Cysteine oxidation in the remodelling of dendritic spines

概述：神经传递反应中的突触可塑性是学习和记忆的关键组成部分，需要细胞外环境和细胞骨架蛋白之间的交流，而细胞骨架蛋白决定了神经元的结构。大多数兴奋性突触后终端位于树突棘中，树突棘被认为是记忆储存的基本单位。通过嗜离子性谷氨酸受体n -甲基- d -天冬氨酸受体（NMDAR）的突触传递是脊柱形态的强调节剂。虽然突触后NMDARs的激活可以加强突触连接，但NMDARs的过度激活会导致细胞骨架修饰，从而促进突触修剪。这些事件之间的平衡控制着大脑中富含棘神经元的区域的突触可塑性，比如学习和记忆的中心海马体。NMDARs的激活导致Ca+快速内流和一氧化氮（NO）合成。总的来说，现有证据支持一种模型，即神经传递后Ca+和NO的第二信使信号赋予神经元通信所需的可塑性。虽然这些第二信使已被广泛研究，但伴随突触传递的脊柱结构形态学变化的机制仍然知之甚少。与Ca+相比，据估计，NO可以通过一种称为s-亚硝基化（SNO）的过程直接调节大脑中高达50%的蛋白质。虽然许多证据支持SNO在突触可塑性中的作用，但尚未对支持和调节神经传递的SNO蛋白质组进行全面分析。方法：本研究旨在通过确定NO在树突棘蛋白直接调节中的作用来探索突触可塑性的基本细胞生物学。为此，我们将利用NMDAR的NR3A亚基的物理特性来调节NO和Ca+信号。虽然NMDAR信号的消融对胚胎是致命的，但NR3A的过表达通过降低通道的Ca+通透性来抑制NMDAR信号，从而减少谷氨酸介导的NO合成。使用以tet依赖方式过表达NR3A的转基因动物或NR3A KO动物，我们有一种促进nmdar介导的NO合成或通过给药多西环素打开或关闭它的方法。通过质谱法鉴定蛋白质s -硝基硫醇，我们将建立海马神经元响应突触激活的sno蛋白质组。这将建立一组关键的NO信号转导效应器，这是突触可塑性所必需的。随后，我们将利用高分辨率活细胞成像技术确定调节NMDAR活性如何影响Ca+内流和随后的NO合成。我们使用crispr生成的突触蛋白的非亚硝基化变体来评估sno蛋白如何控制突触可塑性的机制。

Overview: Synaptic plasticity in response to neurotransmission is a key component of learning and memory, requiring communication between the extracellular environment and the cytoskeletal proteins that dictate the structure of a neuron. Most excitatory postsynaptic terminals reside in dendritic spines, which are thought to serve as basic units of memory storage. Synaptic transmission through the ionotropic glutamate receptor, N-methyl-D-aspartate receptor (NMDAR), is a strong modulator of spine morphology. While activation of post-synaptic NMDARs can strengthen synaptic connections, over-activation of NMDARs results in cytoskeletal modifications that promote synaptic pruning. The equilibrium between these events governs synaptic plasticity in brain regions rich in spiny neurons such as the hippocampus, the center for learning and memory. Activation of NMDARs leads to rapid Ca+ influx and nitric oxide (NO) synthesis. Collectively, the existing evidence supports a model in which second messenger signaling by Ca+ and NO following neurotransmission confers on neurons the plasticity required for neuronal communication. While these second messengers have been extensively studied, mechanisms underlying the morphological changes to spine structure that accompany synaptic transmission are still poorly understood. By contrast to Ca+, it is estimated that NO can directly modulate up to 50% of proteins in the brain, through a process called s-nitrosylation (SNO). While much evidence supports a role for SNO in synaptic plasticity, a comprehensive analysis of the SNO-proteome that supports and regulates neurotransmission has not been performed. Approach: This proposal aims to explore the fundamental cell biology of synaptic plasticity by identifying a the role of NO in direct modulation of proteins in dendritic spines. To this end, we will capitalize on the physical properties of the NR3A subunit of NMDAR to modulate NO and Ca+ signaling. While ablation of NMDAR signaling is embryonic lethal, overexpression of NR3A dampens NMDAR signaling by decreasing the Ca+ permeability of the channel, thus reducing glutamate mediated NO synthesis. Using transgenic animals over-expressing NR3A in a tet dependant manner or NR3A KO animals, we have a means of either promoting NMDAR-mediated NO synthesis or of turning it on and off through doxycycline administration. By employing a mass spectrometry method for identification of protein S-nitrothiols, we will establish the SNO-proteome of hippocampal neurons in response to synaptic activation. This will establish a critical set of effectors of NO signal transduction, necessary for synaptic plasticity. We will subsequently determine how modulating NMDAR activity influences Ca+ influx and subsequent NO synthesis using high resolution live cell imaging. We use Crispr-generate, non-nitrosylatable variants of synaptic proteins to assess mechanism of how SNO-proteins govern synaptic plasticity.