Mechanistic studies of novel factors regulating axon regeneration in the PNS/CNS

描述(由申请人提供)：我的长期职业目标是阐明在生理和病理条件下神经回路的维持和功能的细胞和分子基础。因此，了解神经系统如何应对损伤提供了一个独特的机会来挖掘这些问题，并确定了这项建议的目标-识别和描绘调节轴突再生的新因素，并根据这些新的靶点制定有效的策略来促进轴突再生。为了为实现这一目标奠定坚实的基础，我加入了世界知名的神经电路组装和功能研究先驱Yeh Nung Jan博士的实验室。我开始了我的博士后研究，建立了一个在表型和分子水平上与哺乳动物损伤模型相似的果蝇感觉神经元损伤模型，以筛选轴突再生的新调节因子。我的研究已经确定RNA处理酶RTCA是中枢神经系统(CNS)轴突再生的抑制因子。这些研究的成功完成将验证以RTCA通路为靶点治疗神经损伤和神经退行性疾病的新概念，并推动我未来的独立研究，以揭示控制再生的分子体系。成人中枢神经系统损伤，如脊髓损伤(SCI)，常因成熟轴突损伤后不能再生而导致持续性损伤。在病理情况下，如多发性硬化症(MS)，这是导致年轻人残疾的第二常见神经疾病，受损轴突的再生失败导致不可逆转的神经功能障碍。目前，只有少数几种疗效有限的疗法可用，这突显了确定新的分子靶点和开发靶向疗法的迫切需要。为此，我的博士后研究重点是建立一个平台，利用苍蝇遗传学的力量发现新的因素，并与哺乳动物损伤模型一起研究它们的同源物和功能恢复。在具体目标1中，我将确定轴突变性与再生之间的相互作用，验证扰动变性延迟再生的假说；在特定目标2和3中，我将利用苍蝇和小鼠损伤模型来确定dRtca/RTCA对轴突再生的调控机制，并验证去除dRtca/RTCA促进电路重组和功能恢复的假说。我的广泛研究已经导致最近在2012年发表了第一作者论文《基因与发育》(封面文章)，两篇共同第一作者论文正在修订《神经元》和《NAT Neurosci》。我产生了许多新的试剂(如果蝇损伤模型、转基因和基因敲除果蝇品系以及基因敲除小鼠)，并组建了一个由共同导师组成的非凡的顾问团队(加州大学旧金山分校神经外科常驻教授、加州大学脑和脊髓损伤中心(Basic)研究主任Michael S.Beattie博士；加州大学旧金山分校神经外科教授兼国家神经创伤学会主席Jacqueline C.Bresnahan博士)以及顾问(加州大学旧金山分校细胞药理学和生物化学教授、HMI研究员和国家科学院院士Jonathan S.Weissman博士)。我还与加州大学旧金山分校的BASIC成员建立了密切的合作关系，该组织专门研究脑和脊髓损伤的动物模型。BASIC的文化是高度协作的，包括神经创伤的基础和临床研究项目。因此，我独一无二地准备承担这项申请中提出的具有巨大翻译潜力的小说研究。在我的K99指导阶段(第一年和第二年)提出的研究主要集中在研究果蝇变性和dRtca对轴突再生的调节，以及建立小鼠损伤模型。我将在Yeh Nung Jan博士的监督下和我的顾问团队的支持下进行这些拟议的实验。在R00独立阶段(第3至第5年)，我将重点研究从我的变性筛选中产生的四个新因子在调节苍蝇再生中的作用，以及研究RTCA在调节小鼠再生中的作用。 在我的咨询/协作团队的持续支持下。在这个项目完成时，我希望(1)建立轴突退化和再生之间的机制联系，(2)确定移除dRtca/RTCA如何促进苍蝇和小鼠中枢神经系统轴突再生的分子机制，以及(3)确定轴突退化/再生的其他新调节因子。我提议的研究具有很高的创新性，因为我们对轴突退化/再生的潜在机制的了解还远远不完整，我的研究将带来新的方法来系统地揭示新的调节因子，并有可能提供新的治疗靶点。我拥有发育神经生物学的博士背景，并在生物信息学、小鼠遗传学和外科方面接受过博士后培训，拥有独特的技能，因此我在完成拟议的新颖研究方面处于非常有利的地位。简博士对我的职业目标给予了全力支持，并鼓励我在未来将这些项目带到我的独立实验室。建议的研究建立在我之前和正在进行的工作的逻辑基础上，正如我最近的出版物所证明的那样，毫无疑问，这将为我在不久的将来领导一个新颖而强大的独立研究计划做好准备。

DESCRIPTION (provided by applicant): My long-term career goal is to elucidate the cellular and molecular basis governing the maintenance and function of neural circuits under physiological and pathological conditions. To understand how the nervous system copes with injury thus offers a unique opportunity to tap into these questions and defines the objective of this proposal-to identify and delineate novel factors regulating axon regeneration, and develop effective strategies to promote axon regeneration based on these new targets. To lay a solid foundation for achieving this goal, I joined the laboratory of Dr. Yuh Nung Jan, a world-renowned pioneer in studying neural circuits assembly and function. I initiated my postdoctoral research by establishing a Drosophila sensory neuron injury model that resembles the mammalian injury model at the phenotypical and molecular level, to screen for novel regulators of axon regeneration. My studies have already led to the identification of the RNA processing enzyme Rtca as an inhibitory factor for axon regeneration in the central nervous system (CNS). Successful completion of the proposed studies will validate the novel concept of targeting the Rtca pathway in treating neural injury and neurodegenerative diseases, and spur my future independent research to uncover the repertoire of molecules governing regeneration. Damage to the adult CNS, such as spinal cord injury (SCI) often leads to persistent deficits due to the inability of mature axons to regenerate after injury. Under pathological situations such as multiple sclerosis (MS), the second most common neurological disorder leading to disability in young adults, the failure of damaged axons to regenerate contributes to non-reversible neurologic dysfunction. Currently, only a few therapies with limited efficacy are available, highlighting the urgent need to identify novel molecular targets and develop targeted therapies. To this end, I have focused my postdoctoral research on building a platform to take advantages of the power of fly genetics in discovering novel factors together with the mammalian injury model to study their homologs and functional recovery. I propose two directions to go after the regeneration regulators: in specific aim 1, I will determine the interplay between axon degeneration and regeneration, test the hypothesis that perturbing degeneration delays regeneration; in specific aims 2 and 3, I will determine the mechanisms underlying dRtca/Rtca's regulation on axon regeneration using fly and mouse injury models, and test the hypothesis that removal of dRtca/Rtca enhances circuit reassembly and functional recovery. My extensive study has already resulted in a recent first author publication in Genes and Development in 2012 (cover article), with two co-first author papers in revision in Neuron and Nat Neurosci. I have generated many novel reagents (e.g. the Drosophila injury model, transgenic and knockout fly strains, and knockout mice), and assembled an extraordinary advisory team consisting of co-mentors (Dr. Michael S. Beattie, Professor in Residence of Neurological Surgery at UCSF, Director of Research of UCSF Brain and Spinal Injury Center (BASIC); Dr. Jacqueline C. Bresnahan, Professor of Neurological Surgery at UCSF and President of the National Neurotrauma Society), and consultant (Dr. Jonathan S. Weissman, Professor of Cellular Pharmacology and Biochemistry at UCSF, an HHMI investigator and a member of the National Academy of Science). I have also established close collaborations with members of the BASIC at UCSF, which specializes in animal models of brain and spinal cord injury. The culture of BASIC is highly collaborative, and includes both basic and clinical research projects in neurotrauma. I am therefore uniquely poised to undertake the novel studies proposed in this application with great translational potential. Research proposed in my K99 mentored phase (year 1 and 2) is mainly focused on studying the regulation of axon regeneration by degeneration and dRtca in flies, as well as establishing the mice injury model. I will carry out these proposed experiments with the supervision of Dr. Yuh Nung Jan and support from my advisory team. In the R00 independent phase (year 3 to 5), I will focus on characterizing the roles of four new factors, which came out from my degeneration screen, in modulating regeneration in flies, as well as studying the function of Rtca in regulating regeneration in mice, with the continual support from my advisory/collaborative team. At the completion of this project I expect to have (1) established mechanistic links between axon degeneration and regeneration, (2) determined the molecular mechanisms of how removal of dRtca/Rtca promotes CNS axon regeneration in flies and mice, and (3) identified additional new regulators of axon degeneration/regeneration. My proposed research is highly innovative because our knowledge of the underlying mechanisms is still far from complete for axon degeneration/regeneration, and my studies will bring in new approaches to systematically uncover novel regulators, with the potential to provide new therapeutic targets. With a Ph.D. background in developmental neurobiology, and postdoctoral training in bioinformatics, mouse genetics and surgery, I possess a unique skillset and hence am in a highly advantageous position to accomplish the proposed novel studies. Dr. Jan has extended his full support towards my career goals and has encouraged me to take these projects to my independent lab in the future. The proposed studies build logically on my prior and ongoing works as evidenced by my recent publication, and will undoubtedly prepare me to lead a novel and strong independent research program in the near future.