Synapse Formation in the C. elegans Nervous System

描述(申请人提供)：适当的突触沟通对于正常的大脑功能和信息处理是必要的，突触沟通中断与许多神经疾病有关。AMPA型谷氨酸受体(AMPAR)是突触交流的重要介体，介导脑内许多兴奋性突触后反应。AMPAR以均相或多相亚基组成的四聚体形式存在。虽然AMPAR的特定亚基组成可以决定通道的生物物理性质，但这些不同的亚基在体内的作用尚不清楚。据推测，学习和记忆是由完整神经回路中突触上AMPAR的经验依赖的运输来编码的，然而先前的感觉经验如何调节AMPAR的运输，以及AMPAR运输的变化如何反过来调节突触的效能和支持完整动物的行为可塑性仍然是一个悬而未决的问题。利用线虫的正向遗传学，我的实验室一直在根据它们在体内的功能需求来研究AMPAR运输的调节因素。最近我们发现，AMPAR是通过受RAB-6.2(一种Rab6型GTP酶)和逆聚体复合体调控的内体到高尔基体的逆行循环途径来循环的。虽然对Rab6/Retmer通路在神经系统中的功能知之甚少，但我们发现该通路维持特定AMPAR亚单位组合的突触水平。我们发现另一个Rab6家族成员RAB-6.1调节着一组不同的AMPAR亚单位组合的循环。利用酵母双杂交，我们还确定了候选效应子和这些RAB的缺口。在正向遗传筛查中，我们在一个新基因中发现了突变，该基因负向调节AMPAR逆行转运，很可能是通过负调节一个或两个Rab6蛋白。我们假设这两个Rab6蛋白促进不同的AMPAR亚单位组合的循环。我们认为，先前的感觉经验调节了这两条循环途径的平衡，这反过来又决定了AMPAR亚单位的组成定位于突触，从而决定了突触的习服动力学。我们将通过四个具体目标来检验这一假设。首先，我们将表征RAB-6.1在调节AMPAR循环中的作用。其次，我们将研究TBC-1作为两种Rab6蛋白潜在的GAP负调节因子的作用。第三，我们将表征一种新的磷脂酰肌醇磷酸酶作为潜在的Rab6效应因子的作用，该效应调节脂膜组成。第四，我们将克隆和鉴定新发现的似乎负调控Rab6活性和AMPAR逆行循环的基因。这些目标将包括分子、细胞、行为、生化和电生理分析，并将为谷氨酸受体的调节机制提供线索。

DESCRIPTION (provided by applicant): Proper synaptic communication is necessary for normal brain function and information processing, and disrupted synaptic communication is associated with many neurological disorders. AMPA-type glutamate receptors (AMPARs) are critical mediators of synaptic communication, mediating much of the excitatory postsynaptic responses in the brain. AMPARs exist as tetramers of either homogeneous or heterogeneous subunit composition. While the specific subunit composition of an AMPAR can dictate the biophysical properties of the channel, the role of these different subunits in vivo is unclear. It as been hypothesized that learning and memory is encoded by experience-dependent trafficking of AMPARs at synapses in intact neural circuits, yet how prior sensory experience modulates AMPAR trafficking and how changes in AMPAR trafficking in turn modulate synaptic efficacy and underlie behavioral plasticity in the intact animal remains an open question. Using C. elegans forward genetics, my lab has been studying regulators of AMPAR trafficking based on their functional requirements in vivo. Recently we found that AMPARs are recycled through an endosome-to-Golgi retrograde recycling pathway regulated by RAB-6.2 (a Rab6-type GTPase) and the retromer complex. Whereas little is known about Rab6/retromer pathway function in the nervous system, we found that this pathway maintains the synaptic level of specific AMPAR subunit combinations. We found that another Rab6 family member, RAB-6.1, regulates the recycling of a different set of AMPAR subunit combinations. Using yeast two-hybrid, we also identified candidate effectors and a GAP for theses Rabs. In a forward genetic screen, we identified mutants in a new gene that negatively regulates AMPAR retrograde transport, mostly likely by negatively regulating one or both Rab6 protein. We hypothesize that these two Rab6 proteins promote the recycling of distinct AMPAR subunit combinations. We propose that prior sensory experience regulates the balance of these two recycling pathways, which in turn determines which AMPAR subunit composition is localized at the synapse and thus the habituation kinetics at that synapse. We will test this hypothesis through four specific aims. Firs, we will characterize the role of RAB-6.1 in regulating AMPAR recycling. Second, we will characterize the role of TBC-1 as a potential GAP negative regulator of the two Rab6 proteins. Third, we will characterize the role of a new phosphoinositide phosphatase enzyme as a potential Rab6 effector that modulates lipid membrane composition. Fourth, we will clone and characterize the newly identified gene that appears to negatively regulate Rab6 activity and AMPAR retrograde recycling. These aims will include molecular, cellular, behavioral, biochemical, and electrophysiological analyses, and will provide clues to the mechanisms by which glutamate receptors are regulated.