Imaging cholesterol metabolic flux and transport underlying brain function

Lead Research Organisation: Swansea University
Department Name: Institute of Life Science Medical School

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Griffiths W (2019) Oxysterol research: a brief review in Biochemical Society Transactions

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Griffiths WJ (2017) Sterolomics: State of the art, developments, limitations and challenges. in Biochimica et biophysica acta. Molecular and cell biology of lipids

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Griffiths WJ (2018) An update on oxysterol biochemistry: New discoveries in lipidomics. in Biochemical and biophysical research communications

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Griffiths WJ (2017) Cholesterolomics: An update. in Analytical biochemistry

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Griffiths WJ (2019) Additional pathways of sterol metabolism: Evidence from analysis of Cyp27a1-/- mouse brain and plasma. in Biochimica et biophysica acta. Molecular and cell biology of lipids

 
Description The idea of on-tissue derivatisation combined with liquid extraction for surface analysis (LESA) and capillary-liquid chromatography (cap-LC) linked with mass spectrometry imaging (MSI) has been validated for the analysis of cholesterol and its metabolites in mouse brain. We have been able to investigate how the levels of different cholesterol metabolites vary in different regions of mouse brain. The major cholesterol metabolite 24S-hydroxycholesterol is found to be most abundant in the striatum and thalamus and least abundant in cerebellum. On the other hand, the neuroprotective cholesterol-derived acid, 3ß,7a-dihydroxycholestenoic acid, is most abundant in the grey matter of the cerebellum, although being at least a factor of ten less abundant that 24S-hydroxycholesterol.
By analysing brain tissue from the Cyp46a1 knock-out mouse, CYP46A1 oxidises cholesterol to 24S-hydroxycholesterol, we were able to confirm the absence of 24S-hydroxycholesterol from this mouse, but instead identify low levels of its isomers, 12a-, 20S-, 24R- and 25-hydroxycholesterol and increased amounts of 3ß,7a-dihydroxycholestenoic acid in grey matter of the cerebellum. 20S-Hydroxycholesterol is an elusive metabolite being an important agonist towards the hedgehog signalling process defining stem cell fate.
A limitation of LESA is the spatial resolution of the process (0.2 - 0.3 mm diameter spot). To achieve improved resolution we have exploited on-tissue derivatisation with MALDI (matrix-assisted laser desorption/ionisation)-MSI giving spot diameters of 0.05 mm. This has proved successful to image highly abundant cholesterol in mouse brain and its precursors desmosterol and 7-dehydrocholesterol in new born mouse brain.
Exploitation Route The next step will be to use the developed methodology to investigate human brain and see how the distribution of different cholesterol metabolites vary between health and disease.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL https://www.biorxiv.org/content/10.1101/450973v1
 
Title EADSA-LESA-LC-MSI 
Description We have developed a novel technology for mass spectrometry imaging (MSI) of cholesterol, its precursors and metabolites (i.e. sterols) in tissue samples. We perform on-tissue derivatisation, which we call Enzyme-Assisted Derivatisation for Sterol Analysis (EADSA), to specifically target sterols in tissue, we then extract them from small tissue areas (0.2 - 0.3 mm diameter) by Liquid Extraction for Surface Analysis (LESA), separate isomers by liquid-chromatography (LC) and analyse the separated sterols by mass spectrometry (MS). By repeatedly performing extractions and LC-MS over the entire tissue surface we are able to reconstruct an image for each sterol present. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Using this methodology we are able to define the regions of maximum cholesterol turnover in mouse brain. 
URL https://doi.org/10.1101/450973
 
Description Cardiff LipidMaps 
Organisation Cardiff University
Country United Kingdom 
Sector Academic/University 
PI Contribution Expert input on sterol biochemistry.
Collaborator Contribution Expert input on lipid biochemistry
Impact Revision of lipid nomenclature.
Start Year 2017
 
Description Case Western Reserve University 
Organisation Case Western Reserve University
Country United States 
Sector Academic/University 
PI Contribution Analysis of brain tissue from transgenic mouse
Collaborator Contribution Provision of transgenic mouse material
Impact Manuscript in biorxiv
Start Year 2018
 
Description Karolinska 
Organisation Karolinska Institute
Country Sweden 
Sector Academic/University 
PI Contribution We have brought essential data and insight to the collaboration. We are bringing new technology
Collaborator Contribution They have brought essential data and insight to the collaboration. Our partners are bringing transgenic animal material and expertise to the collaboration.
Impact This is a multidisciplinary collaboration between neuroscientists and clinical chemists at Karolinska Institute and analytical scientists in Swansea. This colaboration has resulted in papers in Nature Chemical Biology and the Journal of Clinical Investigation.
Start Year 2006
 
Description Mass Spectrometry Imaging 
Organisation Sheffield Hallam University
Country United Kingdom 
Sector Academic/University 
PI Contribution Provide expertise in sterol/steroid analysis and derivatisation protocols.
Collaborator Contribution Provide expertise in MALDI imaging
Impact None yet.
Start Year 2016
 
Description Peroxisome 
Organisation University of Leuven
Department Zoological Institute
Country Belgium 
Sector Academic/University 
PI Contribution Expertise in sterol and oxysterol analysis
Collaborator Contribution Expertise in biochemistry of the peroxisome
Impact Publications in Steroids (2015), Biochemical Journal (2014)
Start Year 2014
 
Description SPG5 at Athens Medical Center 
Organisation Athens Medical Center
PI Contribution Analysis of plasma and urine samples from SPG5 patients under a new treatment regime.
Collaborator Contribution Disease diagnosis. Treatment of patients.
Impact Manuscript submitted. Collaboration between Medicine and Bioanalysis.
Start Year 2015
 
Description University of Houston 
Organisation University of Houston
Country United States 
Sector Academic/University 
PI Contribution Analysis of human and transgenic mouse material.
Collaborator Contribution Provision of human and transgenic mouse material.
Impact Three publications.
Start Year 2013
 
Description Zurich 
Organisation University Hospital Zurich
Country Switzerland 
Sector Hospitals 
PI Contribution Analysis of tissue and blood samples from human and mouse samples.
Collaborator Contribution Provision of human and mouse samples.
Impact Manuscript in preparation. Multi-disciplinary- Medicine, analytical biochemistry
Start Year 2016