An interneuronal signaling network governs the fate of retinal ganglion cells after optic nerve injury

项目摘要 视网膜和视神经已被广泛研究，以了解抑制或促进细胞存活的因素 中枢神经系统损伤后轴突再生。视神经损伤后，视网膜神经节细胞(RGCs)，促进 眼睛的投射神经元不能再生轴突，几天后开始死亡。尽管有这样的发展 在改善RGC存活和/或轴突再生的治疗中，迄今所取得的视力恢复水平仍然存在 强调需要更好地了解导致细胞死亡和再生失败的机制。 我们最近报道(Li等人，PNAS，2017，参考文献1)视神经损伤导致移动性/游离性快速升高 无长突细胞突触末梢的锌离子，随后是胞吐作用和视网膜神经节细胞内锌离子的积累； 这种锌离子的络合作用导致许多视网膜节细胞的长期存活和大量轴突的再生。我们的预赛 数据表明，锌离子升高的机制涉及一种以前未知的多细胞网络 这利用了非经典的信号机制，最终决定了RGC的命运。此网络AP- 梨树通过一个信号传递到受损的轴突，使视网膜节细胞中的K+通道磷酸化；逆行信号- 受损伤的视网膜节细胞和中间神经元(或穆勒胶质细胞)之间通过细胞外K+升高而发生的内化，导致 谷氨酸转运体GLT-1和谷氨酸外流；这反过来激活NMDA受体，导致钙内流， 神经元型一氧化氮合酶-1(NOS1)的激活，以及NO介导的锌离子从金属中的释放. 硫蛋白(S)。这一序列基于使用药物抑制剂和免疫组织化学的初步结果。 STY，但关于具体细胞类型和涉及的信号的准确知识仍有待建立。基于 观察到NOS1介导的NO生成直接位于锌离子释放的上游，目标1和2将起作用 回到这一点，以确定将视神经损伤与RGC死亡联系起来的细胞群体和信号。目标 1将检验这样的假设：谷氨酸从双极细胞或Mueller细胞流出(通过逆转谷氨酸转运体 GLT-1)和NOS1阳性无长突细胞上NMDA受体的激活直接位于NO生成的上游。 目标2将检验假设，即进一步的上游步骤涉及激活MAP激酶级联和/或 损伤的视网膜节细胞中的钙信号，导致钾通道的磷酸化和激活(以及可能的其他 通道)，导致细胞外K+升高，导致Glu- 双极细胞或Mueller细胞中的Tamate转运蛋白。目标3将检验这一途径对RGC有贡献的假设 青光眼小鼠模型的死亡。为了与美国国立卫生研究院的指导方针保持一致，拟议的研究将同时使用男性和 雌性小鼠正在进行一项关键实验，以确定在我们尚未发现的信号通路中是否存在性别差异。 这可能会对细胞死亡和再生失败的潜在机制提供进一步的见解。这些 研究将在视网膜中定义一个新的多细胞信号网络，它调节视网膜的活性和再生 视神经损伤后视网膜神经节细胞的能力，可能与青光眼有关。

Project Summary The retina and optic nerve have been widely studied for insights into factors that suppress or promote cell survival and axon regeneration after CNS injury. Following injury to the optic nerve, retinal ganglion cells (RGCs), the pro- jection neurons of the eye, cannot regenerate their axons and begin to die after a few days. Despite the development of treatments that improve RGC survival and/or axon regeneration, levels of visual recovery achieved to date remain modest, underscoring the need to better understand the mechanisms that produce cell death and regenerative failure. We recently reported (Li et al., PNAS, 2017, ref. 1) that optic nerve injury leads to a rapid elevation of mobile/free zinc (Zn2+) in synaptic terminals of amacrine cells, followed by exocytosis and Zn2+ accumulation within RGCs; and that Zn2+ chelation leads to long-term survival of many RGCs and considerable axon regeneration. Our preliminary data indicate that the mechanisms underlying Zn2+ elevation involve a previously unknown, multi-cellular network that utilizes non-classical signaling mechanisms and that ultimately determines the fate of RGCs. This network ap- pears to involve phosphorylation of a K+ channel in RGCs by a signal conveyed up the injured axons; retrograde sig- naling between injured RGCs and interneurons (or Muller glia) via elevation of extracellular K+, causing reversal of the glutamate transporter GLT-1 and glutamate efflux; this in turn activates NMDA receptors, leading to Ca2+ entry, activation of neuronal nitric oxide (NO) synthase-1 (NOS1), and NO-mediated liberation of Zn2+ from metal- lothionein(s). This sequence is based on preliminary results using pharmacological inhibitors and immunohistochem- istry, but precise knowledge of the specific cell types and signals involved remains to be established. Based on the observation that NOS1- mediated NO generation lies directly upstream of Zn2+ liberation, Aims 1 and 2 will work back from this point to identify the cellular populations and signals that link optic nerve damage to RGC death. Aim 1 will test the hypothesis that glutamate efflux from bipolar or Mueller cells (via reversal of the glutamate transporter GLT-1) and activation of NMDA receptors on NOS1-positive amacrine cells lie directly upstream of NO generation. Aim 2 will test the hypothesis that the further upstream steps involve activation of a MAP kinase cascade and/or Ca2+ signaling in injured RGCs, leading to phosphorylation and activation of potassium channels (and possibly other channels) in RGCs, causing an elevation of extracellular K+ that leads to a reversal of the normal operation of glu- tamate transporters in bipolar or Mueller cells. Aim 3 will test the hypothesis that this pathway contributes to RGC death in a mouse model of glaucoma. In keeping with NIH guidelines, the proposed studies will use both male and female mice in a key experiment to determine whether sex differences exist in the signaling pathway we have un- covered that might provide further insights into the mechanisms underlying cell death and regenerative failure. These studies will define a novel multi-cellular signaling network in the retina that regulates the viability and regenerative capacity of RGCs after optic nerve injury and perhaps in glaucoma.