Molecular genetic mechanisms of spontaneous spinal cord regeneration

摘要 在哺乳动物中，脊髓损伤经常导致不可逆的损伤，主要是由于非常有限的 损伤的中枢神经系统(CNS)轴突与损伤前的目标重新连接。功能再生 需要受损的中枢神经系统轴突延伸很长一段距离，并与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。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步 参与脊髓再生过程的途径和机制留下了许多基本的 未回答的问题。例如，再生轴突的生长速度是均匀的吗，它们是预先编程的吗 当它们向受伤部位延伸或进入受伤部位时，它们是不变的还是有调节的？如果是这样，是什么机制 基因调控和调节再生生长速度？与哺乳动物相比，非哺乳类脊椎动物 包括斑马鱼在内，都保留了显著的中枢神经系统自发再生能力。我们已经开发出 基于激光的轴突切断术研究斑马鱼幼体脊髓在单一轴突分辨率下的再生 在其他完好无损的动物身上。从一个候选筛选中，我们确定了Cherin EGF LAG受体celsr3为 在中枢神经系统再生中起关键作用。我们的初步数据显示，在野生型动物中，再生M-ell 一旦轴突穿过损伤部位，它们的生长速度就会提高3倍。Celsr3突变的M细胞轴突反应 受伤，并以与野生型兄弟姐妹难以区分的生长速度穿过受伤部位，但随后未能 加快它们的生长速度，并经常在受伤前约25%的长度处过早失速。因此，我们的 初步结果确定了一个基因切入点，进入了一个基本但研究不足的问题，即是否和 如果是这样的话，再生脊髓轴突的分子机制是通过什么来调节它们沿 随着环境的变化，它们的再生路径。最后，我们发现Celsr3也是视神经所必需的。 再生，但对于周围神经的再生是可有可无的，强烈表明Celsr3在 中枢神经系统轴突再生中的选择性作用。该方案中的实验将(1)确定细胞和 Celsr3选择性生长中枢神经系统轴突的分子机制；(2)鉴定 分子信号级联，通过它，celsr3促进再生；和(3)识别额外的入口点 转化为促进脊髓自发再生的途径。总而言之，我们的结果预计将使 对促进体内脊髓自发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了基础。虽然 在哺乳动物中，自发脊髓再生在很大程度上是不存在的，自发脊髓的机制 再生可能被生长抑制的存在和优势所掩盖，因此无法被检测到 机制。因此，我们的研究是对哺乳动物模型研究的补充，主要集中在 克服生长抑制的策略。

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.