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

Nerve cells communicate with each other using electrical signals, which require large amounts of energy, making the brain the most fuel-demanding structure in the body. Brains therefore use a great deal of oxygen to generate the energy required for processing information, controlling behavior and for cognition. Brains are also very sensitive to ageing and most of us have experience of ageing relatives with faulty memories. By using a lot of oxygen to generate energy, the brain also produces a by-product, which becomes toxic when allowed to accumulate. This by-product is toxic forms of oxygen and is termed 'Reactive Oxygen Species', or ROS for short. Normally, the brain can cope with low levels of ROS that are a by-product of normal metabolism, because it has a battery of protective mechanisms to neutralise ROS. But as the brain ages, these self-repair mechanisms become less efficient, and as a consequence ROS levels become excessive and signs of ROS-caused damage more evident. ROS chemically react with and damage the building blocks of cells. As waste material accumulates, it can also generate a second source of ROS, generated by metals within the accumulated waste material reacting with oxygen to produce more ROS. Thus, a self-perpetuating cycle of damage ensues. Because of this cyclical nature, it has remained unclear precisely how ROS affect the nervous system in the first instance, as opposed to secondary or tertiary knock-on effects. We previously found that the connections between nerve cells, called synapses, grow excessively when ROS are excessive. Synapses are known to adjust their size in response to neuronal activity, though how nerve cells sense activity levels is incompletely understood.

With this proposal we are tackling these fundamental questions. First, we are investigating the hypothesis that during normal nervous system development and function, cells use ROS as signals that inform them about their activity levels. Next, we will study the molecular mechanisms by which ROS regulate the size and function of synapses. Last, we will test the hypothesis that ROS signaling is evolutionary conserved and we will examine how discoveries made in the genetic model system of the fruitfly also apply to vertebrate nerve cells.

We use the fruitfly, Drosophila, as an experimental system, because the high evolutionary conservation of basic cellular processes has meant that most discoveries made in the fly, including mechanisms that underlie learning and memory, are directly pertinent to our understanding of the human brain. Importantly, the fruitfly is one of the most powerful genetic experimental organisms and allows unprecedented precision for genetically manipulating identified neurons and their connecting partners.
In our experimental system, when we induce activity in neurons, but reduce ROS at the same time, we prevent the synapse overgrowth that we see when we induce activity or ROS alone. This suggests that neurons use ROS generated during energy manufacturing as a readout for their activity and that ROS signal synapse growth. We have discovered the function of one protein, DJ1 (aka Parkinson Disease Protein 7) as important for sensing ROS levels and then activating a known cell growth promoting pathway to enlarge synapses. These DJ1 mediated processes that we have observed are very likely of critical importance to our understanding of the decline in brain function as we age, and this proposal aims to investigate DJ1 function further.

In summary, this work will help us to understand the mechanisms, events and molecules that cause change and, ultimately, failure in nerve cell function in the ageing brain. The results of this work have every potential to aid the discovery of drugs and treatments to alleviate adverse effects of ageing and will thus, in time, benefit society as a whole.

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.