Dissecting populations of PrRP neurone in conditional transgenic mice

In order to survive, we need to balance our energy intake (the food we eat) with metabolic demands. The latter includes the energy we require to keep warm, exercise and maintain body functions. We must ensure that there is adequate energy easily available in the form of glucose and stored in the form of fat, and predict any changes in demand. To do this, the brain and particularly regions of the hypothalamus and brainstem, collect information from other organs regarding whether we have recently eaten, the time of day, how much glucose is in our blood and how much fat is laid down in adipose tissue. All of this information is integrated by specific brain cells and translated into actions, again by peripheral organs: should we eat, produce more warmth, release more energy from stores? Our problem is that we know relatively little about the kinds of brain cell (neurone) which carry out this key integrative function. However, we have demonstrated that a novel brain transmitter, called PrRP, is capable of regulating many of these critical responses to changing energy demands, and that mice lacking the receptor for PrRP are obese. More interestingly, PrRP is produced in distinct populations of neurone in the exact regions of the hypothalamus and brainstem mentioned. However, the problem remains how to study or manipulate these distinct neurones in their different environments. We have a unique opportunity to bring together the very best physiological and behavioural analyses with cutting-edge genetics. Using the latest techniques we will produce transgenic mice with harmless alterations to the genes expressed in PrRP neurones. The first type of mouse will express a fluorescent marker only in PrRP neurones which will allow us to visualise them amongst the millions of cells in the brain. We will then be able to record their electrical activity and determine how they respond to different stimuli. This mouse will also allow us to selectively silence signalling molecules, thus demonstrating the importance of different signals specifically to PrRP neurones. The second mouse will have a small piece of DNA inserted into the PrRP gene to disrupt its expression. We predict that this mouse will be obese because it cannot respond to metabolic signals to produce the normal regulatory responses. However, we can cross this mouse with others which will result in the insert being removed from the PrRP gene in specific populations of PrRP neurone. This will allow us to study mice in which PrRP now functions normally in the brainstem alone and then also in the hypothalamus. Through these studies we will gain important insight into the brain circuitry controlling energy balance and that will perhaps allow us to develop new strategies to fight the burgeoning obesity epidemic.

Hypothalamic neurones of the arcuate and ventromedial nuclei are important in sensing and responding to changes in metabolic status. However, outputs from these cells need to first be integrated with other internal signals; state of appetite (for example, sated or hungry), time of day, metabolic demand (for example, thermoregulatory need). These roles are played to a large extent by unidentified neurones in the dorsomedial hypothalamic nucleus (DMN) and brainstem nucleus of the tractus solitarius (NTS). Cells here integrate feeding, metabolic, endocrine and circadian signals and project this information to autonomic and behavioural output structures, such as the paraventricular nucleus and brainstem motor columns. This project will demonstrate that distinct populations of prolactin-releasing peptide (PrRP)-containing neurones in the DMN and NTS can integrate various signals to determine regulatory responses. The distinct populations will be dissected by genetic manipulation of the PrRP gene locus to allow cell-specific targeting. The first transgenic line will express Cre-recombinase under control of the PrRP gene promoter, and this will be crossed with other lines to produce PrRP neurones containing enhanced green-fluorescent protein or that lack leptin receptors. Phenotyping these mice will include an electrophysiological characterisation of the integrative actions of PrRP neurones and a demonstration of their responsiveness to leptin, as well as other metabolic signals, including glucose. The second knock-in will have the PrRP coding sequence separated from its promoter by a loxP-STOP-loxP cassette. This results in the equivalent of a PrRP null mouse. However, when it is crossed with other Cre-expressing mice, the transcriptional stop sequence is removed, and PrRP can be rescued in selective neurones. This will allow us to study gain in function in the brainstem population alone and then in the hypothalamic population.

The work proposed will be immediately relevant to many academics in the field of metabolism and energy balance. Our laboratory has an established identity within the research community and we will continue to disseminate our knowledge through the publication of our work in top scientific journals (open access whenever possible) and in reviews. We also actively encourage all research staff to present their latest finding at scientific meetings. For example, in 2005-2008, we published twelve peer-reviewed research papers, were invited to write four reviews and to give oral presentations at eleven international scientific conferences. In addition to this, we organised two international symposia ourselves. In addition, our basic research has been presented and discussed at a 'Healthy Lifestyles and Obesity' meeting organised by the Child Health Research Network, and at meetings of the Diabetes and Obesity Research Network and the Association for the Study of Obesity. These meetings are forums for basic researchers, psychologists, clinicians, community nurses and other health professionals, as well as patient group representatives. Prof Luckman has provided consultations to a number of bodies regarding obesity research and Integrative Mammalian Biology (IMB; e.g. The Department for Trade and Industry). He served for nine years on the steering committee of the British Society for Neuroendocrinology, and is currently a convenor of a Physiologcial Society special interest group. He is a member of the Journal of Neuroendocrinology editorial board. Outreach work is encouraged at all levels. So, in the last year, Prof Luckman has given a lecture at a local school, and members of our laboratory have taken part in Brain Awareness Week. For The University of Manchester, Prof Luckman is academic lead on a cross-Faculty and cross-University initiative in IMB, responsible for increasing capacity in this field. The main aim of the initiative is to increase training to fill the skills gap in in vivo sciences at all levels: undergraduate, MRes and PhD and postdoctoral. We also run continuing professional development courses in experimental design and statistics. Through an additional grant from the North West Development Agency, The University has employed an IMB-dedicated Business Development Manager, who works with companies of all sizes in setting up collaborations whereby they can utilise our facilities and expertise in IMB. Our laboratory has been involved in collaborative projects with a number of industrial partners on specific metabolic targets. During this time we have been able to provide evidence for several novel targets as possibilities for drug development that has underpinned programmes on these targets by AstraZeneca, Servier, Novo Nordisk and Merck. Additionally, Prof Luckman has supervised four CASE studentships with UK-based pharma. We are hoping that a successful application for this Industrial Partnership Award will start a long-term collaborative effort with Eli Lilly, which has a major neuroscience research capability within the UK and obesity focus in the US. The high standard of our in vivo research enables us to maintain strong collaborations with other groups. Thus, currently we have joint grant funding with Prof Steve Williams on functional magnetic resonance imaging in rats and with Prof Andrew Loudon on torpor and physiological mechanisms of energy conservation. In addition, we collaborate with Dr Cath Lawrence, Prof Hugh Piggins and Prof Brad Lowell.