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

神经退行性疾病发生时，神经元出现故障，死亡速度和范围比健康老化期间更快、更广泛。这些疾病已经成为日益老龄化的人口的标志之一。一氧化氮(NO)是一种高度可扩散的信号分子，参与多种细胞通路，具有重要的生理功能，但在病理条件和脑老化过程中也可促进亚硝化应激。多种神经退行性疾病，包括阿尔茨海默病(AD)，由于NO介导的翻译后修饰增加，如3-硝基酪氨酸化(3-NT)，导致异常的NO信号。这些修饰与疾病发病机制之间的联系尚不清楚。为了解决这个问题，这个项目将研究3-NT信号如何影响突触内的蛋白质功能、整个动物的行为和生存。这项工作的目的是确定蛋白质靶点以减轻突触的病理变化，并将检验3-NT通过损害与AD相关的突触功能而导致神经退行性变的假说。该项目将进一步探索将3-NT水平降至最低的方法，并评估与神经退行性变和神经元功能障碍相关的影响。这个模型系统在应用电生理学、活体成像、生物化学和整体动物行为和存活研究等技术方面提供了独特的优势。我们建议研究NO改变突触功能的机制，例如通过调节囊泡招募和/或其他释放机制(例如，钙依赖/SNARE蛋白)。目的：1：确定NO信号在果蝇神经肌肉接头(NMJ)突触的功能后果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和释放的动员。一系列转基因果蝇将被用来表征蛋白介导的SNARE释放的影响。在这些不同的菌株中，我们将诱导3-NT识别NO的靶点，并揭示它们参与神经元功能障碍的情况。蛋白质的表达水平将通过免疫印迹得到确认。我们将在长寿和负地理趋向性研究中使用NO合成酶活性改变的苍蝇系来展示3-NT对神经元健康/功能的影响，并利用免疫细胞化学(ICC)和电子显微镜分析年龄诱导的成人大脑突触的超微结构、分子和功能组织的变化。研究NO对神经元功能的影响在AD模型中的病理作用具有重要意义。这就是为什么我们将使用表达神经毒性形式的淀粉样β蛋白(AB1-42)的苍蝇来研究NO在AD相关病理中的作用。我们将通过调节这些果蝇中神经元NO的生成或氧化应激水平来评估AD病理对NO的依赖性。3-NT和氧化应激的水平将通过ICC和免疫印迹在成人大脑中得到证实。我们将评估神经元形态、飞行运动活动和研究生存。除了在成年果蝇身上进行实验外，还将通过NMJ突触的电生理学来评估AB1-42过表达幼虫内源性NO和抗氧化剂水平的调节作用。修饰后的蛋白质将通过质谱分析进行鉴定。我们的初步研究使用了NOS‘Null’突变体和NO处理的幼虫，并鉴定了与钙信号相关的亚硝基酪化候选蛋白和参与囊泡运输的细胞骨架/马达蛋白。此外，该项目将产生功能数据来说明NO信号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，这可能使神经元易于发展进一步的病理学。

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.