Neuroprotection and Retinal Ganglion Cell Death

视神经挤压和青光眼后，视网膜神经节细胞通过细胞凋亡而死亡，导致不可逆转的视力丧失。目前的治疗方法降低了眼压，但并不是专门为防止视网膜神经节细胞死亡和失明而设计的。细胞凋亡至少可以通过两条不同的途径启动：一条是激活Fas等死亡受体，导致FADD的募集；另一条是通过caspase 8形成含有死亡诱导信号复合体的活性caspase 8。这会导致下游caspase的裂解和激活，导致细胞死亡。或者，促凋亡的bcl2家族成员(去磷酸化的pBAD)可以移位到线粒体，导致细胞色素c、APAF-1的释放和caspase-9的激活，再次启动包括caspase 3在内的下游caspase。我们观察到视神经挤压导致视网膜神经节细胞快速、刻板的死亡，这两条途径都很可能参与其中。尽管它们被概念化为不同的路径，但它们很可能是这些上游启动现象的交叉点和调节点。我们认为，钙调神经磷酸酶的活性是caspase上游的一个主要调节因子，因为钙调神经磷酸酶抑制可以防止死亡受体启动的细胞凋亡，也可以阻止pBad的磷酸化。我们证明，FK506是一种临床上广泛使用的免疫抑制剂，是一种有效的钙调神经磷酸酶抑制剂，治疗大鼠在视神经挤压后导致统计上显著的视网膜神经节细胞保护。目前应用的目的是(目的1)确定钙调神经磷酸酶的抑制是否是观察到的FK506神经保护作用的基础，以及(目的2)检测FK506对caspase 8/Death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在几周的过程中因细胞凋亡而丢失，与我们在亚急性视神经挤压模型中观察到的许多特征相同。我们现在将进一步描述这一模型，并确定在这种情况下，FK506对钙调神经磷酸酶的抑制是否具有神经保护作用。综上所述，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所涉及的细胞死亡途径，以便为视网膜神经节细胞死亡的疾病开发神经保护性治疗方法，如青光眼。

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.