Search

Ocular levels of IL-1β, TNFα, IL-8, and IL-6 correlate with progression of diabetic retinopathy (DR). Müller cells (MC), which are crucial to maintaining retinal homeostasis, are targets and sources of these cytokines. We explored the relative capacities of these four DR-associated cytokines to amplify inflammatory signal expression both in and between human MC (hMC) and retinal microvascular endothelial cells (hRMEC) and in the mouse retina. Of the four cytokines, IL-1β was the most potent stimulus of transcriptomic alterations in hMC and hRMEC in vitro, as well as in the mouse retina after intravitreal injection in vivo. Stimulation with IL-1β significantly induced expression of all four transcripts in hMC and hRMEC. TNFα significantly induced expression of some, but not all, of the four transcripts in each cell, while neither IL-8 nor IL-6 showed significant induction in either cell. Similarly, conditioned media (CM) derived from hMC or hRMEC treated with IL-1β, but not TNFα, upregulated inflammatory cytokine transcripts in the reciprocal cell type. hRMEC responses to hMC-derived CM were dependent on IL-1R activation. In addition, we observed a correlation between cytokine expression changes following direct and CM stimulation and NFκB-p65 nuclear translocation in both hMC and hRMEC. Finally, in mice, intravitreal injections of IL-1β, but not TNFα, induced retinal expression of Il1b and CXCL8 homologues Cxcl1, Cxcl2, Cxcl3, and Cxcl5, encoding pro-angiogenic chemokines. Our results suggest that expression of IL-1β, TNFα, IL-8, and IL-6 may be initiated, propagated, and sustained by autocrine and paracrine signals in hRMEC and hMC through a process involving IL-1β and NFκB. Targeting these signals may help thwart inflammatory amplification, preventing progression to vision-threatening stages and preserving sight., Competing Interests: Declaration of competing interest There are no financial or non-financial competing interests declared by any author., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Sialylation plays an important role in self-recognition, as well as keeping the complement and innate immune systems in check. It is unclear whether the reduced sialylation seen during aging and in mice heterozygous for the null mutant of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (Gne+/-), an essential enzyme for sialic acid biosynthesis, contributes to retinal inflammation and degeneration. We found a reduction of polysialic acid and trisialic acid expression in several retinal layers in Gne+/- mice at 9 months of age compared to Gne+/+ wildtype (WT) mice, which was associated with a higher microglial expression of the lysosomal marker CD68. Furthermore, the total number of rod bipolar cells was reduced in 12 months old Gne+/- mice in comparison to WT mice, demonstrating loss of these retinal interneurons. Transcriptome analysis showed up-regulation of complement, inflammation, and apoptosis-related pathways in the retinas of Gne+/- mice. Particularly, increased gene transcript levels of the complement factors C3 and C4 and the pro-inflammatory cytokine Il-1β were observed by semi-quantitative real-time polymerase chain reaction (sqRT-PCR) in 9 months old Gne+/- mice compared to WT mice. The increased expression of CD68, loss of rod bipolar cells, and increased gene transcription of complement factor C4, were all prevented after crossing Gne+/- mice with complement factor C3-deficient animals. In conclusion, our data show that retinal hyposialylation in 9 and 12 months old Gne+/- mice was associated with complement-related inflammation and lysosomal microglia response, as well as rod bipolar cells loss, which was absent after genetic deletion of complement factor C3., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

Aims: Nerve growth factor is a well characterised neurotrophic factor that play a critical role in the survival, growth and differentiation of neurons both in central and peripheral nervous system. However, it is difficult for the conventional exogenous nerve growth factor administration delivery to the central nervous system due to the biological barrier in human bodies. Results: We validated a series of cell penetrating peptides and found that L-PenetraMax significantly enhanced the efficiency of recombinant human nerve growth factor entry into the rat retina. In the optic nerve crush mice model, eye drop administration of recombinant human nerve growth factor alone promoted retinal ganglion cell survival and axon regeneration at high dose, while the combination of recombinant human nerve growth factor with L-PenetraMax significantly enhanced the neuroprotective efficacy at lower dose, thus potentially enhancing the availability of recombinant human nerve growth factor eye drops in patients with optic neuropathy. Conclusions: This study provides the evidence that the noncovalent coadministration of recombinant human nerve growth factor with L-PenetraMax could be a potent strategy for the non-invasive and sustained ocular delivery of therapeutic proteins for improving the optic nerve injury.

Microglia are resident immune cells in the central nervous system, including the retina that surveil the environment for damage and infection. Following retinal damage, microglia undergo morphological changes, migrate to the site of damage, and express and secrete pro-inflammatory signals. In the zebrafish retina, inflammation induces the reprogramming and proliferation of Müller glia and the regeneration of neurons following damage or injury. Immunosuppression or pharmacological ablation of microglia reduce or abolish Müller glia proliferation. We evaluated the retinal architecture and retinal regeneration in adult zebrafish irf8 mutants, which have significantly depleted numbers of microglia. We show that irf8 mutants have normal retinal structure at 3 months post fertilization (mpf) and 6 mpf but fewer cone photoreceptors by 10 mpf. Surprisingly, light-induced photoreceptor ablation induced Müller glia proliferation in irf8 mutants and cone and rod photoreceptor regeneration. Light-damaged retinas from both wild-type and irf8 mutants show upregulated expression of mmp-9, il8, and tnfβ pro-inflammatory cytokines. Our data demonstrate that adult zebrafish irf8 mutants can regenerate normally following acute retinal injury. These findings suggest that microglia may not be essential for retinal regeneration in zebrafish and that other mechanisms can compensate for the reduction in microglia numbers., (© 2024. The Author(s).)

The mammalian retina is considered an autonomous circuit, yet work dating back to Ramon y Cajal indicates that it receives inputs from the brain. How such inputs affect retinal processing has remained unknown. We confirmed brain-to-retina projections of histaminergic neurons from the mouse hypothalamus. Histamine application ex vivo altered the activity of various retinal ganglion cells (RGCs), including direction-selective RGCs that gained responses to high motion velocities. These results were reproduced in vivo with optic tract recordings where histaminergic retinopetal axons were activated chemogenetically. Such changes could improve vision of fast-moving objects (e.g., while running), which fits with the known increased activity of histaminergic neurons during arousal. An antihistamine drug reduced optomotor responses to high-speed moving stimuli in freely moving mice. In humans, the same antihistamine nonuniformly modulated visual sensitivity across the visual field, indicating an evolutionary conserved function of the histaminergic system. Our findings expose a previously unappreciated role for brain-to-retina projections in modulating retinal function.

The blood-retina barrier (BRB), which is disrupted in diabetic retinopathy (DR) and uveitis, is an important anatomical characteristic of the retina, regulating nutrient, waste, water, protein, and immune cell flux. The BRB is composed of endothelial cell tight junctions, pericytes, astrocyte end feet, a collagen basement membrane, and perivascular macrophages. Despite the importance of the BRB, retinal perivascular macrophage function remains unknown. We found that retinal perivascular macrophages resided on postcapillary venules in the superficial vascular plexus and expressed MHC class II. Using single-cell RNA-Seq, we found that perivascular macrophages expressed a prochemotactic transcriptome and identified platelet factor 4 (Pf4, also known as CXCL4) as a perivascular macrophage marker. We used Pf4Cre mice to specifically deplete perivascular macrophages. To model retinal inflammation, we performed intraocular CCL2 injections. Ly6C+ monocytes crossed the BRB proximal to perivascular macrophages. Depletion of perivascular macrophages severely hampered Ly6C+ monocyte infiltration. These data suggest that retinal perivascular macrophages orchestrate immune cell migration across the BRB, with implications for inflammatory ocular diseases including DR and uveitis.

The incorporation of gold nanoparticles (GNPs) into retinal imaging signifies a notable advancement in ophthalmology, offering improved accuracy in diagnosis and patient outcomes. This review explores the synthesis and unique properties of GNPs, highlighting their adjustable surface plasmon resonance, biocompatibility, and excellent optical absorption and scattering abilities. These features make GNPs advantageous contrast agents, enhancing the precision and quality of various imaging modalities, including photoacoustic imaging, optical coherence tomography, and fluorescence imaging. This paper analyzes the unique properties and corresponding mechanisms based on the morphological features of GNPs, highlighting the potential of GNPs in retinal disease diagnosis and management. Given the limitations currently encountered in clinical applications of GNPs, the approaches and strategies to overcome these limitations are also discussed. These findings suggest that the properties and efficacy of GNPs have innovative applications in retinal disease imaging.

Uveitis is characterised by breakdown of the blood-retinal barrier (BRB), allowing infiltration of immune cells that mediate intraocular inflammation, which can lead to irreversible damage of the neuroretina and the loss of sight. Treatment of uveitis relies heavily on corticosteroids and systemic immunosuppression due to limited understanding of disease pathogenesis. We performed single-cell RNA-sequencing of retinas, as well as bulk RNA-sequencing of retinal pigment epithelial (RPE) cells from mice with experimental autoimmune uveitis (EAU) versus healthy control. This revealed that the Th1/Th17-driven disease induced strong gene expression changes in response to inflammation in rods, cones, Müller glia and RPE. In particular, Müller glia and RPE cells were found to upregulate expression of chemokines, complement factors, leukocyte adhesion molecules and MHC class II, thus highlighting their contributions to immune cell recruitment and antigen presentation at the inner and outer BRB, respectively. Additionally, ligand-receptor interaction analysis with CellPhoneDB revealed key interactions between Müller glia and T cell / natural killer cell subsets via chemokines, galectin-9 to P4HB/TIM-3, PD-L1 to PD-1, and nectin-2/3 to TIGIT signalling axes. Our findings elucidate mechanisms contributing to breakdown of retinal immune privilege during uveitis and identify novel targets for therapeutic interventions., (© 2024. The Author(s).)

In vertebrate retina, individual neurons of the same type are distributed regularly across the tissue in a pattern known as a mosaic. Establishment of mosaics during development requires cell-cell repulsion among homotypic neurons, but the mechanisms underlying this repulsion remain unknown. Here, we show that two mouse retinal cell types, OFF and ON starburst amacrine cells, establish mosaic spacing by using their dendritic arbors to repel neighboring homotypic somata. Using transgenic tools and single-cell labeling, we identify a developmental period when starburst somata are contacted by neighboring starburst dendrites; these serve to exclude somata from settling within the neighbor's dendritic territory. Dendrite-soma exclusion is mediated by MEGF10, a cell-surface molecule required for starburst mosaic patterning. Our results implicate dendrite-soma exclusion as a key mechanism underlying starburst mosaic spacing and raise the possibility that this could be a general mechanism for mosaic patterning across many cell types and species., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The pericyte coverage of microvessels is altered in metabolic diseases, but the mechanisms regulating pericyte-endothelial cell communication remain unclear. This study investigated the formation and function of pericyte tunneling nanotubes (TNTs) and their impact on endothelial cell metabolism. TNTs were analyzed in vitro in retinas and co-cultures of pericytes and endothelial cells. Using mass spectrometry, the influence of pericytes on endothelial cell metabolism was examined. TNTs were present in the murine retina, and although diabetes was associated with a decrease in pericyte coverage, TNTs were longer. In vitro, pericytes formed TNTs in the presence of PDGF, extending toward endothelial cells and facilitating mitochondrial transport from pericytes to endothelial cells. In experiments with mitochondria-depleted endothelial cells displaying defective TCA cycle metabolism, pericytes restored the mitochondrial network and metabolism. 19,20-Dihydroxydocosapentaenoic acid (19,20-DHDP), known to disrupt pericyte-endothelial cell junctions, prevented TNT formation and metabolic rescue in mitochondria-depleted endothelial cells. 19,20-DHDP also caused significant changes in the protein composition of pericyte-endothelial cell junctions and involved pathways related to phosphatidylinositol 3-kinase, PDGF receptor, and RhoA signaling. Pericyte TNTs contact endothelial cells and support mitochondrial transfer, influencing metabolism. This protective mechanism is disrupted by 19,20-DHDP, a fatty acid mediator linked to diabetic retinopathy.

Protocadherin 19 (Pcdh19) is a homophilic cell adhesion molecule and is involved in a variety of neuronal functions. Here, we tested whether Pcdh19 has a regulatory role in axon guidance using the developing Xenopus retinotectal system. We performed targeted microinjections of a translation blocking antisense morpholino oligonucleotide to knock down the expression of Pcdh19 selectively in the central nervous system. Knocking down Pcdh19 expression resulted in navigational errors of retinal ganglion cell (RGC) axons specifically at the optic chiasm. Instead of projecting to the contralateral optic tectum, RGC axons in the Pcdh19-depleted embryo misprojected ipsilaterally. Although incorrectly delivered into the ipsilateral brain hemisphere, these axons correctly reached the optic tectum. These data suggest that Pcdh19 has a critical role in preventing mixing of RGC axons originating from the opposite eyes at the optic chiasm, highlighting the importance of cell adhesion in bundling of RGC axons., (© 2024. The Author(s).)

The retina is light-sensitive neuronal tissue in the back of the eye. The phospholipid composition of the retina is unique and highly enriched in polyunsaturated fatty acids, including docosahexaenoic fatty acid (DHA). While it is generally accepted that a high DHA content is important for vision, surprisingly little is known about the mechanisms of DHA enrichment in the retina. Furthermore, the biological processes controlled by DHA in the eye remain poorly defined as well. Here, we combined genetic manipulations with lipidomic analysis in mice to demonstrate that acyl-CoA synthetase 6 (Acsl6) serves as a regulator of the unique composition of retinal membranes. Inactivation of Acsl6 reduced the levels of DHA-containing phospholipids, led to progressive loss of light-sensitive rod photoreceptor neurons, attenuated the light responses of these cells, and evoked distinct transcriptional response in the retina involving the Srebf1/2 (sterol regulatory element binding transcription factors 1/2) pathway. This study identifies one of the major enzymes responsible for DHA enrichment in the retinal membranes and introduces a model allowing an evaluation of rod functioning and pathology caused by impaired DHA incorporation/retention in the retina., (© 2024. The Author(s).)

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in several physiological light responses. In this study, we generate an improved Opn4 cre knockin allele (Opn4 cre(DSO) ), which faithfully reproduces endogenous Opn4 expression and improves compatibility with widely used reporters. We evaluated the efficacy and sensitivity of Opn4 cre(DSO) for labeling in retina and brain and provide an in-depth comparison with the extensively utilized Opn4 cre(Saha) line. Through this characterization, Opn4 cre(DSO) demonstrated higher specificity in labeling ipRGCs with minimal recombination escape. Leveraging a combination of electrophysiological, molecular, and morphological analyses, we confirmed its sensitivity in detecting all ipRGC types (M1-M6) and defined their unique topographical distribution across the retina. In the brain, the Opn4 cre(DSO) line labels ipRGC projections with minimal labeling of cell bodies. Overall, the Opn4 cre(DSO) mouse line represents an improved tool for studying ipRGC function and distribution, offering a means to selectively target these cells to study light-regulated behaviors and physiology., Competing Interests: Declaration of interests The authors declare no competing financial or non-financial interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The retina is increasingly recognised as a potential source of biomarkers for neurodegenerative diseases. Hallmark protein aggregates in the retinal neuronal tissue could be imaged through light non-invasively. Post-mortem studies have already shown the presence of specific hallmark proteins in Alzheimer's disease, primary tauopathies, synucleinopathies and frontotemporal lobar degeneration. This study aims to assess proteinopathy in a post-mortem cohort with different neurodegenerative diseases and assess the presence of the primary pathology in the retina. Post-mortem eyes were collected in collaboration with the Netherlands Brain Bank from donors with Alzheimer's disease (n = 17), primary tauopathies (n = 8), synucleinopathies (n = 27), frontotemporal lobar degeneration (n = 8), mixed pathology (n = 11), other neurodegenerative diseases (n = 6), and cognitively normal controls (n = 25). Multiple cross sections of the retina and optic nerve tissue were immunostained using antibodies against pTau Ser202/Thr205 (AT8), amyloid-beta (4G8), alpha-synuclein (LB509), pTDP-43 Ser409/410 and p62-lck ligand (p62) and were assessed for the presence of aggregates and inclusions. pTau pathology was observed as a diffuse signal in Alzheimer's disease, primary tauopathies and controls with Alzheimer's disease neuropathological changes. Amyloid-beta was observed in the vessel wall and as cytoplasmic granular deposits in all groups. Alpha-synuclein pathology was observed as Lewy neurites in the retina in synucleinopathies associated with Lewy pathology and as oligodendroglial cytoplasmic inclusions in the optic nerve in multiple system atrophy. Anti-pTDP-43 generally showed typical neuronal cytoplasmic inclusion bodies in cases with frontotemporal lobar degeneration with TDP-43 and also in cases with later stages of limbic-associated TDP-43 encephalopathy. P62 showed inclusion bodies similar to those seen with anti-pTDP-43. Furthermore, pTau and alpha-synuclein pathology were significantly associated with increasing Braak stages for neurofibrillary tangles and Lewy bodies, respectively. Mixed pathology cases in this cohort consisted of cases (n = 6) with high Braak LB stages (> 4) and low or moderate AD pathology, high AD pathology (n = 1, Braak NFT 6, Thal phase 5) with moderate LB pathology, or a combination of low/moderate scores for different pathology scores in the brain (n = 4). There were no cases with advanced co-pathologies. In seven cases with Braak LB ≥ 4, LB pathology was observed in the retina, while tau pathology in the retina in the mixed pathology group (n = 11) could not be observed. From this study, we conclude that the retina reflects the presence of the major hallmark proteins associated with neurodegenerative diseases. Although low or moderate levels of copathology were found in the brains of most cases, the retina primarily manifested protein aggregates associated with the main neurodegenerative disease. These findings indicate that with appropriate retinal imaging techniques, retinal biomarkers have the potential to become highly accurate indicators for diagnosing the major neurodegenerative diseases of the brain., (© 2024. The Author(s).)

Neurite outgrowth is a crucial process for organizing neuronal circuits in neuronal development and regeneration after injury. Regenerative failure in the adult mammalian central nervous system (CNS) is attributed to axonal growth inhibitors such as the Nogo protein that commonly binds to Nogo receptor-1 (NgR1). We previously reported that lateral olfactory tract usher substance (LOTUS) functions as an endogenous antagonist for NgR1 in forming neuronal circuits in the developing brain and improving axonal regeneration in the adult injured CNS. However, another molecular and cellular function of LOTUS remains unknown. In this study, we found that cultured retinal explant neurons extend their neurites on the LOTUS-coating substrate. This action was also observed in cultured retinal explant neurons derived from Ngr1 -deficient mouse embryos, indicating that the promoting action of LOTUS on neurite outgrowth may be mediated by unidentified LOTUS-binding protein(s). We therefore screened the binding partner(s) of LOTUS by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS analysis and pull-down assay showed that LOTUS interacts with Teneurin-4 (Ten-4), a cell adhesion molecule. RNAi knockdown of Ten-4 inhibited neurite outgrowth on the LOTUS substrate in retinoic acid (RA)-treated Neuro2A cells. Furthermore, a soluble form of Ten-4 attenuates the promoting action on neurite outgrowth in cultured retinal explant neurons on the LOTUS substrate. These results suggest that LOTUS promotes neurite outgrowth by interacting with Ten-4. Our findings may provide a new molecular mechanism of LOTUS to contribute to neuronal circuit formation in development and to enhance axonal regeneration after CNS injury.

This study investigated the therapeutic effects of exosomes derived from human-induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) on corneal epithelial wound healing. Exosomes were isolated from the culture medium of the hiPSC-derived ROs (Exo-ROs) using ultracentrifugation, and then they were characterized by a nanoparticle tracking analysis and transmission electron microscopy. In a murine model of corneal epithelial wounds, these exosomes were topically applied to evaluate their healing efficacy. The results demonstrated that the exosome-treated eyes showed significantly enhanced wound closures compared with the controls at 24 h post-injury. The 5-ethyl-2'-deoxyuridine assay and quantitative reverse transcription polymerase chain reaction revealed a substantial increase in cell proliferation and a decrease in inflammatory marker contents in the exosome-treated group. The RNA sequencing and exosomal microRNA analysis revealed that the Exo-RO treatment targeted various pathways related to inflammation and cell proliferation, including the PI3K-Akt, TNF, MAPK, and IL-17 signaling pathways. Moreover, the upregulation of genes related to retinoic acid and eicosanoid metabolism may have enhanced corneal epithelial healing in the eyes treated with the Exo-ROs. These findings suggest that hiPSC-derived RO exosomes could be novel therapeutic agents for promoting corneal epithelial wound healing.

The pupillary light reflex (PLR) adapts the amount of light reaching the retina, protecting it and improving image formation. Two PLR mechanisms have been described in vertebrates. First, the pretectum receives retinal inputs and projects to the Edinger-Westphal nucleus (EWN), which targets the ciliary ganglion through the oculomotor nerve (nIII). Postganglionic fibers enter the eye-globe, traveling to the iris sphincter muscle. Additionally, some vertebrates exhibit an iris-intrinsic PLR mechanism mediated by sphincter muscle cells that express melanopsin inducing muscle contraction. Given the high degree of conservation of the lamprey visual system, we investigated the mechanisms underlying the PLR to shed light onto their evolutionary origins. Recently, a PLR mediated by melanopsin was demonstrated in lampreys, suggested to be brain mediated. Remarkably, we found that PLR is instead mediated by direct retino-iridal cholinergic projections. This retina-mediated PLR acts synergistically with an iris-intrinsic mechanism that, as in other vertebrates, is mediated by melanopsin and has contribution of gap junctions between muscle fibers. In contrast, we show that lampreys lack the brain-mediated PLR. Our results suggest that two eye-intrinsic PLR mechanisms were present in early vertebrate evolution, whereas the brain-mediated PLR has a more recent origin., (© 2024. The Author(s).)

This study asked whether the P2X7 receptor was necessary and sufficient to trigger astrocyte polarization into neuroinflammatory activation states. Intravitreal injection of agonist BzATP increased gene expression of pan-astrocyte activation markers Gfap , Steap4 , and Vim and A1-type astrocyte activation markers C3 , Serping1 , and H2T23 , but also the Cd14 and Ptx3 genes usually associated with the A2-type astrocyte activation state and Tnfa , IL1a , and C1qa , assumed to be upstream of astrocyte activation in microglia. Correlation analysis of gene expression suggested the P2X7 receptor induced a mixed A1/A2-astrocyte activation state, although A1-state genes like C3 increased the most. A similar pattern of mixed glial activation genes occurred one day after intraocular pressure (IOP) was elevated in wild-type mice, but not in P2X7 -/- mice, suggesting the P2X7 receptor is necessary for the glial activation that accompanies IOP elevation. In summary, this study suggests stimulation of the P2X7R is necessary and sufficient to trigger the astrocyte activation in the retina following IOP elevation, with a rise in markers for pan-, A1-, and A2-type astrocyte activation. The P2X7 receptor is expressed on microglia, optic nerve head astrocytes, and retinal ganglion cells (RGCs) in the retina, and can be stimulated by the mechanosensitive release of ATP that accompanies IOP elevation. Whether the P2X7 receptor connects this mechanosensitive ATP release to microglial and astrocyte polarization in glaucoma remains to be determined.

Microglia (μG), the resident immune cells in the central nervous system, surveil the parenchyma to maintain the structural and functional homeostasis of neurons. Besides, they influence neurogenesis and synaptogenesis through complement-mediated phagocytosis. Emerging evidence suggests that μG may also influence development through proinflammatory cytokines. Here, we examined the premise that tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), the two most prominent components of the μG secretome, influence retinal development, specifically the morphological and functional differentiation of human retinal ganglion cells (hRGCs). Using controlled generation of hRGCs and human μG (hμG) from pluripotent stem cells, we demonstrate that TNF-α and IL-1β secreted by unchallenged hμG did not influence hRGC generation. However, their presence significantly facilitated neuritogenesis along with the basal function of hRGCs, which involved the recruitment of the AKT/mTOR pathway. We present ex vivo evidence that proinflammatory cytokines may play an important role in the morphological and physiological maturation of hRGCs, which may be recapitulated for regeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Biallelic mutations in DRAM2 lead to an autosomal recessive cone-rod dystrophy known as CORD21, which typically presents between the third and sixth decades of life. Although DRAM2 localizes to the lysosomes of photoreceptor and retinal pigment epithelium (RPE) cells, its specific role in retinal degeneration has not been fully elucidated. In this study, we generated and characterized retinal organoids (ROs) and RPE cells from induced pluripotent stem cells (iPSCs) derived from two CORD21 patients. Our investigation revealed that CORD21-ROs and RPE cells exhibit abnormalities in lipid metabolism, defects in autophagic flux, accumulation of aberrant lysosomal content, and reduced lysosomal enzyme activity. We identified potential interactions of DRAM2 with vesicular trafficking proteins, suggesting its involvement in this cellular process. These findings collectively suggest that DRAM2 plays a crucial role in maintaining the integrity of photoreceptors and RPE cells by regulating lysosomal function, autophagy, and potentially vesicular trafficking., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Subretinal fibrosis is a major untreatable cause of poor outcomes in neovascular age-related macular degeneration. Mouse models of subretinal fibrosis all possess a degree of invasiveness and tissue damage not typical of fibrosis progression. This project characterises JR5558 mice as a model to study subretinal fibrosis. Fundus and optical coherence tomography (OCT) imaging was used to non-invasively track lesions. Lesion number and area were quantified with ImageJ. Retinal sections, wholemounts and Western blots were used to characterise alterations. Subretinal lesions expand between 4 and 8 weeks and become established in size and location around 12 weeks. Subretinal lesions were confirmed to be fibrotic, including various cell populations involved in fibrosis development. Müller cell processes extended from superficial retina into subretinal lesions at 8 weeks. Western blotting revealed increases in fibronectin (4 wk and 8 wk, p < 0.001), CTGF (20 wks, p < 0.001), MMP2 (12 wks and 20 wks p < 0.05), αSMA (12 wks and 20 wks p < 0.05) and GFAP (8 wk and 12 wk, p ≤ 0.01), consistent with our immunofluorescence results. Intravitreal injection of Aflibercept reduced subretinal lesion growth. Our study provides evidence JR5558 mice have subretinal fibrotic lesions that grow between 4 and 8 weeks and confirms this line to be a good model to study subretinal fibrosis development and assess treatment options., (© 2024. Crown.)

Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases., Competing Interests: The study was funded by Santen Pharmaceutical Co. Ltd and Tokiyoshi Matsushita, Takahiro Matsuyama, and Masaaki Kageyama are employed by Santen Pharmaceutical Co., Ltd. Tokiyoshi Matsushita, Akishi Onishi, and Masayo Takahashi are co-inventors of a patent related to this work, filed by RIKEN and Santen Pharmaceutical Co., Ltd [WO2022138803A1]. None of these alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Matsushita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The development of the retina is under tight temporal and spatial control. To gain insights into the molecular basis of this process, we generate a single-nuclei dual-omic atlas of the human developing retina with approximately 220,000 nuclei from 14 human embryos and fetuses aged between 8 and 23-weeks post-conception with matched macular and peripheral tissues. This atlas captures all major cell classes in the retina, along with a large proportion of progenitors and cell-type-specific precursors. Cell trajectory analysis reveals a transition from continuous progression in early progenitors to a hierarchical development during the later stages of cell type specification. Both known and unrecorded candidate transcription factors, along with gene regulatory networks that drive the transitions of various cell fates, are identified. Comparisons between the macular and peripheral retinae indicate a largely consistent yet distinct developmental pattern. This atlas offers unparalleled resolution into the transcriptional and chromatin accessibility landscapes during development, providing an invaluable resource for deeper insights into retinal development and associated diseases., (© 2024. The Author(s).)

Shp2, a critical SH2-domain-containing tyrosine phosphatase, is essential for cellular regulation and implicated in metabolic disruptions, obesity, diabetes, Noonan syndrome, LEOPARD syndrome, and cancers. This study focuses on Shp2 in rod photoreceptor cells, revealing its enrichment, particularly in rods. Deletion of Shp2 in rods leads to age-dependent photoreceptor degeneration. Shp2 targets occludin (OCLN), a tight junction protein, and its deletion reduces OCLN expression in the retina and retinal pigment epithelium (RPE). The isolation of actively translating mRNAs from rods lacking Shp2, followed by RNA sequencing, reveals alterations in cell cycle regulation. Additionally, altered retinal metabolism is observed in retinal cells lacking Shp2. Our studies indicate that Shp2 is crucial for maintaining the structure and function of photoreceptors., (© 2024. The Author(s).)

Due to the large number of genes and mutations that result in inherited retinal degenerations (IRD), there has been a paucity of therapeutic options for these patients. There is a large unmet need for therapeutic approaches targeting shared pathophysiologic pathways in a mutation-independent manner. The Fas receptor is a major activator and regulator of retinal cell death and inflammation in a variety of ocular diseases. We previously reported the activation of Fas-mediated photoreceptor (PR) cell death in two different IRD mouse models, rd10 and P23H, and demonstrated the protective effect of genetic Fas inhibition. The purpose of this study was to examine the effects of pharmacologic inhibition of Fas in these two models by intravitreal injection with a small peptide inhibitor of the Fas receptor, ONL1204. A single intravitreal injection of ONL1204 was given to one eye of rd10 mice at P14. Two intravitreal injections of ONL1204 were given to the P23H mice, once at P14 and again at 2-months of age. The fellow eyes were injected with vehicle alone. Fas activation, rate of PR cell death, retinal function, and the activation of immune cells in the retina were evaluated. In both rd10 and P23H mice, ONL1204 treatment resulted in decreased number of TUNEL (+) PRs, decreased caspase 8 activity, enhanced photoreceptor cell counts, and improved visual function compared with vehicle treated fellow eyes. Treatment with ONL1204 also reduced immune cell activation in the retinas of both rd10 and P23H mice. The protective effect of pharmacologic inhibition of Fas by ONL1204 in two distinct mouse models of retinal degeneration suggests that targeting this common pathophysiologic mechanism of cell death and inflammation represents a potential therapeutic approach to preserve the retina in patients with IRD, regardless of the genetic underpinning., (© 2024. The Author(s).)

Primary cilia are antenna-like structures protruding from the surface of various eukaryotic cells, and have distinct protein compositions in their membranes. This distinct protein composition is maintained by the presence of the transition zone (TZ) at the ciliary base, which acts as a diffusion barrier between the ciliary and plasma membranes. Defects in cilia and the TZ are known to cause a group of disorders collectively called the ciliopathies, which demonstrate a broad spectrum of clinical features, such as perinatally lethal Meckel syndrome (MKS), relatively mild Joubert syndrome (JBTS), and nonsyndromic nephronophthisis (NPHP). Proteins constituting the TZ can be grouped into the MKS and NPHP modules. The MKS module is composed of several transmembrane proteins and three soluble proteins. TMEM218 was recently reported to be mutated in individuals diagnosed as MKS and JBTS. However, little is known about how TMEM218 mutations found in MKS and JBTS affect the functions of cilia. In this study, we found that ciliary membrane proteins were not localized to cilia in TMEM218-knockout cells, indicating impaired barrier function of the TZ. Furthermore, the exogenous expression of JBTS-associated TMEM218 variants but not MKS-associated variants in TMEM218-knockout cells restored the localization of ciliary membrane proteins. In particular, when expressed in TMEM218-knockout cells, the TMEM218(R115H) variant found in JBTS was able to restore the barrier function of cells, whereas the MKS variant TMEM218(R115C) could not. Thus, the severity of symptoms of MKS and JBTS individuals appears to correlate with the degree of their ciliary defects at the cellular level., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mutations in ABCA4 are the most common cause of Mendelian retinal disease. Clinical evaluation of this gene is challenging because of its extreme allelic diversity, the large fraction of non-exomic mutations, and the wide range of associated disease. We used patient-derived retinal organoids as well as DNA samples and clinical data from a large cohort of patients with ABCA4-associated retinal disease to investigate the pathogenicity of a variant in ABCA4 (IVS30 + 1321 A>G) that occurs heterozygously in 2% of Europeans. We found that this variant causes mis-splicing of the gene in photoreceptor cells such that the resulting protein contains 36 incorrect amino acids followed by a premature stop. We also investigated the phenotype of 10 patients with compound genotypes that included this mutation. Their median age of first vision loss was 39 years, which is in the mildest quintile of a large cohort of patients with ABCA4 disease. We conclude that the IVS30 + 1321 A>G variant can cause disease when paired with a sufficiently deleterious opposing allele in a sufficiently permissive genetic background., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Neovascular eye diseases, including proliferative diabetic retinopathy and retinopathy of prematurity, is a major cause of blindness. Laser ablation and intravitreal anti-VEGF injection have shown their limitations in treatment of retinal neovascularization. Identification of a new therapeutic strategies is in urgent need. Our study aims to assess the effects of Cryptotanshinone (CPT), a natural compound derived from Salvia miltiorrhiza Bunge, in retina neovascularization and explore its potential mechanism. Our study demonstrated that CPT did not cause retina tissue toxicity at the tested concentrations. Intravitreal injections of CPT reduced pathological angiogenesis and promoted physical angiogenesis in oxygen-induced retinopathy (OIR) model. CPT improve visual function in OIR mice and reduced cell apoptosis. Moreover, we also revealed that CPT diminishes the expression of inflammatory cytokines in the OIR retina. In vitro, the administration of CPT effectively inhibited endothelial cells proliferation, migration, sprouting, and tube formation induced by the stimulation of human retinal vascular endothelial cells (HRVECs) with VEGF165. Mechanistically, CPT blocking the phosphorylation of VEGFR2 and downstream targeting pathway. After all, the findings demonstrated that CPT exhibits potent anti-angiogenic and anti-inflammatory effects in OIR mice, and it has therapeutic potential for the treatment of neovascular retinal diseases., Competing Interests: Declaration of competing interest The authors assert that they do not have any known financial interests or personal relationships that might have influenced the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The photopigment-encoding visual opsin genes that mediate color perception show great variation in copy number and adaptive function across vertebrates. An open question is how this variation has been shaped by the interaction of lineage-specific structural genomic architecture and ecological selection pressures. We contribute to this issue by investigating the expansion dynamics and expression of the duplicated Short-Wavelength-Sensitive-1 opsin (SWS1) in sea snakes (Elapidae). We generated one new genome, 45 resequencing datasets, 10 retinal transcriptomes, and 81 SWS1 exon sequences for sea snakes, and analyzed these alongside 16 existing genomes for sea snakes and their terrestrial relatives. Our analyses revealed multiple independent transitions in SWS1 copy number in the marine Hydrophis clade, with at least three lineages having multiple intact SWS1 genes: the previously studied Hydrophis cyanocinctus and at least two close relatives of this species; Hydrophis atriceps and Hydrophis fasciatus; and an individual Hydrophis curtus. In each lineage, gene copy divergence at a key spectral tuning site resulted in distinct UV and Violet/Blue-sensitive SWS1 subtypes. Both spectral variants were simultaneously expressed in the retinae of H. cyanocinctus and H. atriceps, providing the first evidence that these SWS1 expansions confer novel phenotypes. Finally, chromosome annotation for nine species revealed shared structural features in proximity to SWS1 regardless of copy number. If these features are associated with SWS1 duplication, expanded opsin complements could be more common in snakes than is currently recognized. Alternatively, selection pressures specific to aquatic environments could favor improved chromatic distinction in just some lineages., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

(233/250) Retinal vein occlusion (RVO) causes macular edema and retinal ischemia resulting in visual field and vision loss. A bispecific antibody that blocks VEGF-A and angiopoietin-2 (Ang-2) has been recently launched and applied clinically to treat macular edema, but the role of Ang-2 in the pathogenesis of RVO is still unclear. In this study, we investigated the effects of the anti-VEGF-A/anti-Ang-2 bispecific antibody (BsAb) in a murine RVO model. By using RVO model mice, the expression of Ang-2 gene and protein was examined in the retina through real-time qPCR and Western blotting, respectively. A significant increase in Ang-2 was detected 1 day after occlusion. Immediately after occlusion, control IgG 400 μg/mL, anti-VEGF-A antibody 200 μg/mL, anti-Ang-2 antibody 200 μg/mL, and BsAb 400 μg/mL were intravitreally administered at 2 μL. Visual function was examined using electroretinograms, and apoptosis was examined using TUNEL staining. Interestingly, BsAb partially suppressed the decrease in amplitude of a and b waves compared to control IgG. Anti-Ang-2 antibody and BsAb reduced apoptosis-positive cells 1 day after occlusion. Comprehensive gene expression profiles were also examined using RNA sequencing analysis. RNA sequencing analysis of the retinal tissues showed that BsAb suppressed expression of gene groups associated with inflammatory response and vascular development compared to anti-VEGF-A antibody. Taken together, higher expression of Ang-2 contributes to the pathophysiology of RVO, providing a possible mechanism for the efficacy of BsAb in suppressing retinal dysfunction in RVO., Competing Interests: Declaration of competing interest S. Nakamura, M. Shimazawa, and H. Hara are financial competing interests (Chugai Pharmaceutical Co., Ltd.). Y. Inokuchi, H. Abe, K. Nagaoka, and T. Soeda are employed at Chugai Pharmaceutical Co., Ltd. A. Kuriyama, H. Yasuda and Y. Hidaka declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Diabetic retinopathy (DR) is one of the most prevalent secondary complications associated with diabetes. Specifically, Type 1 Diabetes Mellitus (T1D) has an immune component that may determine the evolution of DR by compromising the immune response of the retina, which is mediated by microglia. In the early stages of DR, the permeabilization of the blood-retinal barrier allows immune cells from the peripheral system to interact with the retinal immune system. The use of new bioactive molecules, such as 3-(2,4-dihydroxyphenyl)phthalide (M9), with powerful anti-inflammatory activity, might represent an advance in the treatment of diseases like DR by targeting the immune systems responsible for its onset and progression. Our research aimed to investigate the molecular mechanisms involved in the interaction of specific cells of the innate immune system during the progression of DR and the reduction in inflammatory processes contributing to the pathology. In vitro studies were conducted exposing Bv.2 microglial and Raw264.7 macrophage cells to proinflammatory stimuli for 24 h, in the presence or absence of M9. Ex vivo and in vivo approaches were performed in BB rats, an animal model for T1D. Retinal explants from BB rats were cultured with M9. Retinas from BB rats treated for 15 days with M9 via intraperitoneal injection were analyzed to determine survival, cellular signaling, and inflammatory markers using qPCR, Western blot, or immunofluorescence approaches. Retinal structure images were acquired via Spectral-Domain-Optical Coherence Tomography (SD-OCT). Our results show that the treatment with M9 significantly reduces inflammatory processes in in vitro, ex vivo, and in vivo models of DR. M9 works by inhibiting the proinflammatory responses during DR progression mainly affecting immune cell responses. It also induces an anti-inflammatory response, primarily mediated by microglial cells, leading to the synthesis of Arginase-1 and Hemeoxygenase-1(HO-1). Ultimately, in vivo administration of M9 preserves the retinal integrity from the degeneration associated with DR progression. Our findings demonstrate a specific interaction between both retinal and systemic immune cells in the progression of DR, with a differential response to treatment, mainly driven by microglia in the anti-inflammatory action. In vivo treatment with M9 induces a switch in immune cell phenotypes and functions that contributes to delaying the DR progression, positioning microglial cells as a new and specific therapeutic target in DR.

Retinitis pigmentosa (RP), an inherited retinal disease, affects 1,5 million people worldwide. The initial mutation-driven photoreceptor degeneration leads to chronic inflammation, characterized by Müller cell activation and upregulation of CD44. CD44 is a cell surface transmembrane glycoprotein and the primary receptor for hyaluronic acid. It is involved in many pathological processes, but little is known about CD44's retinal functions. CD44 expression is also increased in Müller cells from our Pde6b STOP/STOP RP mouse model. To gain a more detailed understanding of CD44's role in healthy and diseased retinas, we analyzed Cd44 -/- and Cd44 -/- Pde6b STOP/STOP mice, respectively. The loss of CD44 led to enhanced photoreceptor degeneration, reduced retinal function, and increased inflammatory response. To understand the underlying mechanism, we performed proteomic analysis on isolated Müller cells from Cd44 -/- and Cd44 -/- Pde6b STOP/STOP retinas and identified a significant downregulation of glutamate transporter 1 (SLC1A2). This downregulation was accompanied by higher glutamate levels, suggesting impaired glutamate homeostasis. These novel findings indicate that CD44 stimulates glutamate uptake via SLC1A2 in Müller cells, which in turn, supports photoreceptor survival and function., (© 2024. The Author(s).)

The transcription factor forkhead/winged-helix domain proteins Foxp1 and Foxp2 have previously been studied in mouse retina, where they are expressed in retinal ganglion cells named F-mini and F-midi. Here we show that both transcription factors are expressed by small subpopulations (on average less than 10%) of retinal ganglion cells in the retina of the marmoset monkey (Callithrix jacchus). The morphology of Foxp1- and Foxp2-expressing cells was revealed by intracellular DiI injections of immunofluorescent cells. Foxp1- and Foxp2-expressing cells comprised multiple types of wide-field ganglion cells, including broad thorny cells, narrow thorny cells, and tufted cells. The large majority of Foxp2-expressing cells were identified as tufted cells. Tufted cells stratify broadly in the middle of the inner plexiform layer. They resemble broad thorny cells but their proximal dendrites are bare of branches and the distal dendrites branch frequently forming dense dendritic tufts. Double labeling with calretinin, a previously established marker for broad thorny and narrow thorny cells, showed that only a small proportion of ganglion cells co-expressed calretinin and Foxp1 or Foxp2 supporting the idea that the two markers are differentially expressed in retinal ganglion cells of marmoset retina., (© 2024 The Author(s). Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Introduction: Diabetic retinopathy is characterised by retinal vascular impairment. A number of aberrant microRNAs (miRNAs) have a role in the pathophysiology of vascular dysfunction. However, the relevance of miR-424 in retinal vascular endothelial cell dysfunction during hyperglycemia stress remains unknown. The purpose of this study is to investigate this issue., Materials and Methods: Rhesus macaque choroid retinal endothelial cell line (RF/6A) cells were cultivated in normal glucose (NG) and high glucose (HG) conditions. The mRNA expression of miR-424 and Cyclin D1 (CCND1) was quantified using qPCR, and the protein quantity of CCND1 was detected using Western Blot. miR-424 mimics, miR-424 inhibitors, miR-424 inhibitor+ siRNA-CCND1 or vehicle molecules were transfected into RF/6A cells. MTT test was used to assess cell proliferation, and flow cytometric analysis was used to assess cell cycle. The interaction between miR-424 and CCND1 was predicted using bioinformatics and validated using dual luciferase reporter analysis., Results: miR-424 was up-regulated, and cell viability was reduced in HG compared to NG. By reversing the expression of miR-424 in certain situations, the phenotypes can be changed. CCND1 has been identified as a miR-424 target gene, and it may be regulated at the transcriptional and translational levels. Manipulation of silencing CCND1 can counteract the effect of transfecting miR-424 inhibitor into RF/6A cells under HG such as proliferation stimulation., Conclusions: Our findings indicate that miR-424 plays an important role in hyperglycemia induced ARPE-19 cells damage, and it could be a new therapeutic target for DR by preventing retinal vascular cells from HG-induced injury.

Branched-chain amino acids (BCAAs) play vital roles in metabolic and physiological processes, with their catabolism initiated by two branched-chain aminotransferase isozymes: cytosolic (BCATc) and mitochondrial (BCATm). These enzymes have tissue and cell-specific compartmentalization and are believed to shuttle metabolites between cells and tissues. Although their expression and localization have been established in most tissues, ocular tissues remain unknown. In this study, we used immunohistochemical analyses to investigate the expression and localization of BCAT enzymes in the normal eye tissues. As expected, BCATc was highly expressed in the neuronal cells of the retina, particularly in the ganglion cell layers, inner nuclear layer, and plexiform layer, with little to no expression in Müller cells. BCATc was also present in the cornea, retinal pigment epithelium (RPE), choroid, ciliary body, and iris but not in the lens. In contrast, BCATm was expressed across all ocular tissues, with strong expression in the Muller cells of the retina, the endothelial and epithelial layers of the cornea, the choroid and iris, and the epithelial cells at the lens's front. The extensive expression and distribution of BCAT isozymes in the ocular tissue, suggests that BCAA transamination is widespread in the eye, potentially aiding in metabolite transport between ocular tissues. The findings provide new insights into the physiological role of BCATs in the eye, particularly within the neuronal retina., Competing Interests: Competing InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met., New Method: This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo., Results: We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN + cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture., Comparison With Existing Methods: Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation., Conclusions: This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Jiansu Chen and Yonglong Guo reports financial support was provided by National Natural Science Foundation of China. Jiansu Chen reports a relationship with National Natural Science Foundation of China that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

To prevent ocular pathologies, new generation of dietary supplements have been commercially available. They consist of nutritional supplement mixing components known to provide antioxidative properties, such as unsaturated fatty acid, resveratrol or flavonoids. However, to date, few data evaluating the impact of a mixture mainly composed of those components (Nutrof Total®) on the retina are available. Only one in-vivo preclinical study demonstrated that dietary supplementation (DS) prevents the retina from light-induced retinal degeneration; and only one in-vitro study on Müller cells culture showed that glutamate metabolism cycle was key in oxidative stress response. Therefore, we raised the question about the in-vivo effect of DS on glutamate metabolism in the retina. Herein, we showed that the dietary supplementation promotes in-vivo increase of retinal glutamine amount through a higher glutamine synthesis as observed in-vitro on Muller cells. Therefore, we can suggest that the promotion of glutamine synthesis is part of the protective effect of DS against retinal degeneration, acting as a preconditioning mechanism against retinal degeneration., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Diabetic retinopathy has a high probability of causing visual impairment or blindness throughout the disease progression and is characterized by the growth of new blood vessels in the retina at an advanced, proliferative stage. Microglia are a resident immune population in the central nervous system, known to play a crucial role in regulating retinal angiogenesis in both physiological and pathological conditions, including diabetic retinopathy. Physiologically, they are located close to blood vessels and are essential for forming new blood vessels (neovascularization). In diabetic retinopathy, microglia become widely activated, showing a distinct polarization phenotype that leads to their accumulation around neovascular tufts. These activated microglia induce pathogenic angiogenesis through the secretion of various angiogenic factors and by regulating the status of endothelial cells. Interestingly, some subtypes of microglia simultaneously promote the regression of neovascularization tufts and normal angiogenesis in neovascularization lesions. Modulating the state of microglial activation to ameliorate neovascularization thus appears as a promising potential therapeutic approach for managing diabetic retinopathy., (© 2024. The Author(s).)

Intimate links between epigenome modifications and metabolites allude to a crucial role of cellular metabolism in transcriptional regulation. Retina, being a highly metabolic tissue, adapts by integrating inputs from genetic, epigenetic, and extracellular signals. Precise global epigenomic signatures guide development and homeostasis of the intricate retinal structure and function. Epigenomic and metabolic realignment are hallmarks of aging and highlight a link of the epigenome-metabolism nexus with aging-associated multifactorial traits affecting the retina, including age-related macular degeneration and glaucoma. Here, we focus on emerging principles of epigenomic and metabolic control of retinal gene regulation, with emphasis on their contribution to human disease. In addition, we discuss potential mitigation strategies involving lifestyle changes that target the epigenome-metabolome relationship for maintaining retinal function., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Ltd.)

Circadian rhythms are biological rhythms that originate from the "master circadian clock," called the suprachiasmatic nucleus (SCN). SCN orchestrates the circadian rhythms using light as a chief zeitgeber, enabling humans to synchronize their daily physio-behavioral activities with the Earth's light-dark cycle. However, chronic/ irregular photic disturbances from the retina via the retinohypothalamic tract (RHT) can disrupt the amplitude and the expression of clock genes, such as the period circadian clock 2, causing circadian rhythm disruption (CRd) and associated neuropathologies. The present review discusses neuromodulation across the RHT originating from retinal photic inputs and modulation offered by endocannabinoids as a function of mitigation of the CRd and associated neuro-dysfunction. Literature indicates that cannabinoid agonists alleviate the SCN's ability to get entrained to light by modulating the activity of its chief neurotransmitter, i.e., γ-aminobutyric acid, thus preventing light-induced disruption of activity rhythms in laboratory animals. In the retina, endocannabinoid signaling modulates the overall gain of the retinal ganglion cells by regulating the membrane currents (Ca 2+ , K + , and Cl - channels) and glutamatergic neurotransmission of photoreceptors and bipolar cells. Additionally, endocannabinoids signalling also regulate the high-voltage-activated Ca 2+ channels to mitigate the retinal ganglion cells and intrinsically photosensitive retinal ganglion cells-mediated glutamate release in the SCN, thus regulating the RHT-mediated light stimulation of SCN neurons to prevent excitotoxicity. As per the literature, cannabinoid receptors 1 and 2 are becoming newer targets in drug discovery paradigms, and the involvement of endocannabinoids in light-induced CRd through the RHT may possibly mitigate severe neuropathologies., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased 'fate bias'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics., Competing Interests: Competing interests J.S.M. holds patents for the NTR inducible cell ablation system (US 7,514,595) and uses thereof (US 8,071,838 and US 8431768)., (© 2024. Published by The Company of Biologists Ltd.)

Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro . Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo .

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are the world's leading causes of blindness. The retinal pigment epithelium (RPE) and vascular endothelial cell exposed to oxidative stress is the major cause of AMD and DR. DJ-1, an important endogenous antioxidant, its overexpression is considered as a promising antioxidant treatment for AMD and DR. Here, we modified the tetrahedral frame nucleic acids (tFNAs) with DJ-1 saRNAs as a delivery system, and synthesized a novel nanocomplex (tFNAs-DJ-1 saRNAs). In vitro studies show that tFNAs-DJ-1 saRNAs can efficiently transfer DJ-1 saRNAs to human umbilical vein endothelial cells (HUVECs) and ARPE-19s, and significantly increased their cellular DJ-1 level. Reactive oxygen species expression in H 2 O 2 -treated HUVECs and ARPE-19s were decreased, cell viability was enhanced and cell apoptosis were inhibited when tFNAs-DJ-1 saRNAs were delivered. Moreover, tFNAs-DJ-1 saRNAs preserved mitochondrial structure and function under oxidative stress conditions. In the aspect of molecular mechanism, tFNAs-DJ-1 saRNAs activated Erk and Nrf2 pathway, which might contribute to its protective effects against oxidative stress damage. To conclude, this study shows the successfully establishment of a simple but effective delivery system of DJ-1 saRNAs associated with antioxidant effects in AMD and DR, which may be a promising agent for future treatment in oxidative stress-related retinal disorders., (© 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Purpose: This study aims to explore the metabolic signature of aging retina and identify the potential metabolic biomarkers for the diagnosis of retinal aging., Methods: Retinal samples were collected from both young (two months) and aging (14 months) mice to conduct an unbiased metabolic profiling. Liquid chromatography-tandem mass spectrometry analysis was conducted to screen for the metabolic biomarkers and altered signaling pathways associated with retinal aging., Results: We identified 166 metabolites differentially expressed between young and aged retinas using a threshold of orthogonal projection to latent structures discriminant analysis variable importance in projection >1 and P < 0.05. These metabolites were significantly enriched in several metabolic pathways, including purine metabolism, citrate cycle, phenylalanine, tyrosine and tryptophan biosynthesis, glycerophospholipid metabolism, and alanine, aspartate and glutamate metabolism. Among these significantly enriched pathways, glycerophospholipid metabolites emerged as promising candidates for retinal aging biomarkers. We assessed the potential of these metabolites as biomarkers through an analysis of their sensitivity and specificity, determined by the area under the receiver-operating characteristic (ROC) curves. Notably, the metabolites like PC (15:0/22:6), PC (17:0/14:1), LPC (P-16:0), PE (16:0/20:4), and PS (17:0/16:1) demonstrated superior performance in sensitivity, specificity, and accuracy in predicting retinal aging., Conclusions: This study sheds light on the molecular mechanisms underlying retinal aging by identifying distinct metabolic profiles and pathways. These findings provide a valuable foundation for developing future clinical applications in diagnosing, identifying, and treating age-related retinal degeneration., Translational Relevance: This study sheds light on novel metabolic profiles and biomarkers in aging retinas, potentially paving the way for targeted interventions in preventing, diagnosing, and treating age-related retinal degeneration and other retinal diseases.

Background: 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., Methods: 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% O 2 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% O 2 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), O 2 with PBS (O 2 -PBS), O 2  + IC100 intravitreal injection (O 2 -IC100-IVT), and O 2  + IC100 intraperitoneal injection (O 2 -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., Results: 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 O 2 . IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O 2 ., Conclusion: 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., (© 2024. The Author(s).)

Current diabetic retinopathy (DR) treatment involves blood glucose regulation combined with laser photocoagulation or intravitreal injection of vascular endothelial growth factor (VEGF) antibodies. However, due to the complex pathogenesis and cross-interference of multiple biochemical pathways, these interventions cannot block disease progression. Recognizing the critical role of the retinal microenvironment (RME) in DR, it is hypothesized that reshaping the RME by simultaneously inhibiting primary and secondary blood-retinal barrier (BRB) injury can attenuate DR. For this, a glucose-responsive hydrogel named Cu-PEI/siMyD88@GEMA-Con A (CSGC) is developed that effectively delivers Cu-PEI/siMyD88 nanoparticles (NPs) to the retinal pigment epithelium (RPE). The Cu-PEI NPs act as antioxidant enzymes, scavenging ROS and inhibiting RPE pyroptosis, ultimately blocking primary BRB injury by reducing microglial activation and Th1 differentiation. Simultaneously, MyD88 expression silence in combination with the Cu-PEI NPs decreases IL-18 production, synergistically reduces VEGF levels, and enhances tight junction proteins expression, thus blocking secondary BRB injury. In summary, via remodeling the RME, the CSGC hydrogel has the potential to disrupt the detrimental cycle of cross-interference between primary and secondary BRB injury, providing a promising therapeutic strategy for DR., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Human interleukin-33 (IL-33) is a 270 amino acid protein that belongs to the IL-1 cytokine family and plays an important role in various inflammatory disorders. Neutrophil proteases (Cathepsin G and Elastase) and mast cell proteases (tryptase and chymase) regulate the activity of IL-33 by processing full-length IL-33 into its mature form. There is little evidence on the role of these mature forms of IL-33 in retinal endothelial cell signaling and pathological retinal angiogenesis. Here, we cloned, expressed, and purified the various mature forms of human IL-33 and then evaluated the effects of IL-33 95-270 , IL-33 99-270 , IL-33 109-270 , and IL-33 112-270 on angiogenesis in human retinal microvascular endothelial cells (HRMVECs). We observed that IL-33 95-270 , IL-33 99-270 , IL-33 109-270 , and IL-33 112-270 significantly induced HRMVEC migration, tube formation and sprouting angiogenesis. However, only IL-33 99-270 could induce HRMVEC proliferation. We used a murine model of oxygen-induced retinopathy (OIR) to assess the role of these mature forms of IL-33 in pathological retinal neovascularization. Our 3'-mRNA sequencing and signaling studies indicated that IL-33 99-270 and IL-33 109-270 were more potent at inducing endothelial cell activation and angiogenesis than the other mature forms. We found that genetic deletion of IL-33 significantly reduced OIR-induced retinal neovascularization in the mouse retina and that intraperitoneal administration of mature forms of IL-33, mainly IL-33 99-270 and IL-33 109-270 , significantly restored ischemia-induced angiogenic sprouting and tuft formation in the hypoxic retinas of IL-33 -/- mice. Thus, our study results suggest that blockade or inhibition of IL-33 cleavage by neutrophil proteases could help mitigate pathological angiogenesis in proliferative retinopathies., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The Omi/HtrA2 inhibitor 5-[5-(2-nitrophenyl) furfuryliodine]-1,3-diphenyl-2-thiobarbituric acid (Ucf-101) has shown neuroprotective effects in the central nervous system. However, whether Ucf-101 can protect retinal ganglion cells (RGCs) after retinal ischemia/reperfusion (IR) has not been investigated. We aimed to investigate the effects of Ucf-101 on RGCs apoptosis and inflammation after IR-induced retinal injury in mice. We injected Ucf-101 into the mouse vitreous body immediately after IR injury. After 7 days, hematoxylin and eosin staining was conducted to assess retinal tissue damage. Next, retrograde labeling with FluoroGold, counting of RGCs and TUNEL staining were conducted to evaluate apoptosis. Immunohistochemistry, immunofluorescence staining, and western blotting were conducted to analyze protein levels. IR injury-induced retinal tissue damage could be prevented by Ucf-101 treatment. The number of TUNEL-positive RGCs was reduced by Ucf-101 treatment in mice with IR injury. Ucf-101 treatment inhibited the upregulation of Bax, cleaved caspase-3 and cleaved caspase-9 and activated the JNK/ERK/P38 signaling pathway. Furthermore, Ucf-101 treatment inhibited the upregulation of glial fibrillary acidic protein (GFAP), vimentin, Iba1 and CD68 in mice with IR injury. Ucf-101 prevents retinal tissue damage, improves the survival of RGCs, and suppresses microglial overactivation after IR injury. Ucf-101 might be a potential target to prevent RGCs apoptosis and inflammation in neurodegenerative eye diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Neuroinflammation and endothelial cell apoptosis are prominent features of blood-brain barrier (BBB) disruption, which have been described in Alzheimer's disease (AD) and can predict cognitive decline. Recent reports revealed vascular β-amyloid (Aβ) deposits, Muller cell degeneration and microglial dysfunction in the retina of AD patients. However, there has been no in-depth research on the roles of inflammation, retinal endothelial cell apoptosis, and blood-retinal barrier (BRB) damage in AD retinopathy. We found that Raddeanin A (RDA) could improve pathological and cognitive deficits in a mouse model of Alzheimer's disease by targeting β-amyloidosis, However, the effects of RDA on AD retinal function require further study. To clarify whether RDA inhibits inflammation and apoptosis and thus improves BRB function in AD-related retinopathy. In vitro we used Aβ-treated HRECs and MIO-M1 cells, and in vivo we used 3×Tg-AD mice to investigate the effect of RDA on BRB in AD-related retinopathy. We found that RDA could improve BRB function in AD-related retinopathy by inhibiting NLRP3-mediated inflammation and suppressing Wnt/β-catenin pathway-mediated apoptosis, which is expected to improve the pathological changes in AD-related retinopathy and the quality of life of AD patients., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aging is a major risk factor for the development or the worsening of retinal degenerative conditions. The intricate network of the neural retina determined that the retinal aging is a complicated process. The aim of this study is to delineate the transcriptomic changes of major retinal neurons during aging in C57BL/6 mice at single-cell level. We analyzed the transcriptional profiles of the photoreceptor, bipolar, amacrine, and Müller glial cells of 1.5-2 and 24-30 months old mice using single-cell RNA sequencing technique. We selectively confirmed the differences in gene expression using immunofluorescence staining and RNA in situ hybridization analysis. We found that each retinal cell type had unique changes upon aging. However, they all showed signs of dysregulated glucose and energy metabolism, and perturbed proteostasis. In particular, old Müller glia exhibited the most profound changes, including the upregulation of cell metabolism, stress-responses, antigen-presentation and immune responses and metal ion homeostasis. The dysregulated gliogenesis and differentiation was confirmed by the presence of Müller glia expressing rod-specific genes in the inner nuclear layer and the outer plexiform layer of the old retina. We further pinpointed the specific loss of GABAergic amacrine cells in old retina. Our study emphasized changes of amacrine and Müller glia during retinal aging, provided resources for further research on the molecular and cellular regulatory mechanisms underlying aging-associated retinal deterioration., Competing Interests: Declaration of competing interest There is no conflict of interest related to the submitted paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)