Effect of Purified Murine NGF on Isolated Photoreceptors of a Rodent Developing Retinitis Pigmentosa

A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies.     

Retinal Ganglion Cells are Resistant to Photoreceptor Loss in Retinal Degeneration

The rapid and massive degeneration of photoreceptors in retinal degeneration might have a dramatic negative effect on retinal circuits downstream of photoreceptors. However, the impact of photoreceptor loss on the morphology and function of retinal ganglion cells (RGCs) is not fully understood, precluding the rational design of therapeutic interventions that can reverse the progressive loss of retinal function. The present study investigated the morphological changes in several identified RGCs in the retinal degeneration rd1 mouse model of retinitis pigmentosa (RP), using a combination of viral transfection, microinjection of neurobiotin and confocal microscopy. Individual RGCs were visualized with a high degree of detail using an adeno-associated virus (AAV) vector carrying the gene for enhanced green fluorescent protein (EGFP), allowed for large-scale surveys of the morphology of RGCs over a wide age range. Interestingly, we found that the RGCs of nine different types we encountered were especially resistant to photoreceptor degeneration, and retained their fine dendritic geometry well beyond the complete death of photoreceptors. In addition, the RGC-specific markers revealed a remarkable degree of stability in both morphology and numbers of two identified types of RGCs for up to 18 months of age. Collectively, our data suggest that ganglion cells, the only output cells of the retina, are well preserved morphologically, indicating the ganglion cell population might be an attractive target for treating vision loss.     

Pluripotent human stem cells for the treatment of retinal disease

Despite advancements made in our understanding of ocular biology, therapeutic options for many debilitating retinal diseases remain limited. Stem cell-based therapies are a potential avenue for treatment of retinal disease, and this mini-review will focus on current research in this area. Cellular therapies to replace retinal pigmented epithelium (RPE) and/or photoreceptors to treat age-related macular degeneration (AMD), Stargardt's macular dystrophy, and retinitis pigmentosa are currently being developed. Over the past decade, significant advancements have been made using different types of human stem cells with varying capacities to differentiate into these target retinal cell types. We review and evaluate pluripotent stem cells, both human embryonic stem cells and human induced pluripotent stem cells, as well as protocols for differentiation of ocular cells, and culture and transplant techniques that might be used to deliver cells to patients.     

Differential Akt activation in the photoreceptors of normal and rd1 mice

Retinitis pigmentosa is a blinding disease in which unknown mechanisms cause the degeneration of retinal photoreceptors. The retinal degeneration (rd1) mouse is a relevant model for this condition, since it carries a mutation also found in some forms of retinitis pigmentosa. To understand the degenerative process in the rd1 mouse, we must identify the survival and apoptosis-related signaling pathways in its photoreceptors and determine whether signaling differs from that in normal mice. The phosphatidylinositol 3-kinase/Akt kinase pathway promotes survival in several different cell types. The purpose of the present study has been to compare Akt activity in retinal cells of normal and rd1 mice. We have found that, in normal mice, Akt becomes activated in the retina in a developmentally regulated and cell-type-specific fashion, encompassing essentially all retinal cells. In most cell types, once Akt activation has begun, it remains in this state throughout life. An exception is seen in the rod photoreceptors, in which Akt is activated only transiently during their development. The rd1 retina behaves identically in all but one respect, namely that the activation of Akt in rod photoreceptors persists until these cells undergo apoptosis. Thus, Akt may participate in constitutive survival processes in retinal neurons, except in rod photoreceptors in which the role of this pathway may be restricted to the developmental period. However, Akt activation in the rods may be part of a defense mechanism initiated in response to insults, such as the retinal degeneration seen in the rd1 mouse.This work was supported by grants from the Foundation for Fighting Blindness, Stiftelsen för synskadade i fd Malmöhus län, 2nd ONCE International Award for New Technologies for the Blind, PRO-AGE-RET: QLK6-CT-2001-00385, PRO-RET: QLK6-CT-2000-00569, KMA, the Crafoord Foundation, and the Segerfalk Foundation.     

Scanning electron microscopy observations of nasal mucosa in patients affected by retinitis pigmentosa.

A controlled study of the ciliated epithelium of olfactory mucosa was performed on 25 patients affected by retinitis pigmentosa and in 5 healthy subjects. Scanning Electron Microscopy showed a significant lack of ciliated epithelial cells. These cells probably represent undifferentiated ciliated cells. These morphological alterations of the olfactory mucosa might correspond to similar changes in the outer segment of retinal photoreceptors in retinitis pigmentosa.     

Reactive oxygen species as mediators of photoreceptor apoptosis in vitro

Retinitis pigmentosa is a heterogeneous group of retinal degenerations characterized by a progressive loss of photoreceptors through the process of apoptosis. The apoptotic cell death of photoreceptors appears to represent a final common pathway in the pathology of retinitis pigmentosa. Previous studies have reported the ability of antioxidants to ameliorate light-induced retinal degeneration, suggesting a role for oxidative stress in photoreceptor cell death. This study demonstrates an early and sustained increase in intracellular reactive oxygen species accompanied by a rapid depletion of intracellular glutathione in an in vitro model of photoreceptor apoptosis. These early changes in the cellular redox state precede disruption of mitochondrial transmembrane potential, nuclear condensation, DNA nicking, and cell shrinkage, all of which are well-characterized events of apoptotic cell death. The ability of zinc chloride and pyrrolidine dithiocarbamate, two established antioxidants, to inhibit photoreceptor apoptosis through the scavenging of intracellular reactive oxygen species establishes a role for reactive oxygen species as possible mediators of in vitro photoreceptor apoptosis. This study provides a molecular basis for the inhibition of photoreceptor apoptosis by antioxidants.     

Therapeutic challenges to retinitis pigmentosa: from neuroprotection to gene therapy

Syndromic retinitis pigmentosa (RP) is the result of several mutations expressed in rod photoreceptors, over 40 of which have so far been identified. Enormous efforts are being made to relate the advances in unraveling the patho-physiological mechanisms to therapeutic approaches in animal models, and eventually in clinical trials on humans. This review summarizes briefly the current clinical management of RP and focuses on the new exciting treatment possibilities. To date, there is no approved therapy able to stop the evolution of RP or restore vision. The current management includes an attempt at slowing down the degenerative process by vitamin supplementation, trying to treat ocular complications and to provide psychological support to blind patients. Novel therapeutic may be tailored dependant on the stage of the disease and can be divided in three groups. In the early stages, when there are surviving photoreceptors, the first approach would be to try to halt the degeneration by correction of the underlying biochemical abnormality in the visual cycle using gene therapy or pharmacological treatment. A second approach aims to cope with photoreceptor cell death using neurotrophic growth factors or anti-apoptotic factors, reducing the production of retino-toxic molecules, and limiting oxidative damage. In advanced stages, when there are few or no functional photoreceptors, strategies that may benefit include retinal transplantation, electronic retinal implants or a newly described optogenetic technique using a light-activated channel to genetically resensitize remnant cone-photoreceptor cells.     

Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa

Protein conformational disorders, which include certain types of retinitis pigmentosa, are a set of inherited human diseases in which mutant proteins are misfolded and often aggregated. Many opsin mutants associated with retinitis pigmentosa, the most common being P23H, are misfolded and retained within the cell. Here, we describe a pharmacological chaperone, 11-cis-7-ring retinal, that quantitatively induces the in vivo folding of P23H-opsin. The rescued protein forms pigment, acquires mature glycosylation, and is transported to the cell surface. Additionally, we determined the temperature stability of the rescued protein as well as the reactivity of the retinal-opsin Schiff base to hydroxylamine. Our study unveils novel properties of P23H-opsin and its interaction with the chromophore. These properties suggest that 11-cis-7-ring retinal may be a useful therapeutic agent for the rescue of P23H-opsin and the prevention of retinal degeneration.     

ER stress in retinal degeneration: a target for rational therapy?

Mutations that cause rhodopsin misfolding and retention within the endoplasmic reticulum (ER) are a prominent cause of retinitis pigmentosa. Here, we discuss the hypothesis that the failure of photoreceptor neurons to adapt to the stress caused by rhodopsin accumulation in the ER leads to a global collapse of homeostasis and to retinal degeneration. We review the molecular mechanisms underlying the activity of local ER conformational sensors and stress-relaying modules and consider how ER-derived stress signals are amplified and implemented to impact on downstream processes, including rhodopsin clearance and cell fate control. The emerging view is that alterations to the systems responsible for the detection, transduction and implementation of ER stress might be used therapeutically to treat retinitis pigmentosa.     

A diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of retinitis pigmentosa

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell‐autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild‐type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild‐type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival‐promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival‐promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival‐promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 475–490, 1999     

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.     

Detection of Retinitis Pigmentosa by Differential Interference Contrast Microscopy

Differential interference contrast microscopy is designed to image unstained and transparent specimens by enhancing the contrast resulting from the Nomarski prism-effected optical path difference. Retinitis pigmentosa, one of the most common inherited retinal diseases, is characterized by progressive loss of photoreceptors. In this study, Differential interference contrast microscopy was evaluated as a new and simple application for observation of the retinal photoreceptor layer and retinitis pigmentosa diagnostics and monitoring. Retinal tissues of Royal College of Surgeons rats and retinal-degeneration mice, both well-established animal models for the disease, were prepared as flatmounts and histological sections representing different elapsed times since the occurrence of the disease. Under the microscopy, the retinal flatmounts showed that the mosaic pattern of the photoreceptor layer was irregular and partly collapsed at the early stage of retinitis pigmentosa, and, by the advanced stage, amorphous. The histological sections, similarly, showed thinning of the photoreceptor layer at the early stage and loss of the outer nuclear layer by the advanced stage. To count and compare the number of photoreceptors in the normal and early-retinitis pigmentosa-stage tissues, an automated cell-counting program designed with MATLAB, a numerical computing language, using a morphological reconstruction method, was applied to the differential interference contrast microscopic images. The number of cells significantly decreased, on average, from 282 to 143 cells for the Royal College of Surgeons rats and from 255 to 170 for the retinal-degeneration mouse. We successfully demonstrated the potential of the differential interference contrast microscopy technique’s application to the diagnosis and monitoring of RP.     

Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa

Synaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. We present evidence linking impaired insulin receptor signaling with RP. We describe a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell terminals in the rd10 mouse model of RP, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin, the insulin precursor, production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function in electroretinogram and optomotor tests. These findings point to a disease-modifying role of insulin receptor and support the therapeutic potential of proinsulin in retinitis pigmentosa.Subject terms: Neurodegeneration, Diseases     

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.     

Characteristics of Rod Regeneration in a Novel Zebrafish Retinal Degeneration Model Using N-Methyl-N-Nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.     

The role of the photoreceptor ABC transporter ABCA4 in lipid transport and Stargardt macular degeneration

ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone–rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of abca4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidene-phosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders.     

Retinal prostheses: Clinical results and future challenges

Retinal prostheses aim at restoring visual perception in blind patients affected by retinal diseases leading to the loss of photoreceptors, such as age-related macular degeneration or retinitis pigmentosa. Recent clinical trials have demonstrated the feasibility of this approach for restoring useful vision. Despite a limited number of electrodes (60), and therefore of pixels, some patients were able to read words and to recognize high-contrast objects. Face recognition and independent locomotion in unknown urban environments imply technological breakthroughs to increase the number and density of electrodes. This review presents recent clinical results and discusses future solutions to answer the major technological challenges.     

Peripherin/rds influences membrane vesicle morphology. Implications for retinopathies

Peripherin/rds is an integral membrane glycoprotein found in the rim regions of vertebrate photoreceptor cell discs. Natural mutations of the encoding gene result in degenerative retinal disorders, such as retinitis pigmentosa. The retinal degeneration slow (rds) phenotype, observed in mice, is considered to be an appropriate model for peripherin/rds-mediated retinitis pigmentosa. Associated abnormalities in the outer segment of photoreceptor cells have implicated peripherin/rds in some aspect of disc morphology, yet it remains unclear whether such morphological effects are the cause or the result of this condition. Here we present the first direct evidence to support a role for peripherin/rds in maintaining the flattened vesicle morphology characteristic of photoreceptor outer segments. In vitro expression yields a 36-kDa immunoreactive species, which is inserted into membranes and undergoes N-glycosylation, inter- and intramolecular disulfide bonding, and dimerization. Electron microscopy reveals that peripherin/rds flattens microsomal vesicles. This effect appears to be dependent on disulfide bond formation but not N-glycosylation. The inability of two pathogenic peripherin/rds mutants (P216L and C165Y) to flatten membrane vesicles implicates such mutations as the primary cause of the retinal degeneration observed in retinitis pigmentosa.