Photoreceptor Outer Segments Research Articles

EVALUATION OF THE NAVIGATION AND PATTERN-TECHNOLOGIES EFFICACY IN THE TREATMENT OF PROLIFERATIVE DIABETIC RETINOPATHY

Purpose: to evaluate the structural changes of the retina after panretinal laser coagulation of the retina in single coagulate mode, pattern and navigation technologies with lasers of different wavelengths and exposures. Materials and methods of research. A prospective study included two main stages, 78 patients with type 2 diabetes mellitus, with the proliferative stage of diabetic retinopathy, who underwent panretinal laser coagulation (PRC). 4 groups of patients were formed. At the first stage, the quality of coagulate formation, structural changes in the surrounding retina and changes in light sensitivity before and after laser coagulation were evaluated. At the second stage, the efficacy of retinal laser coagulation was studied depending on the laser coagulation technology. Optical coherence tomography RTVue XR Avanti (Optovue, USA), Canon was included in the diagnostic examination. The structural changes of the retina were evaluated before and after laser exposure. A study of the light sensitivity of the central parts of the retina was conducted using the Octopus 900 HAAG-STREIT perimeter (Switzerland). The severity of the pain syndrome at the time of the operation was assessed using a visual analog scale 30 minutes after the operationResults. The formation of a full-fledged chorioretinal scar was observed with all types of laser exposure. It has been proven that a laser with a wavelength of 577 nm with an exposure of 20 ms has a minimal damaging effect, as evidenced by the absence of damage to the outer boundary membrane, outer segments of photoreceptors according to OCT data, as well as the absence of depression of average light sensitivity. According to the results of the pain perception study, it was found that patients who underwent coagulation on the Navilas 577s laser system in automatic mode (exposure of 20 msec) practically did not experience pain.Conclusions. An assessment of the structural changes in the retina revealed the least damaging effect on the retinal tissue of laser coagulation with a wavelength of 577 nm with an exposure of 20 msec using pattern and navigation technologies, which allowed the photosensitivity of the peripheral parts of the retina to be maintained at the initial level. When using pattern and navigation technologies, the side effects of surgery (pain, glare,) are not pronounced, which makes panretinal laser coagulation comfortable and increases the patient's commitment to this type of treatment.

Phenotype and genetic spectrum of six Indian patients with bestrophinopathy

Abstract: The aim of this study is to describe genotype and phenotype of patients with bestrophinopathy. The case records were reviewed retrospectively, findings of multimodal imaging such as color fundus photograph, optical coherence tomography (OCT), fundus autofluorescence, electrophysiological, and genetic tests were noted. Twelve eyes of six patients from distinct Indian families with molecular diagnosis were enrolled. Exon 4 of BEST1 was mutated in 3 cases, while exons 2, 3, and 7 in others. Deletion is seen in Exon 7 and missense mutation in other exons. Sporadic autosomal dominant and recessive inheritance was observed in these families. Two patients had primary angle closure glaucoma with a history of consanguineous marriage and glaucoma in the family. Based on our findings, multifocal vitelliform subretinal deposits were the most common fundus finding in patients with autosomal recessive mutation while macular vitelliform lesion was seen with sporadic or autosomal dominant mutation; however, cosegregation analysis was not done. Baseline OCT showed macular and extramacular subretinal exudates, subretinal fluid, intraretinal cystic and schitic spaces, and thickened photoreceptors outer segment tips. Two patients developed abnormal vasculature and focal choroidal excavation in OCT. A severe reduction in the electro-oculogram Ardens ratio was noted while electroretinography was normal. Bestrophinopathy has a varied presentation with complex genotype-phenotype relationships. OCT is a noninvasive tool for monitoring and prognostication. Genetic testing of other family members should be facilitated.

Single-Cell Transcriptomic Dataset of RPGR-associated Retinitis Pigmentosa Patient-Derived Retinal Organoids

X-linked retinitis pigmentosa (XLRP) is a severe hereditary retinal disorder marked by progressive vision loss due to photoreceptor dysfunction. The retinitis pigmentosa GTPase regulator (RPGR) gene, responsible for most XLRP cases, encodes a protein crucial for the transport of visual signal proteins between the photoreceptor inner and outer segments. However, the mechanism of RPGR mutation causing photoreceptor disorder is not clear and effective treatments remain elusive. This study utilized retinal organoids (ROs) derived from normal and RPGR-mutant human induced pluripotent stem cells (hiPSC) at four developmental stages (40, 90, 150, and 200 days). Single-cell RNA sequencing (scRNA-seq) was conducted on 71,096 cells, including 33,839 cells from the control group and 37,257 cells from the RPGR group. Key retinal cell types were identified and the obtained scRNAseq dataset was validated reliable and high -quality. This study has provided data resources and references for exploring the mechanism of RPGR-related retinal degeneration and support the development of targeted therapies.

Predictive risk scores for visual prognosis after photodynamic therapy for central serous chorioretinopathy.

To comprehensively evaluate baseline characteristics of patients with central serous chorioretinopathy (CSC) and develop predictive risk scores to identify visual prognosis. This single-institute, retrospective cohort study included 144 eyes of 144 patients with CSC who underwent photodynamic therapy and achieved serous retinal detachment resolution. We developed and assessed the performance of several risk scores for best-corrected visual acuity (BCVA) outcomes six months post-treatment: i) BCVA improvement (≤-1.0 logMAR), and ii) BCVA deterioration (≥+ 1.0 logMAR). The BCVA improvement models used photoreceptor outer segment thickness, loss of photoreceptor outer segment, and neurosensory retinal thickness (NSRT), while the BCVA deterioration models included outer nuclear layer thickness and NSRT. The BCVA improvement models demonstrated a corrected area under the curve (AUC) of 0.786 (95% confidence interval [CI]: 0.699-0.864), with 80.4% sensitivity, and 71.2% specificity. The BCVA deterioration models achieved a corrected AUC of 0.864 (95% CI: 0.742-0.958), with 85.7% sensitivity, and 83.5% specificity. The predictive models for CSC exhibited favorable performance in predicting individual visual prognoses. A thinner outer nuclear layer may be associated with BCVA deterioration, whereas preservation of the photoreceptor outer segment may be correlated with BCVA improvement. WHAT IS KNOWN : Pre-treatment best-corrected visual acuity, thickness of each sensory retinal layer, time fromonset to treatment, and macular atrophy were each found to be associated with visualprognosis for patients with central serous chorioretinopathy (CSC). The current study comprehensively assessed potential prognostic factors and precisely identified individual likelihood of visual prognosis. The study found that different regions of the sensory retina were associated with either worsening or improving visual acuity. Accurately predicting visual outcomes after photodynamic therapy for CSC would help healthcare providers create personalized treatment plans and enable patients to make informed decisions about their treatment based on their expected visual results.

Complement C3 knockout protects photoreceptors in the sodium iodate model

Complement factor 3 (C3) has emerged as a primary therapeutic target in age-related macular degeneration (AMD) supported by genetic, histologic, and clinical trial evidence. Yet, the site(s) of action are unclear. The purpose of this study was to test the effect of C3 knockout on photoreceptors and retinal pigment epithelial cells (RPE) in the sodium iodate (NaIO3) model, which mirrors some features of AMD. C3−/− and WT mice, both on a C57Bl/6J background, were injected intraperitoneally with 25 mg/kg NaIO3. Electroretinography and optical coherence tomography were performed 7 days later to assess retinal function and structure, respectively. Then, mice were euthanized for retinal immunohistochemistry, quantitative real-time PCR and enzyme-linked immunosorbent assays. NaIO3 increased C3 protein levels in the neural retina but not RPE. WT but not C3−/− mice showed NaIO3-induced iC3b deposition on photoreceptor outer segments. C3−/− mice were partially protected against photoreceptor layer thinning. There was partial preservation of rod and cone function in the C3−/− group. Neither RPE structure nor function was protected. These results suggest outer segment opsonization contributes to photoreceptor death in this model, and that targeting C3 can protect photoreceptor structure and function when RPE cells are stressed.

Near-Infrared Autofluorescence: Early Detection of Retinal Pigment Epithelial Alterations in Inherited Retinal Dystrophies.

Near-infrared autofluorescence (NIA) is a non-invasive retinal imaging technique used to examine the retinal pigment epithelium (RPE) based on the autofluorescence of melanin. Melanin has several functions within RPE cells. It serves as a protective antioxidative factor and is involved in the phagocytosis of photoreceptor outer segments. Disorders affecting the photoreceptor-RPE complex result in alterations of RPE cells which are detectable by alterations of NIA. NIA allows us to detect early alterations in various chorioretinal disorders, frequently before they are ophthalmoscopically visible and often prior to alterations in lipofuscin-associated fundus autofluorescence (FAF) or optical coherence tomography (OCT). Although NIA and FAF relate to disorders affecting the RPE, the findings for both imaging methods differ and the area involved has been demonstrated to be larger in NIA compared to FAF in several disorders, especially inherited retinal dystrophies (IRDs), indicating that NIA detects earlier alterations compared to FAF. Foveal alterations can be much more easily detected using NIA compared to FAF. A reduced subfoveal NIA intensity is the earliest sign of autosomal dominant Best disease, when FAF and OCT are still normal. In other IRDs, a preserved subfoveal NIA intensity is associated with good visual acuity. So far, the current knowledge on NIA in IRD has been presented in multiple separate publications but has not been summarized in an overview. This review presents the current knowledge on NIA in IRD and demonstrates NIA biomarkers.

Genetic and Cellular Basis of Impaired Phagocytosis and Photoreceptor Degeneration in CLN3 Disease.

CLN3 Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease induced pluripotent stem cell-RPE cells show defective phagocytosis of photoreceptor outer segment (POS). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect. Isogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9-mediated biallelic deletion of exons 7 and 8. A transgenic CLN3Δ7-8/Δ7-8 (CLN3) Yucatan miniswine was also used to study the impact of CLN3Δ7-8/Δ7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis of CLN3Δ7-8/Δ7-8 and wild-type miniswine eyes were carried out at 6, 36, or 48 months of age. CLN3Δ7-8/Δ7-8 RPE (CLN3 RPE) displayed decreased POS binding and consequently decreased uptake of POS compared with isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosed CLN3Δ7-8/Δ7-8 POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36months of age and was followed by almost complete loss of photoreceptors at 48months of age. CLN3Δ7-8/Δ7-8 mutation (which affects ≤85% of patients) affects both RPE and POS and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.

The co-expression of the depolarizing and hyperpolarizing mechanosensitive ion channels in mammalian retinal neurons.

The elevation of the intraocular and extraocular pressures is associated with various visual conditions, including glaucoma and traumatic retinal injury. The retina expresses mechanosensitive channels (MSCs), but the role of MSCs in retinal physiology and pathologies has been unclear. Using immunocytochemistry, confocal microscopy, and patch-clamp recording techniques, we studied the co-expression of K+-permeable (K-MSCs) TRAAK and big potassium channel BK with the epithelial sodium channel ENaC and transient receptor potential channel vanilloid TPRV4 and TRPV2 favorably permeable to Ca2+ than Na+ (together named N-MSCs), and TRPV4 activity in the mouse retina. TRAAK immunoreactivity (IR) was mainly located in Müller cells. Photoreceptor outer segments (OSs) expressed BK and ENaCα intensively and TRAAK, TRPV2, and TRPV4 weakly. Somas and axons of retinal ganglion cells (RGCs) retrograde-identified clearly expressed ENaCα, TRPV4, and TRPV2 but lacked TRAAK and BK. Rod bipolar cells (RBCs) showed TRPV4-IR in somas and BK-IR in axonal globules. Horizontal cells were BK-negative, and some cone BCs lacked TRPV4-IR. TRPV4 agonist depolarized RGCs, enhanced spontaneous spikes and excitatory postsynaptic currents, reduced the visual signal reliability (VSR = 1-noise/signal) by ~50%, and resulted in ATP crisis, which could inactivate voltage-gated sodium channels in RGCs. Individual neurons co-express hyperpolarizing K-MSCs with depolarizing N-MSCs to counterbalance the pressure-induced excitation, and the level of K-MSCs relative to N-MSCs (RK/N ratio) is balanced in the outer retina but low in RGCs, bringing out novel determinants for the pressure vulnerability of retinal neurons and new targets for clinical interventions.

Inpp5e Is Critical for Photoreceptor Outer Segment Maintenance.

In humans, inositol polyphosphate-5-phosphatase e (INPP5E) mutations cause retinal degeneration as part of Joubert and MORM syndromes and can also cause non-syndromic blindness. In mice, mutations cause a spectrum of brain, kidney, and other anomalies and prevent the formation of photoreceptor outer segments. To further explore the function of Inpp5e in photoreceptors, we generated conditional and inducible knockouts of mouse Inpp5e where the gene was deleted either during outer segment formation or after outer segments were fully formed. In both cases, the loss of Inpp5e led to severe defects in photoreceptor outer segment morphology and ultimately photoreceptor cell loss. The primary morphological defect consisted of outer segment shortening and reduction in the number of newly forming discs at the outer segment base. This was accompanied by structural abnormalities of the Golgi apparatus, mislocalized rhodopsin, and an accumulation of extracellular vesicles. In addition, knockout cells showed a reduction in the size and prevalence of the actin network at the site of new disc morphogenesis and the occasional formation of membrane whorls instead of discs in a subset of cells. Together, these data demonstrate that Inpp5e plays a critical role in maintaining the outer segment and the normal process of outer segment renewal depends on the activity of this enzyme.

Loss of paired immunoglobin-like type 2 receptor B gene associated with age-related macular degeneration impairs photoreceptor function in mouse retina.

Genome-wide association studies have uncovered mostly non-coding variants at over 60 genetic loci linked to susceptibility for age-related macular degeneration (AMD). To ascertain the causal gene at the PILRB/PILRA locus, we used a CRISPR strategy to produce germline deletions in the mouse paired immunoglobin-like type 2 receptor (Pilr) genes that encode highly related activating (PILRB) and inhibitory (PILRA) receptors. We show that a combined loss of Pilrb1 and Pilrb2, but not Pilra, leads to an early but relatively stationary defect as the electroretinography (ERG) amplitudes of Pilrb1/2-/- mice exhibit a marked reduction as early as postnatal day 15 and do not show additional significant decrease at 3 and 12-months. No alterations are evident in Müller glia, microglia, bipolar, amacrine and horizontal cells based on immunohistochemistry using cell-type specific markers. PILRB immunostaining is specifically detected at the proximal part of photoreceptor outer segment. Reduced expression of select calcium-regulated phototransduction and synapse-associated proteins, including GCAP1 and 2, PDE6b, AIPL1, PSD95, and CTBP1 indicates dysregulation of calcium homeostasis as a possible mechanism of retinal phenotype in Pilrb1/2-/- mice. Our studies suggest a novel function of PILRB in retinal photoreceptors and an association of PILRB, but not PILRA, with AMD pathogenesis.

Glutamylation imbalance impairs the molecular architecture of the photoreceptor cilium.

Microtubules, composed of conserved α/β-tubulin dimers, undergo complex post-translational modifications (PTMs) that fine-tune their properties and interactions with other proteins. Cilia exhibit several tubulin PTMs, such as polyglutamylation, polyglycylation, detyrosination, and acetylation, with functions that are not fully understood. Mutations in AGBL5, which encodes the deglutamylating enzyme CCP5, have been linked to retinitis pigmentosa, suggesting that altered polyglutamylation may cause photoreceptor cell degeneration, though the underlying mechanisms are unclear. Using super-resolution ultrastructure expansion microscopy (U-ExM) in mouse and human photoreceptor cells, we observed that most tubulin PTMs accumulate at the connecting cilium that links outer and inner photoreceptor segments. Mouse models with increased glutamylation (Ccp5-/- and Ccp1-/-) or loss of tubulin acetylation (Atat1-/-) showed that aberrant glutamylation, but not acetylation loss, disrupts outer segment architecture. This disruption includes exacerbation of the connecting cilium, loss of the bulge region, and destabilization of the distal axoneme. Additionally, we found significant impairment in tubulin glycylation, as well as reduced levels of intraflagellar transport proteins and of retinitis pigmentosa-associated protein RPGR. Our findings indicate that proper glutamylation levels are crucial for maintaining the molecular architecture of the photoreceptor cilium.

PRPH2-ASSOCIATED RETINAL DISEASES: A SYSTEMATIC REVIEW OF PHENOTYPIC FINDINGS

PurposePRPH2-associated retinal diseases (PARD) result from pathogenic PRPH2 variants, primarily affecting photoreceptor outer segments and retinal pigment epithelium. The focus of this article is to review and discuss the phenotyping of PARD subtypes. DesignA systematic review MethodsThe review followed PRISMA 2020 guidelines with searches on PubMed, Medline, Web of Science, Google Scholar, and Cochrane Library. Eligible studies were those which discussed molecularly confirmed PARD or described associated diseases such as butterfly pattern dystrophy. Inclusion: cross-sectional, cohort, case-control studies, book chapters. Exclusion: non-English, conference papers, non-peer-reviewed, or non-full text articles. ResultsPARD is responsible for 25% of pattern dystrophy and up to 5% of inherited retinal dystrophies. There is clear evidence of phenotypic variability between individuals carrying the same pathogenic variant. Fundus autofluorescence, fluorescein angiography, optical coherence tomography, while in research adaptive optics reveal detailed phenotypic characteristics, notably in retinal pigment epithelium changes and photoreceptor disruption. The phenotypic of PARD variability presents diagnostic challenges, with phenotypic features often overlapping with other retinal diseases including age-related macular degeneration, Stargardt disease and retinitis pigmentosa. ConclusionThis review emphasizes revising diagnostic criteria by incorporating more recent imaging techniques and confirming diagnosis with the use of genetic testing. Understanding phenotypic diversity and intrafamilial variability in PARD is crucial for developing new treatments and for patient prognosis and future research should focus on larger cohorts studying genotype-phenotype correlations.

Prominin-1-null Xenopus laevis develop subretinal drusenoid-like deposits, cone-rod dystrophy and RPE atrophy.

Prominin-1 (PROM1) variants are associated with inherited, non-syndromic vision loss. We used CRISPR/Cas9 to induce prom1-null mutations in Xenopus laevis and then tracked retinal disease progression from the ages of 6 weeks to 3 years. We found that prom1-null-associated retinal degeneration in frogs was age-dependent and involved retinal pigment epithelium (RPE) dysfunction preceding photoreceptor degeneration. Before photoreceptor degeneration occurred, aging prom1-null frogs developed larger and increasing numbers of cellular debris deposits in the subretinal space and outer segment layer, which resembled subretinal drusenoid deposits (SDDs) in their location, histology and representation as seen by color fundus photography and optical coherence tomography (OCT). Evidence for an RPE origin of these deposits included infiltration of pigment granules into the deposits, thinning of the RPE as measured by OCT, and RPE disorganization as measured by histology and OCT. The appearance and accumulation of SDD-like deposits and RPE thinning and disorganization in our animal model suggests an underlying disease mechanism for prom1-null-mediated blindness that involves death and dysfunction of the RPE preceding photoreceptor degeneration, instead of direct effects upon photoreceptor outer segment morphogenesis, as was previously hypothesized.

Extracellular Vesicles Derived from Adipose-Derived Mesenchymal Stem Cells Alleviate Apoptosis and Oxidative Stress of Retinal Pigment Epithelial Cells Through Activation of Nrf2 Signaling Pathway.

Purpose: To examine the potential protective effects of adipose-derived mesenchymal stem cell-derived extracellular vesicles (ASC-EVs) on ARPE-19 cells exposed to hydrogen peroxide (H2O2) stress and to evaluate their ability to delay retinal degeneration in Royal College of Surgeons (RCS) rats. Methods: ARPE-19 cells were pre-treated with ASC-EVs for 24 h, followed by exposure to 200 μM H2O2 for an additional 24 h. RCS rats received an intravitreal injection of phosphate-buffered saline in one eye and ASC-EVs in the other eye. Results: ASC-EV pretreatment significantly protected against H2O2 in the Cell Counting Kit-8 assay and was also effective in the lactate dehydrogenase-release assay. It notably reduced early apoptosis (Annexin V-fluorescein isothiocyanate/propidium iodide assay) and late apoptosis (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling assay), while significantly decreasing intracellular reactive oxygen species, glutathione levels, and superoxide dismutase activity. NFE2L2, HMOX1, and NQO1 mRNA levels, along with Nrf2, HO-1, and NQO1 protein levels, were significantly elevated with ASC-EV pretreatment. Compared with ARPE-19-derived EVs, 11 miRNAs were upregulated and 34 were downregulated in ASC-EVs. In RCS rats, intravitreal injections of ASC-EVs led to significant preservation of the outer nuclear layer and photoreceptor segments, along with increased nuclear Nrf2 expression and elevated HO-1 and NQO1 levels in the inner retina. Eyes that received intravitreal injections of ASC-EVs demonstrated significantly preserved electroretinography a- and b-wave amplitudes at 1 week post-injection, though this effect faded by 2 weeks. Conclusions: ASC-EVs mitigated apoptosis and oxidative stress in ARPE-19 cells subjected to H2O2 exposure and temporarily slowed retinal degeneration in RCS rats via Nrf2 pathway activation by miRNAs.

Splice Variant of Retinal G-Protein-Coupled Receptor Deletion-Mediated Dysregulation of Autophagy Increases the Susceptibility to Age-Related Macular Degeneration-Like Defects

Introduction: The splice variant of retinal G-protein-coupled receptor deletion (RGR-d) is a persistent component of drusen and may be involved in the pathogenesis of dry age-related macular degeneration (AMD). Increasing evidence has demonstrated the critical role of autophagy in AMD. In this study, we investigated whether RGR-d disrupts autophagy in early dry AMD in vivo and in vitro. Methods: Fundus imaging and fluoroscopy were performed on RGR-d mice created by multiplex gene editing. The retina microstructure was evaluated by performing hematoxylin and eosin (H&E) staining as well as transmission electron microscopy (TEM). Retinal function was assessed by full-field electroretinography (ERG). After lentivirus transfection and stimulation, the permeability, phagocytosis, and tight junctions of ARPE-19 cells were evaluated. Western blotting of ATG5, Beclin-1, LC3II/I, and P62 was performed to detect the changes in autophagy pathways. Results: Atrophy and patchy penetrating hyperfluorescent foci, consistent with early AMD-like defects, were observed in the fundus of 12-month-old RGR-d mice. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. TEM analysis showed some diffuse granular deposits. And the morphology of choroidal microvascular endothelial cells was degraded and distorted. The morphology of the photoreceptor outer segments showed structural damage, and Bruch’s membrane was thickened. ERG indicated that the photoreceptor of RGR-d mice were dysfunctional. Changes in autophagy-related protein expression were observed in the retinal pigment epithelium and retinal neurepithelium, and autophagy regulation was decreased. Palmitic acid (PA) stimulation caused permeability, phagocytosis, and tight junction dysfunction in cells overexpressing RGR-d. Beclin-1 and LC3II/I expression levels were significantly decreased and that of P62 was elevated in RGR-d cells after PA stimulation. Conclusion: RGR-d disrupts the autophagy pathway, causing the development of an early AMD-like pathophysiology.

CYLD Maintains Retinal Homeostasis by Deubiquitinating ENKD1 and Promoting the Phagocytosis of Photoreceptor Outer Segments.

Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is essential for retinal homeostasis. Dysregulation of the phagocytotic process is associated with irreversible retinal degenerative diseases. However, the molecular mechanisms underlying the phagocytic activity of RPE cells remain elusive. In an effort to uncover proteins orchestrating retinal function, the cylindromatosis (CYLD) deubiquitinase is identified as a critical regulator of photoreceptor outer segment phagocytosis. CYLD-deficient mice exhibit abnormal retinal structure and function. Mechanistically, CYLD interacts with enkurin domain containing protein 1 (ENKD1) and deubiquitinates ENKD1 at lysine residues K141 and K242. Deubiquitinated ENKD1 interacts with Ezrin, a membrane-cytoskeleton linker, and stimulates the microvillar localization of Ezrin, which is essential for the phagocytic activity of RPE cells. These findings thus reveal a crucial role for the CYLD-ENKD1-Ezrin axis in regulating retinal homeostasis and may have important implications for the prevention and treatment of retinal degenerative diseases.

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein.

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4. These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis. Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

Emc1 is essential for vision and zebrafish photoreceptor outer segment morphogenesis.

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.

Deciphering the impact of PROM1 alternative splicing on human photoreceptor development and maturation

Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity, which is highly regulated in tissue- and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular, photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs), undergoes exon 4 inclusion from the 12th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy, however its role in photoreceptor maturation and function is unknown. In this study retinal organoids, a valuable model system, were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Retinal organoids were treated with the PMOs for four weeks after which RT-PCR, western blotting and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors. The transcriptome of treated ROs was studied by bulk RNA-Seq. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%, abnormal accumulation of cones in the basal side of the retinal organoids as well as detectable cone photoreceptor cilium defects. Transcriptomic and western blot analyses revealed decreased expression of cone, inner segment and connecting cilium basal body markers, increased expression of genes associated with stress response and the ubiquitin-proteasome system, and downregulation of autophagy. Importantly, the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context, opening avenues for further research and potential therapeutic interventions. Together our data indicate that cones may be more sensitive to PROM1 exon 4 skipping and/or reduced Prominin-1 expression, corroborating the pathogenesis of late-onset mild maculopathy.

Photoreceptor outer segment reflectivity with ultrahigh resolution visible light optical coherence tomography in systemic hydroxychloroquine use.

To evaluate outer retinal organization in normal subjects and those using hydroxychloroquine (HCQ) with ultrahigh resolution visible light optical coherence tomography (VIS-OCT). Forty eyes of 22 adult subjects were recruited from a tertiary care retina practice including controls (20 eyes, 12 subjects, mean age 40±22yrs, mean logMAR BCVA 0.19, 90% female) and subjects with a history of HCQ use (20 eyes, 10 subjects, mean age 62±17yrs, mean logMAR BCVA 0.03, 67% female). Each subject was imaged using a custom-built VIS-OCT device (axial resolution 1.3μm) and FDA-approved OCT devices. Using VIS-OCT, control subjects demonstrate 5 and 6 hyperreflective bands in the foveal and parafoveal regions, respectively, between the outer nuclear layer and Bruch's membrane. These bands demonstrate intensity profiles complementary to the known histopathologic distribution of rods and cones. In comparison to controls, subjects taking HCQ demonstrate blunting of all bands, particularly bands 2-4. In all cases of suspected or known toxicity, VIS-OCT demonstrated attenuation of band 3i and in no cases was there attenuation of other bands that was more severe than band 3i, suggesting that changes in the reflectivity of Band 3i may be the earliest identifiable sign of HCQ toxicity. VIS-OCT of the outer retina demonstrates a unique outer retinal banding pattern corresponding to photoreceptor density profiles. There is a notable attenuation of the photoreceptor outer segment reflectivity profile associated with early HCQ toxicity. This finding may be an early, and possibly reversible, sign of HCQ toxicity, primarily impacting the cones.