Mendelian randomization study and mediation analysis about the relation of inflammatory bowel disease and diabetic retinopathy: the further exploration of gut-retina axis.

Background: The concept of the gut-retinal axis proposed by previous scholars primarily focused on the relationship between intestinal microbiota and retinal diseases, and few further expanded the relationship between intestinal diseases and retinal diseases. To further substantiate the concept of the gut-retinal axis, we analyzed inflammatory bowel disease (IBD) and diabetic retinopathy (DR) using Mendelian randomization (MR), and use mediation analysis to further explore the potential substances that influence this causal relationship. Methods: The genome-wide association study's (GWAS) summary statistics for genetic variations were utilized in a Mendelian randomization (MR) investigation. GWAS data on IBD (including ulcerative colitis (UC), Crohn's disease (CD), and IBD) for non-Finnish Europeans (NFE) were sourced from published articles. In contrast, data on DR (including DR and diabetic maculopathy (DMP)) were obtained from FinnGen R9. The causal relationship has been investigated using inverse variance weighted (IVW), MR-Egger, and weighted median and sensitivity analysis was applied to verify the stability of the results. In addition, we applied mediation analysis to investigate whether circulating inflammatory proteins and plasma lipids played a mediating role, and calculated its effect ratio. Results: The causal relationship between IBD and DR was discovered by employing the inverse variance weighted (IVW) method and weighted median method. In forward MR, UC was significantly associated with lower risk of DR (IVW: OR=0.874; 95%CI= 0.835-0.916; P value= 1.28E-08) (Weighted median: OR=0.893; 95%CI= 0.837-0.954; P value= 7.40E-04). In reverse MR, it was shown that DR (IVW: OR=0.870; 95%CI= 0.828-0.914; P value= 2.79E-08)(Weighted median: OR=0.857; 95%CI= 0.801-0.916; P value= 6.40E-06) and DMP (IVW: OR=0.900; 95%CI= 0.865-0.937; P value= 3.34E-07)(Weighted median: OR=0.882; 95%CI= 0.841-0.924; P value= 1.82E-07) could reduce the risk of CD. What's more, DR is associated with a lower risk of IBD according to genetic prediction (IVW: OR=0.922; 95%CI= 0.873-0.972; P value= 0.002) (Weighted median: OR=0.924; 95%CI= 0.861-0.992; P value= 0.029). Fibroblast growth factor 21 (FGF21), phosphatidylcholine (PC), and triacylglycerol (TG) serve as mediators in these relationships. Conclusions: Our research offers novel insights and sources for investigating the gut-retina axis in the genetic relationship between IBD and DR. We discover four mediators and more about the association between the intestine and retinal disorders and provide more evidence for the gut-retinal axis theory.

Primary cilia formation requires the Leigh syndrome-associated mitochondrial protein NDUFAF2.

Mitochondria-related neurodegenerative diseases have been implicated in the disruption of primary cilia function. Mutation in an intrinsic mitochondrial complex I component NDUFAF2 has been identified in Leigh syndrome, a severe inherited mitochondriopathy. Mutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome, a ciliopathy with defects in the brain, kidney, and eye. Here, we report a mechanistic link between mitochondria metabolism and primary cilia signaling. We discovered that loss of NDUFAF2 caused both mitochondrial and ciliary defects in vitro and in vivo and identified NDUFAF2 as a binding partner for ARMC9. We also found that NDUFAF2 was both necessary and sufficient for cilia formation and that exogenous expression of NDUFAF2 rescued the ciliary and mitochondrial defects observed in cells from patients with known ARMC9 deficiency. NAD+ supplementation restored mitochondrial and ciliary dysfunction in ARMC9-deficient cells and zebrafish and ameliorated the ocular motility and motor deficits of a patient with ARMC9 deficiency. The present results provide a compelling mechanistic link, supported by evidence from human studies, between primary cilia and mitochondrial signaling. Importantly, our findings have significant implications for the development of therapeutic approaches targeting ciliopathies.

Insulin resistance in the retina: possible implications for certain ocular diseases.

Insulin resistance (IR) is becoming a worldwide medical and public health challenge as an increasing prevalence of obesity and metabolic disorders. Accumulated evidence has demonstrated a strong relationship between IR and a higher incidence of several dramatically vision-threatening retinal diseases, including diabetic retinopathy, age-related macular degeneration, and glaucoma. In this review, we provide a schematic overview of the associations between IR and certain ocular diseases and further explore the possible mechanisms. Although the exact causes explaining these associations have not been fully elucidated, underlying mechanisms of oxidative stress, chronic low-grade inflammation, endothelial dysfunction and vasoconstriction, and neurodegenerative impairments may be involved. Given that IR is a modifiable risk factor, it may be important to identify patients at a high IR level with prompt treatment, which may decrease the risk of developing certain ocular diseases. Additionally, improving IR through the activation of insulin signaling pathways could become a potential therapeutic target.

Mechanistic and therapeutic perspectives of non-coding RNA-modulated apoptotic signaling in diabetic retinopathy.

Diabetic retinopathy (DR), a significant and vision-endangering complication associated with diabetes mellitus, constitutes a substantial portion of acquired instances of preventable blindness. The progression of DR appears to prominently feature the loss of retinal cells, encompassing neural retinal cells, pericytes, and endothelial cells. Therefore, mitigating the apoptosis of retinal cells in DR could potentially enhance the therapeutic approach for managing the condition by suppressing retinal vascular leakage. Recent advancements have highlighted the crucial regulatory roles played by non-coding RNAs (ncRNAs) in diverse biological processes. Recent advancements have highlighted that non-coding RNAs (ncRNAs), including microRNAs (miRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs), act as central regulators in a wide array of biogenesis and biological functions, exerting control over gene expression associated with histogenesis and cellular differentiation within ocular tissues. Abnormal expression and activity of ncRNAs has been linked to the regulation of diverse cellular functions such as apoptosis, and proliferation. This implies a potential involvement of ncRNAs in the development of DR. Notably, ncRNAs and apoptosis exhibit reciprocal regulatory interactions, jointly influencing the destiny of retinal cells. Consequently, a thorough investigation into the complex relationship between apoptosis and ncRNAs is crucial for developing effective therapeutic and preventative strategies for DR. This review provides a fundamental comprehension of the apoptotic signaling pathways associated with DR. It then delves into the mutual relationship between apoptosis and ncRNAs in the context of DR pathogenesis. This study advances our understanding of the pathophysiology of DR and paves the way for the development of novel therapeutic strategies.

Diacylglycerol Signaling in Retinal Ganglion Cells.

With impaired retinal ganglion cell (RGC) function and eventual RGC death, there is a heightened risk of experiencing glaucoma-induced blindness or other optic neuropathies. Poor RGC efficiency leads to limited transmission of visual signals between the retina and the brain by RGC axons. Increased focus on studying lipid messengers found in neurons such as endocannabinoids (eCBs) has importance due to their potential axonal pathway regenerative properties. 2-Arachidonoylglycerol (2-AG), a common eCB, is synthesized from an sn-1 hydrolysis reaction between diacylglycerol (DAG) and diacylglycerol lipase (DAGL). Examination of DAG production allows for future downstream analysis in relation to DAGL functionality. Here, we describe protocol guidelines for extracting RGCs from mouse retinas and subsequent mass spectrometry analysis of the DAG content present within the RGCs.

The Prodrug DHED Delivers 17ß-Estradiol into the Retina for Protection of Retinal Ganglion Cells and Preservation of Visual Function in an Animal Model of Glaucoma.

We report a three-pronged phenotypic evaluation of the bioprecursor prodrug 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED) that selectively produces 17ß-estradiol (E2) in the retina after topical administration and halts glaucomatous neurodegeneration in a male rat model of the disease. Ocular hypertension (OHT) was induced by hyperosmotic saline injection into an episcleral vein of the eye. Animals received daily DHED eye drops for 12 weeks. Deterioration of visual acuity and contrast sensitivity by OHT in these animals were markedly prevented by the DHED-derived E2 with concomitant preservation of retinal ganglion cells and their axons. In addition, we utilized targeted retina proteomics and a previously established panel of proteins as preclinical biomarkers in the context of OHT-induced neurodegeneration as a characteristic process of the disease. The prodrug treatment provided retina-targeted remediation against the glaucomatous dysregulations of these surrogate endpoints without increasing circulating E2 levels. Collectively, the demonstrated significant neuroprotective effect by the DHED-derived E2 in the selected animal model of glaucoma supports the translational potential of our presented ocular neuroprotective approach owing to its inherent therapeutic safety and efficacy.

Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development.

The proneural transcription factor atonal basic helix-loop-helix transcription factor 7 (ATOH7) is expressed in early progenitors in the developing neuroretina. In vertebrates, this is crucial for the development of retinal ganglion cells (RGCs), as mutant animals show an almost complete absence of RGCs, underdeveloped optic nerves, and aberrations in retinal vessel development. Human mutations are rare and result in autosomal recessive optic nerve hypoplasia (ONH) or severe vascular changes, diagnosed as autosomal recessive persistent hyperplasia of the primary vitreous (PHPVAR). To better understand the role of ATOH7 in neuroretinal development, we created ATOH7 knockout and eGFP-expressing ATOH7 reporter human induced pluripotent stem cells (hiPSCs), which were differentiated into early-stage retinal organoids. Target loci regulated by ATOH7 were identified by Cleavage Under Targets and Release Using Nuclease with sequencing (CUT&RUN-seq) and differential expression by RNA sequencing (RNA-seq) of wildtype and mutant organoid-derived reporter cells. Additionally, single-cell RNA sequencing (scRNA-seq) was performed on whole organoids to identify cell type-specific genes. Mutant organoids displayed substantial deficiency in axon sprouting, reduction in RGCs, and an increase in other cell types. We identified 469 differentially expressed target genes, with an overrepresentation of genes belonging to axon development/guidance and Notch signaling. Taken together, we consolidate the function of human ATOH7 in guiding progenitor competence by inducing RGC-specific genes while inhibiting other cell fates. Furthermore, we highlight candidate genes responsible for ATOH7-associated optic nerve and retinovascular anomalies, which sheds light to potential future therapy targets for related disorders.

Selective Assembly of TRPC Channels in the Rat Retina during Photoreceptor Degeneration.

Transient receptor potential canonical (TRPC) channels are calcium channels with diverse expression profiles and physiological implications in the retina. Neurons and glial cells of rat retinas with photoreceptor degeneration caused by retinitis pigmentosa (RP) exhibit basal calcium levels that are above those detected in healthy retinas. Inner retinal cells are the last to degenerate and are responsible for maintaining the activity of the visual cortex, even after complete loss of photoreceptors. We considered the possibility that TRPC1 and TRPC5 channels might be associated with both the high calcium levels and the delay in inner retinal degeneration. TRPC1 is known to mediate protective effects in neurodegenerative processes while TRPC5 promotes cell death. In order to comprehend the implications of these channels in RP, the co-localization and subsequent physical interaction between TRPC1 and TRPC5 in healthy retina (Sprague-Dawley rats) and degenerating (P23H-1, a model of RP) retina were detected by immunofluorescence and proximity ligation assays. There was an overlapping signal in the innermost retina of all animals where TRPC1 and TRPC5 physically interacted. This interaction increased significantly as photoreceptor loss progressed. Both channels function as TRPC1/5 heteromers in the healthy and damaged retina, with a marked function of TRPC1 in response to retinal degenerative mechanisms. Furthermore, our findings support that TRPC5 channels also function in partnership with STIM1 in Müller and retinal ganglion cells. These results suggest that an increase in TRPC1/5 heteromers may contribute to the slowing of the degeneration of the inner retina during the outer retinal degeneration.

Genetic and Epigenetic Biomarkers Linking Alzheimer's Disease and Age-Related Macular Degeneration.

Alzheimer's disease (AD) represents a prominent neurodegenerative disorder (NDD), accounting for the majority of dementia cases worldwide. In addition to memory deficits, individuals with AD also experience alterations in the visual system. As the retina is an extension of the central nervous system (CNS), the loss in retinal ganglion cells manifests clinically as decreased visual acuity, narrowed visual field, and reduced contrast sensitivity. Among the extensively studied retinal disorders, age-related macular degeneration (AMD) shares numerous aging processes and risk factors with NDDs such as cognitive impairment that occurs in AD. Histopathological investigations have revealed similarities in pathological deposits found in the retina and brain of patients with AD and AMD. Cellular aging processes demonstrate similar associations with organelles and signaling pathways in retinal and brain tissues. Despite these similarities, there are distinct genetic backgrounds underlying these diseases. This review comprehensively explores the genetic similarities and differences between AMD and AD. The purpose of this review is to discuss the parallels and differences between AMD and AD in terms of pathophysiology, genetics, and epigenetics.

Thyroid Hormone Signaling in Retinal Development and Function: Implications for Diabetic Retinopathy and Age-Related Macular Degeneration.

Thyroid Hormones (THs) play a central role in the development, cell growth, differentiation, and metabolic homeostasis of neurosensory systems, including the retina. The coordinated activity of various components of TH signaling, such as TH receptors (THRs) and the TH processing enzymes deiodinases 2 and 3 (DIO2, DIO3), is required for proper retinal maturation and function of the adult photoreceptors, Müller glial cells, and pigmented epithelial cells. Alterations of TH homeostasis, as observed both in frank or subclinical thyroid disorders, have been associated with sight-threatening diseases leading to irreversible vision loss i.e., diabetic retinopathy (DR), and age-related macular degeneration (AMD). Although observational studies do not allow causal inference, emerging data from preclinical models suggest a possible correlation between TH signaling imbalance and the development of retina disease. In this review, we analyze the most important features of TH signaling relevant to retinal development and function and its possible implication in DR and AMD etiology. A better understanding of TH pathways in these pathological settings might help identify novel targets and therapeutic strategies for the prevention and management of retinal disease.

Single-cell transcriptomic analysis reveals the antiangiogenic role of Mgarp in diabetic retinopathy.

INTRODUCTION: Diabetic retinopathy (DR) is a common vascular complication of diabetes mellitus and a leading cause of vision loss worldwide. Endothelial cell (EC) heterogeneity has been observed in the pathogenesis of DR. Elucidating the underlying mechanisms governing EC heterogeneity may provide novel insights into EC-specific therapies for DR. RESEARCH DESIGN AND METHODS: We used the single-cell data from the Gene Expression Omnibus database to explore EC heterogeneity between diabetic retinas and non-diabetic retinas and identify the potential genes involved in DR. CCK-8 assays, EdU assays, transwell assays, and tube formation assays were conducted to determine the role of the identified gene in angiogenic effects. RESULTS: Our analysis identified three distinct EC subpopulations in retinas and revealed that Mitochondria-localized glutamic acid-rich protein (Mgarp) gene is potentially involved in the pathogenesis of DR. Silencing of Mgarp significantly suppressed the proliferation, migration, and tube formation capacities in retinal endothelial cells. CONCLUSIONS: This study not only offers new insights into transcriptomic heterogeneity and pathological alteration of retinal ECs but also holds the promise to pave the way for antiangiogenic therapy by targeting EC-specific gene.

Mutant mice with rod-specific VPS35 deletion exhibit retinal α-synuclein pathology-associated degeneration.

Vacuolar protein sorting 35 (VPS35), the core component of the retromer complex which regulates endosomal trafficking, is genetically linked with Parkinson's disease (PD). Impaired vision is a common non-motor manifestation of PD. Here, we show mouse retinas with VPS35-deficient rods exhibit synapse loss and visual deficit, followed by progressive degeneration concomitant with the emergence of Lewy body-like inclusions and phospho-α-synuclein (P-αSyn) aggregation. Ultrastructural analyses reveal VPS35-deficient rods accumulate aggregates in late endosomes, deposited as lipofuscins bound to P-αSyn. Mechanistically, we uncover a protein network of VPS35 and its interaction with HSC70. VPS35 deficiency promotes sequestration of HSC70 and P-αSyn aggregation in late endosomes. Microglia which engulf lipofuscins and P-αSyn aggregates are activated, displaying autofluorescence, observed as bright dots in fundus imaging of live animals, coinciding with pathology onset and progression. The Rod∆Vps35 mouse line is a valuable tool for further mechanistic investigation of αSyn lesions and retinal degenerative diseases.

Triptonide protects retinal cells from oxidative damage via activation of Nrf2 signaling.

Age­related macular degeneration (AMD) is an ocular disease that threatens the visual function of older adults worldwide. Key pathological processes involved in AMD include oxidative stress, inflammation and choroidal vascular dysfunction. Retinal pigment epithelial cells and Müller cells are most susceptible to oxidative stress. Traditional herbal medicines are increasingly being investigated in the field of personalized medicine in ophthalmology. Triptonide (Tn) is a diterpene tricyclic oxide, the main active ingredient in the extract from the Chinese herbal medicinal plant Tripterygium wilfordii, and is considered an effective immunosuppressant and anti­inflammatory drug. The present study investigated the potential beneficial role of Tn in retinal oxidative damage in order to achieve personalized treatment for early AMD. An oxidative stress model of retinal cells induced by H2O2 and a retinal injury model of mice induced by light and N­Methyl­D­aspartic acid were constructed. In vitro, JC­1 staining, flow cytometry and apoptosis assay confirmed that low concentrations of Tn effectively protected retinal cells from oxidative damage, and reverse transcription­quantitative PCR and western blotting analyses revealed that Tn reduced the expression of retinal oxidative stress­related genes and inflammatory factors, which may depend on the PI3K/AKT/mTOR­induced Nrf2 signaling pathway. In vivo, by retinal immunohistochemistry, hematoxylin and eosin staining and electroretinogram assay, it was found that retinal function and structure improved and choroidal neovascularization was significantly inhibited after Tn pretreatment. These results suggested that Tn is an efficient Nrf2 activator, which can be expected to become a new intervention for diseases such as AMD, to inhibit retinal oxidative stress damage and pathological neovascularization.

Cell-Penetrating Chaperone Nuc1 for Small- and Large-Molecule Delivery Into Retinal Cells and Tissues.

Purpose: There are currently no means available for the efficient delivery of recombinant proteins into retinal cells in vivo. Although cell-penetrating peptides have been somewhat effective in protein delivery to the retina, they generally require conjugation chemistry with the payload, negatively impacting function of the therapeutic protein. In this study, we developed a novel peptide (Nuc1) that acts as a chaperone for delivery of small and large molecules, including steroids, peptides, antibodies, recombinant proteins, and viruses (adeno-associated viruses [AAVs]) across biological membranes in vivo without the need for conjugation. Methods: Nuc1 peptide was designed based on sequences known to bind heparan sulfate proteoglycans and nucleolin found on the surface of retinal cells. Nuc1 was injected into the vitreous of mice with a variety of molecules and retinas examined for uptake and function of these molecules. Results: Nuc1 engages the process of macropynocytosis for cell entry. The delivery of functional recombinant X-linked inhibitor of apoptosis protein to photoreceptors via the intravitreal route of injection inhibited retinal apoptosis. Nuc1 was found to enhance the delivery of anti-VEGF antibodies delivered intravitreally or topically in models of age-related macular degeneration (AMD). Nuc1 enhanced delivery of decorin, facilitating significant inhibition of neovascularization and fibrosis in a model of AMD. Finally, Nuc1 was found to enhance penetration of retinal cells and tissues by AAV via both the subretinal and intravitreal routes of injection. Conclusions: Nuc1 shows promise as a novel approach for the delivery of recombinant proteins into retinal cells in vivo.

Safety of intravitreally delivered AAV2 vector-mediated multi-characteristic opsin genetic construct in wild type beagle dogs.

BACKGROUND: A novel adeno-associated virus 2 (AAV2)-carried multi-characteristic opsin (MCO) (MCO-010) is undergoing several clinical trials as a novel therapeutic modality for the treatment of degenerative retinal diseases including retinitis pigmentosa and Stargardt disease. The present study aimed to determine the ocular and systemic safety of MCO-010 and the AAV2 vehicle in adult Beagle dogs following intravitreal (IVT) injection. METHODS: The current safety/toxicology studies spanning 13 weeks described here utilized well-documented techniques to assess the effects of IVT injection of MCO-010 up to 2.2 × 1011 genome copies (gc) per eye, or the AAV2 capsid (vehicle control) on gross behavioral and immunogenic changes, alterations in body weights, blood biochemistry, hematology, blood coagulation, gross necropsy lesions, organ weight changes and histopathology in the dogs (n = 4 per group; two males and two females per group). Immunohistochemical and functional electroretinogram studies were also conducted to determine MCO expression in the retina and determine any retinal toxicity associated with MCO-010. RESULTS: There were no significant deleterious effects of the MCO-010 (or the AAV2 at the tested doses) on any of the examined parameters, including the absence of any severe ocular or systemic adverse events. However, as expected, inflammation after IVT delivery of AAV2 and MCO-010 was observed in the conjunctivae of all groups of animals, although this self-resolved within 1 week post-injection. Quantitative immunohistochemical analyses of MCO-010-associated mCherry revealed successful delivery of the gene therapy within the inner retina. CONCLUSIONS: In summary, MCO-010 demonstrated a favorable safety profile when administered to the eyes of adult Beagle dogs of both sexes at dose levels up to 2.2 × 1011 gc per eye, with no adverse effects observed. This dose was identified as the No Observed Adverse Effect Level (i.e. NOAEL) and guided selection of safe doses for human clinical trials.

Characterization of Ocular Morphology in Col4a3-/- Mice as a Murine Model for Alport Syndrome.

Purpose: The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. Methods: The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Results: Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000 µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Conclusions: Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. Translational Relevance: This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.

Deletion of myeloid HDAC3 promotes efferocytosis to ameliorate retinal ischemic injury.

Ischemia-induced retinopathy is a hallmark finding of common visual disorders including diabetic retinopathy (DR) and central retinal artery and vein occlusions. Treatments for ischemic retinopathies fail to improve clinical outcomes and the design of new therapies will depend on understanding the underlying disease mechanisms. Histone deacetylases (HDACs) are an enzyme class that removes acetyl groups from histone and non-histone proteins, thereby regulating gene expression and protein function. HDACs have been implicated in retinal neurovascular injury in preclinical studies in which nonspecific HDAC inhibitors mitigated retinal injury. Histone deacetylase 3 (HDAC3) is a class I histone deacetylase isoform that plays a central role in the macrophage inflammatory response. We recently reported that myeloid cells upregulate HDAC3 in a mouse model of retinal ischemia-reperfusion (IR) injury. However, whether this cellular event is an essential contributor to retinal IR injury is unknown. In this study, we explored the role of myeloid HDAC3 in ischemia-induced retinal neurovascular injury by subjecting myeloid-specific HDAC3 knockout (M-HDAC3 KO) and floxed control mice to retinal IR. The M-HDAC3 KO mice were protected from retinal IR injury as shown by the preservation of inner retinal neurons, vascular integrity, and retinal thickness. Electroretinography confirmed that this neurovascular protection translated to improved retinal function. The retinas of M-HDAC3 KO mice also showed less proliferation and infiltration of myeloid cells after injury. Interestingly, myeloid cells lacking HDAC3 more avidly engulfed apoptotic cells in vitro and after retinal IR injury in vivo compared to wild-type myeloid cells, suggesting that HDAC3 hinders the reparative phagocytosis of dead cells, a process known as efferocytosis. Further mechanistic studies indicated that although HDAC3 KO macrophages upregulate the reparative enzyme arginase 1 (A1) that enhances efferocytosis, the inhibitory effect of HDAC3 on efferocytosis is not solely dependent on A1. Finally, treatment of wild-type mice with the HDAC3 inhibitor RGFP966 ameliorated the retinal neurodegeneration and thinning caused by IR injury. Collectively, our data show that HDAC3 deletion enhances macrophage-mediated efferocytosis and protects against retinal IR injury, suggesting that inhibiting myeloid HDAC3 holds promise as a novel therapeutic strategy for preserving retinal integrity after ischemic insult.

YTHDC1 aggravates high glucose-induced retinal vascular endothelial cell injury via m6A modification of CDK6.

OBJECTIVE: Retinal vascular endothelial cell (RVECs) injury is a major cause of morbidity and mortality among the patients with diabetes. RVECs dysfunction is the predominant pathological manifestation of vascular complication in diabetic retinopathy. N6-methyladenosine (m6A) serves as the most prevalent modification in eukaryotic mRNAs. However, the role of m6A RNA modification in RVECs dysfunction is still unclear. METHODS: RT-qPCR analysis and western blot were conducted to detect the change of m6A RNA modification in diabetic retinopathy. CCK-8 assay, transwell experiment, wound healing assay, tube formation experiment, m6A-IP-qPCR were performed to determine the role of YTHDC1 in RVECs. Retinal trypsin digestion test and H&E staining were used to evaluate histopathological changes. RESULTS: The levels of m6A RNA methylation were significantly up-regulated in HG-induced RVECs, which were caused by increased expression of YTHDC1. YTHDC1 regulated the viability, proliferation, migration and tube formation ability in vitro. YTHDC1 overexpression impaired RVECs function by repressing CDK6 expression, which was mediated by YTHDC1-dependent mRNA decay. Moreover, it showed sh-YTHDC1 inhibited CDK6 nuclear export. Sh-YTHDC1 promotes the mRNA degradation of CDK6 in the nucleus but does not affect the cytoplasmic CDK6 mRNA. In vivo experiments showed that overexpression of CDK6 reversed the protective effect of sh-YTHDC1 on STZ-induced retinal tissue damage. CONCLUSION: YTHDC1-mediated m6A methylation regulates diabetes-induced RVECs dysfunction. YTHDC1-CDK6 signaling axis could be therapeutically targeted for treating DR.

Evolutionary conservation of VSX2 super-enhancer modules in retinal development.

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.

In Vivo Correlation Between Macular Pigment Optical Volume and Retinal Layers Thickness.

Purpose: This study aims to investigate the potential in vivo relationship between macular pigment (MP) and retinal layers thickness in healthy subjects and dry, non-advanced age-related macular degeneration (AMD). Methods: An observational, cross-sectional study was conducted. Healthy subjects >40 years and patients with early or intermediate AMD were recruited. Structural OCT and macular pigment optical volume (MPOV) were collected for each subject. Retinal layers parameters were calculated based on the standard early treatment diabetic retinopathy study (ETDRS) map. Additionally, MPOV within 1°, 2°, and 9° of eccentricity was assessed and associated with retinal layers thickness and volume. Linear mixed-effects models were used to test the relationship between MP and structural OCT parameters, while adjusting for known possible confounding factors. Results: A total of 144 eyes of 91 subjects (60.4% females) were evaluated, comprising 43% normal eyes and 57% with early/intermediate AMD. Among the retinal layers, only the outer nuclear layer (ONL) thickness and volume appeared to be associated to higher MP levels. Specifically, the central ONL thickness was identified as a significant predictor of the MPOV 1°(P = 0.04), while the parafoveal ONL thickness (inner ETDRS subfield) was identified as a significant fixed effect on the MPOV 9° (P = 0.037). Age and the presence of drusen or subretinal drusenoid deposits were also tested without showing significant correlations. Conclusions: Among the retinal layers examined, only the ONL thickness demonstrated a significant association with MPOV. Consequently, ONL thickness might serve as a potential biomarker related to MP levels.