Cytochrome P450 27A1 Deficiency and Regional Differences in Brain Sterol Metabolism Cause Preferential Cholestanol Accumulation in the Cerebellum

Abstract

Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereospecific hydroxylation of different sterols. In humans, complete CYP27A1 deficiency leads to cerebrotendinous xanthomatosis or nodule formation in tendons and brain (preferentially in the cerebellum) rich in cholesterol and cholestanol, the 5α-saturated analog of cholesterol. In Cyp27a1^-/- mice, xanthomas are not formed, despite a significant cholestanol increase in the brain and cerebellum. The mechanism behind cholestanol production has been clarified, yet little is known about its metabolism, except that CYP27A1 might metabolize cholestanol. It also is unclear why CYP27A1 deficiency results in preferential cholestanol accumulation in the cerebellum. We hypothesized that cholestanol might be metabolized by CYP46A1, the principal cholesterol 24-hydroxylase in the brain. We quantified sterols along with CYP27A1 and CYP46A1 in mouse models (Cyp27a1^-/-, Cyp46a1^-/-, Cyp27a1^-/-Cyp46a1^-/-, and two wild type strains) and human brain specimens. In vitro experiments with purified P450s were conducted as well. We demonstrate that CYP46A1 is involved in cholestanol removal from the brain and that several factors contribute to the preferential increase in cholestanol in the cerebellum arising from CYP27A1 deficiency. These factors include (i) low cerebellar abundance of CYP46A1 and high cerebellar abundance of CYP27A1, the lack of which probably selectively increases the cerebellar cholestanol production; (ii) spatial separation in the cerebellum of cholesterol/cholestanol-metabolizing P450s from a pool of metabolically available cholestanol; and (iii) weak cerebellar regulation of cholesterol biosynthesis. We identified a new physiological role of CYP46A1, an important brain enzyme and cytochrome P450 that could be activated pharmacologically.

Keywords: brain; cerebellum; cholesterol; cholesterol metabolism; cholesterol regulation; cytochrome P450.

FIGURE 1.

Proposed pathways of cholestanol biosynthesis and elimination from the brain. The 7α-hydroxy-4-cholesten-3-one-dependent pathway is initiated in the liver and is indicated with blue arrows; cholestanol precursors and metabolites in this pathway are shown in gray. The cholesterol-dependent pathway is initiated in the brain and is indicated with magenta arrows. In both pathways, the known enzymes are indicated; boldface arrows represent the major mechanism for cholesterol or cholestanol elimination from the brain.

FIGURE 2.

Brain levels of CYP27A1, CYP46A1, and their substrates and metabolites. For sterol quantifications, the results are the mean ± S.D. of the independent measurements in five mice per genotype and gender (A), three mice per genotype and gender (B), or duplicate measurements in the specimens from one female and one male donor (C). ND, not detected (the limit of detection is 1 pmol/mg of protein). MRM data from 3–4 transitions/peptide (3 peptides for CYP46A1 and 2 peptides for CYP27A1) were combined to determine the amount of each P450. Data represent mean ± S.D. (error bars) of the independent P450 measurements in three mice or one human donor. NM, not measured. Pink asterisks, significant changes between females of the knock-out strains versus females of the corresponding wild type strain. Blue asterisks, significant changes between males of the knock-out strains and males of the corresponding wild type strain. Gray asterisks, significant changes between female and male mice of the same strain. Black asterisks, significant changes between the genotypes when data were collapsed across genders. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 by two-way analysis of variance followed by Bonferroni correction as described under “Experimental Procedures.”

FIGURE 3.

Levels of cholestanol and 7α-hydroxy-4-cholesten-3-one in mouse plasma and brain. The results are mean ± S.D. of the independent measurements in five mice per genotype and gender (A–C) or three mice per genotype and gender (D). The bar fill pattern, color codes for bars, statistical analysis, and asterisks as well as p values are the same as in Fig. 2.

FIGURE 4.

Cholestanol is an endogenous substrate for CYP46A1 in mice. A, total ion chromatogram of sterol extract (after trimethylsilylation) from the in vitro incubations of cholestanol with purified recombinant CYP46A1. The peak at 29.46 min corresponding to the CYP46A1 product is circled and shown as an inset. B, mass spectrum of the peak at 29.46 min is consistent with the fragmentation pattern of 24-hydroxycholestanol. C, single ion monitoring chromatogram of the oxysterol fraction isolated from the whole brain of wild type (Cyp46a1^+/+) and Cyp46a1^−/− mice.

FIGURE 5.

Cholestanol is an endogenous substrate for CYP27A1 in mice. A, total ion chromatogram of sterol extract (after trimethylsilylation) from the in vitro incubations of cholestanol with purified recombinant CYP27A1. The peak at 30.96 min corresponding to the CYP27A1 product is circled and shown as an inset. B, mass spectrum of the peak at 30.96 min is consistent with the fragmentation pattern of 27-hydroxycholestanol. C, single ion monitoring chromatogram of the oxysterol fraction isolated from the whole brain of wild type (Cyp27a1^+/+) and Cyp27a1^−/− mice.

FIGURE 6.

Levels of lathosterol (A) and desmosterol (B) in mouse whole brain and cerebellum. The results are mean ± S.D. (error bars) of the independent measurements in three mice per genotype and gender. The bar fill pattern, color codes for bars, statistical analysis, and asterisks as well as p values are the same as in Fig. 2.