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

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

William J Griffiths et al. Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Feb.

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.

Keywords: Brain; CYP27A1; Cerebrotendinous xanthomatosis; Cholestenoic acid; Mass spectrometry; Oxysterol.

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Figures

Unlabelled Image — **Graphical abstract**

**Fig. 1**
Bile acid biosynthesis via the neutral, acidic, 25-hydroxylase and (25S)26-hydroxylase pathways. The 25-hydroxylase pathway is shown in inset (i), the mouse (25S)26-hydroxylase pathway in inset (ii). R = OH in acids or SCoA in CoA thioesters. R₁ = H or OH. Where known, mouse enzymes are indicated in **bold**, CYP3A11 was found in the present work to introduce a (25S)26-hydroxy group to 7α-hydroxycholesterol as indicated by
underlining
of the enzyme symbol. The enzyme which converts the (25S)26-primary alcohol to a carboxylic acid is indicated as a sterol oxidase (SO). Abbreviations: CYP, cytochrome P450; HSD, hydroxysteroid dehydrogenase; AKR, aldo-keto reductase; BACS, bile acyl-CoA synthetase (SLC27A5); VLCS, very long chain acyl-CoA synthetase (SLC27A2); AMACR, alpha-methylacyl-CoA racemase; ACOX2, branched chain acyl-CoA oxidase 2, also called branched-chain acyl-CoA oxidase; DBP, D-bifunctional protein or multifunctional enzyme type 2 (HSD17B4); SCPx, sterol carrier protein x. [3,4]. Metabolites of increased or decreased abundance in the *Cyp27a1−/−* mouse are indicated by upward or downward arrows. Red arrows are used to indicate changes in plasma, blue arrows for brain. A solid horizontal line indicates detected but not significantly changed. *, P < 0.05; ** P < 0.01; *** P < 0.001. P < 0.05 is considered significant. The low levels of di- and tri-hydroxycholesterols and of dihydroxycholestenoic acids in brain makes it difficult to distinguish between these compounds and their 3-oxo equivalents using EADSA as their differentiation is based on peak area *difference* between samples treated *with* and *without* cholesterol oxidase (see Fig. S1). Hence, for these metabolites the combined values for the two structures are used.

**Fig. 2**
Concentrations of oxysterols and cholestenoic acids in *Cyp27a1−/−* (n = 3) and *Cyp27a1+/+* (wt, n = 3) mouse plasma. No hydrolysis or solvolysis steps were performed so the values reported are for “free” non-esterified molecules. Sterols are arranged according to mass and chromatographic order of elution of the GP-derivative. To maintain a single y-axis magnification factors have been applied as indicated. Using the EADSA method 24S,25-epoxycholesterol isomerises to 24-oxocholesterol, becomes hydrolysed to 24,25-dihydroxycholesterol and undergoes methanolysis to 3β,24-dihydroxycholest-5-ene-25-methoxide. The total 24S,25-epoxycholesterol corresponds to the sum of the individual forms.

**Fig. 3**
Oxysterols in *Cyp27a1−/−* and *Cyp27a1+/+* (wt) mouse plasma. Each chromatogram is normalised to the most intense peak at 100% relative abundance (RA). Magnification factors are as indicated. The concentration of the indicated analyte (by retention time, Rt) is given in the right-hand corner of each chromatogram. Chromatograms from the oxysterol fractions treated with cholesterol oxidase (combination of sterols with a *native* 3-oxo group and those *oxidised* by cholesterol oxidase to contain a 3-oxo group) are shown. The insets show structures of generic GP derivatives. Many oxysterols elute as twin peaks corresponding to *syn* and *anti* conformers. (A) Monohydroxycholesterols and monohydroxycholest-4-en-3-ones. (B) Dihydroxycholesterols and dihydroxycholest-4-en-3-ones. (C) Trihydroxycholesterols and trihydroxycholest-4-en-3-ones. (D) 3β,24-Dihydroxycholest-5-ene-25-methoxide, the methanolysis product of 24S,25-epoxycholesterol. In (A-C) peaks are labelled with the location of the relevant hydroxy groups on the cholesterol or cholest-4-en-3-one structure. In (D) 3β,24-Dihydroxycholest-5-ene-25-methoxide is abbreviated to 24H,25 M.

**Fig. 4**
Cholestenoic acids in *Cyp27a1−/−* and *Cyp27a1+/+* (wt) mouse plasma. Each chromatogram is normalised to the most intense peak at 100% RA. Magnification factors are as indicated. The concentration of the indicated analyte (by Rt) is given in the right-hand corner of each chromatogram. Chromatograms from the oxysterol fractions treated with cholesterol oxidase (combination of sterols with a *native* 3-oxo group and those *oxidised* by cholesterol oxidase to contain a 3-oxo group) are shown in (A)–(B). The insets show structures of generic GP derivatives. Many cholestenoic acids elute as twin peaks corresponding to *syn* and *anti* conformers. (A) 3β-Hydroxychest-5-enoic and 3-oxocholest-4-enoic acids, and dihydroxycholestenones and hydroxycholestenediones. (B) Dihydroxycholestenoic and hydroxyoxocholestenoic acids. (C) Dihydroxyoxocholestenoic acids. Abbreviations: 3β-HCA, 3β-hydroxycholest-5-en-(25R)26-oic; 7αH,3O-CA(25R or S), 7α-hydroxy-3-oxocholest-4-en-(25R or S)26-oic; 3β,7β-diHCA, 3β,7β-dihydroxycholest-5-en-26-oic; and 7α,12α-diH,3O-CA(25R or S), 7α,12α-dihydroxy-3-oxocholest-4-en-(25R or S)26-oic acids. The peaks at 7.30 min and 7.31 min in (A) are annotated to a combination of 3β,22-dihydroxycholest-5-en-24-one and 22-hydroxycholest-4-ene-3,24-dione. The peaks at 4.90 min and 4.87 min in (B) are annotated to 3β,22,25-trihydroxycholest-5-en-24-one.

**Fig. 5**
Oxysterols in *Cyp27a1−/−* and *Cyp27a1+/+* (wt) mouse brain. Each chromatogram is normalised to the most intense peak at 100% RA. Magnification factors are as indicated. The concentration of the indicated analyte (by Rt) is given in the right-hand corner of each chromatogram. Chromatograms from the oxysterol fractions treated with cholesterol oxidase (combination of sterols with a *native* 3-oxo group and those *oxidised* by cholesterol oxidase to contain a 3-oxo group) are shown. The insets show structures of generic GP derivatives. Many oxysterols elute as twin peaks corresponding to *syn* and *anti* conformers. (A) Monohydroxycholesterols and monohydroxycholest-4-en-3-ones. (B) As in (A) but over an extended gradient. (C) Dihydroxycholesterols and dihydroxycholest-4-en-3-ones. (D) Trihydroxycholesterols and trihydroxycholest-4-en-3-ones. (E) 3β,24-Dihydroxycholest-5-ene-25-methoxide, the methanolysis product of 24S,25-epoxycholesterol. (F) Monohydroxycholestenones, monohydroxydehydrocholesterols and monohydroxycholesta-4,6(or24)-dien-3-ones. In (A-D) peaks are labelled according to the respective location of hydroxy groups on the core cholesterol or cholest-4-en-3-one structure. In (E) and (F) the abbreviations are 24H,25 M, 3β,24-dihydroxycholest-5-ene-25-methoxide; 24,25-EC, 24S,25-epoxycholesterol; 24O-C, 3β-hydroxycholest-5-en-24-one; 7α-HD, 7α-hydroxydesmosterol; 12α-HCdO, 12α-hydroxycholestadien-3-one.

**Fig. 6**
Concentrations of oxysterols and cholestenoic acids in *Cyp27a1−/−* (n = 3) and *Cyp27a1+/+* (wt, n = 3) mouse brain. No hydrolysis or solvolysis steps were performed so the values reported are for “free” non-esterified molecules. Sterols are arranged according to mass and chromatographic order of elution of the GP-derivative. To maintain a single y-axis magnification factors have been applied as indicated. The low levels of di- and tri-hydroxycholesterols and of dihydroxycholestenoic acids in brain made it difficult to distinguish between these compounds and their 3-oxo equivalents using EADSA as their differentiation is based on peak area *difference* between samples treated *with* and *without* cholesterol oxidase. Hence, for these metabolites the combined values for the two structures are given, and for simplicity we just give values for di- and trihydroxycholest-4-en-3-ones and 7α-hydroxy-3-oxocholest-4-enoic acids. Using the EADSA method 24S,25-epoxycholesterol isomerises to 24-oxocholesterol, becomes hydrolysed to 24,25-dihydroxycholesterol and undergoes methanolysis to 3β,24-dihydroxycholest-5-ene-25-methoxide. The total 24S,25-epoxycholesterol corresponds to the sum of the individual forms.

**Fig. 7**
Cholestenoic acids in *Cyp27a1−/−* and *Cyp27a1+/+* (wt) mouse brain. Each chromatogram is normalised to the most intense peak at 100% RA. Magnification factors are as indicated. The concentration of the indicated analyte (by Rt) is given in the right-hand corner of each chromatogram. Chromatograms from the oxysterol fractions treated with cholesterol oxidase (combination of sterols with a *native* 3-oxo group and those *oxidised* by cholesterol oxidase to contain a 3-oxo group) are shown. The insets show structures of generic GP derivatives. Many cholestenoic acids elute as twin peaks corresponding to *syn* and *anti* conformers. (A) 3β-Hydroxychest-5-enoic and 3-oxocholest-4-enoic acids, and dihydroxycholestenones and hydroxycholestenediones. (B) Dihydroxycholestenoic and hydroxyoxocholestenoic acids. Abbreviations: 3β-HCA, 3β-hydroxycholest-5-en-(25R)26-oic; 7αH,3O-CA(25R or S), 7α-hydroxy-3-oxocholest-4-en-(25R or S)26-oic.

**Fig. 8**
Analysis of the PXR activational capacity of sterols and oxysterols of enhanced or changed abundance in *Cyp27a1−/−* mouse. Luciferase activity in SN4741 neural cells transfected with (A) a PXR-responsive luciferase reporter construct (PXRE) and PXR, and (B) an LXR-responsive luciferase reporter construct (LXRE) and LXRα, and stimulated for 24 h with the compounds indicated (10 μM). Cholest-4-en-3-one and 7α,24-dihydroxycholest-4-en-3-one were increased in brain but not in plasma, 7α,12α-dihydroxycholestan-3-one was elevated in plasma but not in brain, 7α,(25S)26-dihydroxycholest-4-en-3-one and 7α,12α,25-tihydroxycholest-4-en-3-one are not commercially available, while 7α-hydroxy-3-oxocholest-4-en-(25S)26-oic is only available as an unresolved mixture with the 25R epimer. (25R)26-Hydroxycholesterol and desmosterol were reduced in both brain and plasma of the *Cyp27a1−/−* mouse, 7α-hydroxy-3-oxocholest-4-en-(25R)26-oic acid was reduced in plasma only.

**Fig. 9**
Additional routes for cholesterol metabolism in the *Cyp27a1−/−* mouse. R = H in acids or SCoA in CoA thioesters. Where known enzymes are indicated in **bold**. CYP3A11 was found in the present work to introduce a (25S)26-hydroxy group to 7α-hydroxycholesterol as indicated by underlining of the enzyme symbol. The enzyme which converts the (25S)26-primary alcohol to a carboxylic acid is indicated as a sterol oxidase (SO). Metabolites of increased or decreased abundance in the *Cyp27a1−/−* mouse are indicated by upward or downward arrow. Red arrows are used to indicate changes in plasma, blue arrows for brain. A horizontal solid line indicates detected but not changed significantly. *, P < 0.05; ** P < 0.01; *** P < 0.001. P < 0.05 is considered significant. The low levels of di- and tri-hydroxycholesterols and of dihydroxycholestenoic acids in brain makes it difficult to distinguish between these compounds and their 3-oxo equivalents using EADSA as their differentiation is based on peak area *difference* between samples treated *with* and *without* cholesterol oxidase. Hence, for these metabolites the combined values for the two structures are considered. The metabolites identified in brain are enclosed within the blue box. Abbreviations are as in Fig. 1.

**Fig. 10**
The PXR ligand 7α-hydroxycholest-4-en-3-one increases the number of Islet-1+ oculomotor neurons in mouse E11.5 midbrain primary cultures, but 3β,7α-dihydroxycholest-5-en-(25S)26-oic acid does not. (A) Representative Islet-1+ and Nkx6.1+ stained cell nuclei in cultures treated with vehicle, cholest-4-en-3-one or 7α-hydroxycholest-4-en-3-one. Quantitation of Islet-1+ neurons in primary cultures from E11.5 embryos treated with (B) vehicle, cholest-4-en-3-one or 7α-hydroxycholest-4-en-3-one, and (C) vehicle, 3β,7α-dihydroxycholest-5-en-(25R)26-oic or 3β,7α-dihydroxycholest-5-en-(25R/S)26-oic acid. * P < 0.05 vs vehicle treatment, Mann-Whitney test.

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References

1. Clayton P.T. Disorders of bile acid synthesis. J. Inherit. Metab. Dis. 2011;34:593–604. - PubMed
1. Bjorkhem I. Cerebrotendinous xanthomatosis. Curr. Opin. Lipidol. 2013;24:283–287. - PubMed
1. Russell D.W. The enzymes, regulation, and genetics of bile acid synthesis. Annu. Rev. Biochem. 2003;72:137–174. - PubMed
1. Ferdinandusse S., Houten S.M. Peroxisomes and bile acid biosynthesis. Biochim. Biophys. Acta. 2006;1763:1427–1440. - PubMed
1. Fakheri R.J., Javitt N.B. 27-Hydroxycholesterol, does it exist? On the nomenclature and stereochemistry of 26-hydroxylated sterols. Steroids. 2012;77:575–577. - PubMed

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