Diabetic retinopathy, a vascular complication of diabetes mellitus, is the leading cause of visual impairment and blindness. circRNAs act as competing endogenous RNA, sponging target miRNA and thus influencing mRNA expression in vascular diseases. We investigated whether and how circDNMT3B is involved in retinal vascular dysfunction under diabetic conditions. qRT-PCR was performed to detect expression of circDNMT3B, miR-20b-5p, and BAMBI in retinal microvascular endothelial cells under diabetic conditions. Western blot, Cell Counting Kit-8, Transwell, Matrigel tube formation, and retinal trypsin digestion assays were conducted to explore the roles of circDNMT3B/miR-20b-5p/BAMBI in retinal vascular dysfunction. Bioinformatics analysis and luciferase reporter, siRNA, and overexpression assays were used to reveal the mechanisms of the circDNMT3B/miR-20b-5p/BAMBI interaction. Electroretinograms were used to evaluate visual function. Upregulation of miR-20b-5p under diabetic conditions promoted proliferation, migration, and tube formation of human retinal microvascular endothelial cells (HRMECs), which was mediated by downregulated BAMBI. Under diabetic conditions, circDNMT3B, which acts as a sponge of miR-20b-5p, is downregulated. circDNMT3B overexpression reduced retinal acellular capillary number and alleviated visual damage in diabetic rats. Changes in expression of circDNMT3B and miR-20b-5p were confirmed in the proliferative fibrovascular membranes of patients with diabetic retinopathy. Downregulation of circDNMT3B contributes to vascular dysfunction in diabetic retinas through regulating miR-20b-5p and BAMBI, providing a potential treatment strategy for diabetic retinopathy. National Natural Science Foundation of China, National Key Basic Research Program of China, Shanghai Municipal Science and Technology Major Project, and ZJLab.

Background: Glaucoma, the leading cause of irreversible blindness, is a retinal neurodegenerative disease, which results from progressive apoptotic death of retinal ganglion cells (RGCs). Although the mechanisms underlying RGC apoptosis in glaucoma are extremely complicated, an abnormal cross-talk between retinal glial cells and RGCs is generally thought to be involved. However, how interaction of Müller cells and microglia, two types of glial cells, contributes to RGC injury is largely unknown. Methods: A mouse chronic ocular hypertension (COH) experimental glaucoma model was produced. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), transwell co-culture of glial cells, flow cytometry assay, ELISA, Ca²⁺ image, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the interaction of Müller cells and microglia, and its underlying mechanisms in COH retina. Results: We first showed that Müller cell activation in mice with COH induced microglia activation through the ATP/P2X7 receptor pathway. The activation of microglia resulted in a significant increase in mRNA and protein levels of pro-inflammatory factors, such as tumor necrosis factor-α and interleukin-6. These inflammatory factors in turn caused the up-regulation of mRNA expression of pro-inflammatory factors in Müller cells through a positive feedback manner. Conclusions: These findings provide robust evidence, for the first time, that retinal inflammatory response may be aggravated by an interplay between activated two types of glial cells. These results also suggest that to reduce the interplay between Müller cells and microglia could be a potential effective strategy for preventing the loss of RGCs in glaucoma.

Müller cell gliosis, which is characterized by upregulated expression of glial fibrillary acidic protein (GFAP), is a universal response in many retinal pathological conditions. Whether down-regulation of inward rectifying K⁺(Kir) channels, which commonly accompanies the enhanced GFAP expression, could contribute to Müller cell gliosis is poorly understood. We investigated changes of Kir currents, GFAP and Kir4.1 protein expression in Müller cells in a rat chronic ocular hypertension (COH) model, and explored the mechanisms underlying Müller cell gliosis. We show that Kir currents and Kir4.1 protein expression in Müller cells were reduced significantly, while GFAP expression was increased in COH rats, and these changes were eliminated by MPEP, a group I metabotropic glutamate receptors (mGluR I) subtype mGluR5 antagonist. In normal isolated Müller cells, the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) suppressed the Kir currents and the suppression was blocked by MPEP. The DHPG effect was mediated by the intracellular Ca²⁺-dependent PLC/IP₃-ryanodine/PKC signaling pathway, but the cAMP-PKA pathway was not involved. Moreover, intravitreal injection of DHPG in normal rats induced changes in Müller cells, similar to those observed in COH rats. The DHPG-induced increase of GFAP expression in Müller cells was obstructed by Ba²⁺, suggesting the involvement of Kir channels. We conclude that overactivation of mGluR5 by excessive extracellular glutamate in COH rats could contribute to Müller cell gliosis by suppressing Kir channels.

In vertebrate retina, melatonin regulates various physiological functions. In this work we investigated the mechanisms underlying melatonin‐induced potentiation of glycine currents in rat retinal ganglion cells (RGCs). Immunofluorescence double labelling showed that rat RGCs were solely immunoreactive to melatonin MT₂receptors. Melatonin potentiated glycine currents of RGCs, which was reversed by the MT₂receptor antagonist 4‐P‐PDOT. The melatonin effect was blocked by intracellular dialysis of GDP‐β‐S. Either preincubation with pertussis toxin or application of the phosphatidylcholine (PC)‐specific phospholipase C (PLC) inhibitor D609, but not the phosphatidylinositol (PI)‐PLC inhibitor U73122, blocked the melatonin effect. The protein kinase C (PKC) activator PMA potentiated the glycine currents and in the presence of PMA melatonin failed to cause further potentiation of the currents, whereas application of the PKC inhibitor bisindolylmaleimide IV abolished the melatonin‐induced potentiation. The melatonin effect persisted when [Ca²⁺]_iwas chelated by BAPTA, and melatonin induced no increase in [Ca²⁺]_i. Neither cAMP‐PKA nor cGMP‐PKG signalling pathways seemed to be involved because 8‐Br‐cAMP or 8‐Br‐cGMP failed to cause potentiation of the glycine currents and both the PKA inhibitor H‐89 and the PKG inhibitor KT5823 did not block the melatonin‐induced potentiation. In consequence, a distinct PC‐PLC/PKC signalling pathway, following the activation of G_i/o‐coupled MT₂receptors, is most likely responsible for the melatonin‐induced potentiation of glycine currents of rat RGCs. Furthermore, in rat retinal slices melatonin potentiated light‐evoked glycine receptor‐mediated inhibitory postsynaptic currents in RGCs. These results suggest that melatonin, being at higher levels at night, may help animals to detect positive or negative contrast in night vision by modulating inhibitory signals largely mediated by glycinergic amacrine cells in the inner retina.

We investigated possible involvement of a calpain/p35-p25/cyclin-dependent kinase 5 (Cdk5) signaling pathway in modifying NMDA receptors (NMDARs) in glutamate-induced injury of cultured rat retinal neurons. Glutamate treatment decreased cell viability and induced cell apoptosis, which was accompanied by an increase in Cdk5 and p-Cdk5^T15 protein levels. The Cdk5 inhibitor roscovitine rescued the cell viability and inhibited the cell apoptosis. In addition, the protein levels of both calpain 2 and calpain-specific alpha-spectrin breakdown products (SBDPs), which are both Ca²⁺-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5^T15, calpain 2 and SBDPs tended to decline with glutamate treatments of more than 9 h. Furthermore, the elevation of SBDPs was attenuated by either D-APV, a NMDAR antagonist, or CNQX, a non-NMDAR antagonist, but was hardly changed by the inhibitors of intracellular calcium stores dantrolene and xestospongin. Moreover, the Cdk5 co-activator p35 was significantly up-regulated, whereas its cleaved product p25 expression showed a transient increase. Glutamate treatment for less than 9 h also considerably enhanced the ratio of the Cdk5-phosphorylated NMDAR subunit NR2A at Ser1232 site (p-NR2A^S1232) and NR2A (p-NR2A^S1232/NR2A), and caused a translocation of p-NR2A^S1232 from the cytosol to the plasma membrane. The enhanced p-NR2A^S1232 was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca²⁺ concentrations ([Ca²⁺]_i) of retinal cells were steadily increased following glutamate treatments of 2 h, 6 h and 9 h. All these results suggest that the activation of the calpain/p35-p25/Cdk5 signaling pathway may contribute to glutamate neurotoxicity in the retina by up-regulating p-NR2A^S1232 expression.