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探讨这些问题并定义该提案的目标——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促进轴突再生的有效策略。为了为实现这一目标打下坚实的基础，我加入了世界著名的神经回路组装和功能研究先驱Yuh Nung Jan博士的实验室。我通过建立果蝇感觉神经元损伤模型开始了我的博士后研究，该模型在表型和分子水平上类似于哺乳动物损伤模型，以筛选轴突再生的新型调节因子。我的研究已经确定 RNA 加工酶 Rtca 是中枢神经系统 (CNS) 轴突再生的抑制因子。拟议研究的成功完成将验证针对 Rtca 通路治疗神经损伤和神经退行性疾病的新概念，并激励我未来的独立研究，以揭示控制再生的分子库。成人中枢神经系统的损伤，例如脊髓损伤（SCI），由于成熟的轴突在损伤后无法再生，通常会导致持续的缺陷。在多发性硬化症（MS）（导致年轻人残疾的第二常见神经系统疾病）等病理情况下，受损轴突无法再生会导致不可逆的神经功能障碍。目前，只有少数疗效有限的疗法可用，这突出表明迫切需要确定新的分子靶点并开发靶向疗法。为此，我的博士后研究重点是建立一个平台，利用果蝇遗传学的力量发现新的因子，并结合哺乳动物损伤模型来研究它们的同源物和功能恢复。我提出了两个研究再生调节因子的方向：具体目标1，我将确定轴突变性和再生之间的相互作用，检验扰动变性延迟再生的假设；在具体目标 2 和 3 中，我将使用果蝇和小鼠损伤模型确定 dRtca/Rtca 对轴突再生调节的机制，并测试去除 dRtca/Rtca 增强电路重组和功能恢复的假设。我的广泛研究已于 2012 年在 Genes and Development 上发表了一篇第一作者论文（封面文章），并在 Neuron 和 Nat Neurosci 上修订了两篇共同第一作者论文。我制作了许多新颖的试剂（例如果蝇损伤模型、转基因和基因敲除蝇品系以及基因敲除小鼠），并组建了一支由共同导师组成的非凡顾问团队（Michael S. Beattie 博士，加州大学旧金山分校神经外科住院教授、加州大学旧金山分校脑和脊髓损伤中心 (BASIC) 研究主任；Jacqueline C. Bresnahan 博士，加州大学旧金山分校神经外科教授兼国家癌症中心主席神经创伤协会）和顾问（Jonathan S. Weissman 博士，加州大学旧金山分校细胞药理学和生物化学教授、霍华德休斯医学研究所 (HHMI) 研究员和美国国家科学院院士）。我还与 UCSF 的 BASIC 成员建立了密切的合作关系，该机构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，我准备好进行本申请中提出的具有巨大转化潜力的新颖研究。我在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中变性和dRtca对轴突再生的调节，以及建立小鼠损伤模型。我将在 Yuh Nung Jan 博士的监督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），我将重点描述从我的变性筛选中得出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， 在我的咨询/合作团队的持续支持下。在该项目完成时，我希望（1）建立轴突变性和再生之间的机制联系，（2）确定去除 dRtca/Rtca 如何促进果蝇和小鼠中枢神经系统轴突再生的分子机制，以及（3）确定轴突变性/再生的其他新调节因子。我提出的研究具有高度创新性，因为我们对轴突变性/再生的潜在机制的了解还远未完成，我的研究将引入新方法来系统地发现新的调节因子，并有可能提供新的治疗靶点。拥有博士学位。凭借发育神经生物学的背景以及生物信息学、小鼠遗传学和外科手术的博士后培训，我拥有独特的技能，因此处于完成拟议的新颖研究的高度有利的位置。 Jan 博士对我的职业目标给予了全力支持，并鼓励我将来将这些项目带到我的独立实验室。正如我最近发表的文章所证明的那样，拟议的研究逻辑上建立在我之前和正在进行的工作的基础上，并且无疑将为我在不久的将来领导一项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.