Reactive Oxygen Species, metabolic by-products of mitochondrial respiration, as conserved regulators of synapse growth and neuronal homeostasis.

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Reactive oxygen species (ROS) accumulate as cells age, and in the brain oxidative stress is thought to be a major factor in cognitive dysfunction associated with ageing and neurodegeneration. The goal of this application is to study ROS signalling during normal nervous system development.
For the most part, we use the Drosophila larva as a model and use genetic expression systems that we and others have developed to genetically manipulate identified, connecting motoneurons their target muscles, but also their presynaptic partner interneurons in the CNS.
1. We will test: a) whether ROS, metabolic by-products of respiration, act to signal neuronal activity levels and are necessary for development and homeostasis of synapses; b) the nature and sites of action of ROS. We will manipulate ROS and/or activity levels in individual cells, image synaptic terminals (neuromuscular junctions - point scanning confocal; central dendrites - field scanning confocal), and use 3D reconstruction software (Amira) for morphometric analysis. We will make electrophysiological recordings from muscles (sharp electrode) and motoneurons (patch clamp) to determine the physiological effects of ROS signalling.
2. To study the underlying molecular mechanisms we will focus on DJ-1b, which we identified as critical for sensing ROS signalling. We will use genetic engineering to determine if oxidation of DJ-1b leads to changes in its subcellular localisation, and/or binding partners. We will apply tandem affinity purification coupled with quantitative mass spectrometry to identify additional DJ-1b interacting proteins regulated by the cellular redox status and characterise their roles in synapse development and function.
3. We will aim to translate observations made in the fruitfly to a vertebrate model system by working with rat hippocampal primary neurons in culture. We will test the requirement for ROS signalling and the rat homologue of DJ-1b, Park7, in regulating synaptic terminal growth.

Who will benefit from this research proposal?
Our research uncovers a role for reactive oxygen species (ROS) as physiological signals in the maintenance of normal synaptic growth and function. A failure to deal with increased ROS and the accumulation of ROS induced damage are hallmarks of ageing and neurodegeneration. Our research therefore is important to our understanding of the formation, functioning and homeostasis of a healthy brain, and through this, the processes that decline and fail as ROS accumulate due to age or disease.
Our work will benefit three main constituencies: 1) ageing individuals, their carers and dedicated health professionals. 2) Academics studying normal neuronal function, 3) Academics and health professionals involved in the study of neurodegenerative disease.

How will they benefit from this research?
We have put in place the following strategies to maximise impact, through efficient communication with potential beneficiaries, for the duration of the grant:
A) Academic Communication: We will work where possible through publication in Open Access journals, or pay premiums to grant open access. We will aim for journals with the highest impact factors to ensure maximum readership and seek broad subject journals to ensure the widest readership. We will attend and publicise our work at prominent conferences and meetings in the neuroscience, ageing, dementia and neurodegeneration fields, through posters and talks. The PDRAs will be encouraged to talk at meetings to generate profile for the study and their own career progression. Reagent generated, e.g. genetic strains and DNA constructs, will be disseminated freely, or lodged in not-for-profit stock centres that allow ready (and cheap) purchase, such as Addgene and the Bloomington stock centre. Our Drosophila data will contribute to Flybase and Flymine entries.
B) Public Communication: STS, SC and ML are well versed at communicating with schools and the general public. STS is well versed with speaking on local radio, having done so on more than one occasion. All three labs have active schools outreach events that run regularly, both to the genera public, e.g. during Science Week, and schools. . We will use our departmental and laboratory public websites to highlight our findings and University press officers to communicate with the press to disseminate news rapidly.
C) Communication with health professionals and relevant societies and organisations: This proposal has longer-term benefits for ageing individuals, carers, the social and healthcare systems and thus our society and its economy in general. Potential benefits are discoveries that will contribute to therapeutic strategies for improving cognitive function in an ageing population, lowered incidence or slowed onset rates for dementia. This proposal will increase our understanding of important cellular and molecular events that are triggered by Alzheimer's, Parkinson's, Motorneuron Disease and related conditions. As we identify gene products and signalling pathways as candidates for therapeutic intervention, economic benefits arise: the market for an anti-neuronal-ageing therapeutic or dietary supplements would be considerable. We have put in place regular quarterly meetings with relevant clinicians to review our data and assess how it may be implemented clinically. STS communicates regularly with the Alzheimer's Society through their York office.

Technological Training and impact: Both PDRAs will be trained in the skills and cutting-edge techniques required for the study. Such skills are highly desirable in the scientific work force and transferable to the pharmaceutical sector.
Creating Industrial Impact: We are aware that at this point we have a fundamental study that would be difficult to transfer to translational impact. However, throughout the course of the grant we will continually review our data and its implications for potential translational impact and industrial interaction.