Identification of a novel ferroptosis-related gene signature associated with retinal degeneration induced by light damage in mice.

Background: Neurodegenerative retinal diseases such as retinitis pigmentosa are serious disorders that may cause irreversible visual impairment. Ferroptosis is a novel type of programmed cell death, and the involvement of ferroptosis in retinal degeneration is still unclear. This study aimed to investigate the related ferroptosis genes in a mice model of retinal degeneration induced by light damage. Methods: A public dataset of GSE10528 deriving from the Gene Expression Omnibus database was analyzed to identify the differentially expressed genes (DEGs). Gene set enrichment analysis between light damage and control group was conducted. The differentially expressed ferroptosis-related genes (DE-FRGs) were subsequently identified by intersecting the DEGs with a ferroptosis genes dataset retrieved from the FerrDb database. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were further performed using the DE-FRGs. A protein-protein interaction (PPI) network was constructed to identify hub ferroptosis-related genes (HFRGs). The microRNAs (miRNAs)-HFRGs, transcription factors (TFs)-HFRGs networks as well as target drugs potentially interacting with HFRGs were analyzed utilizing bioinformatics algorithms. Results: A total of 932 DEGs were identified between the light damage and control group. Among these, 25 genes were associated with ferroptosis. GO and KEGG analyses revealed that these DE-FRGs were mainly enriched in apoptotic signaling pathway, response to oxidative stress and autophagy, ferroptosis, necroptosis and cytosolic DNA-sensing pathway. Through PPI network analysis, six hub ferroptosis-related genes (Jun, Stat3, Hmox1, Atf3, Hspa5 and Ripk1) were ultimately identified. All of them were upregulated in light damage retinas, as verified by the GSE146176 dataset. Bioinformatics analyses predicated that 116 miRNAs, 23 TFs and several potential therapeutic compounds might interact with the identified HFRGs. Conclusion: Our study may provide novel potential biomarkers, therapeutic targets and new insights into the ferroptosis landscape in retinal neurodegenerative diseases.

Dysregulated Arginine Metabolism Is Linked to Retinal Degeneration in Cep250 Knockout Mice.

Purpose: Degeneration of retinal photoreceptors is frequently observed in diverse ciliopathy disorders, and photoreceptor cilium gates the molecular trafficking between the inner and the outer segment (OS). This study aims to generate a homozygous global Cep250 knockout (KO) mouse and study the resulting phenotype. Methods: We used Cep250 KO mice and untargeted metabolomics to uncover potential mechanisms underlying retinal degeneration. Long-term follow-up studies using optical coherence tomography (OCT) and electroretinography (ERG) were performed. Results: OCT and ERG results demonstrated gradual thinning of the outer nuclear layer (ONL) and progressive attenuation of the scotopic ERG responses in Cep250-/- mice. More TUNEL signal was observed in the ONL of these mice. Immunostaining of selected OS proteins revealed mislocalization of these proteins in the ONL of Cep250-/- mice. Interestingly, untargeted metabolomics analysis revealed arginine-related metabolic pathways were altered and enriched in Cep250-/- mice. Mis-localization of a key protein in the arginine metabolism pathway, arginase 1 (ARG1), in the ONL of KO mice further supports this model. Moreover, adeno-associated virus (AAV)-based retinal knockdown of Arg1 led to similar architectural and functional alterations in wild-type retinas. Conclusions: Altogether, these results suggest that dysregulated arginine metabolism contributes to retinal degeneration in Cep250-/- mice. Our findings provide novel insights that increase understanding of retinal degeneration in ciliopathy disorders.

Causal effects of serum lipid biomarkers on early age-related macular degeneration using Mendelian randomization.

BACKGROUND: Age-related macular degeneration (AMD) is one of the major causes of vision loss. Early AMD needs to be taken seriously, but the causal effects of lipid biomarkers on early AMD remain unclear. METHODS: In this study, two-sample Mendelian randomization (MR) analysis was performed to systematically assess the causal relationships between seven serum lipid biomarkers (apolipoprotein A (ApoA), apolipoprotein B (ApoB), total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C), direct low-density lipoprotein cholesterol (LDL-C), lipoprotein A [Lp(a)], and triglycerides (TG)) and risk of early AMD. In total, 14,034 cases and 91,214 controls of European ancestry were included in the analysis (number of SNPs = 11,304,110). RESULTS: MR estimates revealed that a higher HDL-C level is strongly associated with increased risk of early AMD (OR = 1.25, 95% CI: 1.15-1.35, P = 2.61 × 10-8). In addition, level of ApoA is also positively associated with risk of early AMD (OR = 2.04, 95% CI: 1.50-2.77, P = 6.27 × 10-6). Conversely, higher levels of TG significantly decrease the risk of early AMD (OR = 0.77, 95% CI: 0.71-0.84, P = 5.02 × 10-10). Sensitivity analyses further supported these associations. Moreover, multivariable MR analyses, adjusted for the effects of correlated lipid biomarkers, yielded similar results. CONCLUSION: This study identifies causal relationships between elevated circulating HDL-C/ApoA levels and increased risk of early AMD, in addition to finding that TG specifically reduces the risk of early AMD. These findings contribute to a better understanding of the role of lipid metabolism in drusen formation, particularly in early AMD development.

Depletion of miR-96 Delays, But Does Not Arrest, Photoreceptor Development in Mice.

Purpose: Abundant retinal microRNA-183 cluster (miR-183C) has been reported to be a key player in photoreceptor development and functionality in mice. However, whether there is a protagonist in this cluster remains unclear. Here, we used a mutant mouse model to study the role of miR-96, a member of miR-183C, in photoreceptor development and functionality. Methods: The mature miR-96 sequence was removed using the CRISPR/Cas9 genome-editing system. Electroretinogram (ERG) and optical coherence tomography (OCT) investigated the changes in structure and function in mouse retinas. Immunostaining determined the localization and morphology of the retinal cells. RNA sequencing was conducted to observe retinal transcription alterations. Results: The miR-96 mutant mice exhibited cone developmental delay, as occurs in miR-183/96 double knockout mice. Immunostaining of cone-specific marker genes revealed cone nucleus mislocalization and exiguous Opn1mw/Opn1sw in the mutant (MT) mouse outer segments at postnatal day 10. Interestingly, this phenomenon could be relieved in the adult stages. Transcriptome analysis revealed activation of microtubule-, actin filament-, and cilia-related pathways, further supporting the findings. Based on ERG and OCT results at different ages, the MT mice displayed developmental delay not only in cones but also in rods. In addition, a group of miR-96 potential direct and indirect target genes was summarized for interpretation and further studies of miR-96-related retinal developmental defects. Conclusions: Depletion of miR-96 delayed but did not arrest photoreceptor development in mice. This miRNA is indispensable for mouse photoreceptor maturation, especially for cones.

An inexpensive anaerobic chamber for the genetic manipulation of strictly anaerobic bacteria.

Strictly anaerobic bacteria are important to both human health and industrial usage. These bacteria are sensitive to oxygen, therefore, it is preferable to manipulate these microbes in an anaerobic chamber. However, commercial anaerobic chambers (CACs) are expensive, making them less accessible to scientists with a limited budget, especially to those in developing countries. The high price of commercial chambers has hindered, at least partially, the progress of research on anaerobes in developing countries. In the research presented here, we developed an inexpensive and reliable anaerobic chamber and successfully achieved routine maintenance of eleven strictly anaerobic bacterial strains. Furthermore, genetic manipulation examples have been set for both Clostridioidesdifficile 630 and Clostridiumbeijerinckii NCIMB 8052 strains to validate that the chamber could applied to advanced genetic engineering of strictly anaerobes. C. difficile and C. beijerinckii were both genetically manipulated in this chamber, showing it's utility for the genetic engineering of anaerobes. Most importantly, the anaerobic chamber was 76% - 88% less expensive than a CACs and has similar functionality with regards to the cultivation and manipulation of strictly anaerobic bacteria. The anaerobic chamber described in this study will promote the research of anaerobes in developing counties and scientists who have limited research budgets.

Mutations in LRP5,FZD4, TSPAN12, NDP, ZNF408, or KIF11 Genes Account for 38.7% of Chinese Patients With Familial Exudative Vitreoretinopathy.

Purpose: Familial exudative vitreoretinopathy (FEVR) is a severe hereditary retinal disorder characterized by defects in retinal vascular development. To date, six genes have been reported to be responsible for this disease, including LRP5, FZD4, TSPAN12, NDP, ZNF408, and KIF11. The purpose of our study was to investigate the genetic defects in Chinese patients with FEVR through mutational analyses of 31 pedigrees. Methods: Clinical data and peripheral blood were collected from 31 pedigrees with FEVR. All coding sequences and intron/exon junctions were amplified and sequenced comprehensively, followed by cosegregation testing to verify suspected variants in the family members. Finally, we assessed clinical relevance of the identified mutations, according to the standards and guidelines from the American College of Medical Genetics and Genomics. Results: Twelve index cases (12/31, 38.7%) were confirmed to harbor mutations in the known genes, including one previously reported mutation and 11 novel mutations. Among the detected mutations, LRP5 accounted for the largest proportion with a mean mutation rate of 16.1% (5/31, 16.1%), followed by NDP (3/31, 9.7%), FZD4 (2/31, 6.5%), TSPAN12 (1/31, 3.2%), and KIF11 (1/31, 3.2%). All the novel changes were predicted to be pathogenic by a series of bioinformatics analyses. Conclusions: We comprehensively screened six known disease-causing genes in 31 pedigrees with FEVR and achieved a clear picture of the mutation spectrum in Chinese patients with FEVR, which highlights the importance and utility of clinical genetic diagnosis.

Genome-Wide Detection of Copy Number Variations in Unsolved Inherited Retinal Disease.

Purpose: Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of Mendelian disorders that plays a crucial role in the etiology of blindness across the world. Molecular genetic diagnosis of IRD remains extremely complex and challenging because mutations are only detected in 40% to 60% of cases. In this study, we aimed to dissect the contributions of copy number variations (CNVs) in IRD patients. Methods: A total of 50 patients were diagnosed with IRD, all of whom previously tested negative for pathogenic mutations in known disease genes. Single-nucleotide polymorphism array analysis was performed by using the HumanCoreExome BeadChip. Analyses of CNVs were carried out by using GenomeStudio, KaryoStudio, and cnvPartition. The putative pathogenic CNVs were further confirmed by real-time quantitative PCR. Results: We identified four novel CNVs in three different genes (one duplication in USH2A gene, two duplications in CEP290 gene, and one duplication in RIMS2 gene) in total four families, at a detection rate of 8% (4/50). All of these CNVs are currently absent in all databases. Three variations are located in genes that are already known to cause inherited retinal disease: USH2A and CEP290, while the association between mutation in the RIMS2 gene and IRD is reported for the first time. Conclusions: We performed whole-genome-wide CNV analyses in a large cohort as an alternative approach to molecular diagnosis of IRDs. This study dissected the contributions of CNVs of IRDs, not only increasing the yield in genetic testing but also suggesting the CNVs should be analyzed in the patients with IRDs.