Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

process has been evaluated as a therapeutic approach for RP, with varying results. For example, overexpression of Bcl-2 was shown to provide transient protection in mouse models of retinal degeneration (77,78). In contrast, however, other studies have reported no protection by either Bcl-2 or Bcl-XL expression (79). Furthermore, different combinations of Bcl-2 anti-apoptotic proteins have been evaluated and again, although some protection has been observed in photoreceptors, effects were transient (80,81). In support of this approach, overexpression of Bcl-XL has been shown to protect against leadinduced photoreceptor apoptosis up to postnatal day 90 (P90) in mice (82). Overall, however, results from studies of exploring Bcl-2 family members as potential therapeutics for RP have not been encouraging. This view is supported by a recent observation in which Bax, the target of Bcl-2, was found to be downregulated during normal retinal development, possibly explaining, at least in part, the lack of protection provided by overexpression of Bcl-2 (81).

It is notable, however, that there are alternative ways to inhibit the formation of the mitochondrial PT pore. For example, cyclosporin A blocks the loss of membrane potential by targeting proteins involved in PT function including cyclophilin D (83,84). It has been shown to be protective in models of Alzheimer’s disease, Parkinson’s disease and ALS (85–87). In addition, this agent was shown to decrease the death of cortical neurons in a model of focal ischemic stroke (88) and has been shown to protect against calciuminduced apoptosis in isolated rat retina (89). Another example of an agent, which has been found to be protective at the level of mitochondria is tauroursodeoxycholic acid (TUDCA), an endogenously produced hydrophilic bile. TUDCA acts, in part, by inhibiting the translocation of pro-apoptotic Bax to the mitochondria and in addition has antioxidant properties (90). It has been shown to be neuroprotective in animal models of Huntington’s disease (91) and also reduced apoptosis in RPE cells (92). Other agents that protect by a similar mode of action are minocyclin and rasagiline, which have been shown to be of therapeutic benefit in models of Parkinson’s disease and ALS (93,94).

Another agent targeting mitochondria, MITO 4565, has been evaluated in a rat model of RP expressing a dominant mutation (Ser344ter) within the rhodopsin gene (95). MITO 4565 is a novel oestrogen analogue that does not inhibit the formation of the PT pore, but rather stabilizes the mitochondrial membrane. MITO 4565 was injected into the left retinas of the Ser344ter model at PD9, and by PD20 the loss of outer nuclear layer (ONL) thickness was shown to be significantly reduced compared to the control right retinas (96). MITO 4565 is believed to intercalate into the mitochondrial membrane, terminating lipid peroxidation and thus maintaining mitochondrial membrane potential.

CALCIUM INVOLVEMENT IN APOPTOSIS

Elevated calcium levels probably play a key role in photoreceptor apoptosis. Calcium overload has been observed in various models of inherited and chemical-induced retinal degenerations (97–99). The role of calcium is well characterized in the rd mouse, which has a mutation in the gene encoding the β-subunit of cGMP phosphodiesterase (100). When cGMP levels rise, channels regulated by cGMP remain open, resulting in the build up of toxic levels of calcium within the photoreceptors. A recent study demonstrated in 661W cone photoreceptor cells, that calcium-induced apoptosis is mediated by calpain activation, resulting in caspase-3 dependent cell death and that this apoptotic pathway

could be inhibited by a calpain inhibitor, SJA6017. In the same study, activation of calpain and caspase-3 were observed in the retinas of the rd mouse, indicating that the pathway of apoptosis observed in 661W cells is the possible mechanism of photoreceptor degeneration in the rd model (51). As elevated calcium has been shown to play a key role in mediating photoreceptor apoptosis, modulation of calcium levels is another potential strategy for slowing cell death in the retina. Calcium enters the cell via voltagedependent calcium channels, and channel blockers have been evaluated as a potential therapy, with varying results. D-cis-diltiazem, one such channel blocker, has been reported to be protective in the rd mouse model and in a light-induced model of retinal degeneration (70,101). In contrast, D-cis-diltiazem was not found to be protective in other studies using the rd mouse, in a Pro23His rat model, or in a canine model of retinal degeneration (102–105). Similar results have been obtained with nilvadipine, which has been shown to be protective in the rd mouse retina, and in another model of retinal degeneration, the Royal College of Surgeons (RCS) rat (104,106). Micorarray analysis of gene expression in the rd mouse following nilvadipine administration suggests that protection is likely mediated through suppression of caspases, and upregulation of fibroblast growth factor (FGF), a neuroprotective cytokine (104). Considering the observation of therapeutic benefit in a number of studies, calcium-channel blockers warrant further investigation as potential therapeutic agents for degenerative retinopathies.

OXIDATIVE STRESS INVOLVEMENT IN APOPTOSIS

Another factor suggested to be involved in photoreceptor degeneration is oxidative stress, resulting from the generation of damaging ROS within retinal tissue. As the retina is one of the highest oxygen-consuming tissues in the body, it is particularly sensitive to oxidative stress (107). Oxidative stress in photoreceptor apoptosis has been studied predominantly using light-induced models of retinal degeneration, in which short exposure to bright light induces retinal damage (20). The role of oxidative stress in mediating apoptosis in light-induced models is supported by various in vitro and in vivo studies demonstrating that increased levels of ROS represents an early event in photoreceptor apoptosis, which can be inhibited by antioxidants (108,109).

In addition to indicating an involvement of ROS in retinal degeneration, these results suggested a possible therapeutic strategy for RP because light has been shown to be a cofactor accelerating disease progression (110). Antioxidants such as dimethylthiourea (DMTU) and phenyl-N-tert-butylnitrone (PBN) have been evaluated using inherited rodent models of retinal degeneration exposed to damaging levels of light (111,112). Protection from the deleterious effects of light has been observed with DMTU in Pro23His and Ser344ter transgenic rats and with PBN in the Pro23His rat model. However, PBN was found to have no effect on the rate of degeneration of the photoreceptors in either model in the absence of additional light insult, indicating that such therapies may be of benefit in limited cases of RP, in which light-accelerated damage is more significant. Another potential protective agent in the context of photodegeneration is thioredoxin, an endogenous protein with various activities including elimination of ROS and regulation of the apoptotic pathway. Thioredoxin has been shown to protect against light-induced retinal degeneration in several studies, but has yet to be evaluated in inherited models of retinal degeneration (113,114).

Fig. 1. Schematic diagram of basic proposed caspase- (marked in blue) and calpain- (marked in green) pathways of cell death in photoreceptor cells with potential therapeutic strategies (marked in red). Apoptotic pathways in RP are ill defined to date but ER stress is known to play a significant role, particularly in cases of RP as a result of mutated rhodopsin (168,169). Other factors that may be involved in degeneration are calcium and ROS (100,108,109).

Oxidative stress may also have a more fundamental role in retinal degenerations, in that it may be responsible for the secondary loss of cone photoreceptors, which almost invariably follows initial rod cell death in conditions such as RP. Understanding processes involved in cone degeneration is of vital importance because cone loss is responsible for the main visual handicap in RP. Several theories have been advanced to explain cone photoreceptor loss, including oxidative damage (115). Markers for oxidative stress, indicating damage to proteins, lipid, and DNA were detected in cone photoreceptors of the transgenic pig model of RP, with a Pro347Leu rhodopsin mutation (116). The data suggest that as the rod photoreceptors degenerate, there is a reduction in oxygen consumption, resulting in increased oxygen levels within the retina (hyperoxia), oxidative stress, and finally cone degeneration. The hypothesis is supported by studies demonstrating increased oxygen levels in models of retinal degeneration (117,118). It remains to be seen whether antioxidants may slow down cone photoreceptor loss in RP, but the potential therapeutic benefit observed in animals supports further evaluation of agents that reduce oxidative stress (see Fig. 1).

TROPHIC FACTORS AND THE POTENTIAL FOR GENE THERAPY

A parallel therapeutic approach to inhibiting cell death is promoting cell survival. One way of achieving this is through use of neurotrophic factors that modulate neuronal growth during development to maintain existing cells and aid recovery of injured neurons. In developing retinal neurons, correct synaptic connections are reinforced by trophic factors, whereas cells with inappropriate connections receive no trophic support and die by apoptosis (119). This observation has lead to the premise that because the removal of neurotrophic factors stimulates cell death, the addition of exogenous trophic factors may have neuroprotective effects in the retina.

In the first study demonstrating that growth factors might protect against photoreceptor degeneration the use of basic FGF (bFGF or FGF-2) was explored in a rat model of inherited retinal dystrophy (120). However, despite the observation that FGF-2 treatment rescued degenerating photoreceptors (121–124), FGF-2 has also been shown to trigger pathological retinal neovascularisation, making it unacceptable for human therapy.

Other trophic factors have also been shown to protect against photoreceptor degeneration. The most well characterized of these is ciliary neurotrophic factor (CNTF), a member of the interleukin (IL)-6 family of cytokines, which has been shown to delay photoreceptor apoptosis in several models of retinal degeneration (125–131). However, CNTF therapy also has its drawbacks. Despite inducing a morphological rescue, functional analysis by electroretinogram (ERG) showed a decreased response in CNTFtreated retinas (129,130). In contrast a study by Cayouette et al. (127) showed a significant preservation of the ERG response in the rds mouse after adenovirus-mediated gene delivery of a gene-encoding CNTF and it has been suggested that this might be in part because of a lower level of expression, whereas previous studies were suspected to have delivered toxic dose levels of CNTF. To provide controlled, continuous, long-term delivery of CNTF, Tao et al. (21) developed an encapsulated cell therapy (ECT) device, specifically designed for intraocular implantation. This involved loading a polymer membrane capsule with mammalian cells genetically engineered to secrete CNTF that was surgically implanted into the vitreous of 7-wk-old rcd1 dogs. After 7 wk, the ECT treated eyes had significantly higher levels of nuclei in the ONL, but retinal function was not evaluated in the study (14). To study the effects of dose of CNTF on normal retinal function, ECT devices secreting a high or low dose of CNTF were implanted into white albino rabbits (132). Low (5 ng/d) doses had no adverse effects, whereas the higher (22 ng/day) dose showed morphological changes in the ONL. but caused no reduction in the ERG, leading the authors to suggest that low-therapeutic doses are not toxic.

Another neurotrophic factor, cardiotrophin (CT)-1, also a member of the IL-6 family, has been shown to protect photoreceptors of the S334ter transgenic rat (133). Repeated intravitreal injection of CT-1 every 4 or 5 d resulted in a significant rescue of ONL cells of the retina. The biological effects of CT-1 are mediated thorough a signal transducer and activator of transcription 3 (STAT3) pathway and the marked increase in phosphorylated STAT3 observed in Müller cells suggests that these cells probably mediate the protective effect (61).

Glial derived neurotrophic factor (GDNF) has also been shown to slow photoreceptor degeneration and preserve visual function in an rd mouse model. Intraocular injections

of GDNF were efficient at delaying photoreceptor cell death and detectable ERG responses were recorded in 4 out of 10 GNDF-treated animals (134). Use of a recombinant adeno-associated virus vector to deliver GDNF has been shown to significantly increase rod photoreceptor survival and substantially increase the amplitude of the ERG response in Ser334ter transgenic rats (135). GDNF-replacement therapy has also been shown to slow cell death and enhance retinal function in the rd2 mouse and the RCS rat in combination with gene replacement therapy (173).

Despite the fact that the mechanisms underlying cone cell death remain relatively unknown, it is important to note that degeneration of cones occurs in patients with rhodopsin mutations after rod cell death exceeds 75% (110). This has also been observed in several animal models of retinal degeneration (136). As the cones are not directly affected by the mutation, they therefore degenerate in a “non-cell autonomous” mechanism, which results in progressive loss of cone function. One proposed hypothesis to account for the degeneration of cones suggests that rod photoreceptors release a trophic factor or factors that are essential for cone cell preservation. Evidence for the loss of trophic support theory has been described in an elegant set of experiments by Thierry Leveillard and colleagues (23).

Mohand-Said et al. (137) demonstrated that retinas from rd mice cultured in the presence of normal retinas showed significantly (15–20%) greater numbers of surviving cones compared with controls. These data suggested the existence of a diffusable trophic factor released by rods that protects cones (137). This factor was eventually identified using an expression cloning approach, and named rod-derived cone viability factor (RdCVF). RdCVF is a truncated thioredoxin-like protein, expressed and secreted specifically by rod photoreceptors (23). These data suggest a novel mode of trophic interactions and should in principle allow for the development of unique therapies aimed at preventing secondary cone cell death and subsequent loss of central vision in degenerative retinopathies. Because only 5% of the normal complement of cone cells is still compatible with visual discrimination and orientation, and 50% cone survival is compatible with normal, 20/20 visual acuity, RdCVF could, in principle, provide substantial therapeutic benefit even if delivered at a relatively advanced stage of disease.

ON THE SIGNIFICANCE OF LIGHT-INDUCED RETINAL APOPTOSIS AS A MODEL OF RP

Although many transgenic mouse lines that mimic human retinal disease are now available (138–140), much of the research carried out to date on photoreceptor apoptosis has used light-induced models. Although lifelong exposure to bright light is known to be a contributing element to retinal disease progression, the light-induced model of retinal degeneration does not necessarily mimic the human condition and potentially activates alternative apoptotic pathways. However, this model does have the advantages of being significantly quicker and more convenient than using transgenic models. It takes only hours of light exposure to significantly alter the appearance of the photoreceptors, whereas with many transgenic animal models degeneration may often not occur for several months.

It has been firmly established that light-induced apoptosis in mice is dependent on a functional visual cycle. Photoreceptors lacking rhodopsin are completely protected

against light-induced apoptosis (141) and mice with a Leu450Met mutation in the Rpe65 gene with slow rhodopsin regeneration kinetics are more resistant to light damage than wild-type (WT) mice (142). Rhodopsin-deficient mice can also be generated by knocking out genes needed for the synthesis of 11-cis-retinal, such as Rpe65, and as expected, mice lacking Rpe65 are also completely protected against light-induced apoptosis. This is also consistent with the finding that vitamin A deficiency, which also prevents rhodopsin synthesis, also protects against light damage (143).

Another gene known to play an important role in retinal light damage is the c-fos gene, which codes for a proto-oncoprotein. Although it has no direct role in the visual cycle, aberrant expression of the c-fos transcript was observed in the rd mouse (144). This led to the investigation of its role in light-induced damage, and the subsequent discovery that c-fos KO mice were protected against light damage (145). This implicated the activator protein (AP)-1 transcription factor, of which c-fos is a component, in the lightinduced cell death cascade. However, c-fos does not protect against photoreceptor cell death in the rhodopsin KO mouse, which suggests that the AP-1 pathway has no role in some, or possibly all mutation-induced forms of photoreceptor apoptosis (146). A study by Hao et al. (147) has demonstrated that at least two different biochemical pathways mediated light damage, one pathway for bright light, and a second for low-level light. Gnat1 KO mice lacking the gene coding for the α-subunit of rod transducin were used to distinguish between the roles of activated rhodopsin and phototransduction. In this way, it was possible to investigate whether light-induced activation alone leads to apoptosis, or if a downstream event in the phototransduction cascade might be involved. Both transducin-deficient and WT mice were equally susceptible to light damage induced by bright light, whereas absence of phototransduction was protective against low-light intensities. These results define a second phototransduction-dependent light-induced mechanism of photoreceptor cell death.

The direct relevance of the light-induced model of retinal degeneration to genetic models of retinal disease has still to be fully established. In this regard, it is notable that in a number of studies (102–104,106,148,149) neuroprotective agents that were found to be protective in light-induced models of retinal degeneration did not translate through to neuroprotection in genetic models of retinal disease.

ON THE INFLUENCE OF GENETIC AND ENVIRONMENTAL FACTORS ON PHOTORECEPTOR CELL DEATH

Light is also known to modify the severity of disease progression in human retinal degenerations. A human case report of two families with Pro23His rhodopsin mutations provided the first indication that light phototoxicity may be an accelerator of RP (150). Studies in transgenic rodent models (111,151) with rhodopsin mutations lent support to this theory, showing that light activation of rhodopsin contributes to the severity of the disease, leading to the suggestion that minimizing exposure to light might delay retinal degenerations arising from rhodopsin mutations.

A recent report using a naturally occurring canine model of ADRP caused by a rhodopsin mutation again tested this hypothesis, exploiting the similarity in eye-size and preretinal light transmission characteristics between dog and human (152). Investigation into the illuminating effects of clinical retinal photography led to the surprising observation

of circular-degenerating areas of retina that matched the pattern of the light flashes. Changes in retinal tissue were visible minutes after light exposure. By titrating transient increases in neuronal stress by light exposure, a dose–response relationship between light exposure and long-term outcomes of these early alterations was established. High doses of light caused a rapid degeneration of neurons, whereas low doses revealed mechanisms acting over several weeks or months that repair the damage. This study represents the first report of repair of retinal injury in an inherited retinal degeneration and establishes a useful in vivo assay to study the balance between proand anti-apoptotic signaling and repair-compensation mechanisms. Taken together, these findings suggest that limiting light exposure in patients with rhodopsin mutations may slow disease progression.

Another factor contributing to the variability in RP phenotypes is the influence of genetic modifier loci. In principle, modifiers cause variation in phenotype by interacting in the same or a parallel biological pathway as that of the disease gene. This modifier effect may suppress the mutant phenotype even to the extent of completely restoring the WT phenotype. Alternatively, expression of the modifier gene may lead to a more severe mutant phenotype or affect the pleiotropy of a given disease resulting in a different combination of traits. In addition, combinations of modifier genes may act together to create cumulative effects on the expression of a phenotype (153). Clearly, the further characterization of genetic modifier loci should provide insights into the biological pathways in which these genes act to cause disease as well as providing novel therapeutic targets. For such reasons, interest in modifier genes has grown rapidly.

MODIFIERS OF RP IN HUMANS

A classic example of the effect of a strong modifier in RP was the occurrence of three separate phenotypes within a single nuclear family with a novel three-base deletion of codon 153 or 154 in the peripherin/rds gene. These patients developed either RP, pattern dystrophy, or fundus flavimaculatus (154). Although the genetic modifier locus or loci underlying these particular modifications have yet to be identified, one gene product known to interact with peripherin/rds is ROM1. Rare individuals heterozygous for a Leu85Pro allele of peripherin/rds who also carry a null ROM1 allele develop digenic RP, whereas individuals with either mutation alone are unaffected (155). In another study of 1941 probands with a clinical diagnosis of RP (who had previously been screened for mutations in rhodopsin and peripherin/rds and found to be negative), 17 families were shown to harbor mutations in the RP1 gene. Patients with a premature stop codon, Arg677ter, demonstrated wide variability in the severity of visual field loss both within and between families, which led the authors to suggest an important role for modifier genes or environmental factors in RP1 releated disease (156).

In a study of RP9, the mutation causing ADRP was shown to lead to regional retinal dysfunction with greatly variable expressivity. Family members with this mutation were reported to be either minimally effected, with normal electrophysiological responses, moderately affected with abnormal ERG responses, or severely effected with no ERG response (157). RP9 has since been shown to be caused by a mutation in the splicing factor gene, PAP-1 (158,159).

Despite a wealth of studies reporting phenotypic differences between individuals with the same genotype, very few modifier loci have been chromosomally localized. In

humans, this difficulty may be largely the result of the genetic variation in the population. In contrast, this problem can be reduced significantly in inbred mice as discussed earlier.

MODIFIERS OF RP IN MOUSE MODELS

The existence of modifiers in mice was originally recognized when the spontaneous obesity mutations Lepob and Lepdb were shown to cause diabetes on a C57BL/KsJ, but not on a C57BL/6J genetic background (160). Recent advances in generating targeted mutant mouse models have also revealed many important examples of modifiers. Such mutants are usually propagated in stem cells derived from 129 mice that were made chimeric with the C57BL/6J strain and then crossed onto a specific genetic background. Alterations in the initial targeted phenotypes have been reported when mice with retinopathies were back crossed onto specific genetic backgrounds (161,162). Modifier effects of RP can result from a single gene at a locus independent of the disease gene (163–165) or can be caused by the combined effects of several genes at different loci, as is typical of quantitative trait loci (QTL) (165). The first report of variable phenotypic expression in a mouse model of retinal degeneration was observed in the rd3 mouse. A mutation at the rd3 locus led to a unique retinal degeneration whereby photoreceptor cell death starts at 3 wk postnatally and is complete at 5 wk (166). However, significant variation in the onset and progression of the disease was observed when mice were crossed onto different background strains. The ocular phenotypes of mice carrying a targeted disruption of the p53 tumor suppressor gene (p53–/– mice) have also been shown to be radically different on C57BL/6J and 129 genetics backgrounds. p53 KO mice bred onto a C57 background, but not on the 129 background, exhibited vitreal opacities, retinal folds, and vitreal neovascularisation, possibly as a result of abnormal developmental retinal apoptosis; although, as pointed out by the authors, angiogenic factors could also be involved in modulation of the p53 phenotype (161). Mice with a targeted disruption in rhodopsin (Rho–/– mice) have also been reported to be protected by modifiers on the C57BL/6J background when compared to the 129 background (162). In the latter investigation, C57BL/6J mice were found to have a significantly greater number of ONL nuclei by 3 months of age and TUNEL staining, over various time points, showed more positive labeling in the ONL of 129 retinas. Both amplitude and waveform features of electroretinographic analysis were remarkably different in the two strains.

Taken together, these results suggest the presence of genetic modifiers on the C57BL/6J background that significantly protect photoreceptors against the retinopathy; however, whether such modifiers directly influence apoptotic mechanisms remains to be established. Two recent studies by Danciger et al. (163,167) have resulted in localization of QTL that contribute to the protection of photoreceptor cells against damage induced by constant light. In the first of these studies, a genome-wide scan on the progeny from backcrossed mice using the thickness of the ONL as the quantitative trait reflecting retinal damage revealed a strong QTL on mouse chromosome 3 that contributes almost 50% of the protective effect (163). A high LOD score linked the Rpe65 gene to the apex of this QTL and sequencing revealed a single base change in codon 450, coding for a methionine in c2J mice and a leucine in the BALB/c strain (163). In a second study, rhodopsin was measured spectrophotometrically subsequent to light-induced apoptosis, and this was used as the quantitative trait to reveal new QTL on mouse chromosomes

1 and 4, with suggestive QTL on chromosomes 6 and 2 (167). More recently, the Rpe65 gene was determined to be a modifier for an inherited retinal degeneration in the VPP mouse model with the authors suggesting that the variation in the gene may modulate rhodopsin regeneration kinetics, therefore affecting light-damage susceptibility (174). Identification of such QTL and the associated modifiers may provide important information needed to further understand human retinal degenerations. However this will be challenging. In a second study rhodopsin was measured spectrophotometrically subsequent to light-induced apoptosis, and this was used as the quantitative trait to reveal significant QTL on mouse chromosomes 1 and 4, with suggestive QTL on chromosomes 6 and 2 (167). Identification of such QTL and the associated modifiers may provide important information needed to further understand human retinal degenerations. However, this will be challenging, especially if more than one gene contributes to the modification of a given phenotype. Nonetheless, the real promise of substantial therapeutic potential remains once the functions of modifier genes associated with a suppression of photoreceptor cell death are elucidated.

CONCLUSION

In the light of significant recent progress, it is tempting to speculate that several therapies for RP may be available within the next few years. Such therapies will be based either on direct intervention at the genetic level, using the technique of gene replacement or suppression of transcripts derived from dominant-acting genes, either through using techniques aimed at suppressing secondary molecular pathological effects, such as apoptosis, or by enhancing neuroprotection. Although significant progress in being made in both gene replacement and in suppression of mutant transcripts (4), targeting of apoptotic or survival mechanisms holds much appeal in the sense that such strategies will be largely independent of the vast number of mutations now known to cause RP-related conditions. The most readily attainable goal of RP research is the elucidation and functional evaluation of all RP genes. Up to 40 RP genes are known to date, but it is possible that many more remain to be identified (http://www.sph.uth.tmc. edu/RetNet/). Why mutations in such genes lead to photoreceptor cell death, sometimes many years after birth, is as yet an unresolved question. It is highly unlikely that there are many different gene-specific pre-apoptotic pathways, all individually activating apoptosis. A more probable scenario is that a smaller number of such pathways, shared by many RP loci, converge toward a few pre-apoptotic initiators. A major endeavor for future RP research will be to identify molecules and interactions in such pathways, and to understand the “switch” that occurs from normal aging to that of disease. Never before have so many avenues been available through which therapeutic interventions for this group of conditions might be achieved.

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