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.     

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.     

Pathways to photoreceptor cell death in inherited retinal degenerations.

The mutations that cause many forms of inherited retinal degenerations have been identified, yet the mechanisms by which these mutations lead to death of photoreceptor cells of the retina are not completely understood. Investigations of the pathways from mutation to retinal degeneration have focused on spontaneous and engineered animal models of disease. Based on the studies performed to date, four major categories of degeneration mechanism can be identified. These include disruption of photoreceptor outer segment morphogenesis, metabolic overload, dysfunction of retinal pigment epithelial cells, and chronic activation of phototransduction. Future investigations will likely identify additional mechanisms of photoreceptor damage. This review will summarize what has been learned from studying animal models of non-syndromic inherited retinal degenerations.     

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     

Inherited retinal degeneration: exceptional genetic and clinical heterogeneity

The function of the retina is to detect light and to send appropriate signals to the brain in response. Inherited diseases that cause the retina to degenerate, leading to either partial or total blindness, affect approximately 1 in 3000 people. Rapid progress is being made in identifying the genetic causes of common, inherited retinal diseases, such as retinitis pigmentosa and macular degeneration, as well as some of the rare forms of retinal disease. Linkage studies of large families and candidate-gene screening of known retinal genes have already identified 59 independent genetic loci that can cause retinal degeneration. The astounding genetic and clinical heterogeneity that is being revealed is a ‘nightmare’ for those interested in molecular diagnostics but, at the same time, provides great insight into functional aspects of the normal retina.     

视网膜色素变性的分子遗传学研究进展

视网膜色素变性（RP）是常见的遗传性眼病,具有高度的遗传异质性,患者常有进行性夜盲和视野缺损。对于常染色体显性遗传RP已经发现有12个基因座与之有关,其中6个致病基因已被克隆,对于这些致病基因的结构、突变及其功能目前已有了新的研究进展。 Abstract:Retinitis pigmentosa (RP) describes a genetically and clinically heterogeneous group of disorders that are characterized by gradual degeneration of photoreceptor cells.Common clinical features include a progressive loss of night vision,leading to night blindness and peripheral-visual-field loss.At least 12 loci have been mapped to chromosomes,and mutations in an ever increasing number of genes have been found to cause autosomal dominant retinitis pigmentosa (ADRP).Six of the 12 genes known to cause ADRP have been cloned.New progress has been made on the studies of structure,mutation and function of these genes.     

Organoid technology for retinal repair

A major cause for vision impairment and blindness in industrialized countries is the loss of the light-sensing retinal tissue in the eye. Photoreceptor damage is one of the main characteristics found in retinal degeneration diseases, such as Retinitis Pigmentosa or age-related macular degeneration. The lack of effective therapies to stop photoreceptor loss together with the absence of significant intrinsic regeneration in the human retina converts such degenerative diseases into permanent conditions that are currently irreversible. Cell replacement by means of photoreceptor transplantation has been proposed as a potential approach to tackle cell loss in the retina. Since the first attempt of photoreceptor transplantation in humans, about twenty years ago, several research groups have focused in the development and improvement of technologies necessary to bring cell transplantation for retinal degeneration diseases to reality. Progress in recent years in the generation of human tissue derived from pluripotent stem cells (PSCs) has significantly improved our tools to study human development and disease in the dish. Particularly the availability of 3D culture systems for the generation of PSC-derived organoids, including the human retina, has dramatically increased access to human material for basic and medical research. In this review, we focus on important milestones towards the generation of transplantable photoreceptor precursors from PSC-derived retinal organoids and discuss recent pre-clinical transplantation studies using organoid-derived photoreceptors in context to related in vivo work using primary photoreceptors as donor material. Additionally, we summarize remaining challenges for developing photoreceptor transplantation towards clinical application.     

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.     

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.     

Lysophosphatidylcholine acyltransferase 1 controls mitochondrial reactive oxygen species generation and survival of retinal photoreceptor cells

Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration–related retinal diseases.     

ATP-binding cassette transporter ABCA4: Molecular properties and role in vision and macular degeneration

ABCA4, also known as ABCR or the rim protein, is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters expressed in vertebrate rod and cone photoreceptor cells and localized to outer segment disk membranes. ABCA4 is organized in two tandem halves, each consisting of a transmembrane segment followed successively by a large exocytoplasmic domain, a multispanning membrane domain, and a nucleotide-binding domain. Over 400 mutations in ABCA4 have been linked to Stargardt macular degeneration and related retinal degenerative diseases that cause severe vision loss in affected individuals. Direct binding studies and ATPase activation measurements have identified N-retinylidene-phosphatidylethanolamine, a product generated from the photobleaching of rhodopsin, as the substrate for ABCA4. Mice deficient in ABCA4 accumulate phosphatidylethanolamine, all-trans retinal, and N-retinylidene-phosphatidylethanolamine in photoreceptors and the diretinal pyridinium compound A2E in retinal pigment epithelial cells. On the basis of these studies, ABCA4 is proposed to actively transport or flip N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic side of disc membranes following the photobleaching of rhodopsin. This transport activity insures that retinoids do not accumulate in disc membranes. Disease-linked mutations in ABCA4 that result in diminished transport activity lead to an accumulation of all-trans retinal and N-retinylidene-PE in disc membranes which react to produce A2E precursors. A2E progressively accumulates as lipofuscin deposits in retinal pigment epithelial cells as a result of phagocytosis of outer segment discs. A2E and photo-oxidation products cause RPE cell death and consequently photoreceptor degeneration resulting in a loss in vision in individuals with Stargardt macular degeneration and other retinal degenerative diseases associated with mutations in ABCA4.     

Human adult retinal pigment epithelial cells as potential cell source for retina recovery

The retinal pigment epithelium (RPE), as well as the neural retina, develops from the neuroectoderm and plays a key role in photoreceptor functions. Several degenerative eye diseases, e.g., macular degeneration or retinitis pigmentosa, associated with an impaired RPE function cause the loss of the photoreceptor and partial or complete blindness. Cultured RPE cells obtained from human cadaver eyes could be a valuable source for transplantation to cure retinal degenerative diseases. The paper describes RPE cell isolation, maintenance in culture, and immunohistochemical characteristics of dedifferentiated cells. It was found that RPE cells from human adults exhibit neural cell properties in vitro.     

The Wnt signaling pathway in retinal degenerations

The retina is a complex tissue composed of multiple interconnected cell layers, highly specialized for transforming light and color into electrical signals perceived by the brain. Damage or death of the primary light-sensing cells, the photoreceptors, results in devastating effects on vision. Despite the identification of numerous mutations that cause inherited retinal degenerations, the cellular and molecular mechanisms leading from the primary mutations to photoreceptor apoptosis are not understood. Wnt signaling has essential regulatory functions in a wide variety of critical developmental processes. Our research and others' have suggested that the Wnt pathway may be involved in retinal degeneration. Wnt ligands regulate developmental death of Drosophila photoreceptors, dysregulated Wnt signaling is involved in neuronal degeneration elsewhere in the central nervous system and Wnts control the expression of pro-survival growth factors in mammalian tissues. Additionally, altered expression of Wnt pathway genes, including the anti-apoptotic Wnt signaling regulator Dickkopf 3 (Dkk3), were observed during photoreceptor loss. This review examines the evidence and develops a model proposing a pro-survival role for Wnt signaling during photoreceptor injury. Because manipulating Wnt signaling has been demonstrated to have therapeutic potential for the treatment of Alzheimers disease, understanding the involvement of Wnts in photoreceptor death will determine whether targeting the Wnt pathway should also be considered as a possible therapeutic strategy for retinal degenerations.     

Otx2 enhances transdifferentiation of Müller cells‐derived retinal stem cells into photoreceptor‐like cells

Retinal Müller glial cells have the potential of neurogenic retinal progenitor cells, and could reprogram into retinal‐specific cell types such as photoreceptor cells. How to promote the differentiation of Müller cells into photoreceptor cells represents a promising therapy strategy for retinal degeneration diseases. This study aimed to enhance the transdifferentiation of rat Müller cells‐derived retinal stem cells (MC‐RSCs) into photoreceptor‐like cells and explore the signalling mechanism. We dedifferentiated rat Müller cells into MC‐RSCs which were infected with Otx2 overexpression lentivirus or control. The positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus was significantly higher compared to control. Furthermore, pre‐treatment with Crx siRNA, Nrl siRNA, or GSK‐3 inhibitor SB‐216763 reduced the positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus. Finally, Otx2 induced photoreceptor precursor cells were injected into subretinal space of N‐methyl‐N‐nitrosourea induced rat model of retinal degeneration and partially recovered retinal degeneration in the rats. In conclusion, Otx2 enhances transdifferentiation of MC‐RSCs into photoreceptor‐like cells and this is associated with the inhibition of Wnt signalling. Otx2 is a potential target for gene therapy of retinal degenerative diseases.     

The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. Ultrastructure expansion microscopy on mouse retina reveals the presence of a novel structure inside the photoreceptor connecting cilium, the inner scaffold, that protects the outer segment against degeneration.     

Genetic Expression of Cyclic GMP Phosphodiesterase Activity Defines Abnormal Photoreceptor Differentiation in Neurological Mutants of Inherited Retinal Degeneration

We have examined cyclic GMP concentrations, guanylate cyclase activities, and cyclic GMP phosphodiesterase (PDE) activities in developing retinas of congenic mice with different allelic combinations at the retinal degeneration (rd) and retinal degeneration slow (rds) loci. Although guanylate cyclase activities were found to be uniformly low in the mutant retinas, striking differences in PDE activity and cyclic GMP levels were observed in retinas of the various genotypes. Homozygous rds mice, which lack receptor outer segments, showed reduced retinal PDE activity and cyclic GMP concentration in comparison to normal animals. In heterozygous rds/+ mice with abnormal outer segments, the levels were intermediate. In retinas of homozygous rd mice, PDE activity was lower than in rds retinas and cyclic GMP levels were much higher. In mice homozygous for both rd and rds genes, retinal PDE activities were even lower than in single homozygous rd mice; the cyclic GMP level reached the same high value as in the rd animals, persisted for a longer time at this high level, and did not correlate with the rate of photoreceptor cell loss. Thus, a marked variation in PDE activity appears to be the major manifestation of abnormal outer segment differentiation and eventual degeneration of photoreceptor cells in these neurological mutants. An increased cyclic GMP level seems to be an essential corollary in the expression of the rd gene even in the absence of outer segments, but it appears unlikely that an abnormally high nucleotide level in itself causes photoreceptor cell death.     

Inhibiting autophagy reduces retinal degeneration caused by protein misfolding

Mutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHO^P23H variant (hereafter called P23H) exhibited elevated levels of autophagy flux, and that pharmacological stimulation of autophagy accelerated retinal degeneration. In contrast, reducing autophagy flux pharmacologically or by rod-specific deletion of the autophagy-activating gene Atg5, improved photoreceptor structure and function. Furthermore, proteasome levels and activity were reduced in the P23H retina, and increased when Atg5 was deleted. Our findings suggest that autophagy contributes to photoreceptor cell death in P23H mice, and that decreasing autophagy shifts the degradation of misfolded RHO protein to the proteasome and is protective. These observations suggest that modulating the flux of misfolded proteins from autophagy to the proteasome may represent an important therapeutic strategy for reducing proteotoxicity in adRP and other diseases caused by protein folding defects.     

Unfolding retinal dystrophies: a role for molecular chaperones?

Inherited retinal dystrophy is a major cause of blindness worldwide. Recent molecular studies have suggested that protein folding and molecular chaperones might play a major role in the pathogenesis of these degenerations. Incorrect protein folding could be a common consequence of causative mutations in retinal degeneration disease genes, particularly mutations in the visual pigment rhodopsin. Furthermore, several retinal degeneration disease genes have recently been identified as putative facilitators of correct protein folding, molecular chaperones, on the basis of sequence homology. We also consider whether manipulation of chaperone levels or chaperone function might offer potential novel therapies for retinal degeneration.