Neuroprotection and Retinal Ganglion Cell Death

视网膜神经节细胞在视神经挤压后和青光眼中通过凋亡而死亡，导致不可逆的视力丧失。目前的治疗方法可以降低眼内压，但并不是专门用于预防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：死亡受体如Fas的活化，导致FADD的募集，以及半胱天冬酶8形成含有活性半胱天冬酶8的死亡诱导信号传导复合物。这导致下游半胱天冬酶的裂解和激活以及细胞死亡。或者，促凋亡Bcl 2家族成员（去磷酸化的pBAD）可以易位到胞浆，导致细胞色素c、APAF-1的释放和半胱天冬酶-9的活化，再次启动下游半胱天冬酶，包括半胱天冬酶3。我们已经观察到视神经挤压诱导视网膜神经节细胞快速、刻板的死亡，这两种途径都可能参与其中。虽然被概念化为不同的途径，但它们可能是这些上游启动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剂可防止死亡受体启动的细胞凋亡，也可防止pBad磷酸化。我们证明，用FK 506（一种临床上广泛使用的免疫抑制剂，是一种有效的钙调磷酸酶抑制剂）治疗大鼠，可在视神经挤压后产生统计学显著的视网膜神经节细胞保护作用。本申请的目的是（目的1）确定钙调磷酸酶抑制是否是观察到的FK 506神经保护作用的基础，以及（目的2）检查FK 506对胱天蛋白酶8/死亡受体和pBad/细胞色素c启动级联的影响。目的3建立在前两个目的的基础上，并利用大鼠实验性青光眼的显微手术血管硬化模型。我们观察到RGC在几周的过程中由于凋亡而丢失，具有与我们在亚急性视神经挤压模型中观察到的许多相同的特征。我们现在将进一步表征该模型，并确定FK 506对钙调磷酸酶的抑制是否在这种情况下具有神经保护作用。总之，本申请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中涉及的细胞死亡途径，使得可以开发用于视网膜神经节细胞死亡的疾病（例如青光眼）的神经保护性治疗。

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.