Metabolic reprogramming of the retinal pigment epithelium by cytokines associated with age-related macular degeneration.

The complex metabolic relationship between the retinal pigment epithelium (RPE) and photoreceptors is essential for maintaining retinal health. Recent evidence indicates the RPE acts as an adjacent lactate sink, suppressing glycolysis in the epithelium in order to maximize glycolysis in the photoreceptors. Dysregulated metabolism within the RPE has been implicated in the pathogenesis of age-related macular degeneration (AMD), a leading cause of vision loss. In the present study, we investigate the effects of four cytokines associated with AMD, TNFα, TGF-ß2, IL-6, and IL-1ß, as well as a cocktail containing all four cytokines, on RPE metabolism using ARPE-19 cells, primary human RPE cells, and ex vivo rat eyecups. Strikingly, we found cytokine-specific changes in numerous metabolic markers including lactate production, glucose consumption, extracellular acidification rate, and oxygen consumption rate accompanied by increases in total mitochondrial volume and ATP production. Together, all four cytokines could potently override the constitutive suppression of glycolysis in the RPE, through a mechanism independent of PI3K/AKT, MEK/ERK, or NF-κB. Finally, we observed changes in glycolytic gene expression with cytokine treatment, including in lactate dehydrogenase subunit and glucose transporter expression. Our findings provide new insights into the metabolic changes in the RPE under inflammatory conditions and highlight potential therapeutic targets for AMD.

Causal association between rheumatoid arthritis and an increased risk of age-related macular degeneration: A Mendelian randomization study.

This study's goal is to evaluate if there is a causal connection between rheumatoid arthritis (RA) and age-related macular degeneration (AMD), despite past epidemiological studies suggesting an association between the 2 disorders. The impact of RA on AMD is still unknown. Mendelian randomization (MR) was utilized in this study to assess the two-sample causal relationship between RA and AMD. Summary data from GWAS for RA and AMD in individuals with all European ancestries were gathered using the IEU GWAS database. The GWAS summary statistics of RA (14,361 RA patients and 43,923 healthy controls) and AMD (14,034 AMD patients and 91,214 controls participated) were obtained from the IEU GWAS database. After identifying suitable instrumental variables in line with the 3 MR assumptions, we conducted MR using the Mendelian randomization-Egger (MR-Egger), weighted median, and inverse variance weighting techniques. The MR-Egger intercept and MR-Polyvalent Residuals and Outliers methods were used to investigate the effects of horizontal pleiotropy. The leave-one-out strategy was used to prevent bias caused by certain single nucleotide polymorphisms. Sensitivity analysis was used to detect the heterogeneity. Using 50 single nucleotide polymorphisms as instrumental variables, this study examined the relationship between RA and AMD and discovered that RA increased the risk of AMD (inverse variance weighting odds ratio [OR] = 1.056, 95% confidence interval [CI] = 1.02-1.09, P = 5.44E-04; weighted median OR = 1.085, 95% CI = 1.04-1.14, P = 4.05E-04; MR-Egger OR = 1.074, 95% CI = 1.01-1.14, P = 2.18E-2). The current investigation demonstrated a causal link between AMD and RA. RA increased the risk of AMD. It is advised that future research concentrate on the processes underlying the relationship between RA and AMD.

QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype-environment interaction in retina.

Understanding and Rescuing the Splicing Defect Caused by the Frequent ABCA4 Variant c.4253+43G>A Underlying Stargardt Disease.

Pathogenic variants in ABCA4 are the underlying molecular cause of Stargardt disease (STGD1), an autosomal recessive macular dystrophy characterized by a progressive loss of central vision. Among intronic ABCA4 variants, c.4253+43G>A is frequently detected in STGD1 cases and is classified as a hypomorphic allele, generally associated with late-onset cases. This variant was previously reported to alter splicing regulatory sequences, but the splicing outcome is not fully understood yet. In this study, we attempted to better understand its effect on splicing and to rescue the aberrant splicing via antisense oligonucleotides (AONs). Wild-type and c.4253+43G>A variant-harboring maxigene vectors revealed additional skipping events, which were not previously detected upon transfection in HEK293T cells. To restore exon inclusion, we designed a set of 27 AONs targeting either splicing silencer motifs or the variant region and screened these in maxigene-transfected HEK293T cells. Candidate AONs able to promote exon inclusion were selected for further testing in patient-derived photoreceptor precursor cells. Surprisingly, no robust splicing modulation was observed in this model system. Overall, this research helped to adequately characterize the splicing alteration caused by the c.4253+43G>A variant, although future development of AON-mediated exon inclusion therapy for ABCA4 is needed.

Research progress of RP1L1 gene in disease.

Retinitis pigmentosa 1-like 1 (RP1L1) is a component of photoreceptor cilia. Pathogenic variants in RP1L1 cause photoreceptor diseases, suggesting that RP1L1 plays an important role in photoreceptor biology, although its exact function is unknown. To date, RP1L1 variants have been associated with occult macular dystrophy (cone degeneration) and retinitis pigmentosa (rod degeneration). Here, we summarize the reported RP1L1-associated photoreceptor pathogenic mutations. The association between RP1L1 and other diseases (mainly several tumors) is also summarized and RP1L1 is included in a wider range of diseases. Finally, it is necessary to further explore the influence mechanism of RP1L1 gene on the health of photoreceptors and how it participates in the occurrence and development of tumors.

Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Identification of Genetic Variants for Risk Prediction and Early Diagnosis of Age-Related Macular Degeneration in the Taiwanese Population.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly worldwide. The prevalence and phenotypes of AMD differ among populations, including between people in Taiwan and other regions. We performed a genome-wide association study to identify genetic variants and to develop genetic models to predict the risk of AMD development and progression in the Taiwanese population. In total, 4039 patients with AMD and 16,488 non-AMD controls (aged ≥ 65 years) were included. We identified 31 AMD-associated variants (p < 5 × 10-8) on chromosome 10q26, surrounding PLEKHA1-ARMS2-HTRA1. Two genetic models were constructed using the clump and threshold method. Model 1 included the single nucleotide polymorphism rs11200630 and showed a 1.31-fold increase in the risk of AMD per risk allele (95% confidence interval (CI) = 1.20-1.43, p < 0.001). In model 2, 1412 single-nucleotide polymorphisms were selected to construct a polygenic risk score (PRS). Individuals with the top 5% PRS had a 1.40-fold higher AMD risk compared with that of individuals with a PRS in the bottom quartile (95% CI = 1.04-1.89, p = 0.025). Moreover, the PRS in the upper quartile was related to a decreased age at AMD diagnosis by 0.62 years (95% CI = -1.15, -0.09, p = 0.023). Both genetic models provide useful predictive power for populations at high risk of AMD, affording a basis for identifying patients requiring close follow-up and early intervention.

Mitochondrial damage and clearance in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a devastating eye disease that causes permanent vision loss in the central part of the retina, known as the macula. Patients with such severe visual loss face a reduced quality of life and are at a 1.5 times greater risk of death compared to the general population. Currently, there is no cure for or effective treatment for dry AMD. There are several mechanisms thought to underlie the disease, for example, ageing-associated chronic oxidative stress, mitochondrial damage, harmful protein aggregation and inflammation. As a way of gaining a better understanding of the molecular mechanisms behind AMD and thus developing new therapies, we have created a peroxisome proliferator-activated receptor gamma coactivator 1-alpha and nuclear factor erythroid 2-related factor 2 (PGC1α/NFE2L2) double-knockout (dKO) mouse model that mimics many of the clinical features of dry AMD, including elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in retinal pigment epithelial cells (RPE). In addition, a human RPE cell-based model was established to examine the impact of non-functional intracellular clearance systems on inflammasome activation. In this study, we found that there was a disturbance in the autolysosomal machinery responsible for clearing mitochondria in the RPE cells of one-year-old PGC1α/NFE2L2-deficient mice. The confocal immunohistochemical analysis revealed an increase in autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as multiple mitophagy markers such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN), along with signs of damaged mitochondria. However, no increase in autolysosome formation was detected, nor was there a colocalization of the lysosomal marker LAMP2 or the mitochondrial marker, ATP synthase ß. There was an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells, together with autofluorescent aggregates. Additionally, we observed an increase in the numbers of Toll-like receptors 3 and 9, while those of NOD-like receptor 3 were decreased in PGC1α/NFE2L2 dKO retinal specimens compared to wild-type animals. There was a trend towards increased complement component C5a and increased involvement of the serine protease enzyme, thrombin, in enhancing the terminal pathway producing C5a, independent of C3. The levels of primary acute phase C-reactive protein and receptor for advanced glycation end products were also increased in the PGC1α/NFE2L2 dKO retina. Furthermore, selective proteasome inhibition with epoxomicin promoted both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial-mediated oxidative stress, leading to the release of mitochondrial DNA to the cytosol, resulting in potassium efflux-dependent activation of the absent in melanoma 2 (AIM2) inflammasome and the subsequent secretion of interleukin-1ß in ARPE-19 cells. In conclusion, the data suggest that there is at least a relative decrease in mitophagy, increases in the amounts of C5 and thrombin and decreased C3 levels in this dry AMD-like model. Moreover, selective proteasome inhibition evoked mitochondrial damage and AIM2 inflammasome activation in ARPE-19 cells.

Genetics and Age-Related Macular Degeneration: A Practical Review for Clinicians.

Age-related macular degeneration (AMD) is a multifactorial genetic disease, with at least 52 identifiable associated gene variants at 34 loci, including variants in complement factor H (CFH) and age-related maculopathy susceptibility 2/high-temperature requirement A serine peptidase-1 (ARMS2/HTRA1). Genetic factors account for up to 70% of disease variability. However, population-based genetic risk scores are generally more helpful for clinical trial design and stratification of risk groups than for individual patient counseling. There is some evidence of pharmacogenetic influences on various treatment modalities used in AMD patients, including Age-Related Eye Disease Study (AREDS) supplements, photodynamic therapy (PDT), and anti-vascular endothelial growth factor (anti-VEGF) agents. However, there is currently no convincing evidence that genetic information plays a role in routine clinical care.

Identification of key lncRNAs in age-related macular degeneration through integrated bioinformatics and experimental validation.

This study aimed to identify key long noncoding RNAs (lncRNAs) in age-related macular degeneration (AMD) patients and to identify relevant pathological mechanisms of AMD development. We identified 407 differentially expressed mRNAs and 429 differentially expressed lncRNAs in retinal pigment epithelium (RPE) and retina in the macular region of AMD patients versus controls (P < 0.05 and |log2FC| > 0.585) from GSE135092. A total of 14 key differentially expressed mRNAs were obtained through external data validation from GSE115828. A miRNA-mRNA and miRNA-lncRNA network containing 52 lncRNA nodes, 49 miRNA nodes, 14 mRNA nodes and 351 edges was constructed via integrated analysis of these components. Finally, the LINC00276-miR-619-5p-IFIT3 axis was identified via protein-protein network analysis. In the t-BH-induced ARPE-19 senescent cell model, LINC00276 and IFIT3 were downregulated. Overexpression of LINC00276 could accelerate cell migration in combination with IFIT3 upregulation. This compelling finding suggests that LINC00276 plays an influential role in the progression of AMD, potentially through modulating senescence processes, thereby setting a foundation for future investigative efforts to verify this relationship.

Targeting ZIP8 mediated ferroptosis as a novel strategy to protect against the retinal pigment epithelial degeneration.

The degeneration of retinal pigment epithelium (RPE) plays an important role in the development of age-related macular degeneration (AMD). However, the underlying mechanism remains elusive. In this study, we identified that ZIP8, a metal-ion transporter, plays a crucial role in the degeneration of RPE cells mediated by ferroptosis. ZIP8 was found to be upregulated in patients with AMD through transcriptome analysis. Upregulated ZIP8 was also observed in both oxidative-stressed RPE cells and AMD mouse model. Importantly, knockdown of ZIP8 significantly inhibited ferroptosis in RPE cells induced by sodium iodate-induced oxidative stress. Blocking ZIP8 with specific antibodies reversed RPE degeneration and restored retinal function, improving visual loss in a mouse model of NaIO3-induced. Interestingly, the modification of the N-glycosylation sites N40, N72 and N88, but not N273, was essential for the intracellular iron accumulation mediated by ZIP8, which further led to increased lipid peroxidation and RPE death. These findings highlight the critical role of ZIP8 in RPE ferroptosis and provide a potential target for the treatment of diseases associated with retinal degeneration, including AMD.

Age-Related Macular Degeneration and Mitochondria-Associated Autoantibodies: A Review of the Specific Pathogenesis and Therapeutic Strategies.

Age-related macular degeneration (AMD) is a severe retinal disease that causes irreversible visual loss and blindness in elderly populations worldwide. The pathological mechanism of AMD is complex, involving the interactions of multiple environmental and genetic factors. A poor understanding of the disease leads to limited treatment options and few effective prevention methods. The discovery of autoantibodies in AMD patients provides an opportunity to explore the pathogenesis and treatment direction of the disease. This review focuses on the mitochondria-associated autoantibodies and summarizes the functional roles of mitochondria under physiological conditions and their alterations during the pathological states. Additionally, it discusses the crosstalk between mitochondria and other organelles, as well as the mitochondria-related therapeutic strategies in AMD.

CRISPR Manipulation of Age-Related Macular Degeneration Haplotypes in the Complement System: Potential Future Therapeutic Applications/Avenues.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss among the elderly in the developed world. Whilst AMD is a multifactorial disease, the involvement of the complement system in its pathology is well documented, with single-nucleotide polymorphisms (SNPs) in different complement genes representing an increased risk factor. With several complement inhibitors explored in clinical trials showing limited success, patients with AMD are still without a reliable treatment option. This indicates that there is still a gap of knowledge in the functional implications and manipulation of the complement system in AMD, hindering the progress towards translational treatments. Since the discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool, the field of molecular biology has been revolutionised. Genetic variants in the complement system have long been associated with an increased risk of AMD, and a variety of haplotypes have been identified to be predisposing/protective, with variation in complement genes believed to be the trigger for dysregulation of the cascade leading to inflammation. AMD-haplotypes (SNPs) alter specific aspects of the activation and regulation of the complement cascade, providing valuable insights into the pathogenic mechanisms of AMD with important diagnostic and therapeutic implications. The effect of targeting these AMD-related SNPs on the regulation of the complement cascade has been poorly explored, and the CRISPR/Cas system provides an ideal tool with which to explore this avenue. Current research concentrates on the association events of specific AMD-related SNPs in complement genes without looking into the effect of targeting these SNPs and therefore influencing the complement system in AMD pathogenesis. This review will explore the current understanding of manipulating the complement system in AMD pathogenesis utilising the genomic manipulation powers of the CRISPR/Cas systems. A number of AMD-related SNPs in different complement factor genes will be explored, with a particular emphasis on factor H (CFH), factor B (CFB), and complement C3 (C3).

Genetic associations of central serous chorioretinopathy subtypes, neovascular age-related macular degeneration, and polypoidal choroidal vasculopathy.

PURPOSE: To evaluate the genetic associations of different subtypes of central serous chorioretinopathy (CSCR), neovascular age-related macular degeneration (nAMD), and polypoidal choroidal vasculopathy (PCV). DESIGN: A case-control genetic association study. METHODS: This study enrolled 217 CSCR, 341 nAMD, 288 PCV patients, and 1380 controls. The CSCR patients were classified into those with focal or diffuse leakage, with or without pigment epithelial detachment (PED), and with or without macular neovascularization (MNV). Associations between 11 variants from 8 genes, ADAMTS9, ANGPT2, ARMS2, CFH, NR3C2, PGF, TNFRSF10A and VIPR2, and diseases/subtypes were analyzed by logistic regression analysis adjusted for age and sex, and inter-phenotype comparison by heterogeneity test. RESULTS: The CFH rs800292-A conferred a protective effect for CSCR with MNV (OR=0.44, P = 0.002) and a risk effect for CSCR without MNV (OR=1.31, P = 0.023). CSCR patients carrying rs800292-G had a 3.23-fold of increased risk towards developing secondary MNV (P = 1.45 ×10-4). CFH rs3753394, rs800292 and rs1329428 showed similar effects among CSCR with MNV, nAMD and PCV, but opposite effects on CSCR without MNV. TNFRSF10A rs13278062-T was associated with overall CSCR but not with CSCR subtypes, nAMD or PCV. Moreover, CFH and ARMS2 SNPs showed heterogeneous effects in CSCR without MNV against CSCR with MNV, nAMD and PCV. CONCLUSIONS: Genetic associations of CSCR with MNV resembled nAMD and PCV compared to CSCR without MNV, indicating differential genetic effects on neovascularization and choroidopathy. Further investigation of the functional roles of CFH, ARMS2, and TNFRSF10A in CSCR, nAMD and PCV should help elucidate the mechanisms of these maculopathies.

The identification of signature genes and their relationship with immune cell infiltration in age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a prevalent source of visual impairment among the elderly population, and its incidence has risen in tandem with the increasing longevity of humans. Despite the progress made with anti-VEGF therapy, clinical outcomes have proven to be unsatisfactory. METHOD: We obtained differentially expressed genes (DEGs) of AMD patients and healthy controls from the GEO database. GO and KEGG analyses were used to enrich the DEGs. Weighted gene coexpression network analysis (WGCNA) was used to identify modules related to AMD. SVM, random forest, and least absolute shrinkage and selection operator (LASSO) were employed to screen hub genes. Gene set enrichment analysis (GSEA) was used to explore the pathways in which these hub genes were enriched. CIBERSORT was utilized to analyze the relationship between the hub genes and immune cell infiltration. Finally, Western blotting and RT‒PCR were used to explore the expression of hub genes in AMD mice. RESULTS: We screened 1084 DEGs in GSE29801, of which 496 genes were upregulated. These 1084 DEGs were introduced into the WGCNA, and 94 genes related to AMD were obtained. Seventy-nine overlapping genes were obtained by the Venn plot. These 79 genes were introduced into three machine-learning methods to screen the hub genes, and the genes identified by the three methods were TNC, FAP, SREBF1, and TGF-ß2. We verified their diagnostic function in the GSE29801 and GSE103060 datasets. Then, the hub gene co-enrichment pathways were obtained by GO and KEGG analyses. CIBERSORT analysis showed that these hub genes were associated with immune cell infiltration. Finally, we found increased expression of TNC, FAP, SREBF1, and TGF-ß2 mRNA and protein in the retinas of AMD mice. CONCLUSION: We found that four hub genes, namely, FAP, TGF-ß2, SREBF1, and TNC, have diagnostic significance in patients with AMD and are related to immune cell infiltration. Finally, we determined that the mRNA and protein expression of these hub genes was upregulated in the retinas of AMD mice.

Causal associations of refractive error and early age-related macular degeneration: A Mendelian randomization study.

This study aims to determine the risk associated with early age-related macular degeneration (AMD) due to refractive errors (RE) using an analysis of genome-wide association study (GWAS) data through the two-sample Mendelian randomization approach. Single-nucleotide polymorphisms (SNPs) linked to refractive errors (RE) were obtained from numerous GWAS studies involving individuals of European descent. The data for early AMD was obtained from a diverse, multiethnic GWAS meta-analysis that included 105,248 participants (14,034 cases and 91,214 controls). The primary outcome measure focused on the rise in early AMD risk corresponding to a 1-diopter alteration in spherical power and cylindrical power. In the main Mendelian randomization analysis, inverse-variance weighting (IVW) methods were applied for the evaluation. Mendelian Randomization (MR) study revealed a substantial impact of refractive error (RE) on early AMD risk, with a 1-diopter increase in hypermetropia being related to a 1.16 odds ratio (OR) for a greater risk of early AMD (95% CI, 1.10-1.23; P < 0.01). This conclusion was further supported by four supplementary approaches, namely, Weighted mode, Weighted-median, Simple mode, and MR-Egger. The results suggest a heightened risk of early AMD correlated with hyperopia, necessitating further research to thoroughly elucidate this potential causal relationship.

Splenic monocytes drive pathogenic subretinal inflammation in age-related macular degeneration.

Age-related macular degeneration (AMD) is invariably associated with the chronic accumulation of activated mononuclear phagocytes in the subretinal space. The mononuclear phagocytes are composed of microglial cells but also of monocyte-derived cells, which promote photoreceptor degeneration and choroidal neovascularization. Infiltrating blood monocytes can originate directly from bone marrow, but also from a splenic reservoir, where bone marrow monocytes develop into angiotensin II receptor (ATR1)+ splenic monocytes. The involvement of splenic monocytes in neurodegenerative diseases such as AMD is not well understood. Using acute inflammatory and well-phenotyped AMD models, we demonstrate that angiotensin II mobilizes ATR1+ splenic monocytes, which we show are defined by a transcriptional signature using single-cell RNA sequencing and differ functionally from bone marrow monocytes. Splenic monocytes participate in the chorio-retinal infiltration and their inhibition by ATR1 antagonist and splenectomy reduces the subretinal mononuclear phagocyte accumulation and pathological choroidal neovascularization formation. In aged AMD-risk ApoE2-expressing mice, a chronic AMD model, ATR1 antagonist and splenectomy also inhibit the chronic retinal inflammation and associated cone degeneration that characterizes these mice. Our observation of elevated levels of plasma angiotensin II in AMD patients, suggests that similar events take place in clinical disease and argue for the therapeutic potential of ATR1 antagonists to inhibit splenic monocytes for the treatment of blinding AMD.

Benefits and Caveats in the Use of Retinal Pigment Epithelium-Specific Cre Mice.

The retinal pigment epithelium (RPE) is an important monolayer of cells present in the outer retina, forming a major part of the blood-retina barrier (BRB). It performs many tasks essential for the maintenance of retinal integrity and function. With increasing knowledge of the retina, it is becoming clear that both common retinal disorders, like age-related macular degeneration, and rare genetic disorders originate in the RPE. This calls for a better understanding of the functions of various proteins within the RPE. In this regard, mice enabling an RPE-specific gene deletion are a powerful tool to study the role of a particular protein within the RPE cells in their native environment, simultaneously negating any potential influences of systemic changes. Moreover, since RPE cells interact closely with adjacent photoreceptors, these mice also provide an excellent avenue to study the importance of a particular gene function within the RPE to the retina as a whole. In this review, we outline and compare the features of various Cre mice created for this purpose, which allow for inducible or non-inducible RPE-specific knockout of a gene of interest. We summarize the various benefits and caveats involved in the use of such mouse lines, allowing researchers to make a well-informed decision on the choice of Cre mouse to use in relation to their research needs.

Distinct Clinical Effects of Two RP1L1 Hotspots in East Asian Patients With Occult Macular Dystrophy (Miyake Disease): EAOMD Report 4.

Purpose: To characterize the clinical effects of two RP1L1 hotspots in patients with East Asian occult macular dystrophy (OMD). Methods: Fifty-one patients diagnosed with OMD harboring monoallelic pathogenic RP1L1 variants (Miyake disease) from Japan, South Korea, and China were enrolled. Patients were classified into two genotype groups: group A, p.R45W, and group B, missense variants located between amino acids (aa) 1196 and 1201. The clinical parameters of the two genotypes were compared, and deep learning based on spectral-domain optical coherence tomographic (SD-OCT) images was used to distinguish the morphologic differences. Results: Groups A and B included 29 and 22 patients, respectively. The median age of onset in groups A and B was 14.0 and 40.0 years, respectively. The median logMAR visual acuity of groups A and B was 0.70 and 0.51, respectively, and the survival curve analysis revealed a 15-year difference in vision loss (logMAR 0.22). A statistically significant difference was observed in the visual field classification, but no significant difference was found in the multifocal electroretinographic classification. High accuracy (75.4%) was achieved in classifying genotype groups based on SD-OCT images using machine learning. Conclusions: Distinct clinical severities and morphologic phenotypes supported by artificial intelligence-based classification were derived from the two investigated RP1L1 hotspots: a more severe phenotype (p.R45W) and a milder phenotype (1196-1201 aa). This newly identified genotype-phenotype association will be valuable for medical care and the design of therapeutic trials.

Fluorescence Lifetime Imaging Ophthalmoscopy of Mouse Models of Age-related Macular Degeneration.

Purpose: To investigate fluorescence lifetime of mouse models of age-related macular degeneration (AMD) by fluorescence lifetime imaging ophthalmoscopy (FLIO). Methods: Two AMD mouse models, apolipoprotein E knockout (ApoE-/-) mice and NF-E2-related factor-2 knockout (Nrf2-/-) mice, and their wild-type mice underwent monthly ophthalmic examinations including FLIO from 3 months of age. After euthanasia at the age of 6 or 11 months, blood plasma was collected to determine total antioxidant capacity and eyes were enucleated for Oil red O (ORO) lipid staining of chorioretinal tissue. Results: In FLIO, the mean fluorescence lifetime (τm) of wild type shortened with age in both spectral channels. In short spectral channel, τm shortening was observed in both AMD models as well, but its rate was more pronounced in ApoE-/- mice and significantly different from the other strains as months of age progressed. In contrast, in long spectral channel, both model strains showed completely opposite trends, with τm becoming shorter in ApoE-/- and longer in Nrf2-/- mice than the others. Oil red O staining at Bruch's membrane was significantly stronger in ApoE-/- mice at 11 months than the other strains. Plasma total antioxidant capacity was highest in ApoE-/- mice at both 6 and 11 months. Conclusions: The two AMD mouse models exhibited largely different fundus fluorescence lifetime, which might be related to the different systemic metabolic state. FLIO might be able to indicate different metabolic states of eyes at risk for AMD. Translational Relevance: This animal study may provide new insights into the relationship between early AMD-associated metabolic changes and FLIO findings.