Cone Degeneration Research Articles

Loss of Class III Phosphoinositide 3-Kinase Vps34 Results in Cone Degeneration.

Simple SummaryCone photoreceptors are the class of neurons in the retina that support daylight and color vision. In humans and rodents, the cone photoreceptors constitute a small percentage of total retinal photoreceptors; in some retinal diseases, these cells malfunction over time and cease to work, and eventually die. Class III phosphoinositide 3-kinase, also known as vacuole protein sorting 34 (Vps34), generates phosphoinositide 3-phosphate (PI(3)P), a lipid molecule that transmits information inside of the cell. PI(3)P plays an essential role in removing injured cells, a process called autophagy, which maintains a healthy environment, as well as in protein trafficking inside of the cell. Furthermore, PI(3)P can act as a bridging molecule for proteins to bind to each other. We eliminated the class III phosphoinositide 3-kinase in mouse cones, which resulted in the loss of visual function and death of cone cells. Our studies suggest that PI(3)P generated by class III phosphoinositide 3-kinase is essential for cone photoreceptor function and survival.The major pathway for the production of the low-abundance membrane lipid phosphatidylinositol 3-phosphate (PI(3)P) synthesis is catalyzed by class III phosphoinositide 3-kinase (PI3K) Vps34. The absence of Vps34 was previously found to disrupt autophagy and other membrane-trafficking pathways in some sensory neurons, but the roles of phosphatidylinositol 3-phosphate and Vps34 in cone photoreceptor cells have not previously been explored. We found that the deletion of Vps34 in neighboring rods in mouse retina did not disrupt cone function up to 8 weeks after birth, despite diminished rod function. Immunoblotting and lipid analysis of cones isolated from the cone-dominant retinas of the neural retina leucine zipper gene knockout mice revealed that both PI(3)P and Vps34 protein are present in mouse cones. To determine whether Vps34 and PI(3)P are important for cone function, we conditionally deleted Vps34 in cone photoreceptor cells of the mouse retina. Overall retinal morphology and rod function appeared to be unaffected. However, the loss of Vps34 in cones resulted in the loss of structure and function. There was a substantial reduction throughout the retina in the number of cones staining for M-opsin, S-opsin, cone arrestin, and peanut agglutinin, revealing degeneration of cones. These studies indicate that class III PI3K, and presumably PI(3)P, play essential roles in cone photoreceptor cell function and survival.

Noninvasive Imaging of Cone Ablation and Regeneration in Zebrafish.

PurposeTo observe and characterize cone degeneration and regeneration in a selective metronidazole-mediated ablation model of ultraviolet-sensitive (UV) cones in zebrafish using in vivo optical coherence tomography (OCT) imaging.MethodsTwenty-six sws1:nfsB-mCherry;sws2:eGFP zebrafish were imaged with OCT, treated with metronidazole to selectively kill UV cones, and imaged at 1, 3, 7, 14, 28, or 56 days after ablation. Regions 200 × 200 µm were cropped from volume OCT scans to count individual UV cones before and after ablation. Fish eyes were fixed, and immunofluorescence staining was used to corroborate cone density measured from OCT and to track monocyte response.ResultsHistology shows significant loss of UV cones after metronidazole treatment with a slight increase in observable blue cone density one day after treatment (Kruskal, Wallis, P = 0.0061) and no significant change in blue cones at all other timepoints. Regenerated UV cones measured from OCT show significantly lower density than pre-cone-ablation at 14, 28, and 56 days after ablation (analysis of variance, P < 0.01, P < 0.0001, P < 0.0001, respectively, 15.9% of expected nonablated levels). Histology shows significant changes to monocyte morphology (mixed-effects analysis, P < 0.0001) and retinal position (mixed-effects analysis, P < 0.0001).ConclusionsOCT can be used to observe loss of individual cones selectively ablated by metronidazole prodrug activation and to quantify UV cone loss and regeneration in zebrafish. OCT images also show transient changes to the blue cone mosaic and inner retinal layers that occur concomitantly with selective UV cone ablation.Translational RelevanceProfiling cone degeneration and regeneration using in vivo imaging enables experiments that may lead to a better understanding of cone regeneration in vertebrates.

Validating Fluorescent Chrnb4.EGFP Mouse Models for the Study of Cone Photoreceptor Degeneration.

PurposeTo validate the application of a known transgenic mouse line with green fluorescent cones (Chrnb4.EGFP) to study cone photoreceptor biology and function in health and disease.MethodsChrnb4.EGFP retinas containing GFP+ cones were compared with retinas without the GFP transgene via immunohistochemistry, quantitative real-time polymerase chain reaction, electroretinograms, and flow cytometry. The Chrnb4.EGFP line was backcrossed to the mouse models of cone degeneration, Pde6ccpfl1 and Gnat2cpfl3, generating the new lines Gnat2.GFP and Pde6c.GFP, which were also studied as described.ResultsGFP expression spanned the length of the cone cell in the Chrnb4.EGFP line, as well as in the novel Gnat2.GFP and Pde6c.GFP lines. The effect of GFP expression showed no significant changes to outer nuclear layer cell death, cone-specific gene expression, and immune response activation. A temporal decrease in GFP expression over time was observed, but GFP fluorescence was still detected through flow cytometry as late as 6 months. Furthermore, a functional analysis of photopic and scotopic electroretinogram responses of the Chrnb4 mouse showed no significant difference between GFP− and GFP+ mice, whereas electroretinogram recordings for the Pde6c.GFP and Gnat2.GFP lines matched previous reports from the original lines.ConclusionsThis study demonstrates that the Chrnb4.EGFP mouse can be a powerful tool to overcome the limitations of studying cone biology, including the use of this line to study different types of cone degeneration.Translational RelevanceThis work validates research tools that could potentially offer more reliable preclinical data in the development of treatments for cone-mediated vision loss conditions, shortening the gap to clinical translation.

Yap haploinsufficiency leads to M\xfcller cell dysfunction and late-onset cone dystrophy

Hippo signalling regulates eye growth during embryogenesis through its effectors YAP and TAZ. Taking advantage of a Yap heterozygous mouse line, we here sought to examine its function in adult neural retina, where YAP expression is restricted to Müller glia. We first discovered an unexpected temporal dynamic of gene compensation. At postnatal stages, Taz upregulation occurs, leading to a gain of function-like phenotype characterised by EGFR signalling potentiation and delayed cell-cycle exit of retinal progenitors. In contrast, Yap+/− adult retinas no longer exhibit TAZ-dependent dosage compensation. In this context, Yap haploinsufficiency in aged individuals results in Müller glia dysfunction, late-onset cone degeneration, and reduced cone-mediated visual response. Alteration of glial homeostasis and altered patterns of cone opsins were also observed in Müller cell-specific conditional Yap-knockout aged mice. Together, this study highlights a novel YAP function in Müller cells for the maintenance of retinal tissue homeostasis and the preservation of cone integrity. It also suggests that YAP haploinsufficiency should be considered and explored as a cause of cone dystrophies in human.

Microglia modulation by TGF-β1 protects cones in mouse models of retinal degeneration.

Retinitis pigmentosa (RP) is a genetically heterogenous group of eye diseases in which initial degeneration of rods triggers secondary degeneration of cones, leading to significant loss of daylight, color, and high-acuity vision. Gene complementation with adeno-associated viral (AAV) vectors is one strategy to treat RP. Its implementation faces substantial challenges, however; for example, the tremendous number of loci with causal mutations. Gene therapy targeting secondary cone degeneration is an alternative approach that could provide a much-needed generic treatment for many patients with RP. Here, we show that microglia are required for the upregulation of potentially neurotoxic inflammatory factors during cone degeneration in RP, creating conditions that might contribute to cone dysfunction and death. To ameliorate the effects of such factors, we used AAV vectors to express isoforms of the antiinflammatory cytokine transforming growth factor beta (TGF-β). AAV-mediated delivery of TGF-β1 rescued degenerating cones in 3 mouse models of RP carrying different pathogenic mutations. Treatment with TGF-β1 protected vision, as measured by 2 behavioral assays, and could be pharmacologically disrupted by either depleting microglia or blocking the TGF-β receptors. Our results suggest that TGF-β1 may be broadly beneficial for patients with cone degeneration, and potentially other forms of neurodegeneration, through a pathway dependent upon microglia.

Homeostatic Plasticity Shapes the Retinal Response to Photoreceptor Degeneration.

Homeostatic plasticity stabilizes input and activity levels during neural development, but whether it can restore connectivity and preserve circuit function during neurodegeneration is unknown. Photoreceptor degeneration is the most common cause of blindness in the industrialized world. Visual deficits are dominated by cone loss, which progresses slowly, leaving a window during which rewiring of second-order neurons (i.e., bipolar cells) could preserve function. Here we establish a transgenic model to induce cone degeneration with precise control and analyze bipolar cell responses and their effects on vision through anatomical reconstructions, invivo electrophysiology, and behavioral assays. In young retinas, we find that three bipolar cell types precisely restore input synapse numbers when 50% of cones degenerate but one does not. Of the three bipolar cell types that rewire, two contact new cones within stable dendritic territories, whereas one expands its dendrite arbors to reach new partners. In mature retinas, only one of four bipolar cell types rewires homeostatically. This steep decline in homeostatic plasticity is accompanied by reduced light responses of bipolar cells and deficits in visual behaviors. By contrast, light responses and behavioral performance are preserved when cones degenerate in young mice. Our results reveal unexpected cell type specificity and a steep maturational decline of homeostatic plasticity. The effect of homeostatic plasticity on functional outcomes identify it as a promising therapeutic target for retinal and other neurodegenerative diseases.

Coordinated Intervention of Microglial and Müller Cells in Light-Induced Retinal Degeneration.

PurposeTo analyze the role of microglial and Müller cells in the formation of rings of photoreceptor degeneration caused by phototoxicity.MethodsTwo-month-old Sprague-Dawley rats were exposed to light and processed 1, 2, or 3 months later. Retinas were dissected as whole-mounts, immunodetected for microglial cells, Müller cells, and S- and L/M-cones and analyzed using fluorescence, thunder imaging, and confocal microscopy. Cone populations were automatically counted and isodensity maps constructed to document cone topography.ResultsPhototoxicity causes a significant progressive loss of S- and L/M-cones of up to 68% and 44%, respectively, at 3 months after light exposure (ALE). One month ALE, we observed rings of cone degeneration in the photosensitive area of the superior retina. Two and 3 months ALE, these rings had extended to the central and inferior retina. Within the rings of cone degeneration, there were degenerating cones, often activated microglial cells, and numerous radially oriented processes of Müller cells that showed increased expression of intermediate filaments. Between 1 and 3 months ALE, the rings coalesced, and at the same time the microglial cells resumed a mosaic-like distribution, and there was a decrease of Müller cell gliosis at the areas devoid of cones.ConclusionsLight-induced photoreceptor degeneration proceeds with rings of cone degeneration, as observed in inherited retinal degenerations in which cone death is secondary to rod degeneration. The spatiotemporal relationship of cone death microglial cell activation and Müller cell gliosis within the rings of cone degeneration suggests that, although both glial cells are involved in the formation of the rings, they may have coordinated actions and, while microglial cells may be more involved in photoreceptor phagocytosis, Müller cells may be more involved in cone and microglial cell migration, retinal remodeling and glial seal formation.

Potential contribution of ryanodine receptor 2 upregulation to cGMP/PKG signaling-induced cone degeneration in cyclic nucleotide-gated channel deficiency.

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca2+ influx in rod and cone photoreceptors. Mutations in cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. Mice lacking functional cone CNG channel show endoplasmic reticulum (ER) stress-associated cone degeneration. The elevated cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) signaling and upregulation of the ER Ca2+ channel ryanodine receptor 2 (RyR2) have been implicated in cone degeneration. This work investigates the potential contribution of RyR2 to cGMP/PKG signaling-induced ER stress and cone degeneration. We demonstrated that the expression and activity of RyR2 were highly regulated by cGMP/PKG signaling. Depletion of cGMP by deleting retinal guanylate cyclase 1 or inhibition of PKG using chemical inhibitors suppressed the upregulation of RyR2 in CNG channel deficiency. Depletion of cGMP or deletion of Ryr2 equivalently inhibited unfolded protein response/ER stress, activation of the CCAAT-enhancer-binding protein homologous protein, and activation of the cyclic adenosine monophosphate response element-binding protein, leading to early-onset cone protection. In addition, treatment with cGMP significantly enhanced Ryr2 expression in cultured photoreceptor-derived Weri-Rb1 cells. Findings from this work demonstrate the regulation of cGMP/PKG signaling on RyR2 in the retina and support the role of RyR2 upregulation in cGMP/PKG signaling-induced ER stress and photoreceptor degeneration.

The porcine iodoacetic acid model of retinal degeneration: Morpho-functional characterization of the visual system

Porcine models of ophthalmological diseases are often used in pre-clinical translational studies due to pigs’ similarities to humans. In particular, the iodoacetic acid (IAA) model of photoreceptor degeneration seems to mimic well the endstage phenotype of human pathologies as retinitis pigmentosa and age-related macular degeneration, with high potential for prosthesis/retinal devices testing. IAA is capable of inducing photoreceptor death by blockage of glycolysis, and its effects on the retina have been described. Nonetheless, up to date, literature lacks of a comprehensive morpho-functional characterization of the entire visual system of this model. This gap is particularly critical for prosthesis testing as inner retinal structures and optic pathways must be preserved to elicit cortical responses and restore vision. In this study, we investigated the functional and anatomical features of the visual system of IAA-treated pigs and compared them to control animals. IAA was administered intravenously at 12 mg/kg; control animals received saline solution (NaCl 0.9% w/v). Electrophysiological analyses included full-field (ffERGs) and pattern (PERGs) electroretinograms and flash visually evoked potentials (fVEPs). Histological evaluations were performed on the retina and the optic pathways and included thickness of the different retinal layers, ganglion cells count, and immunohistochemistry for microglial cells, macroglial cells, and oligodendrocytes. The histological results indicate that IAA treatment does not affect the morphology of the inner retina and optic pathways. Electrophysiology confirms the selective rod and partial cone degeneration, but is ambiguous as to the functionality of the optic pathways, seemingly preserved as indicated by the still detectable fVEPs. Overall, the work ameliorates the characterization of such rapid and cost-effective model, providing more strength and reliability for future pre-clinical translational trials.

Deletion of M-Opsin Prevents M Cone Degeneration in a Mouse Model of Leber Congenital Amaurosis

Mutations in retinoid isomerase (RPE65) or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA). Despite the success of recent RPE65 gene therapy, follow-up studies show that patients continue to experience photoreceptor degeneration and lose vision benefit over time. In Lrat-/- mouse model, mislocalized medium (M)-wavelength opsin was degraded, whereas mislocalized short (S)-wavelength opsin accumulated before the onset of cone degeneration. The mechanism for the foveal M/long-wavelength cone photoreceptor degeneration in LCA is unknown. By crossing Lrat-/- mice with a proteasome reporter mouse strain, this study showed that M-opsin-enriched dorsal cones in Lrat-/- mice exhibit proteasome stress because of the degradation of large amounts of M-opsin. Deletion of M-opsin relieves the proteasome stress and completely prevents M cone degeneration in Lrat-/-Opn1sw-/- mice (a pure M cone LCA model, Opn1sw encoding S-opsin) for at least 12 months. These results suggest that M-opsin degradation-associated proteasome stress plays a major role in M cone degeneration in Lrat-/- model. This finding may represent a general mechanism for M cone degeneration in multiple forms of cone degeneration because of M-opsin mislocalization and degradation. These results have important implications for the current gene therapy strategy for LCA that emphasizes the need for combinatorial therapies to both improve vision and slowphotoreceptor degeneration.

CD36 Deficiency Inhibits Retinal Inflammation and Retinal Degeneration in Cx3cr1 Knockout Mice.

Background: CD36, a member of the class B scavenger receptor family, participates in Toll-like receptor signaling on mononuclear phagocytes (MP) and can promote sterile pathogenic inflammation. We here analyzed the effect of CD36 deficiency on retinal inflammation and photoreceptor degeneration, the hallmarks of age-related macular degeneration (AMD), that characterize Cx3cr1−/−mice.Methods: We analyzed subretinal MP accumulation, and cone- and rod-degeneration in light-challenged and aged, CD36 competent or deficient, hyper-inflammatory Cx3cr1−/− mice, using histology and immune-stained retinal flatmounts. Monocytes (Mo) were subretinally adoptively transferred to evaluate their elimination rate from the subretinal space and Interleukin 6 (IL-6) secretion from cultured Mo-derived cells (MdCs) of the different mouse strains were analyzed.Results: CD36 deficient Cx3cr1−/− mice were protected against age- and light-induced subretinal inflammation and associated cone and rod degeneration. CD36 deficiency in Cx3cr1−/− MPs inhibited their prolonged survival in the immune-suppressive subretinal space and reduced the exaggerated IL-6 secretion observed in Cx3cr1−/− MPs that we previously showed leads to increased subretinal MP survival.Conclusion: Cd36 deficiency significantly protected hyperinflammatory Cx3cr1−/− mice against subretinal MP accumulation and associated photoreceptor degeneration. The observed CD36-dependent induction of pro-inflammatory IL-6 might be at least partially responsible for the prolonged MP survival in the immune-suppressive environment and its pathological consequences on photoreceptor homeostasis.

IL-1\u03b2 induces rod degeneration through the disruption of retinal glutamate homeostasis

BackgroundAge-related macular degeneration is characterized by the accumulation of subretinal macrophages and the degeneration of cones, but mainly of rods. We have previously shown that Mononuclear Phagocytes-derived IL-1β induces rod photoreceptor cell death during experimental subretinal inflammation and in retinal explants exposed to IL-1β but the mechanism is unknown.MethodsRetinal explants were culture in the presence of human monocytes or IL-1β and photoreceptor cell survival was analyzed by TUNEL labeling. Glutamate concentration and transcription levels of gene involved in the homeostasis of glutamate were analyzed in cell fractions of explant cultured or not in the presence of IL-1β. Glutamate receptor antagonists were evaluated for their ability to reduce photoreceptor cell death in the presence of IL1-β or monocytes.ResultsWe here show that IL-1β does not induce death in isolated photoreceptors, suggesting an indirect effect. We demonstrate that IL-1β leads to glutamate-induced rod photoreceptor cell death as it increases the extracellular glutamate concentrations in the retina through the inhibition of its conversion to glutamine in Müller cells, increased release from Müller cells, and diminished reuptake. The inhibition of non-NMDA receptors completely and efficiently prevented rod apoptosis in retinal explants cultured in the presence of IL-1β or, more importantly, in vivo, in a model of subretinal inflammation.ConclusionsOur study emphasizes the importance of inflammation in the deregulation of glutamate homeostasis and provides a comprehensive mechanism of action for IL-1β-induced rod degeneration.

Oxidative stress-induced KLF4 activates inflammatory response through IL17RA and its downstream targets in retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness worldwide. Oxidative stress (OS), inflammation and genetics are considered the key pathogenic factors contributing to AMD development. Recent evidence shows the pro-inflammatory interleukin 17 (IL17) signaling is activated in AMD patients and promotes disease pathogenesis. However, the interplay between OS and IL17 signaling, and the regulatory mechanism of IL17 pathway are largely unknown. OS-induced retinal pigment epithelial cell (RPE) damage causes both the initial pathogenesis of AMD and secondary degeneration of rods and cones. Healthy RPE is essential for ocular immune privilege, however, damaged RPE cells can activate inflammatory response. In the present study, we identified IL17RA, the principle receptor of IL17 signaling, is one of the most upregulated inflammatory genes in human RPE cells upon OS exposure. The prominent increase of IL17RA was also observed in RPE and retina of an AMD-like mouse model. Knockdown of IL17RA in RPE cells prevented OS-induced RPE cell apoptosis and reduced the inflammatory response in both RPE and macrophages. Furthermore, we found that transcription factor KLF4 directly activates IL17RA expression, therefore, promotes the production of IL1β and IL8 in an IL17RA-dependent manner. In addition, the mRNA level of KLF4 isoform 2 was positively correlated with that of IL17RA in AMD patients. Together, our study demonstrates an unrevealed relationship between IL17RA and OS, and a new regulatory mechanism of IL17RA by KLF4 in RPE cells. These findings suggest that inhibition of IL17RA as a new potential therapeutic target for AMD through RPE protection and inflammatory suppression upon OS exposure.

Mechanism of cone cells death in retinitis pigmentosa

AbstractRetinitis pigmentosa (RP) is a group of diseases in which one of a large number of mutations causes death of rod photoreceptors. After rods die, cone photoreceptors slowly degenerate in a characteristic pattern. Rod cell death causes night blindness, but visual disability and blindness result from cone degeneration and therefore it is critical to determine the mechanisms by which it occurs. The death of rods reduces oxygen consumption resulting high tissue levels of oxygen in the outer retina. The excess oxygen stimulates superoxide radical production by mismatches in the electron transport chain in mitochondria and by stimulation of NADPH oxidase activity in cytoplasm. The high levels of superoxide radicals overwhelm the antioxidant defense system and generate more reactive species including peroxynitrite which is extremely damaging and difficult to detoxify. This results in progressive oxidative damage in cones which contributes to cone cell death and loss of function because drugs or gene transfer that reduce oxidative stress promote cone survival and maintenance of function. Oral administration of N‐acetylcysteine (NAC) has shown good efficacy in mouse models of RP prompting the FIGHT RP1 open label clinical trial, the results of which will be presented.

Systematic spatiotemporal mapping reveals divergent cell death pathways in three mouse models of hereditary retinal degeneration.

Calcium (Ca2+ ) dysregulation has been linked to neuronal cell death, including in hereditary retinal degeneration. Ca2+ dysregulation is thought to cause rod and cone photoreceptor cell death. Spatial and temporal heterogeneities in retinal disease models have hampered validation of this hypothesis. We examined the role of Ca2+ in photoreceptor degeneration, assessing the activation pattern of Ca2+ -dependent calpain proteases, generating spatiotemporal maps of the entire retina in the cpfl1 mouse model for primary cone degeneration, and in the rd1 and rd10 models for primary rod degeneration. We used Gaussian process models to distinguish the temporal sequences of degenerative molecular processes from other variability sources.In the rd1 and rd10 models, spatiotemporal pattern of increased calpain activity matched the progression of primary rod degeneration. High calpain activity coincided with activation of the calpain-2 isoform but not with calpain-1, suggesting differential roles for both calpain isoforms. Primary rod loss was linked to upregulation of apoptosis-inducing factor, although only a minute fraction of cells showed activity of the apoptotic marker caspase-3. After primary rod degeneration concluded, caspase-3 activation appeared in cones, suggesting apoptosis as the dominant mechanism for secondary cone loss. Gaussian process models highlighted calpain activity as a key event during primary rod photoreceptor cell death. Our data suggest a causal link between Ca2+ dysregulation and primary, nonapoptotic degeneration of photoreceptors and a role for apoptosis in secondary degeneration of cones, highlighting the importance of the spatial and temporal location of key molecular events, which may guide the evaluation of new therapies.

Cone Structure Persists Beyond Margins of Short-Wavelength Autofluorescence in Choroideremia.

PurposeWe studied the relationship between structure and function of the choriocapillaris (CC), retinal pigment epithelium (RPE), and photoreceptors in patients with choroideremia (CHM).MethodsSix CHM patients (12 eyes) and four normal subjects (six eyes) were studied with fundus-guided microperimetry, confocal and nonconfocal adaptive optics scanning laser ophthalmoscopy (AOSLO), near-infrared and color fundus photos, short wavelength fundus autofluorescence (SW-AF), and swept-source optical coherence tomography (SS-OCT) and angiography (SS-OCTA) images. Cone spacing was represented using Z-scores (standard deviations from the mean at that eccentricity). CC flow voids were defined using a threshold of 1 SD below the normal mean.ResultsCone spacing Z-scores were not significantly correlated with distance from the borders of preserved RPE, determined using either the SS-OCT or SW-AF scans. Cone spacing Z-scores were significantly correlated with CC flow voids and retinal sensitivity. Flow voids were abnormal in regions of preserved RPE and increased progressively from within −2° of the preserved area to +2° beyond the border. Visual sensitivity decreased as CC flow voids increased approaching and beyond the border of preserved structure.ConclusionsIn CHM, cone spacing Z-scores correlated with CC flow voids, and were negatively correlated with retinal sensitivity, suggesting cone degeneration accompanied reduced CC perfusion. Functional cones were found outside the presumed borders of preserved outer-retina/RPE as defined by SW-AF, but not outside the borders determined by SS-OCT. The use of SW-AF to identify the border of preserved structures may underestimate regions with cells that may be amenable to treatment.

Investigations Into Bioenergetic Neuroprotection of Cone Photoreceptors: Relevance to Retinitis Pigmentosa.

Recent studies suggest cone degeneration in retinitis pigmentosa (RP) may result from intracellular energy depletion. We tested the hypothesis that cones die when depleted of energy by examining the effect of two bioenergetic, nutraceutical agents on cone survival. The study had three specific aims: firstly, we, studied the neuroprotective efficacies of glucose and creatine in an in vitro model of RP. Next, we utilized a well-characterized mouse model of RP to examine whether surviving cones, devoid of their inner segments, continue to express genes vital for glucose, and creatine utilization. Finally, we analyzed the neuroprotective properties of glucose and creatine on cone photoreceptors in a mouse model of RP. Two different bioenergy-based therapies were tested in rd1 mice: repeated local delivery of glucose and systemic creatine. Optomotor responses were tested and cone density was quantified on retinal wholemounts. The results showed that glucose supplementation increased survival of cones in culture subjected to mitochondrial stress or oxidative insult. Despite losing their inner segments, surviving cones in the rd1 retina continued to express the various glycolytic enzymes. Following a single subconjunctival injection, the mean vitreous glucose concentration was significantly elevated at 1 and 8 h, but not at 16 h after injection; however, daily subconjunctival injection of glucose neither enhanced spatial visual performance nor slowed cone cell degeneration in rd1 mice relative to isotonic saline. Creatine dose-dependently increased survival of cones in culture subjected to mitochondrial dysfunction, but not to oxidative stress. Despite the loss of their mitochondrial-rich inner segments, cone somas and axonal terminals in the rd1 retina were strongly positive for both the mitochondrial and cytosolic forms of creatine kinase at each time point examined. Creatine-fed rd1 mice displayed enhanced optomotor responses compared to mice fed normal chow. Moreover, cone density was significantly greater in creatine-treated mice compared to controls. The overall results of this study provide tentative support for the hypothesis that creatine supplementation may delay secondary degeneration of cones in individuals with RP.

Allele Frequency of the C.5G>A Mutation in the PRCD Gene Responsible for Progressive Retinal Atrophy in English Cocker Spaniel Dogs.

Simple SummaryProgressive retinal atrophy (PRA) in English cocker spaniels (ECSs) is associated with progressive rod–cone degeneration (prcd-PRA), an inherited autosomal recessive disease caused by the c.5G>A mutation in the progressive rod–cone degeneration (PRCD) gene. Data regarding the prevalence of the mutated allele are scarce in the global literature, and there is no study evaluating this frequency in Brazil. Therefore, the aim of this study was to evaluate the allele frequency of the c.5G>A mutation in the PRCD gene responsible for progressive retinal atrophy (prcd-PRA) in ECS dogs.Progressive retinal atrophy (PRA) due to the c.5G>A mutation in the progressive rod–cone degeneration (PRCD) gene is an important genetic disease in English cocker spaniel (ECS) dogs. Because the prevalence of this disease has not been verified in Brazil, this study aimed to evaluate the allele frequency of the c.5G>A mutation in the PRCD gene. Purified DNA from 220 ECS dogs was used for genotyping, of which 131 were registered from 18 different kennels and 89 were unregistered. A clinical eye examination was performed in 28 of the genotyped animals; 10 were homozygous mutants. DNA fragments containing the mutation region were amplified by PCR and subjected to direct genomic sequencing. The prcd-PRA allele frequency was 25.5%. Among the registered dogs, the allele frequency was 14.9%; among the dogs with no history of registration, the allele frequency was 41%. Visual impairment was observed in 80% (8/10) of the homozygous mutant animals that underwent clinical eye examination. The high mutation frequency found in this study emphasizes the importance of genotyping ECSs as an early diagnostic test, especially as part of an informed breeding program, to avoid clinical cases of PRA.

Spatio-temporal characterization of S- and M/L-cone degeneration in the Rd1 mouse model of retinitis pigmentosa

BackgroundThe Pde6brd1 (Rd1) mouse is widely used as a murine model for human retinitis pigmentosa. Understanding the spatio-temporal patterns of cone degeneration is important for evaluating potential treatments. In the present study we performed a systematic characterization of the spatio-temporal patterns of S- and M/L-opsin+ cone outer segment and cell body degeneration in Rd1 mice, described the distribution and proportion of dual cones in Rd1 retinas, and examined the kinetics of microglial activation during the period of cone degeneration.ResultsOuter segments of S- and M/L-cones degenerated far more rapidly than their somas. Loss of both S- and M/L-opsin+ outer segments was fundamentally complete by P21 in the central retina, and 90% complete by P45 in the peripheral retina. In comparison, degeneration of S- and M/L-opsin+ cell bodies proceeded at a slower rate. There was a marked hemispheric asymmetry in the rate of S-opsin+ and M/L-opsin+ cell body degeneration. M/L-opsin+ cones were more resilient to degeneration in the superior retina, whilst S-opsin+ cones were relatively preserved in the inferior retina. In addition, cone outer segment and cell body degeneration occurred far more rapidly in the central than the peripheral retina. At P14, the superior retina comprised a minority of genuine S-cones with a much greater complement of genuine M/L-opsin cones and dual cones, whilst the other three retinal quadrants had broadly similar numbers of genuine S-cones, genuine M/L-cones and dual cones. At P60, approximately 50% of surviving cones in the superior, nasal and temporal quadrants were dual cones. In contrast, the inferior peripheral retina at P60 contained almost exclusively genuine S-cones with a tiny minority of dual cones. Microglial number and activity were stimulated during rod breakdown, remained relatively high during cone outer segment degeneration and loss of cone somas in the central retina, and decreased thereafter in the period coincident with slow degeneration of cone cell bodies in the peripheral retina.ConclusionThe results of the present study provide valuable insights into cone degeneration in the Rd1 mouse, substantiating and extending conclusions drawn from earlier studies.

Rescue of M-cone Function in Aged Opn1mw-/- Mice, a Model for Late-Stage Blue Cone Monochromacy.

PurposePreviously we showed that AAV5-mediated expression of either human M- or L-opsin promoted regrowth of cone outer segments and rescued M-cone function in the treated M-opsin knockout (Opn1mw−/−) dorsal retina. In this study, we determined cone viability and window of treatability in aged Opn1mw−/− mice.MethodsCone viability was assessed with antibody against cone arrestin and peanut agglutinin (PNA) staining. The rate of cone degeneration in Opn1mw−/− mice was quantified by PNA staining. AAV5 vector expressing human L-opsin was injected subretinally into one eye of Opn1mw−/− mice at 1, 7, and 15 months old, while the contralateral eyes served as controls. M-cone–mediated retinal function was analyzed 2 and 13 months postinjection by full-field ERG. L-opsin transgene expression and cone outer segment structure were examined by immunohistochemistry.ResultsWe showed that dorsal M-opsin dominant cones exhibit outer segment degeneration at an early age in Opn1mw−/− mice, whereas ventral S-opsin dominant cones were normal. The remaining M-opsin dominant cones remained viable for at least 15 months, albeit having shortened or no outer segments. We also showed that AAV5-mediated expression of human L-opsin was still able to rescue function and outer segment structure in the remaining M-opsin dominant cones when treatment was initiated at 15 months of age.ConclusionsOur results showing that the remaining M-opsin dominant cones in aged Opn1mw−/− mice can still be rescued by gene therapy is helpful for establishing the window of treatability in future blue cone monochromacy clinical trials.