Molecular genetic mechanisms of spontaneous spinal cord regeneration

脊髓自发再生的分子遗传学机制

• 批准号: 10681837
• 负责人: Michael Granato
• 金额: $ 47.26万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-15 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681837
• 关键词: Adult; Animal Model; Animals; Astrocytes; Axon; Axotomy; Cadherins; Cells; Central Nervous System; Complement; Data; Development; Dystroglycan; EGF gene; Environment; Foundations; Functional Regeneration; Gene Silencing; Genes; Genetic; Growth; Hour; Injury; Knowledge; Lasers; Left; Length; Libraries; Mammals; Masks; Modeling; Molecular; Molecular Genetics; Molecular Target; Natural regeneration; Nerve Regeneration; Neuroglia; Neurons; Oligodendroglia; Pathway interactions; Phenotype; Play; Process; Recovery; Reporting; Resolution; Role; Siblings; Signal Pathway; Signal Transduction; Site; Spinal Cord; Spinal cord injury; Synapses; System; Testing; Time; Transgenic Organisms; Vertebrates; Zebrafish; axon regeneration; candidate selection; cell regeneration; cell type; central nervous system injury; compound 30; experimental study; in vivo; in vivo regeneration; live cell imaging; mRNA Expression; mutant; neurodevelopment; optic nerve regeneration; peripheral nerve regeneration; planar cell polarity; premature; receptor; regenerative; regenerative growth; small molecule; spinal cord regeneration; tool; treatment strategy

ABSTRACT In mammals, spinal cord injury frequently leads to irreversible damage mainly due to the very limited capacity of injured central nervous system (CNS) axons to reconnect with their preinjury targets. Functional regeneration requires injured CNS axons to extend over long distances and reconnect with their original synaptic targets, however even in animal models current treatment strategies produce only modest levels of recovery. Despite enormous progress over the past decades, our knowledge and understanding of the fundamental molecular pathways and mechanisms that contribute to the process of spinal cord regeneration has left many fundamental questions unanswered. For example, are growth rates of regenerating axons uniform, are they preprogramed and invariable or are they modulated as they extend towards and into the injury site? And if so, what mechanisms and genes regulate and tune regenerating growth rates? In contrast to mammals, non-mammalian vertebrates including zebrafish have retained a remarkable capacity for spontaneous CNS regeneration. We have developed a laser-based axotomy approach to study spinal cord regeneration in larval zebrafish at single axon resolution in otherwise intact animals. From a candidate screen we identified the Cadherin EGF LAG receptor celsr3 to play a critical role in CNS regeneration. Our preliminary data reveal that in wild type animals regenerating M-ell axons switch to 3 fold higher growth rates once they cross the injury site. Celsr3 mutant M-cell axons respond to injury and grow across the injury site at growth rates indistinguishable from wildtype siblings, but then fail to increase their growth rates and frequently stall prematurely at about 25% of pre-injury length. Thus, our preliminary results identified a genetic entry point into the fundamental yet understudied question of whether and if so through which molecular mechanisms regenerating spinal cord axons regulate their growth rates along their regenerative path as their environment changes. Finally, we find that Celsr3 is also required for optic nerve regeneration but is dispensable for peripheral nerve regeneration, strongly suggesting that Celsr3 plays a selective role in CNS axon regeneration. The experiments in this proposal will (1) determine cellular and molecular mechanism by which Celsr3 growth rates selectively of regenerating CNS axons; (2) identify the molecular signaling cascade through which celsr3 promotes regeneration; and (3) Identify additional entry points into pathways that promote spontaneous spinal cord regeneration. Combined, our results are expected to make significant contributions to fundamental mechanisms that promote spontaneous spinal cord regeneration in vivo, and lay the foundation for a comprehensive analysis of spontaneous spinal cord regeneration. Although spontaneous spinal cord regeneration is largely absent in mammals, mechanisms of spontaneous spinal cord regeneration might be masked and thus undetectable by the presence and dominance of growth inhibitory mechanism. Our studies therefore complement studies in mammalian models that focus predominantly on strategies to overcome growth inhibition.

抽象的 在哺乳动物中，脊髓损伤经常导致不可逆的损伤，这主要是由于脊髓损伤的能力非常有限。 受伤的中枢神经系统（CNS）轴突与受伤前的目标重新连接。功能再生 需要受伤的中枢神经系统轴突长距离延伸并与其原始突触目标重新连接， 然而，即使在动物模型中，目前的治疗策略也只能产生适度的恢复水平。尽管 过去几十年的巨大进步，我们对基本分子的知识和理解 有助于脊髓再生过程的途径和机制留下了许多基本的 未解答的问题。例如，再生轴突的生长速度是否均匀，是否经过预先编程 当它们延伸到受伤部位并进入受伤部位时，它们是不变的还是受到调节？如果是的话，是什么机制 基因调节和调节再生生长速度？与哺乳动物相比，非哺乳动物脊椎动物 包括斑马鱼在内的动物保留了非凡的中枢神经系统自发再生能力。我们开发了 基于激光的轴切术方法以单轴突分辨率研究斑马鱼幼虫的脊髓再生 在其他完好的动物中。从候选筛选中，我们鉴定出钙粘蛋白 EGF LAG 受体 celsr3 在中枢神经系统再生中发挥重要作用。我们的初步数据表明，在野生型动物中再生 M-ell 一旦轴突穿过损伤部位，其生长速度就会提高 3 倍。 Celsr3 突变体 M 细胞轴突做出反应 受伤并在受伤部位以与野生型兄弟姐妹无法区分的生长速度生长，但随后未能 增加其生长速度，并且经常过早地停滞在受伤前长度的 25% 左右。因此，我们的 初步结果确定了一个尚未得到充分研究的基本问题的遗传切入点：是否和 如果是这样的话，再生脊髓轴突通过哪些分子机制调节它们的生长速率 随着环境变化的再生路径。最后，我们发现Celsr3也是视神经所必需的 但对于周围神经再生来说是可有可无的，这强烈表明 Celsr3 发挥着重要作用 CNS 轴突再生中的选择性作用。本提案中的实验将（1）确定细胞和 Celsr3选择性地促进中枢神经系统轴突再生的分子机制； (2) 识别 celsr3 通过分子信号级联促进再生； (3) 确定其他切入点 进入促进脊髓自发再生的途径。综合起来，我们的结果预计将 对促进体内自发脊髓再生的基本机制做出了重大贡献， 为全面分析脊髓自发再生奠定基础。虽然 哺乳动物中基本上不存在自发性脊髓再生，自发性脊髓再生的机制 再生可能被掩盖，因此无法被生长抑制的存在和优势检测到 机制。因此，我们的研究补充了主要关注哺乳动物模型的研究 克服生长抑制的策略。