apoptosis in glaucoma will not necessarily be the same. This implies that the death pathways for different ganglion cells dying at various stages in glaucoma may not be the same.

Mitochondrial functions and apoptosis

Mitochondria are involved in numerous metabolic functions that include oxidative energy metabolism, the related production of ROS, and a role in promoting and regulating apoptotic death of the cell (Schon and Manfredi, 2003; Chan, 2006). Among the numerous stresses known to participate in mitochondrion-mediated apoptosis, at least in vitro, bioenergetic failure and elevated ROS figure prominently (Kroemer and Reed, 2000; Schon and Manfredi, 2003). Moreover, it is now established that light can be absorbed by mitochondrial photosensitizers, such as cytochrome oxidase and flavin-containing oxidases, causing a production of ROS (King et al., 2004; Godley et al., 2005). It is also known that the rate of ROS production from mitochondria is increased in a variety of pathologic conditions that include aging, hypoxia, and ischemia (Osborne et al., 1999; Chan, 2006; Morin and Simon, 2006).

Of key importance is the role of mitochondria in oxidative energy metabolism. Oxidative phosphorylation generates most of the cell’s ATP, and any impairment of the organelle’s ability to produce energy can have catastrophic consequences, not only due to the primary loss of ATP, but also due to indirect impairment of downstream functions, such as maintenance of organellar and cellular calcium homeostasis. Moreover, deficient mitochondrial metabolism will generate ROS that can wreak havoc in the cell. It is for such reasons that all evidence points to inadequate mitochondrial function being linked to apoptosis. This is supported by laboratory findings showing that substances that can maintain mitochondrial function (e.g., to allow ATP to be generated efficiently) or scavenge excessive ROS production blunt the process of apoptosis.

The hallmarks of apoptosis are condensation of nuclear and cytoplasmic contents, nuclear DNA fragmentation, cell blebbing, and autophagy of

343

membrane-bound bodies. Apoptosis appears to occur via different pathways. A mitochondrionmediated (intrinsic) pathway has been described where an external insult –– elevated cytosolic calcium, to cite one example –– acts to cause a release of cytochrome c, located in the inner membrane space of mitochondria. Cytosolic cytochrome c can then bind apoptotic proteaseactivating factor 1 (APAF-1), which then binds to the inactive form of caspase-9 (see Fig. 3). This complex ‘‘apoptosome’’ can then activate a cascade of events, which includes caspase-3, ultimately resulting in the hallmarks of apoptosis. Mitochondrion-mediated activation of caspase-9 can also occur via extracellular receptor-mediated signals (extrinsic pathway) to target various ligands (growth factor deprivation) –– e.g., Bad, Bax, and Bik –– to the mitochondrion, thereby causing cytochrome c release. Under certain circumstances, a separate mitochondrial-indepen- dent pathway also operates, which involves the activation of caspase-8 and caspase-3. In addition, a caspase-independent/mitochondrial-dependent pathway exists where apoptosis-inducing factor (AIF), confined to mitochondria, is released and translocates to the nucleus to cause chromatin condensation and DNA degradation (see Fig. 3).

Mitochondrial function enhancement and the attenuation of ganglion cell death

Mitochondria provide the bulk of a neurone’s energy by oxidation of reducing equivalents (e.g., NADH and FADH2), via the electron transport chain, to ultimately yield ATP. In addition, mitochondria contribute to cytosolic calcium buffering (Steeghs et al., 1997), apoptosis (Green and Reed, 1998; Kroemer and Reed, 2000; Schon and Manfredi, 2003), excitotoxicity (Peng et al., 1998), and generation of superoxide (McLennan and Degli Esposti, 2000). Also, mitochondrial flavin and cytochrome oxidases are affected by light to stimulate a production of ROS. Thus, an alteration in any aforementioned process or a combination of the processes maybe a cause of retinal ganglion cells dying in glaucoma.

Substances that enhance mitochondrial function might therefore benefit glaucoma patients because ganglion cell axons have many mitochondria and these organelles are implicated in their apoptosis. Targeting impaired energy production is a realistic possibility. This will necessitate the use of an agent that can reach the retina, enhance the metabolic state of energetically compromised ganglion cells, and not affect healthy retinal cells adversely. Substances that might fulfil these criteria include creatine, a-lipoic acid, nicotinamide, and epigallocatechin gallate (EGCG), and each will be considered in turn. All four substances can be taken orally at regular intervals by humans without any obvious detrimental affects.

Creatine

Creatine is a guanidine compound that is ubiquitous among mammalian cells. Its concentration appears to reflect the energy requirements of various tissues, being highest in retinal

photoreceptors with intermediate levels in the brain (Wyss and Kaddurah-Daouk, 2000). Creatine is released from the liver or consumed in the diet and transported into cells by specific transporters (Wyss and Kaddurah-Daouk, 2000). Within the cell, ATP and ADP and creatine are metabolized to form phosphocreatine by mitochondrial creatine kinase. Phosphocreatine then serves not only as an intermediate temporary energy buffer, but also as an energy shuttle from subcellular sites of energy production (mitochondria) to sites of energy consumption (Brewer and Wallimann, 2000). Thus, the phosphocreatine/ creatine kinase system has been suggested to be of physiological importance in tissues with high energy and fluctuating energy requirements. This should apply to the intraretinal ganglion cell axons in particular because of their many mitochondria and presumed high level of creatine kinase. It has also been hypothesized that supplementation with creatine will result in cells making more phosphocreatine (Brewer and

Wallimann, 2000), and this has been supported by experimental studies. For example, creatine supplementation attenuates accumulation of oxidative stress markers and is shown to scavenge superoxide and peroxynitrite (Lawler et al., 2002). In cell culture experiments, creatine protects against toxicity induced by glutamate or b-amyloid (Brewer and Wallimann, 2000). Creatine supplementation has also been shown to exhibit remarkable neuroprotection in animal models of amyotropic lateral sclerosis (Klivenyi et al., 1998), Huntington’s disease, Parkinson’s disease, and traumatic brain injury (Zhu et al., 2004). Thus, the idea that creatine supplementation is beneficial for the treatment of glaucoma by attenuating the death process to ganglion cells seems to be a real possibility.

In preliminary studies, rats were injected subcutaneously twice a day with creatine (1 g/kg body weight/day) for six days, which was followed a day later for another five days (because of Sunday break from the laboratory). Controls received

345

saline. On the sixth day, NMDA (concentration within the vitreous estimated to be 100 mM) was injected into the vitreous humor of one eye while the other received vehicle. On the 12th day, the animals were killed. Retinas were fixed and optic nerves analyzed by Western blotting for neurofilament light (NF-L) protein content relative to actin. Sections of fixed retinas were cut in areas of similar eccentricities. Eyes injected with NMDA showed a clear reduction of amacrine (ChAT) and ganglion (Thy-1) cell markers when compared with vehicleinjected eyes. However, in those animals that had been prophylactically treated with creatine, the effect of the NMDA injection was less evident (Fig. 4). Moreover, analysis of NF-L content in optic nerves showed that the reduction caused by NMDA treatment was significantly less in rats treated with creatine. These preliminary studies therefore provide support for the idea that creatine supplementation can attenuate an NMDA insult to inner retinal neurones, which include ganglion cells.

346

a-Lipoic acid

a-Lipoic acid (1,2-dithiolane-3-pentanoic acid) is a mitochondrial dithiol compound that functions as a coenzyme for pyruvate dehydrogenase and a- ketoglutarate dehydrogenase. It is a cofactor in the mitochondrial dehydrogenase complex that catalyzes the oxidative decarboxylation of a-keto acids such as pyruvate and a-ketoglutarate. In the process, a-lipoic acid is reduced to dihydrolipoic acid and the two substances operate as a redox couple. Besides quenching of free radicals, lipoic acid/dihydrolipoic acid chelate transition metals and also assist in the regeneration of other antioxidants, such as glutathione, a-tocopherol, and ascorbate (Biewenga et al., 1997). Administration of a-lipoic acid to rodents has been demonstrated to reduce the damage that occurs after ischemic-reperfusion injuries in the cerebral cortex (Packer et al., 1997), heart (Freisleben, 2000), and peripheral nerve (Mitsui et al., 1999). a- Lipoic acid has also been found to exert protective effects in cell culture models of hypoxia and excitotoxicity (Tirosh et al., 1999). Despite such positive results, the effectiveness of a-lipoic acid as a neuroprotectant in the retina has largely been ignored to date. It is known that in the early diabetic rat retina, decreased free cytosolic and mitochondrial NAD+/NADH ratios and increased levels of 4-hydroxyalkenals, which are indicative of retinal hypoxia and increased lipid peroxidation, respectively, are reversed by

treatment with a-lipoic acid (Obrosova et al., 2001). It is also important to note that a-lipoic acid is well tolerated by humans with no known side effects reported when taken daily (500–600 mg).

In order to test whether a-lipoic acid might be a candidate drug for the prophylactic use in attenuating ganglion cell death in glaucoma, we examined this possibility in a rat model given ischemia (Chidlow et al., 2002). Rats were injected intraperitoneally with either vehicle or a-lipoic acid (100 mg/kg) once daily for 11 days. On the third day, ischemia was delivered to the rat retina by raising the intraocular pressure above systolic blood pressure for 45 min. The electroretinogram (ERG) was measured prior to ischemia and five days after reperfusion. Rats were killed five or eight days after reperfusion, and the retinas were processed for immunohistochemistry and for determination of mRNA levels by RT-PCR. Ischemia –reperfusion caused a significant reduction in the a- and b-wave amplitudes of the ERG (Fig. 5), a decrease in nitric oxide synthase and Thy-1 immunoreactivities, a decrease in retinal ganglion cell-specific mRNAs (Thy-1 and NF-L), and an increase in bFGF and CNTF mRNA levels. All of these changes were clearly counteracted by a-lipoic acid. Moreover, in mixed rat retinal cultures, a-lipoic acid partially counteracted the loss of GABA-immunoreactive neurons induced by anoxia. The results of the study demonstrate that a-lipoic acid provides