Regulation of neuronal plasticity by NADPH oxidases

The ability to remain plastic is a fundamental property of nerve cells. It is critical for learning and adaptation, as well as for protective responses to over-excitation caused by injury or disease. We study basic questions, such as: "How do nerve cells measure how active they are?" or: "What mechanisms transform changes in nerve cell activation into changes of nerve cell structure and connectivity?" We work with the fruit fly, Drosophila. The high evolutionary conservation of basic cellular processes has meant that most discoveries made in this model system are directly pertinent to our understanding of the human brain, including mechanisms that underlie learning and memory. Importantly, the fruit fly is one of the most powerful genetic experimental organisms. For example the larva of the fruit fly is one of the few model systems whose brain circuitry has been charted close to completion and where one can genetically manipulate individual connecting neurons with unrivalled precision.

We recently discovered that nerve cells use reactive oxygen species (ROS) as signals to monitor their own activity levels. ROS levels are well known to increase in the brain with ageing and to reach pathological levels in many neurodegenerative conditions, including Alzheimer's, Parkinsonism or Motorneuron Disease. Nerve cells closely regulate the size of their synaptic terminals and the number of connections to other nerve cells. We found that at normal physiological levels ROS regulate this process. Some ROS are produced by mitochondria as constitutive by-products of their energy/ATP metabolism. This finding suggests that nerve cells might use metabolic ROS signals as a readout of their activity levels. As a cellular sensor for ROS we identified the conserved redox sensitive protein DJ-1b (Park7), which regulates synaptic terminal size through the PI3Kinase growth pathway.

More recently we discovered that ROS produced in different sub-cellular compartments of nerve cells regulate different aspects of synaptic terminal plasticity. In this proposal we focus on ROS generated at the plasma membrane by NADPH oxidases. These regulate the size of synaptic terminals, as contrasting with mitochondrially produced ROS, which control how densely such terminals are populated with synapses. NADPH oxidases are best known for their roles in the immune system. In the nervous system they are thought to be required for learning associated plasticity, and a recent study suggests that their dysregulation could cause psychiatric conditions, such as schizophrenia.

The first aim of our proposal is to determine how structural changes of synaptic terminals are paralleled by physiological functional alterations. Using state of the art imaging methods we will determine the role of NADPH oxidases in regulating activity-dependent connectivity in the central nervous system (CNS). Next, to determine how neuronal activity regulates NADPH oxidase activation, we will seek to identify interacting regulatory proteins and probe interactions with other signalling pathways known to regulate synaptic terminal growth. Our third aim explores the potential of NADPH oxidases to orchestrate the complex inter-cellular interactions that take place during neuronal remodelling: between neurons, their extracellular environment and adjacent glial cells. By producing ROS signals within the inter-cellular space, NADPH oxidases are ideally placed for coordinating local intercellular interactions, potentially signalling pathways conserved between the immune and nervous system.

In summary, we aim to understand fundamental mechanisms of plasticity, which might be compromised in the ageing brain. Though our work is basic science it has the potential to aid the discovery of drugs and treatments that could alleviate adverse effects of ageing and might thus, in time, benefit society.

We use the Drosophila larva as a model because it provides unique genetic access to manipulate and study in vivo identified connected cells: motoneurons, their pre-motor interneurons in the CNS and postsynaptic target muscles.

1. To determine how NADPH oxidase-generated ROS shape synaptic function and connectivity, we will make sharp electrode recordings from muscles and determine which aspects of activity-regulated changes in synapse physiology are regulated by NADPH oxidases. In the CNS, we will characterize synaptic connectivity using expansion microscopy in combination with a novel endogenous postsynaptic reporter (Drep2-YPET); and a new GRASP method to mark synapses between pairs of partner neurons. As a behavioural correlate, locomotor network output will be measured using larval crawling.

2. We will investigate how NADPH oxidase activity is regulated by neuronal activation and other synaptic plasticity pathways (AP-1, Wnt, BMP, Neurotrophins). Interactors of dDuox and dNox will be identified by immuno-precipitation. Interactors identified in non-neural cells will serve as contingency. Candidate regulatory interactors will be tested genetically by epistasis and by RNAi knockdown experiments against defined synaptic phenotypes. We will test regulation of dDuox and dNox transcription by other plasticity pathways via qRT-PCR. Conversely, activation of AP-1, Wnt, BMP, Neurotrophin pathways by NADPH oxidase-generated ROS will be assayed by established indicators.

3. We will test the hypothesis that NADPH oxidase-generated extracellular ROS orchestrate activity-dependent neuronal remodelling by signalling to adjacent neurons and glia. ECM modification requires joint activity of peroxidases, which we will test genetically by RNAi knockdown. ROS signalling between neurons and glia will be investigated by targeted RNAi knockdown and imaging. We will probe in the nervous system conserved NADPH oxidase-ROS signalling pathways critical to immune function.

Who will benefit from this research proposal?

Our research has uncovered 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. We further discovered a highly conserved protein as critical for sensing changes in neuronal ROS levels and that ROS generated in different sub-cellular compartments by different mechanisms regulate distinct aspects of synaptic plasticity. Our research is therefore important to our understanding of the formation, functioning and homeostasis of a healthy brain. Through this, it will inform us about 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 strategies to maximise impact, through efficient communication with potential beneficiaries:

A) Academic Communication: Publications will be published in Open Access journals, seeking the highest impact and widest readership. We will publicise our work at prominent conferences in the neuroscience, ageing, dementia and neurodegeneration fields. The PDRA will be encouraged to talk at meetings to generate profile for the study and their own career progression. Reagents generated, e.g. genetic strains and DNA constructs, will be disseminated freely, or lodged in not-for-profit stock centres, such as Addgene and the Bloomington stock centre. Our Drosophila data will contribute to Flybase and Flymine entries.

B) Public Communication: ML is well versed at communicating with schools and the general public. He has been active with schools outreach and events open to the general public, e.g. during Science Week. 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: We will have regular review meetings with academics active in translational neuroscience research. We discovered ROS signalling as critical to neuronal adjustment mechanisms. Because breakdown in the tightly controlled regulation of cellular ROS levels are symptomatic of the ageing brain and neurodegenerative conditions, this proposal might have 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.

Technological Training and impact: The PDRA will be trained in new skills and cutting-edge super-resolution imaging and electrophysiology techniques, which 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, though it has potential to spawn work with translational impact. Throughout the course of the grant we will therefore regularly review our data and its implications for potential translational impact and industrial interaction.