The daily gene transcription cycle in mouse retina.

Many physiological retinal processes, such as outer segment disk shedding and visual sensitivity, exhibit a daily rhythm. However, the detailed transcriptome dynamics and related biological processes of the retina are not fully understood. Retinal tissues were collected from C57BL/6J male mice housed in a 12h light/12h dark (LD) cycle for 4 weeks, at Zeitgeber time (ZT) 0, 4, 8, 12, 16, and 20. Total RNA was extracted from the tissues and used for unique identifier RNA sequencing experiments. The rhythmicity of gene expression was determined using the MetaCycle R package. We found that 1741 genes (10.26%) were rhythmically expressed in the retina. According to the expression patterns, the rhythmically expressed genes were assigned to four clusters, each with about 361-492 genes, using the Mfuzz R package. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were conducted to identify pathways and biological processes of the profiled genes. Genes in Clusters 1 and 4 were associated with glycolysis and energy production, showed higher activity at night (from ZT16 to ZT20), and were enriched in the Hif-1α signaling pathway and low-oxygen-related terms. Genes in Cluster 2 were predominantly involved in cilium assembly and organization and were relatively upregulated during the day. Genes in Cluster 3 were associated with ribosome biosynthesis and were highly expressed during the day-night transition period. Taken together, these results demonstrate that a large proportion of retinal genes are expressed rhythmically. Genes involved in energy production and glycolysis are highly expressed at night, leading to relative hypoxia and activation of the Hif-1α signaling pathway. Genes associated with the formation of photoreceptor cilia are expressed during the day.

Integrated Analysis of DNA methylation and transcriptome profile to identify key features of age-related macular degeneration.

Age-related macular degeneration (AMD) is a common vision-threatening disease. The current study sought to integrate DNA methylation with transcriptome profile to explore key features in AMD. Gene expression data were obtained from the Gene Expression Omnibus (GEO, accession ID: GSE135092) and DNA methylation data were obtained from the ArrayExpress repository (E-MTAB-7183). A total of 456 differentially expressed genes (DEGs) and 4827 intragenic differentially methylated CpGs (DMCs) were identified between AMD and controls. DEGs and DMCs were intersected and 19 epigenetically induced (EI) genes and 15 epigenetically suppressed (ES) genes were identified. Immune cell infiltration analysis was performed to estimate the abundance of different types of immune cell in each sample. Enrichment scores of inflammatory response and tumor necrosis factor-alpha (TNFα) signaling via nuclear factor kappa B (NF-κb) were positively correlated with abundance of activated memory CD4 T cells and M1 macrophages. Subsequently, two significant random forest classifiers were constructed based on DNA methylation and transcriptome data. SMAD2 and NGFR were selected as key genes through functional epigenetic modules (FEM) analysis. Expression level of SMAD2, NGFR and their integrating proteins was validated in hydrogen peroxide (H2O2) and TNFα co-treated retinal pigment epithelium (RPE) in vitro. The findings of the current study showed that local inflammation and systemic inflammatory host response play key roles in pathogenesis of AMD. SMAD2 and NGFR provide new insight in understanding the molecular mechanism and are potential therapeutic targets for development of AMD therapy.

Clock Gene Nr1d1 Alleviates Retinal Inflammation Through Repression of Hmga2 in Microglia.

PURPOSE: Retinal inflammation is involved in the pathogenesis of several retinal diseases. As one of the core clock genes, Nr1d1 has been reported to suppress inflammation in many diseases. We investigated whether pharmacological activation of Nr1d1 can inhibit retinal inflammation and delineated the mechanisms of Nr1d1 in alleviating microglia activation. METHODS: Lipopolysaccharide (LPS) induced mice models were used to examine the effects of SR9009 (agonist of NR1D1) treatment on inflammatory phenotypes in vivo. Anti-inflammatory effects of Nr1d1 and associated mechanisms were investigated in the BV2 microglia cell line, and in primary retinal microglia in vitro. RESULTS: SR9009 treatment alleviated LPS-induced inflammatory cell infiltration, elevated cytokine levels and morphological changes of the microglia in mice models. In LPS-stimulated BV2 cells and primary retinal microglia, SR9009 suppressed cytokine expressions by inhibiting the NF-κB signaling pathway. Moreover, SR9009 treatment increased the levels of the M2 phenotype marker (CD206) and the proportions of ramified microglia. Suppression of Nr1d1 with siRNA reversed the inhibitory effects of SR9009 on cytokine production in BV2 cells. RNA-seq analysis showed that genes that were upregulated following Nr1d1 knockdown were enriched in inflammatory-associated biological processes. Subsequently, ChIP-seq of NR1D1 in BV2 was performed, and the results were integrated with RNA-seq results using the Binding and Expression Target Analysis (BETA) tool. Luciferase assays, electrophoretic mobility shift assay (EMSA), qPCR and Western blotting assays revealed that NR1D1 binds the promoter of Hmga2 to suppress its transcription. Notably, overexpressed Hmga2 in activated microglia could partly abolish the anti-inflammatory effects of Nr1d1. CONCLUSION: The clock gene Nr1d1 protects against retinal inflammation and microglia activation in part by suppressing Hmga2 transcription.

Clinical Characteristics and Surgical Safety in Congenital Cataract Eyes with Three Pathological Types of Posterior Capsule Abnormalities.

PURPOSE: To observe the clinical characteristics of 3 pathological types of posterior capsule abnormalities (PCAs) in congenital cataracts (CCs) and evaluate the surgical safety in these eyes. METHODS: This study involved 239 children (367 eyes) with CC who underwent cataract surgery at the Eye Hospital of Wenzhou Medical University. All surgery videos were collected for detailed reviews. Intraoperative and postoperative complications (within 3 months) were all recorded. RESULTS: The 3 pathological types of PCAs, namely, persistent fetal vasculature (PFV), posterior capsule defect (PCD), and posterior lenticonus (PLC), presented in 129 (35.1%) CC eyes, while 238 (64.9%) eyes were recorded as CC without PCA. The percentages of PFV, PCD, and PLC were 10.9%, 26.7%, and 5.4% in CC eyes (n = 367), respectively. The most common concomitant of PFV eyes was PCD (42.5%), and PFV was the most frequent (17.3%) one in PCD eyes. PLC was only associated with PFV (15%) and PCD (50%). The occurrence rates of surgical complications ranged from 0 to 5.4%, and no statistical difference was found between the eyes with and without PCA (all P > 0.05). CONCLUSIONS: PFV, PCD, and PLC play a very important role in the CCs. The effect of fetal vessels in PFV eyes might be an abnormally strong attachment on the posterior capsule, leading to PLC and PCD. Even in PCA patients, severe surgical complication can also be avoided with well-designed and skilled operation. This trial is registered with NCT03905044 at http://ClinicalTrials.gov.