CX3CL1/CX3CR1 Signaling Mediated Neuroglia Activation Is Implicated in the Retinal Degeneration: A Potential Therapeutic Target to Prevent Photoreceptor Death.

Purpose: Retinal degeneration (RD) is a large cluster of retinopathies that is characterized by the progressive photoreceptor death and visual impairments. CX3CL1/CX3CR1 signaling has been documented to mediate the microglia activation and gliosis reaction during neurodegeneration. We intend to verify whether the CX3CL1/CX3CR1 signaling is involved in the RD pathology. Methods: A pharmacologically induced RD mice model was established. AZD8797, a CX3CR1 antagonist, was injected into the vitreous cavity of an RD model to modulate the neuroglia activation. Then, the experimental animals were subjected to functional, morphological, and behavioral analysis. Results: The CX3CL1/CX3CR1 signaling mediated neuroglia activation was implicated in the photoreceptor demise of an RD model. Intravitreal injection of AZD8797 preserved the retinal structure and enhanced the photoreceptor survival through inhibiting the CX3CL1/CX3CR1 expressions. Fundus photography showed that the distribution of retinal vessel was clear, and the severity of lesions was alleviated by AZD8797. In particular, these morphological benefits could be translated into remarkable functional improvements, as evidenced by the behavioral test and electroretinogram (mf-ERG) examination. A mechanism study showed that AZD8797 mitigated the microglia activation and migration in the degenerative retinas. The Müller cell hyper-reaction and secondary gliosis response were also suppressed by AZD8797. Conclusions: The neuroinflammation is implicated in the photoreceptor loss of RD pathology. Targeting the CX3CL1/CX3CR1 signaling may serve as an effective therapeutic strategy. Future refinements of these findings may cast light into the discovery of new medications for RD.

Ferroptosis in the ageing retina: A malevolent fire of diabetic retinopathy.

Ageing retina is prone to ferroptosis due to the iron accumulation and impaired efficiency of intracellular antioxidant defense system. Ferroptosis acts as a cell death modality that is characterized by the iron-dependent accumulation of lipid peroxidation. Ferroptosis is distinctively different from other types of regulated cell death (RCD) at the morphological, biochemical, and genetic levels. Diabetic retinopathy (DR) is a common microvascular complication of diabetes. Its prevalence and severity increase progressively with age. Recent reports have shown that ferroptosis is implicated in the pathophysiology of DR. Under hyperglycemia condition, the endothelial cell and retinal pigment epithelium (RPE) cell will undergo ferroptosis, which contributes to the increased vascular permeability and the disrupted blood retinal barrier (BRB). The underlying etiology of DR can be attributed to the impaired BRB integrity and subsequent damages of the neurovascular units. In the absence of timely intervention, the compromised BRB can ultimately cause profound visual impairments. In particular, the ageing retina is vulnerable to ferroptosis, and hyperglycemia will accelerate the progression of this pathological process. In this article, we discuss the contributory role of ferroptosis in DR pathogenesis, and summarize recent therapeutic trials that targeting the ferroptosis. Further study on the ferroptosis mediated damage would enrich our knowledge of DR pathology, and promote the development of clinical treatment for this degenerative retinopathy.

Intravitreal Injection of ZYAN1 Restored Autophagy and Alleviated Oxidative Stress in Degenerating Retina via the HIF-1α/BNIP3 Pathway.

Mitochondrial autophagy plays a contributary role in the pathogenesis of retina degeneration (RD). ZYAN1 is a novel proline hydroxylase domain (PHD) inhibitor that can enhance the expression of hypoxia-inducible factor 1-alpha (HIF-1α). This study investigated whether ZYAN1 could alleviate progressive photoreceptor loss and oxidative damage in a pharmacologically induced RD model via the modulation of mitophagy. ZYAN1 was injected into the vitreous body of the RD model, and the retinal autophagy level was analyzed. The therapeutic effects of ZYAN1 were evaluated via a function examination, a morphological assay, in situ reactive oxygen species (ROS) detection, and an immunofluorescence assay. It was shown that the thickness of the outer nuclear layer (ONL) increased significantly, and visual function was efficiently preserved via ZYAN1 treatment. The mitochondria structure of photoreceptors was more complete in the ZYAN1-treated mice, and the number of autophagosomes also increased significantly. Membrane disc shedding and ROS overproduction were alleviated after ZYAN1 treatment, and the axonal cilia were more structurally intact. A Western blot analysis showed that the expression levels of the autophagy-related proteins LC3-B, Beclin-1, and ATG5 increased significantly after ZYAN1 treatment, while the expression of P62 was down-regulated. Moreover, the expression levels of HIF-1α and BNIP3 were up-regulated after ZYAN1 treatment. Therefore, an intravitreal injection of ZYAN1 can act as part of the pharmacologic strategy to modulate mitophagy and alleviate oxidative stress in RD. These findings enrich our knowledge of RD pathology and provide insights for the discovery of a therapeutic molecule.

Epigenetic mechanisms of Müller glial reprogramming mediating retinal regeneration.

Retinal degenerative diseases, characterized by retinal neuronal death and severe vision loss, affect millions of people worldwide. One of the most promising treatment methods for retinal degenerative diseases is to reprogram non-neuronal cells into stem or progenitor cells, which then have the potential to re-differentiate to replace the dead neurons, thereby promoting retinal regeneration. Müller glia are the major glial cell type and play an important regulatory role in retinal metabolism and retinal cell regeneration. Müller glia can serve as a source of neurogenic progenitor cells in organisms with the ability to regenerate the nervous system. Current evidence points toward the reprogramming process of Müller glia, involving changes in the expression of pluripotent factors and other key signaling molecules that may be regulated by epigenetic mechanisms. This review summarizes recent knowledge of epigenetic modifications involved in the reprogramming process of Müller glia and the subsequent changes to gene expression and the outcomes. In living organisms, epigenetic mechanisms mainly include DNA methylation, histone modification, and microRNA-mediated miRNA degradation, all of which play a crucial role in the reprogramming process of Müller glia. The information presented in this review will improve the understanding of the mechanisms underlying the Müller glial reprogramming process and provide a research basis for the development of Müller glial reprogramming therapy for retinal degenerative diseases.

The role of epigenetic methylation/demethylation in the regulation of retinal photoreceptors.

Photoreceptors are integral and crucial for the retina, as they convert light into electrical signals. Epigenetics plays a vital role in determining the precise expression of genetic information in space and time during the development and maturation of photoreceptors, cell differentiation, degeneration, death, and various pathological processes. Epigenetic regulation has three main manifestations: histone modification, DNA methylation, and RNA-based mechanisms, where methylation is involved in two regulatory mechanisms-histone methylation and DNA methylation. DNA methylation is the most studied form of epigenetic modification, while histone methylation is a relatively stable regulatory mechanism. Evidence suggests that normal methylation regulation is essential for the growth and development of photoreceptors and the maintenance of their functions, while abnormal methylation can lead to many pathological forms of photoreceptors. However, the role of methylation/demethylation in regulating retinal photoreceptors remains unclear. Therefore, this study aims to review the role of methylation/demethylation in regulating photoreceptors in various physiological and pathological situations and discuss the underlying mechanisms involved. Given the critical role of epigenetic regulation in gene expression and cellular differentiation, investigating the specific molecular mechanisms underlying these processes in photoreceptors may provide valuable insights into the pathogenesis of retinal diseases. Moreover, understanding these mechanisms could lead to the development of novel therapies that target the epigenetic machinery, thereby promoting the maintenance of retinal function throughout an individual's lifespan.

The molecular mechanism of human stem cell-derived extracellular vesicles in retinal repair and regeneration.

Extracellular vesicles (EVs), including microvesicles (MVs) and exosomes, play a critical role in metabolic regulation and intracellular communication. Stem cell-derived EVs are considered to have the potential for regeneration, like stem cells, while simultaneously avoiding the risk of immune rejection or tumour formation. The therapeutic effect of stem cell-derived EVs has been proven in many diseases. However, the molecular mechanism of stem cell-derived EVs in retinal repair and regeneration has not been fully clarified. In this review, we described the biological characteristics of stem cell-derived EVs, summarized the current research on stem cell-derived EV treatment in retinal repair and regeneration, and discussed the potential and challenges of stem cell-derived EVs in translational medicine.

Repopulated retinal microglia promote Müller glia reprogramming and preserve visual function in retinal degenerative mice.

Rationale: Müller glia (MG) play a key role in maintaining homeostasis of the retinal microenvironment. In zebrafish, MG reprogram into retinal progenitors and repair the injured retina, while this MG regenerative capability is suppressed in mammals. It has been revealed that microglia in zebrafish contribute to MG reprogramming, whereas those in mammals are over-activated during retinal injury or degeneration, causing chronic inflammation, acceleration of photoreceptor apoptosis, and gliosis of MG. Therefore, how to modulate the phenotype of microglia to enhance MG reprogramming rather than gliosis is critical. Methods: PLX3397, a colony-stimulating factor 1 receptor inhibitor, was applied to deplete microglia in the retinas of retinal degeneration 10 (rd10) mice, and withdrawal of PLX3397 was used to induce the repopulated microglia (Rep-MiG). The protective roles of the Rep-MiG on the degenerative retina were assessed using a light/dark transition test, and scotopic electroretinogram recordings. Immunofluorescence, western blot, transcriptomic sequencing, and bioinformatics analysis were performed to investigate the effects and mechanisms of microglia on MG reprogramming. Results: Following PLX3397 withdrawal, Rep-MiG replenished the entire retina with a ramified morphology and significantly improved the retinal outer nuclear layer structure, the electroretinography response, and the visual behavior of rd10 mice. Coincidentally, MG were activated, de-differentiated, and showed properties of retina progenitors in a spatial correlation with Rep-MiG. Morphological and transcriptomic analyses revealed Rep-MiG significantly enhanced protease inhibitor activity and suppressed extracellular matrix (ECM) levels during retinal degeneration. Conclusions: It suggested that Rep-MiG with the homeostasis characteristic stimulated the progenitor cell-like properties of MG, probably through regulating ECM remodeling, which protected photoreceptors and improved visual function of rd10 mice. It might be a potential protocol to reprogram MG and delay mammal retinal degeneration.

Complement C3a receptor inactivation attenuates retinal degeneration induced by oxidative damage.

Retinal degeneration causes vision loss and threatens the health of elderly individuals worldwide. Evidence indicates that the activation of the complement system is associated with retinal degeneration. However, the mechanism of complement signaling in retinal degeneration needs to be further studied. In this study, we show that the expression of C3 and C3a receptor (C3ar1) is positively associated with the inflammatory response and retinal degeneration. Genetic deletion of C3 and pharmacological inhibition of C3ar1 resulted in the alleviation of neuroinflammation, prevention of photoreceptor cell apoptosis and restoration of visual function. RNA sequencing (RNA-seq) identified a C3ar1-dependent network shown to regulate microglial activation and astrocyte gliosis formation. Mechanistically, we found that STAT3 functioned downstream of the C3-C3ar1 pathway and that the C3ar1-STAT3 pathway functionally mediated the immune response and photoreceptor cell degeneration in response to oxidative stress. These findings reveal an important role of C3ar1 in oxidative-induced retinal degeneration and suggest that intervention of the C3ar1 pathway may alleviate retinal degeneration.

Complement C3 deficiency alleviates alkylation-induced retinal degeneration in mice.

BACKGROUND: It has been found that the extensive use of anticancer drugs containing DNA-alkylating agents not only target cancer cells but also cause retinal inflammation through toxic intermediates. Complement C3 (C3) is a core component of the complement activation pathway, and dysregulation of the complement pathway is involved in several retinal degenerative diseases. However, whether C3 plays a critical role in alkylation-induced retinal degeneration is unclear. METHODS: Following treatment with the alkylating agent methyl methane sulfonate (MMS), the C3 mRNA and protein level was measured, DNA damage and photoreceptor cell death were assessed in both wild-type (WT) C57BL/6J and C3 knockout (KO) mice. RESULTS: We determined that complement pathway is activated following MMS treatment, and C3 knockout (KO) increased the rate of photoreceptor cell survival and preserved visual function. The mRNA levels of nuclear erythroid-related factor 2 (Nrf2) and related genes were higher after MMS application in C3 KO mice. CONCLUSION: In summary, our study found that C3 KO promotes photoreceptor cell survival and activates the Nrf2 signaling pathway in the context of alkylation-induced retinal degeneration.

Rapamycin Improved Retinal Function and Morphology in a Mouse Model of Retinal Degeneration.

The retina is an important visual organ, which is responsible for receiving light signals and transmitting them to the optic nerve center step by step. The retina contains a variety of cells, among which photoreceptor cells receive light signals and convert them into nerve signals, and are mainly responsible for light and dark vision. Retinal degeneration is mainly the degeneration of photoreceptor cells, and retinitis pigmentosa (RP) is characterized by rod degeneration followed by cone degeneration. So far, there is still a lack of effective drugs to treat RP. Here, we established a stable RP model by tail vein injection of methyl methanesulfonate to study the mechanism of retinal photoreceptor degeneration. Mechanistic target of rapamycin (mTOR) is located in the central pathway of growth and energy metabolism and changes in a variety of diseases in response to pathological changes. We found that the mTOR was activated in this model. Therefore, the inhibitor of mTOR, rapamycin was used to suppress the expression of mTOR and interfere with photoreceptor degeneration. Electroretinogram assay showed that the function of mice retina was improved. Hematoxylin and eosin staining results displayed that retinal photoreceptor thickness and morphology were improved. Also, the autophagy in rapamycin group was activated, which revealed that rapamycin may protect the retinal photoreceptor by inhibiting mTOR and then activating autophagy.

Decitabine improves MMS-induced retinal photoreceptor cell damage by targeting DNMT3A and DNMT3B.

Introduction: Retinitis pigmentosa (RP) is a group of neurodegenerative retinopathies causing blindness due to progressive and irreversible photoreceptor cell death. The alkylating agent methyl methanesulfonate (MMS) can induce selective photoreceptor cell death, which is used to establish RP animal models. MMS induces DNA base damage by adding alkyl groups to DNA, and epigenetic modifications influence DNA damage response. Here, we aimed to explore the relationship between DNA methylation and DNA damage response in dying photoreceptors of RP. Methods: The mouse RP model was established by a single intraperitoneal injection of MMS. The retinal structure and function were assessed by H&E, OCT, TUNEL, and ERG at several time points. The expression of DNA methylation regulators was assessed by qPCR and Western blot. DNMT inhibitor 5-aza-dC was applied to inhibit the activity of DNA methyltransferases and improve the retinal photoreceptor damage. Results: The outer nuclear layer (ONL) and IS/OS layer were significantly thinner and the retinal function was impaired after MMS treatment. The cell death was mainly located in the ONL. The retinal damage induced by MMS was accompanied by hyperexpression of DNMT3A/3B. The application of DNMT inhibitor 5-aza-dC could suppress the expression level of DNMT3A/3B, resulting in the remission of MMS-induced photoreceptor cell damage. The ONL and IS/OS layers were thicker than that of the control group, and the retinal function was partially restored. This protective effect of 5-aza-dC was associated with the down-regulated expression of DNMT3A/3B. Conclusion: These findings identified a functional role of DNMT3A/3B in MMS-induced photoreceptor cell damage and provided novel evidence to support DNMTs as potential therapeutic targets in retinal degenerative diseases.Graphical Abstract.

Regulatory Mechanisms of Retinal Photoreceptors Development at Single Cell Resolution.

Photoreceptors are critical components of the retina and play a role in the first step of the conversion of light to electric signals. With the discovery of the intrinsically photosensitive retinal ganglion cells, which regulate non-image-forming visual processes, our knowledge of the photosensitive cell family in the retina has deepened. Photoreceptor development is regulated by specific genes and proteins and involves a series of molecular processes including DNA transcription, post-transcriptional modification, protein translation, and post-translational modification. Single-cell sequencing is a promising technology for the study of photoreceptor development. This review presents an overview of the types of human photoreceptors, summarizes recent discoveries in the regulatory mechanisms underlying their development at single-cell resolution, and outlines the prospects in this field.

Identification of an intraocular microbiota.

The current dogma in ophthalmology and vision research presumes the intraocular environment to be sterile. However, recent evidence of intestinal bacterial translocation into the bloodstream and many other internal organs including the eyes, found in healthy and diseased animal models, suggests that the intraocular cavity may also be inhabited by a microbial community. Here, we tested intraocular samples from over 1000 human eyes. Using quantitative PCR, negative staining transmission electron microscopy, direct culture, and high-throughput sequencing technologies, we demonstrated the presence of intraocular bacteria. The possibility that the microbiome from these low-biomass communities could be a contamination from other tissues and reagents was carefully evaluated and excluded. We also provide preliminary evidence that a disease-specific microbial signature characterized the intraocular environment of patients with age-related macular degeneration and glaucoma, suggesting that either spontaneous or pathogenic bacterial translocation may be associated with these common sight-threatening conditions. Furthermore, we revealed the presence of an intraocular microbiome in normal eyes from non-human mammals and demonstrated that this varied across species (rat, rabbit, pig, and macaque) and was established after birth. These findings represent the first-ever evidence of intraocular microbiota in humans.

Longitudinal single-cell RNA-seq of hESCs-derived retinal organoids.

Human retina development involves multiple well-studied signaling pathways that promote the genesis of a wide arrange of different cell types in a complex architectural structure. Human embryonic stem cells (hESCs)-derived retinal organoids could recapitulate the human retinal development. We performed single-cell RNA-seq of retinal organoids from 5 time points (D36, D66, D96, D126, D186) and identified 9 distinct populations of cells. In addition, we analyzed the molecular characteristics of each main population and followed them from genesis to maturity by pseudotime analysis and characterized the cell-cell interactions between different cell types. Interestingly, we identified insulin receptor (INSR) as a specifically expressed receptor involved in the genesis of photoreceptors, and pleiothropin (PTN)-protein tyrosine phosphatase receptor type Z1 (PTPRZ1) as a mediator of a previously unknown interaction between Müller and retinal progenitor cells. Taken together, these findings provide a rich transcriptome-based lineage map for studying human retinal development and modeling developmental disorders in retinal organoids.

The Role of Histone Acetyltransferases and Histone Deacetylases in Photoreceptor Differentiation and Degeneration.

Photoreceptors are critical components of the retina and play a role in the first step of the conversion of light to electrical signals. The differentiation and degeneration of photoreceptors are regulated by specific genes and proteins. With the development of epigenetic approaches, scientists have discovered that histone modifications, such as acetylation, methylation, ubiquitylation, and phosphorylation, may modulate the processes of photoreceptor differentiation and degeneration. Histone acetylation is regulated by two opposing classes of enzymes, namely, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which add and remove acetyl groups to and from target histones, respectively, causing changes in transcriptional activity. Herein, we review the effects of HATs and HDACs on the differentiation and degeneration of photoreceptors and discuss the underlying mechanisms of these effects.

Optogenetic stimulation inhibits the self-renewal of mouse embryonic stem cells.

Modulation of the embryonic stem cell state is beneficial for elucidating the innate mechanisms of development and regenerative medicine. Ion flux plays important roles in modulating the transition between stemness and differentiation in mouse embryonic stem cells (mESCs). Optogenetics is a novel tool for manipulating ion flux. To investigate the impact of optical stimulation on embryonic stem cells, optogenetically engineered V6.5 mESCs were used to measure the depolarization mediated by ChR2 on the proliferation, self-renewal, and differentiation of mESCs. Blue light stimulation significantly inhibited ChR2-GFP-V6.5 ESC proliferation and disrupted the cell cycle progression, reducing the proportion of cells in the S phase. Interestingly, optical stimulation could inhibit ChR2-GFP-V6.5 ESC self-renewal and trigger differentiation by activating the extracellular regulated protein kinase (ERK) signaling pathway. Our data suggest that membrane potential changes play pivotal roles in regulating the proliferation, self-renewal and initiation of differentiation of mESCs.

The Influence of Age and Sex on Ocular Surface Microbiota in Healthy Adults.

Purpose: A growing body of evidence suggests that the microbiome of the ocular surface confers potent immunoregulatory functions and has a key role in the physiologic maintenance of healthy eyes and in the pathogenesis of ocular diseases. Although the microbiome is known to be affected by age and sex, the influence of these factors on ocular surface microbiota in healthy adults remains largely unknown. Methods: Ocular surface microbiome samples were obtained from the inferior bulbar conjunctiva of 48 young and 42 old adults at Zhongshan Ophthalmic Center. Using metagenomic shotgun sequencing, we characterized the sex- and age-differences in conjunctival microbiome profiles of healthy adults. Results: Male and female groups differed only in the ß diversity of bacterial communities, while there were significant differences in bacterial composition, metabolic functions, and the abundance of antibiotic resistance genes between young and old adult groups. Conclusions: Our findings suggest that age and sex collectively shape the conjunctival microbiome, and may change the immune homeostasis of the ocular surface through alterations of its commensal microbiome.

Experimental Study of the Biological Properties of Human Embryonic Stem Cell-Derived Retinal Progenitor Cells.

Retinal degenerative diseases are among the leading causes of blindness worldwide, and cell replacement is considered as a promising therapeutic. However, the resources of seed cells are scarce. To further explore this type of therapy, we adopted a culture system that could harvest a substantial quantity of retinal progenitor cells (RPCs) from human embryonic stem cells (hESCs) within a relatively short period of time. Furthermore, we transplanted these RPCs into the subretinal spaces of Royal College of Surgeons (RCS) rats. We quantified the thickness of the treated rats' outer nuclear layers (ONLs) and explored the visual function via electroretinography (ERG). It was found that the differentiated cells expressed RPC markers and photoreceptor progenitor markers. The transplanted RPCs survived for at least 12 weeks, resulting in beneficial effects on the morphology of the host retina, and led to a significant improvement in the visual function of the treated animals. These therapeutic effects suggest that the hESCs-derived RPCs could delay degeneration of the retina and partially restore visual function.

Topographic Quantification of the Transcorneal Electrical Stimulation (TES)-Induced Protective Effects on N-Methyl-N-Nitrosourea-Treated Retinas.

PURPOSE: To quantify the transcorneal electrical stimulation (TES)-induced effects on regional photoreceptors and visual signal pathway of N-methyl-N-nitrosourea (MNU)-treated retinas via topographic measurements. METHODS: N-methyl-N-nitrosourea-administered mice received TES or sham stimulations and were subsequently subjected to electroretinography (ERG), multielectrode array (MEA), and histologic and immunohistochemistry examinations. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses were also performed to determine the mRNA levels of Bax, Bcl-2, Calpain-2, Caspase-3, brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF). RESULTS: Amplitudes of ERG b-wave in the TES-treated mice were significantly larger than those in the sham controls (P < 0.01). Microelectrode array examination revealed that the photoreceptors in TES-treated retina were efficiently preserved (P < 0.01). Morphologic measurements showed that the central retina region was more consolidated than the other areas in the TES-treated mice. Together with the disproportionate distribution of immunostaining in retinal flat mounts, these findings indicated that different rescuing kinetics existed among regional photoreceptors. Compared with the sham controls, a significantly increased signal-to-noise ratio was also found in the TES-treated mice (TES100: 2.02 ± 1.12; TES200: 4.42 ± 1.51; sham: 0.25 ± 0.13; P < 0.01). Moreover, qRT-PCR measurements suggested that the altered expression of several apoptotic factors and neurotrophic cytokines was correlated with TES-induced protection. CONCLUSIONS: Regional photoreceptors in the MNU-administered retinas exhibit different sensitivities to TES. Transcorneal electrical stimulation is capable of ameliorating MNU-induced photoreceptor degeneration and rectifying abnormalities in the inner visual signal pathways.