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%的艾滋病毒/艾滋病患者失明和失明 患者在开发联合抗逆转录病毒治疗(CART)之前，但视网膜进展缓慢 疾病仍然是全球艾滋病毒/艾滋病患者的一个严重的眼科问题，这些患者没有反应 或停止治疗的人。慢性感染人巨细胞病毒是年龄进展的危险因素- 相关性黄斑变性(AMD)。人巨细胞病毒治疗和预防进展的关键障碍 视网膜炎是对未感染的旁观者细胞死亡的机制的有限理解和缺乏 预防性战略。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RIP3)在先天性免疫反应和未感染的旁观者视网膜细胞的死亡 巨细胞病毒感染。我们的中心假设是RIP1/RIP3在未感染的死亡中起关键作用 旁观者细胞通过AIF介导，caspase3非依赖的凋亡/坏死下垂。我们的目标是使用 小鼠体内巨细胞病毒视网膜炎模型、脉络膜巨细胞病毒感染小鼠体内模型和小鼠视网膜病变模型 体外器官型视网膜培养模型1)展示RIP激酶如何促进未感染患者的死亡 旁观者视网膜细胞直接激活坏死性下垂/凋亡途径或间接通过凋亡 Caspase 1或NFkB激活的下游诱导物；2)确定 脉络膜后RIP1/RIP3活化和炎症诱导光感受器细胞死亡 MCMV感染和3)开始开发防止细胞死亡的策略。我们的预期结果 包括1)受Bax和Caspase 12负向调节RIP1和RIP3的激活 AIF介导的caspase 3非依赖性在视网膜神经元，尤其是光感受器死亡中的中心作用 细胞凋亡/坏死性下垂；以及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视网膜炎的发病机制，2)加深对细胞死亡复杂相互作用的理解 控制神经细胞存活的途径；3)建立药理学发展的基础 预防或减少巨细胞病毒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，其发病机制依赖于或与某些 相同的分子途径；4)增加我们对AMD在人类中的发病机制的了解 病人。目的1验证以下假设：激活RIP3可诱导AIF介导的caspase 3- 通过炎症体和核因子κB下游效应直接或间接地实现独立的细胞凋亡/坏死性下垂。 目的2将验证假设，即Bax抑制RIP1的激活和随后AIF介导的caspase 3- 通过促进caspase12的激活实现独立的细胞凋亡/坏死性下垂。目标3将检验这一假设 抑制RIP激酶可减轻脉络膜巨细胞病毒感染后光感受器的死亡。

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.