Genetic Ablation of Pannexin1 Protects Retinal Neurons from Ischemic Injury

Pannexin1 (Panx1) forms large nonselective membrane channel that is implicated in paracrine and inflammatory signaling. In vitro experiments suggested that Panx1 could play a key role in ischemic death of hippocampal neurons. Since retinal ganglion cells (RGCs) express high levels of Panx1 and are susceptible to ischemic induced injury, we hypothesized that Panx1 contributes to rapid and selective loss of these neurons in ischemia. To test this hypothesis, we induced experimental retinal ischemia followed by reperfusion in live animals with the Panx1 channel genetically ablated either in the entire mouse (Panx1 KO), or only in neurons using the conditional knockout (Panx1 CKO) technology. Here we report that two distinct neurotoxic processes are induced in RGCs by ischemia in the wild type mice but are inactivated in Panx1KO and Panx1 CKO animals. First, the post-ischemic permeation of RGC plasma membranes is suppressed, as assessed by dye transfer and calcium imaging assays ex vivo and in vitro. Second, the inflammasome-mediated activation of caspase-1 and the production of interleukin-1β in the Panx1 KO retinas are inhibited. Our findings indicate that post-ischemic neurotoxicity in the retina is mediated by previously uncharacterized pathways, which involve neuronal Panx1 and are intrinsic to RGCs. Thus, our work presents the in vivo evidence for neurotoxicity elicited by neuronal Panx1, and identifies this channel as a new therapeutic target in ischemic pathologies.

潘宁蛋白1（Pannexin1，Panx1）可形成大型非选择性膜通道，参与旁分泌信号与炎症信号传导。此前体外实验提示，Panx1可能在海马神经元的缺血性细胞死亡中发挥关键作用。鉴于视网膜神经节细胞（retinal ganglion cells，RGCs）高表达Panx1且易受缺血性损伤，我们提出假说：Panx1可介导缺血状态下此类神经元的快速选择性丢失。为验证该假说，我们分别通过两种方式构建活体动物的实验性视网膜缺血再灌注模型：一是在全小鼠体内敲除Panx1通道（Panx1 KO，即全基因敲除），二是仅在神经元中利用条件性基因敲除（Panx1 CKO）技术敲除Panx1通道。本研究发现，野生型小鼠的视网膜神经节细胞在缺血后会激活两种截然不同的神经毒性进程，而该进程在Panx1 KO与Panx1 CKO动物中均被阻断。其一，通过离体与体外的染料转移及钙成像实验评估发现，缺血后视网膜神经节细胞的质膜通透性异常升高现象受到抑制；其二，Panx1 KO小鼠视网膜中，炎性小体介导的半胱天冬酶-1激活与白细胞介素-1β生成均被显著抑制。我们的研究结果表明，视网膜缺血后的神经毒性由此前未被阐明的信号通路介导，该通路依赖神经元源性Panx1且为视网膜神经节细胞所固有。综上，本工作首次提供了神经元Panx1诱发神经毒性的在体实验证据，并将该通道确立为缺血性病理状态下的全新治疗靶点。