Knockout of the Cholesterol 24-Hydroxylase Gene in Mice Reveals a Brain-specific Mechanism of Cholesterol Turnover

Erik G Lund,Chonglun Xie,Tiina Kotti,Stephen D Turley,John M Dietschy,David W Russell

doi:10.1074/jbc.m303415200

Abstract

Most cholesterol turnover takes place in the liver and involves the conversion of cholesterol into soluble and readily excreted bile acids. The synthesis of bile acids is limited to the liver, but several enzymes in the bile acid biosynthetic pathway are expressed in extra-hepatic tissues and there also may contribute to cholesterol turnover. An example of the latter type of enzyme is cholesterol 24-hydroxylase, a cytochrome P450 (CYP46A1) that is expressed at 100-fold higher levels in the brain than in the liver. Cholesterol 24-hydroxylase catalyzes the synthesis of the oxysterol 24(S)-hydroxycholesterol. To assess the relative contribution of the 24-hydroxylation pathway to cholesterol turnover, we performed balance studies in mice lacking the cholesterol 24-hydroxylase gene (Cyp46a1-/- mice). Parameters of hepatic cholesterol and bile acid metabolism in the mutant mice remained unchanged relative to wild type controls. In contrast to the liver, the synthesis of new cholesterol was reduced by approximately 40% in the brain, despite steady-state levels of cholesterol being similar in the knockout mice. These data suggest that the synthesis of new cholesterol and the secretion of 24(S)-hydroxycholesterol are closely coupled and that at least 40% of cholesterol turnover in the brain is dependent on the action of cholesterol 24-hydroxylase. We conclude that cholesterol 24-hydroxylase constitutes a major tissue-specific pathway for cholesterol turnover in the brain.

Highlights

The major site for the turnover of cholesterol1 in the body is the liver
The synthesis of bile acids is limited to the liver, but several enzymes in the bile acid biosynthetic pathway are expressed in extra-hepatic tissues and there may contribute to cholesterol turnover
The transfer of cholesterol from the peripheral tissues to circulating lipoprotein particles occurs at the surfaces of cells and represents the major pathway by which extra-hepatic tissues turn over cholesterol [2]; an exception is the brain, where the blood-brain barrier prevents transfer of cholesterol to circulating lipoproteins

Summary

EXPERIMENTAL PROCEDURES

Construction of Cyp46a1 Knockout Mouse—Routine molecular biology methods, including restriction enzyme digestion, agarose and polyacrylamide gel electrophoresis, DNA ligation, transformation of bacterial cells, electroporation of cultured cells, and Southern and RNA blotting were done as described previously [28]. This ligation positioned the 3Ј-end of the cholesterol 24-hydroxylase genomic DNA fragment (the “short arm”) immediately upstream of the ATG codon of the bovine tau-Escherichia coli ␤-galactosidase (lacZ) fusion gene of pBS-tau-lacZ. This intermediate plasmid was modified further to contain an additional SalI site just 3Ј to the tau-lacZ fusion gene. To assemble the final targeting vector, the DNA fragment containing the “short arm” composed of the Cyp46a1 promoter fused to the tau-lacZ gene was released by SalI digestion and inserted into an XhoI site in the plasmid containing the “long arm.”. Values represent the mean Ϯ 1 S.M. for 7-10 animals in each group

Metabolic parameter

RESULTS

Plasma cholesterol levels as determined by an enzymatic

DISCUSSION