Non-canonical NLRP3 Inflammasome Activation in Geographic Atrophy

描述（由申请人提供）：这笔K99/R00资助将促进申请人（Nagaraj Kerur博士）成为视觉研究领域独立研究者的长期目标。地理萎缩（GA）是年龄相关性黄斑变性（AMD）的一种晚期形式，其特征是由于视网膜色素上皮（RPE）变性导致中央视力丧失。由于其病因不明，目前尚无治疗GA的方法。我们最近发现DICER1缺陷导致人类GA眼RPE中Alu RNA转录物的病理积累，积累的Alu RNA激活NLRP3炎性体，进而导致IL- 18/ myd88依赖性RPE细胞死亡（Kaneko等人，Nature 2011； Tarallo等人，cell 2012）。在令人兴奋的新初步研究中，我们发现Alu rna诱导的NLRP3炎性小体激活以非规范方式发生-一种新描述的途径，其中caspase 4（在小鼠中又称为caspase 11）是NLRP3炎性小体激活所必需的（相反，规范炎性小体激活独立于caspase 4）。本研究的中心目标是确定caspase 4介导的非规范NLRP3炎性体激活在GA背景下的作用，并通过以下目的确定调节caspase 4激活的关键信号通路：(1)确定Alu RNA激活caspase 4（在小鼠中称为caspase 11）的信号通路；(2)阐明caspase 4（小鼠caspase 11）激活NLRP3炎性体的机制；(3)测量人类GA中这些非典型炎性体信号通路的激活。这些研究将阐明GA的分子基础，揭示NLRP3炎性小体活化的新调控检查点，并为开发AMD的新治疗靶点提供强有力的分子基础。在指导阶段，（K99）申请人将在肯塔基大学导师Jayakrishna Ambati博士的直接监督下进行拟议的研究。此外，由共同导师凯瑟琳·菲茨杰拉德博士和眼科和视觉科学系的教职员工组成的咨询委员会将监督申请人的研究进展，并为申请人向独立研究者过渡提供帮助。肯塔基大学的安巴蒂集团以及马萨诸塞大学医学院的菲茨杰拉德实验室都为申请人的科学事业发展提供了必要的科学丰富的环境和资源。K99期的主要重点将包括：(1)指导研究，以确定caspase 4激活机制的分子见解，并为R00期的研究优化实验条件；(2)培训活动，包括正式的课程作业、研讨会、关于拨款写作和获得成功开发和领导独立研究项目所需的其他技能的讲习班。在R00阶段，重点将放在：(1)破译caspase 4调节NLRP3炎性体激活的机制；(2)量化人眼非典型炎性体信号通路的激活。总的来说，这一提议不仅将揭示GA中RPE变性的新分子机制，而且将为申请人提供适当的技能，以过渡到独立的视觉研究职业。

DESCRIPTION (provided by applicant): 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 int an independent career in vision research.