Restoration of Rod-Derived Metabolic and Redox Signaling to Prevent Blindness.

Vision is initiated by capturing photons in highly specialized sensory cilia known as the photoreceptor outer segment. Because of its lipid and protein composition, the outer segments are prone to photo-oxidation, requiring photoreceptors to have robust antioxidant defenses and high metabolic synthesis rates to regenerate the outer segments every 10 days. Both processes required high levels of glucose uptake and utilization. Retinitis pigmentosa is a prevalent form of inherited retinal degeneration characterized by initial loss of low-light vision caused by the death of rod photoreceptors. In this disease, rods die as a direct effect of an inherited mutation. Following the loss of rods, cones eventually degenerate, resulting in complete blindness. The progression of vision loss in retinitis pigmentosa suggested that rod photoreceptors were necessary to maintain healthy cones. We identified a protein secreted by rods that functions to promote cone survival, and we named it rod-derived cone viability factor (RdCVF). RdCVF is encoded by an alternative splice product of the nucleoredoxin-like 1 (NXNL1) gene, and RdCVF was found to accelerate the uptake of glucose by cones. Without RdCVF, cones eventually die because of compromised glucose uptake and utilization. The NXNL1 gene also encodes for the thioredoxin RdCVFL, which reduces cysteines in photoreceptor proteins that are oxidized, providing a defense against radical oxygen species. We will review here the main steps of discovering this novel intercellular signaling currently under translation as a broad-spectrum treatment for retinitis pigmentosa.

Modulating antioxidant systems as a therapeutic approach to retinal degeneration.

The human retina is facing a big challenge of reactive oxygen species (ROS) from endogenous and exogenous sources. Excessive ROS can cause damage to DNA, lipids, and proteins, triggering abnormal redox signaling, and ultimately lead to cell death. Thus, oxidative stress has been observed in inherited retinal diseases as a common hallmark. To counteract the detrimental effect of ROS, cells are equipped with various antioxidant defenses. In this review, we will focus on the antioxidant systems in the retina and how they can protect retina from oxidative stress. Both small antioxidants and antioxidant enzymes play a role in ROS removal. Particularly, the thioredoxin and glutaredoxin systems, as the major antioxidant systems in mammalian cells, exert functions in redox signaling regulation via modifying cysteines in proteins. In addition, the thioredoxin-like rod-derived cone viability factor (RdCVFL) and thioredoxin interacting protein (TXNIP) can modulate metabolism in photoreceptors and promote their survival. In conclusion, elevating the antioxidant capacity in retina is a promising therapy to curb the progress of inherited retinal degeneration.

The metabolic signaling of the nucleoredoxin-like 2 gene supports brain function.

The nucleoredoxin gene NXNL2 encodes for two products through alternative splicing, rod-derived cone viability factor-2 (RdCVF2) that mediates neuronal survival and the thioredoxin-related protein (RdCVF2L), an enzyme that regulates the phosphorylation of TAU. To investigate the link between NXNL2 and tauopathies, we studied the Nxnl2 knockout mouse (Nxnl2-/-). We established the expression pattern of the Nxnl2 gene in the brain using a Nxnl2 reporter mouse line, and characterized the behavior of the Nxnl2-/- mouse at 2 months of age. Additionally, long term potentiation and metabolomic from hippocampal specimens were collected at 2 months of age. We studied TAU oligomerization, phosphorylation and aggregation in Nxnl2-/- brain at 18 months of age. Finally, newborn Nxnl2-/- mice were treated with adeno-associated viral vectors encoding for RdCVF2, RdCVF2L or both and measured the effect of this therapy on long-term potential, glucose metabolism and late-onset tauopathy. Nxnl2-/- mice at 2 months of age showed severe behavioral deficiency in fear, pain sensitivity, coordination, learning and memory. The Nxnl2-/- also showed deficits in long-term potentiation, demonstrating that the Nxnl2 gene is involved in regulating brain functions. Dual delivery of RdCVF2 and RdCVF2L in newborn Nxnl2-/- mice fully correct long-term potentiation through their synergistic action. The expression pattern of the Nxnl2 gene in the brain shows a predominant expression in circumventricular organs, such as the area postrema. Glucose metabolism of the hippocampus of Nxnl2-/- mice at 2 months of age was reduced, and was not corrected by gene therapy. At 18-month-old Nxnl2-/- mice showed brain stigmas of tauopathy, such as oligomerization, phosphorylation and aggregation of TAU. This late-onset tauopathy can be prevented, albeit with modest efficacy, by recombinant AAVs administrated to newborn mice. The Nxnl2-/- mice have memory dysfunction at 2-months that resembles mild-cognitive impairment and at 18-months exhibit tauopathy, resembling to the progression of Alzheimer's disease. We propose the Nxnl2-/- mouse is a model to study multistage aged related neurodegenerative diseases. The NXNL2 metabolic and redox signaling is a new area of therapeutic research in neurodegenerative diseases.

A Splice Variant in SLC16A8 Gene Leads to Lactate Transport Deficit in Human iPS Cell-Derived Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) is a blinding disease for which most of the patients remain untreatable. Since the disease affects the macula at the center of the retina, a structure specific to the primate lineage, rodent models to study the pathophysiology of AMD and to develop therapies are very limited. Consequently, our understanding relies mostly on genetic studies highlighting risk alleles at many loci. We are studying the possible implication of a metabolic imbalance associated with risk alleles within the SLC16A8 gene that encodes for a retinal pigment epithelium (RPE)-specific lactate transporter MCT3 and its consequences for vision. As a first approach, we report here the deficit in transepithelial lactate transport of a rare SLC16A8 allele identified during a genome-wide association study. We produced induced pluripotent stem cells (iPSCs) from the unique patient in our cohort that carries two copies of this allele. After in vitro differentiation of the iPSCs into RPE cells and their characterization, we demonstrate that the rare allele results in the retention of intron 2 of the SLC16A8 gene leading to the absence of MCT3 protein. We show using a biochemical assay that these cells have a deficit in transepithelial lactate transport.

The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.

Metabolic and Redox Signaling of the Nucleoredoxin-Like-1 Gene for the Treatment of Genetic Retinal Diseases.

The loss of cone photoreceptor function in retinitis pigmentosa (RP) severely impacts the central and daily vision and quality of life of patients affected by this disease. The loss of cones follows the degeneration of rods, in a manner independent of the causing mutations in numerous genes associated with RP. We have explored this phenomenon and proposed that the loss of rods triggers a reduction in the expression of rod-derived cone viability factor (RdCVF) encoded by the nucleoredoxin-like 1 (NXNL1) gene which interrupts the metabolic and redox signaling between rods and cones. After providing scientific evidence supporting this mechanism, we propose a way to restore this lost signaling and prevent the cone vision loss in animal models of RP. We also explain how we could restore this signaling to prevent cone vision loss in animal models of the disease and how we plan to apply this therapeutic strategy by the administration of both products of NXNL1 encoding the trophic factor RdCVF and the thioredoxin enzyme RdCVFL using an adeno-associated viral vector. We describe in detail all the steps of this translational program, from the design of the drug, its production, biological validation, and analytical and preclinical qualification required for a future clinical trial that would, if successful, provide a treatment for this incurable disease.

Is Retinal Metabolic Dysfunction at the Center of the Pathogenesis of Age-related Macular Degeneration?

The retinal pigment epithelium (RPE) forms the outer blood⁻retina barrier and facilitates the transepithelial transport of glucose into the outer retina via GLUT1. Glucose is metabolized in photoreceptors via the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) but also by aerobic glycolysis to generate glycerol for the synthesis of phospholipids for the renewal of their outer segments. Aerobic glycolysis in the photoreceptors also leads to a high rate of production of lactate which is transported out of the subretinal space to the choroidal circulation by the RPE. Lactate taken up by the RPE is converted to pyruvate and metabolized via OXPHOS. Excess lactate in the RPE is transported across the basolateral membrane to the choroid. The uptake of glucose by cone photoreceptor cells is enhanced by rod-derived cone viability factor (RdCVF) secreted by rods and by insulin signaling. Together, the three cells act as symbiotes: the RPE supplies the glucose from the choroidal circulation to the photoreceptors, the rods help the cones, and both produce lactate to feed the RPE. In age-related macular degeneration this delicate ménage à trois is disturbed by the chronic infiltration of inflammatory macrophages. These immune cells also rely on aerobic glycolysis and compete for glucose and produce lactate. We here review the glucose metabolism in the homeostasis of the outer retina and in macrophages and hypothesize what happens when the metabolism of photoreceptors and the RPE is disturbed by chronic inflammation.

Mechanisms Underlying the Visual Benefit of Cell Transplantation for the Treatment of Retinal Degenerations.

The transplantation of retinal cells has been studied in animals to establish proof of its potential benefit for the treatment of blinding diseases. Photoreceptor precursors have been grafted in animal models of Mendelian-inherited retinal degenerations, and retinal pigmented epithelial cells have been used to restore visual function in animal models of age-related macular degeneration (AMD) and recently in patients. Cell therapy over corrective gene therapy in inherited retinal degeneration can overcome the genetic heterogeneity by providing one treatment for all genetic forms of the diseases. In AMD, the existence of multiple risk alleles precludes a priori the use of corrective gene therapy. Mechanistically, the experiments of photoreceptor precursor transplantation reveal the importance of cytoplasmic material exchange between the grafted cells and the host cells for functional rescue, an unsuspected mechanism and novel concept. For transplantation of retinal pigmented epithelial cells, the mechanisms behind the therapeutic benefit are only partially understood, and clinical trials are ongoing. The fascinating studies that describe the development of methodologies to produce cells to be grafted and demonstrate the functional benefit for vision are reviewed.

Maintaining Cone Function in Rod-Cone Dystrophies.

Retinal degenerative diseases are a major cause of untreatable blindness due to a loss of photoreceptors. Recent advances in genetics and gene therapy for inherited retinal dystrophies (IRDs) showed that therapeutic gene transfer holds a great promise for vision restoration in people with currently incurable blinding diseases. Due to the huge genetic heterogeneity of IRDs that represents a major obstacle for gene therapy development, alternative therapeutic approaches are needed. This review focuses on the rescue of cone function as a therapeutic option for maintaining central vision in rod-cone dystrophies. It highlights recent developments in better understanding the mechanisms of action of the trophic factor RdCVF and its potential as a sight-saving therapeutic strategy.

Otx2-Genetically Modified Retinal Pigment Epithelial Cells Rescue Photoreceptors after Transplantation.

Inherited retinal degenerations are blinding diseases characterized by the loss of photoreceptors. Their extreme genetic heterogeneity complicates treatment by gene therapy. This has motivated broader strategies for transplantation of healthy retinal pigmented epithelium to protect photoreceptors independently of the gene causing the disease. The limited clinical benefit for visual function reported up to now is mainly due to dedifferentiation of the transplanted cells that undergo an epithelial-mesenchymal transition. We have studied this mechanism in vitro and revealed the role of the homeogene OTX2 in preventing dedifferentiation through the regulation of target genes. We have overexpressed OTX2 in retinal pigmented epithelial cells before their transplantation in the eye of a model of retinitis pigmentosa carrying a mutation in Mertk, a gene specifically expressed by retinal pigmented epithelial cells. OTX2 increases significantly the protection of photoreceptors as seen by histological and functional analyses. We observed that the beneficial effect of OTX2 is non-cell autonomous, and it is at least partly mediated by unidentified trophic factors. Transplantation of OTX2-genetically modified cells may be medically effective for other retinal diseases involving the retinal pigmented epithelium as age-related macular degeneration.

Cell Signaling with Extracellular Thioredoxin and Thioredoxin-Like Proteins: Insight into Their Mechanisms of Action.

Thioredoxins are small thiol-oxidoreductase enzymes that control cellular redox homeostasis. Paradoxically, human thioredoxin (TXN1) was first identified as the adult T cell leukemia-derived factor (ADF), a secreted protein. ADF has been implicated in a wide variety of cell-to-cell communication systems acting as a cytokine or a chemokine. TRX80 is a truncated TXN1 protein with cytokine activity. The unconventional secretion mechanism of these extracellular thioredoxins is unknown. The thioredoxin system is relying on glucose metabolism through the pentose phosphate pathway that provides reducing power in the form of NADPH, the cofactor of thioredoxin reductase (TXNRD). While a complete extracellular TXN system is present in the blood in the form of circulating TXN1 and TXNDR1, the source of extracellular NADPH remains a mystery. In the absence of redox regenerating capacity, extracellular thioredoxins may rather be prooxidant agents. Rod-derived cone viability factor (RdCVF) is the product of intron retention of the nucleoredoxin-like 1 (NXNL1) gene, a secreted truncated thioredoxin-like protein. The other product encoded by the gene, RdCVFL, is an enzymatically active thioredoxin. This is a very singular example of positive feedback of a superthioredoxin system encoded by a single gene likely emerging during evolution from metabolic constraints on redox signaling.

Metabolic and redox signaling in the retina.

Visual perception by photoreceptors relies on the interaction of incident photons from light with a derivative of vitamin A that is covalently linked to an opsin molecule located in a special subcellular structure, the photoreceptor outer segment. The photochemical reaction produced by the photon is optimal when the opsin molecule, a seven-transmembrane protein, is embedded in a lipid bilayer of optimal fluidity. This is achieved in vertebrate photoreceptors by a high proportion of lipids made with polyunsaturated fatty acids, which have the detrimental property of being oxidized and damaged by light. Photoreceptors cannot divide, but regenerate their outer segments. This is an enormous energetic challenge that explains why photoreceptors metabolize glucose through aerobic glycolysis, as cancer cells do. Uptaken glucose produces metabolites to renew that outer segment as well as reducing power through the pentose phosphate pathway to protect photoreceptors against oxidative damage.

Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells.

To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene.

Viral-mediated RdCVF and RdCVFL expression protects cone and rod photoreceptors in retinal degeneration.

Alternative splicing of nucleoredoxin-like 1 (Nxnl1) results in 2 isoforms of the rod-derived cone viability factor. The truncated form (RdCVF) is a thioredoxin-like protein secreted by rods that promotes cone survival, while the full-length isoform (RdCVFL), which contains a thioredoxin fold, is involved in oxidative signaling and protection against hyperoxia. Here, we evaluated the effects of these different isoforms in 2 murine models of rod-cone dystrophy. We used adeno-associated virus (AAV) to express these isoforms in mice and found that both systemic and intravitreal injection of engineered AAV vectors resulted in RdCVF and RdCVFL expression in the eye. Systemic delivery of AAV92YF vectors in neonates resulted in earlier onset of RdCVF and RdCVFL expression compared with that observed with intraocular injection using the same vectors at P14. We also evaluated the efficacy of intravitreal injection using a recently developed photoreceptor-transducing AAV variant (7m8) at P14. Systemic administration of AAV92YF-RdCVF improved cone function and delayed cone loss, while AAV92YF-RdCVFL increased rhodopsin mRNA and reduced oxidative stress by-products. Intravitreal 7m8-RdCVF slowed the rate of cone cell death and increased the amplitude of the photopic electroretinogram. Together, these results indicate different functions for Nxnl1 isoforms in the retina and suggest that RdCVF gene therapy has potential for treating retinal degenerative disease.

Genome-wide association study of age-related macular degeneration identifies associated variants in the TNXB-FKBPL-NOTCH4 region of chromosome 6p21.3.

Age-related macular degeneration (AMD) is a leading cause of visual loss in Western populations. Susceptibility is influenced by age, environmental and genetic factors. Known genetic risk loci do not account for all the heritability. We therefore carried out a genome-wide association study of AMD in the UK population with 893 cases of advanced AMD and 2199 controls. This showed an association with the well-established AMD risk loci ARMS2 (age-related maculopathy susceptibility 2)-HTRA1 (HtrA serine peptidase 1) (P =2.7 × 10(-72)), CFH (complement factor H) (P =2.3 × 10(-47)), C2 (complement component 2)-CFB (complement factor B) (P =5.2 × 10(-9)), C3 (complement component 3) (P =2.2 × 10(-3)) and CFI (P =3.6 × 10(-3)) and with more recently reported risk loci at VEGFA (P =1.2 × 10(-3)) and LIPC (hepatic lipase) (P =0.04). Using a replication sample of 1411 advanced AMD cases and 1431 examined controls, we confirmed a novel association between AMD and single-nucleotide polymorphisms on chromosome 6p21.3 at TNXB (tenascin XB)-FKBPL (FK506 binding protein like) [rs12153855/rs9391734; discovery P =4.3 × 10(-7), replication P =3.0 × 10(-4), combined P =1.3 × 10(-9), odds ratio (OR) = 1.4, 95% confidence interval (CI) = 1.3-1.6] and the neighbouring gene NOTCH4 (Notch 4) (rs2071277; discovery P =3.2 × 10(-8), replication P =3.8 × 10(-5), combined P =2.0 × 10(-11), OR = 1.3, 95% CI = 1.2-1.4). These associations remained significant in conditional analyses which included the adjacent C2-CFB locus. TNXB, FKBPL and NOTCH4 are all plausible AMD susceptibility genes, but further research will be needed to identify the causal variants and determine whether any of these genes are involved in the pathogenesis of AMD.

Complement factor H genetic variant and age-related macular degeneration: effect size, modifiers and relationship to disease subtype.

BACKGROUND: Variation in the complement factor H gene (CFH) is associated with risk of late age-related macular degeneration (AMD). Previous studies have been case-control studies in populations of European ancestry with little differentiation in AMD subtype, and insufficient power to confirm or refute effect modification by smoking. METHODS: To precisely quantify the association of the single nucleotide polymorphism (SNP rs1061170, 'Y402H') with risk of AMD among studies with differing study designs, participant ancestry and AMD grade and to investigate effect modification by smoking, we report two unpublished genetic association studies (n = 2759) combined with data from 24 published studies (26 studies, 26,494 individuals, including 14,174 cases of AMD) of European ancestry, 10 of which provided individual-level data used to test gene-smoking interaction; and 16 published studies from non-European ancestry. RESULTS: In individuals of European ancestry, there was a significant association between Y402H and late-AMD with a per-allele odds ratio (OR) of 2.27 [95% confidence interval (CI) 2.10-2.45; P = 1.1 x 10(-161)]. There was no evidence of effect modification by smoking (P = 0.75). The frequency of Y402H varied by ancestral origin and the association with AMD in non-Europeans was less clear, limited by paucity of studies. CONCLUSION: The Y402H variant confers a 2-fold higher risk of late-AMD per copy in individuals of European descent. This was stable to stratification by study design and AMD classification and not modified by smoking. The lack of association in non-Europeans requires further verification. These findings are of direct relevance for disease prediction. New research is needed to ascertain if differences in circulating levels, expression or activity of factor H protein explain the genetic association.

Transcriptomic analysis of human retinal detachment reveals both inflammatory response and photoreceptor death.

BACKGROUND: Retinal detachment often leads to a severe and permanent loss of vision and its therapeutic management remains to this day exclusively surgical. We have used surgical specimens to perform a differential analysis of the transcriptome of human retinal tissues following detachment in order to identify new potential pharmacological targets that could be used in combination with surgery to further improve final outcome. METHODOLOGY/PRINCIPAL FINDINGS: Statistical analysis reveals major involvement of the immune response in the disease. Interestingly, using a novel approach relying on coordinated expression, the interindividual variation was monitored to unravel a second crucial aspect of the pathological process: the death of photoreceptor cells. Within the genes identified, the expression of the major histocompatibility complex I gene HLA-C enables diagnosis of the disease, while PKD2L1 and SLCO4A1 -which are both down-regulated- act synergistically to provide an estimate of the duration of the retinal detachment process. Our analysis thus reveals the two complementary cellular and molecular aspects linked to retinal detachment: an immune response and the degeneration of photoreceptor cells. We also reveal that the human specimens have a higher clinical value as compared to artificial models that point to IL6 and oxidative stress, not implicated in the surgical specimens studied here. CONCLUSIONS/SIGNIFICANCE: This systematic analysis confirmed the occurrence of both neurodegeneration and inflammation during retinal detachment, and further identifies precisely the modification of expression of the different genes implicated in these two phenomena. Our data henceforth give a new insight into the disease process and provide a rationale for therapeutic strategies aimed at limiting inflammation and photoreceptor damage associated with retinal detachment and, in turn, improving visual prognosis after retinal surgery.

Gene therapy in ophthalmology: validation on cultured retinal cells and explants from postmortem human eyes.

Gene therapy studies in primates can provide important information regarding vector tropism, specific cellular expression, biodistribution, and safety prior to clinical trials. In this study, we report the assessment of transduction efficiency of recombinant adeno-associated virus (rAAV) vectors using human postmortem retina. Transductions were performed using two in vitro models prepared from human tissue: dissociated cell cultures and retinal explants. These models were used to assess cellular tropism and selectivity of rAAV vectors encoding for fluorescent proteins under the control of different promoters. These promoters were a ubiquitous cytomegalovirus promoter and a cell type-specific promoter targeting expression in ON bipolar cells. The results demonstrate that this in vitro approach can limit the use of living primates for the validation of gene therapy in vision and ophthalmology.