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.     

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.     

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.     

Localization of retina/pineal-expressed sequences: identification of novel candidate genes for inherited retinal disorders.

More than 100 genes causing inherited retinal diseases have been mapped to chromosomal locations, but less than half of these genes have been cloned. Mutations in many retina/pineal-specific genes are known to cause inherited retinal diseases. Examples include mutations in arrestin, rhodopsin kinase, and the cone-rod homeobox gene, CRX. To identify additional candidate genes for inherited retinal disorders, novel retina/pineal-expressed EST clusters were identified from the TIGR Human Gene Index database and mapped to specific chromosomal sites. After known human gene sequences were excluded, and repeat sequences were masked, 26 novel retina and pineal gland cDNA clusters were identified. The retinal expression of each novel EST cluster was confirmed by PCR assay of a retinal cDNA library, and each cluster was localized in the genome using the GeneBridge 4.0 radiation hybrid panel. In silico expression data from the TIGR database suggest that these EST clusters are retina/pineal-specific or predominantly expressed in these tissues. This combination of database analysis and laboratory investigation has localized several EST clusters that are potential candidates for genes causing inherited retinopathy.     

Cytokine interplay among the diseased retina,inflammatory cells and mesenchymal stem cells - a clue to stem cell-based therapy

Retinal degenerative disorders, such as diabetic retinopathy, retinitis pigmentosa, age-related macular degeneration or glaucoma, represent the most common causes of loss of vision and blindness. In spite of intensive research, treatment options to prevent, stop or cure these diseases are limited. Newer therapeutic approaches are offered by stem cell-based therapy. To date, various types of stem cells have been evaluated in a range of models. Among them, mesenchymal stem/stromal cells (MSCs) derived from bone marrow or adipose tissue and used as autologous cells have been proposed to have the potential to attenuate the negative manifestations of retinal diseases. MSCs delivered to the vicinity of the diseased retina can exert local anti-inflammatory and repair-promoting/regenerative effects on retinal cells. However, MSCs also produce numerous factors that could have negative impacts on retinal regeneration. The secretory activity of MSCs is strongly influenced by the cytokine environment. Therefore, the interactions among the molecules produced by the diseased retina, cytokines secreted by inflammatory cells and factors produced by MSCs will decide the development and propagation of retinal diseases. Here we discuss the interactions among cytokines and other factors in the environment of the diseased retina treated by MSCs, and we present results supporting immunoregulatory and trophic roles of molecules secreted in the vicinity of the retina during MSC-based therapy.     

Treatment of human disease by adeno-associated viral gene transfer

During the past decade, in vivo administration of viral gene transfer vectors for treatment of numerous human diseases has been brought from bench to bedside in the form of clinical trials, mostly aimed at establishing the safety of the protocol. In preclinical studies in animal models of human disease, adeno-associated viral (AAV) vectors have emerged as a favored gene transfer system for this approach. These vectors are derived from a replication-deficient, non-pathogenic parvovirus with a single-stranded DNA genome. Efficient gene transfer to numerous target cells and tissues has been described. AAV is particularly efficient in transduction of non-dividing cells, and the vector genome persists predominantly in episomal forms. Substantial correction, and in some instances complete cure, of genetic disease has been obtained in animal models of hemophilia, lysosomal storage disorders, retinal diseases, disorders of the central nervous system, and other diseases. Therapeutic expression often lasted for months to years. Treatments of genetic disorders, cancer, and other acquired diseases are summarized in this review. Vector development, results in animals, early clinical experience, as well as potential hurdles and challenges are discussed.     

Gene therapy: prospects for glycolipid storage diseases

Lysosomal storage diseases comprise a group of about 40 disorders, which in most cases are due to the deficiency of a lysosomal enzyme. Since lysosomal enzymes are involved in the degradation of various compounds, the diseases can be further subdivided according to which pathway is affected. Thus, enzyme deficiencies in the degradation pathway of glycosaminoglycans cause mucopolysaccharidosis, and deficiencies affecting glycopeptides cause glycoproteinosis. In glycolipid storage diseases enzymes are deficient that are involved in the degradation of sphingolipids. Mouse models are available for most of these diseases, and some of these mouse models have been used to study the applicability of in vivo gene therapy. We review the rationale for gene therapy in lysosomal disorders and present data, in particular, about trials in an animal model of metachromatic leukodystrophy. The data of these trials are compared with those obtained with animal models of other lysosomal diseases.     

Prospectives for Gene Therapy of Retinal Degenerations

Retinal degenerations encompass a large number of diseases in which the retina and associated retinal pigment epithelial (RPE) cells progressively degenerate leading to severe visual disorders or blindness. Retinal degenerations can be divided into two groups, a group in which the defect has been linked to a specific gene and a second group that has a complex etiology that includes environmental and genetic influences. The first group encompasses a number of relatively rare diseases with the most prevalent being Retinitis pigmentosa that affects approximately 1 million individuals worldwide. Attempts have been made to correct the defective gene by transfecting the appropriate cells with the wild-type gene and while these attempts have been successful in animal models, human gene therapy for these inherited retinal degenerations has only begun recently and the results are promising. To the second group belong glaucoma, age-related macular degeneration (AMD) and diabetic retinopathy (DR). These retinal degenerations have a genetic component since they occur more often in families with affected probands but they are also linked to environmental factors, specifically elevated intraocular pressure, age and high blood sugar levels respectively. The economic and medical impact of these three diseases can be assessed by the number of individuals affected; AMD affects over 30 million, DR over 40 million and glaucoma over 65 million individuals worldwide. The basic defect in these diseases appears to be the relative lack of a neurogenic environment; the neovascularization that often accompanies these diseases has suggested that a decrease in pigment epithelium-derived factor (PEDF), at least in part, may be responsible for the neurodegeneration since PEDF is not only an effective neurogenic and neuroprotective agent but also a potent inhibitor of neovascularization. In the last few years inhibitors of vascularization, especially antibodies against vascular endothelial cell growth factors (VEGF), have been used to prevent the neovascularization that accompanies AMD and DR resulting in the amelioration of vision in a significant number of patients. In animal models it has been shown that transfection of RPE cells with the gene for PEDF and other growth factors can prevent or slow degeneration. A limited number of studies in humans have also shown that transfection of RPE cells in vivo with the gene for PEDF is effective in preventing degeneration and restore vision. Most of these studies have used virally mediated gene delivery with all its accompanying side effects and have not been widely used. New techniques using non-viral protocols that allow efficient delivery and permanent integration of the transgene into the host cell genome offer novel opportunities for effective treatment of retinal degenerations.     

Pathophysiology of stroke and stroke-induced retinal ischemia: emerging role of stem cells

The current review focuses on pathophysiology, animal models and molecular analysis of stroke and retinal ischemia, and the role of stem cells in recovery of these disease conditions. Research findings associated with ischemic stroke and retinal ischemia have been discussed, and efforts towards prevention and limiting the recurrence of ischemic diseases, as well as emerging treatment possibilities with endothelial progenitor cells (EPCs) in ischemic diseases, are presented. Although most neurological diseases are still not completely understood and reliable treatment is lacking, animal models provide a major step in validating novel therapies. Stem cell approaches constitute an emerging form of cell-based therapy to treat ischemic diseases since it is an attractive source for regenerative therapy in the ischemic diseases. In this review, we highlight the advantages and limitations of this approach with a focus on key observations from preclinical animal studies and clinical trials. Further research, especially on treatment with EPCs is warranted.     

Gene therapy and retinitis pigmentosa: advances and future challenges.

It may be possible, one day, to use gene therapy to treat diseases whose genetic defects have been discerned. Because many genes responsible for inherited eye disorders within the retina have been identified, diseases of the eye are prime candidates for this form of therapy. The eye also has the advantage of being highly accessible with altered immunological properties, important considerations for easy delivery of virus and avoidance of systemic immune responses. Currently, adenovirus, adeno-associated virus and lentivirus have been used to successfully transfer genetic material to retinal pigment epithelium and photoreceptor cells. By harnessing therapeutic genes to these viruses, researchers have been able to demonstrate rescue in rodent models of retinitis pigmentosa, providing evidence that this form of therapy can be effective in delaying photoreceptor cell death. Future challenges include confirming therapeutic effects in animal models with eyes more anatomically similar to those of humans and demonstrating long-term rescue with minimal toxicity.     

Dawn of ocular gene therapy: implications for molecular diagnosis in retinal disease

Personalized medicine aims to utilize genomic information about patients to tailor treatment. Gene replacement therapy for rare genetic disorders is perhaps the most extreme form of personalized medicine, in that the patients’ genome wholly determines their treatment regimen. Gene therapy for retinal disorders is poised to become a clinical reality. The eye is an optimal site for gene therapy due to the relative ease of precise vector delivery, immune system isolation, and availability for monitoring of any potential damage or side effects. Due to these advantages, clinical trials for gene therapy of retinal diseases are currently underway. A necessary precursor to such gene therapies is accurate molecular diagnosis of the mutation(s) underlying disease. In this review, we discuss the application of Next Generation Sequencing (NGS) to obtain such a diagnosis and identify disease causing genes, using retinal disorders as a case study. After reviewing ocular gene therapy, we discuss the application of NGS to the identification of novel Mendelian disease genes. We then compare current, array based mutation detection methods against next NGS-based methods in three retinal diseases: Leber’s Congenital Amaurosis, Retinitis Pigmentosa, and Stargardt’s disease. We conclude that next-generation sequencing based diagnosis offers several advantages over array based methods, including a higher rate of successful diagnosis and the ability to more deeply and efficiently assay a broad spectrum of mutations. However, the relative difficulty of interpreting sequence results and the development of standardized, reliable bioinformatic tools remain outstanding concerns. In this review, recent advances NGS based molecular diagnoses are discussed, as well as their implications for the development of personalized medicine.     

Preventing retinal apoptosis — Is there a common therapeutic theme?

There is an urgent need for therapies for retinal diseases; retinitis pigmentosa sufferers have no treatment options available and those targeted at other retinopathies have shown limited effectiveness. The process of programmed cell death or apoptosis although complex, remains a possible target for the treatment of retinal diseases. Having identified apoptosis in the vertebrate retina in populations of immature neurons as an essential part of development it was proposed that re-activation of these developmental cell death pathways might provide insight into the death mechanisms operating in retinal diseases. However, the discovery that numerous factors initiate and mediate the apoptotic cascade in mature photoreceptors has resulted in a relatively untargeted approach to examining and arresting apoptosis in the retina. In the last 5 years, mouse models have been treated with a diverse range of drugs or factors including anti-oxidants, growth factors, steroid hormones, calcium/calpain inhibitors and tetracycline antibiotics. Therefore to draw a unifying theme from these broad research areas is challenging. However, this review focusses on two targets which are currently under investigation, reactive oxygen species and mammalian target of rapamycin, drawing together the common themes of these research areas.     

Expression and signaling of NGF in the healthy and injured retina

This review summarizes the present knowledge concerning the retinal localization of the nerve growth factor (NGF), its precursor proNGF, and the receptors TrkA and p75^NTR in the developing and mature rodent retina. We further discuss the changes in the expression of NGF and the receptors in experimental models of retinal disorders and diseases like inherited retinitis pigmentosa, retinal detachment, glaucoma, and diabetic retinopathy. Since proNGF is now recognized as a bioactive signaling molecule which induces cell death through p75^NTR activation, the role of proNGF in the induction of retinal cell loss under neurodegenerative conditions is also highlighted. In addition, we present the evidences for a potential therapeutic intervention with NGF for the treatment of retinal neurodegenerative diseases. Different strategies have been developed and experimentally tested in mice and rats in order to reduce cell loss and Müller cell gliosis, e.g., increasing the availability of endogenous NGF, administration of exogenous NGF, activation of TrkA, and inhibition of p75^NTR. Here, we discuss the several lines of evidence supporting a protective effect of NGF on retinal cell loss, with specific emphasis on photoreceptor and retinal ganglion cell degeneration. A better understanding of the mechanisms underlying the effects of NGF and proNGF in the modulation of neurodegeneration and gliosis in the retina will help to develop efficient therapeutic strategies for various retinal diseases.     

Protein misfolding and prion diseases.

The prion diseases provide an intriguing connection between protein folding and neurodegenerative disease. In this review, I explore that importance of protein folding and misfolding in the prion diseases. Thermodynamic and kinetic models are examined in an effort to understand infectious, inherited and sporadic forms of these diseases. These concepts can be generalized to gain insight into other disorders of protein aggregation and deposition such as Alzheimer's disease.     

Involvement of FSP1-CoQ10-NADH and GSH-GPx-4 pathways in retinal pigment epithelium ferroptosis

Retinal pigment epithelium (RPE) degeneration plays an important role in a group of retinal disorders such as retinal degeneration (RD) and age-related macular degeneration (AMD). The mechanism of RPE cell death is not yet fully elucidated. Ferroptosis, a novel regulated cell death pathway, participates in cancer and several neurodegenerative diseases. Glutathione peroxidase 4 (GPx-4) and ferroptosis suppressor protein 1 (FSP1) have been proposed to be two main regulators of ferroptosis in these diseases; yet, their roles in RPE degeneration remain elusive. Here, we report that both FSP1-CoQ₁₀-NADH and GSH-GPx-4 pathways inhibit retinal ferroptosis in sodium iodate (SIO)-induced retinal degeneration pathologies in human primary RPE cells (HRPEpiC), ARPE-19 cell line, and mice. GSH-GPx-4 signaling was compromised after a toxic injury caused by SIO, which was aggravated by silencing GPx-4, and ferroptosis inhibitors robustly protected RPE cells from the challenge. Interestingly, while inhibition of FSP1 caused RPE cell death, which was aggravated by SIO exposure, overexpression of FSP1 effectively protected RPE cells from SIO-induced injury, accompanied by a significant down-regulation of CoQ₁₀/NADH and lipid peroxidation. Most importantly, in vivo results showed that Ferrostatin-1 not only remarkably alleviated SIO-induced RPE cell loss, photoreceptor death, and retinal dysfunction but also significantly ameliorated the compromised GSH-GPx-4 and FSP1-CoQ₁₀-NADH signaling in RPE cells isolated from SIO-induced RPE degeneration. These data describe a distinct role for ferroptosis in controlling RPE cell death in vitro and in vivo and may provide a new avenue for identifying treatment targets for RPE degeneration.Subject terms: Apoptosis, Neurodegenerative diseases, Experimental models of disease     

Metformin and retinal diseases in preclinical and clinical studies: Insights and review of literature

Metformin is one of the most prescribed drugs in the world giving potential health benefits beyond that of type 2 diabetes (T2DM). Emerging evidence suggests that it may have protective effects for retinal/posterior segment diseases including diabetic retinopathy (DR), age-related macular degeneration (AMD), inherited retinal degeneration such as retinitis pigmentosa (RP), primary open angle glaucoma (POAG), retinal vein occlusion (RVO), and uveitis. Metformin exerts potent anti-inflammatory, antiangiogenic, and antioxidative effects on the retina in response to pathologic stressors. In this review, we highlight the broad mechanism of action of metformin through key preclinical studies on animal models and cell lines used to simulate human retinal disease. We then explore the sparse but promising retrospective clinical data on metformin’s potential protective role in DR, AMD, POAG, and uveitis. Prospective clinical data is needed to clarify metformin’s role in management of posterior segment disorders. However, given metformin’s proven broad biochemical effects, favorable safety profile, relatively low cost, and promising data to date, it may represent a new therapeutic preventive and strategy for retinal diseases.     

North Carolina macular dystrophy: exclusion map using RFLPs and microsatellites.

The autosomal dominant macular dystrophies are a confusing group of poorly understood diseases. Linkage studies will greatly aid our classification of these disorders and hopefully provide insight into central retinal function and dysfunction such as occurs in age-related macular degeneration. North Carolina macular dystrophy is one such disease that has been amenable to linkage analysis because of the large pedigree size. Seventy-six polymorphic markers have been tested for linkage and exclusion data are presented.     

Ceramide inhibits connective tissue growth factor expression by human retinal pigment epithelial cells

Connective tissue growth factor (CTGF) is known to be involved in retinal fibrotic disorders. We used human retinal pigment epithelial cells (HRPE), which play critical roles in retinal fibrosis, to examine the expression of CTGF and its regulation by ceramide and TGF-β. Real-time PCR analysis showed downregulation of CTGF mRNA by C₂ ceramide and upregulation by TGF-β. C₂ ceramide also inhibited constitutive and TGF-β-enhanced CTGF secretion by HRPE cells. Predominant secretion (>80% of total) of CTGF from the apical side was observed in highly polarized HRPE cells. Fumonosin, an inhibitor of ceramide synthesis, stimulated CTGF secretion while 4HPR, an activator of ceramide synthesis, downregulated CTGF secretion. Based on these results demonstrating ceramide regulation of CTGF secretion by HRPE, we suggest that ceramide may have therapeutic potential for the treatment of retinal fibrotic diseases by inhibiting CTGF production.