Development of a C. elegans model for axonal regeneration

描述（由申请人提供）：很少有研究利用遗传学和功能基因组学的力量来了解轴突损伤后再生的机制。我们开发了飞秒激光轴突切开术，以切割完整的秀丽隐杆线虫活体动物的单个轴突。一些秀丽隐杆线虫细胞类型的切断轴突表现出强劲的再生和功能恢复。我们发现，细胞类型、轴突切除位置和生命阶段等因素可调节损伤后轴突是否再生。保守的信号通路，包括环AMP信号通路和ephrin信号通路，调节轴突的再生生长。我们还发现了突触分支在调节轴突再生中的一个意想不到的作用。可用于秀丽隐杆线虫的易于操作的遗传和基因组工具有助于大规模筛选新的再生基因。初步筛选发现了几种促进或抑制再生生长的新基因。我们的三个具体目标建立在这些初步结果的基础上：首先，我们将剖析突触分支调节机械感觉神经元再生的机制。我们假设突触分支点包含一个在损伤后调节膜和细胞器运输的分选区域。我们将分析再生所需的马达和货物的运输，并将具体测试Liprin通路在促进再生中的作用。其次，我们将定义cAMP信号如何促进秀丽隐杆线虫的神经元再生。我们将测试再生是否需要cAMP或其效应物。我们将研究肛门切开术对体内cAMP动力学的影响。我们将测试一种假定的camp调节转录因子的作用，我们发现这种转录因子对再生至关重要。第三，我们将进行大规模的功能基因组筛选，以确定在再生轴突生长中起作用的新基因。具有强促或抗再生作用的基因的机制将被详细研究。相关性：这项工作将在一个简单的模型系统中对损伤后调节轴突再生的途径产生系统的理解。了解控制轴突再生的保守机制将使它们能够在神经系统疾病和损伤的治疗中得到操纵。公共卫生相关性：我们将分析秀丽隐杆线虫神经再生的分子和遗传机制。具体来说，我们将定义轴突分支点的运输作用和循环AMP信号的功能。我们将进行大规模筛选，以发现参与神经元再生生长的新基因。这一结果将进一步加深我们对神经再生的理解，并将有助于治疗创伤和神经系统疾病。

DESCRIPTION (provided by applicant): Few studies have exploited the power of genetics and functional genomics to understand the mechanisms of regrowth of axons following injury. We have developed femtosecond laser axotomy to cut single axons in intact living C. elegans animals. Severed axons of several C. elegans cell types show robust regrowth and functional recovery. We have shown that several factors, including cell type, position of axotomy and life stage, can regulate whether axons regrow after injury. Conserved signaling pathways, including cyclic AMP signaling and ephrin signaling, regulate regenerative growth of axons. We also found an unexpected role for synaptic branches in regulating axon regrowth. The tractable genetic and genomic tools available in C. elegans facilitate large scale screens for new regeneration genes. A pilot screen has uncovered several new genes that promote or repress regenerative growth. Our three specific Aims build on these preliminary results: First, we will dissect the mechanism by which the synaptic branch regulates regeneration in mechanosensory neurons. We hypothesize that the synaptic branch point contains a sorting area that regulates membrane and organelle traffic after injury. We will analyze the transport of motors and cargoes required for regrowth and will specifically test the role of the Liprin pathway in promoting regrowth. Second we will define how cAMP signaling promotes C. elegans neuronal regeneration. We will test whether cAMP or its effectors are required for regrowth. We will examine the effects of axotomy on cAMP dynamics in vivo. We will test the role of a putative cAMP-regulated transcription factor that we have found is essential for regeneration. Third, we will perform a large scale functional genomic screen to identify new genes with roles in regenerative axon growth. The mechanisms of genes with strong pro- or anti-regeneration roles will be studied in detail. Relevance: This work will yield a systematic understanding of the pathways that regulate axon regeneration after injury in a simple model system. Knowledge of the conserved mechanisms controlling axon regeneration will allow their manipulation in therapies for nervous system disease and injury. PUBLIC HEALTH RELEVANCE: We will analyze the molecular and genetic mechanisms underlying nerve regeneration in the nematode C. elegans. Specifically, we will define the roles of trafficking at axonal branch points and the function of cyclic AMP signaling. We will perform a large scale screen to discover novel genes involved in regenerative growth of neurons. The results will further our understanding of nerve regrowth and will aid therapies for trauma and disease of the nervous system.