In vivo cellular imaging of various stress/response pathways using AAV following axonal injury in mice.
In vivo cellular imaging of various stress/response pathways using AAV following axonal injury in mice.
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DOI：
10.1038/srep18141
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发表时间：
2015-12-16
影响因子：
4.6
通讯作者：
Nakazawa T
中科院分区：
文献类型：
--
作者：
Fujita K;Nishiguchi KM;Yokoyama Y;Tomiyama Y;Tsuda S;Yasuda M;Maekawa S;Nakazawa T
青光眼是世界范围内致盲的主要原因，由多种因素引起，包括轴突损伤，其最终导致视网膜神经节细胞（RGC）的进行性丧失。为了研究据报道参与由轴突损伤引起的RGC死亡的发病机制的各种途径，研究了七种途径。将途径特异性荧光蛋白编码的报告基因各自包装到腺相关病毒（AAV）中。在眼睛中产生轴突损伤后，注射AAV以诱导RGC死亡，通过使用共聚焦检眼镜检测荧光RGC在体内监测每个应激相关途径的时间活性。我们确定了参与内质网应激和巨噬细胞募集的ATF 6和MCP-1通路的激活，分别作为神经元死亡前RGC应激的早期标志物。相反，NF-κB和细胞死亡相关通路p53探测的炎症反应在晚期最为突出，此时RGC死亡已经在进行中。AAV介导的应激/应答报告基因的递送，随后进行体内细胞成像是表征视网膜疾病中涉及的复杂分子途径的时间方面的有力策略。识别在RGCs死亡之前激活的启动子元件使得能够开发专门针对患病细胞的早期阶段的先发制人的基因治疗。
Glaucoma, a leading cause of blindness worldwide, is instigated by various factors, including axonal injury, which eventually leads to a progressive loss of retinal ganglion cells (RGCs). To study various pathways reportedly involved in the pathogenesis of RGC death caused by axonal injury, seven pathways were investigated. Pathway-specific fluorescent protein-coded reporters were each packaged into an adeno-associated virus (AAV). After producing axonal injury in the eye, injected with AAV to induce RGC death, the temporal activity of each stress-related pathway was monitored in vivo through the detection of fluorescent RGCs using confocal ophthalmoscopy. We identified the activation of ATF6 and MCP-1 pathways involved in endoplasmic reticulum stress and macrophage recruitment, respectively, as early markers of RGC stress that precede neuronal death. Conversely, inflammatory responses probed by NF-κB and cell-death-related pathway p53 were most prominent in the later phases, when RGC death was already ongoing. AAV-mediated delivery of stress/response reporters followed by in vivo cellular imaging is a powerful strategy to characterize the temporal aspects of complex molecular pathways involved in retinal diseases. The identification of promoter elements that are activated before the death of RGCs enables the development of pre-emptive gene therapy, exclusively targeting the early phases of diseased cells.