Mo Juan, Atherton Sally S, Wang Liya, Liu Susu
Henan Eye Institute, Henan Eye Hospital, Henan Provincial People's Hospital, People's Hospital of Zhengzhou University, Zhengzhou, Henan Province, 450003, People's Republic of China.
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, 1120 15th Street, Augusta, GA, 30912, USA.
BMC Ophthalmol. 2019 Jul 10;19(1):146. doi: 10.1186/s12886-019-1141-y.
Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis (CMV). Our previous results have shown that there is a functional relationship between autophagy and apoptosis during MCMV infection of retinal pigment epithelium (RPE). The purpose of this study was to determine whether autophagy plays a significant role in the death of retinal cells during MCMV retinitis.
The retinas of adult BALB/c mice were infected with MCMV via supraciliary injection. Rapamycin, a mTOR inhibitor, was injected to MCMV-infected BALB/c mice intraperitoneally. Immunohistochemistry and western blot were performed to observe the spread pattern of virus in retinas and the levels of targeted proteins. Plaque assay was performed to determine the virus titer in different groups. Since Atg5 is a key gene regulating autophagy, we bred Atg5; Nestin-Cre mice to deeply elucidate the role of autophagy during MCMV retinitis. Atg5; Nestin-Cre mice were genotyped and infected with MCMV. Immunohistochemistry was performed to observe the type of virus-infected cells and apoptosis in retinas during MCMV retinitis.
In MCMV mouse model, MCMV infection in outer nuclear layer (ONL) and inner nuclear layer (INL) in the retinas caused cleaved caspase 3 positive apoptosis, which is not co-localized with early antigen (EA) positive virus infected cells in rapamycin treated group. Rapamycin treatment increased the levels of LC3B-II by inhibiting mTOR and decreased the levels of cleaved caspase-3 during MCMV retinitis. However, virus propagation was not affected by rapamycin. In Atg5; Nestin-Cre mice, RPE and glial cells were the main targets of viral infection, and number of EA positive retinal cells and TUNEL positive retinal cells was significantly increased compared to Atg5; Nestin-Cre mice though there was no difference of virus propagation between Atg5; Nestin-Cre mice and Atg5; Nestin-Cre mice.
Autophagy protects retinal cells from MCMV infection induced apoptosis through mTOR-mediated signaling pathway.
未感染的旁观者神经元细胞大量死亡是巨细胞病毒性视网膜炎（CMV）发病机制的重要组成部分。我们之前的研究结果表明，在视网膜色素上皮（RPE）的小鼠巨细胞病毒（MCMV）感染过程中，自噬与凋亡之间存在功能关系。本研究的目的是确定自噬在MCMV视网膜炎期间视网膜细胞死亡中是否起重要作用。
通过睫状体上腔注射将MCMV感染成年BALB/c小鼠的视网膜。将mTOR抑制剂雷帕霉素腹腔注射到感染MCMV的BALB/c小鼠体内。进行免疫组织化学和蛋白质印迹以观察病毒在视网膜中的传播模式和靶向蛋白的水平。进行噬斑测定以确定不同组中的病毒滴度。由于Atg5是调节自噬的关键基因，我们培育了Atg5；Nestin-Cre小鼠以深入阐明自噬在MCMV视网膜炎期间的作用。对Atg5；Nestin-Cre小鼠进行基因分型并感染MCMV。进行免疫组织化学以观察MCMV视网膜炎期间视网膜中病毒感染细胞的类型和凋亡情况。
在MCMV小鼠模型中，视网膜外核层（ONL）和内核层（INL）中的MCMV感染导致裂解的半胱天冬酶3阳性凋亡，在雷帕霉素治疗组中，这与早期抗原（EA）阳性病毒感染细胞不共定位。雷帕霉素治疗通过抑制mTOR增加了LC3B-II的水平，并在MCMV视网膜炎期间降低了裂解的半胱天冬酶-3的水平。然而，病毒传播不受雷帕霉素影响。在Atg5；Nestin-Cre小鼠中，RPE和神经胶质细胞是病毒感染的主要靶标，与Atg5；Nestin-Cre小鼠相比，EA阳性视网膜细胞和TUNEL阳性视网膜细胞的数量显著增加，尽管Atg5；Nestin-Cre小鼠与Atg5；Nestin-Cre小鼠之间的病毒传播没有差异。
自噬通过mTOR介导的信号通路保护视网膜细胞免受MCMV感染诱导的凋亡。
