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

IV

DEVELOPING THERAPEUTIC STRATEGIES

FOR RETINAL DEGENERATIVE DISEASES

e.g., retinal pigment epithelial protein (RPE) 65 (4). Interestingly, a number of RP genes are widely expressed but only cause disease pathology within the retina, highlighting the unique and complex biochemistry of photoreceptor cells. Included in the latter category are the genes HPRP3 and PRPC8 encoding pre-messenger RNA splicing factors and the gene encoding inosine monophosphate dehydrogenase type 1 (IMPDH1), the rate-limiting enzyme of the de novo pathway of guanine nucleotide biosynthesis (5–7). Despite such genetic heterogeneity, photoreceptors degenerate in RP, and indeed in other inherited retinal degenerations, by a common form of cell death, apoptosis (8,9). Apoptosis is a regulated mode of cell death that is essential for normal development and homeostasis (10,11). However, abnormal regulation of apoptosis contributes to many disease pathologies, including cancer, autoimmune disorders, and neurodegenerative diseases, for example, Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) (12–16). Numerous studies in cell culture, and in various animal models of retinal degeneration, including inherited and light-induced models of retinal damage support the initial observation by Chang et al. (8) that photoreceptors die by apoptosis in retinal degenerations (17–20). One of the key aims of RP research is the development of effective therapeutics, and modulation of apoptosis clearly represents a potential therapeutic approach. It is unlikely that each RP mutation initiates an equivalent number of separate apoptotic pathways, so what is more probable is that such events converge and progress via one, or a limited number of apoptotic cascades, providing an alternative therapeutic approach to targeting the underlying primary mutations. Therapeutic invervention for primary mutations may involve either gene replacement for autosomal recessive (AR) RP or alternatively some form of gene suppression for ADRP. With respect to ADRP, targeting of primary mutations presents a particularly formidable challenge, since multiple mutations are routinely encountered in any given disease-causing gene, e.g., more than 100 different rhodopsin mutations have been identified (4). On the other hand, the goal of inhibiting apoptosis is to modulate the course of the disease in an entirely mutation independent fashion, providing therapeutic benefit by targeting a common pathway. In addition to modulating apoptotic programs, other therapeutic strategies may include promoting photoreceptor survival using neurotrophic factors (21–23) or replacing lost photoreceptor cells by retinal transplantation or stem cell therapy (24,25). For recent reviews of RP therapy, see Delyfer, et al. and Doonan et al. (26,27). None of these therapeutic approaches is mutually exclusive and indeed it is likely that a combination of therapies may ultimately be used to treat this group of conditions. A summary of therapeutic strategies for RP is provided in Table 1.

The focus of this chapter is on how apoptosis can be modulated for potential therapeutic benefit in RP, including the inhibition of key proteases involved in mediating apoptosis and the reduction of reactive oxygen species (ROS) that may play a role in photoreceptor degeneration. Recent exciting developments in the area of cell survival factors will also be discussed. In addition, the role of light in apoptosis will be reviewed: how light-induced animal models of retinal degeneration have provided insights into mechanisms of degeneration in models of RP and how such discoveries may impact on the development of therapeutic strategies. Finally, it is clear from studies of the segregation pattern of genetic disorders in humans and from studies in animal models that so

AAV, adeno-associated virus; Ac-DEVD-CHO, N-Ac-Asp-Glu-Val-Asp-CHO; Ad, adenovirus; BAG-1, Bcl-2 associated anthogene-1; Bcl-2, B-cell leukemia/lymphoma 2; Bcl-XL, homologue of Bcl-2; BNDF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic growth factor; CR-6, 3,4-dihydro-6- hydroxy-7-methoxy-2,2-dimethy1-1(2H)-benzopyran; DMTU, dimethylnitrourea; ECT, encapsulated cell technology; EPO, erythropoietin; ERG, electroretinogram; FADD, FAS-associating death domain-contain- ing protein; FGF2, fibroblast growth factor-2; GDNF, glial-derived neurotrophic factor; LI, light-induced; L-NAME, N(G)-nitro-L-arginie methyl ester; NOS, nitric oxide synthase; ONL, outer nuclear layer; p35, baculoviral anti-apoptotic protein; PBN, phenyl-N-tert-butyInitrone; PR, photoreceptor; rcdl, rod-cone-dys- plasia type 1; RCS, Royal College of Surgeons; rd, retinal degeneration; rdl, retinal degeneration 1 (same as rd); rd2, retinal degeneration 2 (previously known as rds); RdCVF, rod-derived cone viability factor; rds, rential degeneration slow; ROS, reactive oxygen species; TUNEL, terminal dUTP nick-end labeling; VPP, mutant transgene for opsin (V20G, P23H, P27L).

Adapted from ref. 170.

called genetic modifiers influence progression of the disease. Identification of such modifiers, some of which are likely to regulate pathways of apoptosis and cell survival, may possibly illuminate novel therapeutic targets.

APOPTOSIS

On the Mechanism of Apoptosis

Apoptosis can be mediated by caspases, a group of cysteine-aspartyl-specific proteases (28–31). To date, 14 mammalian caspases have been identified, a subset of which are involved in apoptosis, whereas the remainder are involved in processing pro-inflammatory cytokines (32). Apoptotic caspases fall broadly into two categories, initiators and effectors. Initiator caspases, such as caspase-8, -10, and -12 are the first to be activated in response to a death stimulus, which in turn activate the effector caspases, namely caspase-3, -6, and-7 (33). Once activated, these caspases mediate cell destruction by degrading a broad range of structural and regulatory proteins (34). Apoptosis can be initiated from both outside and within the cell, depending on the pro-apoptotic stimulus. The extrinsic pathway is triggered via the activation of cell surface death receptors, e.g., Fas (or CD95) receptor and tumor necrosis factor receptor 1 (TNFR1), which in turn, activate caspase-8 within the cell (35,36). The intrinsic pathway can be activated by a variety of stimuli, including ultraviolet light, chemotherapeutic agents, or growth factor deprivation, which trigger mitochondrial outer membrane permeabilization (MOMP), releasing cytochrome-c and other pro-apoptotic factors (37–39). MOMP is a central event in cell death, and is tightly regulated by the Bcl-2 family of proteins, comprising both proand anti-apoptotic members (40,41). An intrinsic pathway that centres on the endoplasmic reticulum (ER) has also been identified, in which insults that induce ER stress including misfolded proteins and oxidative stress, lead to caspase-12 activation (42,43). Thus, it is clear that apoptosis is a complex process with numerous potential

points for modulation. In the context of therapeutic development, initial targets to be explored were the caspases, in which inhibition of these proteins was used as a therapeutic approach for conditions including neurodegenerative disorders, myocardial infarction, and acute brain injury (44–47). In this context, there have been notable successes, and several drugs are now at the clinical trial stage of development. For example, novel caspase inhibitors (Idun Pharmaceuticals, Inc., IDN-1965 and IDN-6556) have been shown to be protective in instances of heart and liver injury (47,48).

Caspase-Dependent Mechanisms of Photoreceptor Cell Death

The possible involvement of caspases in RP has been explored to assess whether caspase inhibition is a potential therapeutic strategy for the disease. For example, there is substantial evidence to support the activation of caspase-3 in various models of retinal degeneration, the rd mouse (49–51), tubby mouse (19), ser334ter rhodopsin mutant rat (18), and in chemically induced models of retinal degeneration (52). In contrast, however, results from other studies suggest that caspase-independent apoptosis may be occurring (53,54). Although there is significant evidence to support caspase-3 activation, the impact of caspase-3 ablation in knockout (KO) mice has been shown to provide only minimal protection against photoreceptor degeneration in the rd model of retinal degeneration (55). This supports a transient protective effect previously observed using the caspase-3 inhibitor Ac-DEVD-CHO in the rd mouse (56). Clearly, caspase-3 is activated in such systems but it may not play a critical role in the mediation of apoptosis, its function perhaps being compensated for by other caspases. Considering the complex nature of the pathways that lead from the numerous primary mutations encountered in RP to the death of photoreceptor cells, it is premature at this stage to discount caspase inhibition as a therapeutic strategy.

In contrast to these aforementioned studies involving caspase-3 inhibitors, successes have been achieved with pan-caspase inhibitors, most notably the p35 protein. p35 was originally identified in baculoviruses and is a pan-caspase inhibitor targeting both initiator (caspase-2, -8, and -10,) and effector (caspase-3, -6, and -7) caspases and it has been shown to rescue photoreceptor degeneration in Drosophila models of retinal degeneration (57,58). Furthermore, p35 has also been shown to protect against chemically induced apoptosis in the cone photoreceptor cell line, 661W (17). p35 inhibits several caspases, in contrast to the specific caspase-3 inhibitors, possibly explaining its greater protective effect. Clearly further evaluation in animal models will be required before any conclusions can be made regarding the use of caspase inhibitors as therapeutic agents in RP.

Caspase-Independent Mechanisms of Photoreceptor Cell Death

Caspase-mediated apoptosis may not be the only pathway of photoreceptor degeneration in RP. Caspases were long considered the key executioners of apoptosis, but research has shown that caspase-independent mechanisms of cell death exist, where dying cells retain many morphological characteristics of apoptosis (59). Caspaseindependent pathways have been demonstrated in neuronal systems in response to ischaemia, traumatic brain injury and in neurodegenerative diseases such as Huntington’s and Alzheimer’s diseases (60–63). Proteases involved in caspaseindependent pathways of cell death include cathepsins, calpains, and serine proteases such as granzyme B (64–66). Calpains are a family of calcium-dependent proteases,

comprising at least 15 members, the best characterized of which are - and m-calpain (65). Although much remains to be learned about the regulation and function of calpains, these proteases have been implicated in the pathogenesis of cell death in cerebral ischaemia (67), cataract formation (68), and neurodegenerative disorders including Huntington’s disease (69). In reference to photoreceptor cell death, calpain activation has been shown in light-induced and inherited models of retinal degeneration (51,70,71). In one study, a calpain inhibitor prevented calcium-induced death in cone photoreceptor-derived 661W cells, further supporting a possible role for calpains in photoreceptor cell death (51). The successful inhibition of cell death in 661W cells calpains using a calpain inhibitor, warrants further exploration of calpains as novel therapeutic targets for modulation of apoptosis in degenerative retinopathies. It is notable that in the previously mentioned study caspase-3 activation was also detected, indicating cross talk between the two proteolytic systems of caspases and calpains. Interaction between these different proteases has been demonstrated in previous studies, including the activation of caspase-3 and -12 by calpains (72,73). Activation of both systems in photoreceptor degeneration suggests a possible explanation for the limited success of caspase inhibitors in preventing apoptosis in models of RP. However, results from other studies suggested no caspase activation in the rd inherited and light-induced model of retinal degeneration, indicating that the complex pathways of cell death in RP remain to be fully elucidated (53,54).

A recent study by the same group showed that although treatment of rd retinal explants with a calpain inhibitor successfully inhibited calpain-induced alpha-fodrin cleavage, it did not protect against photoreceptor degeneration, suggesting the involvement of multiple cell death pathways (171). Lohr and colleagues reached a similar conclusion by comparing three photoreceptor degenerations caused by different events: calcium overload (rd mouse), structural defects (rds mouse), and light induced retinal degeneration (172). By comparing caspase, lysozyme and cathepsin activity, as well as the expression of several other apoptotic marker genes, they concluded that multiple parallel cell death mechanisms are involved in retinal cell death (172). Until a common upstream initiator of cell death can be determined, each of these components must be addressed for successful inhibition of photoreceptor degeneration.

Mitochondria

Caspases and calpains represent some possible therapeutic targets, in respect of photoreceptor protection, but there are several others within the apoptotic pathway, most notably those centring on the mitochondria. Apoptosis proceeding through the mitochondria represents an important pathway of cell death, which is characterized by a central event, that of MOMP (39). Following MOMP, factors mediating apoptosis are released including cytochrome-c, apoptosis inducing factor, and Smac/Diablo (74–76). As a result, the mitochondrial potential is dissipated and the essential functions of the mitochondria are lost. Initiation of this process is tightly regulated by the Bcl-2 family of proteins, comprising both proand anti-apoptotic members and they modulate the formation of permeability transition (PT) pores on the surface of the outer membrane. Anti-apoptotic members block MOMP by preventing the formation of the PT pores, whereas pro-apoptotic members facilitate opening of the pores. Modulation of this