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One explanation for the relative protection of glia against TNF-a cytotoxicity could be differential expression of different receptor subtypes. However, although the expression level of TNF receptors may vary between RGCs and glial cells, they are expressed by both cell types (Tezel et al., 2001). Considerable evidence rather suggests that diverse responses of RGCs and glial cells to TNF-a are associated with differential regulation of many signaling molecules involved in TNF-a signaling between these cell types. Since the functional activation of several adaptive/protective or pathogenic proteins involved in TNF-a signaling is known to require phosphorylation, kinase activity appears to be crucial in determining the ultimate cell fate in response to TNF-a. For example, similar to other cell types, functional activation of TNF-a-induced survival signaling through NF-kB in RGCs and glia is phosphorylation dependent (Tezel and Yang, 2005). On the other hand, JNK activity following optic nerve injury has been associated with the amplification of TNF-a-mediated cell death signaling, thereby switching the life balance toward cell death (Tezel et al., 2004). Findings of a recent histopathological study in human donor eyes support a prominent and persistent activation of glial ERK signaling in glaucomatous retinas, although the most prominent immunolabeling for phospho-JNK or phos- pho-p38 in these eyes is localized to RGCs (Tezel et al., 2003). Such a differential kinase activity between RGCs and glia appears to be associated with the different susceptibility of these cell types to TNF-a, since a critical balance between the survival-promoting ERK pathway and the deathpromoting JNK and p38 pathways regulates cell fate (Xia et al., 1995).

Consistent with immunohistochemical observations, a recent comparative gene array analysis revealed differential regulation of MAPK or NF- kB signaling pathways between RGCs and glial cells exposed to TNF-a (Tezel and Yang, 2005). Findings of this in vitro study support that phosphorylation cascades are important components of TNF-a signaling and that a cross-talk between deathor survival-promoting signals determines whether an RGC should live or die in

response to TNF-a. Besides the differential activity of MAPK and NF-kB signaling between RGCs and glial cells exposed to TNF-a, differentially regulated genes in RGCs and glial cells exposed to TNF-a also included HSP27. Upregulation and phosphorylation of this chaperonin protein selectively in glial cells suggests that various intrinsic adaptive/protective mechanisms, including HSPs (as well as MAPK and NF-kB signaling pathways), are important for relative protection of these cells against TNF-a-mediated cell death. Better understanding of the factors determining diverse responses of RGCs and glia to TNF-a can provide clues for therapeutic manipulation of TNF-a signaling for the gain of RGC survival, similar to that naturally seen in glia.

Conclusions on neuroprotective treatment targets in glaucoma

Growing evidence supports that TNF-a-mediated neurotoxicity is a component of the neurodegenerative injury in glaucoma. It is clear that a critical balance between diverse signaling pathways determines RGC fate after increased exposure to TNF- a in glaucomatous eyes. Based on our current understanding of diverse bioactivities of TNF-a signaling, which can promote both cell death and survival, specific inhibition of cell death signaling and/or amplification of survival signaling, rather than the inhibition of death receptor binding, is expected to accomplish RGC protection. Since such strategies are expected to not impede the survival-promoting signaling triggered by TNF-a, their neuroprotective outcome should be superior. Current understanding of TNF-a-mediated cell death signaling in RGCs suggests that neuroprotective strategies targeting TNF-a signaling for RGC rescue should include tools to block the caspase cascade and also improve the ability of these neurons to survive cytotoxic consequences of mitochondrial dysfunction. In addition, several molecules involved in TNF-a signaling, such as MAPKs, NF-kB, and HSPs, appear to be promising treatment targets for manipulation of the life

balance between cell death and survival signals. Neuroprotective ability of anti-TNF-a strategies against axonal injury also needs to be clarified. Because the main source of TNF-a is glial cells, targeting key glial activation pathways should also be determined for attenuation of the gliaassociated component of neurodegeneration, while maintaining glial neurosupportive functions. Evidently, multiple neurodegenerative processes are involved in RGC death, and current strategies are not expected to individually provide complete neuroprotection in glaucoma patients. Elucidation of specific signaling pathways is therefore crucial to define new treatment targets for effective neuroprotective interventions. Ongoing efforts utilizing targeted proteomic approaches are expected to identify signaling molecules associated with glaucomatous neurodegeneration. RNA interference technology also offers a powerful tool through which specific siRNAs can be used to determine functional significance of newly identified molecules as treatment targets. This technology can also serve as an intervention strategy along with other genomic or pharmacologic treatments to provide neuroprotection in glaucoma.

Acknowledgments

Dr. Tezel’s work is supported by National Eye Institute (2R01 EY013813, 1R01 EY017131, R24 EY015636), Bethesda, MD, and an unrestricted grant to University of Louisville Department of Ophthalmology & Visual Sciences from Research to Prevent Blindness Inc., New York, NY.

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