Mechanisms of Synaptic Specificity in C. elegans

描述（申请者提供）：化学突触是一种特殊的细胞连接结构，对神经元之间的交流至关重要。在发育过程中，突触在特定的亚细胞区室的特定神经元之间形成。突触目标选择、轴突运输和突触前组装是突触形成的重要步骤，但人们对这些步骤知之甚少。在这里，我建议扩大我们的研究，以了解突触形成的两个基本方面：极化轴突运输和如何调节活性区蛋白的聚集。突触通常在远端轴突和树突上形成，这给细胞体和突触之间有效的胞内物质交换带来了挑战。微管和MT相关马达介导细胞内运输。一般认为，传输的方向取决于两个因素：mt的极性和所涉及的电机类型。基于我们已发表和未发表的数据，我们已经确定了两种周期蛋白依赖性激酶途径，它们对突触前成分的运输至关重要。在缺乏这两种通路的情况下，绝大多数突触囊泡蛋白和活性区标记物不能定位到轴突，而是由于运动蛋白马达的错误调节而在树突中发现。突触形成的另一个鲜为人知的问题是突触囊泡池的大小是如何确定的。许多突触显示出固定大小的突触囊泡簇，这表明分子机制在突触前末端局部调节突触囊泡前体的组装。当适当数量的囊泡被招募时，可能存在一个负反馈系统来关闭组装途径。我们推断，如果这种反馈机制是有缺陷的，我们应该期待看到突变突触与异常的囊泡池。事实上，在正向遗传筛选中，我们分离出一个突变体，其中近端突触异常大，而远端突触含有很少的物质。我们还发现了一种类似于Arf的GTPase，它负责调节突触前特化的位置和大小。为了深入了解这些基因的功能机制，我特别计划了解：1)周期蛋白依赖性激酶途径如何通过调节分子马达来控制轴突运输和突触定位，以及2)像GTPase， ARL8这样的ARF如何调节突触囊泡池大小和突触前组装。鉴于许多神经疾病与突触连通性的改变有关，并且周期蛋白依赖激酶与神经退行性疾病有关，该项目将有助于了解生理和病理条件下的大脑发育。

DESCRIPTION (provided by applicant): Chemical synapses are specialized cellular junction structures that are essential for communication between neurons. During development, synapses form between specific neurons at defined subcellular compartments. Synaptic target selection, axonal transport and presynaptic assembly are integral steps of synapse formation that are poorly understood. Here, I propose to expand our research to understand two essential aspects of synapse formation: polarized axonal trafficking and how aggregation of active zone proteins is regulated. Synapses are usually formed on distal axon and dendrites, creating a challenging problem for effective exchange of intracellular material between cell bodies and synapses. Microtubules and MT associated motors mediate intracellular trafficking. It is generally believed that the direction of transport depends on two factors: the polarity of MTs and the type of motor involved. Based on our published and unpublished data, we have identified two cyclin-dependent kinase pathways that are essential for the trafficking of presynaptic components. In the absence of both pathways, the vast majority of synaptic vesicle proteins and active zone markers fail to localize to axon and instead are found in dendrites due to misregulation of kinesin motors. Another poorly understood question in synapse formation is how the pool size of synaptic vesicles is determined. Many synapses display stereotyped size of synaptic vesicle clusters, suggesting that molecular mechanisms regulate the assembly of synaptic vesicle precursors locally at the presynaptic terminals. When the appropriate number of vesicles is recruited, there might be a negative feedback system to shut down the assembly pathway. We reasoned that if this feedback mechanism is defective, one should expect to see mutant synapses with abnormal vesicle pool. Indeed in a forward genetic screen, we isolated a mutant in which the proximal synapses are abnormally large while the distal synapses contain little material. We have also identified an Arf like GTPase to be responsible for the regulation the location and size of presynaptic specializations. To gain mechanistic insights on the functions of these genes, I specifically plan to understand: 1) how the cyclin-dependent kinases pathways control axonal transport and synapse localization through the regulation of molecular motors, and 2) how an ARF like GTPase, ARL8 regulate the synaptic vesicle pool size and presynaptic assembly. Given that many neural disorders are associated with alterations in synaptic connectivity and that cyclin dependent kinases have been implicated in neurodegenerative diseases, it is hopeful that this project will help to understand brain development under both physiological and pathological conditions. PUBLIC HEALTH RELEVANCE: Synapses are critical for the communication between neurons. During synapse formation, proteins and vesicle are transported to presynaptic terminals in distal axons. The proposed study characterizes critical regulatory mechanisms for trafficking synaptic components to axons as well as locally synaptic assembly. This project will potentially shed light on the pathogenesis mechanisms of neurodegenerative diseases such as the Alzheimer's disease.