Targeting BAX as a Therapeutic for Protection of Retinal Ganglion Cells

Bax是一种促凋亡蛋白，在视网膜神经节细胞的内源性凋亡过程中起关键作用。 视神经损伤后的视网膜神经节细胞(RGCs)，提示靶向Bax可能是一种有效的治疗策略 神经保护。然而，据报道，降低Bax水平仅提供了对 青光眼小鼠模型中的轴突。这种瞬时保护是否足以让轴突 恢复后消除原来的压力源，还没有经过测试。 目标1将测试Bax还原对轴突存活和/或恢复能力的保护作用 模拟高眼压(OHT)患者经历的降眼压治疗的模型。二 小鼠诱导性OHT模型，微球注射和类固醇诱导OHT，将在两种野生动物上使用 类型和Bax+/-小鼠(其中减少的Bax更有可能模仿人类的治疗)。在这两种模型中，眼压水平 可在所需间隔后恢复正常。我们将测试降低Bax蛋白水平是否都会降低胞体 和轴突病理，或者保护轴突功能，或者允许功能恢复。我们还将研究 控制性实验性眼压(CEI)的影响。CEI的优势在于它使眼压侮辱正常化，这 消除了与其他OHT模型相关的可变眼压的混杂因素。 背根神经节细胞包含一条Bax依赖的退变通路，该通路在早期和之前被激活 其他更有特点的分解代谢反应，涉及钙内流。该途径由BH3激活- 只有蛋白质在胞体中产生，然后运输到轴突。RGC轴突也有类似的机制 可能解释了BAX还原的部分保护作用。AIM 2将评估这一途径在 在体外诱导视神经变性的RGC轴突。我们还将审问轴突以了解 激活Bax的关键分子以及作用于Bax下游的分子的存在和定位 激活。初步研究表明，仅有BH3的蛋白noxa可能是bax的重要调节因子。 轴突的激活。我们将使用NOXA缺乏的小鼠进行诱导的OHT来详细探讨这一点。 受保护的RGC沉默功能所需基因的转录。新的研究表明，承诺的 在Bax缺乏的细胞中死亡的过程，在8周后关闭。因此，细胞可以分为两类 类别，那些活跃的垂死(AD-模式)和那些已经静止(Q-模式)。AIM 3将调查 如果处于Q模式的细胞对重新激活它们的3次尝试有不同的反应，包括使用HDAC抑制剂 减少RGC特异性基因表达的转录沉默，添加酵母多糖和CPT-cAMP 诱导再生，或上调可指定视网膜前体细胞的转录因子 RGC。这些实验将在视神经急性损伤后的视网膜神经节细胞上进行，视神经的作用是 一种模式，在这种模式中，再生对治疗结果具有高度的优先权。

BAX is a proapoptotic protein, which is critical for the execution of intrinsic apoptosis in retinal ganglion cells (RGCs) after optic nerve damage, suggesting that targeting BAX may be an effective therapeutic strategy for neuroprotection. It has been reported, however, that reducing BAX levels only provides a transient protection to axons in a mouse model of glaucoma. Whether or not this transient protection is sufficient to allow axons to recover after elimination of the original stressor, has not been tested. Aim 1 will test the protective effect of BAX reduction on the ability of axons to survive and/or recover in models that mimic the IOP-lowering therapy experienced by patients with ocular hypertension (OHT). Two models of inducible OHT in mice, microbead injection and steroid induced OHT, will be employed on both wild type and Bax+/- mice (where reduced BAX more likely mimics a human treatment). In both models IOP levels can be returned to normal after a desired interval. We will test if reducing BAX protein levels both reduces soma and axon pathology, and either protects axon function, or allows functional recovery. We will also examine the effects of controlled experimental IOP (CEI). The advantage of CEI is that it normalizes the IOP insult, which eliminates the confounding factors of variable IOP associated with other models of OHT. Dorsal Root Ganglion cells contain a BAX-dependent degenerative pathway that is activated early and before other more well-characterized catabolic reactions involving calcium influx. This pathway is activated by a BH3- only protein produced in the cell soma and then transported to the axon. A similar mechanism in RGC axons may explain the partial protective effect of BAX reduction. Aim 2 will evaluate the presence of this pathway in RGC axons where degeneration will be induced in optic nerves ex vivo. We will also interrogate axons for the presence and localization of key molecules that activate BAX, and molecules that act down-stream of BAX activation. Preliminary studies suggest that the BH3-only protein NOXA may be an important regulator of BAX activation in axons. We will explore this in detail using Noxa-deficient mice subjected to induced OHT. Protected RGCs silence transcription of genes required for function. New studies show that the committed process of dying in Bax-deficient cells, is shut down after 8 weeks. Thus cells can be classified into two categories, those actively dying (AD-mode) and those that have gone quiescent (Q-mode). Aim 3 will investigate if cells in Q-mode respond differently to 3 attempts to reactivate them, including using HDAC inhibitors to attenuate the transcriptional silencing of RGC-specific gene expression, the addition of Zymosan and CPT-cAMP to induce regeneration, or the up-regulation of transcription factors that can specify retinal precursor cells as RGCs. These experiments will be conducted on RGCs after acute damage to the optic nerve, which serves as a model where regeneration has a high priority for a therapeutic outcome.