Search

Microglia are resident immune cells in the central nervous system, including the retina that surveil the environment for damage and infection. Following retinal damage, microglia undergo morphological changes, migrate to the site of damage, and express and secrete pro-inflammatory signals. In the zebrafish retina, inflammation induces the reprogramming and proliferation of Müller glia and the regeneration of neurons following damage or injury. Immunosuppression or pharmacological ablation of microglia reduce or abolish Müller glia proliferation. We evaluated the retinal architecture and retinal regeneration in adult zebrafish irf8 mutants, which have significantly depleted numbers of microglia. We show that irf8 mutants have normal retinal structure at 3 months post fertilization (mpf) and 6 mpf but fewer cone photoreceptors by 10 mpf. Surprisingly, light-induced photoreceptor ablation induced Müller glia proliferation in irf8 mutants and cone and rod photoreceptor regeneration. Light-damaged retinas from both wild-type and irf8 mutants show upregulated expression of mmp-9, il8, and tnfβ pro-inflammatory cytokines. Our data demonstrate that adult zebrafish irf8 mutants can regenerate normally following acute retinal injury. These findings suggest that microglia may not be essential for retinal regeneration in zebrafish and that other mechanisms can compensate for the reduction in microglia numbers., (© 2024. The Author(s).)

We develop a mathematical model for photoreceptors in the retina. We focus on rod and cone outer segment dynamics and interactions with a nutrient source associated with the retinal pigment epithelium cells. Rod and cone densities (number per unit area of retinal surface) are known to have significant spatial dependence in the retina with cones located primarily near the fovea and the rods located primarily away from the fovea. Our model accounts for this spatial dependence of the rod and cone photoreceptor density as well as for the possibility of nutrient diffusion. We present equilibrium and dynamic solutions, discuss their relation to existing models, and estimate model parameters through comparisons with available experimental measurements of both spatial and temporal photoreceptor characteristics. Our model compares well with existing data on spatially-dependent regrowth of photoreceptor outer segments in the macular region of Rhesus Monkeys. Our predictions are also consistent with existing data on the spatial dependence of photoreceptor outer segment length near the fovea in healthy human subjects. We focus primarily on the healthy eye but our model could be the basis for future efforts designed to explore various retinal pathologies, eye-related injuries, and treatments of these conditions., Competing Interests: Declaration of competing interest We declare that the submitted manuscript and associated research is correct and that no situation of real, potential, or apparent conflict of interest is known to any of us., (Published by Elsevier Ltd.)

Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases., Competing Interests: Competing interest statement: J.W.K. declares that he is a board member and shareholder of Proteostasis Therapeutics Inc., Protego BioPharma, and Yumanity, which may develop ATF6 activators to treat degenerative diseases, although not for stem cell–associated purposes at this time. R.L.W. is a shareholder and board member of Protego BioPharma. J.W.K. and R.L.W. are inventors on a patent describing the ATF6 activating compound used in this study. All other authors declare they have no competing interests.

Background: In adult rats we study the short- and long-term effects of focal blue light-emitting diode (LED)-induced phototoxicity (LIP) on retinal thickness and Iba-1 + activation., Methods: The left eyes of previously dark-adapted Sprague Dawley (SD) rats were photoexposed to a blue LED (20 s, 200 lux). In vivo longitudinal monitoring of retinal thickness, fundus images, and optical retinal sections was performed from 1 to 30 days (d) after LIP with SD-OCT. Ex vivo, we analysed the population of S-cone and Iba-1 + cells within a predetermined fixed-size circular area (PCA) centred on the lesion., Results: LIP resulted in a circular focal lesion readily identifiable in vivo by fundus examination, which showed within the PCAs a progressive thinning of the outer retinal layer, and a diminution of the S-cone population to 19% by 30 d. In parallel to S-cone loss, activated Iba-1 + cells delineated the lesioned area and acquired an ameboid morphology with peak expression at 3 d after LIP. Iba-1 + cells adopted a more relaxed-branched morphology at 7 d and by 14-30 d their morphology was fully branched., Conclusion: LIP caused a progressive reduction of the outer retina with loss of S cones and a parallel dynamic activation of microglial cells in the lesioned area.

Bassoon (BSN) is a presynaptic cytomatrix protein ubiquitously present at chemical synapses of the central nervous system, where it regulates synaptic vesicle replenishment and organizes voltage-gated Ca 2+ channels. In sensory photoreceptor synapses, BSN additionally plays a decisive role in anchoring the synaptic ribbon, a presynaptic organelle and functional extension of the active zone, to the presynaptic membrane. In this study, we functionally and structurally analyzed two mutant mouse lines with a genetic disruption of Bsn-Bsn gt and Bsn ko -using electrophysiology and high-resolution microscopy. In both Bsn mutant mouse lines, full-length BSN was abolished, and photoreceptor synaptic function was similarly impaired, yet synapse structure was more severely affected in Bsn gt/gt than in Bsn ko/ko photoreceptors. The synaptic defects in Bsn gt/gt retina coincide with remodeling of the outer retina-rod bipolar and horizontal cell sprouting, formation of ectopic ribbon synaptic sites-and death of cone photoreceptors, processes that did not occur in Bsn ko/ko retina. An analysis of Bsn gt/ko hybrid mice revealed that the divergent retinal phenotypes of Bsn gt/gt and Bsn ko/ko mice can be attributed to the expression of the Bsn gt allele, which triggers cone photoreceptor death and neurite sprouting in the outer retina. These findings shed new light on the existing Bsn mutant mouse models and might help to understand mechanisms that drive photoreceptor death., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Rhegmatogenous retinal detachment (RD) is a threatening visual condition and a human disease model for retinal degenerations. Despite successful reattachment surgery, vision does not fully recover, due to subretinal fluid accumulation and subsequent photoreceptor cell death, through mechanisms that recapitulate those of retinal degenerative diseases. Hydrophilic bile acids are neuroprotective in animal models, but whether they can be used orally for retinal diseases is unknown. Ursodeoxycholic acid (UDCA) being approved for clinical use (e.g., in cholestasis), we have evaluated the ocular bioavailability of oral UDCA, administered to patients before RD surgery. The level of UDCA in ocular media correlated with the extent of blood retinal barrier disruption, evaluated by the extent of detachment and the albumin concentration in subretinal fluid. UDCA, at levels measured in ocular media, protected photoreceptors from apoptosis and necrosis in rat retinal explants, an ex vivo model of RD. The subretinal fluid from UDCA-treated patients, collected during surgery, significantly protected rat retinal explants from cell death, when compared to subretinal fluid from control patients. Pan-transcriptomic analysis of the retina showed that UDCA upregulated anti-apoptotic, anti-oxidant, and anti-inflammatory genes. Oral UDCA is a potential neuroprotective adjuvant therapy in RD and other retinal degenerative diseases and should be further evaluated in a clinical trial., (© 2021. The Author(s).)

INPP5E, also known as pharbin, is a ubiquitously expressed phosphatidylinositol polyphosphate 5-phosphatase that is typically located in the primary cilia and modulates the phosphoinositide composition of membranes. Mutations to or loss of INPP5E is associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans-a syndromic ciliopathy affecting multiple tissues including the brain, liver, kidney, and retina. In contrast to other primary cilia, photoreceptor INPP5E is prominently expressed in the inner segment and connecting cilium and absent in the outer segment, which is a modified primary cilium dedicated to phototransduction. To investigate how loss of INPP5e causes retina degeneration, we generated mice with a retina-specific KO (Inpp5e F/F ;Six3Cre, abbreviated as ret Inpp5e -/- ). These mice exhibit a rapidly progressing rod-cone degeneration resembling Leber congenital amaurosis that is nearly completed by postnatal day 21 (P21) in the central retina. Mutant cone outer segments contain vesicles instead of discs as early as P8. Although P10 mutant outer segments contain structural and phototransduction proteins, axonemal structure and disc membranes fail to form. Connecting cilia of ret Inpp5e -/- rods display accumulation of intraflagellar transport particles A and B at their distal ends, suggesting disrupted intraflagellar transport. Although INPP5E ablation may not prevent delivery of outer segment-specific proteins by means of the photoreceptor secretory pathway, its absence prevents the assembly of axonemal and disc components. Herein, we suggest a model for INPP5E-Leber congenital amaurosis, proposing how deletion of INPP5E may interrupt axoneme extension and disc membrane elaboration., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sanfilippo syndrome is an untreatable form of childhood-onset dementia. Whilst several therapeutic strategies are being evaluated in human clinical trials including i.v. delivery of AAV9-based gene therapy, an urgent unmet need is the availability of non-invasive, quantitative measures of neurodegeneration. We hypothesise that as part of the central nervous system, the retina may provide a window through which to 'visualise' degenerative lesions in brain and amelioration of them following treatment. This is reliant on the age of onset and the rate of disease progression being equivalent in retina and brain. For the first time we have assessed in parallel, the nature, age of onset and rate of retinal and brain degeneration in a mouse model of Sanfilippo syndrome. Significant accumulation of heparan sulphate and expansion of the endo/lysosomal system was observed in both retina and brain pre-symptomatically (by 3 weeks of age). Robust and early activation of micro- and macroglia was also observed in both tissues. There was substantial thinning of retina and loss of rod and cone photoreceptors by ~ 12 weeks of age, a time at which cognitive symptoms are noted. Intravenous delivery of a clinically relevant AAV9-human sulphamidase vector to neonatal mice prevented disease lesion appearance in retina and most areas of brain when assessed 6 weeks later. Collectively, the findings highlight the previously unrecognised early and significant involvement of retina in the Sanfilippo disease process, lesions that are preventable by neonatal treatment with AAV9-sulphamidase. Critically, our data demonstrate for the first time that the advancement of retinal disease parallels that occurring in brain in Sanfilippo syndrome, thus retina may provide an easily accessible neural tissue via which brain disease development and its amelioration with treatment can be monitored.

Here, we evaluated the effects of PEDF (pigment epithelium-derived factor) and PEDF peptides on cone-photoreceptor cell damage in a mouse model of focal LED-induced phototoxicity (LIP) in vivo. Swiss mice were dark-adapted overnight, anesthetized, and their left eyes were exposed to a blue LED placed over the cornea. Immediately after, intravitreal injection of PEDF, PEDF-peptide fragments 17-mer, 17-mer[H105A] or 17-mer[R99A] (all at 10 pmol) were administered into the left eye of each animal. BDNF (92 pmol) and bFGF (27 pmol) injections were positive controls, and vehicle negative control. After 7 days, LIP resulted in a consistent circular lesion located in the supratemporal quadrant and the number of S-cones were counted within an area centered on the lesion. Retinas treated with effectors had significantly greater S-cone numbers (PEDF (60%), 17-mer (56%), 17-mer [H105A] (57%), BDNF (64%) or bFGF (60%)) relative to their corresponding vehicle groups (≈42%). The 17-mer[R99A] with no PEDF receptor binding and no neurotrophic activity, PEDF combined with a molar excess of the PEDF receptor blocker P1 peptide, or with a PEDF-R enzymatic inhibitor had undetectable effects in S-cone survival. The findings demonstrated that the cone survival effects were mediated via interactions between the 17-mer region of the PEDF molecule and its PEDF-R receptor.

Inherited photoreceptor degenerations are not treatable diseases and a frequent cause of blindness in working ages. In this study we investigate the safety, integration and possible rescue effects of intravitreal and subretinal transplantation of adult human bone-marrow-derived mononuclear stem cells (hBM-MSCs) in two animal models of inherited photoreceptor degeneration, the P23H-1 and the Royal College of Surgeons (RCS) rat. Immunosuppression was started one day before the injection and continued through the study. The hBM-MSCs were injected in the left eyes and the animals were processed 7, 15, 30 or 60 days later. The retinas were cross-sectioned, and L- and S- cones, microglia, astrocytes and Müller cells were immunodetected. Transplantations had no local adverse effects and the CD45+ cells remained for up to 15 days forming clusters in the vitreous and/or a 2-3-cells-thick layer in the subretinal space after intravitreal or subretinal injections, respectively. We did not observe increased photoreceptor survival nor decreased microglial cell numbers in the injected left eyes. However, the injected eyes showed decreased GFAP immunoreactivity. We conclude that intravitreal or subretinal injection of hBM-MSCs in dystrophic P23H-1 and RCS rats causes a decrease in retinal gliosis but does not have photoreceptor neuroprotective effects, at least in the short term. However, this treatment may have a potential therapeutic effect that merits further investigation.

Major depressive disorder, bipolar disorder and schizophrenia are currently among the most common psychiatric disorders, known to constitute a serious public health issue in terms of morbidity, mortality and functional handicap. Their pathophysiology is still unclear, but there is now increasing evidence supporting the existence of abnormalities of neurotransmission. As the retina is an extension of the central nervous system, it may be an interesting site of study which might provide a better understanding of the pathophysiology of psychiatric disorders. Several studies have demonstrated retinal abnormalities, with abnormal cone and rod responses on electroretinography (ERG), suggesting a process of functional neuronal loss, structurally supported by a decrease in the retinal nerve fiber layer thickness (RNFL) on optical coherence tomography (OCT), which suggests involvement of the molecular signal pathways of neurotransmission. These tests could be useful tools for diagnosing and monitoring psychiatric disorders. This article is an overview of the literature on retinal abnormalities observed in patients with major depressive disorder, bipolar disorder or schizophrenia, and discusses how they could be pathophysiologic markers., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Lighting is rapidly changing with the introduction of light-emitting diodes (LEDs) in our homes, workplaces, and cities. This evolution of our optical landscape raises major concerns regarding phototoxicity to the retina since light exposure is an identified risk factor for the development of age-related macular degeneration (AMD). In this disease, cone photoreceptors degenerate while the retinal pigment epithelium (RPE) is accumulating lipofuscin containing phototoxic compounds such as A2E. Therefore, it remains unclear if the light-elicited degenerative process is initiated in cones or in the RPE. Using purified cone photoreceptors from pig retina, we here investigated the effect of light on cone survival from 390 to 510 nm in 10 nm steps, plus the 630 nm band. If at a given intensity (0.2 mW/cm²), the most toxic wavelengths are comprised in the visible-to-near-UV range, they shift to blue-violet light (425-445 nm) when exposing cells to a solar source filtered by the eye optics. In contrast to previous rodent studies, this cone photoreceptor phototoxicity is not related to light absorption by the visual pigment. Despite bright flavin autofluorescence of cone inner segment, excitation-emission matrix of this inner segment suggested that cone phototoxicity was instead caused by porphyrin. Toxic light intensities were lower than those previously defined for A2E-loaded RPE cells indicating cones are the first cells at risk for a direct light insult. These results are essential to normative regulations of new lighting but also for the prevention of human retinal pathologies since toxic solar light intensities are encountered even at high latitudes.

Genome duplication leads to an emergence of gene paralogs that are essentially free to undergo the process of neofunctionalization, subfunctionalization or degeneration (gene loss). Onecut1 (Oc1) and Onecut2 (Oc2) transcription factors, encoded by paralogous genes in mammals, are expressed in precursors of horizontal cells (HCs), retinal ganglion cells and cone photoreceptors. Previous studies have shown that ablation of either Oc1 or Oc2 gene in the mouse retina results in a decreased number of HCs, while simultaneous deletion of Oc1 and Oc2 leads to a complete loss of HCs. Here we study the genetic redundancy between Oc1 and Oc2 paralogs and focus on how the dose of Onecut transcription factors influences abundance of individual retinal cell types and overall retina physiology. Our data show that reducing the number of functional Oc alleles in the developing retina leads to a gradual decrease in the number of HCs, progressive thinning of the outer plexiform layer and diminished electrophysiology responses. Taken together, these observations indicate that in the context of HC population, the alleles of Oc1/Oc2 paralogous genes are mutually interchangeable, function additively to support proper retinal function and their molecular evolution does not follow one of the typical routes after gene duplication., Competing Interests: The authors have declared that no competing interests exist.

USH2A variants are the most common cause of Usher syndrome type 2, characterized by congenital sensorineural hearing loss and retinitis pigmentosa (RP), and also contribute to autosomal recessive non-syndromic RP. Several treatment strategies are under development; however, sensitive clinical trial endpoint metrics to determine therapeutic efficacy have not been identified. In the present study, we have performed longitudinal retrospective examination of the retinal and auditory symptoms in (i) 56 biallelic molecularly confirmed USH2A patients and (ii) ush2a mutant zebrafish to identify metrics for the evaluation of future clinical trials and rapid preclinical screening studies. The patient cohort showed a statistically significant correlation between age and both rate of constriction for the ellipsoid zone length and hyperautofluorescent outer retinal ring area. Visual acuity and pure tone audiograms are not suitable outcome measures. Retinal examination of the novel ush2au507 zebrafish mutant revealed a slowly progressive degeneration of predominantly rods, accompanied by rhodopsin and blue cone opsin mislocalization from 6 to 12 months of age with lysosome-like structures observed in the photoreceptors. This was further evaluated in the ush2armc zebrafish model, which revealed similar changes in photopigment mislocalization with elevated autophagy levels at 6 days post fertilization, indicating a more severe genotype-phenotype correlation and providing evidence of new insights into the pathophysiology underlying USH2A-retinal disease., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Variants in interphotoreceptor matrix proteoglycans (IMPG2) have been reported in retinitis pigmentosa (RP) and vitelliform macular dystrophy (VMD) patients. However, the underlying molecular mechanisms remain elusive due to a lack of suitable disease models. We developed two independent Impg2 knockout (KO) mouse models using the CRISPR/Cas9 technique to assess the in vivo functions of Impg2 in the retina. Impg2 ablation in mice recapitulated the RP phenotypes of patients, including an attenuated electroretinogram (ERG) response and the progressive degeneration of photoreceptors. The histopathological examination of Impg2-KO mice revealed irregularly arranged rod cells and mislocalized rhodopsin protein in the inner segment at 6 months of age. In addition to the pathological changes in rod cells, cone cells were also affected in KO retinas. KO retinas exhibited progressive cone cell death and impaired cone cell elongation. Further immunoblotting analysis revealed increased levels of endoplasmic reticulum (ER) stress-related proteins, including C/EBP homologous protein (CHOP), immunoglobulin heavy-chain-binding protein (BIP) and protein disulfide isomerase (PDI), in Impg2-KO mouse retinas. Increased gliosis and apoptotic cell death were also observed in the KO retinas. As autophagy is closely associated with ER stress, we then checked whether autophagy was disturbed in Impg2-KO mouse retinas. The results showed that autophagy was impaired in KO retinas, as revealed by the increased accumulation of SQSTM1 and other proteins involved in autophagy. Our results demonstrate the essential roles of Impg2 in the retina, and this study provides novel models for mechanistic investigations and development of therapies for RP caused by IMPG2 mutations., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The high-energy demands of the retina are thought to contribute to its particular vulnerability to mitochondrial dysfunction. Photoreceptors are the cells with the higher oxygen consumption within the retina, and among these, the cones contain more mitochondria and have a higher energy demand than rods. A cohort of twenty-two patients with genetically-defined mitochondrial diseases (MDs) were enrolled to determine if the macula is functionally and anatomically impaired in these metabolic disorders. Visual acuity and fERG amplitude of patients with primary mitochondrial dysfunction were reduced compared to controls. Furthermore, SD-OCT layer segmentation showed a reduction of retinal and outer nuclear layer (ONL) volume in the macula of the patients. fERG amplitude showed a positive correlation with both ONL volume and thickness. A negative relationship was noted between fERG amplitude and disease severity assessed with Newcastle Mitochondrial Disease Adult Scale. In conclusion, MDs are associated with functional and anatomical alteration of macular cone system, characterized by its strong correlation with clinical disease severity suggesting a role as a potential biomarker of primary mitochondrial disorders.

Purpose: To analyze the role of microglial and Müller cells in the formation of rings of photoreceptor degeneration caused by phototoxicity., Methods: Two-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., Results: Phototoxicity 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., Conclusions: Light-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.

Purpose: The aim of the study was to evaluate retinal sensitivity and stereoacuity (SA) in retinitis pigmentosa (RP) patients., Methods: Twenty-six patients with RP were examined, mean age 36.4 ± 7.21 (SD) years old and best corrected visual acuity better than 0.15 logMAR. The control group (CG) included 25 healthy subjects matching the RP group by age and sex. Every patient and healthy control underwent a complete ophthalmologic examination: Titmus, Lang, TNO stereotests and microperimetry (MP-1) (Nidek Technologies). Results were subjected to factor analysis using Varimax rotation, and p values < 0.05 were considered statistically significant., Results: With the Titmus stereotest, the mean SA was 136.52 ± 26.5 (SD) arcsec in the RP group and 67.2 ± 11.5 (SD) in CG; Lang SA was 391.39 ± 53.72 (SD) in RP group and 1150 ± 33.4 (SD) in CG; and TNO SA was 69.3 ± 14.39 (SD) in the RP group and 15.97 ± 3.7 (SD) in CG. Factor analysis showed significant correlation between visual acuity and SA (p = 0.0001) in RP group. MP-1 demonstrated that in RP patients, inter-ocular difference in retinal sensitivity and fixation stability was related to anomalous stereopsis (p values < 0.05)., Conclusion: Progressive RP degeneration in the cone system could determine a significant impairment in the binocular vision due to anomalous inter-ocular retinal sensitivity and incomplete Panum's area utilization, causing an incongruent retinal localization. These findings suggest a possible reason why RP patients with a central retinal involvement, even if minimal, perceive a damaged stereoscopic perception that produces a severe disability.

To study short and long-term effects of acute ocular hypertension (AOHT) on inner and outer retinal layers, in adult Sprague-Dawley rats AOHT (87mmHg) was induced for 90min and the retinas were examined longitudinally in vivo with electroretinogram (ERG) recordings and optical coherent tomography (OCT) from 1 to 90 days (d). Ex vivo, the retinas were analyzed for rod (RBC) and cone (CBC) bipolar cells, with antibodies against protein kinase Cα and recoverin, respectively in cross sections, and for cones, horizontal (HZ) and ganglion (RGC) cells with antibodies against arrestin, calbindin and Brn3a, respectively in wholemounts. The inner retina thinned progressively up to 7d with no further changes, while the external retina had a normal thickness until 30d, with a 20% thinning between 30 and 90d. Functionally, the a-wave showed an initial reduction by 24h and a further reduction from 30 to 90d. All other main ERG waves were significantly reduced by 1d without significant recovery by 90d. Radial sections showed a normal population of RBCs but their terminals were reduced. The CBCs showed a progressive decrease with a loss of 56% by 30d. In wholemount retinas, RGCs diminished to 40% by 3d and to 16% by 30d without further loss. Cones diminished to 58% and 35% by 3 and 7d, respectively and further decreased between 30 and 90d. HZs showed normal values throughout the study. In conclusion, AOHT affects both the inner and outer retina, with a more pronounced degeneration of the cone than the rod pathway., (© 2020 Elsevier B.V. All rights reserved.)

Purpose: To investigate structural changes in the retinal outer layers and choroid using adaptive optics (AO) and optical coherence tomography (OCT) in eyes with myopia, and to correlate the changes with decreased macular light sensitivity (MLS)., Methods: This prospective study included 27 subjects with emmetropia and low myopia (EM/LM), 25 with moderate myopia (MM), and 25 with high myopia (HM). Microperimetry was used to quantify MLS in each subject, while AO and OCT images of fundus were analyzed to quantify cone density and regularity and thickness of outer retinal sublayers and choroid. Differences of MLS, cone distribution, and chorioretinal thicknesses were compared among the three groups, and the associations among photoreceptor morphological alterations, MLS, and other parameters were analyzed., Results: In HM, the MLS, cone density and regularity, and thicknesses of the myoid and ellipsoid zone (MEZ), Henle fiber layer and outer nuclear layer, interdigitation zone and RPE/Bruch complex, and choroid were lower than in EM/LM. Decreased MLS was correlated with lower cone density and regularity, and thinner MEZ and choroid in the inner region, and with lower cone density, thinner MEZ and choroid, and longer axial length in the outer region. Multivariate regression showed that better MLS was correlated with thicker MEZ in the inner region and with higher cone density in the outer region., Conclusions: Altered cone distribution and outer retinal thickness, especially cone density and MEZ thickness, were significantly correlated with decline of MLS in HM, which may help to evaluate and monitor visual impairment in HM.

The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca 2+ concentrations decline, and decelerates the cyclase in the dark, when Ca 2+ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 ( GUCA1A ) gene in a family with a dominant retinopathy. The G86R substitution in a "hinge" region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca 2+ -dependent activator-to-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca 2+ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca 2+ concentrations characteristic of dark-adapted photoreceptors. Ca 2+ -dependent increase in Trp 94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca 2+ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca 2+ , and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca 2+ sensitivity of the cyclase within the physiological range of intracellular Ca 2+ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly 86 , leading to abnormally high affinity for the target enzyme and reduced Ca 2+ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca 2+ influx in photoreceptors in the dark., (© 2019 Peshenko et al.)

Purpose: To examine the features of the tapetal-like reflex (TLR) in female carriers of RPGR-associated retinopathy by means of adaptive optics scanning light ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography., Methods: Nine molecularly confirmed RPGR carriers and three healthy controls underwent ocular examination and the following retinal imaging modalities: color photography, near-infrared reflectance, fundus autofluorescence, spectral domain optical coherence tomography, and AOSLO. After identifying TLR areas across all imaging modalities, normalized local contrast of outer retinal bands on spectral domain optical coherence tomography was calculated and AOSLO-acquired photoreceptor mosaic analysis was performed., Results: Seven carriers had TLR areas, which colocalized with increased rod photoreceptor reflectivity on confocal AOSLO and reduced cone photoreceptor densities. Parafoveal TLR areas also exhibited reduced local contrast (i.e., increased reflectivity) of the outer retinal bands on spectral domain optical coherence tomography (inner segment ellipsoid zone and outer segment interdigitation zone). Healthy controls did not show TLR., Conclusion: The cellular resolution provided by AOSLO affords the characterization of the photoreceptor mosaic in RPGR carriers with a TLR. Features revealed include reduced cone density, increased cone inner segment diameter, and increased rod outer segment reflectivity.

Retinal photoreceptors are important in visual signaling for normal eye growth in animals. We used Gnat2 cplf3/cplf3 (Gnat2 -/- ) mice, a genetic mouse model of cone dysfunction to investigate the influence of cone signaling in ocular refractive development and myopia susceptibility in mice. Refractive development under normal visual conditions was measured for Gnat2 -/- and age-matched Gnat2 +/+ mice, every 2 weeks from 4 to 14 weeks of age. Weekly measurements were performed on a separate cohort of mice that underwent monocular form-deprivation (FD) in the right eye from 4 weeks of age using head-mounted diffusers. Refraction, corneal curvature, and ocular biometrics were obtained using photorefraction, keratometry and optical coherence tomography, respectively. Retinas from FD mice were harvested, and analyzed for dopamine (DA) and 3,4-dihydroxyphenylacetate (DOPAC) using high-performance liquid chromatography. Under normal visual conditions, Gnat2 +/+ and Gnat2 -/- mice showed similar refractive error, axial length, and corneal radii across development (p > 0.05), indicating no significant effects of the Gnat2 mutation on normal ocular refractive development in mice. Three weeks of FD produced a significantly greater myopic shift in Gnat2 -/- mice compared to Gnat2 +/+ controls (-5.40 ± 1.33 D vs -2.28 ± 0.28 D, p = 0.042). Neither the Gnat2 mutation nor FD altered retinal levels of DA or DOPAC. Our results indicate that cone pathways needed for high acuity vision in primates are not as critical for normal refractive development in mice, and that both rods and cones contribute to visual signalling pathways needed to respond to FD in mammalian eyes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Retinitis pigmentosa is a devastating, blinding disorder that affects 1 in 4000 people worldwide. During the progression of the disorder, phagocytic clearance of dead photoreceptor cell bodies has a protective role by preventing additional retinal damage from accumulation of cellular debris. However, the cells responsible for the clearance remain unidentified. Taking advantage of a mouse model of retinitis pigmentosa ( Rho P23H/P23H ), we clarified the roles of Müller glia in the phagocytosis of rod photoreceptor cells. During the early stage of retinal degeneration, Müller glial cells participated in the phagocytosis of dying or dead rod photoreceptors throughout the outer nuclear layer. Nearly 50% of Müller glia engaged in phagocytosis. Among the Müller phagosomes, >90% matured into phagolysosomes. Those observations indicated that Müller glial cells are the primary contributor to phagocytosis. In contrast, macrophages migrate to the inner part of the outer nuclear layer during photoreceptor degeneration, participating in the phagocytosis of a limited population of dying or dead photoreceptor cells. In healthy retinas of wild-type mice, Müller glial cells phagocytosed cell bodies of dead rod photoreceptors albeit at a lower frequency. Taken together, the phagocytic function of Müller glia is responsible for retinal homeostasis and reorganization under normal and pathologic conditions.-Sakami, S., Imanishi, Y., Palczewski, K. Müller glia phagocytose dead photoreceptor cells in a mouse model of retinal degenerative disease.

Purpose: To determine whether high-resolution retinal imaging measures of macular structure correlate with visual function over 36 months in retinal degeneration (RD) patients and normal subjects., Methods: Twenty-six eyes of 16 RD patients and 16 eyes of 8 normal subjects were studied at baseline; 15 eyes (14 RD) and 11 eyes (6 normal) were studied 36 months later. Adaptive Optics Scanning Laser Ophthalmoscopy (AOSLO) was used to identify regions of interest (ROIs) with unambiguous cones at baseline to measure cone spacing. AOSLO images were aligned with spectral-domain optical coherence tomography (SD-OCT) and fundus-guided microperimetry results to correlate structure and function at the ROIs. SD-OCT images were segmented to measure inner segment (IS) and outer segment (OS) thickness. Correlations between cone spacing, IS and OS thickness and sensitivity were assessed using Spearman correlation coefficient ρ with bootstrap analyses clustered by person., Results: Cone spacing (ρ = 0.57, P < 0.001) and macular sensitivity (ρ = 0.19, P = 0.14) were significantly correlated with eccentricity in patients. Controlling for eccentricity, cone spacing Z-scores were inversely correlated with IS (ρ = -0.29, P = 0.002) and OS thickness (ρ = -0.39, P < 0.001) in RD patients only, and with sensitivity in normal subjects (ρ = -0.22, P < 0.001) and RD patients (ρ = -0.38, P < 0.001). After 36 months, cone spacing increased (P < 0.001) and macular sensitivity decreased (P = 0.007) compared to baseline in RD patients., Conclusions: Cone spacing increased and macular sensitivity declined significantly in RD patients over 36 months. High resolution images of cone structure correlated with retinal sensitivity, and may be appropriate outcome measures for clinical trials in RD.

Glucose metabolism in vertebrate retinas is dominated by aerobic glycolysis (the "Warburg Effect"), which allows only a small fraction of glucose-derived pyruvate to enter mitochondria. Here, we report evidence that the small fraction of pyruvate in photoreceptors that does get oxidized by their mitochondria is required for visual function, photoreceptor structure and viability, normal neuron-glial interaction, and homeostasis of retinal metabolism. The mitochondrial pyruvate carrier (MPC) links glycolysis and mitochondrial metabolism. Retina-specific deletion of MPC1 results in progressive retinal degeneration and decline of visual function in both rod and cone photoreceptors. Using targeted-metabolomics and 13 C tracers, we found that MPC1 is required for cytosolic reducing power maintenance, glutamine/glutamate metabolism, and flexibility in fuel utilization., Competing Interests: The authors declare no conflict of interest.

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.

Retinal imaging has advanced to enable noninvasive in vivo visualization of macular photoreceptors with cellular resolution. Images of retinal structure are best interpreted in the context of visual function, but clinical measures of visual function lack resolution on the scale of individual cells. Combined with cross-sectional measures of retinal structure acquired with optical coherence tomography (OCT), macular photoreceptor function can be evaluated using visual acuity and fundus-guided microperimetry, but the resolution of these measures is limited to relatively large retinal areas. By incorporating adaptive optics correction of aberrations in light entering and exiting the pupil, individual photoreceptors can be visualized and stimulated to assess structure and function. Discrepancy between structural images and visual function can shed light on the origin of visible features and their relation to visual function. Dysflective cones, cones with abnormal waveguiding properties on confocal adaptive optics scanning laser ophthalmoscopy (AOSLO) images and measurable function, provide insight into the visual significance of features in retinal images and may facilitate identification of patients who could benefit from therapies.

The mammalian retina contains an autonomous circadian clock system that controls many physiological functions within this tissue. Previous studies on young mice have reported that removal of the key circadian clock gene Bmal1 from the retina affects the circadian regulation of visual function, but does not affect photoreceptor viability. Because dysfunction in the circadian system is known to affect cell viability during aging in other systems, we compared the effect of Bmal1 removal from the retina on visual function, inner retinal structure, and photoreceptor viability in young (1 to 3 months) and aged (24 to 26 months) mice. We found that removal of Bmal1 from the retina significantly affects visual information processing in both rod and cone pathways, reduces the thickness of inner retinal nuclear and plexiform layers, accelerates the decline of visual functions during aging, and reduces the viability of cone photoreceptors. Our results thus suggest that circadian clock dysfunction, caused by genetic or other means, may contribute to the decline of visual function during development and aging., Competing Interests: The authors declare no conflict of interest.

RPE65 is the essential trans-cis isomerase of the classical retinoid (visual) cycle. Mutations in RPE65 give rise to severe retinal dystrophies, most of which are associated with loss of protein function and recessive inheritance. The only known exception is a c.1430G>A (D477G) mutation that gives rise to dominant retinitis pigmentosa with delayed onset and choroidal and macular involvement. Position 477 is distant from functionally critical regions of RPE65. Hence, the mechanism of D477G pathogenicity remains unclear, although protein misfolding and aggregation mechanisms have been suggested. We characterized a D477G knock-in mouse model which exhibited mild age-dependent changes in retinal structure and function. Immunoblot analysis of protein extracts from the eyes of these knock-in mice demonstrated the presence of ubiquitinated RPE65 and reduced RPE65 expression. We observed an accumulation of retinyl esters in the knock-in mice as well as a delay in rhodopsin regeneration kinetics and diminished electroretinography responses, indicative of RPE65 functional impairment induced by the D477G mutation in vivo. However, a cell line expressing D477G RPE65 revealed protein expression levels, cellular localization and retinoid isomerase activity comparable to cells expressing wild-type protein. Structural analysis of an RPE65 chimera suggested that the D477G mutation does not perturb protein folding or tertiary structure. Instead, the mutation generates an aggregation-prone surface that could induce cellular toxicity through abnormal complex formation as suggested by crystal packing analysis. These results indicate that a toxic gain-of-function induced by the D477G RPE65 substitution may play a role in the pathogenesis of this form of dominant retinitis pigmentosa.

Gene therapy is a promising approach in the treatment of inherited and common complex disorders of the retina. Preclinical and clinical studies have validated the use of adeno-associated viral vectors (AAV) as a safe and efficient delivery vehicle for gene transfer. Retinal pigment epithelium and rods-and to a lesser extent, cone photoreceptors-can be efficiently targeted with AAV. Other retinal cell types however are more challenging targets. The aim of this study was to characterize the transduction profile and efficiency of in silico designed, synthetic Anc80 AAVs for retinal gene transfer. Three Anc80 variants were evaluated for retinal targeting in mice and primates following subretinal delivery. In the murine retina Anc80L65 demonstrated high level of retinal pigment epithelium and photoreceptor targeting with comparable cone photoreceptor affinity compared to other AAVs. Remarkably, Anc80L65 enhanced transduction kinetics with visible expression as early as day 1 and steady state mRNA levels at day 3. Inner retinal tropism of Anc80 variants demonstrated distinct transduction patterns of Müller glia, retinal ganglion cells and inner nuclear layer neurons. Finally, murine findings with Anc80L65 qualitatively translated to the Rhesus macaque in terms of cell targets, levels and onset of expression. Our findings support the use of Anc80L65 for therapeutic subretinal gene delivery.

In healthy beings, age is the ultimate reason of cellular malfunction and death. In the rat retina, age causes a functional decline and loss of specific neuronal populations. In this regard, controversial conclusions have been reported for the innermost retina. Here, we have studied the albino and pigmented retina for the duration of the rat life-span. Independent of age (21 days-22 months), the electroretinographic recordings and the volume of the retina and its layers are smaller in albinos. Functionally, aging causes in both strains a loss of cone- and rod-mediated responses. Anatomically, cell density decreases with age because the retina grows linearly with time; no cell loss is observed in the ganglion cell layer; and only in the pigmented rat, there is a decrease in cone photoreceptors. In old animals of both strains, there is gliosis in the superior colliculi and a diminution of the area innervated by retinal ganglion cells. In conclusion, this work provides the basis for further studies linking senescence to neurodegenerative retinal diseases., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Studies of antigen presentation in retina using mice that expressed green fluorescent protein (GFP) from a transgenic CD11c promoter found that retinal GFP hi cells possessed antigen presentation function. Subsequent studies found that these high GFP hi cells preferentially localized to sites of retinal injury, consistent with their APC function. Interest in the roles of macrophages in degenerative CNS diseases led us to study the GFP hi cells in a retinal model of neurodegeneration. We asked if apoptotic cone photoreceptor cell death in Rpe65 -/- knockout mice induced the GFP hi cells, explored their relationship to resident microglia (MG), and tested their role in cone survival., Methods: Rpe65 -/- mice were bred to CD11c GFP mice on the B6/J background. CD11c GFP Rpe65 -/- mice were also backcrossed to CX3CR1 YFP-creER ROSA DTA mice so that CX3CR1 + mononuclear cells could be depleted by Tamoxifen. Retinas were analyzed by immunohistochemistry, confocal microscopy, fluorescence fundoscopy and flow cytometry., Results: Elevated numbers of GFP hi cells were concentrated in photoreceptor cell layers of CD11c GFP Rpe65 -/- mice coinciding with the peak of cone death at 2 to 4weeks of age, and persisted for at least 14months. After the initial wave of cone loss, a slow progressive loss of cones was found that continued to retain GFP hi cells in the outer retina. Sustained, four-week Tamoxifen depletions of the GFP hi cells and MG in Rpe65 -/- mice from day 13 to day 41, and from day 390 to day 420 promoted a small increase in cone survival. We found no evidence that the GFP hi cells were recruited from the circulation; all data pointed to a MG origin. MG and GFP hi cells were well segregated in the dystrophic retina; GFP hi cells were foremost in the photoreceptor cell layer, while MG were concentrated in the inner retina., Conclusions: The expression of GFP on a subset of retinal mononuclear cells in CD11c GFP mice identified a distinct population of cells performing functions previously attributed to MG. Although GFP hi cells dominated the macrophage response to cone death in the photoreceptor cell layer, their ablation led to only an incremental increase in cone survival. The ability to identify, ablate, and isolate these cells will facilitate analysis of this activated, antigen-presenting subset of MG., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphatidylserine (PS) is asymmetrically distributed between the outer and inner leaflets of the plasma membrane in eukaryotic cells. PS asymmetry on the plasma membrane depends on the activities of P4-ATPases, and disruption of PS distribution can lead to various disease conditions. Folding and transporting of P4-ATPases to their cellular destination requires the β subunit TMEM30A proteins. However, the in vivo functions of Tmem30a remain unknown. To this end, we generated retinal-specific Tmem30a-knockout mice to investigate its roles in vivo for the first time. Our data demonstrated that loss of Tmem30a in mouse cone cells leads to mislocalization of cone opsin, loss of photopic electroretinogram (ERG) responses and loss of cone cells. Mechanistically, Tmem30a-mutant mouse embryonic fibroblasts (MEFs) exhibited diminished PS flippase activity and increased exposure of PS on the cell surface. The broad loss of Tmem30a in adult mice led to a reduced scotopic photoresponse, mislocalization of ATP8A2 to the inner segment and cell body, and increased apoptosis in the retina. Our data demonstrated novel essential roles of Tmem30a in the retina.

Purpose: Choroideremia is a progressive X-linked recessive dystrophy, characterized by degeneration of the retinal pigment epithelium (RPE), choroid, choriocapillaris, and photoreceptors. We examined photoreceptor structure in a series of subjects with choroideremia with particular attention to areas bordering atrophic lesions., Methods: Twelve males with clinically-diagnosed choroideremia and confirmed hemizygous mutations in the CHM gene were examined. High-resolution images of the retina were obtained using spectral domain optical coherence tomography (SD-OCT) and both confocal and non-confocal split-detector adaptive optics scanning light ophthalmoscope (AOSLO) techniques., Results: Eleven CHM gene mutations (3 novel) were identified; three subjects had the same mutation and one subject had two mutations. SD-OCT findings included interdigitation zone (IZ) attenuation or loss in 10/12 subjects, often in areas with intact ellipsoid zones; RPE thinning in all subjects; interlaminar bridges in the imaged areas of 10/12 subjects; and outer retinal tubulations (ORTs) in 10/12 subjects. Only split-detector AOSLO could reliably resolve cones near lesion borders, and such cones were abnormally heterogeneous in morphology, diameter and density. On split-detector imaging, the cone mosaic terminated sharply at lesion borders in 5/5 cases examined. Split-detector imaging detected remnant cone inner segments within ORTs, which were generally contiguous with a central patch of preserved retina., Conclusions: Early IZ dropout and RPE thinning on SD-OCT are consistent with previously published results. Evidence of remnant cone inner segments within ORTs and the continuity of the ORTs with preserved retina suggests that these may represent an intermediate state of retinal degeneration prior to complete atrophy. Taken together, these results supports a model of choroideremia in which the RPE degenerates before photoreceptors., Competing Interests: The authors have declared that no competing interests exist.

Background and Objective: To report the clinical features of Japanese patients at Stage 1 and 2 of central areolar choroidal dystrophy (CACD)., Patients and Methods: Five family members had comprehensive ophthalmic examinations including adaptive optics (AO) retinal imaging. Mutation analysis of the PRPH2 gene was performed by Sanger sequencing. The protocol conformed to the tenets of the Declaration of Helsinki and was approved by the institutional review board of The Jikei University School of Medicine., Results: Four family members had a heterozygous PRPH2 mutation, p.R172Q; however, one member with a mutation did not show any ophthalmological abnormalities. Two patients had mild parafoveal retinal dystrophy and a reduction of cone density determined by AO analysis., Conclusion: The results indicate that the parafoveal cone photoreceptors can be affected even at the early stage of CACD. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:1115-1126.]., (Copyright 2016, SLACK Incorporated.)

Purpose: Mutations in rod photoreceptor genes can cause retinitis pigmentosa (RP). Rod gene expression is regulated by the nuclear hormone receptor, Nr2e3. Genetic deletion of Nr2e3 reprograms rods into cells that resemble cone photoreceptors, and might therefore prevent their death from some forms of RP. There are no identified ligands for Nr2e3; however, reverse agonists might mimic the genetic rescue effect and may be therapeutically useful for the treatment of RP., Methods: We screened for small molecule modulators of Nr2e3 using primary retinal cell cultures and characterized the most potent, which we have named photoregulin1 (PR1), in vitro and in vivo. We also tested the ability of PR1 to slow the progression of photoreceptor degeneration in two common mouse models of autosomal dominant RP, the RhoP23H and the Pde6brd1 mutations., Results: In developing retina, PR1 causes a decrease in rod gene expression and an increase in S opsin+ cones. Photoregulin1 continues to inhibit rod gene expression in adult mice. When applied to two mouse models of RP, PR1 slows the degeneration of photoreceptors., Conclusions: Chemical compounds identified as modulators of Nr2e3 activity may be useful for the treatment of RP through their effects on expression of disease-causing mutant genes.

Ciliary defects can result in severe disorders called ciliopathies. Mutations in NPHP5 cause a ciliopathy characterized by severe childhood onset retinal blindness, Leber congenital amaurosis (LCA), and renal disease. Using the canine NPHP5-LCA model we compared human and canine retinal phenotypes, and examined the early stages of photoreceptor development and degeneration, the kinetics of photoreceptor loss, the progression of degeneration and the expression profiles of selected genes. NPHP5-mutant dogs recapitulate the human phenotype of very early loss of rods, and relative retention of the central retinal cone photoreceptors that lack function. In mutant dogs, rod and cone photoreceptors have a sensory cilium, but develop and function abnormally and then rapidly degenerate; L/M cones are more severely affected than S-cones. The lack of outer segments in mutant cones indicates a ciliary dysfunction. Genes expressed in mutant rod or both rod and cone photoreceptors show significant downregulation, while those expressed only in cones are unchanged. Many genes in cell-death and -survival pathways also are downregulated. The canine disease is a non-syndromic LCA-ciliopathy, with normal renal structures and no CNS abnormalities. Our results identify the critical time points in the pathogenesis of the photoreceptor disease, and bring us closer to defining a potential time window for testing novel therapies for translation to patients., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Inherited retinal dystrophies are a group of genetically heterogeneous conditions with broad phenotypic heterogeneity. We analyzed a large five-generation pedigree with early-onset recessive retinal degeneration to identify the causative mutation. Linkage analysis and homozygosity mapping combined with exome sequencing were carried out to map the disease locus and identify the p.G178R mutation in the asparaginase like-1 gene (ASRGL1), segregating with the retinal dystrophy phenotype in the study pedigree. ASRGL1 encodes an enzyme that catalyzes the hydrolysis of L-asparagine and isoaspartyl-peptides. Studies on the ASRGL1 expressed in Escherichia coli and transiently transfected mammalian cells indicated that the p.G178R mutation impairs the autocatalytic processing of this enzyme resulting in the loss of functional ASRGL1 and leaving the inactive precursor protein as a destabilized and aggregation-prone protein. A zebrafish model overexpressing the mutant hASRGL1 developed retinal abnormalities and loss of cone photoreceptors. Our studies suggest that the p.G178R mutation in ASRGL1 leads to photoreceptor degeneration resulting in progressive vision loss., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: To assess outer retinal tubulation (ORT) morphology from spectral-domain optical coherence tomography (SD-OCT) volumes and donor eye histology, analyze ORT reflectivity, and estimate the number of cones surviving in ORT., Methods: In SD-OCT volumes from nine patients with advanced AMD, ORT was analyzed en face and in B-scans. The hyperreflective ORT border in cross-section was delineated and surface area calculated. Reflectivity was compared between ORT types (Closed, Open, Forming, and Branching). A flatmount retina from a donor with neovascular AMD was labeled to visualize the external limiting membrane that delimits ORT and allow measurements of cross-sectional cone area, center-to-center cone spacing, and cone density. The number of cones surviving in ORT was estimated., Results: By en face SD-OCT, ORT varies in complexity and shape. Outer retinal tubulation networks almost always contain Closed cross-sections. Spectral-domain OCT volumes containing almost exclusively Closed ORTs showed no significant direction-dependent differences in hyperreflective ORT border intensity. The surface areas of partial ORT assessed by SD-OCT volumes ranged from 0.16 to 1.76 mm2. From the flatmount retina, the average cross-sectional area of cone inner segments was 49.1 ± 7.9 μm2. The average cone spacing was 7.5 ± 0.6 μm. Outer retinal tubulation cone density was 20,351 cones/mm2. The estimated number of cones in ORT in a macula ranged from 26,399 to 186,833 cones, which is 6% to 44% of the cones present in a healthy macula., Conclusions: These first estimates for cone density and number of cones surviving in ORT suggest that ORT formation considerably distorts the photoreceptor mosaic. Results provide additional insight into the reflectivity characteristics and number of ORT cones observable in living patients by SD-OCT, as cones persist and disease progresses.

The purpose of this study was to correlate features on flood-illuminated adaptive optics (AO) images with color fundus, fundus autofluorescence (FAF) and spectral domain optical coherence tomography (SD-OCT) images in patients with retinitis pigmentosa (RP). We imaged 39 subjects diagnosed with RP using the rtx1™ flood-illuminated AO camera from Imagine Eyes (Orsay, France). We observed a correlation between hyper-autofluoresence changes on FAF, disruption of the interdigitation zone (IZ) on SD-OCT and loss of reflective cone profiles on AO. Four main patterns of cone-reflectivity were seen on AO: presumed healthy cone mosaics, hypo-reflective blurred cone-like structures, higher frequency disorganized hyper-reflective spots, and lower frequency hypo-reflective spots. These regions were correlated to progressive phases of cone photoreceptor degeneration observed using SD-OCT and FAF. These results help provide interpretation of en face images obtained by flood-illuminated AO in subjects with RP. However, significant ambiguity remains as to what truly constitutes a cone, especially in areas of degeneration. With further refinements in technology, flood illuminated AO imaging has the potential to provide rapid, standardized, longitudinal and lower cost imaging in patients with retinal degeneration.

Little is known about the mechanisms underlying macular degenerations, mainly for the scarcity of adequate experimental models to investigate cone cell death. Recently, we generated R91W;Nrl(-/-) double-mutant mice, which display a well-ordered all-cone retina with normal retinal vasculature and a strong photopic function that generates useful vision. Here we exposed R91W;Nrl(-/-) and wild-type (wt) mice to toxic levels of blue light and analyzed their retinas at different time points post illumination (up to 10 days). While exposure of wt mice resulted in massive pyknosis in a focal region of the outer nuclear layer (ONL), the exposure of R91W;Nrl(-/-) mice led to additional cell death detected within the inner nuclear layer. Microglia/macrophage infiltration at the site of injury was more pronounced in the all-cone retina of R91W;Nrl(-/-) than in wt mice. Similarly, vascular leakage was abundant in the inner and outer retina in R91W;Nrl(-/-) mice, whereas it was mild and restricted to the subretinal space in wt mice. This was accompanied by retinal swelling and the appearance of cystoid spaces in both inner and ONLs of R91W;Nrl(-/-) mice indicating edema in affected areas. In addition, basal expression levels of tight junction protein-1 encoding ZO1 were lower in R91W;Nrl(-/-) than in wt retinas. Collectively, our data suggest that exposure of R91W;Nrl(-/-) mice to blue light not only induces cone cell death but also disrupts the inner blood-retinal barrier. Macular edema in humans is a result of diffuse capillary leakage and microaneurysms in the macular region. Blue light exposure of the R91W;Nrl(-/-) mouse could therefore be used to study molecular events preceding edema formation in a cone-rich environment, and thus potentially help to develop treatment strategies for edema-based complications in macular degenerations.

Human RPE65 mutations cause a spectrum of blinding retinal dystrophies from severe early-onset disease to milder manifestations. The RPE65 P25L missense mutation, though having <10% of wild-type (WT) activity, causes relatively mild retinal degeneration. To better understand these mild forms of RPE65-related retinal degeneration, and their effect on cone photoreceptor survival, we generated an Rpe65/P25L knock-in (KI/KI) mouse model. We found that, when subject to the low-light regime (∼100 lux) of regular mouse housing, homozygous Rpe65/P25L KI/KI mice are morphologically and functionally very similar to WT siblings. While mutant protein expression is decreased by over 80%, KI/KI mice retinae retain comparable 11-cis-retinal levels with WT. Consistently, the scotopic and photopic electroretinographic (ERG) responses to single-flash stimuli also show no difference between KI/KI and WT mice. However, the recovery of a-wave response following moderate visual pigment bleach is delayed in KI/KI mice. Importantly, KI/KI mice show significantly increased resistance to high-intensity (20 000 lux for 30 min) light-induced retinal damage (LIRD) as compared with WT, indicating impaired rhodopsin regeneration in KI/KI. Taken together, the Rpe65/P25L mutant produces sufficient chromophore under normal conditions to keep opsins replete and thus manifests a minimal phenotype. Only when exposed to intensive light is this hypomorphic mutation manifested physiologically, as its reduced expression and catalytic activity protects against the successive cycles of opsin regeneration underlying LIRD. These data also help define minimal requirements of chromophore for photoreceptor survival in vivo and may be useful in assessing a beneficial therapeutic dose for RPE65 gene therapy in humans., (Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

In advanced retinal degeneration loss of rods and cones leaves melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) as the only source of visual information. ipRGCs drive non-image-forming responses (e.g., circadian photoentrainment) under such conditions but, despite projecting to the primary visual thalamus [dorsal lateral geniculate nucleus (dLGN)], do not support form vision. We wished to determine what precludes ipRGCs supporting spatial discrimination after photoreceptor loss, using a mouse model (rd/rd cl) lacking rods and cones. Using multielectrode arrays, we found that both RGCs and neurons in the dLGN of this animal have clearly delineated spatial receptive fields. In the retina, they are typically symmetrical, lack inhibitory surrounds, and have diameters in the range of 10-30° of visual space. Receptive fields in the dLGN were larger (diameters typically 30-70°) but matched the retinotopic map of the mouse dLGN. Injections of a neuroanatomical tracer (cholera toxin β-subunit) into the dLGN confirmed that retinotopic order of ganglion cell projections to the dLGN and thalamic projections to the cortex is at least superficially intact in rd/rd cl mice. However, as previously reported for deafferented ipRGCs, onset and offset of light responses have long latencies in the rd/rd cl retina and dLGN. Accordingly, dLGN neurons failed to track dynamic changes in light intensity in this animal. Our data reveal that ipRGCs can convey spatial information in advanced retinal degeneration and identify their poor temporal fidelity as the major limitation in their ability to provide information about spatial patterns under natural viewing conditions., (Copyright © 2015 the American Physiological Society.)

To investigate the long-term effects of laser-photocoagulation (LP)-induced ocular hypertension (OHT) in the innermost and outermost (outer-nuclear and outer segment)-retinal layers (ORL). OHT was induced in the left eye of adult rats. To investigate the ganglion cell layer (GCL) wholemounts were examined at 1, 3 or 6 months using Brn3a-immunodetection to identify retinal ganglion cells (RGCs) and DAPI-staining to detect all nuclei in this layer. To study the effects of LP on the ORL up to 6 months, retinas were: i) fresh extracted to quantify the levels of rod-, S- and L-opsin; ii) cut in cross-sections for morphometric analysis, or; iii) prepared as wholemounts to quantify and study retinal distributions of entire populations of RGCs (retrogradely labeled with fluorogold, FG), S- and L-cones (immunolabeled). OHT resulted in wedge-like sectors with their apex on the optic disc devoid of Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. The levels of all opsins diminished by 2 weeks and further decreased to 20% of basal-levels by 3 months. Cross-sections revealed focal areas of ORL degeneration. RGC survival at 15 days represented approximately 28% and did not change with time, whereas the S- and L-cone populations diminished to 65% and 80%, or to 20 and 35% at 1 or 6 months, respectively. In conclusion, LP induces in the GCL selective RGCs loss that does not progress after 1 month, and S- and L-cone loss that progresses for up to 6 months. Thus, OHT results in severe damage to both the innermost and the ORL., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Huntington's disease (HD) is an inherited neurodegenerative disorder that primarily affects the medium-size GABAergic neurons of striatum. The R6/2 mouse line is one of the most widely used animal models of HD. Previously the hallmarks of HD-related pathology have been detected in photoreceptors and interneurons of R6/2 mouse retina. Here we aimed to explore the survival of retinal ganglion cells (RGCs) and functional integrity of distinct retinal cell populations in R6/2 mice. The pattern electroretinography (PERG) signal was lost at the age of 8 weeks in R6/2 mice in contrast to the situation in wild-type (WT) littermates. This defect may be attributable to a major reduction in photopic ERG responses in R6/2 mice which was more evident in b- than a-wave amplitudes. At the age of 4 weeks R6/2 mice had predominantly the soluble form of mutant huntingtin protein (mHtt) in the RGC layer cells, whereas the aggregated form of mHtt was found in the majority of those cells from the 12-week-old R6/2 mice and onwards. Retinal astrocytes did not contain mHtt deposits. The total numbers of RGC layer cells, retinal astrocytes as well as optic nerve axons did not differ between 18-week-old R6/2 mice and their WT controls. Our data indicate that mHtt deposition does not cause RGC degeneration or retinal astrocyte loss in R6/2 mice even at a late stage of HD-related pathology. However, due to functional deficits in the rod- and cone-pathways, the R6/2 mice suffer progressive deficits in visual capabilities starting as early as 4 weeks; at 8 weeks there is severe impairment. This should be taken into account in any behavioral testing conducted in R6/2 mice.

Differential cell death is a common feature of aging and age-related disease. In the retina, 30% of rod photoreceptors are lost over life in humans and rodents. However, studies have failed to show age-related cell death in mouse cone photoreceptors, which is surprising because cone physiological function declines with age. Moreover in human, differential loss of short wavelength cone function is an aspect of age-related retinal disease. Here, cones are examined in young (3-month-old) and aged (12-month-old) C57 mice and also in complement factor H knock out mice (CFH-/-) that have been proposed as a murine model of age-related macular degeneration. In vivo imaging showed significant age-related reductions in outer retinal thickness in both groups over this period. Immunostaining for opsins revealed a specific significant decline of >20% for the medium/long (M/L)-wavelength cones but only in the periphery. S cones numbers were not significantly affected by age. This differential cell loss was backed up with quantitative real-time polymerase chain reaction for the 2 opsins, again showing S opsin was unaffected, but that M/L opsin was reduced particularly in CFH-/- mice. These results demonstrate aged cone loss, but surprisingly, in both genotypes, it is only significant in the peripheral ventral retina and focused on the M/L population and not S cones. We speculate that there may be fundamental differences in differential cone loss between human and mouse that may question the validity of mouse models of human outer retinal aging and pathology., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in CACNA1F encoding the α1-subunit of the retinal Cav1.4 L-type calcium channel have been linked to Cav1.4 channelopathies including incomplete congenital stationary night blindness type 2A (CSNB2), Åland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients via X-chromosomal recessive inheritance. Occasionally, clinical symptoms have been observed in female carriers, too. It is currently unknown how these mutations lead to symptoms in carriers and how the retinal network in these females is affected. To investigate these clinically important issues, we compared retinal phenotypes in Cav1.4-deficient and Cav1.4 heterozygous mice and in human female carrier patients. Heterozygous Cacna1f carrier mice have a retinal mosaic consistent with differential X-chromosomal inactivation, characterized by adjacent vertical columns of affected and non-affected wild-type-like retinal network. Vertical columns in heterozygous mice are well comparable to either the wild-type retinal network of normal mice or to the retina of homozygous mice. Affected retinal columns display pronounced rod and cone photoreceptor synaptopathy and cone degeneration. These changes lead to vastly impaired vision-guided navigation under dark and normal light conditions and reduced retinal electroretinography (ERG) responses in Cacna1f carrier mice. Similar abnormal ERG responses were found in five human CACNA1F carriers, four of which had novel mutations. In conclusion, our data on Cav1.4 deficient mice and human female carriers of mutations in CACNA1F are consistent with a phenotype of mosaic CSNB2.