Cerebrospinal fluid proteome mediated signaling in the developing CNS

描述(申请人提供)：我对神经系统疾病的兴趣源于我在宾夕法尼亚大学的本科教育，在那里，作为阿特·麦克莫里斯博士的Wistar研究所本科生，我研究了触发胶质细胞cAMP信号的外部信号。作为洛克菲勒大学布鲁斯·麦克尤恩博士实验室的一名本科生研究员，我研究了甲状腺功能减退是如何改变大脑中的信号分子的。这些经历让我去了哈佛大学的研究生院，在那里我和阿扎德·邦尼博士一起接受了分子神经科学方面的培训，并阐明了在哺乳动物神经元和线虫体内调节生存和死亡信号的信号通路。在完成我的博士学位后，我在芬兰赫尔辛基的Folkhdlsan遗传学研究所与Anna-Elina Lehejoki博士一起进行了早期的博士后培训，在那里我发现氧化还原平衡受损是引发进行性肌阵挛癫痫-Unverricht-Lundborg病(EPM1)神经退化的关键机制。从这些研究经验中，我获得了分子神经科学及其在神经系统疾病中的应用经验。我加入了克里斯·沃尔什博士的实验室，这是我的第二个博士后，我希望在这个奖项的指导阶段，发育神经生物学的全面培训将补充我在分子神经科学方面的培训，并为终生神经疾病的潜在机制提供独特的视角。沃尔什博士的研究在大脑皮层发育研究领域处于领先地位。我将在实验室学习基本技术，从定量组织分析到宫内电穿孔。沃尔什博士组建了一支才华横溢的科学家团队，他们将继续在非正式对话、每周实验室会议和期刊俱乐部中提供独特的支持和灵感来源。沃尔什实验室位于遗传学分部，该分部还与神经科学分部每周举办一次数据会谈，以促进技术和理论问题的合作交流和协助。在该奖项的指导阶段，这种具有智力吸引力的环境将提供一个极好的讨论和反馈来源。重要的是，遗传学分部致力于支持博士后向独立研究岗位的过渡。为此，我将参加一些职业培训研讨会和会议，为我开始独立研究生涯做准备。我研究的长期目标是阐明脑脊液(CSF)如何发挥信号利基的作用，协调信号因子的丰富相互作用，在健康和疾病期间远距离发挥作用，调节靶细胞的行为。在大脑皮层发育过程中，神经上皮细胞的广大区域几乎同时发生增殖的祖细胞的快速变化。脑脊液中神经上皮纤毛的位置延长，表明脑脊液可能作为引导祖细胞增殖的外部信号源发挥作用。由于脑脊液每天翻转几次，脑脊液-脉络丛系统非常适合触发长距离分子信号的快速和空间同步变化。我在这项提议中的直接研究目标是调查胚胎脑脊液蛋白质组在发育过程中调节皮质前体细胞增殖的作用。Aim1的实验将使用异种外植体和培养的干细胞来测试脑脊液支持皮质祖细胞存活、生长和增殖的能力。由于脑脊液含有数百种蛋白质，我将使用生化和遗传方法来研究在我们的初步质谱分析中确定的候选因子Igf2(胰岛素样生长因子2)如何被脑脊液主动分布以影响祖细胞的增殖(目标2)。该奖项的指导阶段(目标1和2)将有助于完善实验模型、技术和专业技能，以启动成功的独立研究计划，检查脑脊液分布因素影响健康和疾病目标细胞的机制。在AIMS 1和AIMS 2中学习的基本技术，包括神经球培养、定量组织学分析和宫内电穿孔，将使我为AIMS 3中建议的实验做好准备，该实验将探索其他脑脊液携带的信号因子，如维甲酸和Sonic Hedgehog，受脑脊液调节作用于脑室表面靶细胞的作用和机制。到这个获奖期结束时，我的独立研究实验室将开创发育生物学的新概念，其中脑脊液在大脑皮层发育中发挥积极作用。拟议的实验为未来研究脑脊液干细胞生态位在衰老和老年相关神经疾病中的变化奠定了坚实的基础。由于在人类和小鼠模型中，脑脊液是一种可通过手术获得的介质，拟议的实验将为开发强大的诊断和治疗方法铺平道路。

DESCRIPTION (provided by applicant): My interest in diseases of the nervous system stems from my undergraduate education at the University of Pennsylvania, where as a Wistar Institute undergraduate fellow with Dr. Art McMorris, I investigated extrinsic cues triggering cAMP signaling in glia. As an undergraduate research fellow at Rockefeller University in Dr. Bruce McEwen's laboratory, I investigated how hypothyroidism altered signaling molecules in the brain. These experiences led me to graduate school at Harvard University, where I trained with Dr. Azad Bonni in molecular neuroscience and elucidated signaling pathways that mediate survival and death signaling in mammalian neurons and in vivo in C. elegans. Upon completion of my PhD, I carried out my early postdoctocal training with Dr. Anna-Elina Lehesjoki at the Folkhdlsan Institute of Genetics in Helsinki, Finland, where I discovered that impaired redox homeostasis is a key mechanism triggering neurodegeneration in the progressive myoclonus epilepsy, Unverricht-Lundborg disease (EPM1). From these research experiences, I have gained experience in molecular neuroscience and its applications to neurologic disease. I joined Dr. Chris Walsh's lab for my second postdoc, in hopes that comprehensive training in developmental neurobiology during the mentored phase of this award would complement my training in molecular neuroscience, and provide unique perspective on the mechanisms underlying neurologic disease throughout life. Dr. Walsh's is a leader in the field of cerebral cortical development research. I will learn fundamental techniques in the lab, ranging from quantitative histological analyses to in utero electroporations. Dr. Walsh has assembled a highly talented group of scientists, which will continue to provide a unique source of support and inspiration in informal conversations and weekly lab meetings and journal clubs. The Walsh lab is located in the Division of Genetics, which also hosts joint weekly data talks with the Division of Neuroscience, to promote collaborative exchanges and assistance with technical and theoretical issues. This intellectually engaging environment will provide an excellent source of discussion and feedback during the mentored phase of the award. Importantly, the Division of Genetics is committed to supporting the transition of postdocs to independent research positions. To this end, I will attend a number of career training seminars and meetings geared at preparing me for launching my independent research career. The long-term goal of my research is to elucidate how the cerebrospinal fluid (CSF) functions as a signaling niche that coordinates a rich interaction of signaling factors, acting at long distances to regulate target cell behavior during health and disease. During cerebral cortical development, rapid changes in proliferating progenitor cells occur almost synchronously across vast areas of the neuroepithelium. The protracted location of neuroepithelial cilia in the CSF suggests a potential role for the CSF as a source of extrinsic signals guiding progenitor proliferation. Since the CSF turns over several times per day, the CSF-choroid-plexus system is ideally suited for triggering rapid and spatially synchronized changes in molecular signaling across large distances. My immediate research goal in this proposal is to investigate the role of embryonic CSF proteome in regulating cortical progenitor proliferation during development. The experiments in Aim1 will use heterochronic explants and cultured stem cells to test the ability of CSF to support the survival, growth, and proliferation of cortical progenitor cells. Since the CSF contains hundreds of proteins, I will then use biochemical and genetic approaches to examine how Igf2 (Insulin-like growth factor 2), a candidate factor identified in our preliminary mass spectrometry analyses, may be actively distributed by the CSF to influence progenitor proliferation (Aim 2). The mentored phase of the award (Aims 1&2) will help refine the experimental models, techniques, and professional skills needed to launch a successful, independent research program examining the mechanisms by which CSF-distributed factors influence target cells in health and disease. The fundamental techniques including neurosphere cultures, quantitative histological analyses, and in utero electroporations, all learned during Aims 1&2, will prepare me for the experiments proposed in Aim 3, which will explore the roles and mechanisms by which other CSF-borne signaling factors, such as retinoic acid and Sonic hedgehog, are regulated by the CSF to act on target cells at the ventricular surface. By the end of this award period, my independent research laboratory will have pioneered a new concept in developmental biology in which the CSF plays an active role in cerebral cortical development. The proposed experiments represent a solid foundation for future studies investigating changes in the CSF stem cell niche in aging and age-associated neurologic disease. Since the CSF is a surgically accessible medium in humans and mouse models, the proposed experiments will pave the way towards development of powerful diagnostic and therapeutic approaches.