Growing evidence supports that TNF-α, through the binding of TNF receptor-1 (TNF-R1), a death receptor, is involved in mediating RGC death during glaucomatous neurodegeneration. Glial production of TNF-α is increased in the retina and optic nerve head, and TNF-R1 is up-regulated in RGCs and their axons in glaucomatous human donor eyes (Tezel et al., 2001
; Yan et al., 2000
; Yuan and Neufeld, 2000
). Findings of ongoing in vivo studies support that TNF-α and TNF-R1 are also up-regulated following experimental elevation of intraocular pressure (IOP), and TNF-α signaling is involved in the RGC death process during neurodegeneration in ocular hypertensive eyes. TNF-α secreted by stressed-glial cells during the neurodegenerative process of glaucoma can induce RGC death through receptor-mediated caspase cascade, mitochondrial dysfunction, and oxidative damage (Tezel and Wax, 2000a
; Tezel and Yang, 2004
An exciting finding of our earlier in vitro studies was the activation of retinal caspase-8 in response to different stress stimuli evident in glaucomatous eyes (Tezel and Wax, 1999
; Tezel and Wax, 2000b
). Since caspase-8 is associated with the apoptosis pathway triggered by TNF death receptor binding (Hsu et al., 1996
), this finding stimulated interest in the role of TNF-α signaling in glaucoma. Following this initial observation, a series of experiments provided evidence that the TNF-α signaling is indeed involved in the death of RGCs during glaucomatous neurodegeneration. A direct evidence comes from primary co-culture experiments. These experiments proved that following exposure to different glaucomatous stimuli, glial cells adversely affect the survival of co-cultured RGCs through the increased production of TNF-α. A similar neurotoxic effect was replicated by the transfer of the conditioned medium obtained from stressed glia cultures, and inhibition of TNF-α bioactivity by treatment of co-cultures with a neutralizing antibody resulted in a decreased rate of apoptosis in RGCs (Tezel and Wax, 2000a
Another series of in vitro experiments also revealed that in addition to caspase activity, mitochondrial dysfunction accompanies RGC death induced by TNF-α (Tezel and Yang, 2004
). These studies demonstrated that the inhibition of caspase activity is not adequate to block RGC death in primary cultures exposed to TNF-α if the mitochondrial membrane potential is lost and mitochondrial cell death mediators, cytochrome c and apoptosis inducing factor, are released. RGCs exposed to TNF-α also accumulated reactive oxygen species (ROS) over time, and when combined with caspase inhibition, a free radical scavenger treatment reduced the production of ROS and provided an additional increase in RGC survival (Tezel and Yang, 2004
). These findings support that in addition to receptor-mediated caspase cascade, RGC death induced by TNF-α also involves both caspase-dependent and caspase-independent components of the mitochondrial cell death pathway and generation of ROS ().
Figure 1 Binding of TNF-α to TNF-R1, a death receptor, can induce RGC death through receptor-mediated caspase activation, and caspase-dependent and -independent components of the mitochondrial cell death pathway, which includes increased generation of (more ...)
Parallel studies through immunohistochemical analysis of human donor eyes revealed an increased immunolabeling for TNF-α and TNF-R1 in the optic nerve head (Yan et al., 2000
) and retina (Tezel et al., 2001
) of glaucomatous eyes compared to age-matched controls. Using in situ hybridization, mRNA signals for TNF-α or TNF-R1 were also found to be similarly more intense in glaucomatous eyes relative to controls (Tezel et al., 2001
). Up-regulation of TNF-α in the glaucomatous optic nerve head and retina was mostly detectable in glial cells. However, TNF-R1 up-regulation was also prominent in nerve bundles and RGC bodies. The up-regulation of TNF-α and TNF-R1 in glaucomatous tissues supports the association of TNF-α signaling with glaucomatous neurodegeneration. The predominant localization of TNF-R1, a death receptor, to RGCs and their axons indicates that they are sensitive targets for the cytotoxic effects of TNF-α.
A series of in vivo observations also support the involvement of TNF-α signaling in the neurodegenerative process of glaucoma. In vivo studies utilizing a chronic pressure-induced rat model of glaucoma demonstrated retinal caspase activation in ocular hypertensive eyes, which include the activation of caspase-8 (available at www.iovs.org
, 2005; 46:E-Abstract 3772). Caspase-8 activation after IOP elevation was also detected by another study using two different rat glaucoma models (McKinnon et al., 2002
). Activation of caspase-8 is an important early step following death receptor binding (Hsu et al., 1996
), although there is in vitro evidence suggesting that caspase-8 activation may also occur downstream of mitochondria (Slee et al., 1999
). The activation of not only the receptor-mediated caspase cascade, but also the TNF-α-mediated mitochondrial cell pathway has been associated with caspase-8 activation, since caspase-8 cleaves a pro-apoptotic member of the bcl-2 family of proteins, Bid, which then participates in the activation of the mitochondrial cell death pathway (Luo et al., 1998
). Further studies also indicate Bid-independent mechanisms for the involvement of mitochondria during TNF-α-mediated cell death (Chen et al., 2007
In vivo experiments also determined the expression and cellular localization of TNF-α and TNF-R1, and IOP-dependent regulation of these molecules in ocular hypertensive rat eyes. Findings of these experiments demonstrated an up-regulation of TNF-α signaling in ocular hypertensive eyes relative to controls. This up-regulation exhibited a close association with the cumulative IOP exposure and neuronal damage (available at www.iovs.org
, 2005; 46:E-Abstract 3772). Similar to glaucomatous human eyes (Tezel et al., 2001
; Yan et al., 2000
), increased TNF-α immunolabeling in ocular hypertensive rat eyes compared with the controls was mainly localized to glial cells. However, retinal TNF-R1 immunolabeling in the ocular hypertensive eyes was most prominent on RGCs and their axons. These findings further support the association of TNF-α signaling with the experimental paradigm of glaucomatous neurodegeneration.
The increased gene expression for TNF-α and TNF-R1 detected in ocular hypertensive eyes is consistent with other studies using gene microarray analysis in experimental models of glaucoma, which have also identified differential regulation of genes associated with TNF-α signaling. For example, a study detected up-regulation of a transcription factor regulating TNF-α gene expression, Litaf, in the ocular hypertensive rat retina (Ahmed et al., 2004
). Another study using Affymetrix analysis of rat retinal RNA identified multiple genes differentially regulated in eyes with ocular hypertension or optic nerve transaction, and its findings were also consistent with the participation of TNF-α in glaucomatous injury in association with JNK signaling (available at www.iovs.org
, 2007; 48:E-Abstract 3279).
To assess the specific role of TNF death receptor signaling in the induction of RGC death, in vivo studies also utilized another experimental model, the optic nerve crush injury model, in TNF-R1 knockout mice (Tezel et al., 2004
). Although not a perfect simulation of glaucomatous conditions, optic nerve degeneration in the crush injury model mimics many of the features of glaucomatous optic nerve degeneration. Most importantly, spreading of damage by secondary degeneration of RGCs is likely similar in crush injury and glaucomatous injury of the optic nerve. Counts of RGCs and their axons 6 weeks after the injury demonstrated that their loss was significantly less in TNF-R1 knockout mice compared with the controls. The most prominent decrease in neuronal loss detected in these animals was beyond the initial 2-week period after the injury. This time period was correlated with the period of glial activation and increased glial immunolabeling for TNF-α in these eyes. No further protection against neuronal loss was detectable in TNF-R1 knockout mice treated with D-JNKI1, which is a specific peptide inhibitor of JNK. However, anti-JNK treatment of control animals provided significant protection against neuronal loss during the same secondary degeneration period. Phospho-JNK immunolabeling of RGCs in control mice subjected to optic nerve crush significantly decreased following their treatment with D-JNKI1, and anti-JNK treatment protected RGCs from degeneration, similar to the lack of TNF-R1 (Tezel et al., 2004
). These findings provide evidence that TNF death receptor signaling is involved in the secondary degeneration of RGCs following optic nerve injury and is associated with JNK signaling.
TNF-α has also been suggested to mediate oligodendrocyte death and delayed RGC loss in a mouse model of glaucoma (Nakazawa et al., 2006
). Intravitreal TNF-α injections in normal mice mimicked these effects. Conversely, treatment with an antibody neutralizing TNF-α activity or deleting the genes encoding TNF-α or its receptor, TNF-R2, blocked the deleterious effects of ocular hypertension.
Optineurin gene mutations detected in glaucoma patients (Sarfarazi and Rezaie, 2003
) provide another line of evidence supporting the role of TNF-α signaling in glaucoma. In addition to TNF-α gene polymorphism detected in different ethnic populations (Lin et al., 2003
), a possible interaction between polymorphisms in optineurin and TNF-α genes has been suggested to increase the risk of glaucoma in the Japanese population (Funayama et al., 2004
). Optineurin, which is expressed by RGCs (Wang et al., 2007
), has been proposed to be associated with TNF-α signaling pathway leading to RGC death based on its direct interaction with adenovirus E3 14.7 kDa protein, which utilizes TNF receptor pathways to mediate apoptosis (Sarfarazi and Rezaie, 2003
). However, a mutated form of optineurin, E50K, identified in normal-tension glaucoma patients, loses its ability to translocate to the nucleus and when over-expressed compromises the mitochondrial membrane integrity, resulting in cells that are less fit to survive under stress conditions (De Marco et al., 2006
). In a recent study, a glaucoma-associated mutant of optineurin has been shown to selectively induce oxidative stress-mediated RGC death, and optineurin has been suggested to be a likely component of the TNF-α signaling pathway leading to RGC death (Chalasani et al., 2007
). More recently, microRNA silencing of optineurin has resulted in markedly enhanced TNF-α-induced nuclear factor kappaB (NF-KB) activity, thereby supporting a physiologic role of optineurin in dampening TNF-α signaling in association with glaucoma (Zhu et al., 2007