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生理和病理条件下控制神经回路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，并定义了本提案的目标-识别和描述调节轴突再生的新因素，并根据这些新目标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Yuh Nung Jan博士的实验室，他是研究神经回路组装和功能的世界知名先驱。我的博士后研究是通过建立一个在表型和分子水平上类似于哺乳动物损伤模型的果蝇感觉神经元损伤模型，来筛选新的轴突再生调节因子。我的研究已经鉴定出RNA加工酶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靶向Rtca通路治疗神经损伤和神经退行性疾病的新概念，并刺激我未来的独立研究，以揭示控制再生的分子库。成人中枢神经系统的损伤，如脊髓损伤（SCI），由于成熟轴突在损伤后无法再生，通常会导致持续的缺陷。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大常见神经系统疾病，受损轴突无法再生导致不可逆转的神经功能障碍。目前，仅有少数几种疗效有限的治疗方法可用，这突出表明迫切需要确定新的分子靶点并开发靶向治疗。为此，我的博士后研究重点是建立一个平台，利用果蝇遗传学发现新因素的力量，结合哺乳动物损伤模型，研究它们的同源性和功能恢复。我提出了两个研究再生调控因子的方向：在具体目标1中，我将确定轴突退化与再生之间的相互作用，验证干扰退化延迟再生的假设；在具体目标2和3中，我将通过果蝇和小鼠损伤模型确定dRtca/Rtca调控轴突再生的机制，并验证去除dRtca/Rtca可增强电路重组和功能恢复的假设。我的广泛研究已经在2012年的《基因与发展》杂志上发表了一篇第一作者论文（封面文章），并在《神经元》和《Nat神经科学》上发表了两篇共同第一作者论文。我已经产生了许多新颖的试剂（例如果蝇损伤模型，转基因和基因敲除蝇株，基因敲除小鼠），并组建了一个非凡的顾问团队，由共同导师组成(Michael S. Beattie博士，UCSF神经外科住院教授，UCSF脑和脊髓损伤中心（BASIC）研究主任；Jacqueline C. Bresnahan博士，UCSF神经外科教授，国家神经创伤学会主席)和顾问（Jonathan S. Weissman博士，UCSF细胞药理学和生物化学教授，HHMI研究员和国家科学院成员）。我还与UCSF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，我有独特的准备承担在这个应用程序中提出的具有巨大转化潜力的新研究。我在K99指导阶段（1、2年）提出的研究主要是研究果蝇变性和dRtca对轴突再生的调控，以及建立小鼠损伤模型。我将在Yuh 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年至第5年），我将重点描述从我的退化屏幕中得到的四个新因子在调节果蝇再生中的作用，以及研究Rtca在调节小鼠再生中的功能。

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.