Uncovering the role of RIN1 in coordinating AMPA-receptor membrane trafficking and structural spine changes in bidirectional synaptic plasticity

大脑中的突触连接随着神经元活动的特定变化而不断减弱或加强。这个过程被称为突触可塑性，是学习和记忆的细胞基础，在一些脑部疾病中受到损害。长期增强（LTP）和长期抑制（LTD）是研究最多的活动依赖性突触可塑性形式。LTD和LTP一方面是由ampa型谷氨酸受体（AMPA-Rs）的高动态和严格控制的运输和突触靶向支持的，AMPA-Rs是大脑中快速兴奋传递的主要介质。另一方面，肌动蛋白细胞骨架的动态重塑支持伴随的树突棘形态和稳定性的变化。尽管AMPA-R在突触可塑性中起着重要的作用，但在突触可塑性过程中协调AMPA-R转运和细胞骨架重塑的分子机制尚不完全清楚。Ras效应蛋白RIN1在前脑的兴奋性神经元中高度特异性表达，特别是在大脑皮层、海马和杏仁核中。重要的是，RIN1具有双重作用，它增强酪氨酸激酶Abl和Arg的信号传导，这些酪氨酸激酶调节肌动蛋白细胞骨架重塑，并激活小GTPase Rab5促进受体内吞。最近，在海马神经元中，我们发现RIN1破坏突触连接的稳定性，并且是突触后AMPA受体内吞作用的关键参与者。我们进一步发现，在LTD期间，活动依赖性脊柱可塑性需要RIN1。基于我们之前的工作，本项目的总体目标是全面了解在兴奋性突触的双向突触可塑性中，RIN1的双重功能如何协调AMPA受体运输和活动依赖的脊柱结构变化。为此，我们将结合一些最先进的技术，包括RIN1的分子操作，生化分析，亚细胞定位研究，活细胞成像实验和电生理记录。生化研究将解决RIN1及其蛋白-蛋白相互作用的调控。成像方法将揭示可塑性驱动的亚细胞调节和下游信号通路激活的变化，包括它们对结构可塑性和AMPA-R运输的贡献。最后，电生理学方法将探究不同的RIN1功能如何在确定的海马回路中促进双向突触强度的调节。

Synaptic connections in the brain are continuously weakened or strengthened in response to specific changes in neuronal activity. This process, known as synaptic plasticity, is the cellular basis for learning and memory, and is impaired in several brain disorders. Long-term potentiation (LTP) and long-term depression (LTD) are the most studied forms of activity-dependent synaptic plasticity. LTD and LTP are on one hand supported by the highly dynamic and tightly controlled trafficking and synaptic targeting of AMPA-type glutamate receptors (AMPA-Rs), which are the major mediators of fast excitatory transmission in the brain. On the other hand, dynamic remodeling of the actin cytoskeleton supports the accompanying changes in dendritic spine morphology and stability. Despite their important function in synaptic plasticity, the molecular mechanisms coordinating AMPA-R trafficking and cytoskeletal remodeling during synaptic plasticity are still not fully understood. The Ras effector protein RIN1 is highly and specifically expressed in excitatory neurons of the forebrain, particularly in the cerebral cortex, hippocampus, and amygdala. Importantly, RIN1 has a dual role as it enhances signaling of the tyrosine kinases Abl and Arg, which regulate actin cytoskeletal remodeling, and it activates the small GTPase Rab5 to facilitate receptor endocytosis. Recently, in hippocampal neurons we have found that RIN1 destabilizes synaptic connections and is a key player in postsynaptic AMPA receptor endocytosis. We further identified RIN1 to be required for activity-dependent spine plasticity during LTD. Building on our previous body of work, the overarching aim of this project is to gain comprehensive insight in how the dual functions of RIN1 orchestrate AMPA receptor trafficking and activity-dependent structural spine changes during bidirectional synaptic plasticity at excitatory synapses. Towards this end, we will combine a number of state of the art techniques including molecular manipulations of RIN1, biochemical assays, subcellular localization studies, live cell imaging experiments, and electrophysiological recordings. Biochemical studies will address the regulation of RIN1 and its protein-protein interactions. Imaging approaches will unravel plasticity-driven changes in subcellular regulation and activation of downstream signaling pathways, including their contributions to structural plasticity and AMPA-R trafficking. Finally, electrophysiological approaches will interrogate how different RIN1 functions contribute to the regulation of bidirectional synaptic strength in defined hippocampal circuits.