The retina is responsible for converting light into electrical signals that, when transmitted to the brain, create the sensation of vision. The mammalian retina is epigenetically unique since the differentiation of retinal progenitor cells (RPCs) into retinal cells is accompanied by a decrease in DNA methylation in the promoters of many genes important for retinal development and function. However, the pathway responsible for DNA demethylation and its role in retinal development and function were unknown. We hypothesized that the Ten-Eleven Translocation (TET) family of dioxygenases plays a key role in this pathway. To this end, we knocked out the TET family in RPCs and characterized the TET-deficient and control retinas using various approaches including electron microscopy, electroretinogram tests, TUNEL, RNA-seq, WGBS, and 5hmC-Seal. We found that while the TET-dependent DNA demethylation pathway contributes to the development of many retinal cell types, it is the most significant contributor to rod and cone photoreceptor development and function. We found that genetic ablation of TET enzymes in RPCs prevents demethylation and the activity of genes essential for rod specification and for rod and cone maturation. Reduced activity of genes responsible for rod specification results in the TET-deficient retina being depleted of these neurons. Meanwhile, reduced activity of genes responsible for rod and cone maturation leads to the underdevelopment or complete absence of outer segments and synaptic termini in the TET-deficient photoreceptors, which results in loss of their function and leads to blindness. These function-deprived, underdeveloped photoreceptors die over time, leading to retinal dystrophy.

Light-field displays typically consist of a two-dimensional (2D) display panel and a light modulation device. The 2D panel presents synthesized parallax images, with the total information content of the three-dimensional (3D) light field dictated by the panel's total resolution. Angular resolution serves as a critical metric for light-field displays, where higher angular resolution correlates with a more realistic 3D visual experience. However, the improvement of angular resolution is typically accompanied by a reduction in spatial resolution, due to the limitations of the 2D display panel's total resolution. To address this challenge, a light-field display method with enhanced information utilization is introduced, achieved through the independent modulation of chrominance and luminance. A static light-field image display system is proposed to verify the feasibility of this method. The system employs a bidirectional angular modulation grating (BAMG) and a collimated light source (CLS) to create uniformly distributed viewpoints in space. A luminance modulation film (LMF) and a chrominance modulation film (CMF) are utilized to modulate the light-field information, with chrominance and luminance synthesized images printed at pixel densities of 720 pixels per inch (PPI) and 8000 dots per inch (DPI), respectively, to align with the differential sensitivities of the human visual system. In the experiment, the proposed display system achieves a full-parallax, high-fidelity color display with a 98.2° horizontal and 97.7° vertical field of view (FOV). So, the light-field display method of modulating chrominance and luminance separately has been proven to achieve high-fidelity display effects.

The coordinated spatial arrangement of organelles within a tissue plane, known as planar cell polarity (PCP), is critical for organ development and function. Gradients of morphogens and their receptors typically set-up PCP, but whether non-molecular cues, akin to phototropism in plants, also play a part remains unknown. Here, we report that basal bodies of newborn photoreceptor cells in the mouse retina are positioned centrally on the apical surface but then move laterally during the first postnatal week, generating cell-intrinsic asymmetry in the retinal plane. After 1 week, when the eyes open, basal bodies of cone cilia, but not rods, become coordinated across the plane to face the center of the retina. We further show that light is essential for cone PCP, triggering a cascade in which cone transducin interacts with the G-protein-signaling modulator protein 2 (GPSM2) to establish PCP. This work identifies a non-canonical PCP pathway initiated by light.

Within the reductionist framework, researchers in the special sciences formulate key terms and concepts and try to explain them with lower-level science terms and concepts. For example, behavioural vision scientists describe contrast perception with a psychometric function, in which the perceived brightness increases logarithmically with the physical contrast of a light patch (the Weber-Fechner law). Visual neuroscientists describe the output of neural circuits with neurometric functions. Intuitively, the key terms from two adjacent scientific domains should map onto each other; for instance, psychometric and neurometric functions may map onto each other. Identifying such mappings has been the very goal of neuroscience for nearly two centuries. Yet mapping behaviour to brain measures has turned out to be difficult. Here, we provide various arguments as to why the conspicuous lack of robust brain-behaviour mappings is rather a rule than an exception. First, we provide an overview of methodological and conceptual issues that may stand in the way of successful brain-behaviour mapping. Second, extending previous theoretical work (Herzog, Doerig and Sachse, 2023), we show that brain-behaviour mapping may be limited by complexity barriers. In this case, reduction may be impossible.

Damage-regulated autophagy modulator 2 (DRAM2) is a homologue of the DRAM family protein, which can induce autophagy process. In the retina, DRAM2 is located to the inner segment of photoreceptors, the apical surface of retinal pigment epithelial (RPE) cells, and the lysosome. Pathogenic variants of DRAM2 lead to autosomal recessive Cone-rod dystrophy 21 (CORD21). Cone-rod dystrophy is characterised by primary cone involvement, or sometimes simultaneous cone and rod loss, thus leading to decreased visual acuity, colour vision deficits, photophobia, and decreased sensitivity of the central visual field. However, the mechanisms underlying DRAM2 related retinal diseases remained unclear. To further explore the role of Dram2 in the retina, we generated Dram2 knockout mice (KO) by CRISPR/Cas-9 technology and demonstrated that expression of DRAM2 was abolished in KO retinas. Dram2 ablation failed to manifest any retinal degenerative phenotypes. Dram2 KO did not exhibit visible defect in photo response and the overt structure of the retinas. Immunostaing analysis using antibodies against cone opsins revealed no detectable loss of cone cells. Moreover, no visible change was observed in the expression and localisation of rhodopsin and other membrane disc proteins in Dram2 KO retinas and no gliosis and apoptosis were detected in KO mice. In summary, these data revealed lack of overt retinal degeneration in Dram2 KO model and emphasized the importance of further investigation of the mechanisms underlying Cone-rod dystrophy 21.

Mutations in the Rhodopsin (RHO) gene are the main cause of autosomal dominant retinitis pigmentosa (adRP), 84% of which are pathogenic gain-of-function point mutations. Treatment strategies for adRP typically involve silencing or ablating the pathogenic allele, while normal RHO protein replacement has no meaningful therapeutic benefit. Here, we present an adenine base editor (ABE)-mediated therapeutic approach for adRP caused by RHO point mutations in vivo. The correctable pathogenic mutations are screened and verified, including T17M, Q344ter, and P347L. Two adRP animal models are created carrying the class 1 (Q344ter) and class 2 (T17M) mutations, and dual AAV-delivered ABE can effectively repair both mutations in vivo. The early intervention of ABE8e efficiently corrects the Q344ter mutation that causes a severe form of adRP, delays photoreceptor death, and restores retinal function and visual behavior. These results suggest that ABE is a promising alternative to treat RHO mutation-associated adRP. Our work provides an effective spacer-mediated point mutation correction therapy approach for dominantly inherited ocular disorders.

New frontiers about retinal cell transplantation for retinal degenerative diseases start from the idea that acting on stem cells can help regenerate retinal layers and establish new synapses among retinal cells. Deficiency or alterations of synaptic input and neurotrophic factors result in trans-neuronal degeneration of the inner retinal cells. Thus, the disruption of photoreceptors takes place. However, even in advanced forms of retinal degeneration, a good percentage of the ganglion cells and the inner nuclear layer neurons remain intact. This phenomenon provides evidence for obtaining retinal circuitry through the transplantation of photoreceptors into the subretinal region. The eye is regarded as an optimal organ for cell transplantation because of its immunological privilege and the relatively small number of cells collaborating to carry out visual activities. The eyeball's immunological privilege, characterized by the suppression of delayed-type hypersensitivity responses in ocular tissues, is responsible for the low rate of graft rejection in transplant patients. The main discoveries highlight the capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells to regenerate damaged retinal regions. Recent progress has shown significant enhancements in transplant procedures and results. The research also explores the ethical ramifications linked to the utilization of stem cells, emphasizing the ongoing issue surrounding ESCs. The analysis centers on recent breakthroughs, including the fabrication of three-dimensional retinal organoids and the innovation of scaffolding for cell transportation. Moreover, researchers are currently assessing the possibility of CRISPR and other advanced gene editing technologies to enhance the outcomes of retinal transplantation. The widespread use of universally recognized safe surgical and imaging methods enables retinal transplantation and monitoring of transplanted cell growth toward the correct location. Currently, most therapy approaches are in the first phases of development and necessitate further research, including both pre-clinical and clinical trials, to attain favorable visual results for individuals suffering from retinal degenerative illnesses.

Sunlight exposure is recognized as a risk factor for the development of age-related macular degeneration (AMD), a common neurodegenerative retinal disease in the elderly. Specifically, the blue light wavelengths within sunlight can negatively impact the physiology of light-sensitive retinal cells, including retinal pigmented epithelium (RPE) and photoreceptors. This review explores blue light-induced retinal degeneration, emphasizing the structural and functional impairments in RPE. The initial section provides a brief overview of blue light's effects on photoreceptors, followed by a comprehensive analysis of its detrimental impact on RPE. In vitro studies reveal that blue light exposure induces morphological alterations and functional impairments in RPE, including reduced phagocytic activity, disrupted secretion of neurotrophic factors, and compromised barrier function. Mechanisms of retinal damage, including oxidative stress, inflammation, lipofuscin accumulation, mitochondrial dysfunction and ER stress in RPE, are also explored. The strengths and limitations of in vitro, animal and ex vivo models for studying blue light exposure are discussed, with recommendations for improving reproducibility in future studies.

Medicinal plants, also known as herbs, have been discovered and utilized in traditional medical practice since prehistoric times. Medicinal plants have been proven rich in thousands of natural products that hold great potential for the development of new drugs. Previously, we reviewed the types of Chinese traditional medicines that a Tang Dynasty monk Jianzhen (Japanese: Ganjin) brought to Japan from China in 742. This article aims to review the origin of Kampo (Japanese traditional medicine), and to present the overview of neurodegenerative diseases and retinitis pigmentosa as well as medicinal plants in some depth. Through the study of medical history of the origin of Kampo, we found that herbs medicines contain many neuroprotective ingredients. It provides us a new perspective on extracting neuroprotective components from herbs medicines to treat neurodegenerative diseases. Retinitis pigmentosa (one of the ophthalmic neurodegenerative diseases) is an incurable blinding disease and has become a popular research direction in global ophthalmology. To date, treatments for retinitis pigmentosa are very limited worldwide. Therefore, we intend to integrate the knowledge and skills from different disciplines, such as medical science, pharmaceutical science and plant science, to take a new therapeutic approach to treat neurodegenerative diseases. In the future, we will use specific active ingredients extracted from medicinal plants to treat retinitis pigmentosa. By exploring the potent bioactive ingredients present in medicinal plants, a valuable opportunity will be offered to uncover novel approaches for the development of drugs which target for retinitis pigmentosa.

Light exposure triggers a range of physiological and behavioural responses that can improve and challenge health and well-being. Insights from laboratory studies have recently culminated in standards and guidelines for measuring and assessing healthy light exposure, and recommendations for healthy light levels. Implicit to laboratory paradigms is a simplistic input-output relationship between light and its effects on physiology. This simplified approach ignores that humans actively shape their light exposure through behaviour. This article presents a novel framework that conceptualises light exposure as an individual behaviour to meet specific, person-based needs. Key to healthy light exposure is shaping behaviour, beyond shaping technology.

INPP5E encodes inositol polyphosphate-5-phosphatase E, an enzyme involved in regulating the phosphatidylinositol (PIP) makeup of the primary cilium membrane. Pathogenic variants in INPP5E hence cause a variety of ciliopathies: genetic disorders caused by dysfunctional cilia. While the majority of these disorders are syndromic, such as the neuronal ciliopathy Joubert syndrome, in some cases patients will present with an isolated phenotype-most commonly non-syndromic retinitis pigmentosa (RP). Here, we report two novel variants in INPP5E identified in two patients with non-syndromic RP: patient 1 with compound heterozygous variants (c.1516C > T, p.(Q506*), and c.847G > A, p.(A283T)) and patient 2 with a homozygous variant (c.1073C > T, p.(P358L)). To determine whether these variants were causative for the phenotype in the patients, automated ciliary phenotyping of patient-derived dermal fibroblasts was performed for percent ciliation, cilium length, retrograde IFT trafficking, and INPP5E localization. In both patients, a decrease in ciliary length and loss of INPP5E localization in the primary cilia were seen. With these molecular findings, we can confirm functionally that the novel variants in INPP5E are causative for the RP phenotypes seen in both patients. Additionally, this study demonstrates the usefulness of utilizing ciliary phenotyping as an assistant in ciliopathy diagnosis and phenotyping.

Smartphone-based colorimetry has been widely applied in clinical analysis, although significant challenges remain in its practical implementation, including the need to consider biases introduced by the ambient imaging environment, which limit its potential within a clinical decision pathway. In addition, most commercial devices demonstrate variability introduced by manufacturer-to-manufacturer differences. Here, we undertake a systematic characterization of the potential imaging interferences that lead to this limited performance in conventional smartphones and, in doing so, provide a comprehensive new understanding of smartphone color imaging. Through derivation of a strongly correlated parameter for sample quantification, we enable real-time imaging, which for the first time, takes the first steps to turning the mobile phone camera into an analytical instrument - irrespective of model, software, and the operating systems used. We demonstrate clinical applicability through the imaging of patients' skin, enabling rapid and convenient diagnosis of cyanosis and measurement of local oxygen concentration to a level that unlocks clinical decision-making for monitoring cardiovascular disease and anemia. Importantly, we show that our solution also accounts for the differences in individuals' skin tones as measured across the Fitzpatrick scale, overcoming potential clinically significant errors in current optical oximetry.

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1-/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1-/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated.

The mammalian retina contains high amounts of metals/metalloid-selenium. Their dyshomeostases are associated with certain retinal diseases. We carried out this bioinformatics study to identify the relationships between putative retinal metal/selenium binding proteins, their molecular functions, and biological processes. Identification of putative mouse metal/selenium binding proteins was based on known binding motifs, domains, patterns, and profiles. Annotations were obtained from Uniprot keywords 'metal binding', 'metal ion co-factors', 'selenium proteins'. Protein functions were estimated by associative frequency with key words in UniProt annotations. The raw data of five mouse proteomics PRIDE datasets (available to date) were downloaded and processed with Mascot against the mouse taxa of Uniprot (SwissProt/Trembl) and MaxQuant (version 1.6.10.43) for qualitative and quantitative datasets, respectively. Clinically relevant variants were evaluated using archives and aggregated information in ClinVar. The 438 proteins common to all the retina proteomics datasets were used to identify over-represented Gene Ontology categories. The putative mouse retinal metal/metalloid binding proteins identified are mainly involved in: (1) metabolic processes (enzymes), (2) homeostasis, (3) transport (vesicle mediated, transmembrane, along microtubules), (4) cellular localization, (5) regulation of signalling and exocytosis, (6) organelle organization, (7) (de)phosphorylation, and (8) complex assembly. Twenty-one proteins were identified as involved in response to light stimulus and/or visual system development. An association of metal ion binding proteins rhodopsin, photoreceptor specific nuclear receptor, calcium binding protein 4 with disease-related mutations in inherited retinal conditions was identified, where the mutations affected an area within or in close proximity to the metal binding site or domain. These findings suggest a functional role for the putative metal/metalloid binding site in retinal proteins in certain retinal disorders.

Tight homeostatic control of cholesterol concentration within the complex tissue microenvironment of the retina is the hallmark of a healthy eye. By contrast, dysregulation of biochemical mechanisms governing retinal cholesterol homeostasis likely contributes to the aetiology and progression of age-related macular degeneration (AMD). While the signalling mechanisms maintaining cellular cholesterol homeostasis are well-studied, a systems-level description of molecular interactions regulating cholesterol balance within the human retina remains elusive. Here, we provide a comprehensive overview of all currently-known molecular-level interactions involved in cholesterol regulation across the major compartments of the human retina, encompassing the retinal pigment epithelium (RPE), photoreceptor cell layer, Müller cell layer and Bruch's membrane. We develop a comprehensive chemical reaction network (CRN) of these interactions, involving 71 molecular species, partitioned into 10 independent subnetworks. These subnetworks collectively ensure robust homeostasis of 14 forms of cholesterol across distinct retinal cellular compartments. We provide mathematical evidence that three independent antithetic integral feedback controllers tightly regulate ER cholesterol in retinal cells, with additional independent mechanisms extending this regulation to other forms of cholesterol throughout the retina. Our novel mathematical model of retinal cholesterol regulation provides a framework for understanding the mechanisms of cholesterol dysregulation in diseased eyes and for exploring potential therapeutic strategies.

Aging is the greatest risk factor for chronic human diseases, including many eye diseases. Geroscience aims to understand the effects of the aging process on these diseases, including the genetic, molecular, and cellular mechanisms that underlie the increased risk of disease over the lifetime. Understanding of the aging eye increases general knowledge of the cellular physiology impacted by aging processes at various biological extremes. Two major diseases, age-related cataract and age-related macular degeneration (AMD) are caused by dysfunction of the lens and retina, respectively. Lens transparency and light refraction are mediated by lens fiber cells lacking nuclei and other organelles, which provides a unique opportunity to study a single aging hallmark, i.e., loss of proteostasis, within an environment of limited metabolism. In AMD, local dysfunction of the photoreceptors/retinal pigmented epithelium/Bruch's membrane/choriocapillaris complex in the macula leads to the loss of photoreceptors and eventually loss of central vision, and is driven by nearly all the hallmarks of aging and shares features with Alzheimer's disease, Parkinson's disease, cardiovascular disease, and diabetes. The aging eye can function as a model for studying basic mechanisms of aging and, vice versa, well-defined hallmarks of aging can be used as tools to understand age-related eye disease.

In AMD, rod-mediated dark adaptation (RMDA) at 5° eccentricity is slower in eyes with subretinal drusenoid deposits (SDDs) than in eyes without. Here we quantified SDD burden using supervised deep learning for comparison to vision and photoreceptor topography.

In persons ≥60 years from the Alabama Study on Early Age-Related Macular Degeneration 2, normal, early AMD, and intermediate AMD eyes were classified by the AREDS nine-step system. A convolutional neural network was trained on 55°-wide near-infrared reflectance images for SDD segmentation. Trained graders annotated ground truth (SDD yes/no). Predicted and true datasets agreed (Dice coefficient, 0.92). Inference was manually proofread using optical coherence tomography. The mean SDD area (mm2) was compared among diagnostic groups (linear regression) and to vision (age-adjusted Spearman correlations). Fundus autofluorescence images were used to mask large vessels in SDD maps.

In 428 eyes of 428 persons (normal, 218; early AMD, 120; intermediate AMD, 90), the mean SDD area differed by AMD severity (P < 0.0001): 0.16 ± 0.87 (normal), 2.48 ± 11.23 (early AMD), 11.97 ± 13.33 (intermediate AMD). Greater SDD area was associated with worse RMDA (r = 0.27; P < 0.0001), mesopic (r = -0.13; P = 0.02) and scotopic sensitivity (r = -0.17; P < 0.001). SDD topography peaked at 5° superior, extended beyond the Early Treatment of Diabetic Retinopathy Study grid and optic nerve, then decreased.

SDD area is associated with degraded rod-mediated vision. RMDA 5° (superior retina) probes where SDD is maximal, closer to the foveal center than the rod peak at 3 to 6 mm (10.4°-20.8°) superior and the further eccentric peak of rod:cone ratio. Topographic data imply that factors in addition to rod density influence SDD formation.

Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation.

We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR.

ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2.

These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Advancements in ocular imaging have significantly broadened our comprehension of mitochondrial retinopathies and optic neuropathies by examining the structural and pathological aspects of the retina and optic nerve in these conditions. This article aims to review the prominent imaging characteristics associated with mitochondrial retinopathies and optic neuropathies, aiming to deepen our insight into their pathogenesis and clinical features. Preceding this exploration, the article provides a detailed overview of the crucial genetic and clinical features, which is essential for the proper interpretation of in vivo imaging. More importantly, we will provide a critical analysis on how these imaging modalities could serve as biomarkers for characterization and monitoring, as well as in guiding treatment decisions. However, these imaging methods have limitations, which will be discussed along with potential strategies to mitigate them. Lastly, the article will emphasize the potential advantages and future integration of imaging techniques in evaluating patients with mitochondrial eye disorders, considering the prospects of emerging gene therapies.

We previously reported that 2-arachidonoylglycerol (2-AG) synthesis by diacylglycerol lipase (DAGL) and lysophosphatidate phosphohydrolase (LPAP) and hydrolysis by monoacylglycerol lipase (MAGL) in rod outer segments (ROS) from bovine retina were differently modified by light applied to the retina. Based on these findings, the aim of the present research was to evaluate whether 2-AG metabolism could be modulated by proteins involved in the visual process. To this end, ROS kept in darkness (DROS) or obtained in darkness and then subjected to light (BROS) were treated with GTPγS and GDPβS, or with low and moderate ionic strength buffers for detaching soluble and peripheral proteins, or soluble proteins, respectively. Only DAGL activity was stimulated by the application of light to the ROS. GTPγS-stimulated DAGL activity in DROS reached similar values to that observed in BROS. The studies using different ionic strength show that (1) the highest decrease in DROS DAGL activity was observed when both phosphodiesterase (PDE) and transducin α (Tα) are totally membrane-associated; (2) the decrease in BROS DAGL activity does not depend on PDE association to membrane, and that (3) MAGL activity decreases, both in DROS and BROS, when PDE is not associated to the membrane. Our results indicate that the bioavailability of 2-AG under light conditions is favored by G protein-stimulated increase in DAGL activity and hindered principally by Tα/PDE association with the ROS membrane, which decreases DAGL activity.

The functionality of photoreceptors, rods, and cones is highly dependent on their outer segments (POS), a cellular compartment containing highly organized membranous structures that generate biochemical signals from incident light. While POS formation and degeneration are qualitatively assessed on microscopy images, reliable methodology for quantitative analyses is still limited. Here, we developed methods to quantify POS (QuaPOS) maturation and quality on retinal sections using automated image analyses. POS formation was examined during the development and in adulthood of wild-type mice via light microscopy (LM) and transmission electron microscopy (TEM). To quantify the number, size, shape, and fluorescence intensity of POS, retinal cryosections were immunostained for the cone POS marker S-opsin. Fluorescence images were used to train the robust classifier QuaPOS-LM based on supervised machine learning for automated image segmentation. Characteristic features of segmentation results were extracted to quantify the maturation of cone POS. Subsequently, this quantification method was applied to characterize POS degeneration in "cone photoreceptor function loss 1" mice. TEM images were used to establish the ultrastructural quantification method QuaPOS-TEM for the alignment of POS membranes. Images were analyzed using a custom-written MATLAB code to extract the orientation of membranes from the image gradient and their alignment (coherency). This analysis was used to quantify the POS morphology of wild-type and two inherited retinal degeneration ("retinal degeneration 19" and "rhodopsin knock-out") mouse lines. Both automated analysis technologies provided robust characterization and quantification of POS based on LM or TEM images. Automated image segmentation by the classifier QuaPOS-LM and analysis of the orientation of membrane stacks by QuaPOS-TEM using fluorescent or TEM images allowed quantitative evaluation of POS formation and quality. The assessments showed an increase in POS number, volume, and membrane coherency during wild-type postnatal development, while a decrease in all three observables was detected in different retinal degeneration mouse models. All the code used for the presented analysis is open source, including example datasets to reproduce the findings. Hence, the QuaPOS quantification methods are useful for in-depth characterization of POS on retinal sections in developmental studies, for disease modeling, or after therapeutic interventions affecting photoreceptors.

Activated microglia play an important role in driving photoreceptor degeneration-associated neuroinflammation in the retina. Controlling pro-inflammatory activation of microglia holds promise for mitigating the progression of photoreceptor degeneration. Our previous study has demonstrated that pre-light damage treatment of hyperoside, a naturally occurring flavonol glycoside with antioxidant and anti-inflammatory activities, prevents photooxidative stress-induced photoreceptor degeneration and neuroinflammatory responses in the retina. However, the direct impact of hyperoside on microglia-mediated neuroinflammation during photoreceptor degeneration remains unknown. Upon verifying the anti-inflammatory effects of hyperoside in LPS-stimulated BV-2 cells, our results here further demonstrated that post-light damage hyperoside treatment mitigated the loss of photoreceptors and attenuated the functional decline of the retina. Meanwhile, post-light damage hyperoside treatment lowered neuroinflammatory responses and dampened microglial activation in the illuminated retinas. With respect to microglial activation, hyperoside mitigated the pro-inflammatory responses in DNA-stimulated BV-2 cells and lowered DNA-stimulated production of 2'3'-cGAMP in BV-2 cells. Moreover, hyperoside was shown to directly interact with cGAS and suppress the enzymatic activity of cGAS in a cell-free system. In conclusion, the current study suggests for the first time that the DNA sensor cGAS is a direct target of hyperoside. Hyperoside is effective at mitigating DNA-stimulated cGAS-mediated pro-inflammatory activation of microglia, which likely contributes to the therapeutic effects of hyperoside at curtailing neuroinflammation and alleviating neuroinflammation-instigated photoreceptor degeneration.

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.

The retina consists of various cell types arranged in eight cell layers and two membranes that originate from the neuroectodermal cells. In this study, the timing of differentiation and distribution of the cellular components and the layers of the rabbit retina are investigated using light and electron microscopy and immunohistochemical techniques. There were 32 rabbit embryos and 12 rabbits used. The rabbit retina begins its prenatal development on the 10th day of gestation in the form of optic cup. The process of neuro- and gliogenesis occurs in several stages: In the first stage, the ganglionic cells are differentiated at the 15th day. The second stage includes the differentiation of Muller, amacrine, and cone cells on the 23rd day. The differentiation of bipolar, horizontal, and rod cells and formation of the inner segments of the photoreceptors consider the late stage that occurs by the 27th and 30th day of gestation. On the first week of age postnatally, the outer segments of the photoreceptors are developed. S100 protein is expressed by the Muller cells and its processes that traverse the retina from the outer to the inner limiting membranes. Calretinin is intensely labeled within the amacrine and displaced amacrine cells. Ganglionic cells exhibited moderate immunoreactivity for calretinin confined to their cytoplasm and dendrites. In conclusion, all stages of neuro- and gliogenesis of the rabbit retina occur during the embryonic period. Then, the retina continues its development postnatally by formation of the photoreceptor outer segments and all layers of the retina become established. RESEARCH HIGHLIGHTS: The aim of this study is to investigate the morphogenesis of the rabbit retina during pre- and postnatal life. The primordia of the retina could be observed in the form of the optic cup. The ganglionic cells are the first cells to differentiate, while the photoreceptor cells are the last. S100 protein is expressed by the Muller cells and its processes. Calretinin is intensely labeled in the amacrine and displaced amacrine cells and moderately expressed in the cytoplasm and dendrites of ganglionic cells.

The retina is part of the central nervous system specialized for vision. Inherited retinal diseases (IRD) are a group of clinically and genetically heterogenous disorders that lead to progressive vision impairment or blindness. Although each disorder is rare, IRD accumulatively cause blindness in up to 5.5 million individuals worldwide. Currently, the pathophysiological mechanisms of IRD are not fully understood and there are limited treatment options available. Most IRD are caused by degeneration of light-sensitive photoreceptors. Genetic mutations that abrogate the structure and/or function of photoreceptors lead to visual impairment followed by blindness caused by loss of photoreceptors. In healthy retina, photoreceptors structurally and functionally interact with retinal pigment epithelium (RPE) and Müller glia (MG) to maintain retinal homeostasis. Multiple IRD with photoreceptor degeneration as a major phenotype are caused by mutations of RPE- and/or MG-associated genes. Recent studies also reveal compromised MG and RPE caused by mutations in ubiquitously expressed ciliary genes. Therefore, photoreceptor degeneration could be a direct consequence of gene mutations and/or could be secondary to the dysfunction of their interaction partners in the retina. This review summarizes the mechanisms of photoreceptor-RPE/MG interaction in supporting retinal functions and discusses how the disruption of these processes could lead to photoreceptor degeneration, with an aim to provide a unique perspective of IRD pathogenesis and treatment paradigm. We will first describe the biology of retina and IRD and then discuss the interaction between photoreceptors and MG/RPE as well as their implications in disease pathogenesis. Finally, we will summarize the recent advances in IRD therapeutics targeting MG and/or RPE.

Rod and cone photoreceptors are named for the distinct morphologies of their outer segment organelles, which are either cylindrical or conical, respectively. The morphologies of the stacked disks that comprise the rod and cone outer segments also differ: rod disks are completely sealed and are discontinuous from the plasma membrane, while cone disks remain partially open to the extracellular space. These morphological differences between photoreceptor types are more prominent in non-mammalian vertebrates, whose cones typically possess a greater proportion of open disks and are more tapered in shape. In mammals, the tetraspanin prph2 generates and maintains the highly curved disk rim regions by forming extended oligomeric structures with itself and a structurally similar paralog, rom1. Here we determined that in addition to these two proteins, there is a third Prph2 family paralog in most non-mammalian vertebrate species, including X. laevis: Glycoprotein 2-like protein or "Gp2l". A survey of multiple genome databases revealed a single invertebrate Prph2 'pro-ortholog' in Amphioxus, several echinoderms and in a diversity of protostomes indicating an ancient divergence from other tetraspanins. Based on phylogenetic analysis, duplication of the vertebrate predecessor likely gave rise to the Gp2l and Prph2/Rom1 clades, with a further duplication distinguishing the Prph2 and Rom1 clades. Mammals have lost Gp2l and their Rom1 has undergone a period of accelerated evolution such that it has lost several features that are retained in non-mammalian vertebrate Rom1. Specifically, Prph2, Gp2l and non-mammalian Rom1 encode proteins with consensus N-linked glycosylation and outer segment localization signals; mammalian rom1 lacks these motifs. We determined that X. laevis gp2l is expressed exclusively in cones and green rods, while X. laevis rom1 is expressed exclusively in rods, and prph2 is present in both rods and cones. The presence of three Prph2-related genes with distinct expression patterns as well as the rapid evolution of mammalian Rom1, may contribute to the more pronounced differences in morphology between rod and cone outer segments and rod and cone disks observed in non-mammalian versus mammalian vertebrates.

Retinal degenerative diseases, including glaucoma, age-related macular degeneration, diabetic retinopathy, and a broad range of inherited retinal diseases, are leading causes of irreversible vision loss and blindness. Gene therapy is a promising and fast-growing strategy to treat both monogenic and multifactorial retinal disorders. Vectors for gene delivery are crucial for efficient and specific transfer of therapeutic gene(s) into target cells. AAV vectors are ideal for retinal gene therapy due to their inherent advantages in safety, gene expression stability, and amenability for directional engineering. The eye is a highly compartmentalized organ composed of multiple disease-related cell types. To determine a suitable AAV vector for a specific cell type, the route of administration and choice of AAV variant must be considered together. Here, we provide a brief overview of AAV vectors for gene transfer into important ocular cell types, including retinal pigment epithelium cells, photoreceptors, retinal ganglion cells, Müller glial cells, ciliary epithelial cells, trabecular meshwork cells, vascular endothelial cells, and pericytes, via distinct injection methods. By listing suitable AAV vectors in basic research and (pre)clinical studies, we aim to highlight the progress and unmet needs of AAV vectors in retinal gene therapy.

Expansion microscopy (ExM) is a highly effective technique for super-resolution fluorescence microscopy that enables imaging of biological samples beyond the diffraction limit with conventional fluorescence microscopes. Despite the development of several enhanced protocols, ExM has not yet demonstrated the ability to achieve the precision of nanoscopy techniques such as Single Molecule Localization Microscopy (SMLM). Here, to address this limitation, we have developed an iterative ultrastructure expansion microscopy (iU-ExM) approach that achieves SMLM-level resolution. With iU-ExM, it is now possible to visualize the molecular architecture of gold-standard samples, such as the eight-fold symmetry of nuclear pores or the molecular organization of the conoid in Apicomplexa. With its wide-ranging applications, from isolated organelles to cells and tissue, iU-ExM opens new super-resolution avenues for scientists studying biological structures and functions.

Mucopolysaccharidoses (MPSs) make up a group of lysosomal storage diseases characterized by the aberrant accumulation of glycosaminoglycans throughout the body. Patients with MPSs display various signs and symptoms, such as retinopathy, which is also observed in patients with MPS II. Unfortunately, retinal disorders in MPS II are resistant to conventional intravenous enzyme-replacement therapy because the blood-retinal barrier (BRB) impedes drug penetration. In this study, we show that a fusion protein, designated pabinafusp alfa, consisting of an antihuman transferrin receptor antibody and iduronate-2-sulfatase (IDS), crosses the BRB and reaches the retina in a murine model of MPS II. We found that retinal function, as assessed by electroretinography (ERG) in MPS II mice, deteriorated with age. Early intervention with repeated intravenous treatment of pabinafusp alfa decreased heparan sulfate deposition in the retina, optic nerve, and visual cortex, thus preserving or even improving the ERG response in MPS II mice. Histological analysis further revealed that pabinafusp alfa mitigated the loss of the photoreceptor layer observed in diseased mice. In contrast, recombinant nonfused IDS failed to reach the retina and hardly affected the retinal disease. These results support the hypothesis that transferrin receptor-targeted IDS can penetrate the BRB, thereby ameliorating retinal dysfunction in MPS II.

The possible applications for human retinal organoids (HROs) derived from human induced pluripotent stem cells (hiPSC) rely on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare, and optimize organoid protocols are starting to be established, but are not yet complete. To advance this, we explored the efficiency and reliability of a differentiation method, called CYST protocol, that facilitates retina generation by forming neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines which reproducibly generated HROs. Histological and ultrastructural analyses indicate that HRO differentiation and maturation are regulated. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Although previous reports have shown that HROs in several other protocols contain a rather low number of cones, HROs from the CYST protocol are consistently richer in cones and with a comparable ratio of cones, rods, and Müller glia. To provide further insight into HRO cell composition, we studied single cell RNA sequencing data and applied CaSTLe, a transfer learning approach. Additionally, we devised a potential strategy to systematically evaluate different organoid protocols side-by-side through parallel differentiation from the same hiPSC batches: In an explorative study, the CYST protocol was compared to a conceptually different protocol based on the formation of cell aggregates from single hiPSCs. Comparing four hiPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the CYST protocol provided a higher HRO yield. So far, our data suggest that CYST-derived HROs remained stable up to at least day 200, while single hiPSC-derived HROs showed spontaneous pathologic changes by day 200. Overall, our data provide insights into the efficiency, reproducibility, and stability of the CYST protocol for generating HROs, which will be useful for further optimizing organoid systems, as well as for basic and translational research applications.

Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic.

To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa.

T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat.

Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

Enhancers function with a basal promoter to control the transcription of target genes. Enhancer regulatory activity is often studied using reporter-based transgene assays. However, unmatched results have been reported when selected enhancers are silenced in situ. In this study, using genomic deletion analysis in mice, we investigated the roles of two previously identified enhancers and the promoter of the Rho gene that codes for the visual pigment rhodopsin. The Rho gene is robustly expressed by rod photoreceptors of the retina, and essential for the subcellular structure and visual function of rod photoreceptors. Mutations in RHO cause severe vision loss in humans. We found that each Rho regulatory region can independently mediate local epigenomic changes, but only the promoter is absolutely required for establishing active Rho chromatin configuration and transcription and maintaining the cell integrity and function of rod photoreceptors. To our surprise, two Rho enhancers that enable strong promoter activation in reporter assays are largely dispensable for Rho expression in vivo. Only small and age-dependent impact is detectable when both enhancers are deleted. Our results demonstrate context-dependent roles of enhancers and highlight the importance of studying functions of cis-regulatory regions in the native genomic context.

Photoreceptors in the retina are highly specialized neurons with photosensitive molecules in the outer segment that transform light into chemical and electrical signals, and these signals are ultimately relayed to the visual cortex in the brain to form vision. Photoreceptors are composed of rods and cones. Rods are responsible for dim light vision, whereas cones are responsible for bright light, color vision, and visual acuity. Photoreceptors undergo progressive degeneration over time in many hereditary and age-related retinal diseases. Despite the remarkable heterogeneity of disease-causing genes, environmental factors, and pathogenesis, the progressive death of rod and cone photoreceptors ultimately leads to loss of vision/blindness. There are currently no treatments available for retinal degeneration. Cyclic guanosine 3', 5'-monophosphate (cGMP) plays a pivotal role in phototransduction. cGMP governs the cyclic nucleotide-gated (CNG) channels on the plasma membrane of the photoreceptor outer segments, thereby regulating membrane potential and signal transmission. By gating the CNG channels, cGMP regulates cellular Ca2+ homeostasis and signal transduction. As a second messenger, cGMP activates the cGMP-dependent protein kinase G (PKG), which regulates numerous targets/cellular events. The dysregulation of cGMP signaling is observed in varieties of photoreceptor/retinal degenerative diseases. Abnormally elevated cGMP signaling interferes with various cellular events, which ultimately leads to photoreceptor degeneration. In line with this, strategies to reduce cellular cGMP signaling result in photoreceptor protection in mouse models of retinal degeneration. The potential mechanisms underlying cGMP signaling-induced photoreceptor degeneration involve the activation of PKG and impaired Ca2+ homeostasis/Ca2+ overload, resulting from overactivation of the CNG channels, as well as the subsequent activation of the downstream cellular stress/death pathways. Thus, targeting the cellular cGMP/PKG signaling and the Ca2+-regulating pathways represents a significant strategy for photoreceptor protection in retinal degenerative diseases.

Transparent polymers and plastics are used to create molded parts and films for many applications. The colors of these products are of great importance for the suppliers, manufacturers, and end-users. However, for simplicity of the processing, the plastics are produced in the form of small pellets or granules. The predictive measurement of the color of such materials is a challenging process and needs consideration of a complex set of factors. A combination of color measurement systems in transmittance and reflectance modes need to be used for such materials, along with the techniques for minimizing the artifacts based on surface texture and particle sizes. This article provides an extensive overview and discussion of the various factors that can affect the perceptive colors and the methods used for the characterization of the colors and minimizing the measuring artifacts.

c-Jun activation domain binding protein-1 (JAB1) is a multifunctional regulator that plays vital roles in diverse cellular processes. It regulates AP-1 transcriptional activity and also acts as the fifth component of the COP9 signalosome complex. While JAB1 is considered an oncoprotein that triggers tumor development, recent studies have shown that it also functions in neurological development and disorders. In this review, we summarize the general features of the JAB1 gene and protein, and present recent updates on the regulation of JAB1 expression. Moreover, we also highlight the functional roles and regulatory mechanisms of JAB1 in neurodevelopmental processes such as neuronal differentiation, synaptic morphogenesis, myelination, and hair cell development and in the pathogenesis of some neurological disorders such as Alzheimer's disease, multiple sclerosis, neuropathic pain, and peripheral nerve injury. Furthermore, current challenges and prospects are discussed, including updates on drug development targeting JAB1.

Organisms have developed different features to capture or sense sunlight. Vertebrates have evolved specialized organs (eyes) which contain a variety of photosensor cells that help them to see the light to aid orientation. Opsins are major photoreceptors found in the vertebrate eye. Fungi, with more than five million estimated members, represent an important clade of living organisms which have important functions for the sustainability of life on our planet. Light signalling regulates a range of developmental and metabolic processes including asexual sporulation, sexual fruit body formation, pigment and carotenoid production and even production of secondary metabolites. Fungi have adopted three groups of photoreceptors: (I) blue light receptors, White Collars, vivid, cryptochromes, blue F proteins and DNA photolyases, (II) red light sensors, phytochromes and (III) green light sensors and microbial rhodopsins. Most mechanistic data were elucidated on the roles of the White Collar Complex (WCC) and the phytochromes in the fungal kingdom. The WCC acts as both photoreceptor and transcription factor by binding to target genes, whereas the phytochrome initiates a cascade of signalling by using mitogen-activated protein kinases to elicit its cellular responses. Although the mechanism of photoreception has been studied in great detail, fungal photoreception has not been compared with vertebrate vision. Therefore, this review will mainly focus on mechanistic findings derived from two model organisms, namely Aspergillus nidulans and Neurospora crassa and comparison of some mechanisms with vertebrate vision. Our focus will be on the way light signalling is translated into changes in gene expression, which influences morphogenesis and metabolism in fungi.

The fatty acid composition of photoreceptor outer segment (POS) phospholipids diverges from other membranes, being highly enriched in polyunsaturated fatty acids (PUFAs). The most abundant PUFA is docosahexaenoic acid (DHA, C22:6n-3), an omega-3 PUFA that amounts to over 50% of the POS phospholipid fatty acid side chains. Interestingly, DHA is the precursor of other bioactive lipids such as elongated PUFAs and oxygenated derivatives. In this review, we present the current view on metabolism, trafficking and function of DHA and very long chain polyunsaturated fatty acids (VLC-PUFAs) in the retina. New insights on pathological features generated from PUFA deficient mouse models with enzyme or transporter defects and corresponding patients are discussed. Not only the neural retina, but also abnormalities in the retinal pigment epithelium are considered. Furthermore, the potential involvement of PUFAs in more common retinal degeneration diseases such as diabetic retinopathy, retinitis pigmentosa and age-related macular degeneration are evaluated. Supplementation treatment strategies and their outcome are summarized.

Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD), characterized by atrophic lesions that first start in the outer retina and progressively expand to cover the macula and the fovea, the center of the macula, leading to irreversible loss of vision over time. GA is distinct from wet or neovascular AMD (nAMD), the other form of advanced AMD. Neovascular AMD is characterized by new invading leaky blood vessels in the macula that can lead to acute vision loss. GA and nAMD may coexist in the same eye. The underlying pathophysiology of GA is complex and thought to involve chronic inflammation due to overactivation of the complement system that leads to the loss of photoreceptors, retinal pigment epithelium (RPE), and the underlying choriocapillaris. The disappearance of these structures appears as sharply demarcated atrophic lesions that are typical of GA. Researchers have reported about 1 million reported cases of GA in the United States, and about 160,000 cases occur per year. The most important risk factors for GA are increasing age and family history. Diagnosis of GA is usually made by using multimodal imaging techniques. Lesions associated with GA are highly heterogeneous, and the growth rate may differ from patient to patient. Despite the progressive nature of GA, the fovea may be spared until much later in the disease, thereby retaining central vision in patients. With time, atrophic lesions may progressively grow to involve the fovea, thereby severely impairing central vision. Vision loss can happen rapidly once the lesions reach the fovea. However, even without the involvement of the fovea, ongoing vision impairment impacting daily life may be present. Median time from GA not involving the center of the fovea (without subfoveal involvement) to GA with lesion boundary affecting the foveal center (subfoveal involvement) ranges from 1.4 to 2.5 years. GA can greatly impact patients' functioning and quality of life and limit their independence by interfering with activities of daily living, including difficulties with reading, driving, watching television, recognizing faces, and being unable to do household chores. No treatments have been available until intravitreal pegcetacoplan was recently approved by the US Food and Drug Administration for GA secondary to AMD. DISCLOSURES: Dr Bakri serves as a consultant to Apellis Pharmaceuticals, as well as AbbVie, Adverum, Eyepoint, iLumen, Iveric Bio, Genentech, Novartis, Outlook Therapeutics, Pixium, Regeneron, Roche, and Regenxbio. Drs Sharp, Luo, and Sarda are employees of Apellis Pharmaceuticals. Dr Bektas and Ms Khan are employees of RTI Health Solutions. Apellis developed and led the concept design of this publication, review and interpretation, approval, and decision to publish. This research was developed under a research contract between RTI Health Solutions and Apellis Pharmaceuticals and was funded by Apellis Pharmaceuticals. This supplement is to describe the disease of geographic atrophy and was funded by Apellis. Apellis Pharmaceuticals has developed Syfovre (pegcetacoplan), the first and only treatment for geographic atrophy.

Light scattering and inability to uniformly expose the cuvette contents to an incident light beam are significant limitations of traditional spectrophotometers. The first of these drawbacks limits their usefulness in studies of turbid cellular and tissue suspensions; the second limits their use in photodecomposition studies. Our strategy circumvents both problems. Although we describe its potential usefulness in vision sciences, application of spherical integrating cuvettes has broad application. Absorbance spectra of turbid bovine rod outer segments and dispersed living frog retina were studied using a standard single-pass 1 cm cuvettes, or a spherical integrating cuvette (DeSa Presentation Chamber, DSPC). The DSPC was mounted on an OLIS Rapid Scanning Spectrophotometer configured to generate 100 spectral scans/sec. To follow rhodopsin bleaching kinetics in living photoreceptors, portions of dark-adapted frog retina were suspended in the DSPC. The incoming spectral beam at 2 scans/sec entered the chamber through a single port. Separate ports contained a 519 nm light emitting diode (LED), or window to the photomultiplier tube. The surface of the DSPC was coated with a highly reflective coating allowing the chamber to act as a multi-pass cuvette. The LED is triggered to flash and the PMT shutter temporarily closed during a "Dark-Interval" between each spectral scan. By interleafing scans with LED pulses, spectra changes can be followed in real time. Kinetic analysis of the 3-dimensional data was performed by Singular Value Decomposition. For crude bovine rod outer segment suspensions, the 1 cm single-pass traditional cuvette gave non-informative spectra dominated by high absorbances and Rayleigh scattering. In contrast, spectra generated using the DSPC showed low overall absorbance with peaks at 405 and 503 nm. The later peak disappeared with exposure to white light in presence of 100 mM hydroxylamine. For the dispersed living retinal, the sample was pulsed at 519 nm between the spectra. The 495 nm rhodopsin peak gradually reduced in size concomitant with the emergence of a 400 nm peak, probably representing Meta II. A conversion mechanism of two species, A → B with rate constant of 0.132 sec^-1 was fit to the data. To our knowledge this is the first application of integrating sphere technology to retinal spectroscopy. Remarkably, the spherical cuvette designed for total internal reflectance to produce diffused light was efffectively immune to light scattering. Furthermore, the higher effective path length enhanced sensitivity and could be accounted for mathematically allowing determination of absorbance/cm. The approach, which complements the use of the CLARiTy RSM 1000 for photodecomposition studies (Gonzalez-Fernandez et al. Mol Vis 2016, 22:953), may facilitate studies of metabolically active photoreceptor suspensions or whole retinas in physiological assays.

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye.