Additional pathways of sterol metabolism: Evidence from analysis of Cyp27a1−/− mouse brain and plasma

William J Griffiths,Peter J Crick,Anna Meljon,Spyridon Theofilopoulos,Jonas Abdel-Khalik,Eylan Yutuc,Josie E Parker,Diane E Kelly,Steven L Kelly,Ernest Arenas,Yuqin Wang

doi:10.1016/j.bbalip.2018.11.006

William J Griffiths, Peter J Crick + Show 9 more

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https://doi.org/10.1016/j.bbalip.2018.11.006

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Abstract

Cytochrome P450 (CYP) 27A1 is a key enzyme in both the acidic and neutral pathways of bile acid biosynthesis accepting cholesterol and ring-hydroxylated sterols as substrates introducing a (25R)26-hydroxy and ultimately a (25R)26-acid group to the sterol side-chain. In human, mutations in the CYP27A1 gene are the cause of the autosomal recessive disease cerebrotendinous xanthomatosis (CTX). Surprisingly, Cyp27a1 knockout mice (Cyp27a1−/−) do not present a CTX phenotype despite generating a similar global pattern of sterols. Using liquid chromatography – mass spectrometry and exploiting a charge-tagging approach for oxysterol analysis we identified over 50 cholesterol metabolites and precursors in the brain and circulation of Cyp27a1−/− mice. Notably, we identified (25R)26,7α- and (25S)26,7α-dihydroxy epimers of oxysterols and cholestenoic acids, indicating the presence of an additional sterol 26-hydroxylase in mouse. Importantly, our analysis also revealed elevated levels of 7α-hydroxycholest-4-en-3-one, which we found increased the number of oculomotor neurons in primary mouse brain cultures. 7α-Hydroxycholest-4-en-3-one is a ligand for the pregnane X receptor (PXR), activation of which is known to up-regulate the expression of CYP3A11, which we confirm has sterol 26-hydroxylase activity. This can explain the formation of (25R)26,7α- and (25S)26,7α-dihydroxy epimers of oxysterols and cholestenoic acids; the acid with the former stereochemistry is a liver X receptor (LXR) ligand that increases the number of oculomotor neurons in primary brain cultures. We hereby suggest that a lack of a motor neuron phenotype in some CTX patients and Cyp27a1−/− mice may involve increased levels of 7α-hydroxycholest-4-en-3-one and activation PXR, as well as increased levels of sterol 26-hydroxylase and the production of neuroprotective sterols capable of activating LXR.

Highlights

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease caused by a defective sterol (25R)26-hydroxylase enzyme, known as sterol 27-hydroxylase, [1,2]
Similar observations with respect to 7α-hydroxycholesterol and (25R)26-hydroxycholesterol were made by Rosen et al in serum and by Honda et al with respect to (25R)26-hydroxycholesterol in liver mitochondria and to 7α-hydroxycholesterol and 7α-hydroxycholest-4-en3-one in liver microsomes in their studies on Cyp27a1−/− animals [13,29]
Levels of brain-derived 24S-hydroxycholesterol [3] show a small but significant increase in the Cyp27a1−/− mouse (6.34 ± 0.01 ng/mL cf. 5.40 ± 0.37 ng/mL) perhaps as a consequence of reduced metabolism [31], as do levels of 22R-hydroxycholesterol, the intermediate formed during side-chain shortening in steroid hormone biosynthesis by the enzyme CYP11A1. 25-Hydroxycholesterol did not vary between genotypes (< 2.5 ng/mL)

Summary

Introduction

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease caused by a defective sterol (25R)26-hydroxylase enzyme, known as sterol 27-hydroxylase, (cytochrome P450 27A1, CYP27A1) [1,2]. In early infancy it can present with cholestatic liver disease, in early childhood with chronic diarrhoea and cataracts, in later childhood with tendon xanthomata, learning difficulties or psychiatric illness and in adult life with spastic paraparesis, a fall in IQ or frank dementia, ataxia and/or dysarthia [1]. CYP27A1 is an essential enzyme of the neutral pathway of bile acid biosynthesis oxidising ring hydroxylated sterols at C-26 to 25R-acids.

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