Award details

Imaging cholesterol metabolic flux and transport underlying brain function

Reference	BB/N015932/1
Principal Investigator / Supervisor	Professor William Griffiths
Co-Investigators / Co-Supervisors	Professor Malcolm Clench, Dr Owain Howell, Dr Jillian Rose Ann Newton, Professor Yuqin Wang
Institution	Swansea University
Department	Institute of Life Science Medical School
Funding type	Research
Value (£)	460,386
Status	Completed
Type	Research Grant
Start date	03/10/2016
End date	02/10/2019
Duration	36 months

Abstract

To visualise molecules of the cholesterol pathways in brain according to location and age we will develop "next gerneration" mass spectrometry imaging (MSI) technology. While the detection of a biomolecule in a tissue can be made by MS analysis of a tissue homogenate, all spatial information is lost. To maintain spatial information MSI techniques have been developed where the analytes are ionised by MALDI directly from the tissue surface and an image recorded by rastering of the ionisation beam relative to the sample stage. A major limitation of MSI is that molecules which are in low abundance, or are poorly ionised, are discriminated against. This limitation can be overcome by the use of derivatisation chemistry, where a specific chemical property of the target analyte is exploited to enhance ionisation of the analyte. Sterols including oxysterols are mainly neutral molecules which ionise poorly and are mostly present in low abundance in biological samples. We will develop on-tissue enzyme-assisted derivatisation for MSI of sterols, where a hydroxy group on a sterol is specifically converted by an enzyme (either cholesterol oxidase for 3beta-hydroxy groups or 3alpha-HSD for 3alpha-hydroxy groups) to a 3-oxo group which is then reacted with a hydrazine reagent carrying a positive charge e.g. Girard P (GP) reagent, to give the sterol a net positive charge and make it very favourable for MS analysis by MALDI or ESI. This method provides specificity and sensitivity revealing many molecules otherwise invisible to MS analysis. For MALDI-MSI we will first apply enzyme to the tissue surface using a sprayer/spotter and after a suitable time period apply GP reagent then matrix. MALDI-MSI will be achieved on a Synapt G2 MS utilising ion-mobility for gas-phase separations. Liquid Extraction for Surface Analysis (LESA) will be performed on similarly treated tissue but in the absence of matrix utilising an Orbitrap for ESI-MSn analysis, with or without prior LC separation.

Summary

Cholesterol in the central nervous system is separated from the rest of the body by the blood brain barrier. Disturbances in the synthesis, transport or metabolism of cholesterol in the central nervous system are associated with severe neurological and cognitive defects. Although the level of cholesterol in adult brain is at a steady-state there is a continuous turnover with an exquisite balance between de novo synthesis and metabolism. Brain cells synthesise cholesterol throughout life. Mounting evidence suggest that not only is the steady-state concentration of cholesterol important, but that cholesterol precursors and metabolites are also critical for effective brain activity, including learning, memory and motor function. Therefore, a continuous flow of metabolites through the cholesterol synthesis and metabolic pathways is essential to maintain brain function and measuring the spatial flux of metabolites in these pathways will provide further insight into the involvement of cholesterol in proper brain function. How the turnover of cholesterol differs in distinct brain regions and varies with age also affecting brain function is unclear. Furthermore, it is not known if local de novo cholesterol biosynthesis and generation of cholesterol-related molecules is essential for adult neurogenesis, as is the case in the embryo. In this project we will develop "next generation" mass spectrometry imaging technologies to answer these quandaries. Results from this study will be particularly important with the growing use of the statin class of drugs as inhibitors of cholesterol biosynthesis.

Impact Summary

As cholesterol is implicated in various neurological disorders the proposed research has the potential to generate considerable public interest and social impact. Importantly, statins are widely prescribed as inhibitors of cholesterol biosynthesis in order to reduce the risk of coronary heart disease. However, their effect on brain function and what if any effects they have on neurodegeneration have still to be uncovered. Significantly, following our publication that 24S,25-epoxycholesterol is important for dopaminergic neurogenesis we received a number of queries from the general public questioning the potential danger of taking statins. Much more research into the involvement of cholesterol metabolism in healthy brain function is still required. In the long term, the proposed research will contribute to future guidance to the NHS on the prescription of statins. A new technology will be developed in this project aimed at visualising molecules in tissues using mass spectrometry imaging (MSI). No molecule, whatever its abundance, can be detected by mass spectrometry if it cannot be ionised. Hence the key element of this proposal is to enhance the ionisation of otherwise invisible molecules by enzyme-assisted chemical derivatisation allowing their observation by MSI. We will exemplify the developed technology in brain in the study of cholesterol precursors and metabolites, which are important for neurogenesis and neuronal survival and whose biosynthesis is crucial for memory and learning. The methods developed will be equally applicable to other classes of molecules and in other tissues. The techniques developed will be of further interest to the pharmaceutical industry as it will enable drug molecules and their metabolites to be imaged in tissue providing information about their local bioavailability. The beneficiaries of this research will include neuroscientists studying cognition, neurodegeneration or autoimmune disease, three areas where oxysterols (oxidised forms of cholesterol) are important. A follow-on impact will be to the pharmaceutical industry in the development of new drug targets. A small clinical trial is already underway trying to limit the biosynthesis of the neurotoxic oxysterol 3b-HCA. Interpretation of trial results will be facilitated by a better understanding of where this oxysterol and its precursors are synthesised in healthy brain. Other than colour chromatography, mass spectrometry imaging is perhaps the most visually appealing of all analytical techniques. By colour coding molecular abundance, a digital mass spectrum can be converted to a colour image depicting molecular abundance and location in a tissue. This has great benefits for the public engagement in science, particularly to children. A final important impact of this project will be the broad education it will give to the employed PDRA, who will become educated in sterol analysis and MSI from two of the leader groups in Europe. In fact both groups will benefit significantly from the proposal as the expertise of the two groups will be shared and most importantly exchanged.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Ageing, 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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