Mechanism of death of bystander retinal cells during MCMV infection

项目摘要 人类巨细胞病毒（HCMV）视网膜炎最初导致约30%的HIV/AIDS患者视力丧失和失明 在联合抗逆转录病毒疗法（cART）开发之前， 疾病仍然是一个重要的眼科问题，在世界各地的艾滋病毒/艾滋病患者谁不响应 或停止治疗的人。HCMV慢性感染是年龄增长的危险因素- 相关性黄斑变性（AMD）。治疗和预防HCMV进展的关键障碍 视网膜炎是对未感染的旁观者细胞死亡机制的有限理解， 预防战略。我们的目标是确定受体相互作用蛋白激酶1和3的作用 (RIP1和RIP 3）在先天免疫应答中的作用，以及 CMV感染。我们的中心假设是RIP 1/RIP 3在未感染者的死亡中起关键作用。 旁观者细胞通过AIF介导的、半胱天冬酶3非依赖性凋亡/坏死性凋亡。我们的目标是利用 CMV视网膜炎的体内小鼠模型、脉络膜MCMV感染的体内小鼠模型和 体外器官型视网膜培养模型，以1）证明RIP激酶如何促进未感染的死亡 旁观者视网膜细胞通过直接激活坏死性凋亡/凋亡途径或间接通过凋亡 诱导剂下游激活caspase 1或NF κ B; 2）确定的机制， 感光细胞死亡由RIP 1/RIP 3激活和脉络膜炎后炎症诱导 MCMV感染和3）开始开发防止细胞死亡的策略。我们的预期成果 包括1）RIP 1和RIP 3激活，其由Bax和caspase 12负调控， 通过AIF介导的、不依赖半胱天冬酶3的视网膜神经元（尤其是光感受器）死亡的中心作用 细胞凋亡/坏死性凋亡;和2）RIP激酶的特异性抑制剂可以大大减少未感染的死亡 旁观者视网膜细胞我们项目的影响包括：1）提供有关 CMV视网膜炎的机制，2）进一步了解细胞死亡的复杂相互作用 控制神经元细胞存活的途径; 3）建立药理学发展的基础 预防或减少CMV患者视网膜神经元死亡和组织损伤的药物 视网膜炎或其他视网膜疾病，其中发病机制取决于或交叉于一些 相同的分子途径;和4）增加我们对人类AMD发病机制的理解 患者目的1将检验RIP 3的激活诱导AIF介导的caspase 3- 直接或间接通过炎性小体和NFκB下游效应子的独立凋亡/坏死性凋亡。 目的2将检验Bax抑制RIP 1和随后的AIF介导的caspase 3的激活的假设。 通过促进半胱天冬酶12活化的独立凋亡/坏死性凋亡。目标3将检验假设 RIP激酶的抑制减轻脉络膜MCMV感染后的感光细胞死亡。

Project Summary Human cytomegalovirus (HCMV) retinitis initially resulted in vision loss and blindness in ~30% of HIV/AIDS patients before development of combination antiretroviral therapy (cART), yet this slowly progressive retinal disease remains a significant ophthalmologic problem in HIV/AIDS patients worldwide who do not respond to cART or who discontinue therapy. Chronic infection with HCMV is a risk factor for the progression of age- related macular degeneration (AMD). A critical barrier to progress in treating and preventing HCMV retinitis is limited understanding of the mechanism of uninfected bystander cell death and the lack of a preventative strategy. Our goal is to determine the role of receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) in innate immune responses and the death of uninfected bystander retinal cells during CMV infection. Our central hypothesis is that RIP1/RIP3 play critical roles in the death of uninfected bystander cells via AIF-mediated, caspase 3-independent apoptosis/necroptosis. Our objectives are to use an in vivo mouse model of CMV retinitis, an in vivo mouse model of choroidal MCMV infection and an in vitro organotypic retinal culture model to 1) demonstrate how RIP kinases promote death of uninfected bystander retinal cells either by directly activating necroptosis/apoptosis pathways or indirectly via apoptosis inducers downstream to activation of caspase 1 or NFkB; 2) identify the mechanism by which photoreceptor cell death is induced by RIP1/RIP3 activation and inflammation subsequent to choroidal MCMV infection and 3) begin development of a strategy to prevent cell death. Our expected outcomes include 1) activation of RIP1 and RIP3, which is negatively regulated by Bax and caspase 12, plays a central role in death of retinal neurons, especially photoreceptors, via AIF-mediated, caspase 3-independent apoptosis/necroptosis; and 2) specific inhibitors of RIP kinases can greatly reduce death of uninfected bystander retinal cells. The impact of our project includes 1) providing new information about the mechanism of CMV retinitis, 2) furthering our understanding of the complex interaction of cell death pathways which govern neuronal cell survival; 3) establishing a basis for development of pharmacological agents which prevent or reduce retinal neuron death and tissue damage in human patients with CMV retinitis or with other retinal diseases in which pathogenesis depends on or intersects with some of the same molecular pathways; and 4) increasing our understanding of the pathogenesis of AMD in human patients. Aim 1 will test the hypothesis that activation of RIP3 induces AIF-mediated, caspase 3- independent apoptosis/necroptosis directly or indirectly via inflammasome and NFκB downstream effectors. Aim 2 will test the hypothesis that Bax inhibits activation of RIP1 and subsequent AIF-mediated, caspase 3- independent apoptosis/necroptosis via promotion of caspase 12 activation. Aim 3 will test the hypothesis that inhibition of RIP kinases relieves photoreceptor death following choroidal MCMV infection.