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.

Differential roles of ERK1/2 and JNK in retinal development and degeneration.

Programmed cell death is well established as a key factor in the development of the vertebrate nervous system of which the retina is a unique sensory component. However, it is of utmost importance for the survival of post-mitotic tissues such as the retina that the execution of the cell death program is kept under stringent control once development is complete. This is exemplified by the many retinal dystrophies where aberrant apoptosis results in loss of distinct cell layers in the mature retina and often culminates in blindness. In this study, we report that the extracellular signal-regulated kinase (ERK1/2) pathway plays a key role in the regulation of apoptosis during retinal development. We show that as the retina matures, the emphasis shifts towards survival and ERK1/2 is activated resulting in phosphorylation of the potent BH3-only protein Bim(EL) and a dramatic decline in Bim(EL) expression via proteasomal degradation. We find that activation of ERK1/2 also occurs in response to injury in retinal explants. However, this is a transient response and appears to be overcome by Jun N-terminal kinase activation resulting in induction of Bim(EL) mRNA and photoreceptor apoptosis. Our findings provide new insights into the intracellular pathways responsible for regulating apoptosis during neuronal development and degeneration.

Enhancing survival of photoreceptor cells in vivo using the synthetic progestin Norgestrel.

Retinal degenerations such as Retinitis Pigmentosa remain difficult to treat given the diverse array of genes responsible for their aetiology. Rather than concentrate on specific genes, our focus is on identifying therapeutic avenues for the treatment of retinal disease that target general survival mechanisms or pathways. Norgestrel is a synthetic progestin commonly used in hormonal contraception. Here, we report a novel anti-apoptotic role for Norgestrel in diseased mouse retinas in vivo. Dosing with Norgestrel protects photoreceptor cells from undergoing apoptosis in two distinct models of retinal degeneration; the light damage model and the Pde6b(rd10) model. Photoreceptor rescue was assessed by analysis of cell number, structural integrity and function. Improvements in cell survival of up to 70% were achieved in both disease models, indicating that apoptosis had been halted or at least delayed. A speculative mechanism of action for Norgestrel involves activation of survival pathways in the retina. Indeed, Norgestrel increases the expression of basic fibroblast growth factor which is known to both promote cell survival and inhibit apoptosis. In summary, our results demonstrate significant protection of photoreceptor cells which may be attributed to Norgestrel mediated activation of endogenous survival pathways within the retina.

A novel free radical scavenger rescues retinal cells in vivo.

The benzopyran BP (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) is a free radical scavenger that is structurally similar to alpha-tocopherol and has provided neuro-protection in a number of disease models where oxidative stress is a causative factor. A novel derivative of BP with improved lipid solubility, which we have designated BP3, was synthesized and its neuro-protective efficacy subsequently analyzed in three mouse models of retinal disease in vivo. In the acute light damage model, balb/c mice received a single intra-peritoneal injection (200 mg/kg) of BP3 one hour prior to phototoxicity, reducing photoreceptor degeneration for up to 48 h post insult. In the rd10/rd10 mouse, a chronic model of inherited retinal degeneration, systemic dosing with BP3 on alternate days between post-natal day 18 and 25 preserved rod photoreceptor numbers and cone photoreceptor morphology. Finally, NMDA induced toxicity in retinal ganglion cells was diminished for at least 72 h after the initial insult by a single dose of BP3. In each disease model, BP3 alleviated cellular oxidative burden as MDA levels were markedly reduced. These results demonstrate that systemically administered BP3 has potent free radical scavenging capacity in the retina and may represent a single therapeutic strategy applicable across several retinopathies.

Rosiglitazone acts as a neuroprotectant in retinal cells via up-regulation of sestrin-1 and SOD-2.

Rosiglitazone is a member of the thiazolidinedione family of synthetic peroxisome proliferator-activated receptor (PPAR) agonists. It is a selective ligand of the PPARgamma subtype and functions by regulating the transcription of insulin-responsive genes. A screen of FDA-approved compounds identified rosiglitazone as a novel anti-apoptotic agent in retinal cells both in vitro and in vivo, functioning as a neuroprotectant in response to oxidative and calcium stress. We have found that the likely mechanism of action is via increased protein expression of the antioxidant enzymes superoxide dismutase 2 (SOD-2) and sestrin-1, boosting antioxidant defences. Transcription of both genes appears to be mediated by PPARgamma as their up-regulation is reversed by the PPARgamma antagonist GW9662 and proliferator hormone response elements were found in the putative promoter regions of mouse SOD-2 and sestrin-1. However, further investigation revealed that p53 expression was also induced in response to rosiglitazone and chromatin immunoprecipitation assays confirm that it is a bona fide target of PPARgamma. Furthermore, inhibition of p53 partially blocks the observed increase in SOD-2 and sestrin-1 expression indicating that p53 expression is upstream of both antioxidants. We conclude that rosiglitazone may increase cell survival in retinal diseases and potentially other neuronal diseases in which oxidative stress is a key factor.

Bim expression indicates the pathway to retinal cell death in development and degeneration.

Programmed cell death (PCD) during development of the mouse retina involves activation of the mitochondrial pathway. Previous work has shown that the multidomain Bcl-2 family proteins Bax and Bak are fundamentally involved in this process. To induce mitochondrial membrane permeabilization, Bax and Bak require that prosurvival members of the family be inactivated by binding of "BH3-only" members. We showed previously that the BH3-only protein BimEL is highly expressed during postnatal retinal development but decreases dramatically thereafter. The purpose of this study was to investigate a possible role for Bim, in retinal development and degeneration, upstream of Bax and Bak. Bim-/- mice analyzed for defective retinal development exhibit an increase in retinal thickness and a delay in PCD, thereby confirming a role for Bim. We also demonstrate that in response to certain death stimuli, bim+/+ retinal explants upregulate BimEL leading to caspase activation and cell death, whereas bim-/- explants are resistant to apoptosis. Finally, we analyzed Bim expression in the retinal degeneration (rd) mouse, an in vivo model of retinal degeneration. Bim isoforms, which decrease during development, are not reexpressed during retinal degeneration and ultimately photoreceptor cells die by a caspase-independent mechanism. Thus, we conclude that in cases in which BimEL is reexpressed during pathological cell death, developmental cell death pathways are reactivated. However, the absence of BimEL expression correlates with caspase-independent death in the rd model.

Innovative training networks: overview of the Marie Sklodowska-Curie PhD training model.

Doctoral training has changed in recent years with most PhDs now performed in structured programmes operated by university graduate schools. These schools generally superimpose a training framework onto the traditional research project to improve the education experience of the students and to prepare them for their careers. Many graduates progress to the commercial sector, where there is demand for highly skilled employees. The European Union (EU) promotes the development of transnational, training-focused, PhD programmes called Innovative Training Networks (ITNs) through Marie Sklodowska-Curie Actions. ITNs share many features of thematic PhD programmes, but they only recruit a single cohort of students, and they align with EU policy goals. These training networks are prestigious and very well regarded within European academia. The authors of this article were participants in a yeast biotechnology ITN, YEASTCELL, which finished in 2017. Some interesting insights into the more and less successful aspects of the project arose during discussions at the final project workshop. The views of the participants are distilled here in a discussion of how an ITN could be structured to maximise the benefits for the three main stakeholders: students, supervisors and industry partners.

Caspase-independent photoreceptor apoptosis in mouse models of retinal degeneration.

Apoptosis is the mode of cell death in retinitis pigmentosa, a group of retinal degenerative disorders primarily affecting rod photoreceptors. Although caspases have been demonstrated to play a central role in many incidences of apoptosis, accumulating evidence suggests that they may not be required for all forms of apoptotic cell death. The present study examined the mechanism of cell death in two in vivo models of photoreceptor apoptosis: the retinal degeneration (rd) mouse, a naturally occurring mutant model, and N-methyl-N-nitrosourea-induced retinal degeneration. Specifically, we examined the activation status of caspase-9, -8, -7, -3, and -2 and determined the caspase requirements for cytochrome c release, DNA fragmentation, and apoptosis-associated proteolysis of specific caspase substrates. We show that apoptosis in both in vivo models is independent of caspase-9, -8, -7, -3, and -2 activation. DNA fragmentation occurs in the absence of caspase-mediated ICAD (inhibitor of caspase-activated DNase) proteolysis, suggesting that an alternative endonuclease is responsible for DNA cleavage in these models. Importantly, we show that apoptosome activation is prevented because of an absence of mitochondrial cytochrome c release. Experiments performed using a cell-free system indicate that cytochrome c-dependent proteolysis and activation of caspase-9 can be restored in a neonatal cell-free system. However, we found that cytochrome c-dependent proteolysis and activation of caspase-9 could not be restored in an adult cell-free system because of an age-related decrease in the expression of Apaf-1 in the normal developing mouse retina. In the rd mouse, however, this age-related downregulation of apoptotic proteins was not observed, highlighting a critical feature of this model and the prevention of cytochrome c release as an apical event in caspase-independent apoptosis in this system.

Activation of multiple pathways during photoreceptor apoptosis in the rd mouse.

PURPOSE: The primary purpose of this study was to characterize photoreceptor apoptosis in the rd mouse. Given that apoptosis is the final common pathway in many cases of retinal degeneration, the ability to retard or even arrest this process may ameliorate retinal disorders such as retinitis pigmentosa (RP). The absence of any recognized therapy emphasizes the fact that a detailed knowledge of the molecular events involved is necessary to identify rational targets for therapeutic intervention. METHODS: Flow cytometry was used to measure physical and chemical characteristics in the photoreceptor population. Individual cells flow in suspension past one or more lasers, scattering light and emitting fluorescence. Western blot techniques demonstrated cleavage of calpain-specific substrates. Retinal explant cultures were used for inhibitor studies. Postnatal day 10 (P(10)) rd retinas were cultured without retinal pigment epithelium (RPE) attached up to P(17). RESULTS: This study demonstrated calcium overload in the cytosol and subsequently in mitochondria. Mitochondrial membrane depolarization and reactive oxygen species (ROS) were detected later, during the peak of cell death. Analysis of downstream events indicated early activation of calcium-activated calpains. Treatment of rd retinal explants with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO (ALLN) successfully inhibited calpain-induced alpha-fodrin cleavage, yet it did not protect against photoreceptor degeneration. Finally, the results demonstrate an increase in the levels of both precursor and processed forms of the aspartate protease cathepsin D. CONCLUSIONS: Excessive calcium influx is an early event that initiates the activation of calcium-activated proteases. However, these proteases are not singularly the cause of death, because their inhibition does not prevent apoptosis. Indeed, the results presented herein suggest that multiple pathways are involved and that each of these components may have to be addressed for cell death to be successfully inhibited.

Apoptosis: a potential therapeutic target for retinal degenerations.

Many retinal degenerations both inherited and induced are characterized by a loss of vision that is associated with death of photoreceptors. Inherited retinal diseases, which include Retinitis Pigmentosa (RP), form the largest single cause of blindness in the developed world. The genetics of RP is complex and approximately 48 genes have been implicated in the pathology of this disorder, in addition to the numerous mutations that exist within each gene (e.g. rhodopsin has <100). An attempt to overcome each individual mutation provides an overwhelming challenge. However targeting apoptosis, which represents a highly controlled, final common pathway to photoreceptor cell death, may provide a more practical approach. Caspases have been considered the primary executioners of apoptosis in many systems, however it is now apparent that other proteases such as calpains and cathepsins are affiliated with apoptosis. Conflicting data regarding the role of caspases in the execution of apoptosis in retinal degenerations will be critically discussed in light of reports demonstrating that key components of this pathway are downregulated during retinal development. This may control susceptibility to apoptosis in the developing retina and indeed during the maturation of other post-mitotic cells such as neurons and heart and skeletal muscle. Mitochondria function as central regulators of the intrinsic pathway of apoptosis through their role in energy production, calcium homeostasis and compartmentalization of cell death activators. The potential to control release of these apoptogenic proteins from mitochondria will also be examined with particular emphasis on the role of Bcl-2 family proteins and the regulators of calcium influx.

Detection of DNA fragmentation in retinal apoptosis by TUNEL.

Terminal dUTP nick end labeling (TUNEL) is an invaluable technique used in the study of late-stage apoptosis. The technique is based upon detection of fragmented DNA, a well-recognized characteristic of apoptosis, usually with fluorescent markers. Here, we describe the TUNEL technique (1) employing two different detection techniques, fluorescence microscopy and fluorescence-activated cell sorting (2) which can be applied to the analysis of apoptosis in retinal tissues or retinal cell cultures, respectively.

Norgestrel may be a potential therapy for retinal degenerations.

Retinal degenerations cover a broad spectrum of diseases, retinitis pigmentosa being the most common inherited retinal degeneration. It remains an untreatable disorder, partly owing to its genetic complexity and variability. Gene therapies, stem cell transplantation and administration of slow release growth factors are some of the treatments currently under development for the treatment of this disease. More recently, steroid hormones, now known to have functions within the CNS aside from their traditional targets, have been suggested as potential therapeutic agents. Progestogenic hormones are thought to modulate pro-survival pathways in the retina and since these hormones are produced naturally in the body their value as potential therapeutic agents is clear. Current data detailing the pro-survival effects of progestogens in the brain and particularly in the eye will be discussed.

Reactive oxygen species regulate prosurvival ERK1/2 signaling and bFGF expression in gliosis within the retina.

PURPOSE: Gliosis is the response of glial cells within retinal tissue to injury. It can be beneficial in the short term, but if the response is extended it can lead to scar formation, which contributes to blindness. Phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) is considered to be a hallmark event of gliosis, but the factors involved throughout its associated signaling pathway remain poorly understood, particularly in the retina. Because reactive oxygen species (ROS) can inhibit phosphatases, thereby altering the phosphorylation of proteins, this study tested the hypothesis that ROS regulate the phosphorylation of ERK1/2 (pERK1/2) in gliosis. METHODS: Increases in pERK1/2 were detected using Western blotting and immunofluorescence in three models of retinal stress, specifically the in vivo light induction, the rd1 disease, and the ex vivo retinal explant models. Explanted murine retinas were used to identify the signaling partners of pERK1/2 via Western blotting, in conjunction with inhibitors. The effect of this pathway on cell death was measured with terminal dUTP nick end labeling. RESULTS: It was demonstrated that several inhibitors of ROS greatly reduce the levels of pERK1/2 in the somata of Müller cells and furthermore decrease two other downstream signaling events: the phosphorylation of STAT3 and the upregulation of basic fibroblast growth factor. Using the specific inhibitor of ERK1/2, UO126, the resultant outcomes of this signaling pathway were determined to contribute significantly to cell survival. CONCLUSIONS: The novel finding of this study that ROS contribute to a prosurvival signaling pathway in retinal Müller cell gliosis indicates that some degree of caution should be used when considering antioxidants as therapeutics.

Morphological assessment of apoptosis.

Apoptosis is implicated in biological processes ranging from embryogenesis to ageing, from normal tissue homoeostasis to many human diseases. Apoptotic cells share a number of common features such as cell shrinkage, membrane blebbing, chromatin cleavage, nuclear condensation and formation of pyknotic bodies of condensed chromatin. In the final stages of apoptosis these pyknotic or apoptotic bodies are rapidly engulfed by neighbouring cells. Necrotic cells on the other hand exhibit loss of membrane integrity, cellular and nuclear swelling and an associated inflammatory response. Such characteristics demonstrate that apoptosis is an orderly genetic programme, which could potentially be manipulated or controlled at various points, while necrosis is a form of cell death that lacks these control points. These distinctive morphological differences form the basis of some of the most widely used techniques for the identification and quantification of apoptosis and thus morphologic description using light or electron microscopy remains one of the best ways to define apoptosis and contrast it with necrosis. However, the field of apoptosis or cell death research is advancing rapidly and it is becoming increasingly evident that apoptosis and necrosis represent two extremes of cell death and that many variations now exist. There is often a continuum of apoptosis and necrosis in response to high and low doses of the same stimulus and features of both apoptosis and necrosis may coexist in the same cell. Therefore, it is clear that an increasing amount of care must be taken when assigning the label 'apoptosis' to a dying cell on the basis of morphology.

Decreased expression of pro-apoptotic Bcl-2 family members during retinal development and differential sensitivity to cell death.

Apoptosis plays a crucial role in the sculpture of the mammalian retina during development. However, once the retina is fully differentiated, the emphasis must shift towards survival and mechanisms have to be put in place to prevent inappropriate cell death. In this study, we identify a potential control point at the level of mitochondrial permeability. We show that pro-apoptotic Bcl-2 family members known to be involved in the regulation of permeability transition and physiological cell death in the retina are down regulated during postnatal retinal development. In addition, we demonstrate an age-dependent susceptibility to retinal cell death induced by various stimuli known to target mitochondrion. These results potentially explain why retinal cells employ different death pathways depending on their stage of development. In contrast to developmental apoptosis, pathological retinal cell death in several animal models has been reported to occur independently of caspase activation. Here, we show that not only is cytochrome c release precluded from degenerating retinas but other pro-death molecules such as Omi/HtrA2 and AIF also remain in the mitochondrion. Our results indicate that transcriptional regulation of 'death genes' such as pro-apoptotic Bcl-2 family members during retinal development affords protection in adult post-mitotic neurons by preventing execution of the archetypal mitochondrial death pathway.

Induction of BIM(EL) following growth factor withdrawal is a key event in caspase-dependent apoptosis of 661W photoreceptor cells.

Apoptosis of photoreceptor cells in the early postnatal period is a normal feature of mammalian retinal development. The role of mitochondria and caspases in the process has been well established; however, the identification of key apoptotic mediators still remains elusive. Here we report that BIM(EL), a pro-apoptotic BCL-2 family member, may be one such molecule. Following growth factor deprivation, BIM(EL) was up-regulated in mouse 661W cone photoreceptors. This event correlated with the release of mitochondrial apoptogenic factors into the cytosol, the activation of caspases and apoptosis. Moreover, a similar behaviour was observed in response to UV radiation, ionomycin or H(2)O(2) treatments. We identified the PI3K-Akt-FKHRL1 signalling cascade as the main regulatory pathway of BIM(EL) expression in these cells. Finally, using RNA interference, we were able to silence BIM(EL) expression and subsequently suppress caspase-3 activation. In conclusion, we propose BIM(EL) as a critical factor in mitochondria-dependent apoptosis of 661W photoreceptors.