Molecular Mechanisms of Neural Circuit Formation

描述（申请人提供）：人类中枢神经系统由1000亿个神经元组成，通过100万亿个突触连接互连成精确的电路。这些回路是神经系统功能所必需的，包括感知，思维和行为。我们对神经回路形成的早期步骤有很多了解，在这些步骤中，神经突延伸到包含正确突触伴侣的目标区域。当单个神经元到达具有许多神经元的目标区域时，它们如何选择正确的突触伙伴，这一点知之甚少。我们开发了一种新的方法来可视化特定神经元之间的接触和突触发生。我们建议使用这种方法，并利用简单，良好的特点，神经系统的C。elegans阐明突触伴侣选择的分子机制。这是一个重要的研究领域，因为突触发生的改变被认为在精神分裂症和自闭症等疾病中发挥作用。这一建议与NIGMS的使命有关，即支持增加对生命过程的理解的研究，包括发育生物学领域，为疾病诊断、治疗和预防方面的进展奠定基础，并培训下一代科学家。为了了解正确的突触伴侣选择是如何介导的，我们开发了一种遗传编码的荧光跨突触标记物，用于在复杂环境中视觉标记感兴趣的单个神经元之间的突触接触，称为NLG-1 GRASP，用于神经配素-1介导的GFP跨突触伴侣重建。我们还用红色mCherry荧光团标记了突触前和突触后神经突。总之，这些标记使我们能够通过可视化活体动物中感兴趣的突触前和突触后神经元之间的神经突接触和突触发生来立即评估正确的突触伴侣选择，从而使使用遗传方法来发现介导这一基本过程的基因成为可能。此外，我们还在C. elegans，唯一的模式生物，有一个完整的突触地图，使其成为理想的研究突触伴侣的选择。使用这个标记，我们发现，先前研究过的两种蛋白质在其他系统中的细胞迁移和轴突导向中的作用，即β-40/DCC（在结直肠癌中缺失）和β-6/Netrin，在介导C中感觉神经元和中间神经元之间的突触伴侣选择中具有新的作用。腹神经索我们的研究将进一步描述这种作用，并确定介导突触伴侣选择的途径。我们的具体目标是：1）表征β-40/DCC受体和β-6/Netrin配体在突触伴侣选择中的作用，2）研究β-40/DCC介导的轴突导向和细胞迁移信号在突触伴侣选择中的作用的基因，和3）通过进行正向遗传筛选鉴定介导突触伴侣选择的新基因。了解调节回路形成的机制将使我们更接近理解和治疗神经系统疾病。 公共卫生相关性：为了使神经系统正常工作，神经元必须忠实地识别与之形成突触的细胞伙伴。突触形成的改变被认为是精神分裂症和自闭症等神经系统疾病的基础。我们试图确定突触伴侣选择的分子机制，因为了解这些机制将使我们更接近理解和治疗神经系统疾病。

DESCRIPTION (provided by applicant): The human central nervous system is composed of 100 billion neurons interconnected into precise circuits by 100 trillion synaptic connections. These circuits are required for nervous system functions including perception, thought and behavior. Much is known about the early steps in circuit formation in which neurites extend to target regions containing the correct synaptic partners. Much less is known about how individual neurons choose the correct synaptic partner when they reach a target region with many neurons. We have developed a novel method to visualize contact and synaptogenesis between specific neurons in vivo. We propose to use this method and take advantage of the simple, well-characterized nervous system of C. elegans to elucidate molecular mechanisms that underlie synaptic partner choice. This is an important area of study, as altered synaptogenesis is thought to play a role in disorders such as schizophrenia and autism. This proposal is relevant to the NIGMS mission to support research that increases understanding of life processes including the field of developmental biology, that lay the foundation for advances in disease diagnosis, treatment, and prevention, and to train the next generation of scientists. To understand how correct synaptic partner choice is mediated, we have developed a genetically encoded fluorescent trans-synaptic marker to visually label synaptic contacts between individual neurons of interest in complex environments called NLG-1 GRASP, for Neuroligin-1-mediated GFP Reconstitution Across Synaptic Partners. We have also labeled pre- and postsynaptic neurites with the red mCherry fluorophore. Together, these markers enable us to instantly assess correct synaptic partner choice by visualizing neurite contact and synaptogenesis between pre- and postsynaptic neurons of interest in live animals, making it feasible to use genetic methods to discover genes mediating this fundamental process. In addition, we have developed these markers in C. elegans, the only model organism for which there is a complete synaptic map, making it ideal for the study of synaptic partner choice. Using this marker, we have found that two proteins previously studied for their role in cell migration and axon guidance in other systems, UNC-40/DCC (deleted in colorectal cancer) and UNC-6/Netrin, have a novel role in mediating synaptic partner choice between sensory neurons and interneurons in the C. elegans ventral nerve cord. Our research will further characterize this role and define the pathway(s) that mediate synaptic partner choice. Our specific aims are: 1) to characterize the role of the UNC-40/DCC receptor and the UNC-6/Netrin ligand in synaptic partner choice, 2) to investigate genes that transduce UNC-40/DCC-mediated axon guidance and cell migration signals for roles in synaptic partner choice, and 3) to identify new genes that mediate synaptic partner choice by conducting a forward genetic screen. Understanding the mechanisms that regulate circuit formation will bring us closer to understanding and treating neurological diseases. PUBLIC HEALTH RELEVANCE: For the nervous system to function correctly, neurons must faithfully identify cellular partners with which to form synapses. Altered synapse formation is thought to underlie neurological diseases, such as schizophrenia and autism. We seek to identify the molecular mechanisms that underlie synaptic partner choice, as understanding these mechanisms will bring us closer to understanding and treating neurological diseases.