Oxidative stress induced regulation of synaptic growth in the nervous system - dissection of genetic and cellular mechanisms.

Brains are very sensitive to ageing and most of us have experience of ageing relatives with faulty memories. The brain requires high levels of food and oxygen to function effectively. By using a lot of oxygen to generate energy, the brain produces a by-product. This by-product is toxic forms of oxygen and is termed Reactive Oxygen Species or ROS. Normally the brain can cope with low levels of ROS that are generated as a by-product of normal metabolism, but as the brain ages, the self-repair mechanisms become less effective and ROS become excessive. ROS are destructive to cells by a self-perpetuating cycle of damage. Primarily, ROS generation occurs in the structure within the cell responsible for producing energy from food and oxygen called the mitochondria. We term ROS generated by the mitochondria, mitochondrial ROS (m-ROS). An ageing brain struggles to clear itself of cellular material damaged by ROS. 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. We term these cytoplasmic ROS (c-ROS). Both sources of ROS now contribute to the increasing cycle of damage as neurons age. We found that the connections between nerve cells, called synapses, grow excessively when ROS are excessive. Synapses are normally known to grow while the brain carries out learning and memory functions and the connections between nerve cells improve their communication efficiency. We therefore find it surprising to see synapses growing during a period when we would expect a decline in the efficiency of neuronal communication. In this proposal we will examine and uncover the processes in nerve cells that react to ROS to cause synapse growth. We have already found that nerve cells activate a process of self-renewal when ROS are present in the brain and suspect that this may be inducing synapse growth. Exactly how this happens we aim to determine. The changes that we have observed are very likely of critical importance to our understanding of the decline in brain function as we age. This work will help us to understand the mechanisms, events and molecules that cause 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.

Oxidative stress is a hallmark of ageing and neurodegenerative diseases. This proposal seeks to investigate how reactive oxygen species (ROS) impinge on the nervous system, specifically the growth of synaptic terminals, where increases in ROS unexpectedly cause overgrowth. Using the genetically tractable fruitfly, Drosophila, as a model we will address key issues. 1. We will determine the effects of different sources of oxidative stress on presynaptic terminals, at late larval neuromuscular junction (NMJ) and in the central nervous system (CNS) as well as postsynaptic dendritic arbors of central neurons. 2. We found that different sources of ROS, cytoplasmic versus mitochondrial, induce synaptic terminal growth via different Jun-N-terminal Kinase (JNK) pathway components and will now identify the relevant upstream components and downstream transcriptional codes. 3. We will establish cellular processes and underlying mechanisms mediating ROS-induced synaptic growth, investigating the role of autophagy and associated genes. We will use genetics to identify interactions between genes and recently developed mosaic and intersectional expression systems to target expression of dominant negative, fluorophore tagged and RNAi transgenes to specific neurons. We will visualize pre- and postsynaptic terminals by immunofluorescence antibody staining and targeted expression of fluorophore tagged reporter constructs, imaging these with point and field scanning confocal systems. Live imaging will inform us about changes to the dynamics of ROS induced growth, T.E.M. about the role of autophagy. We will apply 3-D reconstruction algorithms for quantitative analysis of complex branched dendrites. Given the high conservation of the signaling pathways and cellular processes associated with oxidative stress, this proposal is likely to identify new candidate genes as potential targets for therapeutic strategies aimed at ameliorating the effects of ageing in the nervous system.

Who will benefit from this proposal? The UK has an expanding ageing population generating a correspondingly large social and economic burden. Decline in cognitive function in the elderly is a major component of this burden to our society. As yet we do not understand the effect of ageing on the function of the nervous system or how this may predispose the ageing brain to dementia and related conditions, notably neurodegenerative diseases. Dementia is a prominent consequence of ageing. Decreasing cognitive function in ageing individuals is a wider and more general problem and inescapable for most individuals, necessitating an increasing need for care and support (financially and emotionally) from close relatives and society at large. In economic terms, according to figures compiled by the Alzheimer's Research Trust, 820,000 people in the UK live with dementia costing the economy approximately £23 billion per year. A key feature of ageing (and neurodegenerative conditions) is increased oxidative stress. Our proposal focuses on this central element of ageing and aims to understand its effects on neuronal structure and connectivity. It addresses fundamental, as yet unresolved issues and identifies the signaling pathways that lead to alterations of neuronal structure during the ageing process. Thus, our work will increase understanding of the ageing brain at a cellular and mechanistic level and it is conceived to identify potential (new) points for therapeutic intervention. As presented, this proposal has clear longer-term benefits for ageing individuals, carers, the social and healthcare systems and the economy in general. Potential benefits in the long term 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 signaling pathways as candidates for therapeutic intervention, economic benefits arise: the market for an anti-neuronal-ageing therapeutic or dietary supplements would be considerable. We use the fruitfly, Drosophila melanogaster, as the experimental organism for well considered reasons. First, the molecular pathways we investigate are highly conserved and we show that they respond in the same manner as they do in mammals. The cellular, molecular and genetic events we investigate will be of relevance to humans. Secondly, the power of Drosophila genetics is second to none. Use of the fruit fly, with a clear track record of driving discoveries in biology, allows for rapid and effective experimentation, such as the testing of genetic interactions and being able to literally watch the effects of manipulations in living, intact animals and their nervous systems. Third, working with Drosophila means less use of vertebrate models, saving on housing and husbandry costs as well as ethical considerations. This has long been a goal of the UK Research Councils and of society at large and falls under the aims of the 3Rs programme: replacement, reduction and refinement. How might individuals, organisations or society benefit from this research? Ageing impacts us all. To communicate the biological effects of ageing, how they are exacerbated and strategies for counteracting them at the level of lifestyle decisions (e.g. exercise, diet (anti-oxidants) or drugs targeting specific proteins), is very important. M.L. and S.T.S. undertake regular outreach activities with local schools, particularly during Science Week. This proposal has not only the potential for medically relevant discoveries but will also produce simple, yet evocative experimental paradigms for teaching school children and undergraduates alike the effects of environmental and dietary stresses on ageing and neuronal function.