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

负责皮质脊髓神经元发育和治疗增强再生的分子程序的亚细胞研究

• 批准号: 10751204
• 负责人: Maria Alejandra Vicent Allende
• 金额: $ 4.02万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751204
• 关键词: Adult; Animal Model; Axon; Body Size; Cell physiology; Cells; Central Nervous System; Cerebral cortex; Clinical; Cytoskeleton; Data; Development; Disabled Persons; Disease; Elements; Environment; Exhibits; Future; Genetic; Goals; Growth; Growth Cones; Human; In Vitro; Injury; Investigation; Label; Maintenance; Mediating; Methodology; Modeling; Molecular; Motor output; Movement; Mus; Natural regeneration; Nerve Regeneration; Neurons; Output; Parents; Persons; Process; Proteins; Proteome; Proteomics; RNA; Recovery of Function; Regenerative capacity; Regulation; Research; Signal Transduction; Spinal Cord; Spinal cord injury; Synapses; System; Therapeutic; Thoracic spinal cord structure; Traction; Training; Transcript; Translations; Validation; Work; axon growth; axon guidance; candidate identification; candidate selection; combinatorial; comparative; disability; gain of function; genetic manipulation; gray matter; in vivo; injured; insight; meter; nerve supply; neuron development; neuron regeneration; neuronal cell body; neuronal circuitry; neuronal growth; novel strategies; postnatal; postsynaptic; programs; receptor; recruit; regenerative; regenerative growth; repaired; subcellular targeting; synaptogenesis; targeted treatment; trafficking; transcriptome; transcriptome sequencing; translational progress

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.

创伤性脊髓损伤 (SCI) 是一种后天性疾病，由于缺乏营养而导致永久性功能缺陷 中枢神经系统（CNS）的再生能力。中枢神经系统无法再生是显而易见的 与其在开发过程中生成精确电路的能力形成鲜明对比。皮质脊髓神经元 (CSN) 是 皮质投射神经元 (PN) 的亚型，通常将大脑皮层连接到脊髓以进行控制 直接和间接的自愿运动输出。在发育过程中，CSN 轴突穿越很长的距离 沿头尾脊髓建立节段特异性功能回路。设立这样的 特定电路需要严格监管、动态开发程序来逐步完善 CSN 身份及其输入和输出连接。受伤后，CSN 通常不会重新建立功能 电路。尽管经过数十年的研究，并且存在多种增加 CSN 再生的动物模型， SCI 患者的功能恢复程度基本保持不变。临床进展的缺乏是 部分原因是对直接负责局部轴突的分子机制的理解有限 生长和引导（或不引导），无论是在发育过程中还是在尝试再生过程中。在我的提议中 在工作中，我将研究 CSN 生长锥 (GC) 与 somata 的不同转录组和蛋白质组 发育和损伤后，以选择用于功能操作的候选分子。 GC 是生长轴突末端的细胞亚区室，直接产生神经元亚型 发育期间和受伤后的特定轴突生长和指导。直接调查CSN GC 分子机器有望阐明支撑发育和发育的局部亚细胞过程 CSN 再生生长。我的实验室最近开发了实验和分析方法来深入研究 研究体内亚型和阶段特异性 GC。这些方法已导致神经元的鉴定 半球间胼胝体中局部RNA、蛋白质和翻译的亚型特异性调节 皮质丘脑投射神经元。我和我的实验室纯化了胸腰椎 CSN (CSNTL) GC 和体细胞 出生后第 3 天 (P3)、P5 和 P7 小鼠，在轴突伸长、灰质神经支配和分支期间。我 将分析获得的 RNA 测序数据，并通过添加蛋白质组学进行扩展，以选择候选者 用于功能研究（目标 1）。我将使用 SCI 后 CSN 再生增加的模型（Pten 删除） 研究再生 CSNTL 的局部 RNA 蛋白（目标 2）。为什么中枢神经系统再生后不发生 伤害是一个尚未得到解答的关键基本问题，具有巨大的转化意义。我的工作目的是 阐明负责指导适当的 CSN 轴突伸长、分段的发育生长计划 特定的分支和抵押，以及突触靶向。重要的是，我的工作还旨在阐明 负责损伤后中枢神经系统生长的分子机制。更深入地阐明分子 再生生长机制将使未来能够开发针对 SCI 致残的靶向疗法。