Identification of novel functional and post-transcriptional regulatory mechanisms during homeostatic synaptic downscaling

面对网络活动的变化，神经元采用一套嵌套的稳态机制来稳定放电率。突触降尺度是一种形式的稳态可塑性，神经元减弱单一突触强度以响应网络活动的慢性增加，特别是通过减少ampa型谷氨酸受体（AMPA-R）的突触表达。稳态突触降尺度在活动依赖性神经元发育和癫痫等神经系统疾病的病因学中都很重要。然而，稳态突触缩尺的分子基础在很大程度上是未知的。我们最近发现，在大鼠海马培养模型中，神经元microRNA miR-134是通过转录后控制rna结合蛋白（RBP） Pumilio-2实现稳态突触缩减所必需的（Fiore et al., 2014）。我们定量蛋白质组学的初步结果表明，大量突触基因的转录后下调是神经元对网络活性增加的反应的标志，其中许多与突触后钙信号传导和AMPA-R磷酸化有关。此外，我们获得了特异性mirna和rbp在突触基因转录后抑制协调中的作用的初步证据。在本文中，我们计划进一步阐明调节和执行稳态突触降尺度的分子机制：1)表征新发现的在稳态降尺度过程中调节的基因的功能，重点研究候选基因Atp2b4和Dcx及其在钙信号和AMPA-R运输和功能水平上的相互作用。2)确定突触基因转录后调控的协调机制，重点关注活性调控rna结合蛋白Rbfox1与microRNA miR-129-5p之间的串音。该项目的结果有望为神经网络慢性激活时执行和调节稳态可塑性的分子机制提供重要的新见解，对活动依赖性神经元发育和以稳态可塑性受损为特征的神经系统疾病（包括癫痫）具有重要意义。

Neurons employ a nested set of homeostatic mechanisms to stabilize firing rates in the face of changes in network activity. Synaptic downscaling is one form of homeostatic plasticity where neurons weaken unitary synaptic strength in response to a chronic increase in network activity, in particular by decreasing synaptic expression of AMPA-type glutamate receptors (AMPA-R). Homeostatic synaptic downscaling is important both in activity-dependent neuronal development and in the etiology of neurological disorders, such as epilepsy. However, the molecular underpinnings of homeostatic synaptic downscaling are largely unknown. We have recently shown that the neuronal microRNA miR-134 is required for homeostatic synaptic downscaling in a rat hippocampal culture model by post-transcriptional control of the RNA-binding protein (RBP) Pumilio-2 (Fiore et al., 2014). Our preliminary results from quantitative proteomics suggest that the post-transcriptional downregulation of a plethora of synaptic genes, many of which are linked to postsynaptic calcium signaling and AMPA-R phosphorylation, is a hallmark of the neuronal response to increased network activity. Furthermore, we obtained preliminary evidence for a role of specific miRNAs and RBPs in the coordination of post-transcriptional inhibition of synaptic genes.In this proposal we plan to further elucidate the molecular mechanisms that regulate and execute homeostatic synaptic downscaling by1) Characterizing the function of newly identified genes regulated during homeostatic downscaling, focusing on the candidates Atp2b4 and Dcx and their interaction at the level of calcium signaling and AMPA-R trafficking and function. 2) Identifying the coordinated mechanisms underlying the post-transcriptional control of synaptic genes during downscaling, focusing on a crosstalk between the activity-regulated RNA-binding protein Rbfox1 and the microRNA miR-129-5p. The results from this project promise to provide significant new insight into the molecular mechanisms that execute and regulate homeostatic plasticity in response to chronic activation of neural networks, with important implications for activity-dependent neuronal development and neurological disorders characterized by impaired homeostatic plasticity, including epilepsy.