retina. These structural changes are accompanied by a loss of visual acuity and decreased contrast sensitivity. XLRS is caused by mutations in the gene Rs1 which encodes the secreted protein Retinoschisin 1. Young Rs1-mutant mouse models develop key hallmarks of XLRS including intraretinal schisis and abnormal electroretinograms. The electroretinogram (ERG) comprises activity of multiple cellular generators, and it is not known how and when each of these is impacted in Rs1 mutant mice. Here we use an ex vivo ERG system and pharmacological blockade to determine how ERG components generated by photoreceptors, ON-bipolar, and Müller glial cells are impacted in Rs1 mutants and to determine the time course of these changes. We report that ERG abnormalities begin near eye-opening and that all ERG components are involved.

Photoreceptor calyceal processes accompany the developing outer segment, adopting a stable length despite a dynamic core.

Vertebrate photoreceptors detect light through a large cilium-based outer segment, which is filled with photopigment-laden membranous discs. Surrounding the base of the outer segment are microvilli-like calyceal processes (CPs). Although CP disruption has been associated with altered outer segment morphology and photoreceptor degeneration, the role of the CPs remains elusive. Here, we used zebrafish as a model to characterize CPs. We quantified CP parameters and report a strong disparity in outer segment coverage between photoreceptor subtypes. CP length is stable across light and dark conditions, yet heat-shock inducible expression of tagged actin revealed rapid turnover of the CP actin core. Detailed imaging of the embryonic retina uncovered substantial remodeling of the developing photoreceptor apical surface, including a transition from dynamic tangential processes to vertically oriented CPs immediately prior to outer segment formation. Remarkably, we also found a direct connection between apical extensions of the Müller glia and retinal pigment epithelium, arranged as bundles around the ultraviolet sensitive cones. In summary, our data characterize the structure, development and surrounding environment of photoreceptor microvilli in the zebrafish retina.

POSTOPERATIVE PHOTORECEPTOR INTEGRITY AND ANATOMICAL OUTCOMES BASED ON PRESENTING MORPHOLOGIC STAGE OF RHEGMATOGENOUS RETINAL DETACHMENT.

PURPOSE: To evaluate outer retinal recovery on postoperative optical coherence tomography (OCT) based on presenting morphologic stage of rhegmatogenous retinal detachment (RRD). METHODS: Retrospective cohort of consecutive primary fovea-involving RRDs, referred from January 2012 to September 2022. Baseline OCTs were assessed for morphologic stage of RRD. Postoperative OCT scans were graded at 3, 6, and 12 months for external limiting membrane, ellipsoid zone and interdigitation zone discontinuity, epiretinal membrane formation and severity, and residual subfoveal fluid. RESULTS: Three hundred and fifty-one patients were included. Increasing baseline morphologic stage of RRD was significantly associated with external limiting membrane, ellipsoid zone, and interdigitation zone discontinuity at all time points postoperatively ( P < 0.001) and was shown to be an independent predictor of foveal photoreceptor integrity after adjusting for height of detachment, time to surgery, and duration of fovea involvement ( P < 0.001). Earlier stages were associated with residual subfoveal fluid ( P < 0.001). There was no association between the stages of RRD and epiretinal membrane severity. However, late stages presented with earlier development of epiretinal membrane ( P = 0.012). CONCLUSION: Increasing morphologic stage of RRD is associated with delayed recovery of outer retinal bands in the first year and faster development of epiretinal membrane after RRD repair. The results of this study suggest that the stages may serve as a prognostic biomarker for postoperative photoreceptor recovery.

Unusual OCT findings in a patient with CABP4-associated cone-rod synaptic disorder.

PURPOSE: Bi-allelic variants in CABP4 are associated with congenital cone-rod synaptic disorder, which has also been classified, electrophysiologically, as incomplete congenital stationary night blindness (iCSNB). We describe clinical findings in a patient who demonstrated an unusual macular optical coherence tomography (OCT) phenotype, not previously reported in this condition. METHODS: Our patient underwent multimodal retinal imaging, international standard full-field ERG testing and whole genome sequencing. RESULTS: The patient was a 60-year-old woman with non-progressive visual impairment since birth, nystagmus and preference for dim lighting. Clinical fundus examination was unremarkable. OCT imaging revealed a hypo-reflective zone under an elevated fovea in both eyes. ERGs showed an electronegative DA10 response, with severely abnormal light-adapted responses. Whole genome sequencing revealed homozygosity for a known pathogenic variant in CABP4. No variants were found in other genes that could explain the patient's phenotype. CONCLUSIONS: OCT findings of foveal elevation and an underlying hypo-reflective zone are novel in this condition. Whilst the clinical history was similar to achromatopsia and other cone dysfunction syndromes, ERG findings suggested disease associated with CACNA1F or CABP4. As CACNA1F is X-linked, CABP4 was more likely, and confirmed on genetic testing. The patient saw better in dim light, confirming that night blindness is not a feature of CABP4-associated disease. Our case highlights the value of ERGs in discriminating between causes of cone dysfunction, and extends the range of retinal imaging phenotypes reported in this disorder.

Effects of different alkylating agents on photoreceptor degeneration and proliferative response of Müller glia.

Animal models for retinal degeneration are essential for elucidating its pathogenesis and developing new therapeutic strategies in humans. N-methyl-N-nitrosourea (MNU) has been extensively used to construct a photoreceptor-specific degeneration model, which has served to unveil the molecular process of photoreceptor degeneration as well as the mechanisms regulating the protective responses of remaining cells. Methyl methanesulphonate (MMS), also known to cause photoreceptor degeneration, is considered a good alternative to MNU due to its higher usability; however, detailed pathophysiological processes after MMS treatment remain uncharacterized. Here, we analyzed the time course of photoreceptor degeneration, Müller glial proliferation, and expression of secretory factors after MNU and MMS treatments in rats. While the timing of rod degeneration was similar between the treatments, we unexpectedly found that cones survived slightly longer after MMS treatment. Müller glia reentered the cell cycle at a similar timing after the two treatments; however, the G1/S transition occurred earlier after MMS treatment. Moreover, growth factors such as FGF2 and LIF were more highly upregulated in the MMS model. These data suggest that comparative analyses of the two injury models may be beneficial for understanding the complex regulatory mechanisms underlying the proliferative response of Müller glia.

Melatonin suppression by light involves different retinal photoreceptors in young and older adults.

Age-related sleep and circadian rhythm disturbances may be due to altered nonvisual photoreception. Here, we investigated the temporal dynamics of light-induced melatonin suppression in young and older individuals. In a within-subject design study, young and older participants were exposed for 60 min (0030-0130 at night) to nine narrow-band lights (range: 420-620 nm). Plasma melatonin suppression was calculated at 15, 30, 45, and 60 min time intervals. Individual spectral sensitivity of melatonin suppression and photoreceptor contribution were predicted for each interval and age group. In young participants, melanopsin solely drove melatonin suppression at all time intervals, with a peak sensitivity at 485.3 nm established only after 15 min of light exposure. Conversely, in older participants, spectral light-driven melatonin suppression was best explained by a more complex model combining melanopsin, S-cone, and M-cone functions, with a stable peak (~500 nm) at 30, 45, and 60 min of light exposure. Aging is associated with a distinct photoreceptor contribution to melatonin suppression by light. While in young adults melanopsin-only photoreception is a reliable predictor of melatonin suppression, in older individuals this process is jointly driven by melanopsin, S-cone, and M-cone functions. These findings offer new prospects for customizing light therapy for older individuals.

SNAP-25, but not SNAP-23, is essential for photoreceptor development, survival, and function in mice.

SNARE-mediated vesicular transport is thought to play roles in photoreceptor glutamate exocytosis and photopigment delivery. However, the functions of Synaptosomal-associated protein (SNAP) isoforms in photoreceptors are unknown. Here, we revisit the expression of SNAP-23 and SNAP-25 and generate photoreceptor-specific knockout mice to investigate their roles. Although we find that SNAP-23 shows weak mRNA expression in photoreceptors, SNAP-23 removal does not affect retinal morphology or vision. SNAP-25 mRNA is developmentally regulated and undergoes mRNA trafficking to photoreceptor inner segments at postnatal day 9 (P9). SNAP-25 knockout photoreceptors develop normally until P9 but degenerate by P14 resulting in severe retinal thinning. Photoreceptor loss in SNAP-25 knockout mice is associated with abolished electroretinograms and vision loss. We find mistrafficked photopigments, enlarged synaptic vesicles, and abnormal synaptic ribbons which potentially underlie photoreceptor degeneration. Our results conclude that SNAP-25, but not SNAP-23, mediates photopigment delivery and synaptic functioning required for photoreceptor development, survival, and function.

The m6A reader YTHDC2 maintains visual function and retinal photoreceptor survival through modulating translation of PPEF2 and PDE6B.

Inherited retinal dystrophies (IRDs) are major causes of visual impairment and irreversible blindness worldwide, while the precise molecular and genetic mechanisms are still elusive. N6-methyladenosine (m6A) modification is the most prevalent internal modification in eukaryotic mRNA. YTH domain containing 2 (YTHDC2), an m6A reader protein, has recently been identified as a key player in germline development and human cancer. However, its contribution to retinal function remains unknown. Here, we explore the role of YTHDC2 in the visual function of retinal rod photoreceptors by generating rod-specific Ythdc2 knockout mice. Results show that Ythdc2 deficiency in rods causes diminished scotopic ERG responses and progressive retinal degeneration. Multi-omics analysis further identifies Ppef2 and Pde6b as the potential targets of YTHDC2 in the retina. Specifically, via its YTH domain, YTHDC2 recognizes and binds m6A-modified Ppef2 mRNA at the coding sequence and Pde6b mRNA at the 5'-UTR, resulting in enhanced translation efficiency without affecting mRNA levels. Compromised translation efficiency of Ppef2 and Pde6b after YTHDC2 depletion ultimately leads to decreased protein levels in the retina, impaired retinal function, and progressive rod death. Collectively, our finding highlights the importance of YTHDC2 in visual function and photoreceptor survival, which provides an unreported elucidation of IRD pathogenesis via epitranscriptomics.

Peripheral monocytes and neutrophils promote photoreceptor cell death in an experimental retinal detachment model.

Photoreceptor cell death and immune cell infiltration are two major events that contribute to retinal degeneration. However, the relationship between these two events has not been well delineated, primarily because of an inadequate understanding of the immunological processes involved in photoreceptor degeneration, especially that of peripheral leukocytes that infiltrate the subretinal space and retinal tissues. In this work, we characterized the role of leukocyte infiltration within the detached retina. We observed that CD45+ CD11b+ Ly6G+ neutrophils and CD45+ CD11b+ Ly6G- Ly6C+ monocytes are the predominant peripheral immune cell populations that infiltrate the retinal and subretinal space after detachment. Selective depletion of monocytes or neutrophils using cell-specific targeting is neuroprotective for photoreceptors. These results indicate that peripheral innate immune cells contribute to photoreceptor degeneration, and targeting these immune cell populations could be therapeutic during retinal detachment.

Hyperoside alleviates photoreceptor degeneration by preventing cell senescence through AMPK-ULK1 signaling.

Vision loss and blindness are frequently caused by photoreceptor degeneration, for example in age-related macular degeneration and retinitis pigmentosa. However, there is no effective medicine to treat these photoreceptor degeneration-related diseases. Cell senescence is a common phenotype in many diseases; however, few studies have reported whether it occurs in photoreceptor degeneration diseases. Herein, we identified that cell senescence is associated with photoreceptor degeneration induced by N-methyl-N-nitrosourea (MNU, a commonly used photoreceptor degeneration model), presented as increased senescence-associated ß-galactosidase activity, DNA damage, oxidative stress and inflammation-related cytokine Interleukin 6 (IL6), and upregulation of cyclin p21 or p16. These results suggested that visual function might be protected using anti-aging treatment. Furthermore, Hyperoside is reported to help prevent aging in various organs. In this study, we showed that Hyperoside, delivered intravitreally, alleviated photoreceptor cell senescence and ameliorated the functional and morphological degeneration of the retina in vivo and in vitro. Importantly, Hyperoside attenuated the MNU-induced injury and aging of photoreceptors via AMPK-ULK1 signaling inhibition. Taken together, our results demonstrated that Hyperoside can prevent MNU-induced photoreceptor degeneration by inhibiting cell senescence via the AMPK-ULK1 pathway.

Intrinsic signal optoretinography revealing AD-induced retinal photoreceptor hyperexcitability before a detectable morphological abnormality.

Neuronal hyperexcitability promises an early biomarker of Alzheimer's disease (AD). However, in vivo detection of neuronal hyperexcitability in the brain is technically challenging. The retina, one part of the central nervous system, presents a unique window for noninvasive monitoring of the brain function. This study aims to test the feasibility of using intrinsic signal optoretinography (ORG) for mapping retinal hyperexcitability associated with early-stage AD. Custom-designed optical coherence tomography (OCT) was employed for both morphological measurement and functional ORG of wild-type mice and 3xTg-AD mice. Comparative analysis revealed AD-induced retinal photoreceptor hyperexcitability prior to detectable structural degeneration.

REG1A protects retinal photoreceptors from blue light damage.

With the increased use of artificial light and the prolonged use of optoelectronic products, light damage (LD) to the human retina has been identified as a global vision-threatening problem. While there is evidence of a significant correlation between light-induced retinal damage and age-related vision impairment in age-related macular degeneration, it is unclear how light-induced retinal degeneration manifests itself and whether there are agents capable of preventing the development of LD in the retina. This study investigated a mechanism by which blue light leads to photoreceptor death. By observing blue light exposure in retinal organoids and photoreceptor cells, we concluded that there could be significant apoptosis of the photoreceptors. We demonstrate that regenerating islet-derived 1 alpha (REG1A) prevents photoreceptors from undergoing this LD-induced apoptosis by increasing expression of the anti-apoptotic gene Bcl2 and downregulating expression of the pro-apoptotic gene Bax, resulting in reduced mitochondrial damage and improved aerobic capacity in photoreceptor cells. For the first time, REG1A has been shown to restore mitochondrial function and cell apoptosis after LD-induced damage, suggesting its potential application in the prevention and treatment of retinal vision loss.