Cysteine oxidation in the remodelling of dendritic spines

树突棘重塑中的半胱氨酸氧化

• 批准号: RGPIN-2021-02418
• 负责人: Ryan, Scott
• 金额: $ 2.33万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741966
• 关键词: Cysteine; oxidation; remodelling; dendritic; spines

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.

概述：响应神经传递的突触可塑性是学习和记忆的关键组成部分，需要细胞外环境和决定神经元结构的细胞骨架蛋白之间的通信。大多数兴奋性突触后末梢位于树突棘中，树突棘被认为是记忆存储的基本单位。通过离子型谷氨酸受体、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 NMDAR 的激活可以加强突触连接，但 NMDAR 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富含多刺神经元的大脑区域的突触可塑性，例如海马体，海马体是学习和记忆的中心。 NMDAR 的激活导致 Ca+ 快速流入和一氧化氮 (NO) 合成。总的来说，现有证据支持这样一种模型：神经传递后 Ca+ 和 NO 发出的第二信使信号赋予神经元神经元通讯所需的可塑性。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变化的机制仍然知之甚少。与 Ca+ 相比，据估计，NO 可以通过称为 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：本提案旨在通过确定 NO 在直接调节树突棘蛋白质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 NR3A 亚基的物理特性来调节 NO 和 Ca+ 信号传导。虽然 NMDAR 信号传导的消除会导致胚胎死亡，但 NR3A 的过度表达会通过降低通道的 Ca+ 通透性来抑制 NMDAR 信号传导，从而减少谷氨酸介导的 NO 合成。使用以 tet 依赖性方式过度表达 NR3A 的转基因动物或 NR3A KO 动物，我们有一种方法可以促进 NMDAR 介导的 NO 合成或通过施用强力霉素打开和关闭它。通过采用质谱法鉴定蛋白质 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。这将建立突触可塑性所必需的一组关键的 NO 信号转导效应器。随后，我们将使用高分辨率活细胞成像确定调节 NMDAR 活性如何影响 Ca+ 流入和随后的 NO 合成。我们使用 Crispr 生成的不可亚硝基化的突触蛋白变体来评估 SNO 蛋白如何控制突触可塑性的机制。