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

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) synapse
2: Identify the contributions of NO to AD-relevant phenotypes
We 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.