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.

Patient-Specific Retinal Organoids Recapitulate Disease Features of Late-Onset Retinitis Pigmentosa.

Although an increasing number of disease genes have been identified, the exact cellular mechanisms of retinitis pigmentosa (RP) remain largely unclear. Retinal organoids (ROs) derived from the induced pluripotent stem cells (iPSCs) of patients provide a potential but unvalidated platform for deciphering disease mechanisms and an advantageous tool for preclinical testing of new treatments. Notably, early-onset RP has been extensively recapitulated by patient-iPSC-derived ROs. However, it remains a challenge to model late-onset disease in a dish due to its chronicity, complexity, and instability. Here, we generated ROs from late-onset RP proband-derived iPSCs harboring a PDE6B mutation. Transcriptome analysis revealed a remarkably distinct gene expression profile in the patient ROs at differentiation day (D) 230. Changes in the expression genes regulating cGMP hydrolysis prompted the elevation of cGMP levels, which was verified by a cGMP enzyme-linked immunosorbent assay (ELISA) in patient ROs. Furthermore, significantly higher cGMP levels in patient ROs than in control ROs at D193 and D230 might lead to impaired formation of synaptic connections and the connecting cilium in photoreceptor cells. In this study, we established the first late-onset RP model with a consistent phenotype using an in vitro cell culture system and provided new insights into the PDE6B-related mechanism of RP.

Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients.

Retinitis pigmentosa (RP) is an irreversible, inherited retinopathy in which early-onset nyctalopia is observed. Despite the genetic heterogeneity of RP, RPGR mutations are the most common causes of this disease. Here, we generated induced pluripotent stem cells (iPSCs) from three RP patients with different frameshift mutations in the RPGR gene, which were then differentiated into retinal pigment epithelium (RPE) cells and well-structured retinal organoids possessing electrophysiological properties. We observed significant defects in photoreceptor in terms of morphology, localization, transcriptional profiling, and electrophysiological activity. Furthermore, shorted cilium was found in patient iPSCs, RPE cells, and three-dimensional retinal organoids. CRISPR-Cas9-mediated correction of RPGR mutation rescued photoreceptor structure and electrophysiological property, reversed the observed ciliopathy, and restored gene expression to a level in accordance with that in the control using transcriptome-based analysis. This study recapitulated the pathogenesis of RPGR using patient-specific organoids and achieved targeted gene therapy of RPGR mutations in a dish as proof-of-concept evidence.

Toll-Like Receptor 3 Activation Initiates Photoreceptor Cell Death In Vivo and In Vitro.

Purpose: Accumulating evidence has demonstrated that excessive immunoreaction plays a prominent role in the pathogenesis of dry AMD. Toll-like receptor 3 (TLR3) can be activated by double-stranded (ds)RNA in retinal pigment epithelia and trigger an innate immunity-mediated inflammatory response. However, its role in photoreceptor cells, the effectors of AMD geographic atrophy, remains unclear. Methods: The expression of TLR3 was examined in mouse retina and in a murine photoreceptor cell line (661W). Retinal structure, function, and cell death in the polyinosine-polycytidylic acid (poly I:C)-treated retina were investigated by optical coherence tomography, electroretinography (ERG), and immunostaining. Cytokine and chemokine expression as well as cell death were measured in poly I:C-exposed 661W cells and explant retinas. By comparing the RNA sequencing (seq) data of 661W cells and murine retina, we comprehensively investigated the contribution of photoreceptor in poly I:C-induced retinal immune response. Results: Toll-like receptor 3 was highly expressed in the inner segment of the photoreceptor and in 661W cells. We found poly I:C induced significant retinal structural damages and impairment of ERG responses. Focal ERG demonstrated that injected and parainjected zones were functionally damaged by poly I:C. In addition, poly I:C acted on cultured photoreceptor cells directly and evoked an inflammatory response that exhibited similarities with the immune response in mouse retina. Moreover, TLR3 activation initiated cell death in murine photoreceptor cells in vivo and in vitro. Additionally, poly I:C initiated immune response in explant retinas. Conclusions: We deciphered the TLR3-mediated inflammatory response in photoreceptor cells. Our findings suggested TLR3-mediated inflammatory response in photoreceptor cells may play an important role in dry AMD, offering new insights of potential treatments targeting photoreceptor immunity.

Targeted RP9 ablation and mutagenesis in mouse photoreceptor cells by CRISPR-Cas9.

Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells. Genetic mutations in many spliceosome genes confer human eye diseases. Mutations in the pre-mRNA splicing factor, RP9 (also known as PAP1), predispose autosomal dominant retinitis pigmentosa (adRP) with an early onset and severe vision loss. However, underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown. Here, we utilize the CRISPR/Cas9 system to generate both the Rp9 gene knockout (KO) and point mutation knock in (KI) (Rp9, c.A386T, P.H129L) which is analogous to the reported one in the retinitis pigmentosa patients (RP9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells in vitro. We found that proliferation and migration were significantly decreased in the mutated cells. Gene expression profiling by RNA-Seq demonstrated that RP associated genes, Fscn2 and Bbs2, were down-regulated in the mutated cells. Furthermore, pre-mRNA splicing of the Fscn2 gene was markedly affected. Our findings reveal a functional relationship between the ubiquitously expressing RP9 and the disease-specific gene, thereafter provide a new insight of disease mechanism in RP9-related retinitis pigmentosa.

Molecular genetic analysis of patients with sporadic and X-linked infantile nystagmus.

OBJECTIVES: Infantile nystagmus (IN) is a genetically heterogeneous condition characterised by involuntary rhythmic oscillations of the eyes accompanied by different degrees of vision impairment. Two genes have been identified as mainly causing IN: FRMD7 and GPR143. The aim of our study was to identify the genetic basis of both sporadic IN and X-linked IN. DESIGN: Prospective analysis. PATIENTS: Twenty Chinese patients, including 15 sporadic IN cases and 5 from X-linked IN families, were recruited and underwent molecular genetic analysis. We first performed PCR-based DNA sequencing of the entire coding region and the splice junctions of the FRMD7 and GPR143 genes in participants. Mutational analysis and co-segregation confirmation were then performed. SETTING: All clinical examinations and genetic experiments were performed in the Eye Hospital of Wenzhou Medical University. RESULTS: Two mutations in the FRMD7 gene, including one novel nonsense mutation (c.1090C>T, p.Q364X) and one reported missense mutation (c.781C>G, p.R261G), were identified in two of the five (40%) X-linked IN families. However, none of putative mutations were identified in FRMD7 or GPR143 in any of the sporadic cases. CONCLUSIONS: The results suggest that mutations in FRMD7 appeared to be the major genetic cause of X-linked IN, but not of sporadic IN. Our findings provide further insights into FRMD7 mutations, which could be helpful for future genetic diagnosis and genetic counselling of Chinese patients with nystagmus.

Molecular screening of the LPCAT1 gene in patients with retinitis pigmentosa without defined mutations in known retinitis pigmentosa genes.

Retinitis pigmentosa (RP) is an inherited retinopathy, which affects the photoreceptors in the retina. Lysophosphatidylcholine acyltransferase (LPCAT) is a critical phospholipid biosynthesis enzyme, which promotes the conversion of lysophosphatidylcholine into phosphatidylcholine in the remodeling pathway of PC biosynthesis. A previous study reported a homozygous insertion in the LPCAT1 gene in mice exhibiting retinal degeneration (rd11). However, whether genetic mutations in LPCAT1 predispose individuals to RP remains to be elucidated. Therefore, the aim of the present study was to investigate whether LPCAT1 mutations exist in patients with RP. A total of 50 unrelated patients diagnosed with either a sporadic or recessive inheritance pattern of RP were recruited in the present study. All of the patients were comprehensively screened for genes associated with the predisposition of RP, and no pathogenic mutations were identified. Reverse transcription-polymerase chain reaction and Sanger sequencing were performed to investigate the coding regions and exon­intron boundaries of the LPCAT1 gene in the recruited patients. In total, three genetic variations in the coding regions, which lead to amino acid changes, were identified. Although two of these mutations were predicted to be pathogenic, co­segregation analysis in the pedigrees excluded these as disease­causing mutations. In addition, the LPCAT1 gene was screen in a panel of RP patients who exhibited no identifiable mutations in any of the known RP­associated genes. No disease­causing mutations in the LPCAT1 gene were identified, indicating that LPCAT1 either does not confer a genetic predisposition to RP, or that the incidence of mutations in LPCAT1 is particularly rare in patients with RP.