Neuroprotection and Retinal Ganglion Cell Death

视神经挤压和青光眼后视网膜神经节细胞凋亡，造成不可逆的视力丧失。目前的治疗方法降低眼压，但不是专门设计来防止视网膜神经节细胞死亡和失明。细胞凋亡可以通过至少两种不同的途径启动：激活死亡受体如Fas，导致FADD的募集，以及caspase 8形成含有死亡诱导信号复合物的活性caspase 8。这导致下游半胱天冬酶的裂解和激活以及细胞死亡。另外，促凋亡的Bcl2家族成员（去磷酸化的pBAD）可以转移到线粒体，导致细胞色素c、APAF-1的释放和caspase-9的激活，再次启动下游的caspase，包括caspase 3。我们已经观察到视神经挤压引起视网膜神经节细胞的快速、定型死亡，并且这两种途径都可能涉及。虽然被定义为不同的途径，但它们很可能是这些上游初始现象的交叉点和调节点。我们认为钙调磷酸酶活性是上游半胱天冬酶的主要调节因子，因为钙调磷酸酶抑制可以防止死亡受体启动的细胞凋亡，也可以防止pBad磷酸化。我们证明用FK506治疗大鼠，一种广泛应用于临床的免疫抑制剂，是一种有效的钙调磷酸酶抑制剂，导致视神经压迫后视网膜神经节细胞的保护具有统计学意义。当前应用的目标是（目的1）确定是否抑制钙调磷酸酶是FK506观察到的神经保护作用的基础，以及（目的2）检验FK506对caspase 8/死亡受体和pBad/细胞色素c启动级联反应的影响。目标3建立在前两个目标的基础上，并利用了实验性青光眼大鼠显微外科血管硬化模型。我们观察到RGC在数周内因细胞凋亡而丢失，其许多特征与我们在亚急性视神经挤压模型中观察到的相同。我们现在将进一步表征该模型，并确定FK506抑制钙调磷酸酶是否在这种情况下具有神经保护作用。综上所述，本应用程序中概述的研究的广泛、长期目标是表征视网膜神经节细胞死亡的细胞死亡途径，以便开发出视网膜神经节细胞死亡疾病（如青光眼）的神经保护治疗方法。

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.