Non-canonical NLRP3 Inflammasome Activation in Geographic Atrophy

摘要 这笔K99/R00赠款将促进申请者(Nagaraj Kerur博士)的长期目标，使其发展为 视觉研究方面的独立调查者。地理萎缩(GA)是一种与年龄相关的高级形式 黄斑变性(AMD)的特征是视网膜色素上皮(RPE)所致的中心性视力丧失 退化。由于其病因尚不清楚，目前尚无治疗方法。我们最近 发现DICER1缺失导致人类RPE中Alu RNA转录本的病理性积聚 而积累的Alu RNA激活了NLRP3炎症体，进而导致IL-1。 18/MyD88依赖的RPE细胞死亡(Kaneko等人，《自然》2011年；Tarallo等人，Cell 2012)。在激动人心的，新的 初步研究发现，Alu RNA诱导的NLRP3炎症体激活发生在 非规范方式-一种新描述的需要caspase 4(在小鼠中称为caspase 11)的途径 对于NLRP3炎症体激活(相比之下，规范的炎症体激活独立于 Caspase 4)。拟议研究的中心目标是确定caspase 4介导的非正则基因的作用。 NLRP3在GA背景下的炎性小体激活和识别关键信号通路调节 通过以下目的激活caspase 4：(1)定义调节caspase 4(又名 Caspase11)被Alu RNA激活；(2)阐明caspase4(又名caspase11in)的作用机制 小鼠)NLRP3炎症体的激活；以及(3)测量这些非规范的激活 人类GA中的炎症体信号通路。这些研究将阐明GA的分子基础， 解开NLRP3炎症体激活的新的调节检查点，并提供一个强大的分子 为开发AMD新的治疗靶点奠定基础。 在指导阶段，(K99)申请人将在以下人员的直接监督下进行拟议的研究 他的导师是肯塔基大学的贾亚克里希纳·安巴蒂博士。此外，一个由以下成员组成的咨询委员会 共同导师凯瑟琳·菲茨杰拉德博士和眼科和眼科教职员工 视觉科学将监测申请者的研究进展，并为推进 申请人向独立调查员的过渡。肯塔基大学的安巴蒂集团也是如此 作为马萨诸塞大学医学院的菲茨杰拉德实验室，两者都提供了科学的 为申请人的科学事业发展提供必要的环境和资源。这个 K99阶段的主要焦点将包括：(1)定义对caspase 4的分子洞察的指导性研究 活化机理，优化R00阶段研究的实验条件； 和(2)培训活动，包括正式课程工作、研讨会、赠款编写讲习班和 获得成功开发和领导独立研究所需的其他技能 程序。在R00阶段，重点将放在：(1)破译caspase 4 调节NLRP3炎症体的激活；以及(2)量化非典型性炎症体的激活 人类GA眼睛中的信号通路。总体而言，这一提议不仅将解开新的分子 GA中RPE退化的机制，但也将使申请者具备适当的技能以过渡到 独立从事视觉研究。

Abstract This K99/R00 grant will facilitate the applicant's (Dr. Nagaraj Kerur) long-term goal to develop as an independent investigator in vision research. Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD) characterized by central loss of vision due to retinal pigmented epithelium (RPE) degeneration. Currently there is no therapy available for GA because of its unclear etiology. We recently discovered that DICER1 deficit causes a pathologic accumulation of Alu RNA transcripts in the RPE of human GA eyes and that the accumulated Alu RNAs activate the NLRP3 inflammasome, which in turn leads to IL- 18/MyD88-dependent RPE cell death (Kaneko et al., Nature 2011; Tarallo et al., Cell 2012). In exciting, new preliminary studies we have discovered that Alu RNA-induced NLRP3 inflammasome activation occurs in a non-canonical fashion- a newly described pathway in which caspase 4 (aka caspase 11 in mouse) is required for the NLRP3 inflammasome activation (in contrast the canonical inflammasome activation is independent of caspase 4). The central goal of the proposed research is to define role of caspase 4-mediated non-canonical NLRP3 inflammasome activation in the context of GA and to identify critical signaling pathways regulating caspase 4 activation via the following aims: (1) Define the signaling pathways that regulate caspase 4 (aka caspase 11 in mouse) activation by Alu RNA; (2) Elucidate the mechanisms of caspase 4 (aka caspase 11 in mouse) activation of the NLRP3 inflammasome; and (3) Measure the activation of these non-canonical inflammasome signaling pathways in human GA. These studies will illuminate the molecular foundation of GA, unravel novel regulatory checkpoints of NLRP3 inflammasome activation, and provide a strong molecular foundation for exploiting new therapeutic targets in AMD. During the mentored phase, (K99) the applicant will carry out the proposed studies under direct supervision of the mentor Dr. Jayakrishna Ambati at the University of Kentucky. In addition, an advisory committee consisting of co-mentor, Dr. Katherine Fitzgerald and the faculty members at the Department of Ophthalmology and Visual Sciences will monitor the applicant's research progress and offer assistance in advancing the applicant's transition towards independent investigator. The Ambati Group at the University of Kentucky as well as the Fitzgerald Laboratory at the University of Massachusetts Medical School, both offer scientifically enriching environments and resources necessary for the advancement of the applicant's scientific career. The major focus of K99 phase will include: (1) mentored research to define molecular insights into the caspase 4 activation mechanism and to optimize experimental conditions for studies to be carried out in the R00 phase; and (2) training activities consisting of formal course work, seminars, workshops on grant writing and acquisition of other skill-sets required for successfully developing and leading an independent research program. During the R00 phase, the focus will be on: (1) deciphering the mechanisms by which caspase 4 regulates NLRP3 inflammasome activation; and (2) quantifying the activation of non-canonical inflammasome signaling pathways in human GA eyes. Overall this proposal will not only unravel the novel molecular mechanisms of RPE degeneration in GA, but will also equip the applicant with suitable skills to transition into an independent career in vision research.