Placental growth factor stabilizes VEGF receptor-2 protein in retinal pigment epithelial cells by downregulating glycogen synthase kinase 3 activity.

Placental growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family of proteins that participate in angiogenesis and vasculogenesis. Anti-VEGF therapy has become the standard treatment for ocular angiogenic disorders in ophthalmological practice. However, there is emerging evidence that anti-VEGF treatment may increase the risk of atrophy of the retinal pigment epithelium (RPE), which is important for the homeostasis of retinal tissue. Whereas the cytoprotective role of VEGF family molecules, particularly that of VEGF A (VEGFA) through its receptor VEGF receptor-2 (VEGFR-2), has been recognized, the physiological role of PlGF in the retina is still unknown. In this study, we explored the role of PlGF in the RPE using PlGF-knockdown RPE cells generated by retrovirus-based PlGF-shRNA transduction. We show that VEGFA reduced apoptosis induced by serum starvation in RPE cells, whereas the antiapoptotic effect of VEGFA was abrogated by VEGFR-2 knockdown. Furthermore, PlGF knockdown increased serum starvation-induced cell apoptosis and unexpectedly reduced the protein level of VEGFR-2 in the RPE. The antiapoptotic effect of VEGFA was also diminished in PlGF-knockdown RPE cells. In addition, we found that glycogen synthase kinase 3 activity was involved in proteasomal degradation of VEGFR-2 in RPE cells and inactivated by PlGF via AKT phosphorylation. Overall, the present data demonstrate that PlGF is crucial for RPE cell viability and that PlGF supports VEGFA/VEGFR-2 signaling by stabilizing the VEGFR-2 protein levels through glycogen synthase kinase 3 inactivation.

Regulation of Aqueous Humor Secretion by Melatonin in Porcine Ciliary Epithelium.

Secretion of melatonin, a natural hormone whose receptors are present in the ciliary epithelium, displays diurnal variation in the aqueous humor (AH), potentially contributing to the regulation of intraocular pressure. This study aimed to determine the effects of melatonin on AH secretion in porcine ciliary epithelium. The addition of 100 µM melatonin to both sides of the epithelium significantly increased the short-circuit current (Isc) by ~40%. Stromal administration alone had no effect on the Isc, but aqueous application triggered a 40% increase in Isc, similar to that of bilateral application without additive effect. Pre-treatment with niflumic acid abolished melatonin-induced Isc stimulation. More importantly, melatonin stimulated the fluid secretion across the intact ciliary epithelium by ~80% and elicited a sustained increase (~50-60%) in gap junctional permeability between pigmented ciliary epithelial (PE) cells and non-pigmented ciliary epithelial (NPE) cells. The expression of MT3 receptor was found to be >10-fold higher than that of MT1 and MT2 in porcine ciliary epithelium. Aqueous pre-treatment with MT1/MT2 antagonist luzindole failed to inhibit the melatonin-induced Isc response, while MT3 antagonist prazosin pre-treatment abolished the Isc stimulation. We conclude that melatonin facilitates Cl- and fluid movement from PE to NPE cells, thereby stimulating AH secretion via NPE-cell MT3 receptors.

[Adenocarcinoma of nonpigmented ciliary body epithelium (clinical cases)]. / Adenokartsinoma iz bespigmentnogo epiteliya tsiliarnogo tela (klinicheskie nablyudeniya).

The article presents two clinical cases of adenocarcinoma of nonpigmented epithelium of the ciliary body, which is a very rare malignant tumor of the organ of vision with distinctive features. Surgical treatment is necessary to verify this tumor and assess the degree of its aggressiveness in terms of the prognosis of the disease, with subsequent pathomorphological and immunohistochemical studies. The article also discusses the epidemiological aspects, morphological features, clinical manifestations of this pathological condition, as well as possible treatment options and features of follow-up monitoring of this group of patients.

Single-cell RNA-sequencing analysis of the ciliary epithelium and contiguous tissues in the mouse eye.

The ciliary epithelium plays a central role in ocular homeostasis but cells of the pigmented and non-pigmented layers are difficult to isolate physically and study. Here we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptional signatures of cells harvested from the ciliary body and contiguous tissues. Microdissected tissue was dissociated by collagenase digestion and the transcriptomes of individual cells were obtained using a droplet-based scRNA-seq approach. In situ hybridization was used to verify the expression patterns of selected differentially-expressed genes. High quality transcriptomes were obtained from 10,024 cells and unsupervised clustering distinguished 22 cell types. Although efforts were made to specifically isolate the ciliary body, approximately half of the sequenced cells were derived from the adjacent retina. Cluster identities were assigned using expression of canonical markers or cluster-specific genes. The transcriptional signature of cells in the PCE and NPCE were distinct from each other and from cells in contiguous tissues. PCE cell transcriptomes were characterized by genes involved in melanin synthesis and transport proteins such as Slc4a4. Among the most differentially expressed genes in NPCE cells were those encoding members of the Zic family of transcription factors (Zic1, 2, 4), collagen XVIII (Col18a1), and corticotrophin-releasing hormone-binding protein (Crhbp). The ocular melanocyte population was distinguished by expression of the gap junction genes Gjb2 and Gjb6. Two fibroblast signatures were detected in the ciliary body preparation and shown by in situ hybridization to correspond to uveal and scleral populations. This cell atlas for the ciliary body and contiguous layers represents a useful resource that may facilitate studies into the development of the ciliary epithelium, the production of the aqueous and vitreous humors, and the synthesis of the ciliary zonule.

Ultra-soft X-ray system for imaging the early cellular responses to X-ray induced DNA damage.

The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1-proteins involved in the signaling axis of mammalian DSB repair-in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.

A case of bilateral acute depigmentation of the Iris in one of two identical twins.

BACKGROUND: Bilateral Acute Depigmentation of the Iris (BADI) is a condition which was first described in a case series from Turkey by Tugal-Tutkin and Urgancioglu in (Graefes Arch Clin Exp Ophthalmol 244:742-6, 2006). The condition is characterized by bilateral acute depigmentation and discoloration of the iris stroma, pigment dispersion, and deposition of pigment in the angle. In our case we report a patient who developed BADI after receiving pitcher plant extract injections for chronic migraine, while her identical twin sister has normal iris architecture and pigmentation and never received any pitcher plant injections. CASE PRESENTATION: Patient is a 41-year-old female with history of pitcher plant extract injections to her face for chronic migraine, who later developed bilateral depigmentation of the iris. She did not have any signs of anterior segment uveitis or iridocyclitis. She has an identical twin sister who maintained normal iris pigmentation during the entire course. CONCLUSIONS: Bilateral Acute depigmentation of the is a recently discovered condition described in the literature in Turkish patients (Tugal-Tutkun and Urgancioglu, Graefes Arch Clin Exp Ophthalmol 244:742-6, 2006; Tugal-Tutkun et al., Ophthalmology 116(8):1552-7, 2009). This condition affects mainly young females and is characterized by acute bilateral stromal depigmentation, without other pathologic ocular findings. These patients usually maintain normal vision and do not develop significant glaucoma from pigment collecting in the anterior chamber angle. This condition can be mistaken for Fuchs' heterochromic iridocyclitis, pigment dispersion syndrome, pseudoexfoliation syndrome, and viral iridocyclitis. This is the first reported case in North America and is important for differentiation from the above pathologies. Our patient had a history of pitcher plant extract injections to the face but it is unclear if this is associated with our patient's development of BADI. As awareness of this condition progresses, a possible etiology may be elucidated.

In patients with a positive family history of familial adenomatous polyposis can the condition be diagnosed from the presence of congenital hypertrophy of the retinal pigment epithelium detected via an eye examination: A systematic review.

In classic familial adenomatous polyposis (FAP) adenomas become malignant. Congenital hypertrophy of the retinal pigment epithelium (CHRPE) is a retinal pigmented lesion and is the earliest and most common potential extraintestinal manifestation of FAP. This review aims to summarize and analyse all of the published data on CHRPE in patients with classic FAP and then ascertain whether these patients should undergo a relatively cheap and non-invasive dilated fundus examination to screen for CHRPE. Adhering to Preferred Reporting Items for Systematic Reviews and Meta Analyses guidelines our database search identified 102 relevant articles of which 13 were selected for further analysis. The percentage of FAP patients with CHRPE was found to be 80.00%, whereas the percentage of at-risk patients with CHRPE was 31.12%. Despite various statistically significant findings, CHRPE alone cannot be used as a surrogate for diagnosing FAP in those with a positive family history. The authors advocate a combined approach of eye examinations, colonoscopy and genetic testing.

Pigmentation formation and expression analysis of tyrosinase in Siniperca chuatsi.

Animal pigmentation primarily depends on the presence and mixing ratio of chromatophores, functioning in animal survival and communication. For the benthic and carnivorous Siniperca chuatsi, pigmentation pattern is key to concealment and predation. In this study, the formation, distribution, and main pattern of chromatophores were observed in the embryos, larvae, skins, and visceral tissues from S. chuatsi. Melanophores were firstly visualized in the yolk sac at segmentation stage, and then they were migrated to the whole body and further clustered into the black stripes, bands, and patches. In adult S. chuatsi, the head, black band, and body side skins mainly contained melanophores, showing as deep or light black. The abdomen skin mainly contained iridophores, showing as silvery. In the eye, the pigment layers were located in the epithelial layers of iris and retina and shown as black. Then, the pigmentation-related gene, tyrosinase gene from S. chuatsi (Sc-tyr) was analyzed by bioinformatics and quantitative methods. The Sc-tyr gene encoded a protein with 540 amino acids (Sc-TYR). The Sc-TYR contained two copper ion binding sites, which were coordinated by six conserved histidines (H182, H205, H214, H366, H370, H393) and necessary for catalytic activity. The Sc-TYR was well conserved compared with TYR of various species with higher degree of sequence similarity with other fishes (77.6-98.3%). The qRT-PCR test showed that the Sc-tyr mRNA reached the peak value at segmentation stage in the embryo development, the black skins displayed a higher expression level than that in silvery skin, and the eye had the highest expression level compared with other tissues. Further research on enzyme activity showed that the expression patterns of tyrosinase activity were similar to that of the Sc-tyr mRNA. Comparing with the results of molecular and phenotype, it was found that the temporal and spatial distributions of tyrosinase corresponded well with changes in pigmentation patterns and the intensity of skin melanization. This study initially explored the pigmentation formation and tyrosinase expression, which served as a foundation for further insight into the genetics mechanism of body color formation in S. chuatsi.

Evaluation of the iris thickness changes for the Chinese families with GPR143 gene mutations.

PURPOSE: Pathogenic variants of the G-protein coupled receptor 143 (GPR143) gene may result in Ocular albinism type I (OA1). In this study, we describe the clinical features and investigate the GPR143 gene mutations in six Chinese families with OA1 and evaluate the thickness changes of iris for the affected males and female carriers. METHODS: Families were ascertained, and patients underwent complete ophthalmologic examinations, including the best corrected visual acuity (BCVA), anterior segment of the eyes, vitreous and fundus changes. Spectral domain optical coherence tomography (SD-OCT) was used to measure the full iris thickness, the stroma/anterior border (SAB) layer, and the posterior epithelial layer (PEL) at the pupillary and ciliary regions. DNA was extracted from the peripheral blood vessels after confirmed consent information. GPR143 gene was directly sequenced by the Sanger method. RESULTS: The affected males had variable reduced visual acuity, nystagmus and macular hypoplasia. Four novel frameshift mutations and two previously reported missense/nonsense mutations in the GPR143 gene were detected in these families. The thickness of the iris was significantly reduced at the ciliary region in the affected males, compared with that in the normal controls and the female carriers. CONCLUSIONS: Pathogenic variants in the GPR143 gene may disturb the normal melanogenesis in the pigmented tissues of the eye, result in macular hypoplasia, and alter the thickness of the iris.

Case Report: Sequelae of Bilateral Iris Pigment Epithelial Cysts Masquerading as Primary Pigmentary Glaucoma.

SIGNIFICANCE: Patients with pigment dispersion syndrome are frequently encountered in clinical practice. Iris abnormalities and the absence of key features of pigment dispersion syndrome can guide the discovery of secondary causes of pigmentary glaucoma, such as peripheral iris pigment epithelial cysts. PURPOSE: The purpose of this study was to describe a patient initially diagnosed as having primary pigmentary glaucoma found to have multiple bilateral peripheral iris pigment epithelial cysts causing pigment liberation, focal angle closure, and advanced secondary glaucoma. CASE REPORT: A 64-year-old man presented for follow-up after a recent diagnosis of primary pigmentary glaucoma in the left eye. Clinical examination revealed transillumination defects of both irides and a heavily pigmented trabecular meshwork. A midperipheral bulge of the iris was appreciated in the superior temporal quadrant of the left eye. Gonioscopic evaluation showed that the bulge focally obstructed the angle. Anterior segment optical coherence tomography demonstrated a normal iris insertion of both eyes, with the exception of a steepened approach superotemporally in the left eye. Ultrasound biomicroscopy revealed bilateral small- to medium-sized peripheral iris pigment epithelial cysts and confirmed a large cyst superotemporally in the left iris. CONCLUSIONS: This case describes a patient with advanced secondary pigmentary glaucoma from iris pigment epithelial cysts. The mechanisms of glaucoma are likely twofold: (1) pigment liberation from the cysts contacting the lens zonules and, to a lesser extent, (2) focal angle closure at the site of the large peripheral cyst. The key features of pigmentary glaucoma, namely, posterior bowing of the iris, may not be present in secondary pigment dispersion. This case highlights the importance of careful evaluation of the iris and angle in making a correct diagnosis. The choice of topical agent to control intraocular pressure while not increasing the size of these cysts is also an important consideration.

Expression and regulation of alarmin cytokine IL-1α in human retinal pigment epithelial cells.

Human retinal pigment epithelial (hRPE) cells play important immune-regulatory roles in a variety of retinal pathologic processes, including the production of inflammatory cytokines that are essential mediators of the innate immune response within the ocular microenvironment. The pro-inflammatory "alarmin" cytokine IL-1α has been implicated in both infectious and non-infectious retinal diseases, but its regulation in the retina is poorly understood. The purpose of this study was to elucidate the expression and regulation of IL-1α within hRPE cells. To do this, IL-1α mRNA and protein in hRPE cells was assessed by RT-PCR, qPCR, ELISA, Western blot, and immunofluorescence following treatment with a variety of stimuli and inhibitors. ER stress, LPS, IL-1ß, and TLR2 activation all significantly increased intracellular IL-1α protein. Increasing intracellular calcium synergized both LPS- and Pam3CSK4-induced IL-1α protein production. Accordingly, blocking calcium signaling and calpain activity strongly suppressed IL-1α protein expression. Significant but more moderate inhibition occurred following blockage of TLR4, caspase-4, or caspase-1. Neutralizing antibodies to IL-1ß and TLR2 partially eliminated LPS- and TLR2 ligand Pam3CSK4-stimulated IL-1α protein production. IFN-ß induced caspase-4 expression and activation, and also potentiated LPS-induced IL-1α expression, but IFN-ß alone had no effect on IL-1α protein production. Interestingly, all inhibitors targeting the PI3K/Akt pathway, with the exception of Ly294002, strongly increased IL-1α protein expression. This study improves understanding of the complex mechanisms regulating IL-1α protein expression in hRPE cells by demonstrating that TLR4 and TLR2 stimulation and exposure to IL-1ß, ER stress and intracellular calcium all induce hRPE cells to produce intracellular IL-1α, which is negatively regulated by the PI3K/Akt pathway. Additionally, the non-canonical inflammasome pathway was shown to be involved in LPS-induced hRPE IL-1α expression through caspase-4 signaling.

Calf melanin immunomodulates RPE cell attachment to extracellular matrix protein.

PURPOSE: It is widely accepted that RPE melanin has a protective effect against oxidative damage in RPE cells. It is possible that an additional protective characteristic of melanin is the ability to modulate RPE cell immune response. In this study, in vitro modeling was used to probe the relationship between RPE pigmentation and immune response by monitoring IL-6 expression and secretion in calf melanin pigmented ARPE-19 cells seeded onto glycated extracellular matrix as a stressor. METHODS: ARPE-19 cells were left unpigmented or were pigmented with either calf melanin or latex beads, and were then seeded onto RPE-derived extracellular matrix (ECM) or tissue culture-treated plates (no ECM). ECMs were modified by glycation. IL-6 expression was measured using qPCR and IL-6 secretion was determined using an ELISA, both at 30 min and 24 h after seeding. MTT assay was used to quantify cell attachment to glycated matrices 30 min after seeding. In unpigmented ARPE-19 cells, rate of cell attachment to substrate was monitored for 60 min after seeding using a hemacytometer to count unattached cells. Additionally, cell viability was evaluated using the Neutral Red assay 24 h after seeding. RESULTS: A significant increase in IL-6 expression was observed in calf melanin pigmented cells versus latex bead and unpigmented controls (p < 0.0001) 30 min after seeding onto ECM. Twenty-four hours after seeding, a significant decrease in IL-6 expression was observed in calf melanin pigmented cells (p < 0.0001) versus controls, implicating down-regulation of the cytokine. Additionally, calf melanin pigmented cell populations showed significant increase in attachment compared to unpigmented controls on either no ECM or unmodified ECM. CONCLUSIONS: Pigmentation of RPE cells with calf melanin resulted in significant changes in IL-6 expression regardless of ECM modification, in vitro. These findings suggest that melanin in the RPE may participate in immune response modulation in the retina with particular regard to cell attachment to protein substrates. The results of this study further implicate the role of chemical changes to melanin in regulating inflammation in retinal disease.

[Infection-associated Acute Posterior Multifocal Placoid Pigment Epitheliopathy]. / Infektassoziierte akute posteriore multifokale plakoide Pigmentepitheliopathie.

Acute posterior multifocal placoid pigment epitheliopathy (APMPPE) is a rare inflammatory chorioretinopathy, which mainly affects young light-skinned, myopic adults between 20 and 30 years of age. The exact aetiology of APMPPE is unknown. Some patients report a viral or flu-like illness preceding the onset of APMPPE symptoms. This condition is usually bilateral and self-limiting with a good overall prognosis. Visual loss is sudden, but usually temporary. Relapses are very rare. Foveal involvement may lead to a worse visual prognosis. There is no current consensus on treatment. A wait-and-see approach with monitoring at short intervals is often sufficient. Based on a case example from our clinic we will demonstrate symptoms, diagnostic work-up and treatment options.

[Congenital Simple Hamartoma of the Retinal Pigment Epithelium]. / Kongenitales einfaches Hamartom des retinalen Pigmentepithels.

BACKGROUND: Congenital simple hamartoma of the retinal pigment epithelium (CSHRPE) is an uncommon benign lesion with characteristic clinical features. Ophthalmoscopically it appears as a small localized, well circumscribed, pigmented tumor in the foveal region. In contrast to the more common flat congenital hypertrophy of the RPE the CSHRPE has an elevated nodular appearance. PATIENTS AND METHODS: Retrospective case series of three patients with CSHRPE. Clinical morphological features using different imaging techniques are presented. RESULTS: A typical dark lesion was incidentally noted in the macula of two patients. Optical coherence tomography (OCT) demonstrated a nodular preretinal hyperreflectivity with shadowing of deeper structures. In one patient the CSHRPE was hypofluorescent throughout the angiogram. The third patient presented with a reduced visual acuity of 0.3. A characteristic lesion was found at the foveal center. OCT revealed a hyperreflective preretinal lesion with associated moderate disruption of the foveal architecture. Amblyopia treatment slightly improved visual acuity in this case. The lesions remained stationary in two patients (follow-up 8 - 14 months). CONCLUSIONS: CSHRPE are usually detected as an incidental finding. Given its benign character and typically asymptomatic presentation an observational treatment approach is generally recommended. The lesions generally remain stationary and are not known to grow. In cases with visual impairment due to foveal involvement amblyopia treatment should be initiated.

[Free-Floating Intraocular Cysts]. / Frei fluktuierende intraokulare Zysten.

BACKGROUND: Free-floating intraocular cysts may be found in the anterior chamber (FZV) and the vitreous (FZG). The first description of a cyst was 150 years ago, and they are considered to be ocular rarities. MATERIALS AND METHODS: The actual knowledge about FZV and FZG is shown on the basis of two exemplary patients. RESULTS AND DISCUSSION: Patient 1 had a FZV as an incidental finding which had a smooth surface, a slight pigmentation and was translucent. The ultrasound biomicroscopy revealed an echo-free interior space. Without the patient's discomfort and missing treatment indication, a watch-and-wait strategy was chosen. Cysts of the iris can be classified as primary and secondary cysts. Primary cysts of the iris can arise from the stroma as the pigment epithelium wherein it is believed that FZV descend from the pigment epithelium. Secondary cysts and FZV can be generated by tumors, inflammation, epithelial ingrowth, the use of eye-drops or intraocular foreign bodies. Patient 2 showed marked myopic fundus changes and an FZG with a yellowish-greenish surface; the transparency was reduced and the surface was not pigmented. The ultrasound examination also revealed an echo-free interior space. Clinical controls were advised. Congenital and acquired causes are discussed for the formation of FZG. FZG could originate from the pigment epithelium of the iris, but there are conflicting study results. Trauma, inflammation and chorioretinal diseases are considered as a reason for acquired causes of FZG. The genesis, especially of FZG, is still unclear. For the treatment of patients with FZV and FZG, it is important to know the potential causes to be able to make a therapeutic decision. High quality photographic and sonographic documentation is needed in the watch-and-wait strategy.

Congenital ectropion uveae with glaucoma: a case report.

Congenital ectropion uveae (CEU) is a rare anomaly characterized by ectropion uveae, iris hypoplasia, iridotrabecular dysgenesis and glaucoma. The apparent ectropion uveae results from the spread of iris pigment epithelium beyond the iris ruff and onto the anterior surface of the iris. Conclusion Open-angle glaucoma results due to angle dysgenesis, so patients should be carefully examined periodically for its early detection.

Effect of berberine on lipopolysaccharide-induced monocyte chemotactic protein-1 and interleukin-8 expression in a human retinal pigment epithelial cell line.

PURPOSE: In this study, we elucidated the effects of berberine, a major alkaloid component contained in medicinal herbs, such as Phellodendri Cortex and Coptidis Rhizoma, on expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in a human retinal pigment epithelial cell line (ARPE-19) caused by lipopolysaccharide (LPS) stimulation. METHODS: ARPE-19 cells were cultured to confluence. Berberine and LPS were added to the medium. MCP-1 and IL-8 mRNA were measured by real-time polymerase chain reaction. MCP-1 and IL-8 protein concentrations in the media were measured using enzyme-linked immunosorbent assay. RESULTS: After stimulation with LPS, MCP-1 and IL-8 mRNA in ARPE-19 cells reached maximum levels at 3 h, and MCP-1 and IL-8 protein in the culture media reached maximum levels at 24 h. Berberine dose-dependently inhibited MCP-1 and IL-8 mRNA expression of the cells and protein levels in the media stimulated with LPS. CONCLUSIONS: These findings indicate that berberine inhibited the expression of MCP-1 and IL-8 induced by LPS.

Rational Tuning of Visual Cycle Modulator Pharmacodynamics.

Modulators of the visual cycle have been developed for treatment of various retinal disorders. These agents were designed to inhibit retinoid isomerase [retinal pigment epithelium-specific 65 kDa protein (RPE65)], the rate-limiting enzyme of the visual cycle, based on the idea that attenuation of visual pigment regeneration could reduce formation of toxic retinal conjugates. Of these agents, certain ones that contain primary amine groups can also reversibly form retinaldehyde Schiff base adducts, which contributes to their retinal protective activity. Direct inhibition of RPE65 as a therapeutic strategy is complicated by adverse effects resulting from slowed chromophore regeneration, whereas effective retinal sequestration can require high drug doses with potential off-target effects. We hypothesized that the RPE65-emixustat crystal structure could help guide the design of retinaldehyde-sequestering agents with varying degrees of RPE65 inhibitory activity. We found that addition of an isopropyl group to the central phenyl ring of emixustat and related compounds resulted in agents effectively lacking in vitro retinoid isomerase inhibitory activity, whereas substitution of the terminal 6-membered ring with branched moieties capable of stronger RPE65 interaction potentiated inhibition. The isopropyl derivative series produced discernible visual cycle suppression in vivo, albeit much less potently than compounds with a high affinity for the RPE65 active site. These agents were distributed into the retina and formed Schiff base adducts with retinaldehyde. Except for one compound [3-amino-1-(3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)phenyl)propan-1-ol (MB-007)], these agents conferred protection against retinal phototoxicity, suggesting that both direct RPE65 inhibition and retinal sequestration are mechanisms of potential therapeutic relevance.

Quick-freeze/deep-etch electron microscopy visualization of the mouse posterior pole.

The mouse is one of the most commonly used mammalian systems to study human diseases. In particular it has been an invaluable tool to model a multitude of ocular pathologies affecting the posterior pole. The aim of this study was to create a comprehensive map of the ultrastructure of the mouse posterior pole using the quick-freeze/deep-etch method (QFDE). QFDE can produce detailed three-dimensional images of tissue structure and macromolecular moieties, without many of the artifacts introduced by structure-altering post-processing methods necessary to perform conventional transmission electron microscopy (cTEM). A total of 18 eyes from aged C57BL6/J mice were enucleated and the posterior poles were processed, either intact or with the retinal pigment epithelium (RPE) cell layer removed, for imaging by either QFDE or cTEM. QFDE images were correlated with cTEM cross-sections and en face images through the outer retina. Nicely preserved outer retinal architecture was observed with both methods, however, QFDE provided excellent high magnification imaging, with greater detail, of the apical, central, and basal planes of the RPE. Furthermore, key landmarks within Bruch's membrane, choriocapillaris, choroid and sclera were characterized and identified. In this study we developed methods for preparing the outer retina of the mouse for evaluation with QFDE and provide a map of the ultrastructure and cellular composition of the outer posterior pole. This technique should be applicable for morphological evaluation of mouse models, in which detailed visualization of subtle ocular structural changes is needed or in cases where post-processing methods introduce unacceptable artifacts.