Subcellular investigation of molecular programs responsible for corticospinal neuron development and treatment-enhanced regeneration

创伤性脊髓损伤(Sci)是一种后天获得性疾病，由于缺乏而导致永久性功能缺陷。 中枢神经系统(CNS)的再生能力。中枢神经系统无法重新生成是显而易见的 与其在开发过程中生成精确电路的能力形成对比。皮质脊髓神经元(CSN)是 皮质投射神经元(PN)的亚型，通常连接大脑皮层和脊髓以控制 直接和间接的自愿性电机输出。在发育过程中，CSN轴突穿过很远的距离 沿着吻-尾侧脊髓建立节段性功能环路。设立这样的机构 特定的电路需要严格监管的动态发展计划来逐步完善CSN 身份及其输入和输出连接。损伤后，CSN通常不会重新建立功能 电路。尽管经过了几十年的研究，而且存在多种增加CSN再生的动物模型， 脊髓损伤患者的功能恢复程度基本没有变化。这种缺乏临床进展的情况是 部分原因是对直接导致局部轴突形成的分子机制的了解有限 生长和指导(或不)，在发展和尝试再生期间都是如此。在我的建议中 工作中，我将研究CSN生长锥(GC)与体节的不同转录和蛋白质组 发展和损伤后，朝着选择功能操作的候选分子的方向。 GCS是生长轴突末端的细胞亚室，直接形成神经元亚型- 发育过程中和损伤后特定轴突的生长和指导。CSN GC的直接调查 分子机械有望阐明局部亚细胞过程，这些过程支撑着发育和 可再生的CSN增长。我的实验室最近开发了实验和分析方法，以深入 在体内研究特定亚型和特定阶段的GC。这些方法导致了神经元的识别 局部RNA、蛋白质和翻译的亚型特异性调节 皮质丘脑投射神经元。我和我的实验室已经提纯了胸腰段CSN(CSNTL)GC和体细胞 从出生后第3天(P3)、P5和P7小鼠开始，在轴突延长、灰质神经支配和分支期间。我 将分析获得的RNA测序数据，并将其扩展为蛋白质组学，以选择候选 用于功能研究(目标1)。我将使用脊髓损伤后增加CSN再生的模型(Pten缺失) 研究再生CSNTL的局部RNA蛋白(目标2)。为什么中枢神经再生不发生在 伤害是一个关键的、尚未回答的、具有巨大翻译意义的基本问题。我的工作目标是 阐明发育生长程序负责引导适当的CSN轴突延长，节段- 特定的分支和侧化，以及突触靶向。重要的是，我的工作还旨在阐明 损伤后中枢神经系统生长的分子机制。更深层次的分子阐释 再生生长机制将使未来针对脊髓损伤致残的靶向治疗的发展成为可能。

Traumatic spinal cord injury (SCI) is an acquired disorder causing permanent functional deficits due to lack of regenerative ability of the central nervous system (CNS). The inability of the CNS to re-generate is in stark contrast with its ability to generate precise circuitry during development. Corticospinal neurons (CSN) are the subtype of cortical projection neurons (PN) that normally connect the cerebral cortex to the spinal cord to control voluntary motor output directly and indirectly. During development, CSN axons traverse vast distances to establish segmentally-specific functional circuitry along the rostro-caudal spinal cord. Establishment of such specific circuitry necessitates tightly regulated, dynamic developmental programs to progressively refine CSN identity and their input and output connections. After injury, CSN do not normally re-establish functional circuitry. Despite decades of research, and existence of multiple animal models of increased CSN regeneration, the extent of functional recovery for people with SCI remains largely unchanged. This lack of clinical advance is partly due to limitations of understanding of molecular mechanisms directly responsible for locally enacting axon growth and guidance (or not), both during development and during attempted regeneration. In my proposed work, I will investigate the distinct transcriptomes and proteomes of CSN growth cones (GCs) vs. somata during development and after injury, toward selecting molecular candidates for functional manipulation. GCs are the cellular subcompartments at the ends of growing axons that directly enact neuronal subtype- specific axon growth and guidance during development and after injury. Direct investigation of CSN GC molecular machinery promises to elucidate local subcellular processes that underpin developmental and regenerative CSN growth. My lab has recently developed experimental and analytic approaches to deeply investigate subtype- and stage-specific GCs in vivo. These approaches have led to identification of neuronal subtype-specific regulation of local RNA, protein, and translation in interhemispheric callosal and corticothalamic projection neurons. My lab and I have purified thoracolumbar CSN (CSNTL) GCs and somata from postnatal day 3 (P3), P5, and P7 mice, during axon elongation, grey matter innervation, and branching. I will analyze the RNA sequencing data obtained, and will expand by addition of proteomics, to select candidates for functional investigation (Aim 1). I will use a model of increased CSN regeneration after SCI (Pten deletion) to investigate local RNA-protein of regenerating CSNTL (Aim 2). Why CNS regeneration does not occur after injury is a critical unanswered, fundamental question with immense translational implications. My work aims to elucidate developmental growth programs responsible for directing appropriate CSN axon elongation, segment- specific branching and collateralization, and synapse targeting. Importantly, my work also aims to elucidate molecular mechanisms responsible for enabling growth of the CNS after injury. Deeper elucidation of molecular mechanisms of regenerative growth will enable future development of targeted therapies for disability from SCI.