Award details

A predictive biology and transgenic analysis of regulatory systems controlling tachykinin expression in sensory neurones

Reference	BB/D004659/1
Principal Investigator / Supervisor	Dr Alasdair MacKenzie
Co-Investigators / Co-Supervisors	Professor David MacEwan, Professor Iain McEwan, Professor Ruth Ross
Institution	University of Aberdeen
Department	School of Medical Sciences
Funding type	Research
Value (£)	313,676
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	31/01/2009
Duration	40 months

Abstract

Murine gene deletion and antagonist studies have shown that the neuropeptide products of the preprotachykinin-A (PPTA) gene in sensory neurones are essential components of the nociceptive and inflammatory response in animals. However little is known about the regulatory systems, that include the ligand-receptor interactions, signal transduction pathways and transcription factor-DNA interactions, that control the expression of PPTA in sensory neurones. We have recently used a novel combination of predictive bioinformatics and transgenic analysis to identify a component of this regulatory system in the form of a remote enhancer called ECR2. This enhancer is able to drive expression of a LacZ marker gene within PPTA expressing sensory neurones of the dorsal root ganglia (DRG). Excitingly, further bio-informatic analysis has shown the presence of binding consensus sequences of two transcription factors critical to the inflammatory response, AP1 and STAT1, within the most highly conserved areas of ECR2. These compelling observations warrant the following investigations. 1. We will carry out a deletion analysis of the 240bp ECR2 element concentrating on the most conserved regions in combination with transgenic analysis to determine the identity of the nucleotides responsible for modulating its tissue specific properties. 2. We will use predictive bioinformatic analysis to identify key transcription factors that may bind to ECR2. EMSA and DNase1 footprinting analysis will then be used to confirm the identity of transcription factors that bind ECR2 in sensory neurones. 3. We have created transgenic lines in which ECR2 drives expression of the LacZ gene in sensory neurones. This unique resource will allow us to investigate the signal transduction pathways of the regulatory system that controls PPTA expression in sensory neurones. We will expose isolated transgenic DRG in culture to a range of specific signal transduction pathway antagonists. 4. Using the same transgenic DRGculture system will also allow us to identify the key receptors essential to PPTA expression using specific receptor anagonists against cell surface receptors known to be expressed on substance P expressing sensory neurones, 5. We will also use our transgenic lines and transgenic DRGs derived from them to examine how ECR2 responds to inflammatory stimuli. The major outcome of these exciting experiments will be a better understanding of the regulatory systems required for expression of PPTA in sensory neurones and during the inflammatory response. By answering important questions relating to PPTA expression systems in sensory neurones this study will greatly facilitate future understanding of how PPTA expression in sensory neurones can go wrong and exacerbate inflammatory disease.

Summary

Sensory neurones allow animals to respond to injury and infection. Sensory neurones are also involved with inducing the inflammatory response associated with injury. A molecule called substance P is produced in sensory neurones and is a critical component of the molecular events that transmit the sensation of pain and start the inflammatory response following injury. Because substance P is a powerful stimulus of the inflammatory response it is vital that substance P within sensory neurones is produced in the right places and responds properly to signals such as injury and infection. Indeed, inappropriate turning on of substance P in sensory neurone is found in tissues affected with inflammatory diseases like asthma and arthritis. Little is known however about the control mechanisms that turn on the substance P gene in sensory neurones. There are several components of this control mechanism that we must first identify and understand before we can understand how substance P is regulated in sensory neurones. First, a message must be received by a molecule (receptor) at the surface of the sensory neuron. Secondly, this message must be transmitted (signal transduction pathway)to the centre of the neuron where the substance P gene is held. And thirdly, as a result of specific molecules (transcription factors) that bind to a specific DNA switch sequence, called an enhancer, these signals are passed onto the machinery that turns the gene on. Identifying each of these components and how they fit together is the main aim of this project. We have used a novel and powerful predictive way to detect the enhancer (called ECR2) required to turn on the SP gene in sensory neurones. We will now find out how this enhancer fits in with the rest of the control mechanism that turn on the substance P gene in sensory neurones. The exciting discovery of ECR2 in addition to the internationally acknowledged expertise of all of the applicants and the excellent facility represented by the IMS will ensure that a far better understanding of the control mechanisms of the substance P gene in sensory neurones is reached. This greater understanding will, in turn, lead to an improvement in understanding how control of the SP gene in sensory neurones can go wrong and contribute to the symptoms of inflammatory disease.

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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