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。在神经回路形成的早期步骤中，神经突起延伸到包含正确突触伙伴的目标区域，这一点我们知道得很多。当单个神经元到达一个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，我们知道的要少得多。我们开发了一种新的方法来可视化体内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，并利用线虫简单的、特征良好的神经系统来阐明突触伴侣选择的分子机制。这是一个重要的研究领域，因为改变的突触发生被认为在精神分裂症和自闭症等疾病中发挥作用。这项建议与NIGMS的任务有关，该任务旨在支持增加对包括发育生物学领域在内的生命过程的理解的研究，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伙伴选择是如何介导的，我们开发了一种基因编码的荧光跨突触标记物，用于可视地标记复杂环境中感兴趣的单个神经元之间的突触接触，称为NLG-1 GRASP，用于神经连接素-1介导的跨突触伙伴的GFP重建。我们还用红色的mCherry荧光团标记了突触前和突触后的轴突。总之，这些标记使我们能够通过可视化活动物中感兴趣的突触前和突触后神经元之间的轴突接触和突触发生，使我们能够立即评估正确的突触伙伴选择，从而使使用遗传学方法来发现介导这一基本过程的基因成为可能。此外，我们还在线虫身上开发了这些标记，线虫是唯一有完整突触图谱的模式生物，这使其成为研究突触伴侣选择的理想工具。利用这个标记，我们发现了两个先前在其他系统中被研究的在细胞迁移和轴突引导中的作用的蛋白质，UNC-40/DCC(在结直肠癌中缺失)和UNC-6/Netrin，在线虫腹神经索的感觉神经元和中间神经元之间的突触伙伴选择中具有新的作用。我们的研究将进一步描述这一角色，并确定调节突触伴侣选择的途径(S)。我们的具体目标是：1)研究UNC-40/DCC受体和UNC-6/Netrin配体在突触伙伴选择中的作用，2)研究UNC-40/DCC介导的轴突引导和细胞迁移信号的转导基因在突触伙伴选择中的作用，3)通过进行正向遗传筛选来鉴定介导突触伙伴选择的新基因。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。 与公共健康相关：为了让神经系统正常运作，神经元必须忠实地识别与之形成突触的细胞伙伴。突触结构的改变被认为是精神分裂症和自闭症等神经疾病的基础。我们试图确定突触伴侣选择背后的分子机制，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.