Human Retinal Degenerative Diseases Research Articles

Ibuprofen reduces inflammation, necroptosis and protects photoreceptors from light-induced retinal degeneration

BackgroundThe retinal degenerative diseases retinitis pigmentosa (RP) and atrophic age- related macular degeneration (AMD) are characterized by vision loss from photoreceptor (PR) degeneration. Unfortunately, current treatments for these diseases are limited at best. Genetic and other preclinical evidence suggest a relationship between retinal degeneration and inflammation. To further explore this relationship, we tested whether Ibuprofen (IBU), an FDA-approved non-steroidal anti-inflammatory drug (NSAID), could promote PR survival and function in a mouse model of light damage (LD)-induced PR degeneration.MethodsLD was induced by exposing mice to 4000 lx of light for 2–4 hours (h). IBU (100 or 200 mg/kg) or vehicle was administered by daily intraperitoneal injection. Retinal structure and function were evaluated by spectral-domain optical coherence tomography (SD-OCT) and electroretinography (ERG). Cell death genes were analyzed at 24 and 72 h after LD using the Mouse Pan-Cell Death Pathway PCR Array (88 genes). The cellular location and protein expression of key necroptosis genes were assessed by immunohistochemistry.ResultsRetinal outer nuclear layer (ONL) thickness in vehicle-injected LD animals was 8.7 ± 0.6% of retinas without LD (p < 0.0001). In IBU 200 mg/kg treated mice, central ONL thickness was 74.9 ± 7.7% of untreated retinas (p < 0.001). A-wave and b-wave ERG amplitudes were significantly preserved in IBU-treated animals. IBU significantly inhibited retinal inflammation. Twenty-four hour after LD, retinal mRNA expression for the inflammatory-factors tumor necrosis factor (Tnf), interleukin-1 beta (Il1B), and C-C motif chemokine ligand 2 (Ccl2) increased by 10-, 17-, and 533-fold, respectively; in IBU-treated animals, the expression levels of these inflammatory factors were not significantly different from no-LD controls. Expression of key necroptosis genes, including Ripk3 and Mlkl, were upregulated in LD vehicle-treated mice, but dramatically reduced to near no LD levels in LD IBU-treated mice. Microglia activation and MLKL protein upregulation were observed primarily in photoreceptors 12 h after LD, as assessed by immunohistochemistry. IBU reduced the upregulation of MLKL protein and microglia migration in the ONL and outer plexiform layer (OPL) of treated retinas.ConclusionsSystemic administration of the anti-inflammatory drug IBU partially protected mouse retinas from light-induced photochemical damage and inhibited both inflammation and the necroptosis cell death pathways. Our results suggest that NSAIDs may provide a promising therapeutic approach for treatment of the human retinal degenerative diseases.

Alignment, calibration, and validation of an adaptive optics scanning laser ophthalmoscope for high-resolution human foveal imaging.

In prior art, advances in adaptive optics scanning laser ophthalmoscope (AOSLO) technology have enabled cones in the human fovea to be resolved in healthy eyes with normal vision and low to moderate refractive errors, providing new insight into human foveal anatomy, visual perception, and retinal degenerative diseases. These high-resolution ophthalmoscopes require careful alignment of each optical subsystem to ensure diffraction-limited imaging performance, which is necessary for resolving the smallest foveal cones. This paper presents a systematic and rigorous methodology for building, aligning, calibrating, and testing an AOSLO designed for imaging the cone mosaic of the central fovea in humans with cellular resolution. This methodology uses a two-stage alignment procedure and thorough system testing to achieve diffraction-limited performance. Results from retinal imaging of healthy human subjects under 30 years of age with refractive errors of less than 3.5 diopters using either 680 nm or 840 nm light show that the system can resolve cones at the very center of the fovea, the region where the cones are smallest and most densely packed.

Contribution of Interleukin-17A to Retinal Degenerative Diseases.

Retinal degenerative diseases are a leading cause of vision loss and blindness throughout the world, characterized by chronic and progressive loss of neurons and/or myelin. One of the common features of retinal degenerative diseases and central neurodegenerative diseases is chronic neuroinflammation. Interleukin-17A (IL-17A) is the cytokine most closely related to disease in its family. Accumulating evidence suggests that IL-17A plays a key role in human retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and glaucoma. This review aims to provide an overview of the role of IL-17A participating in the pathogenesis of retinal degenerative diseases, which may open new avenues for potential therapeutic interventions.

The Role of Inflammation in Retinal Neurodegeneration and Degenerative Diseases.

Retinal neurodegeneration is predominantly reported as the apoptosis or impaired function of the photoreceptors. Retinal degeneration is a major causative factor of irreversible vision loss leading to blindness. In recent years, retinal degenerative diseases have been investigated and many genes and genetic defects have been elucidated by many of the causative factors. An enormous amount of research has been performed to determine the pathogenesis of retinal degenerative conditions and to formulate the treatment modalities that are the critical requirements in this current scenario. Encouraging results have been obtained using gene therapy. We provide a narrative review of the various studies performed to date on the role of inflammation in human retinal degenerative diseases such as age-related macular degeneration, inherited retinal dystrophies, retinitis pigmentosa, Stargardt macular dystrophy, and Leber congenital amaurosis. In addition, we have highlighted the pivotal role of various inflammatory mechanisms in the progress of retinal degeneration. This review also offers an assessment of various therapeutic approaches, including gene-therapies and stem-cell-based therapies, for degenerative retinal diseases.

Development and characterization of a chronic photoreceptor degeneration model in adult zebrafish that does not trigger a regenerative response

Zebrafish (Danio rerio) have become a highly-utilized model system in the field of regenerative biology because of their endogenous ability to regenerate many tissues and organs, including the retina. The vast majority of previous research on retinal regeneration in adult zebrafish utilizes acute methodologies for retinal damage. Acute retinal cell death triggers a reactive gliosis response of Müller glia (MG), the resident macroglia of the retina. In addition, each activated MG undergoes asymmetric cell division to produce a neuronal progenitor, which continues to divide and ultimately gives rise to new retinal neurons. Studies using these approaches have uncovered many crucial mechanisms by which MG respond to acute damage. However, they may not adequately mimic the chronic neuronal degeneration observed in many human retinal degenerative diseases. The current study aimed to develop a new long-term, chronic photoreceptor damage and degeneration model in adult zebrafish. Comparing the subsequent cellular responses to that of the commonly-used acute high-intensity model, we found that low, continuous light exposure damaged the outer segments of both rod and cone photoreceptors, but did not result in significant apoptotic cell death, MG gliosis, or MG cell-cycle re-entry. Instead, chronic light nearly completely truncated photoreceptor outer segments and resulted in a recruitment of microglia to the area. Together, these studies present a chronic photoreceptor model that can be performed in a relatively short time frame (21 days), that may lend insight into the cellular events underlying non-regenerative photoreceptor degeneration observed in other model systems.

Peptide Derivatives of Retinylamine Prevent Retinal Degeneration with Minimal Side Effects on Vision in Mice.

Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH2) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH2 with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH2 derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4-/-Rdh8-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH2 or other tested amino acid derivatives of Ret-NH2. Ret-NH2-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.

Conflict of interest: the authors declare no conflict of interest.

Retinal degenerations comprise a large heterogeneous class of diseases (dystrophies) of retina leading to a progressive vision loss. The emergence of new experimental therapies (gene and cell therapy, regenerative ophthalmic surgery) generates a demand for new experimental models. The review tackles various animal models of human retinal degenerative diseases. Pros and contras of induced and genetic experimental models are highlighted. An adequate experimental model choice may condition a pathogenetic approach to retinal disease therapy.

Building Preclinical Models of Retinal Degeneration in Non-human Primates

Mutations affecting the expression of cone photoreceptors can lead to retinal degeneration, which in many cases can result in a permanent loss of vision. However, preclinical models for human retinal degenerative diseases are lacking. Dr Zi-Bing Jin and his colleagues study rhesus macaque models of achromatopsia (a congenital disorder characterised by an inability to distinguish colours) and oculocutaneous albinism (characterised by a disorder of melanin synthesis, leading to loss of visual acuity). The animal models utilised in the Jin laboratory offer important opportunities for studies on disease mechanisms as well as therapeutic development.

Limitations and Promise of Retinal Tissue From Human Pluripotent Stem Cells for Developing Therapies of Blindness.

The self-formation of retinal tissue from pluripotent stem cells generated a tremendous promise for developing new therapies of retinal degenerative diseases, which previously seemed unattainable. Together with use of induced pluripotent stem cells or/and CRISPR-based recombineering the retinal organoid technology provided an avenue for developing models of human retinal degenerative diseases “in a dish” for studying the pathology, delineating the mechanisms and also establishing a platform for large-scale drug screening. At the same time, retinal organoids, highly resembling developing human fetal retinal tissue, are viewed as source of multipotential retinal progenitors, young photoreceptors and just the whole retinal tissue, which may be transplanted into the subretinal space with a goal of replacing patient’s degenerated retina with a new retinal “patch.” Both approaches (transplantation and modeling/drug screening) were projected when Yoshiki Sasai demonstrated the feasibility of deriving mammalian retinal tissue from pluripotent stem cells, and generated a lot of excitement. With further work and testing of both approaches in vitro and in vivo, a major implicit limitation has become apparent pretty quickly: the absence of the uniform layer of Retinal Pigment Epithelium (RPE) cells, which is normally present in mammalian retina, surrounds photoreceptor layer and develops and matures first. The RPE layer polarize into apical and basal sides during development and establish microvilli on the apical side, interacting with photoreceptors, nurturing photoreceptor outer segments and participating in the visual cycle by recycling 11-trans retinal (bleached pigment) back to 11-cis retinal. Retinal organoids, however, either do not have RPE layer or carry patches of RPE mostly on one side, thus directly exposing most photoreceptors in the developing organoids to neural medium. Recreation of the critical retinal niche between the apical RPE and photoreceptors, where many retinal disease mechanisms originate, is so far unattainable, imposes clear limitations on both modeling/drug screening and transplantation approaches and is a focus of investigation in many labs. Here we dissect different retinal degenerative diseases and analyze how and where retinal organoid technology can contribute the most to developing therapies even with a current limitation and absence of long and functional outer segments, supported by RPE.

Generation of Nonhuman Primate Model of Cone Dysfunction through In Situ AAV-Mediated CNGB3 Ablation

SummaryA major challenge to the development of therapies for human retinal degenerative diseases is the lack of an ideal preclinical model because of the physiological differences between humans and most model animals. Despite the successful generation of a primate model through germline knockout of a disease-causing gene, the major issues restricting modeling in nonhuman primates (NHPs) are their relatively long lifespan, lengthy gestation, and dominant pattern of singleton births. Herein, we generated three cynomolgus macaques with macular in situ knockout by subretinal delivery of an adeno-associated virus (AAV)-mediated CRISPR-Cas9 system targeting CNGB3, the gene responsible for achromatopsia. The in vivo targeting efficiency of CRISPR-Cas9 was 12%–14%, as shown by both immunohistochemistry and single-cell transcriptomic analysis. Through clinical ophthalmic examinations, we observed a reduced response of electroretinogram in the central retina, which corresponds to a somatic disruption of CNGB3. In addition, we did not detect CRISPR-Cas9 residue in the heart, liver, spleen, kidney, brain, testis, or blood a year after administration. In conclusion, we successfully generated a NHP model of cone photoreceptor dysfunction in the central retina using an in situ CNGB3-knockout strategy.

Cell-Based Therapy for Retinal Disease: The New Frontier.

The availability of noninvasive high-resolution imaging technology, the immune-suppressive nature of the subretinal space, and the existence of surgical techniques that permit transplantation surgery to be a safe procedure all render the eye an ideal organ in which to begin cell-based therapy in the central nervous system. A number of early stage clinical trials are underway to assess the safety and feasibility of cell-based therapy for retinal blindness. Cell-based therapy using embryonic stem cell-derived differentiated cells (e.g., retinal pigment epithelium (RPE)), neural progenitor cells, photoreceptor precursors, and bone marrow-derived hematopoietic stem/progenitor cells has demonstrated successful rescue and/or replacement in preclinical models of human retinal degenerative disease. Additional research is needed to identify the mechanisms that control synapse formation/disjunction (to improve photoreceptor transplant efficacy), to identify factors that limit RPE survival in areas of geographic atrophy (to improve RPE transplant efficacy in eyes with age-related macular degeneration), and to identify factors that regulate immune surveillance of the subretinal space (to improve long-term photoreceptor and RPE transplant survival).

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the "open", transcriptionally active chromatin in developing and postmitotic mouse retina.

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the "open", transcriptionally active chromatin in developing and postmitotic mouse retina.

Quantitative Assessment of Retina Explant Viability in a Porcine Ex Vivo Neuroretina Model.

Given that porcine and human retinas have similar structures and characteristics, ex vivo culture of porcine neuroretina provides an attractive model for studying mechanisms of human retinal injury and degenerative disease. Here, we describe the method that was used to establish and characterize an adult porcine retina culture system as a rapid screening tool for retinal survival in real time. Neuroretina explants 8 mm in diameter were harvested from adult swine and cultured on porous cell culture inserts with adjustable heights. Retina explant viability was evaluated at 1, 4, 7, 11, and 14 days of culture using a resazurin-based metabolic assay. The explants were analyzed morphologically through immunohistochemistry for glial activation and apoptosis. Morphometric analysis was also performed on hematoxylin and eosin-stained retina sections from each time point. The viability of retina explants gradually decreased over time in culture. The laminar structure of the neuroretina was well preserved during the first 7 days. However, by day 14, most explants showed significant loss of cells in each laminar layer and obvious thinning. Overall, the progressive loss of retinal lamination and thickness, and increase in apoptotic nuclei with activated hypertrophic Müller cells were well correlated with the metabolic activity of the ex vivo neuroretina explants. This study was the first report to describe the use of a high-throughput and quantitative method for monitoring retina explant viability in real time. Ex vivo neuroretina cultures closely mimic the functional dynamics of the organ, and can be used efficiently to screen novel therapeutics for retinal neurodegenerative disease.

Adenosine A1 receptor: A neuroprotective target in light induced retinal degeneration.

Light induced retinal degeneration (LIRD) is a useful model that resembles human retinal degenerative diseases. The modulation of adenosine A1 receptor is neuroprotective in different models of retinal injury. The aim of this work was to evaluate the potential neuroprotective effect of the modulation of A1 receptor in LIRD. The eyes of rats intravitreally injected with N6-cyclopentyladenosine (CPA), an A1 agonist, which were later subjected to continuous illumination (CI) for 24 h, showed retinas with a lower number of apoptotic nuclei and a decrease of Glial Fibrillary Acidic Protein (GFAP) immunoreactive area than controls. Lower levels of activated Caspase 3 and GFAP were demonstrated by Western Blot (WB) in treated animals. Also a decrease of iNOS, TNFα and GFAP mRNA was demonstrated by RT-PCR. A decrease of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. Electroretinograms (ERG) showed higher amplitudes of a-wave, b-wave and oscillatory potentials after CI compared to controls. Conversely, the eyes of rats intravitreally injected with dipropylcyclopentylxanthine (DPCPX), an A1 antagonist, and subjected to CI for 24 h, showed retinas with a higher number of apoptotic nuclei and an increase of GFAP immunoreactive area compared to controls. Also, higher levels of activated Caspase 3 and GFAP were demonstrated by Western Blot. The mRNA levels of iNOS, nNOS and inflammatory cytokines (IL-1β and TNFα) were not modified by DPCPX treatment. An increase of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. ERG showed that the amplitudes of a-wave, b-wave, and oscillatory potentials after CI were similar to control values. A single pharmacological intervention prior illumination stress was able to swing retinal fate in opposite directions: CPA was neuroprotective, while DPCPX worsened retinal damage. In summary, A1 receptor agonism is a plausible neuroprotective strategy in LIRD.

Human embryonic stem cells extracellular vesicles and their effects on immortalized human retinal Müller cells.

Extracellular vesicles (EVs) released by virtually every cell of all organisms are involved in processes of intercellular communication through the delivery of their functional mRNAs, proteins and bioactive lipids. We previously demonstrated that mouse embryonic stem cell-released EVs (mESEVs) are able to transfer their content to different target retinal cells, inducing morphological and biochemical changes in them. The main objective of this paper is to characterize EVs derived from human embryonic stem cells (hESEVs) and investigate the effects that they have on cultured retinal glial, progenitor Müller cells, which are known to give rise to retinal neurons under specific conditions. This would allow us to establish if hESEVs have a pro-regenerative potential not yet described that could be used in the future for treatment of human retinal degenerative diseases. Initially, we showed that hESEVs are heterogeneous in size, contain mRNAs and proteins involved in the induction and maintenance of stem cell pluripotency and can be internalized by cultured Müller cells. After a single exposure to hESEVs these cells display changes in their gene expression profile, and with multiple exposures they de-differentiate and trans-differentiate into retinal neuronal precursors. hESEVs were then fractionated into microvesicles (MVs) and exosomes (EXOs), which were characterized by size, specific surface proteins and biochemical/molecular components. We demonstrate that despite the similar internalization of non-fractionated hESEVs, MVs and EXOs by Müller progenitor cells, in vitro, only the release of MVs’ cargo into the cells’ cytoplasm induces specific changes in their levels of pluripotency mRNAs and early retinal proteins. EXOs do not produce any detectable effect. Thus, we conclude that MVs and MVs-containing hESEVs are promising agents that possibly could promote the regeneration of diseased or damaged retinas in vivo through inducing glial Müller cells to become replacement neurons.

Functional ectopic neuritogenesis by retinal rod bipolar cells is regulated by miR-125b-5p during retinal remodeling in RCS rats

Following retinal degeneration, retinal remodeling can cause neuronal microcircuits to undergo structural alterations, which particularly affect the dendrites of bipolar cells. However, the mechanisms and functional consequences of such changes remain unclear. Here, we used Royal College of Surgeon (RCS) rats as a model of retinal degeneration, to study structural changes in rod bipolar cells (RBCs) and the underlying mechanisms of these changes. We found that, with retinal degeneration, RBC dendrites extended into the outer nuclear layer (ONL) of the retina, and the ectopic dendrites formed synapses with the remaining photoreceptors. This ectopic neuritogenesis was associated with brain-derived neurotrophic factor (BDNF) – expression of which was negatively regulated by miR-125b-5p. Overexpression of miR-125b-5p in the retinae of RCS rats diminished RBC ectopic dendrites, and compromised the b-wave of the flash electroretinogram (ERG). In contrast, down-regulation of miR-125b-5p (or exogenous BDNF treatment) increased RBC ectopic dendrites, and improved b-wave. Furthermore, we showed that the regulation of ectopic neuritogenesis by BDNF occurred via the downstream modulation of the TrkB-CREB signaling pathway. Based on these findings, we conclude that ectopic dendrites are likely to be providing functional benefits and that, in RCS rats, miR-125b-5p regulates ectopic neuritogenesis by RBCs through modulation of the BDNF-TrkB-CREB pathway. This suggests that therapies that reduce miR-125b-5p expression could be beneficial in human retinal degenerative disease.

Loss of ift122, a Retrograde Intraflagellar Transport (IFT) Complex Component, Leads to Slow, Progressive Photoreceptor Degeneration Due to Inefficient Opsin Transport

In the retina, aberrant opsin transport from cell bodies to outer segments leads to retinal degenerative diseases such as retinitis pigmentosa. Opsin transport is facilitated by the intraflagellar transport (IFT) system that mediates the bidirectional movement of proteins within cilia. In contrast to functions of the anterograde transport executed by IFT complex B (IFT-B), the precise functions of the retrograde transport mediated by IFT complex A (IFT-A) have not been well studied in photoreceptor cilia. Here, we analyzed developing zebrafish larvae carrying a null mutation in ift122 encoding a component of IFT-A. ift122 mutant larvae show unexpectedly mild phenotypes, compared with those of mutants defective in IFT-B. ift122 mutants exhibit a slow onset of progressive photoreceptor degeneration mainly after 7 days post-fertilization. ift122 mutant larvae also develop cystic kidney but not curly body, both of which are typically observed in various ciliary mutants. ift122 mutants display a loss of cilia in the inner ear hair cells and nasal pit epithelia. Loss of ift122 causes disorganization of outer segment discs. Ectopic accumulation of an IFT-B component, ift88, is observed in the ift122 mutant photoreceptor cilia. In addition, pulse-chase experiments using GFP-opsin fusion proteins revealed that ift122 is required for the efficient transport of opsin and the distal elongation of outer segments. These results show that IFT-A is essential for the efficient transport of outer segment proteins, including opsin, and for the survival of retinal photoreceptor cells, rendering the ift122 mutant a unique model for human retinal degenerative diseases.

Changes in aquaporin-4 and Kir4.1 expression in rats with inherited retinal dystrophy

Muller glial cells (MGC) are essential for normal functioning of retina. They are especially involved in potassium (K+) and water homeostasis, via inwardly rectifying K+ (Kir 4.1) and aquaporin-4 (AQP4) channels respectively. Because MGC appear morphologically and functionally altered in most retinal pathologies, we studied the expression of AQP 4 and Kir 4.1 during the time course of progressive retinal degeneration in Royal College of Surgeons (RCS) rats, an animal model for the hereditary human retinal degenerative disease Retinitis pigmentosa. Simultaneous detection of AQP4 and Kir 4.1 was performed by quantitative real-time polymerase chain reaction (QRT-PCR), Western blot and immunohistochemistry at birth and during progression of the pathology. Although small quantities of AQP4 and Kir 4.1 mRNA were detected at birth (postnatal day (PNd) 0) in both control and dystrophic rat retinas, proteins could not be detected at this age. Detectable proteins appeared in the second week of postnatal life. From PNd15 onwards, the time course in the expression of both AQP4 and Kir 4.1 mRNAs and protein was similar in dystrophic and control rats, with a progressive increase peaking at PNd60 and a subsequent decrease by one year. AQP4 protein and mRNA content were significantly lowered in dystrophic compared to control rats. Kir 4.1 protein levels were also lower in dystrophic retinas, while mRNA concentrations were unchanged and/or slightly higher in dystrophic rats. The discrepancies between Kir4.1 mRNA and protein suggest perturbation in protein translation due to the pathology. AQP4 and Kir 4.1/vimentin co-immunolabeling showed that: 1) apical radial processes of some MGC invaded the subretinal zone, and 2) MGC morphology was distorted in advanced pathology. MGC became hypertrophic both during the pathology and also with age in control rats. In conclusion, our results confirm that this inherited photoreceptor degeneration also leads to progressive alterations in physiological and morphological parameters of MGC which may aggravate retinal impairment.

Cell-based therapy for retinal degenerative disease

The availability of non-invasive high-resolution imaging technology, the immune suppressive nature of the subretinal space, and the existence of surgical techniques that permit transplantation surgery to be a safe procedure all render the eye an ideal organ in which to begin cell-based therapy in the central nervous system. A number of early stage clinical trials are underway to assess the safety and feasibility of cell-based therapy for retinal blindness. Cell-based therapy using embryonic stem cell derived differentiated cells (e.g., retinal pigment epithelium (RPE)), neural progenitor cells, photoreceptor precursors, and bone marrow-derived hematopoietic stem/progenitor cells all have demonstrated successful rescue and/or replacement in preclinical models of human retinal degenerative disease. Additional research is needed to identify the mechanisms that control synapse formation/disjunction (to improve photoreceptor transplant efficacy), to identify factors that limit RPE survival in areas of geographic atrophy (to improve RPE transplant efficacy in eyes with age-related macular degeneration), and to identify factors that regulate immune surveillance of the subretinal space (to improve long-term photoreceptor and RPE transplant survival).