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

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 ∼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. 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 ∼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. The major site for the turnover of cholesterol 1The abbreviations and trivial names used are: cholesterol, 5-cholesten-3β-ol; 24(S)-hydroxycholesterol, 5-cholesten-3β,24(S)-diol; 25-hydroxycholesterol, 5-cholesten-3β,25-diol; 27-hydroxycholesterol, 5-cholesten-3β,27-diol; cholic acid, 5β-cholanic acid-3α,7α,12α-triol; muricholic acid, 5β-cholanic acid-3,6,7-triol; LDL, low density lipoprotein; HDL, high density lipoprotein; LXR, liver X receptor. in the body is the liver. This process involves the secretion of cholesterol and of bile acids derived from cholesterol. A pathway of 16 enzymes is responsible for the conversion of cholesterol to primary bile acids like cholic acid and chenodeoxycholic acid, which are then secreted into the bile and eventually excreted from the body (1Russell D.W. Annu. Rev. Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1406) Google Scholar). Cholesterol for bile acid synthesis is derived from three sources, including de novo synthesis in the liver, absorption from the diet, and delivery from peripheral tissues via lipoprotein-mediated transport. 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 (2Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar); an exception is the brain, where the blood-brain barrier prevents transfer of cholesterol to circulating lipoproteins. The brain is thought to utilize an alternate mechanism of turnover in which cholesterol is converted into 24(S)-hydroxycholesterol, an oxysterol that may be able to diffuse across the blood-brain barrier (3Lutjohann D. Breuer O. Ahlborg G. Nennesmo I. Siden A. Diczfalusy U. Bjorkhem I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9799-9804Crossref PubMed Scopus (569) Google Scholar, 4Bjorkhem I. Lutjohann D. Breuer O. Sakinis A. Wennmalm A. J. Biol. Chem. 1997; 272: 30178-30184Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 5Meaney S. Bodin K. Diczfalusy U. Bjorkhem I. J. Lipid Res. 2002; 43: 2130-2135Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 6Bjorkhem I. J. Clin. Invest. 2002; 110: 725-730Crossref PubMed Scopus (216) Google Scholar). Once in the circulation, 24(S)-hydroxycholesterol is cleared by the liver and therein converted into 7α-hydroxylated intermediates in the bile acid synthetic pathway by a dedicated enzyme (7Bretillon L. Lutjohann D. Stahle L. Widhe T. Bindl L. Eggertsen G. Diczfalusy U. Bjorkhem I. J. Lipid Res. 2000; 41: 840-845Abstract Full Text Full Text PDF PubMed Google Scholar, 8Bjorkhem I. Andersson U. Ellis E. Alvelius G. Ellegard L. Diczfalusy U. Sjovall J. Einarsson C. J. Biol. Chem. 2001; 276: 37004-37010Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 9Li-Hawkins J. Lund E.G. Bronson A.D. Russell D.W. J. Biol. Chem. 2000; 275: 16543-16549Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In humans, the mass of cholesterol converted into 24(S)-hydroxycholesterol by the brain is estimated to be 0.09 mg/day/kg of body weight (10Bjorkhem I. Lutjohann D. Diczfalusy U. Stahle L. Ahlborg G. Wahren J. J. Lipid Res. 1998; 39: 1594-1600PubMed Google Scholar). The role of 24-hydroxylation has not been defined in other species, but the total amount of sterol excreted from the brain has been calculated to equal 0.18 mg/day/kg of body weight in baboons (11Wilson J.D. J. Clin. Invest. 1970; 49: 655-665Crossref PubMed Scopus (53) Google Scholar), and 1.37 mg/day/kg of body weight in mice (12Xie Z. Burns D.K. Turley S.D. Dietschy J.M. J. Neuropathol. Exp. Neurol. 2000; 59: 1106-1117Crossref PubMed Scopus (97) Google Scholar). Although the synthesis of 24(S)-hydroxycholesterol appears to be an evolutionarily conserved process, a direct demonstration of the importance of this pathway in the whole animal has not been reported. It also is not clear whether this is the only mechanism by which cholesterol is turned over in the brain; however, the amount of 24(S)-hydroxycholesterol synthesized in the rat brain is about one-half the amount of cholesterol synthesized, suggesting that additional turnover pathways exist (4Bjorkhem I. Lutjohann D. Breuer O. Sakinis A. Wennmalm A. J. Biol. Chem. 1997; 272: 30178-30184Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Several aspects of brain function may depend on cholesterol turnover, including the formation of axons and dendrites during development, neuronal repair and remodeling (13Mahley R.W. Rall Jr., S.C. Annu. Rev. Genom. Hum. Genet. 2000; 1: 507-537Crossref PubMed Scopus (1336) Google Scholar, 14de Chaves E.I.P. Rusinol A.E. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 1997; 272: 30766-30773Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), the formation of new synapses (15Mauch D.H. Nagler K. Schumacher S. Goritz C. Muller E.-C. Otto A. Pfrieger F.W. Science. 2001; 294: 1354-1357Crossref PubMed Scopus (1260) Google Scholar), and learning and memory (16Xu G. Servatius R.J. Shefer S. Tint G.S. O'Brien W.T. Batta A.K. Salen G. Metabolism. 1998; 47: 878-882Abstract Full Text PDF PubMed Scopus (34) Google Scholar). 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Genet. 2000; 1: 507-537Crossref PubMed Scopus (1336) Google Scholar), influences susceptibility to Alzheimer disease (20Corder E.H. Saunders A.M. Strittmatter W.J. Schmechel D.E. Gaskell P.C. Small G.W. Roses A.D. Haines J.L. Pericak-Vance M.A. Science. 1993; 261: 921-923Crossref PubMed Scopus (7294) Google Scholar, 21Holtzman D.M. Bales K.R. Tenkova T. Fagan A.M. Parsadanian M. Sartorius L.J. Mackey B. Olney J. McKeel D. Wozniak D. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2892-2897Crossref PubMed Scopus (731) Google Scholar). There also is evidence that 24(S)-hydroxycholesterol itself may have a discrete biological function in that this oxysterol is a potent ligand for the liver X 1996; PubMed Scopus Google Scholar, J.M. J. D.E. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, B. J. Jr., Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar), a of the that is expressed in including the brain, and that the of in metabolism A. Science. 2001; 294: PubMed Scopus Google Scholar). To into the role of 24(S)-hydroxycholesterol formation in brain cholesterol turnover and the importance of this process, we the and cholesterol 24-hydroxylase enzymes E.G. J.M. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), which a conserved cytochrome P450 enzyme of the L. T. R. O. M.J. R.W. D.W. 1996; PubMed Scopus Google Scholar). Cholesterol 24-hydroxylase and protein are expressed at low levels in the liver and and at higher levels in the brain. and in that 24-hydroxylase is expressed in of the cells of the of the and but not in cells in the of the brain E.G. J.M. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The and protein are expressed in the brain at and 24(S)-hydroxycholesterol is in the on levels in the and during the of and levels of this oxysterol in the brain. these data that cholesterol 24-hydroxylase may be in brain cholesterol metabolism E.G. J.M. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). To the role of cholesterol 24-hydroxylase in brain and cholesterol turnover, we a of mice that not cholesterol 24-hydroxylase. of these that this enzyme an role in cholesterol in the brain. of including enzyme and of of and and J. Russell D.W. A Scholar). A an of that the of the was used to a The from this was by and into to the A the of the gene and a of the of of the that the was from and into a site in the of C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google This the of the cholesterol 24-hydroxylase of the of the gene of This was to an additional site to the To the of the a a of and of the cholesterol 24-hydroxylase gene was from by the a The was and the was To of the at the of A was that of of for of for and for by 16 of for and for the at for and a of for by to The was by and and then into the site of the K.R. PubMed Scopus Google Scholar). To the the the of the to the gene was by and into an site in the the enzyme and of used to the of the in the and the was by and into cells by cells derived from the on cells B. R. E. the Scholar). cells to of a from of and by of a derived from the of and the and into from a contribution from the cells from to derived from the gene the performed the and of these of of and in to and at for in the The three for in and and then in the for 16 at and the in at for 16 and then a a and was by the at the to and a to an and a the in the on a J. Scholar). of bile was for a from that of and A.M. D. The and Metabolism. Scholar). of bile was at for 16 of in of to bile acids. The of the was to acid, and the was to a which was of The was of and a including bile was of This was to a of in of and to an that was of from the of This was and bile acids of from M.A. D.E. J. Lipid Res. Full Text PDF PubMed Google Scholar). The was to a of and in of acid by of at for The was to and converted to S. Breuer O. Lund E. Diczfalusy U. Biochem. 1995; PubMed Scopus Google Scholar). was performed E.G. J. Russell D.W. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google the the used on the at for by of to a of and then to a of An at for the mass the was and at a of acid from and mice used in these studies in in of and at the of the the a low cholesterol equal of wild type and knockout and the during the the of the of the (12Xie Z. Burns D.K. Turley S.D. Dietschy J.M. J. Neuropathol. Exp. Neurol. 2000; 59: 1106-1117Crossref PubMed Scopus (97) Google Scholar). The and of this experimental of Cholesterol in animal was of the and The tissues and and J. Dietschy J.M. J. Clin. Invest. 1995; 95: PubMed Scopus Google Scholar, M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: Full Text Full Text PDF PubMed Google Scholar). The of sterol synthesis in expressed of into sterol of The of synthesis to for whole animal sterol on studies where it was that into the sterol for the biosynthetic pathway these converted to an of cholesterol and expressed the of sterol synthesized of body weight Dietschy J.D. J. Lipid Res. Full Text PDF PubMed Google Scholar, J.M. D.K. J. Lipid Res. Full Text PDF PubMed Google Scholar). of Cholesterol and from the and the major and Cholesterol was from tissues and by an The from and to the whole animal cholesterol and these expressed of cholesterol of body weight M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: Full Text Full Text PDF PubMed Google Scholar, S.D. Dietschy J.M. Full Text PDF PubMed Scopus Google Scholar). of was from mice over and and by an and and these expressed of sterol excreted of body total sterol was calculated the of these for animal M. Russell D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; 39: Full Text Full Text PDF PubMed Google Scholar). of cholesterol was Lipid in of by protein a of from the and cholesterol and the for cholesterol and a for data in calculated The was used to the of In the and an a that was at this from lacking the cholesterol 24-hydroxylase gene by in cells the in The was to a of of the gene a the of the protein to the E. enzyme Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The from was to place of the gene the of in the gene and to of that cholesterol 24-hydroxylase Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P. C. A. M. J. 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Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), the liver, brain, and unchanged in the mutant was the of body weight by of these tissues of and and of whole animal and of whole animal of liver weight of liver of body of brain weight of brain of body of cholesterol cholesterol cholesterol absorption cholesterol in whole animal and animal cholesterol liver cholesterol cholesterol in the that was from that in the mice of the in the that was from that in the mice of the in a new The of the on the of cholesterol 24-hydroxylase was by and of 24(S)-hydroxycholesterol A of was in the brain of wild type mice the of in an E.G. J.M. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google was also not The levels of reduced by in mice and to levels in mice. The protein an weight of The amount of protein was reduced in mice for the gene and was in for the mutant these data that the of the 24-hydroxylase and was used to the of of 24-hydroxylase enzyme on levels of 24(S)-hydroxycholesterol in the and brain. 24(S)-hydroxycholesterol in wild type mice and this was reduced to in mice The of 24(S)-hydroxycholesterol in the of wild type mice was and was in knockout mice of this levels of the oxysterol in wild type mice and E.G. J.M. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), but there was during this in the knockout mice These data that the enzyme was responsible for the synthesis of of 24(S)-hydroxycholesterol in the The biosynthetic of the 24(S)-hydroxycholesterol in the of the mice was not but may from the sterol which and E. Bjorkhem I. C. K. 1993; PubMed Scopus Google Scholar). The of the latter not wild type and mice not The of 24(S)-hydroxycholesterol in the of wild type mice was of protein but was of in the mice There was a in 24(S)-hydroxycholesterol levels to of protein in the of wild type mice at of but the remained low of in the mutant mice. in the levels of this oxysterol in the mutant mice the data of that the 24-hydroxylase enzyme was responsible for the synthesis of of the 24(S)-hydroxycholesterol in the brain. The used to the cholesterol 24-hydroxylase gene the E. gene the of in the gene To the of the 24-hydroxylase gene during development, of and for enzyme An was in cells of the on in The amount of in the from of the and of the also at and in these to and In contrast to the of in the and in cells of the the of the during this and remained the of the brain wild type a and The of the gene was in of the enzyme was limited to of in the brain of these of in the and of the of the in the of this not The of cells well that in the whole cholesterol 24-hydroxylase is expressed in a of the brain of the it is in the E.G. J.M. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In the liver, 24(S)-hydroxycholesterol is converted into intermediates in the bile acid biosynthetic pathway by the of several enzymes (1Russell D.W. Annu. Rev. Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1406) Google Scholar). To the of cholesterol 24-hydroxylase for whole body sterol balance and bile acid several of cholesterol and oxysterol metabolism These in cholesterol cholesterol amount of cholesterol the wild type and mutant mice The of bile acid by the of in the in mice the of sterol unchanged mice of and the total of in these mice was the acid in knockout from of wild type and the of cholic acid to muricholic acid and in the wild type and knockout major in bile acid cholesterol levels by an not wild type and a was in the mutant of by protein that the of cholesterol and of not the low density LDL, and also not mice of A reduced of was in the mutant but the was not the cholesterol was in but not knockout mice this was calculated and to a body weight of The cholesterol of tissues and the by and to be similar in wild type and 24-hydroxylase knockout mice body cholesterol, calculated the of the cholesterol also was unchanged and These data that steady-state levels of cholesterol in tissues of the mutant including the brain, despite the of cholesterol 24-hydroxylase. The synthesis of cholesterol in tissues was The only in de novo synthesis and knockout mice was in the brain, where the of of was reduced by from to of body cholesterol synthesis was not the brain cholesterol synthesis for only of the The of of the cholesterol 24-hydroxylase gene on sterol metabolism are in this The major of the is that of in a in cholesterol synthesis in the brain. cholesterol synthesis being reduced by ∼40% in the knockout the steady-state of cholesterol in the brain was unchanged relative to wild type mice. These that cholesterol 24-hydroxylase is responsible for the turnover of at least 40% of brain cholesterol, and that in the is by the synthesis of new cholesterol by an The synthesis of 24(S)-hydroxycholesterol in the brain and role in the turnover of cholesterol in this by Bjorkhem and in (3Lutjohann D. Breuer O. Ahlborg G. Nennesmo I. Siden A. Diczfalusy U. Bjorkhem I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9799-9804Crossref PubMed Scopus (569) Google Scholar), and an increasing body of evidence these I. Diczfalusy U. Lutjohann D. PubMed Scopus Google Scholar). The in mice these and a of the contribution of sterol 24-hydroxylase enzyme to cholesterol in the brain. Cholesterol is but not in cholesterol mice. a in de novo cholesterol a in turnover of the this that there be other that of cholesterol from the brain. Several have been proposed to a role in the turnover of cholesterol in the including of the low density lipoprotein J. Annu. Rev. Biochem. 2002; PubMed Scopus Google Scholar), the ligand for these apolipoprotein E (13Mahley R.W. Rall Jr., S.C. Annu. Rev. Genom. Hum. Genet. 2000; 1: 507-537Crossref PubMed Scopus (1336) Google Scholar), and other oxysterol biosynthetic enzymes like sterol in knockout mice that of the the apolipoprotein E gene not cholesterol biosynthetic in the (12Xie Z. Burns D.K. Turley S.D. Dietschy J.M. J. Neuropathol. Exp. Neurol. 2000; 59: 1106-1117Crossref PubMed Scopus (97) Google Scholar). sterol E. G. S. D. D. and J. M. and cholesterol and D. have in brain cholesterol additional enzymes in cholesterol from the brain to be Although steady-state levels of cholesterol in the whole brain unchanged in the mutant mice of cholesterol may and wild type cholesterol is in including and K. Science. 2002; PubMed Scopus Google Scholar), which for and the levels of cholesterol in these the mutant and wild type mice to be but the of in the cholesterol mice suggests a role for the enzyme in cholesterol Cholesterol 24-hydroxylase is expressed in but not of the and and brain E.G. J.M. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and not to be in the of cells in the of Science. Scholar). that a similar of cells in the brain are and from the data that of these cells cholesterol 24-hydroxylase, then it be calculated that of cells in the brain are responsible for ∼40% of cholesterol turnover in this This is on the of cholesterol in the brain, which is in the by than in neuronal and other (12Xie Z. Burns D.K. Turley S.D. Dietschy J.M. J. Neuropathol. Exp. Neurol. 2000; 59: 1106-1117Crossref PubMed Scopus (97) Google Scholar), and it the role of cholesterol turnover in this of cells and the of in the mice. of cholesterol 24-hydroxylase in by and the knockout mice to in which and balance in and to a in these wild type and knockout mice. these data are the of the cholesterol 24-hydroxylase in of the and cells of the E.G. J.M. Russell D.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), which are in of Science. Scholar), it that cholesterol turnover an role in the of the A role also is in that of the mice in are of and not be for a of by that cholesterol 24-hydroxylase. Cholesterol turnover in the and brain may have a role in the in remodeling for higher like learning and which may in sterol neuronal other pathways may for of cholesterol 24-hydroxylase in the mutant mice. A in circulating levels and the total of cholesterol was in the mice and but the mechanism this of the was not The of the 24(S)-hydroxycholesterol is a ligand for the liver X A. Science. 2001; 294: PubMed Scopus Google Scholar), a that the of in It is that the of cholesterol in the knockout mice may be in to the of 24(S)-hydroxycholesterol and a in In this knockout mice have reduced levels of circulating Turley S.D. 1998; 93: Full Text Full Text PDF PubMed Scopus Google Scholar, S. G. P. D. Diczfalusy U. M. B. Bjorkhem I. S. J. Clin. Invest. 2001; PubMed Scopus Google Scholar). mice that and brain including and L. K. Andersson S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). This was not in mice of this genotype L. K. Andersson S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). In we have not brain in cholesterol mice. and E. G. The of the of the of in these mice. In the studies that the synthesis of 24(S)-hydroxycholesterol and secretion from the brain a mechanism of cholesterol turnover in this an in the biosynthetic cholesterol 24-hydroxylase, cholesterol in the by the de novo synthesis of cholesterol. these may a to the of of cholesterol synthesis on in which in cholesterol metabolism are thought to a causative studies on of cholesterol 24-hydroxylase the of type and Alzheimer disease and in the role of cholesterol turnover in We and for for of knockout for a the at for and and for of the