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.

摘要 在哺乳动物中，脊髓损伤经常导致不可逆的损伤，主要是由于脊髓损伤的能力非常有限。 损伤的中枢神经系统（CNS）轴突与其损伤前的靶点重新连接。功能性再生 需要受损的CNS轴突长距离延伸并与其原始突触靶点重新连接， 然而，即使在动物模型中，目前的治疗策略也仅产生适度水平的恢复。尽管 在过去的几十年里，我们对基本分子的认识和理解取得了巨大的进展， 促进脊髓再生过程的途径和机制留下了许多基本的 没有答案的问题例如，再生轴突的生长速度是否一致，它们是否预先编程 或者当它们向损伤部位延伸并进入损伤部位时，它们是被调节的？如果是的话， 基因调节和调整再生生长率？与哺乳动物相比，非哺乳类脊椎动物 包括斑马鱼在内的动物都保留了非凡的自发中枢神经系统再生能力。我们已经开发 激光切断法研究斑马鱼幼鱼脊髓再生的单轴突分辨率 在其他方面完好的动物身上。从候选筛选中，我们鉴定了钙粘蛋白EGF LAG受体celsr 3， 在中枢神经系统再生中起着关键作用。我们的初步数据显示，在野生型动物中， 一旦轴突穿过损伤部位，它们的生长速度就会提高3倍。Celsr 3突变体M细胞轴突响应 损伤并以与野生型同胞无区别的生长速率在损伤部位生长，但随后未能 增加它们的生长速率，并且经常在损伤前长度的约25%处过早地停止。所以我们 初步结果确定了一个基因切入点，进入了一个基本但尚未充分研究的问题， 如果是这样的话，再生脊髓轴突的分子机制是通过沿着它们的 随着环境的变化而变化。最后，我们发现Celsr 3也是视神经所必需的。 但对周围神经再生的影响不大，这强烈表明Celsr 3在神经再生中起着重要作用。 在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 Celsr 3选择性生长CNS轴突的分子机制;（2）鉴定 celsr 3通过其促进再生的分子信号级联;和（3）识别额外的进入点 进入促进脊髓自发再生的途径。综合起来，我们的结果有望使 对促进体内自发脊髓再生的基本机制的重大贡献， 为全面分析脊髓自发性再生奠定基础。虽然 自发性脊髓再生在哺乳动物中基本上不存在，自发性脊髓再生的机制 再生可能被生长抑制剂的存在和优势所掩盖，因此无法检测到。 机制因此，我们的研究补充了哺乳动物模型中的研究，这些研究主要集中在 克服生长抑制的策略。