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

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.

Imaging Sterols and Oxysterols in Mouse Brain Reveals Distinct Spatial Cholesterol Metabolism

“…Both of these oxysterols were detected at low levels in an earlier study of homogenised Cyp46a1-/-mouse brain, as was an unknown hydroxycholesterol eluting later than the peaks of 24R-HC (53). Here we again observe low levels of this oxysterol (0.007 ± 0.005 -0.012 ± 0.006 ng/mm 2 ) which we have previously assigned, based on retention time and MS 3 spectra, but in the absence of an authentic standard, to be 12α-hydroxycholesterol (12α-HC, Figure S9A & S9E) (25).…”

“…The enzymes required to biosynthesise 24R-HC and 20S-HC from cholesterol are unknown. The enhanced abundance of 24R-HC in cerebellum of the Cyp46a1-/-mouse does suggest CYP27A1 to be the relevant cholesterol hydroxylase, but 24R-HC is present in brain of the Cyp27a1-/-mouse arguing against this (25). CYP3A11 is a sterol 24R-hydroxylase and could perhaps convert cholesterol to 24R-HC in brain.…”

“…According to the Allen Mouse Brain Atlas the expression of Cyp27a1, the gene coding the enzyme required to introduce a hydroxy and then a carboxylate group at C-26 of cholesterol, is of highest expression in the cerebellum (57), which is in agreement with data generated here, finding cholestenoic acids to be most abundant in cerebellum ( Figure 6G). 24S-HC is by far the most abundant oxysterol in mouse brain (25,53,54). This makes analysis of closely eluting low abundance isomers challenging.…”

“…Previous studies using MS have defined concentrations of 24S-HC, the major oxysterol in mouse brain, to be of the order of 20 -60 ng/mg (wet weight) (25,(52)(53)(54). For comparison, the concentration of cholesterol is in the range of 10 -20 µg/mg (1,25,53), and after EADSA treatment, in the absence of chromatographic separation, cholesterol totally dominates the µLESA-MS analysis of the mouse brain surface extract (Figure 2A). However, with the addition of LC to give the µLESA-LC-MS platform, analysis of 400 µm diameter spot extracts of brain tissue give clear separation of peaks corresponding to 24S-HC, 24S,25-EC, desmosterol, 8(9)-dehydrocholesterol (8-DHC) and cholesterol ( Figure 2B).…”

“…3β,7α-diHCA has been shown to be biologically active as a ligand to the LXRs and to be protective towards oculomotor neurons (21). The location of the acid has not previously been defined in brain (25).…”