Functional complexomics associated with maturation and activity-dependent plasticity of excitatory synapse

大脑中信息的处理和存储从根本上依赖于兴奋性突触中适当的信号转导和活动依赖的动力学。这些突触的关键参与者是ampa型谷氨酸受体（AMPARs），这是一种大分子复合物，几乎驱动突触生理学的任何方面，从突触发生到电信号转导和突触可塑性，这些都是记忆形成和学习的基础。我们最近发现了AMPARs在ER中作为“多状态装配线”的生物发生，并发现其损伤/破坏会对人类和啮齿动物造成严重后果。在人类中，FRRS1l蛋白（AMPAR组装过程的关键决定因素）的功能丧失突变会导致严重的智力残疾，其中记忆形成、运动技能和认知功能受损最为严重。在小鼠中，敲除FRRS1l会破坏活动依赖的突触可塑性，减少突触形成和成熟，并严重损害学习能力。有趣的是，病毒驱动的FRRS1l的重新表达完全逆转了所有敲除诱导的表型，从而为随意“开启”突触和可塑性行为的形成提供了实验工具。在这个项目中，我们将利用这些最新的见解，首次对构建具有活动驱动可塑性的功能性突触所需的蛋白质进行公正和全面的研究。为此，我们将(i)在立体定向递送病毒开启AMPAR生物发生之前和之后，对FRRS1l敲除小鼠的特定大脑区域进行定量蛋白质组学分析，（ii）研究已确定的关键蛋白质和蛋白质复合物的亚细胞分布和动力学，（iii）研究它们在体外和体内的功能意义和特征。总之，这些分析将破译驱动兴奋性突触形成的分子过程及其活动依赖的可塑性。

Processing and storage of information in the brain fundamentally rely on proper signal transduction and activity-dependent dynamics in excitatory synapses. Key players in these synapses are AMPA-type glutamate receptors (AMPARs), macro-molecular complexes that drive almost any aspect of synapse physiology from synaptogenesis to electrical signal transduction and synaptic plasticity underlying memory formation and learning. We have recently uncovered biogenesis of AMPARs in the ER as a ‘multi-state assembly line’ and found that its impairment/disruption leads to severe consequences in both humans and rodents. In humans loss-of-function mutations in protein FRRS1l, a key determinant of the AMPAR assembly process, lead to severe forms of intellectual disability with strongest impairment in memory formation, motor skills and cognition. In mice, knock-out of FRRS1l abolished activity-dependent synaptic plasticity, reduced synapse formation and maturation and profoundly impaired learning. Interestingly, virally-driven re-expression of FRRS1l fully reversed all knock-out induced phenotypes and thus provided an experimental tool for ‘switching on’ formation of synapses and plastic behavior at will. In this project, we will use these latest insights for a first-time unbiased and comprehensive investigation of proteins that are required for building functional synapses with activity-driven plasticity. For this purpose we will (i) perform quantitative proteomic analyses on defined brain regions from FRRS1l knock-out mice before and after switching on AMPAR biogenesis by stereotactically delivered viruses, (ii) investigate identified key proteins and protein complexes for their subcellular distribution and dynamics and (iii) study their functional significance and characteristics in-vitro and in-vivo. Together, these analyses will decipher the molecular processes driving formation of excitatory synapses and their activity-dependent plasticity.