Programmed Cell Death in Retinal Degeneration: Targeting Apoptosis in Photoreceptors as Potential Therapy for Retinal Degeneration

Retinal degenerations are the major cause of incurable blindness characterized by loss of retinal photoreceptor cells. Several genes causing these genetic diseases have been identified, however the molecular characterization of a high percentage of patients affected by retinitis pigmentosa (RP), a common form of retinal degeneration, is still unknown. The high genetic heterogeneity of these diseases hampers the comprehension of the pathogenetic mechanism causing photoreceptor cell death. Therapies are not available yet and for this reason there is a lot of interest in understanding the etiology and the pathogenesis of these disorders at a cellular and molecular level. Some common features have been identified in different forms of RP. Apoptosis was reported to be the final outcome in all RP animal models and patients analyzed so far. We recently identified two apoptotic pathways co-activated in photoreceptors undergoing cell death in the retinal degeneration (rd1) mouse model of autosomal recessive RP. Our studies opened new perspectives together with many questions that require deeper analyses in order to take advantage of this knowledge and develop new therapeutic approaches. We believe that minimizing cell demise may represent a promising curing strategy that needs to be exploited for retinal degeneration.     

Biology and therapy of inherited retinal degenerative disease: insights from mouse models

Retinal neurodegeneration associated with the dysfunction or death of photoreceptors is a major cause of incurable vision loss. Tremendous progress has been made over the last two decades in discovering genes and genetic defects that lead to retinal diseases. The primary focus has now shifted to uncovering disease mechanisms and designing treatment strategies, especially inspired by the successful application of gene therapy in some forms of congenital blindness in humans. Both spontaneous and laboratory-generated mouse mutants have been valuable for providing fundamental insights into normal retinal development and for deciphering disease pathology. Here, we provide a review of mouse models of human retinal degeneration, with a primary focus on diseases affecting photoreceptor function. We also describe models associated with retinal pigment epithelium dysfunction or synaptic abnormalities. Furthermore, we highlight the crucial role of mouse models in elucidating retinal and photoreceptor biology in health and disease, and in the assessment of novel therapeutic modalities, including gene- and stem-cell-based therapies, for retinal degenerative diseases.KEY WORDS: Mouse mutants, Photoreceptor, Retinal development, Retinal disease     

Disease model: photoreceptor degenerations

Retinal diseases can be caused by genetic or non-genetic factors, and typically involve specific cell layers within the retina. A large number of simple Mendelian gene defects are known to affect the photoreceptor cell layer. As photoreceptor cells cannot be maintained in vitro, the study of animal models is instrumental in understanding the disease process and in testing therapeutic approaches. This review briefly summarizes those animal models of photoreceptor diseases with defined genotypes.     

The splicing factor Prp31 is essential for photoreceptor development in Drosophila

Retinitis pigmentosa is a leading cause of blindness and a progressive retinal disorder, affecting millions of people worldwide. This disease is characterized by photoreceptor degeneration, eventually leading to complete blindness. Autosomal dominant (adRP) has been associated with mutations in at least four ubiquitously expressed genes encoding pre-mRNA splicing factors—Prp3, Prp8, Prp31 and PAP1. Biological function of adRP-associated splicing factor genes and molecular mechanisms by which mutations in these genes cause cell-type specific photoreceptor degeneration in humans remain to be elucidated. To investigate the in vivo function of these adRP-associated splicing factor genes, we examined Drosophila in which expression of fly Prp31 homolog was down-regulated. Sequence analyses show that CG6876 is the likely candidate of Drosophila melanogaster Prp31 homolog (DmPrp31). Predicted peptide sequence for CG6876 shows 57% similarity to the Homo sapiens Prp31 protein (HsPrp31). Reduction of the endogenous Prp31 by RNAi-mediated knockdown specifically in the eye leads to reduction of eye size or complete absence of eyes with remarkable features of photoreceptor degeneration and recapitulates the bimodal expressivity of human Prp31 mutations in adRP patients. Such transgenic DmPrp31RNAi flies provide a useful tool for identifying genetic modifiers or interacting genes for Prp31. Expression of the human Prp31 in these animals leads to a partial rescue of the eye phenotype. Our results indicate that the Drosophila CG6876 is the fly ortholog of mammalian Prp31 gene.     

AAV-mediated gene therapy in mouse models of recessive retinal degeneration

In recent years, more and more mutant genes that cause retinal diseases have been detected. At the same time, many naturally occurring mouse models of retinal degeneration have also been found, which show similar changes to human retinal diseases. These, together with improved viral vector quality allow more and more traditionally incurable inherited retinal disorders to become potential candidates for gene therapy. Currently, the most common vehicle to deliver the therapeutic gene into target retinal cells is the adenoassociated viral vector (AAV). Following delivery to the immuno-privileged subretinal space, AAV-vectors can efficiently target both retinal pigment epithelium and photoreceptor cells, the origin of most retinal degenerations. This review focuses on the AAV-based gene therapy in mouse models of recessive retinal degenerations, especially those in which delivery of the correct copy of the wild-type gene has led to significant beneficial effects on visual function, as determined by morphological, biochemical, electroretinographic and behavioral analysis. The past studies in animal models and ongoing successful LCA2 clinical trials, predict a bright future for AAV gene replacement treatment for inherited recessive retinal diseases.     

Conserved genetic pathways associated with microphthalmia,anophthalmia, and coloboma

The human eye is a complex organ whose development requires extraordinary coordination of developmental processes. The conservation of ocular developmental steps in vertebrates suggests possible common genetic mechanisms. Genetic diseases involving the eye represent a leading cause of blindness in children and adults. During the last decades, there has been an exponential increase in genetic studies of ocular disorders. In this review, we summarize current success in identification of genes responsible for microphthalmia, anophthalmia, and coloboma (MAC) phenotypes, which are associated with early defects in embryonic eye development. Studies in animal models for the orthologous genes identified overlapping phenotypes for most factors, confirming the conservation of their function in vertebrate development. These animal models allow for further investigation of the mechanisms of MAC, integration of various identified genes into common developmental pathways and finally, provide an avenue for the development and testing of therapeutic interventions. Birth Defects Research (Part C) 105:96–113, 2015. © 2015 Wiley Periodicals, Inc.     

Molecular genetics of retinitis pigmentosa.

Retinitis pigmentosa is a model for the study of genetic diseases. Its genetic heterogeneity is reflected in the different forms of inheritance (autosomal dominant, autosomal recessive, or X-linked) and, in a few families, in the presence of mutations in the visual pigment rhodopsin. Clinical and molecular genetic studies of these disorders are discussed. Animal models of retinal degeneration have been investigated for many years with the hope of gaining insight into the cause of photoreceptor cell death. Recently, the genes responsible for two of these animal disorders, the rds and rd mouse genes, have been isolated and characterized. The retinal degeneration of the rd mouse is presented in detail. The possible involvement of human analogues of these mouse genes in human retinal diseases is being investigated.     

The role of mislocalized phototransduction in photoreceptor cell death of retinitis pigmentosa

Most of inherited retinal diseases such as retinitis pigmentosa (RP) cause photoreceptor cell death resulting in blindness. RP is a large family of diseases in which the photoreceptor cell death can be caused by a number of pathways. Among them, light exposure has been reported to induce photoreceptor cell death. However, the detailed mechanism by which photoreceptor cell death is caused by light exposure is unclear. In this study, we have shown that even a mild light exposure can induce ectopic phototransduction and result in the acceleration of rod photoreceptor cell death in some vertebrate models. In ovl, a zebrafish model of outer segment deficiency, photoreceptor cell death is associated with light exposure. The ovl larvae show ectopic accumulation of rhodopsin and knockdown of ectopic rhodopsin and transducin rescue rod photoreceptor cell death. However, knockdown of phosphodiesterase, the enzyme that mediates the next step of phototransduction, does not. So, ectopic phototransduction activated by light exposure, which leads to rod photoreceptor cell death, is through the action of transducin. Furthermore, we have demonstrated that forced activation of adenylyl cyclase in the inner segment leads to rod photoreceptor cell death. For further confirmation, we have also generated a transgenic fish which possesses a human rhodopsin mutation, Q344X. This fish and rd10 model mice show photoreceptor cell death caused by adenylyl cyclase. In short, our study indicates that in some RP, adenylyl cyclase is involved in photoreceptor cell death pathway; its inhibition is potentially a logical approach for a novel RP therapy.     

The potential therapeutic of siRNA eye drops in ocular diseases

In recent years, researchers have expressed an ongoing interest in developing RNA interference (RNAi) technology for therapeutic gene suppression in various diseases. Preclinical studies in animal models and cultured cell studies indicated that RNAi technology was an effective experimental tool against a variety of ocular diseases, and some small interference RNA (siRNA) drugs have been entered into clinical trials in Stage I and Stage II. However, in these studies siRNAs were delivered into ocular tissues via either systemic or subconjunctival/intravitreous injection, which is invasive and harmful if repeated. Based on this evidence, we hypothesize that topical application of siRNA eye drops may be a safe and effective therapeutic option in ocular surface diseases with temporary changes of gene expression. Furthermore, siRNA eye drops targeting different genes may simultaneously treat several ocular surface diseases.     

Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors

Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six- to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.     

Expression of Drosophila rhodopsins during photoreceptor cell differentiation: insights into R7 and R8 cell subtype commitment

The R7 and R8 photoreceptor cells of the Drosophila retina are thought to mediate color discrimination and polarized light detection. This is based on the patterned expression of different visual pigments, rhodopsins, in different photoreceptor cells. In this report, we examined the developmental timing of retinal patterning. There is genetic evidence that over the majority of the eye, patterned expression of opsin genes is regulated by a signal from one subtype of R7 cells to adjacent R8 cells. We examined the onset of expression of the rhodopsin genes to determine the latest time point by which photoreceptor subtype commitment must have occurred. We found that the onset of rhodopsin expression in all photoreceptors of the compound eye occurs during a narrow window from 79% to 84% of pupal development (approximately 8 h), pupal stages P12-P14. Rhodopsin 1 has the earliest onset, followed by Rhodopsins 3, 4, and 5 at approximately the same time, and finally Rhodopsin 6. This sequence mimics the model for how R7 and R8 photoreceptor cells are specified, and defines the timing of photoreceptor cell fate decisions with respect to other events in eye development.     

Genetic and phenotypic variations of inherited retinal diseases in dogs: the power of within- and across-breed studies

Considerable clinical and molecular variations have been known in retinal blinding diseases in man and also in dogs. Different forms of retinal diseases occur in specific breed(s) caused by mutations segregating within each isolated breeding population. While molecular studies to find genes and mutations underlying retinal diseases in dogs have benefited largely from the phenotypic and genetic uniformity within a breed, within- and across-breed variations have often played a key role in elucidating the molecular basis. The increasing knowledge of phenotypic, allelic, and genetic heterogeneities in canine retinal degeneration has shown that the overall picture is rather more complicated than initially thought. Over the past 20?years, various approaches have been developed and tested to search for genes and mutations underlying genetic traits in dogs, depending on the availability of genetic tools and sample resources. Candidate gene, linkage analysis, and genome-wide association studies have so far identified 24 mutations in 18 genes underlying retinal diseases in at least 58 dog breeds. Many of these genes have been associated with retinal diseases in humans, thus providing opportunities to study the role in pathogenesis and in normal vision. Application in therapeutic interventions such as gene therapy has proven successful initially in a naturally occurring dog model followed by trials in human patients. Other genes whose human homologs have not been associated with retinal diseases are potential candidates to explain equivalent human diseases and contribute to the understanding of their function in vision.     

Photoreceptor morphogenesis and retinal degeneration: lessons from Drosophila

Cells exhibit an amazingly wide range of different forms, and in most cases the shape of a cell is crucial for performing its specific function(s). But how does a cell obtain its particular shape during development, how can the shape be adapted to different environmental conditions, and what are the consequences if morphogenesis is impaired? An ideal cell type to study these questions is the photoreceptor cell, a photosensitive cell present in most metazoa, highly specialised to transform the energy from the light into a visual response. In the last few years, studies in the Drosophila eye have led to a considerable increase in understanding of the genetic control of photoreceptor morphogenesis; lessons, which may apply to other cell types as well. Most of the genes involved have been conserved during evolution, and mutations in several of them result in retinal degeneration, both in flies and humans. This makes the fly eye an attractive model to unravel the genetic, molecular and cell biological basis of the mechanisms that prevent retinal dystrophies.     

Gene expression-targeted isoflavone therapy

Lysosomal storage diseases (LSD) form a group of inherited metabolic disorders caused by dysfunction of one of the lysosomal proteins, resulting in the accumulation of certain compounds. Although these disorders are among first genetic diseases for which specific treatments were proposed, there are still serious unsolved problems that require development of novel therapeutic procedures. An example is neuronopathy, which develops in most of LSD and cannot be treated efficiently by currently approved therapies. Recently, a new potential therapy, called gene expression-targeted isoflavone therapy (GET IT), has been proposed for a group of LSD named mucopolysaccharidoses (MPS), in which storage of incompletely degraded glycosaminoglycans (GAGs) results in severe symptoms of virtually all tissues and organs, including central nervous system. The idea of this therapy is to inhibit synthesis of GAGs by modulating expression of genes coding for enzymes involved in synthesis of these compounds. Such a modulation is possible by using isoflavones, particularly genistein, which interfere with a signal transduction process necessary for stimulation of expression of certain genes. Results of in vitro experiments and studies on animal models indicated a high efficiency of GET IT, including correction of behavior of affected mice. However, clinical trials, performed with soy isoflavone extracts, revealed only limited efficacy. This caused a controversy about GET IT as a potential, effective treatment of patients suffering from MPS, especially neuronopathic forms of these diseases. It this critical review, I present possible molecular mechanisms of therapeutic action of isoflavones (particularly genistein) and suggest that efficacy of GET IT might be sufficiently high when using relatively high doses of synthetic genistein (which was employed in experiments on cell cultures and mouse models) rather than low doses of soy isoflavone extracts (which were used in clinical trials). This proposal can be tested in double-blinded, placebo-controlled clinical trials.