Retinal ganglion cells die by apoptosis after optic nerve crush and in glaucoma, causing irreversible visual loss. Current therapies lower intraocular pressure, but are not designed specifically to prevent retinal ganglion cell death and blindness. Apoptosis can be initiated by at least two different pathways: activation of death receptors such as Fas, leading to recruitment of FADD, and caspase 8 to form the active caspase 8 containing death inducing signaling complex. This leads to cleavage and activation of downstream caspases and cell death. Alternatively, pro- apoptotic Bcl2 family members (dephosphorylated pBAD) can translocate to the mitochondrion, leading to release of cytochrome c, APAF-1, and activation of caspase-9, again initiating downstream caspases including caspase 3. We have observed that optic nerve crush induces rapid, stereotyped death of retinal ganglion cells, and that both of these pathways may well be involved. Although conceptualized as distinct pathways, they are likely points of intersection and regulation of these upstream initiating phenomenon. We propose that calcineurin activity is a major regulator of upstream caspases, because calcineurin inhibition protects against death receptor-initiated apoptosis and also prevents pBad phosphorylation. We demonstrated that treatment of rats with FK506, a widely clinically used immunosuppressant agent that is a potent calcineurin inhibitor, led to statistically significant retinal ganglion cell protection after optic nerve crush. The goals of the current application are (Aim 1) to determine whether inhibition of calcineurin underlies the observed neuroprotective effect of FK506, AND (Aim 2) to examine the effect of FK506 on the caspase 8/death receptor and pBad/cytochrome c initiating cascades.
Aim 3 builds on the first 2 aims, and takes advantage of a microsurgical vascular sclerosis model of experimental glaucoma in the rat. We observe that RGC are lost over the course of several weeks by apoptosis, with many of the same characteristics as we observe in the subacute optic nerve crush model. We will now further characterize this model, and determine whether calcineurin inhibition by FK506 is neuroprotective in this setting. Taken together, the broad, long-term aims of the studies outlined in this application are to characterize the cell death pathways involved in retinal ganglion cell death so that neuroprotective treatments can be developed for diseases in which retinal ganglion cells die, such as glaucoma.
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