Severe Retinal Diseases Research Articles

Molecular basis of CRX/DNA recognition and stoichiometry at the Ret4 response element

Two retinal transcription factors, cone-rod homeobox (CRX) and neural retina leucine zipper (NRL), cooperate functionally and physically to control photoreceptor development and homeostasis. Mutations in CRX and NRL cause severe retinal diseases. Despite the roles of NRL and CRX, insight into their functions at the molecular level is lacking. Here, we have solved the crystal structure of the CRX homeodomain in complex with its cognate response element (Ret4) from the rhodopsin proximal promoter region. The structure reveals an unexpected 2:1 stoichiometry of CRX/Ret4 and unique orientation of CRX molecules on DNA, and it explains the mechanisms of pathogenic mutations in CRX. Mutations R41Q and E42K disrupt the CRX protein-protein contacts based on the structure and reduce the CRX/Ret4 binding stoichiometry, suggesting a novel disease mechanism. Furthermore, we show that NRL alters the stoichiometry and increases affinity of CRX binding at the rhodopsin promoter, which may enhance transcription of rod-specific genes and suppress transcription of cone-specific genes.

Self-Supervised Sequence Recovery for Semi-Supervised Retinal Layer Segmentation.

Automated layer segmentation plays an important role for retinal disease diagnosis in optical coherence tomography (OCT) images. However, the severe retinal diseases result in the performance degeneration of automated layer segmentation approaches. In this paper, we present a robust semi-supervised layer segmentation network to relieve the model failures on abnormal retinas. We obtain the lesion features from the labeled images with disease-balanced distribution, and utilize the unlabeled images to supplement the layer structure information. Specifically, in our method, the cross-consistency training is utilized over the predictions of different decoders, and we enforce a consistency between different decoder predictions to improve the encoder's representation. Then, we propose a sequence prediction branch based on self-supervised manner, which is designed to predict the position of each jigsaw puzzle to obtain sensory perception of the retinal layer structure. To this task, a layer spatial pyramid pooling (LSPP) module is designed to extract multi-scale layer spatial features. Furthermore, we use the optical coherence tomography angiography (OCTA) to supplement the information damaged by diseases. The experimental results illustrate that our method achieves more robust results compared with current supervised segmentation methods. Meanwhile, advanced segmentation performance can be obtained compared with state-of-the-art semi-supervised segmentation methods.

Retinal layer segmentation in optical coherence tomography (OCT) using a 3D deep-convolutional regression network for patients with age-related macular degeneration.

Introduction - Retinal layer segmentation in optical coherence tomography (OCT) images is an important approach for detecting and prognosing disease. Automating segmentation using robust machine learning techniques lead to computationally efficient solutions and significantly reduces the cost of labor-intensive labeling, which is traditionally performed by trained graders at a reading center, sometimes aided by semi-automated algorithms. Although several algorithms have been proposed since the revival of deep learning, eyes with severe pathological conditions continue to challenge fully automated segmentation approaches. There remains an opportunity to leverage the underlying spatial correlations between the retinal surfaces in the segmentation approach. Methods - Some of these proposed traditional methods can be expanded to utilize the three-dimensional spatial context governing the retinal image volumes by replacing the use of 2D filters with 3D filters. Towards this purpose, we propose a spatial-context, continuity and anatomical relationship preserving semantic segmentation algorithm, which utilizes the 3D spatial context from the image volumes with the use of 3D filters. We propose a 3D deep neural network capable of learning the surface positions of the layers in the retinal volumes. Results - We utilize a dataset of OCT images from patients with Age-related Macular Degeneration (AMD) to assess performance of our model and provide both qualitative (including segmentation maps and thickness maps) and quantitative (including error metric comparisons and volumetric comparisons) results, which demonstrate that our proposed method performs favorably even for eyes with pathological changes caused by severe retinal diseases. The Mean Absolute Error (MAE) and Root Mean Squared Error (RMSE) for patients with a wide range of AMD severity scores (0-11) were within 0.84±0.41 and 1.33±0.73 pixels, respectively, which are significantly better than some of the other state-of-the-art algorithms. Conclusion - The results demonstrate the utility of extracting features from the entire OCT volume by treating the volume as a correlated entity and show the benefit of utilizing 3D autoencoder based regression networks for smoothing the approximated retinal layers by inducing shape based regularization constraints.

Molecular insights into the maturation of phosphodiesterase 6 by the specialized chaperone complex of HSP90 with AIPL1

Phosphodiesterase 6 (PDE6) is a key effector enzyme in vertebrate phototransduction, and its maturation and function are known to critically depend on a specialized chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Defects in PDE6 and AIPL1 underlie several severe retinal diseases, including retinitis pigmentosa and Leber congenital amaurosis. Here, we characterize the complex of AIPL1 with HSP90 and demonstrate its essential role in promoting the functional conformation of nascent PDE6. Our analysis suggests that AIPL1 preferentially binds to HSP90 in the closed state with a stoichiometry of 1:2, with the tetratricopeptide repeat domain and the tetratricopeptide repeat helix 7 extension of AIPL1 being the main contributors to the AIPL1/HSP90 interface. We demonstrate that mutations of these determinants markedly diminished both the affinity of AIPL1 for HSP90 and the ability of AIPL1 to cochaperone the maturation of PDE6 in a heterologous expression system. In addition, the FK506-binding protein (FKBP) domain of AIPL1 encloses a unique prenyl-binding site that anchors AIPL1 to posttranslational lipid modifications of PDE6. A mouse model with rod PDE6 lacking farnesylation of its PDE6A subunit revealed normal expression, trafficking, and signaling of the enzyme. Furthermore, AIPL1 was unexpectedly capable of inducing the maturation of unprenylated cone PDE6C, whereas mutant AIPL1 deficient in prenyl binding competently cochaperoned prenylated PDE6C. Thus, we conclude neither sequestration of the prenyl modifications is required for PDE6 maturation to proceed, nor is the FKBP-lipid interaction involved in the conformational switch of the enzyme into the functional state.

Comparison of Viscoelastic Substance Injection Versus Air Filling in the Anterior Chamber During Foldable Capsular Vitreous Body (FCVB) Implant Surgery: A Prospective Randomized Controlled Trial.

IntroductionTo compare the outcomes of viscoelastic substance injection with air filling in the anterior chamber during foldable capsular vitreous body (FCVB) implant surgery in patients with severe retinal disease.MethodsThirty eyes with severe retinal diseases were randomly divided into two groups. In the viscoelastic group, 0.15-0.2 mL of 1.7% sodium hyaluronate was injected into the anterior chamber after FCVB implantation; in the air group, the anterior chamber was maintained by filling the air after FCVB implant surgery. The eyes of treated patients were examined during a 24-week follow-up appointment. Data, including intraocular pressure (IOP), the difference between preoperative and postoperative IOP, and postoperative complications, were recorded.ResultsData collected from 27 eyes were used in the final analysis. The IOP of the air group was lower than that of the viscoelastic group from the first to third postoperative day (P < 0.01). Moreover, the difference between preoperative and postoperative IOP in the viscoelastic group was significantly smaller than that in the air group from the first to third postoperative day (P < 0.01). After the 1st postoperative week, postoperative IOP values were similar in the two groups (P > 0.05). Postoperative complications in the air group and the viscoelastic group included corneal blood staining (1 eye vs. 0 eyes), transient postoperative diffuse hemorrhage (5 eyes vs. 1 eye), inflammation reaction (9 eyes vs. 4 eyes), and postoperative fibrin exudation (4 eyes vs. 1 eye), respectively.ConclusionThe use of viscoelastic substances in the anterior chamber during FCVB implant surgery was associated with less fluctuation in postoperative IOP and could reduce postoperative complications.Registration NumberChiCTR-TNC-00000396.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12325-021-01840-5.

The neuroprotective role of Wnt signaling in the retina.

The canonical Wnt/β-catenin signaling pathway has been shown to play a major role during embryonic development and maturation of the central nervous system including the retina. It has a significant impact on retinal vessel formation and maturation, as well as on the establishment of synaptic structures and neuronal function in the central nervous system. Mutations in components of the Wnt/β-catenin signaling cascade may lead to severe retinal diseases, while dysregulation of Wnt signaling can contribute to disease progression. Apart from the angiogenic role of Wnt/β-catenin signaling, research in the last decades leads to the theory of a protective effect of Wnt/β-catenin signaling on damaged neurons. In this review, we focus on the neuroprotective properties of the Wnt/β-catenin pathway as well as its downstream signaling in the retina.

Toward a Bidirectional Communication Between Retinal Cells and a Prosthetic Device - A Proof of Concept.

Background: Significant progress toward the recovery of useful vision in blind patients with severe degenerative retinal diseases caused by photoreceptor death has been achieved with the development of visual prostheses that stimulate the retina electrically. However, currently used prostheses do not provide feedback about the retinal activity before and upon stimulation and do not adjust to changes during the remodeling processes in the retina. Both features are desirable to improve the efficiency of the electrical stimulation (ES) therapy offered by these devices. Accordingly, devices that not only enable ES but at the same time provide information about the retinal activity are beneficial. Given the above, a bidirectional communication strategy, in which inner retinal cells are stimulated and the output neurons of the retina, the ganglion cells, are recorded using penetrating microelectrode arrays (MEAs) is proposed.Methods: Custom-made penetrating MEAs with four silicon-based shanks, each one with three or four iridium oxide electrodes specifically designed to match retinal dimensions were used to record the activity of light-adapted wildtype mice retinas and degenerated retinas from rd10 mice in vitro. In addition, responses to high potassium concentration and to light stimulation in wildtype retinas were examined. Furthermore, voltage-controlled ES was performed.Results: The spiking activity of retinal ganglion cells (RGCs) was recorded at different depths of penetration inside the retina. Physiological responses during an increase of the extracellular potassium concentration and phasic and tonic responses during light stimulation were captured. Moreover, pathologic rhythmic activity was recorded from degenerated retinas. Finally, ES of the inner retina and simultaneous recording of the activity of RGCs was accomplished.Conclusion: The access to different layers of the retina with penetrating electrodes while recording at the same time the spiking activity of RGCs broadens the use and the field of action of multi-shank and multi-site penetrating MEAs for retinal applications. It enables a bidirectional strategy to stimulate inner retinal cells electrically and to record from the spiking RGCs simultaneously (BiMEA). This opens the possibility of a feedback loop system to acknowledge the success of ES carried out by retinal prostheses.

Presence of calpain-5 in mitochondria

Calpains are Ca2+-dependent cysteine proteases that are widely distributed in animal tissues and modulate a variety of cellular processes. There are 15 members of the calpain family in mammals. In animal cells, there are three types of calpains, viz., calpain-1, calpain-2, and calpain-10 in the mitochondria. The three types of calpains have been shown to play significant roles in pathophysiological conditions, including in apoptosis- and necrosis-like cell death. One of the severe retinal diseases, autosomal dominant neovascular inflammatory vitreoretinopathy, is known to be induced by mutations of the calpain-5 gene. However, the distribution of calpain-5 in the retina has not been elucidated. Therefore, in the present study, we determined the localization of calpain-5 in the porcine retina. We detected calpain-5 in the inner segment of photoreceptor cells using immunohistochemistry. With immunoelectron microscopy, calpain-5 was localized in the mitochondria of photoreceptor cells. Western blot analyses showed that calpain-5 was present in each mitochondrial subfraction. Furthermore, we showed that the molecular weight of mitochondrial calpain-5 was slightly smaller than cytosolic one. Our results demonstrated that a novel mitochondrial calpian, calpain-5, was localized in the mitochondria of retinal photoreceptor cells.

Rhythmic Regulation of Photoreceptor and RPE Genes Important for Vision and Genetically Associated With Severe Retinal Diseases.

PurposeThe aim of the present study was to identify candidate genes for mediating daily adjustment of vision.MethodsGenes important for vision and genetically associated with severe retinal diseases were tested for 24-hour rhythms in transcript levels in neuronal retina, microdissected photoreceptors, photoreceptor-related pinealocytes, and retinal pigment epithelium-choroid (RPE-choroid) complex by using quantitative PCR.ResultsPhotoreceptors of wildtype mice display circadian clock-dependent regulation of visual arrestins (Arr1, Arr4) and the visual cycle gene Rdh12, whereas cells of the RPE-choroid exhibit light-dependent regulation of the visual cycle key genes Lrat, Rpe65, and Rdh5. Clock-driven rhythmicity of Arr1, Arr4, and Rdh12 was observed also in rat pinealocytes, to persist in a mouse model of diabetic retinopathy (db/db) and, in the case of Arr1, to be abolished in retinae of mice deficient for dopamine D4 receptors. Therefore, the expression rhythms appear to be evolutionary conserved, to be unaffected in diabetic retinopathy, and, for Arr1, to require dopamine signaling via dopamine D4 receptors.ConclusionsThe data of the present study suggest that daily adjustment of retinal function combines clock-dependent regulation of genes responsible for phototransduction termination (Arr1, Arr4) and detoxification (Rdh12) in photoreceptors with light-dependent regulation of genes responsible for retinoid recycling (Lrat, Rpe65, and Rdh5) in RPE. Furthermore, they indicate circadian and light-dependent regulation of genes genetically associated with severe retinal diseases.

Mechanisms of mutant PDE6 proteins underlying retinal diseases

Mutations in PDE6 genes encoding the effector enzymes in rods and cones underlie severe retinal diseases including retinitis pigmentosa (RP), autosomal dominant congenital stationary night blindness (adCSNB), and achromatopsia (ACHM). Here we examined a spectrum of pathogenic missense mutations in PDE6 using the system based on co-expression of cone PDE6C with its specialized chaperone AIPL1 and the regulatory Pγ subunit as a potent co-chaperone. We uncovered two mechanisms of PDE6C mutations underlying ACHM: (a) folding defects leading to expression of catalytically inactive proteins and (b) markedly diminished ability of Pγ to co-chaperone mutant PDE6C proteins thereby dramatically reducing the levels of functional enzyme. The mechanism of the Rambusch adCSNB associated with the H258N substitution in PDE6B was probed through the analysis of the model mutant PDE6C-H262N. We identified two interrelated deficits of PDE6C-H262N: disruption of the inhibitory interaction of Pγ with mutant PDE6C that markedly reduced the ability of Pγ to augment the enzyme folding. Thus, we conclude that the Rambusch adCSNB is triggered by low levels of the constitutively active PDE6. Finally, we examined PDE6C-L858V, which models PDE6B-L854V, an RP-linked mutation that alters the protein isoprenyl modification. This analysis suggests that the type of prenyl modifications does not impact the folding of PDE6, but it modulates the enzyme affinity for its trafficking partner PDE6D. Hence, the pathogenicity of PDE6B-L854V likely arises from its trafficking deficiency. Taken together, our results demonstrate the effectiveness of the PDE6C expression system to evaluate pathogenicity and elucidate the mechanisms of PDE6 mutations in retinal diseases.

Defining Outcomes for Clinical Trials of Leber Congenital Amaurosis Caused by GUCY2D Mutations

To determine outcome measures for a clinical trial of Leber congenital amaurosis (LCA) associated with mutations in the GUCY2D gene. Retrospective observational case series. Twenty-eight patients with GUCY2D-LCA (aged 2-59 years) were studied clinically and with chromatic full-field sensitivity testing (FST), optical coherence tomography (OCT), pupillometry, and the NEI Visual Function Questionnaire (VFQ). FST permitted quantitation of cone and rod sensitivity in these patients with severe visual impairment. For most patients, the degree of rod and cone sensitivity losses showed a relationship, thereby providing an opportunity to divide patients into cohorts by severity of rod and cone dysfunction. OCT analyses indicated that retinal structure could be used not only as an objective safety measure but also as an exploratory efficacy outcome. A foveal bulge was not present in 67% of patients. The intensity of inner segment/outer segment (ellipsoid zone line) reflectivity was reduced significantly at the fovea and in the rod-dense superior retina. Based on OCT and FST parameters, most patients had dissociation of structure and function. Abnormal pupillometry sensitivity in the majority of GUCY2D-LCA patients provided another objective efficacy outcome. NEI VFQ scores showed a similar range of findings to those of other severe retinal diseases. Conventional outcome measures, such as visual acuity and the NEI VFQ, will need to be complemented by methods more specific to this GUCY2D-LCA population. Any therapeutic strategy should determine ifthere is an effect on rod as wellascone function and structure. FST provides a photoreceptor-based subjective outcome; and OCT in 2 retinal regions, fovea and superior retina, can assess photoreceptor structure. A change in the relationship of structure and function away from baseline becomes evidence of efficacy.

Evolving Knowledge in Pharmacologic Treatments of Age-Related Macular Degeneration.

Modern retinal drug therapy is a result of the recent challenges and breakthroughs in chemistry, physics, genetics, cell biology and biotechnologies. Specific pharmaceutical and pharmacokinetic characteristics of a drug are of major importance and contribute to its ability to penetrate targeted ocular tissues in order to result in effective therapeutic concentrations. In addition, the drugs should maintain a prolonged time of activity and be safe with minimal local and systemic toxicity. The transporter vehicle or drug delivery system is crucial in order to enhance ocular tissue penetration and establish controlled drug release. Administration methods should be local, thereby reducing systemic side effects, and, ideally, treatment should be noninvasive. Within the group of so-called classic therapies, the use of pharmacologic treatments has become widespread for most severe retinal diseases. Thereby, ocular therapy of diseases like exudative age-related macular degeneration has improved markedly. Moreover, new metabolic pathways have been identified, new molecules have emerged, new synthesis technologies have been discovered, and new formulae conceived. These developments have opened new avenues for limiting disease progression.

Extended conformation of the proline-rich domain of human aryl hydrocarbon receptor-interacting protein-like 1: implications for retina disease.

Mutations in the primate-specific proline-rich domain (PRD) of aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) are thought to cause Leber congenital amaurosis or dominant cone-rod dystrophy. The role of PRD and the mechanisms of PRD mutations are poorly understood. Here, we have examined properties of hAIPL1 and effects of the PRD mutations on protein structure and function. Solution structures of hAIPL1, hAIPL11-316 with PRD truncation, and the P351Δ12 and P376S mutants were examined by small angle X-ray scattering. Our analysis suggests that PRD assumes an extended conformation and does not interact with the FK506-binding and tetratricopeptide domains. The PRD truncation, but not PRD mutations, reduced the molecule's radius of gyration and maximum dimension. We demonstrate that hAIPL1 is a monomeric protein, and its secondary structure and stability are not affected by the PRD mutations. PRD itself is an extended monomeric random coil. The PRD mutations caused little or no changes in hAIPL1 binding to known partners, phosphodiesterase-6A and HSP90. We also identified the γ-subunit of phosphodiesterase-6 as a novel partner of hAIPL1 and hypothesize that this interaction is altered by P351Δ12. Our results highlight the complexity of mechanisms of PRD mutations in disease and the possibility that certain mutations are benign variants. Mutations in the proline-rich domain (PRD) of human AIPL1 cause severe retinal diseases, yet the role of PRD and the mechanisms of PRD mutations are unknown. Here, we describe a SAXS-derived solution structure of AIPL1 and functional properties of disease-linked AIPL1-PRD mutants. This structure and functional analyses provide a framework for understanding the mechanisms of PRD in disease.

Involvement of carrier-mediated transport in the retinal uptake of clonidine at the inner blood-retinal barrier.

In the present study, the blood-to-retina transport across the inner BRB was investigated for clonidine, a compound which is expected to exhibit a neuroprotective effect for the treatment of severe retinal diseases. In the in vivo study, the integration plot analysis for [(3)H]clonidine exhibited an apparent influx permeability clearance of 457 μL/(min·g retina) in the retina. The in vivo inhibition study suggests that the blood-to-retina transport of clonidine at the BRB is organic cation-sensitive since clonidine, pyrilamine, and propranolol, at a concentration of 40 mM, significantly reduced the retinal uptake index (RUI) of [(3)H]clonidine, and an inhibitory effect on the RUI was also exhibited by verapamil, at a concentration of 3 mM. The in vitro study with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, suggests that carrier-mediated transport is involved in the blood-to-retina transport of clonidine at the inner BRB since the results obtained demonstrated time-, temperature-, pH-, and concentration-dependent [(3)H]clonidine uptake, with a Km of 286 μM. In the in vitro inhibition study, the [(3)H]clonidine uptake was significantly reduced by several organic cations, such as clonidine, verapamil, pyrilamine, and propranolol, and was competitively inhibited by 200 μM verapamil, in spite of slight or no significant alteration being produced with organic anions. Furthermore, the typical substrates and inhibitors of well-known organic cation transporters had no significant effect on the uptake of [(3)H]clonidine to suggest the involvement of novel transporter molecules in the transport of clonidine across the inner BRB. These results suggest that the blood-to-retina transport of clonidine across the inner BRB involves a carrier-mediated transport manner, suggesting the contribution of a novel organic cation transporter expressed by the retinal capillary endothelial cells.

Severe ischemic retinopathy in a patient with systemic lupus erythematosus without antiphospholipid syndrome: A case report

Systemic lupus erythematosus (SLE) is a chronic, autoimmune, multisystem disease which may affect the eyes and/or visual system in up to a third of patients. Severe retinal vaso-occlusive diseases have been rarely reported as manifestation in patients with SLE. We report the case of a 35-year-old female treated for systemic lupus erythematosus for 6months, presented a sudden loss of vision. Fundus examination and fluorescein angiography revealed severe retinal vascular occlusion. This has motivated the search for antiphospholipid antibody syndrome and this was confirmed without the presence of anticardiolipin antibodies. And the treatment consisted in a laser therapy. The purpose of this case report is to demonstrate that an ocular vascular event can reveal the disease and that its diagnosis is important because this disease generally affects young people and may endanger ocular and vital prognosis.

In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene

The neural retina is subjected to various degenerative conditions. Regenerative stem-cell-based therapy holds great promise for treating severe retinal degeneration diseases, although many drawbacks remain to be overcome. One important problem is to gain authentically differentiated cells for replacement. Paired box 6 protein (5a) (PAX6 (5a)) is a highly conserved master control gene that has an essential role in the development of the vertebrate visual system. Human adipose-tissue-derived stem cell (hADSC) isolation was performed by using fat tissues and was confirmed by the differentiation potential of the cells into adipocytes and osteocytes and by their surface marker profile. The coding region of the human PAX6 (5a) gene isoform was cloned and lentiviral particles were propagated in HEK293T. The differentiation of hADSCs into retinal cells was characterized by morphological characteristics, quantitative real-time reverse transcription plus the polymerase chain reaction (qPCR) and immunocytochemistry (ICC) for some retinal cell-specific and retinal pigmented epithelial (RPE) cell-specific markers. hADSCs were successfully isolated. Flow cytometric analysis of surface markers indicated the high purity (~97 %) of isolated hADSCs. After 30 h of post-transduction, cells gradually showed the characteristic morphology of neuronal cells and small axon-like processes emerged. qPCR and ICC confirmed the differentiation of some neural retinal cells and RPE cells. Thus, PAX6 (5a) transcription factor expression, together with medium supplemented with fibronectin, is able to induce the differentiation of hADSCs into retinal progenitors, RPE cells and photoreceptors.

Hypertension-related eye abnormalities

Hypertension affects a significant proportion of the population, however, it is often diagnosed with a delay. The aim of this article is to review the well known and less known eye abnormalities related to hypertension, and place them in the context of population based studies. Hypertension affects various parts of the eye. The originally classified hypertensive retinopathy (retinal microvascular changes) is still relevant, but new features are visible in cases of controlled hypertension. Signs of mild hypertensive retinopathy are more common than expected occurring in nearly 10–15% of the adult non-diabetic population. Hypertensive retinopathy can be an indicator of other hypertensive complications such as neurologic and cardiac complications. Microvascular changes are reversible in well controlled hypertension. Proper treatment of hypertension can reduce the development and progression of diabetic retinopathy and, thus, visual loss due to severe retinal diseases such as retinal vascular occlusion (artery and vein), retinal arteriolar emboli, macroaneurysm, ischemic optic neuropathy and age-related macular degeneration.Orv. Hetil., 154(45), 1773–1780.

Gene therapy restores vision and delays degeneration in the CNGB1-/- mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(-/-)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (~4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(-/-) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(-/-) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.

Applications of nanoparticles in ophthalmology

Nanocarriers, such as nanoparticles, have the capacity to deliver ocular drugs to specific target sites and hold promise to revolutionize the therapy of many eye diseases. Results to date strongly suggest that ocular medicine will benefit enormously from the use of this nanometric scale technology. One of the most important handicaps of the eye as a target organ for drugs is the presence of several barriers that impede direct and systemic drug access to the specific site of action. Superficial barriers include the ocular surface epithelium and the tear film, and internal barriers include the blood-aqueous and blood-retina barriers. Topical application is the preferred route for most drugs, even when the target tissues are at the back part of the eye where intraocular injections are currently the most common route of administration. Direct administration using any of these two routes faces many problems related to drug bioavailability, including side effects and repeated uncomfortable treatments to achieve therapeutic drug levels. In this regard, the advantages of using nanoparticles include improved topical passage of large, poorly water-soluble molecules such as glucocorticoid drugs or cyclosporine for immune-related, vision-threatening diseases. Other large and unstable molecules, such as nucleic acids, delivered using nanoparticles offer promising results for gene transfer therapy in severe retinal diseases. Also, nanoparticle-mediated drug delivery increases the contact time of the administered drug with its target tissue, such as in the case of brimonidine, one of the standard treatments for glaucoma, or corticosteroids used to treat autoimmune uveitis, a severe intraocular inflammatory process. In addition, nanocarriers permit the non-steroidal anti-inflammatory drug indomethacin to reach inner eye structures using the transmucosal route. Finally, nanoparticles allow the possibility of targeted delivery to reach specific types of cancer, such as melanoma, leaving normal cells untouched. This review summarizes experimental results from our group and others since the beginnings of nanocarrier technology to deliver drugs to different locations in the eye. Also, it explores the future possibilities of nanoparticles not only as drug delivery systems but also as aides for diagnostic purposes.

Otx2 Gene Deletion in Adult Mouse Retina Induces Rapid RPE Dystrophy and Slow Photoreceptor Degeneration

BackgroundMany developmental genes are still active in specific tissues after development is completed. This is the case for the homeobox gene Otx2, an essential actor of forebrain and head development. In adult mouse, Otx2 is strongly expressed in the retina. Mutations of this gene in humans have been linked to severe ocular malformation and retinal diseases. It is, therefore, important to explore its post-developmental functions. In the mature retina, Otx2 is expressed in three cell types: bipolar and photoreceptor cells that belong to the neural retina and retinal pigment epithelium (RPE), a neighbour structure that forms a tightly interdependent functional unit together with photoreceptor cells.Methodology/Principal FindingsConditional self-knockout was used to address the late functions of Otx2 gene in adult mice. This strategy is based on the combination of a knock-in CreERT2 allele and a floxed allele at the Otx2 locus. Time-controlled injection of tamoxifen activates the recombinase only in Otx2 expressing cells, resulting in selective ablation of the gene in its entire domain of expression. In the adult retina, loss of Otx2 protein causes slow degeneration of photoreceptor cells. By contrast, dramatic changes of RPE activity rapidly occur, which may represent a primary cause of photoreceptor disease.ConclusionsOur novel mouse model uncovers new Otx2 functions in adult retina. We show that this transcription factor is necessary for long-term maintenance of photoreceptors, likely through the control of specific activities of the RPE.