Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

of the disease usually progress over many years. Clinical observations, as well as the results obtained from experimental animal models, indicate that it can take months or even years for RGC somata to die after initial damage to the axon. Currently, efforts to lower IOP, which is considered by many to be a “primary” cause of RGC degeneration, are the main strategy to slow the progression of the disease. In patients with normal pressure glaucoma, lowering IOP also usually has a positive effect. However, glaucomatous neuropathy often continues to progress even after intraocular pressure has been alleviated due to “secondary” RGC degeneration. “Secondary” degeneration is the process of spreading degeneration to apparently healthy neurons that escaped the primary insult, but are adjacent to the injured, dying or already dead neurons. It is clear that new strategies are required to reduce the number of neurons degenerating in glaucoma and to preserve the surviving RGCs and their axons. Since the exact molecular pathways of RGC death are not well understood, several directions of RGC neuroprotection are being investigated, including blocking glutamate excitotoxicity and stabilizing Ca2 homeostasis, inhibiting nitric oxide production, supplying neurotrophins, preventing apoptosis, improving blood flow to the optic nerve, and modulating immunologic status via vaccination. Examples of the successful application of some of these neuroprotective strategies in animal models and the rational behind those applications are described below.

II. NEUROTROPHIC FACTORS

PROMOTE RGC SURVIVAL

Neurotrophic growth factors are polypeptides that play critical roles in neuronal development, growth, maintenance, and survival. The most intensively studied neurotrophic factors include members of the nerve growth factor (NGF) family, known

as neurotrophins, ciliary neurotrophic factors (CNTF), and glial-derived neurotrophic factors (GDNF). The neurotrophin family consists of NGF, brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4/5), and neurotrophin 6 (NT6) (Barbacid, 1995). These factors regulate cellular metabolism and maintain cellular and neurotrophin homeostasis by interacting with specific cell–surface receptors of neuronal target cells. Two classes of neurotrophin receptors are involved in this process. The low affinity neurotrophin receptor (LANR), a member of the tumor necrosis factor receptor family, also known as the p75 receptor, is common to all four neurotrophins (Bamji et al., 1998). This receptor has no cytoplasmic tyrosine kinase domain (Chao and Hempstead, 1995; Greene and Kaplan, 1995; Segal and Greenberg, 1996), and plays debatable roles, as it may promote or reduce the cellular response to the neurotrophin. The high affinity receptors include receptor tyrosine kinase proteins: Trk A for NGF, Trk B for BDNF and NT-4, and Trk C for NT-3. With lower affinity, NT-3 can also bind to Trk A and Trk B, and NT-4 to Trk A. There are non-catalytic isoforms of Trk B and Trk C that do not contain the cytoplasmic tyrosine kinase catalytic region. It is unknown whether these receptors act as agonists or antagonists.

Pressure-induced changes in the lamina cribrosa may lead to the inadequate physical or metabolic support of RGC axons and subsequent failure of axonal transport. The blockade of retrograde axonal transport in RGC axons may interrupt the supply of neurotrophins to the cell somata from their target cells in the superior colliculus, leading to the initiation of cell death (Quigley and Anderson, 1977; Isenmann et al., 1999). The survival of RGCs appears to be particularly dependent upon BDNF. Although BDNF is produced in the normal retina, including RGCs, the glaucomatous process may disturb normal levels of BDNF and once it reaches the critical level,

the process of cell death is initiated. It has been reported that retrograde transport of BDNF from outside the eye to the RGCs is obstructed by IOP elevation (Quigley et al., 2000). Several reports on BDNF overexpression suggest its beneficial, but limited, effect on RGC survival in optic nerve axot- omy-induced degeneration, as well as in a glaucoma model induced by IOP elevation (Mansour-Robaey et al., 1994; Takano et al., 2002; Watanabe et al., 1997; Martin et al., 2003). There is also evidence that neuronal injury downregulates responsiveness to BDNF (Shen et al., 1999), potentially decreasing the effect of existing levels of BDNF. The AAV-mediated Trk B (receptor for BDNF) gene transfer into RGCs combined with exogenous BDNF administration reported transiently increased neuronal survival after optic nerve transaction (Cheng et al., 2002). The use of other neurotrophins, such as NT-3, NT-4/5, and NGF, has lower effectiveness in RGC rescue compared to BDNF. Interestingly, these results contrast with the BDNF dependence of RGCs in transgenic mice with null mutations for BDNF or NT4, showing no apparent effect on the number of RGCs that survive beyond the period of normal developmental RGC death (Ernfors et al., 1994; Jones et al., 1994; Conover et al., 1995; Liu et al., 1995; Cellerino et al., 1997). It was suggested that because of the extensive overlap in signaling pathways used by the receptors for different neurotrophic factors, the compensating factors may not act on the same receptor as the knocked-out factor (Heumann, 1994; Ghosh and Greenberg, 1995; Tolkovsky, 1997).

Neurotrophic factor CNTF has been shown to have a cell protective effect in various neurodegenerative diseases of the CNS, including animal models of amyotrophic lateral sclerosis and Huntington’s disease (Lindsay 1994; Sagot et al., 1995; Emerich et al., 1996, 1997; De Almeida et al., 2001). CNTF was also reported to protect retinal cells in several RP models, including rd (Cayouette and Gravel, 1997),

rds-peripherin (Cayouette et al., 1998; Bok et al., 2002), rhodopsin-knockout mice (Liang et al., 2001), transgenic animals expressing mutant rhodopsin (Liang et al., 2001; Tao et al., 2002), and others. CNTF promotes RGC survival and enhances axonal regeneration in vitro (Lehwalder et al., 1989), after axotomy (Mey and Thanos, 1993; Cui et al., 1999; Leaver et al., 2006), and in an experimental glaucoma model induced by laser photocoagulation (Ji et al., 2004). In the retina of an experimental glaucoma model, a significantly increased level of endogenous CNTF was observed compared with that of a normal retina. CNTF overexpression can be viewed as a cellular stress-response mechanism to activate anti-apoptotic signaling pathways for RGC neuroprotection. However, the endogenous CNTF level was not sufficient to prevent RGC death in this model. About 13% and 21% of RGCs were degenerated 2º and 4 weeks, respectively, after IOP elevation. Exogenous treatment with CNTF treatment was reported to have a significant neuroprotective effect observed at 2º (PBS 12.6%s vs CNTF 7.4%) and 4 (PBS 21.7% vs CNTF 4.6%) weeks. The neuroprotective effect of CNTF was observed in the central and peripheral retina.

III. RGC PROTECTION FROM GLUTAMATE INDUCED

EXCITOTOXICITY

Glutamate is an excitatory neurotransmitter that activates different types of metabotropic (G-protein coupled) and ionotropic (ion channel-forming) receptors in the CNS (Dingledine et al., 1999). The metabotropic receptors are subdivided into three main families and can be coupled with phospholipase C or adenylyl cyclise (Pin and Duvoisin, 1995). The ionotropic receptors are divided into three classes named by their selective agonists: AMPA (alpha-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid); kainate; and NMDA (N-methyl- D-aspartic acid) receptors (Nakanishi et al.,

1992; Hollmann and Heinemann, 1994). Both AMPA and kainate receptors activate rapid excitatory neurotransmission by supporting the entry of Na into neurons. Some neurons in the retina, hippocampus, and cortex express AMPA that are also permeable to Ca2 . NMDA receptors are associated with a high-conductance Ca2 channel. In the resting, non-depolarizing condition, NMDA receptors are blocked by Mg2 in a voltage-dependent manner. Their activation is secondary to AMPAor kainate-receptor activation, which depolarizes the neuron and releases the Mg2 blockade. The level of glutamate in the synapse is regulated by active, ATP-dependent transporters in neurons and glia (Tymianski and Tator, 1996). When extracellular glutamate levels are increased due either to increased release or decreased uptake from the synapse, it can lead to glutamate-induced excitotoxicity. A high concentration of glutamate through the excessive activation of NMDA receptors triggers an excessive entry of Ca2 , initiating a series of cytoplasmic and nuclear processes that could promote neuronal cell death (Choi, 1988; Sattler and Tymianski, 2000). The main mechanisms by which the excess of Ca2 leads to cell death are:

1.Ca2 activates proteolytic enzymes and endonucleases that can degrade essential cellular proteins and DNA. This can lead to the fatal disruption of cellular structure and function.

2.Ca2 leads to the generation of a highly reactive free radical species, which can oxidize many substrates. (Information about the generation of free radicals and their roles in cellular processes is included in the “Free radical generation and nitric oxide neurotoxicity” section).

3.The influx of Ca2 activates Ca2 – inducible nitric oxide synthase, producing nitric oxide, which diffuses to neighboring neurons and participates in free radical production causing cell death.

Excitotoxicity has been associated with several acute neurological disorders, such as epileptic convulsions and ischemic stroke. A role for excitotoxicity has also been put forward in neurodegenerative disorders like Parkinson’s disease (Loopuijt and Schmidt, 1998), Alzheimer’s disease (Francis et al., 1993a,b), Huntington’s chorea (Alberch et al., 2004), and amyotrophic lateral sclerosis (Plaitakis and Caroscio, 1987). Glutamate excitotoxicity has also been implicated as one of the factors contributing to RGC death in vitro (Otori et al., 1998) and during the glaucomatous process in animal models (Sucher et al., 1997). Yoles and Schwartz (1998) have proposed a self-perpetuating model for the progressive degeneration of RGCs that involves glutamate excitotoxicity. According to their model, “the primary insult, a crush lesion of the optic nerve (or elevated IOP in glaucoma) causes degeneration of directly injured axons and death of their RGCs”. Toxic agents, including glutamate, free radicals, K and others, will leak from dying cells and lead to the death of the neurons that initially escaped the injury (secondary degeneration). Moreover, the authors showed that MK-801, the NMDAreceptor antagonist, protects optic neurons from secondary degeneration, demonstrating the role of glutamate excitotoxicity in this process.

However, the glutamate excitotoxicity hypothesis does not answer the following questions:

1.Since the NMDA receptors mediating glutamate-induced injury are located on the cell body, and not on the axons, how can high levels of glutamate damage the nerve head (Quigley, 1999)?

2.What is the source of the elevated extracellular glutamate found in the vitreous humor? Since RGCs are known to contain high levels of glutamate (Massey, 1990), it would be reasonable to assume that the glutamate could originate from dead ganglion cells, as suggested by Yoles and Schwartz (1998).

However, if the glutamate is released from the ganglion cells, it should be taken up and metabolized by astrocytes and Müller cells (Derouiche, 1996).

It is possible, however, that glaucoma patients have defects in the glutamate inactivation mechanism in the retina. If so, then the glutamate released from neurons will accumulate, killing retinal neurons containing glutamate receptors. The inactivation of glutamate and other neurotransmitters may be compromised when retinal neurons experience an ischemic-like insult, such as reduced ocular blood flow, which has been suggested to participate in glaucomatous damage (Delbarre et al., 1991; Rego et al., 1996).

Since excitotoxicity is implicated in the pathophysiology of a wide variety of acute and chronic neurodegenerative disorders, it presents an attractive target for neuroprotection. Drugs that decrease glutamate release or block its receptors show attenuation of damage in experimental models with a neurodegenerative disease. However, glutamate is absolutely critical for normal neuronal function, and most of the excitotoxicity blocking drugs also block normal neuronal function. These lead to serious side effects and were therefore abandoned in clinical trials (Osborne et al., 1999a; Lipton and Rosenberg, 1994). Memantine, a drug capable of blocking excitotoxic cell death without interfering with normal neurotransmission, has been approved in Europe and the US for the treatment of Alzheimer’s disease, vascular dementia, Parkinson’s disease, and spasticity. It has also been considered a potential treatment for glaucomatous optic neuropathy and is now a subject of clinical studies to determine its efficacy and safety (Chen et al., 1998; Lipton 1993; Le and Lipton, 2001). Memantine-mediated neuroprotection has been reported in animal models of glaucoma (Hare et al., 2001).

Drugs stabilizing Ca2 homeostasis may also be beneficial in efforts to preserve

RGCs in glaucoma. Beta-adrenoceptor antagonists, or β-blockers, such as betaxolol, levobunolol, and timolol, are commonly used for glaucoma treatment because of their ability to lower IOP. Furthermore, there is evidence that betaxolol (and metipranolol, and timolol to a lesser extent) may have a neuroprotective effect by suppressing glutamate-induced intracellular calcium increases in RGCs (Zhang et al., 2003; Wood et al., 2003). The accumulation of Ca2 within neurons and their axons leading to neuronal injury may also result from persistent activation of voltage-gated sodium channels. The blockade of volt- age-gated sodium channels by phenytoin reduced the loss of RGCs in glaucomatous eyes of phenytoin-treated animals to 8%, compared to a 51% loss with vehicle-treated animals. The axon density in phenytointreated animals was 98% of that in controls, compared to 83% in vehicle-treated animals (Hains and Waxman, 2005).

Activation of alpha-2 receptors by brimonidine has been shown to enhance survival of retinal neurons in chronic ocular hypertension, optic nerve crush, ischemia, photoreceptor degeneration, models (Wheeler et al., 2001; Yoles et al., 1999; Donello et al., 2001; Wen et al., 1996). Alpha-2 receptors have been implicated in activation of various pathways, including the activation of intracellular kinases that enhance cell survival, and the inhibition of glutamate release and calcium influx.

Finally, estrogens have been demonstrated to have neuroprotective effects against glutamate cytotoxicity (Zaulyanov et al., 1999; Green et al., 1998; Bhavnani et al., 2003). The neuroprotective effect of estrogen was associated with its anti-inflammatory and anti-oxidant characteristics (Harms et al., 2001; Behl et al., 1997; Dykens et al., 2003). Estrogen neuroprotection can be achieved via an estrogen receptor-mediated process or independently (Garcia-Segura et al., 2001). Immunohistochemical staining with the anti-estrogen receptor showed expression in the neural retina with extensive

distribution in the nerve fiber layer, the ganglion cell layer, the inner nuclear layer, and the outer plexiform layer (Kobayashi et al., 1998). Recent studies indicate that estrogen promotes RGC survival following glutamate exposure (Kumar et al., 2005) and after optic nerve axotomy (Nakazawa et al., 2006). In the axotomy model, the neuroprotective effect of estrogen was shown to be mediated by estrogen receptor.

IV. PROTECTION FROM FREE RADICALS AND NITRIC OXIDE NEUROTOXICITY

Free radicals are uncharged atomic or molecular species with unpaired electrons in their outer orbits, and these unpaired electrons cause them to react almost instantly with substances in their vicinity. Free radicals play an important function in a variety of biological processes. Some of these processes, such as the intracellular killing of bacteria by neutrophil granulocytes, are necessary and beneficial for life. However, free radicals can also participate in biochemical reactions causing cell damage. Oxygen-free radicals are especially hazardous because of their high reactivity. Reactions between free radicals and DNA could lead to mutations that may adversely affect different biological functions, including the cell cycle, and may consequently be responsible for many forms of cancer. Some consequences of aging, such as atherosclerosis, are also attributed to free radicalinduced oxidation. To deactivate free radicals and minimize their damage, cells exploit several molecules, such as antioxidants (vitamins C and E), glutathione, and superoxide dismutase.

Glutamate excitotoxicity increases the level of intracellular Ca2 , which could lead to the generation of free radicals. Free radicals are also generated as a by-product of normal oxidative metabolism (Boveris and Chance, 1973), especially in tissues with a high metabolic rate, such as the retina.

Mitochondria, the main source of reactive oxygen species (ROS) in the excitotoxic process, as well as many other enzymatic systems, participate in the production of free radicals in the CNS (Dugan et al., 1995). An increased entry of Ca2 into mitochondria, which plays an important role in the regulation of the intracellular calcium concentration, enhances electron transport, increasing the level of ROS. The peroxyand hydroxyl-radicals generated by the peroxidation of fatty acids initiate lipid peroxidation, leading to irreversible changes in the physical and chemical properties of cell membranes. Ca2 -activated phospholipase A2 hydrolyzes membrane phospholipids that generate free radicals, including superoxide anions.

Calcium also activates NO-synthase, increasing the presence of nitric oxide (NO) in the neurons and also in surrounding areas. NO has important physiological and pathological roles (Moncada and Higgs, 1993; Bredt and Snyder, 1994; Garthwaite and Boulton, 1995). It is a free radical that reacts with heme groups on a variety of proteins, leading to the activation or inactivation of proteins (Stamler, 1994). NO has an essential role as a signaling molecule in signal transduction systems, which are especially important in the brain and the cardiovascular system (Moncada, 1992). Nitric oxide neurotoxicity occurs through the reaction of NO with a superoxide anion to form a highly toxic peroxynitrite and other more reactive free radical species. Peroxynitrite is a strong oxidizing agent that causes nitration in proteins and the oxidation of lipids, proteins and DNA, leading to apoptosis. In animal models of human neurodegenerative diseases, NO has been implicated in stroke (Kiechle and Malinski 1993), multiple sclerosis (Bo et al., 1994), and Parkinson’s disease (Hunot et al., 1996). It has also been suggested that excessive NO, found in reactive astrocytes of the lamina cribrosa, leads to irreversible damage of the RGC axons at the level of the optic nerve head (Neufeld and Liu, 2003). Selective

inhibition of inducible NOS-2 (L-N6- (1-iminoethly)lysine 5-tetrazole amide) is shown to be effective in preventing the loss of RGCs in rats with glaucoma (Neufeld et al., 2002). Nitro-memantine (secondgeneration memantine derivative) also shows neuroprotective effects in animal models by reducing the effect of NO on the NMDA channel (Le and Lipton, 2001).

V. ANTI-APOPTOTIC THERAPY

It has become clear in recent years that in different optic neuropathy models, including axon transection, optic nerve crush, ocular ischemia, ocular hypertension and excitotoxin injection, RGCs die by distinct morphologic and biochemical features characteristic of apoptosis, including chromatin condensation, the formation of apoptotic bodies, and DNA fragmentation (Nickells, 1999; Osborne et al., 1999b; Farkas and Grosskreutz, 2001). Although the exact molecular pathways of RGC apoptosis are not understood, anti-apoptotic therapy can offer a new way of RGC protection in glaucomatous neuropathy. Anti-apoptotic therapy is based on inhibiting apoptosis in cells with an activated self-destruction pathway. Once this pathway is blocked, the damaged cells can repair themselves and restore their normal function. Some of the cells will die anyway, since the severity and extent of the damage is beyond repair. There are many reports in the literature describing the protective effect of anti-apoptotic therapies in animal models. To better understand how anti-apoptotic therapeutic strategies can be designed and applied to preserve RGCs, some general information about apoptotic cell death is provided below.

Although the apoptotic death of neurons can be initiated by different stimuli, common morphological and biochemical alterations observed after triggering apoptosis suggest that most apoptotic pathways converge on a limited number of common effector routes (Sastry and Rao, 2000)

(Figure 19.1). There are two principal pathways leading to apoptotic cell death: the extrinsic, death receptor-initiated pathway, and the intrinsic or mitochondrial pathway (Strasser et al., 2000; Green et al., 1998). The extrinsic pathway originates with the binding of death-promoting ligands to their cognate death receptors (Pinkoski and Green, 1999). There is a large family of death ligands and death receptors in different tissues. Two members of the tumor necrosis factor receptor (TNFR) family, Fas and the p75 neurotrophin receptor (p75), were implicated in neuronal death (Raoul et al., 2002). Upon exposure to deathtriggering stimuli, cells express the Fas ligand (Fas-L), which binds Fas and induces its oligomerization. Two pathways involving Jun amino-terminal kinase (JNK) (Le Niculescu et al., 1999) and protein kinase B (PKB or Akt) (Brunet et al., 2001) have been shown to regulate Fas-L expression. Once the Fas/Fas-L complex is formed, the Fasassociated death domain protein (FADD) (Thorburn, 2004) initiates the activation of procaspase 8, which in turn leads to the activation of effector caspase 3 (Blatt and Glick, 2001).

The p75 receptor was discovered as a neurotrophin receptor, but more recent studies have been focused on its putative role in neuronal apoptosis (Rabizadeh and Bredesen, 1994; Barker, 1998). The p75 intracellular signal transduction pathway (Barrett, 2000) is still unknown and probably different from that of Fas or TNFR, since p75 has a different death domain (Liepinsh et al., 1997). Using antisense oligonucleotides targeting p75, it was demonstrated that p75 is required for the post-axotomy death of sensory neurons (Cheema et al., 1996). Reduced neuronal death in the retina, certain spinal cord interneurons, and sympathetic neurons were observed in p75 knockout animals (Bamji et al., 1998; Frade and Barde, 1999). Interestingly, ngf / and bdnf / knockout animals suggest that apoptosis of the retinal and spinal cord neurons is triggered

-95 Fas/CD

Ask1

TNFR1

Ask1

Ikk

by the binding of NGF to p75 (Frade and Barde, 1999), whereas sympathetic neuronal death is initiated by a BDNF/p75 complex formation (Majdan et al., 1997).

The intrinsic or mitochondrial pathway may be induced by a variety of structurally unrelated agents and have several distinct mechanisms (Green, 1998). Elevated cytosolic Ca2 and oxidative stress both contribute to the opening of the mitochondrial permeability transition pore (PTP), which depolarizes the mitochondria and

leads to mitochondrial swelling and the subsequent release of cytochrome c from the intermembrane space. Cytochrome c normally functions as part of the respiratory chain, but when released into the cytosol (as a result of PTP opening), it becomes a critical component of the apoptosis execution machinery (Adrain and Martin, 2001).

The release of cytochrome c may also be assisted by the activation of pro-apoptotic members of the Bcl family (BAD, BAX, and BID) (Zimmermann et al., 2001). The

anti-apoptotic proteins BCL-2 and BCL-XL on the mitochondrial outer membrane protect cells from death by preventing the release of cytochrome c from the mitochondria (Yang et al., 1997; Kluck et al., 1997). During apoptosis, pro-apoptotic molecules translocate from the cytosol to the mitochondria, where they induce the release of cytochrome c (and other proteins) from the intermembrane space. Once cytochrome c is released into the cytoplasm, it binds to Apaf-1 (apoptotic protease-activating factor 1) to form the caspase-activating complex known as apoptosome (Salvesen and Renatus, 2002). Apoptosome recruits procaspase 9 and activates effector caspases 3 and 7.

Another protein that may be released into the cytoplasm from the mitochondria is apoptosis-inducing factor (AIF). The overactivation of poly (ADP-ribose) polymerase 1 (PARP-1), an important activator of cas- pase-independent cell death (Smith, 2001), initiates a nuclear signal that propagates to mitochondria and triggers the release of AIF. In contrast to cytochrome c, AIF acts in a caspase-independent fashion (Figure 19.1) (Daugas et al., 2000). Upon death signaling, AIF, containing putative NLS (nuclear localization signal), translocates to the nucleus, binds to DNA, and leads to chromatin condensation and large scale ( 50 kb) DNA fragmentation. The inactivation of AIF appears to abolish the early neuronal death of proliferating precursor cells and young postmitotic neuroblasts (Joza et al., 2001). Recently, the caspase-independent cell death pathway was implicated in different types of neurodegeneration, including photoreceptor degeneration (Doonan et al., 2003).

PARP-1 is an important factor implicated not only in caspase-independent, but in cas- pase-mediated apoptosis and necrosis as well. The cleavage of PARP-1 by caspases, the main executors of apoptosis, is now accepted as the hallmark of apoptosis. PARP is also involved in the regulation of various biological processes, such as replication,

transcription, and protein degradation. The role of PARP-1 in DNA repair is still considered to be its primary function.

p53 is involved in both the extrinsic and the intrinsic pathways of apoptosis. One of the many responsibilities of p53 is to survey cellular stress and damage, and, if necessary, to initiate cell death (Hofseth et al., 2004). Biochemical mechanisms underlying p53-dependent apoptotic responses are not completely characterized. It was suggested that p53 activates apoptosis through a three-step process: (1) the transcriptional induction of redox-related genes; (2) the formation of reactive oxygen species; and

(3) the oxidative degradation of mitochondrial components, culminating in cell death (Polyak et al., 1997). p53 has also been implicated in the increase of the expression of cellular death receptors, and the stimulation of apoptotic infrastructure by increasing the expression of APAF-1. Activated p53 can directly or indirectly modulate the expression of the proteins that control mitochondrial membrane permeability and can modulate the release of mitochondrial proteins during apoptosis. p53 has a significant role in apoptosis that follows DNA damage in vivo (Wood and Youle, 1995). p53 also functions in the p75 apoptotic signal cascade (Aloyz et al., 1998).

In an ocular hypertensive rat model of glaucoma, the apoptotic death of RGCs was associated with the activation of members of the caspase family, including caspases 3, 8, (McKinnon et al., 2002a) and 9 (Hanninen et al., 2002). It has also been suggested that a caspase-independent mechanism may play a role in RGC death in vitro (Tezel and Yang, 2004). Bonfanti et al. (1996) have shown that RGCs in transgenic mice overexpressing Bcl2 are rescued from cell death during retinal development and after axotomy. The expression of baculoviral IAP repeatcontaining 4 (BIRC4), a caspase 3 inhibitor, prevented optic nerve axon loss in a rat glaucoma model (McKinnon et al., 2002b).

Tezel et al. (2001) analyzed neuronal loss following optic nerve crush in mice deficient

for TNF receptor-1 (TNF-R1). Both TNF-α and TNF-R1 are constitutively expressed in the normal retina and optic nerve head and were found to be upregulated in the glaucomatous retina, suggesting the involvement of TNF-mediated cell death in glaucoma (Tezel et al., 2001; Yan et al., 2000). TNF-R1 was shown to be localized predominantly to RGCs and their axons. It was proposed that TNF-α, mostly produced by activated glial cells (Lieberman et al., 1989), has cytotoxic effects on RGCs (Tezel et al., 2001) and their axons. A significant reduction of RGC loss was observed in TNF-R1 deficient mice compared to control animals during 6 weeks after the injury. Axonal loss 6 weeks after optic nerve injury was 42% in TNFR1 deficient mice compared to 69% in ageand gender-matched controls. RGC loss in TNF-R1 / animals was 37% versus 61% in control mice. Furthermore, similar protection of RGCs was shown with the specific inhibition of JNK. Results of these studies also demonstrate the involvement of the TNF pathway in association with JNK signaling in RGC cell death resulting from optic nerve injury (Tezel et al., 2004).

Huang et al. (2005) showed that calcineurin (CaN), a Ca2 calmodulindependent protein phosphatase, is involved in RGC apoptosis in the glaucoma model, and its inhibition by oral FK506 is neuroprotective for RGCs and the optic nerve in eyes with elevated IOP. CaN is known to mediate the dephosphorylation of the proapoptotic Bcl-2 family member, Bad, which subsequently leads to the release of cytochrome c, caspase activation, and apoptotic cell death (Wang et al., 1999).

VI. RGC PROTECTION WITH

HSP70

The overexpression of an inducible form of heat shock protein 70 (HSP70) has been shown to increase the rate of cell survival in different forms of neurodegeneration. The HSP70 family is the most abundant

HSP group of molecular chaperones in eukaryotes and includes both constitutive (HSC70) and inducible (HSP70 or HSP72) members. HSP70 is involved in a wide spectrum of cellular activity, including protein synthesis, folding, translocation, and oligomerization. As molecular chaperones, they recognize and bind to unfolded or misfolded proteins, prevent their aggregation, and assist them to obtain a proper native conformation. The involvement of HSP70 in stimulating a cell survival process in different types of stress-induced apoptosis, such as ischemia, irradiation, and heat shock, is well described (Gabai et al., 2000; Jaattela and Wissing, 1993; Mosser et al., 1997; Sharp et al., 1999). Although numerous papers have been published on the neuroprotective role of HSP70, the exact mechanism of neuroprotection by this stress protein remains unknown. It may be explained by the chaperoning function of HSP70 on proteins that are important for the survival of cells. HSPs participate in the folding and assembly of nascent and unfolded peptides, and they facilitate protein transport to specific subcellular compartments and disposal by degradation (Hartle 1996; Rokutan et al., 1998). The overexpression of HSP70 protects mitochondria from the deleterious effect of ROS (Polla et al., 1996). The HSP70 cell protection effect can be explained by its ability to inhibit several apoptotic signaling pathways, including p38 MAPK apoptosis signaling and the JNK-dependent pathway (Gabai et al., 1997; Park et al., 2001a). It can also block the assembly of a functional apoptosome by binding to Apaf-1 and preventing the recruitment of caspases to the apoptosome complex (Beere et al., 2000). Moreover, HSP70 may inhibit caspaseindependent cell death by interacting with the apoptosis inducing factor (Figure 19.2) (Matsumori et al., 2005).

HSP70 induction in a rat glaucoma model by a systemic administration of the divalent cation zinc showed increased survival of RGCs compared to the control

ROS

NECROSIS

Apaf1

Bcl-2

Cyto C

Cyto C

HSP70

Apaf1

AIF

Caspase 9

HSP70

Caspase 3

Caspase independent

APOPTOSIS

APOPTOSIS

group (Figure 19.3) (Park et al., 2001b). The neuroprotective effect of zinc treatment was reversed by an inhibitor of HSP expression, quercetin. Zinc is involved in most cellular metabolic processes as an essential co-factor of many enzymes (Karcioglu, 1982; Barcelouz, 1999). The dosage (10 mg/kg of zinc twice a week) used in this experiment

to stimulate HSP70 expression showed no systemic side effects during the 4 weeks of the study period. Zinc generally has lower toxicity than other transition metals (Choi and Koh, 1998). Rats receiving 16 mg/kg zinc daily for 32 weeks showed no pathologic changes (Denkert et al., 2002). In a human clinical study, zinc has been tested