glaucomatous Bax / mice exhibited complete abrogation of ganglion cell soma loss (Fig. 3). Thus, regardless of what combinations of apoptotic pathways are activated in ganglion cells, the initial pathology in the retina must occur through a Bax-dependent, intrinsic pathway. Second, even though ganglion cell somas were spared, glaucomatous Bax / mice still exhibited maximal damage to the optic nerve, including the complete loss of axons. This observation has helped to formulate the concept that RGCs die using compartmentalized self-destruct pathways (Whitmore et al., 2005). In this model, axons, dendrites, somas, and synapses may all die independent of each other. Such a model may help explain the link between elevated IOP and the initial activation of ganglion cell loss in glaucoma (Whitmore et al., 2005; Nickells, 2007). Third, this study also revealed that Bax gene dosage could markedly affect cell survival. Previous studies using Bax mutant mice had suggested that only a single functioning Bax allele was sufficient for normal activation of neuronal death (Deckwerth et al., 1996). In the DBA/2J genetic background, however, Bax+/ mice exhibited the same attenuated

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ganglion cell death observed in completely Bax- deficient mice. Recent studies using the Bax mutant allele in different genetic strains have also shown that reducing Bax gene dosage reduces transcript levels from this gene (Fig. 3). Strain differences in the cell death response to Bax gene dosage may be related to polymorphisms in the Bax gene promoter that affect transcriptional activity of this gene (S. Semaan and R. Nickells, unpublished). These studies suggest that therapeutically reducing BAX levels by just 50% could have a pronounced effect on the survival of ganglion cells.

BH3-only proteins and the early signaling of ganglion cell apoptosis

As noted in an earlier section of this review, the Bcl2 gene family is divided into three main subgroups based on the presence of common amino acid domains. We have already discussed the potential functions of the BclX and Bax genes and products, but the critical element for the activation of intrinsic apoptosis is the interactions of these proteins with the members of the BH3-only family

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of molecules. Evidence exists for the function of BIM, BAD, and proteolytically cleaved BID (tBID) peptides in the process of RGC death. In one respect, evidence for an active role of each may seem contradictory to a common final pathway because the known activation events of these BH3only proteins are dramatically different. In this section, we will suggest a model that incorporates all three of these peptides in the overall process of ganglion cell death. This model is based on a recent hypothesis that BH3-only proteins are really sentinels of the apoptotic program, which are recruited to help amplify and accelerate the process (Adams and Cory, 2007).

The actual function of the BH3-only proteins is controversial. By virtue of the BH3 domain, they are able to interact with the globular BH domains of other Bcl2 family proteins. In many cells, the presence of BH3-only proteins dramatically facilitates the cell death process. This lead to early hypotheses that these small proteins interacted directly with pro-apoptotic Bcl2 family proteins, like BAX, possibly targeting them to organellar membranes or allowing these proteins to change conformation and insert into lipid bilayers. Current experimental evidence, however, suggests that this model is not correct. In vivo, BH3-only proteins show a distinct affinity for anti-apoptotic members, such as BCL2 and BCL-X (Adams and Cory, 2007). A new model of indirect activation has emerged that suggests that the activation of BH3-only proteins allows them to interact and neutralize anti-apoptotic proteins, thus limiting their ability to antagonize their pro-apoptotic counterparts.

Studies using Bax / mice clearly show that a Bax-mediated program of cell death must be initiated in order to achieve complete retinal pathology (Li et al., 2000; Libby et al., 2005b). In the compartmentalized cell death model of glaucoma, the initiating event of ganglion cell soma death is the deficit of neurotrophins due to the blockade of axoplasmic transport in the lamina cribrosa. This process is biochemically similar to serum deprivation in cultured neurons. Studies using primary cultures of neurons showed that this traumatic episode activated both the transcriptional upregulation, and then the post-translational

phosphorylation, of the BIM BH3-only protein (Putcha et al., 2003). This study also showed that the activating phosphorylation of BIM was mediated by c-Jun N-Terminal Kinases (JNKs) localized to the mitochondria. Several studies have identified BIM regulation and activation as one of the critical molecules participating in ganglion cell death after experimental trauma to the optic nerve (Na¨pa¨nkangas et al., 2003; McKernan and Cotter, 2007). The role of BIM in glaucoma is less clear, but several investigators have observed JNK upregulation and activation in rat models of experimental glaucoma (Kwong and Caprioli, 2006; LevkovitchVerbin et al., 2007), suggesting a link with BIM activation.

Other studies have also found associations between the activation of the BH3-only protein BAD and RGC death. Like BAX, the proapoptotic action of BAD requires interaction with charged lipids in the mitochondrial outer membrane. BAD is often present as a latent phosphorylated molecule in cells. Phosphorylation appears to block its interaction with lipids and facilitates an interaction with 14-3-3 inhibitory peptides (Hekman et al., 2006). The activation of BAD generally requires the activity of a protein phosphatase such as Calcineurin, which removes these inhibitory phosphates. Recently, Huang and colleagues showed that activated Calcineurin was present in ganglion cells of rat models of optic nerve trauma, including experimental glaucoma, and in aged DBA/2J mice (Huang et al., 2005b). This led to a decrease in the level of phosphorylated BAD and was temporally associated with increased ganglion cell death. In addition, pharmacologic inhibition of Calcineurin using FK506 was able to attenuate the process of cell death, at least in a short-term analysis of 10 days post ocular hypertension. Overall, the activation of Calcineurin, by proteolytic cleavage, is linked to increased levels of intracellular calcium ions and the activation of calmodulin. In dying cells, the most likely source of free calcium ions is from stores in the endoplasmic reticulum (ER), which are extruded during phases of ER stress. Intrinsically, the mediator of this release is the secondary integration of activated BAX protein into the ER lipid bilayer (Nutt et al., 2002; Breckenridge et al.,

2003). In Bax / neurons, for example, Ca2+ ions are not released from ER stores. Thus, even though the activity of BAD is to neutralize antiapoptotic proteins, presumably allowing BAX to function, the activation of BAD may be reliant on the initial activation of BAX by some other mechanism.

The third BH3-only protein that has been found to play a role in ganglion cell death is BID. Like BIM and BAD, the putative function of BID is to neutralize anti-apoptotic proteins. Unlike its two counterparts, however, BID predominantly plays a role in amplifying the extrinsic apoptotic pathway (Adams and Cory, 2007). As discussed above, extrinsic apoptosis initiates from an extracellular interaction between a death-inducing ligand, such as TNFa, with a death receptor located on the membrane of the target cell. This interaction directly activates the caspase cascade, initially through the proteolytic cleavage of procaspase 8. One of the proteolytic targets of caspase 8 is inactive BID, which when cleaved to become tBID, is able to interact with multiple antiapoptotic Bcl2 family members (Adams and Cory, 2007). This sequence of events is not essential for the full activation of the caspase cascade and cell death, but instead helps amplify and accelerate the process by recruiting the intrinsic pathway as well. There is a great deal of interest in this pathway in the process of RGC death and glaucoma. Several studies have helped establish that TNFa levels are elevated in eyes after optic nerve trauma, including glaucoma (Tezel et al., 2001; Tezel et al., 2004), and there is an increase in the level of tBID in ganglion cells (Huang et al., 2005a). The relative importance of the TNFa pathway has been established using mice carrying mutations in the TNFa receptor-1 and 2 genes. TNFa-R1 mutants, after optic nerve crush, show no immediate change in the rate of ganglion cell loss, but a significant attenuation of loss 2 weeks after the injury (Tezel et al., 2004), supporting a role for TNF-mediated cell death in secondary degeneration. Surprisingly, TNFa-R1 mutants show no effect in a mouse model of ocular hypertension, but TNFa-R2 mutants do (Nakazawa et al., 2006). The delayed reliance on TNF-mediated ganglion cell death suggests that it plays a more significant role in late

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stages of retinal pathology. This is even more likely since the principal source of TNFa is activated glia, particularly microglia, in the retina (Tezel and Wax, 2000; Tezel et al., 2001; Nakazawa et al., 2006).

Given the available evidence, the following hypothetical model of BH3-only protein mediated activation of ganglion cell death arises (Fig. 4). Initially, neurotrophin deprivation stimulates activating kinases that lead to the upregulation and modification of BIM. BIM neutralizes some of the BCL-X in ganglion cells, allowing for BAX, which has been translocated and inserted into the mitochondrial membrane, to facilitate an increase in membrane permeability. If this step is blocked, such as in Bax / or Bim / mice, there is complete and long-term rescue of cell death (Li et al., 2000; Libby et al., 2005b; McKernan and Cotter, 2007). If BAX does become active in this initial stage, then not only can it insert into mitochondrial membranes, but it can also insert into ER membranes, precipitating ER stress and the release of calcium ions. The release of Ca2+ is potentially cytotoxic by itself. If calcium levels become too high, cell death can ensue by a variety of mechanisms, many of which are not as controlled as apoptosis and thus deleterious to the overall health of the organism or surrounding tissue. Since apoptosis is in the best interest of the organism, elevated calcium levels are sensed by another BH3-only protein, BAD, which becomes activated via a calcium-calmodulin dependent proteolytic mechanism. Activated BAD then collaborates with BIM by also competing for and neutralizing BCL-X. This could be considered as a second gear in the apoptotic machinery. As ganglion cell somas begin to die, the overall pathology of the retina is sensed by resident glial cells. In some cases, glia, such as astrocytes may try to prevent further ganglion cell death. Other cells, however, may exacerbate the pathology. It is suspected that TNFa release principally originates from activated microglia. The presence of TNFa activates concomitant receptors on ganglion cells, thus activating extrinsic apoptosis and the tBID BH3-only sensor. As with BAD, tBID likely functions to help neutralize BCL-X along with BIM. An interesting caveat to the TNFa-mediated

pathway, however, is that unlike BAD, tBID activation may occur in cells that do not have already activated BAX protein in them. Thus, TNFa could be a critical component of the suspected secondary degeneration of healthy

ganglion cells not directly affected by pathology at the lamina cribrosa (Schwartz, 2005; Tezel, 2006; Nickells, 2007). The evidence that there is no ganglion cell loss in Bax / mice may seem to contradict this hypothesis, since once extrinsic