Exploring the role and therapeutic potential of 3-nitrosynation in Alzheimer's disease

探索 3-亚硝基化在阿尔茨海默病中的作用和治疗潜力

• 批准号: 2432878
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2432878
• 关键词: Exploring; role; therapeutic; potential; nitrosynation

Neurodegenerative disorders occur when neurons malfunction and die much more quickly and extensively than during healthy aging. These disorders have become one of the hallmarks of an increasingly aging population. Nitric oxide (NO) a highly diffusible signaling molecule, is involved in a multitude of cellular pathways with crucial functions in physiology but can also promote nitrosative stress during pathological conditions and brain aging. Multiple neurodegenerative diseases, including Alzheimer's disease (AD), are exacerbated by abnormal NO signalling associated with increased NO-mediated post-translational modifications, such as 3-nitrotyrosination (3-NT). The mechanistic link between these modifications and disease pathogenesis is not clear. To address this question, this project will investigate how 3-NT signalling affects protein function within a synapse, whole animal behaviour and survival. The aim of this work is to identify protein targets to mitigate pathology at the synapse and will test the hypothesis that 3-NT contributes to neurodegeneration by compromising synaptic functions associated with AD. The project will further explore approaches to minimise 3-NT levels and assess effects associated with neurodegeneration and neuronal dysfunction. This model system provides unique advantages to apply techniques such as electrophysiology, live-imaging, biochemistry and whole animal behaviour and survival studies.We propose to investigate the mechanisms by which NO changes synaptic function such as by modulation of vesicular recruitment and/or other release mechanisms (e.g. Ca2+ dependency/SNARE proteins).Aims:1: Identify functional consequences of NO signalling at the Drosophila Neuromuscular Junction (NMJ) synapse2: Identify the contributions of NO to AD-relevant phenotypesWe will initially use electrophysiological and fluorescence approaches to assess synapse function and mobilisation of vesicle pools and release.A range of genetically modified Drosophila lines will be used to characterise effects on SNARE protein-mediated release. In these different strains we will induce 3-NT to identify targets of NO and reveal their involvement I neuronal dysfunction. Expression levels of proteins will be confirmed by immunoblotting.We will employ fly lines with altered NO synthase activity (to manipulate neuronal NO levels) in longevity and negative geotaxis studies to show the impact of 3-NT upon neuronal health/function and analyse age-induced changes in the ultrastructural, molecular and functional organization of synapses using immunocytochemistry (ICC) and Electron Microscopy in adult brains.It is important to investigate how the effects of NO on neuronal function contribute to any pathology induced in an AD model. That's why we will employ flies pan-neuronally expressing a neurotoxic form of amyloid beta (Ab1-42) to investigate the contributions of NO in AD-relevant pathology. We will assess the dependency of AD pathology on NO by modulating either neuronal NO generation or oxidative stress levels in these flies. Levels of 3-NT and oxidative stress will be confirmed by ICC and immunoblotting in adult brains. We will assess neuronal morphology, fly locomotor activity and study survival. In addition to experiments in adult flies, the effects of modulation of endogenous NO and antioxidant levels in Ab1-42 overexpressing larvae will be assessed by electrophysiology at the NMJ synapse.Modified proteins will be identified using Mass Spectrometry analysis. Our preliminary studies have employed NOS 'null' mutants and NO-treated larvae and identified nitrotyrosinated candidate proteins associated with Ca2+ signalling and cytoskeletal/motor proteins involved in vesicular transport.Together, this project will generate functional data to illustrate the interactions between NO signalling and oxidative stress levels and identify connections between Abeta and NO which can predispose neurons to develop further pathology.

神经退行性疾病发生时，神经元功能障碍和死亡比健康衰老更快和更广泛。这些疾病已经成为人口日益老龄化的标志之一。一氧化氮（NO）是一种高度扩散的信号分子，参与多种细胞通路，在生理上具有重要功能，但也可以在病理状态和脑衰老过程中促进亚硝化应激。多种神经退行性疾病，包括阿尔茨海默病（AD），与NO介导的翻译后修饰（如3-硝基酪氨酸化（3-NT））增加相关的异常NO信号传导加剧。这些修饰与疾病发病机制之间的机制联系尚不清楚。为了解决这个问题，该项目将研究3-NT信号如何影响突触内的蛋白质功能，整个动物行为和生存。这项工作的目的是确定减轻突触病理的蛋白靶点，并将验证3-NT通过损害与AD相关的突触功能来促进神经退行性变的假设。该项目将进一步探索最小化3-NT水平的方法，并评估与神经变性和神经元功能障碍相关的影响。该模型系统为应用电生理学、活体成像、生物化学、全动物行为和生存研究等技术提供了独特的优势。我们建议研究NO改变突触功能的机制，如通过调节囊泡募集和/或其他释放机制（例如Ca2+依赖性/SNARE蛋白）。目的：1：确定NO信号在果蝇神经肌肉连接（NMJ）突触中的功能后果2：确定NO对ad相关表型的贡献我们将首先使用电生理和荧光方法来评估突触功能和囊泡池的动员和释放。一系列转基因果蝇将被用于表征对SNARE蛋白介导释放的影响。在这些不同的菌株中，我们将诱导3-NT来识别NO的靶点并揭示它们在神经元功能障碍中的作用。蛋白表达水平将通过免疫印迹法确认。我们将在长寿和负地向性研究中使用NO合成酶活性改变的果蝇系（以操纵神经元NO水平），以显示3-NT对神经元健康/功能的影响，并使用免疫细胞化学（ICC）和电子显微镜分析成人大脑中突触超微结构、分子和功能组织的年龄诱导变化。在AD模型中，研究NO对神经元功能的影响如何导致任何病理是很重要的。这就是为什么我们将使用泛神经表达β淀粉样蛋白（a1 -42）的果蝇来研究NO在ad相关病理中的作用。我们将通过调节这些果蝇的神经元NO生成或氧化应激水平来评估AD病理对NO的依赖性。3-NT和氧化应激水平将通过ICC和免疫印迹法证实。我们将评估神经元形态，苍蝇运动活动和研究存活。除了在成虫身上的实验外，我们还将通过NMJ突触的电生理来评估Ab1-42过表达幼虫对内源性NO和抗氧化水平的调节作用。修改后的蛋白质将使用质谱分析进行鉴定。我们的初步研究采用NOS ‘null’突变体和no处理的幼虫，并鉴定了与Ca2+信号和参与囊泡运输的细胞骨架/运动蛋白相关的硝基酪氨酸化候选蛋白。总之，该项目将生成功能数据，以说明NO信号传导和氧化应激水平之间的相互作用，并确定Abeta和NO之间的联系，这些联系可能使神经元更易发生进一步的病理变化。