Systems-analysis of the Nf-kappaB signalling networks that control levels of reactive oxygen species

Reactive Oxygen Species (ROS) are oxygen-containing free radicals that are a natural by-product of cellular metabolism. ROS levels also dramatically increase in response to cellular stresses such as heat or UV exposure, tissue wounding, or infection. ROS are highly damaging to cell structures and can result in genetic mutation. Importantly, ROS accumulation is thought to be the cause of ageing, and underlies health problems associated with ageing including impairment of memory and cognitive function. High levels of ROS are also thought to promote the onset of neurodegenerative diseases, diabetes, and cancer. Due to the toxic effects of ROS, cells have evolved a number of genes and biochemical mechanisms in order to prevent the build-up of ROS, and many of these genes are in fact activated as part of the normal stress response. Mutation of these genes can cause the stress response to breakdown, leading to ROS accumulation and subsequent acceleration of the ageing process, and/or the development of diverse pathologies. Through genetic screens, we have found that a gene called Nf-kappaB (or Nf-kB) has a very important role in controlling ROS levels. Nf-kB is a transcription factor that regulates ROS levels by controlling the levels of other genes. Our experiments reveal that loss of Nf-kB activity raises ROS levels, but we also found that excessive Nf-kB activity has the same effect. Therefore, Nf-kB activity must be finely-tuned in order to keep ROS levels low. Using novel genetic screening technology we have developed, we also discovered approximately 200 genes that work together with Nf-kB to control ROS. We predict these genes act as a complex network to modulate the concentration of cellular ROS. While our studies have identified these critical genes, it is currently unknown how the genes work to control ROS levels. Using a number of different technologies and methods, we aim to study the function of these genetic network towards the goal of developing therapeutics that could be used to maintain the proper balance of Nf-kB activity, and thus improve wellbeing during the ageing process. (1) By inhibiting the function of each of these individually we will study how the genes might act to control Nf-kB entry into the nucleus and thus activate transcription. We will also study how inhibition of these genes controls the activity of another important transcription factor called FOXO that we have found regulates metabolism in response to increases in ROS levels. (2) We plan to study how the genes we have discovered are involved in regulating and responding to endoplasmic reticulum (ER) stress and a process called autophagy. Both ER stress and autophagy have been previously linked to the regulation of cellular ROS levels. We will also study how these genes are involved in regulating the metabolic changes in cells that are caused by the exposure of cells to insulin as insulin has also been implicated in controlling ROS levels. (3) Although we identified 213 genes that work together with Nf-kB to regulate ROS levels, there remain many more to be discovered. We will continue to perform further genetic screening in order to determine all the genes that work together with Nf-kB to control the generation of ROS. Many of the genes could novel targets for therapeutics that will improve wellbeing and/or prevent disease. (4) We will develop and implement computational methods in order to map all the relationships that exist between the genes we have discovered. Thus we will generate a map of the Nf-kB gene network that control ROS levels. By developing this map we will be able to determine critical points or 'nodes' in the network that can be targeted by therapeutics to control ROS levels.

Reactive Oxygen Species (ROS), such as superoxide (O2-) and peroxides, are natural by-products of metabolism, and are generated as a means to communicate cellular signals. However, ROS accumulation is highly damaging to cellular structures and genome integrity. But little is known regarding the architecture and dynamics of the genetic and biochemical systems that cells have evolved in order to modulate ROS levels. In order to understand how ROS levels are genetically controlled, we have performed a genome-wide RNAi screen for regulators of superoxide levels in Drosophila cells and identified a number of known and novel genes that are enhancers or suppressors of ROS generation. Notably inhibition of both Drosophila Nf-kB and IkB led to increases in ROS levels, which demonstrates a balance of Nf-kB activity is essential to modulate ROS levels. To begin to understand how Nf-kB activity is tailored by signaling networks to regulate ROS levels, we performed a subsequent series of screens in Drosophila cells for regulators of ROS in sensitized genetic backgrounds where Nf-kB or IkB were also inhibited by RNAi. Many of the genes isolated in these sensitized screen are regulators of insulin signaling, metabolism, ER stress, and autophagy which strongly suggests these cellular processes are involved in Nf-kB-mediated ROS production. Using a series of 14 quantitative RNAi screens, we aim to; (a) determine how Nf-kB transcriptional dynamics are regulated by genetic interactors identified in sensitized RNAi screens for regulators of ROS (b) determine how Nf-kB and interactors of Nf-kB may be involved in the control of ROS levels by acting as regulators of ER stress, insulin signaling and autophagy; (c) assemble of comprehensive genome-wide list of ROS regulators that interact with Nf-kB and IkB. We also will; (d) Perform computational integration to develop systems-level models of the Nf-kB signaling network which act to regulate ROS levels.

ROS accumulation is considered the primary cause of ageing, and underlies the development of diseases such as neurodegenerative pathologies, cancer, and diabetes, but little is known has to how cellular ROS is regulated by a complex network of genes. The research described in this proposal aims to provide fundamental insight into the role of the genetic networks that regulate ROS, and thus a number of groups will potentially benefit from this research including: - staff working on the project will develop which develop research and professional they could apply in all employment sectors - academic researchers studying ROS, the ageing process, neurodegenerative diseases, cancer, and diabetes - academic and industrial organizations devising therapies to control ROS levels and/or modulate Nf-kB activity - an ageing British and international population - the British economy as a whole (1) Academic Researchers. Dr. Julia Sero and other laboratory staff who will work on this project will gain valuable and highly useful experience in: robotics, high-throughput screening techniques, image-analysis, statistical and computational analysis. Moreover, Dr. Sero will gain experience in project and budget management, industrial collaboration, and public speaking. A number of academic researchers in the greater scientific community will benefit immediately from this work including those studying; Nf-kB, ROS generation, metabolism, the molecular basis of ageing diabetes, nutritional sciences, and immunology. (2) Commercial Private Sector Beneficiaries. The project outlined could potentially lead to the development of both single and combinatorial therapeutic targets to modulate ROS and/or Nf-kB activity by academic or industrial organizations. The Institute of Cancer Research Technology Transfer Enterprise Unit is well equipped to protect any Intellectual Property generated by this project and further pursue it in terms of commercial exploitation. (3) Public Sector. Completion of these studies pay potentially lead to therepuetic treatments, and/or intuition as to how to make lifestyle choices (e.g. nutrional changes) to control ROS accumulation and thus improve the quality of life of an ageing British populace. (4) Potential to impact on the nation's health, wealth or culture. By providing novel insights into the regulation of ROS this research could potentially lead to therapies and treatments which could dramatically improve the well-being, health, and creative output of an increasingly larger geriatric population. Improving the health of the aged has the potential to lower the ever-rising health care costs. The potential benefits for the academic and commercial sector will be realized almost immediately after completion of this research (3-5 years). Potential benefits to the health of the public sector would be realized over a longer period (5-15 years). Research findings will be disseminated information through peer reviewed public journals, and public presentations (both to academic and public audiences). All datasets will be made publicly available via an ICR-maintained website, and data will be stored on the ICR server indefinitely.