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

Erik G. Lund {ddagger} §, Chonglun Xie ¶, Tiina Kotti {ddagger}, Stephen D. Turley ¶, John M. Dietschy ¶ and David W. Russell {ddagger} ||

From the {ddagger}Department of Molecular Genetics and Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, April 2, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The major site for the turnover of cholesterol1 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 (1). 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 (2); 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 (36). Once in the circulation, 24(S)-hydroxycholesterol is cleared by the liver and therein converted into 7{alpha}-hydroxylated intermediates in the bile acid synthetic pathway by a dedicated enzyme (79). 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 (10). 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 (11), and 1.37 mg/day/kg of body weight in mice (12). 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 (4).

Several aspects of brain function may depend on cholesterol turnover, including the formation of axons and dendrites during development, neuronal repair and remodeling (13, 14), the formation of new synapses (15), and learning and memory (16). In addition to these proposed roles, an increasing body of experimental evidence suggests that cholesterol levels in the brain may influence the cleavage of the amyloid precursor protein into {beta}-amyloid, the causative agent of Alzheimer disease (1719), and it is well established that the genotype of the apolipoprotein E locus, which encodes a cholesterol transport protein expressed in the brain (13), influences susceptibility to Alzheimer disease (20, 21). 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 receptor (2224), a member of the nuclear hormone receptor family that is expressed in many tissues, including the brain, and that activates the transcription of genes involved in lipid metabolism (25).

To gain further insight into the role of 24(S)-hydroxycholesterol formation in brain cholesterol turnover and the physiological importance of this process, we cloned cDNAs encoding the mouse and human cholesterol 24-hydroxylase enzymes (26), which encoded a highly conserved cytochrome P450 enzyme of the endoplasmic reticulum designated CYP46A1 (27). Cholesterol 24-hydroxylase mRNA and protein are expressed at low levels in the liver and testis and at much higher levels in the adult brain. Immunohistochemical and in situ mRNA hybridization experiments indicate that 24-hydroxylase is expressed in pyramidal neurons of the mouse cerebral cortex, Purkinje cells of the cerebellum, neurons of the thalamus, dentate gyrus, and hippocampus but not in support cells or in the white matter of the adult brain (26). The mRNA and protein are both expressed in the brain at birth, and 24(S)-hydroxycholesterol is detected in the serum on postnatal day 1. 24(S)-Hydroxycholesterol levels in the serum peak between days 1 and 21 during the time of myelination, and thereafter, levels of this oxysterol gradually increase in the brain. Together these data imply that cholesterol 24-hydroxylase may be involved in brain cholesterol metabolism (26).

To determine the role of cholesterol 24-hydroxylase in brain development and cholesterol turnover, we produced a line of mice that do not express cholesterol 24-hydroxylase. Characterization of these animals demonstrated that this enzyme normally plays an important role in cholesterol metabolism, specifically in the brain.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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). A bacteriophage {lambda} clone containing an insert of mouse genomic DNA (129S6/SvEv strain) that spanned the 5'-flanking region of the Cyp46a1 locus was used to assemble a targeting vector. The insert from this vector was released by NotI digestion and cloned into pBluescript-SK(+) to produce the plasmid pBS-mg24.2. A 2.2-kb DNA fragment containing the immediate 5'-flanking region of the gene and a portion of the 5'-untranslated region of exon 1 (14 base pairs upstream of the ATG codon that specifies the initiator methionine) was isolated from pBS-mg24.2 and ligated into a SalI site in the pBS-tau-lacZ plasmid (a gift of C. Callahan (29)). This ligation positioned the 3'-end of the cholesterol 24-hydroxylase genomic DNA fragment (the "short arm") immediately upstream of the ATG codon of the bovine tau-Escherichia coli {beta}-galactosidase (lacZ) fusion gene of pBS-tau-lacZ. This intermediate plasmid was modified further to contain an additional SalI site just 3' to the tau-lacZ fusion gene.

To isolate the "long arm" of the targeting vector, a DNA fragment encompassing a portion of intron 1, exon 2, and intron 2 of the cholesterol 24-hydroxylase gene was amplified from pBS-mg24.2 by the polymerase chain reaction using a TaKaRa LA PCR kit, version 2 (Takara Bio, Shiga, Japan). The 5'-primer was 5'-AAAGGATCCTCAGAGCCAGCGCCCAGACGGGGGTCCA-3', and the 3'-primer was 5'-AAAGGATCCCCTCAGGACCAGGAGAGGTGGTCTT-3'. To facilitate cloning of the amplified DNA, BamHI sites were placed at the 5'-ends of both primers. A thermocycler program was employed that consisted of 1 cycle of 94 °C for 1 min, 14 cycles of 98 °C for 20 s, and 72 °C for 20 min, followed by 16 cycles of 98 °C for 20 s and 72 °C for 20 min with the extension time at 72 °C prolonged for 5 s each cycle, and a final elongation step of 72 °C for 10 min followed by cooling to 4 °C. The ~8-kb, amplified DNA product was purified by agarose gel electrophoresis and recovered, digested with BamHI, and then ligated into the BamHI site of the plasmid pPolIIshort-neobPA-HSVTK (30). 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." Restriction enzyme mapping and DNA sequencing of ligation junctions were used to confirm the orientations of the various DNA fragments in the targeting vector (see Fig. 1), and thereafter the plasmid DNA was linearized by SalI digestion and introduced into embryonic stem cells by electroporation.



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FIG. 1.
Strategy for inactivating the Cyp46a1 gene in mouse. A schematic of the 5'-end of the Normal Allele is shown illustrating the first two exons (blocks) and introns (thin connecting lines) of the cholesterol 24-hydroxylase gene. Diagonal hashed lines indicate coding regions within the exons. The Targeting Vector for homologous recombination contained two copies of a viral thymidine kinase gene (TK), a short arm corresponding to an ~2.2-kb fragment of DNA derived from the 5'-flanking region of the Cyp46a1 gene and the non-coding region of exon 1, a segment of the bovine tau gene, the E. coli {beta}-galactosidase gene (lacZ), a neomycin resistance cassette (Neo), and an ~8-kb-long arm derived from intron 1, exon 2, and intron 2 of the Cyp46a1 gene. Homologous recombination in embryonic stem cells was expected to produce a Mutant Allele in which the coding region of exon 1 was replaced with the tau-lacZ fusion gene and the neomycin resistance cassette. The locations of three EcoRI sites (R1) and a probe (black box) used for genotyping are indicated on the schematic.

 

Mouse AB-1 embryonic stem cells derived from the 129S6/SvEv strain were cultured on STO feeder cells as described (31). Approximately 3 x 107 cells were subjected to electroporation with 100 µg/ml of SalI-linearized targeting vector using a device ("CellPorator") from Invitrogen (Bethesda, MD). Cells were seeded onto feeder layers of irradiated (10,000 rads) STO cells. After selection with 210 µg/ml G418 and 0.5 µM gancyclovir, recombinant clones were identified by Southern blotting of genomic DNA digested with EcoRI using a hybridization probe derived from the 5'-flanking region of Cyp46a1 (see Fig. 1). From ~800 stem cell colonies screened, two independent lines (5G3 and 7G5) with the desired homologous recombination event were identified, and their predicted genotypes were confirmed using PCR. Both embryonic stem cell lines were injected into blastocysts isolated from C57Bl/6J females, yielding 12 chimeric males whose coat color (agouti) indicated a contribution from the stem cells ranging from 20 to 95%. Of these, two animals (both derived from clone 5G3) transmitted the disrupted Cyp46a1 gene through the germ line. All experiments reported here were performed with the mixed strain (C57Bl/6J;129S6/SvEv) descendants (F2 and subsequent generations) of these Cyp46a1+/ animals.

{beta}-Galactosidase Staining of Embryos—Mouse embryos of gestational ages 11.5, 12.5, and 13.5 days were dissected in phosphate-buffered saline, transferred to 10% (v/v) neutral buffered formalin (Sigma Diagnostics, St. Louis, MO), and incubated at 4 °C for 1 h in the dark. The embryos were rinsed three times for 15 min each in 10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 2 mM MgCl2, 5 mM EGTA, 0.01% (w/v) deoxycholate, and 0.02% (v/v) Nonidet P40, and then stained in the dark for 16 h at 37 °Cin10mM Na2HPO4,2mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 5 mM EGTA, 0.01% (w/v) deoxycholate, 0.02% (v/v) Nonidet P-40, 2 mM MgCl2, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside. Following staining, the embryos were again incubated in 10% (v/v) neutral buffered formalin at 4 °C for 16 h and then photographed under a dissecting microscope (Leica, Wild MZ8) with a Nikon 35-mm camera and Kodak Ektachrome 160T film. Cell type-specific staining was determined by sectioning the fixed embryos at ~5-µm thickness, transferring the sections to glass slides, staining with nuclear fast red, and photographing using a Nikon (Melville, NY) Eclipse E1000 M microscope linked to an Optronics Engineering (Goleta, CA) DEI750 video camera and a computer running the Image Pro Plus, version 3.0 software (Media Cybernetics, Silver Spring, MD). Cell types in the developing cerebral cortex were identified based on a standard reference text (32).

Chemical Analysis of Biliary Bile Acids—Gallbladder bile was processed for gas chromatography-mass spectrometry using a protocol modified from that of Lawson and Setchell (33). Briefly, 10 µl of gallbladder bile was incubated at 37 °C for 16 h with 10 units of choloylglycine hydrolase (C-4018, Sigma, St. Louis, MO) in 0.5 ml of 0.2 M sodium phosphate buffer, pH 5.6, to deconjugate bile acids. The pH of the reaction was adjusted to ~2.5 with 1 M phosphoric acid, and the sample was applied to a 100-mg Isolute MF-C18 cartridge column (International Sorbent Technology Ltd., Mid Glamorgan, UK), which was equilibrated with 2 ml of 0.2 M sodium phosphate buffer, pH 2.5. The column was washed with 1 ml of water, and a lipid fraction, including deconjugated bile acids, was eluted with 1 ml of methanol. This eluate was taken to dryness under a stream of nitrogen, redissolved in 0.5 ml of chloroform, and applied to an Isolute NH2 column (International Sorbent Technology Ltd.) that was equilibrated with 2 ml of hexane. Neutral lipids were eluted from the column with 2 ml of chloroform-isopropanol (2:1, v/v). This fraction was discarded, and bile acids were eluted with 2 ml of acetic acid/diethyl ether/methanol (2:50: 50, v/v) (modified from Ref. 34). The eluate was taken to dryness under a stream of nitrogen and resuspended in 10 µl of methanol. Free acid groups were derivatized by treatment with 50 µl of trimethylsilyl-diazomethane at room temperature for 30 min. The sample was taken to dryness under nitrogen, and hydroxyl groups were converted to trimethylsilyl ethers (35).

Gas chromatography-mass spectrometry was performed (36) with the following modifications: the program used on the gas chromatograph involved elution at 60 °C for 1 min, followed by elution with temperature gradients of 40 °C/min to a final temperature of 220 °C and then 2 °C/min to a final temperature of 280 °C. An elution at 280 °C for 15 min ended the run. For mass spectrometry, the quadrupole was scanned between m/z 100 and m/z 650 at a rate of 1 scan/1.5 s. Bile acid standards were obtained from Steraloids (Wilton, NH).

Animals and Diets—The mice used in these studies were housed in plastic colony cages in rooms with alternating 12-h periods of light and dark. After weaning at the end of the third week, the animals were fed ad libitum a low cholesterol pelleted diet (7001, Harlan Teklad, Madison, WI) until they were studied. Experimental groups contained nearly equal numbers of wild type and knockout mice, and the various measurements were carried out during the fed state near the end of the dark phase of the light cycle (12). The Institutional Animal Care and Research Advisory Committee of this institution approved all experimental protocols.

Measurement of Cholesterol Synthesis Rates in Vivo—Each animal was injected intraperitoneally with ~50 mCi of 3H-labeled water. One hour later the animals were anesthetized and exsanguinated. The tissues and remaining carcass were saponified, and digitonin-precipitable sterols were isolated as described previously (37, 38). The rates of sterol synthesis in each tissue were expressed as nanomoles of 3H-water incorporated into sterol per hour per gram of tissue. The organ rates of synthesis were added to give values for whole animal sterol synthesis. Based on previous studies where it was determined that 0.69 3H atoms were incorporated into the sterol molecule for each carbon atom entering the biosynthetic pathway as acetyl-CoA, these rates were converted to an equivalent milligram quantity of cholesterol and expressed as the milligrams of sterol synthesized per day per kilogram of body weight (39, 40).

Measurement of Tissue Cholesterol Concentrations—Animals were anesthetized and exsanguinated from the inferior vena cava, and the major organs and remaining carcass were saponified. Cholesterol was extracted from tissues with petroleum ether and quantitated by gas chromatography using stigmasterol as an internal standard. The values from each organ and remaining carcass were added to give the whole animal cholesterol pools, and these values were expressed as milligrams of cholesterol per kilogram of body weight (38, 41).

Measurement of Fecal Sterol Excretion—Stool was collected from individually housed mice over 72 h, dried, weighed, and ground. Acidic and neutral sterols were measured by an enzymatic method and gas chromatography, respectively, and these values were expressed as milligrams of sterol excreted per day per kilogram of body weight. Fecal total sterol excretion was calculated as the sum of these two values for each animal (38).

Measurement of Plasma Lipids—Total plasma cholesterol concentration was measured enzymatically (kit 1127771, Roche Applied Science, Indianapolis, IN). Lipid distribution profiles were determined in 200-µl aliquots of pooled plasma separated by fast protein liquid chromatography using a Superose 6 column. Fractions of 150 µl were collected from the column, and their cholesterol and triglyceride concentrations were determined enzymatically using the above kit for cholesterol and a separate kit for triglycerides (Sigma, INFINITYTM triglyceride reagent).

Calculations—The data in all experiments were calculated as means ± 1 S.E. The Student's unpaired t test was used to compare the various sets of data. In the figures and table, an asterisk indicates a value that was significantly different (p ≤ 0.05) at this level from its appropriate control value.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice lacking the cholesterol 24-hydroxylase gene (Cyp46a1) were produced by homologous recombination in embryonic stem cells using the strategy outlined in Fig. 1. The targeting vector was constructed to replace a portion of exon 1 of the Cyp46a1 gene with a chimeric cDNA encoding the amino terminus of the bovine tau protein fused to the E. coli {beta}-galactosidase enzyme (29). The replacement mutation arising from homologous recombination was predicted to place expression of the tau-{beta}-galactosidase gene under the control of regulatory sequences in the Cyp46a1 gene and thus to facilitate histochemical detection of neurons that express cholesterol 24-hydroxylase (29, 42). Electroporation of the linearized targeting vector into the AB-1 line of 129S6/SvEv embryonic stem cells followed by selection and screening identified two independent clones out of 800 with the desired homologous recombination event. Injection of these clones into C57Bl/6J blastocysts produced 12 chimeric males that had contributions from the embryonic stem cells ranging from 20 to 95% based on the amount of agouti color in the animal's coats. Of these mice, two transmitted the disrupted Cyp46a1 gene through the germ line. The mutant allele was inherited in Mendelian fashion and expected numbers of wild type, heterozygous, and homozygous offspring were born and survived to adulthood. Equal numbers of male and female pups were obtained in crosses between Cyp46a1+/ males and females. Fertility and fecundity were normal in mice heterozygous and homozygous for the introduced mutation.

Mice lacking the cholesterol 24-hydroxylase gene were outwardly normal. No differences were observed between the growth rates of wild type and knockout mice (data not shown), and the weights of the mutant mice did not differ significantly from those of age-matched controls (Table I). Unlike sterol 27-hydroxylase-deficient mice, which exhibited enlarged livers and adrenal glands (43), the liver, brain, and adrenal gland weights were unchanged in the mutant Cyp46a1/ mice, as was the proportion of body weight occupied by each of these tissues (Table I).


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TABLE I
Characterization of male and female Cyp46a1+/+ and Cyp46a1-/- mice

Seven-week-old mice were maintained on the basal rodent diet after weaning. Values represent the mean ± 1 S.M. for 7-10 animals in each group.

 

The effect of the mutation on the expression of cholesterol 24-hydroxylase was determined by RNA blotting, immunoblotting, and measurement of 24(S)-hydroxycholesterol levels. A CYP46A1 mRNA of ~2.4 kb was detected in the brain of wild type mice (Fig. 2A). With longer exposures, the less abundant mRNA of ~3.0 kb reported in an earlier study (26) was detected also (data not shown). The levels of both mRNAs were reduced by half in heterozygous Cyp46a1+/ mice and to undetectable levels in homozygous Cyp46a1/ mice. The CYP46A1 protein migrated with an apparent molecular weight of ~52,000 (Fig. 2B). The amount of CYP46A1 protein was reduced in mice heterozygous for the disrupted Cyp46a1 gene and was undetectable in animals homozygous for the mutant allele. Together, these data indicated that the introduced mutation eliminated expression of the 24-hydroxylase mRNA and protein.



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FIG. 2.
Cholesterol 24-hydroxylase mRNA and protein, and 24(S)-hydroxycholesterol levels in mice of different Cyp46a1 genotypes. A, aliquots of poly(A)+ RNA (5 µg) from the brains of six animals of the indicated Cyp46a1 genotype were separated by agarose gel electrophoresis and analyzed by blot hybridization using radiolabeled probes derived from the cholesterol 24-hydroxylase and cyclophilin cDNAs. B, microsomal membrane proteins were isolated from the brains of six mice with different Cyp46a1 genotypes and aliquots (30 µg) were subjected to immunoblotting using a polyclonal antiserum raised against a peptide sequence (residues 254–270) in the mouse CYP46A1 protein (26). Staining of the membrane with Ponceau S indicated that equal amounts of microsomal protein were electrophoresed in each lane. C, concentrations of 24(S)-hydroxycholesterol were determined in the serum of Cyp46a1 wild type and knockout mice of the indicated ages by gas chromatography-mass spectrometry. The histogram bars indicate the means ± S.E. for values determined in 4–20 animals of each genotype and age. D, concentrations of 24(S)-hydroxycholesterol in the brains of mice of differing Cyp46a1 genotypes. Animal numbers, methods, and key to shading of histogram bars is as in C.

 

Isotope dilution gas chromatography-mass spectrometry was used to measure the effect of loss of 24-hydroxylase enzyme activity on levels of 24(S)-hydroxycholesterol in the serum and brain. Serum 24(S)-hydroxycholesterol concentrations in suckling, 15-day-old wild type mice averaged 65.8 ng/ml, and this level was reduced to 11.4 ng/ml in age-matched Cyp46a1/ mice (Fig. 2C). The concentration of 24(S)-hydroxycholesterol in the serum of sexually mature, adult 90-day-old wild type mice was 20.1 ng/ml and was again lower in knockout mice of this age (7.4 ng/ml). Serum levels of the oxysterol declined in wild type mice between days 15 and 90 as expected (26), but there was little if any decline observed during this period in the knockout mice (Fig. 2C). These data indicated that the CYP46A1 enzyme was responsible for the synthesis of ~60–80% of 24(S)-hydroxycholesterol present in the serum. The biosynthetic origin of the residual 24(S)-hydroxycholesterol in the serum of the Cyp46a1/ mice was not determined but may arise from the CYP27A1 sterol 27-hydroxylase, which can produce 24(S)-, 25-, and 27-hydroxycholesterol (44). The serum concentrations of the latter two oxysterols were not significantly different between wild type and 24-hydroxylase-deficient mice (data not shown).

The level of 24(S)-hydroxycholesterol in the brains of 15-day-old wild type mice was 196 ng/mg of protein but was substantially lower (4.4 ng/mg of protein) in the Cyp46a1/ mice (Fig. 2D). There was a slight increase in 24(S)-hydroxycholesterol levels to 234 ng/mg of protein in the brains of wild type mice at 90 days of age, but again, the level remained low (3.2 ng/mg of protein) in the mutant mice. Because no significant changes in the serum levels of this oxysterol were observed with age in the mutant mice (Fig. 2C), the data of Fig. 2D indicated that the 24-hydroxylase enzyme was responsible for the synthesis of almost all (~98–99%) of the 24(S)-hydroxycholesterol present in the brain.

The strategy used to knock out the cholesterol 24-hydroxylase gene placed the E. coli {beta}-galactosidase gene (lacZ) under the control of transcriptional regulatory sequences in the Cyp46a1 gene (Fig. 1). To elucidate the expression pattern of the 24-hydroxylase gene during development, mouse embryos of different Cyp46a1 genotypes and gestational ages were stained for {beta}-galactosidase enzyme activity (Fig. 3). An intense blue stain was detected in cells of the spinal cord beginning on day 11.5 (E11.5) in embryos viewed laterally (Fig. 3A, panels a–c) or dorsally (panels d–f). The amount of staining in the spinal cord decreased gradually from E11.5 through E13.5. Cells of the midbrain and hindbrain regions of the developing central nervous system also exhibited intense staining at E11.5, and again, expression in these regions appeared to decrease between E11.5 and E13.5 (panels d–i). In contrast to the gradual decrease of staining with age in the midbrain and hindbrain, {beta}-galactosidase activity in cells of the telencephalon (forebrain), the anlage of the cerebral cortex, intensified during this time period, and remained concentrated near the midline of the developing brain (panels g–i). Control experiments with wild type embryos revealed no {beta}-galactosidase staining (Fig. 3B, panels a and b).



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FIG. 3.
{beta}-Galactosidase staining in Cyp46a1+/+ and Cyp46a1/ embryos. Mice of the indicated gestational age were stained for {beta}-galactosidase enzyme activity as described under "Experimental Procedures" and photographed under a dissecting microscope (Leica, Wild MZ8) with a Nikon 35-mm camera and Kodak Ektachrome 160T film. A, panels a–i, staining in Cyp46a1/ embryos. Bars, 1 mm. B, panels a and b, staining in Cyp46a1+/+ embryos. Bars, 1 mm.

 

The cell type-specific expression pattern of the lacZ gene was examined next in E13.5 embryos. Expression of the tau-lacZ enzyme was limited to subpopulations of neurons in the developing brain of these embryos (Fig. 4). Cell bodies of dividing neurons in the neuroepithelium and post-mitotic neurons of the primordial plexiform layer of the telencephalon were stained, whereas post-mitotic migrating neurons in the intermediate/subventricular zone of this region did not stain (Fig. 4, panels e–g). The overall distribution of positively stained cells corresponded well with that observed in the whole mount embryos. Thus, cholesterol 24-hydroxylase is expressed in a restricted set of neurons within the brain of the developing mouse embryo, as it is in the adult (26).



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FIG. 4.
{beta}-Galactosidase staining in histological sections from an embryonic day 13.5 Cyp46a1/ embryo. The animal shown at the left was stained in whole mount for {beta}-galactosidase enzyme activity, sectioned at ~5 µm, transferred to glass slides, stained with nuclear fast red, and subjected to photomicroscopy (magnification = 0.5x) as indicated under "Experimental Procedures." The approximate locations of the four sections shown in panels a–d are indicated in the intact embryo at the left (bar = 1 mm). Cells judged to be neurons in the telencephalon (b), hindbrain (b), cerebellar primordium (c), and choroid plexus (d) stained positive for {beta}-galactosidase. In contrast, most neurons of the diencephalon (a), which is the primordium of the thalamus, and the midbrain (c), were negative. When examined at higher power (e, x20; f and g, x40), staining in the telencephalon is localized to the cell bodies of dividing neurons in the neuroepithelium adjacent to the ventricle (e and f) and those of the primordial plexiform layer (e and g) but not in the migrating neurons of the intermediate/subventricular zone (e–g).

 

In the liver, 24(S)-hydroxycholesterol is converted into intermediates in the bile acid biosynthetic pathway by the actions of several enzymes (1). To determine the consequences of cholesterol 24-hydroxylase loss for whole body sterol balance and bile acid synthesis, several parameters of cholesterol and oxysterol metabolism were examined. These experiments revealed no differences in food consumption, dietary cholesterol intake, intestinal cholesterol absorption, or amount of dietary cholesterol absorbed between the wild type and mutant mice (Table I).

The rates of bile acid synthesis, as reflected by the excretion of acidic sterols in the feces, were no different in Cyp46a1/ mice (Fig. 5, top panel). Similarly, the rates of neutral sterol excretion were unchanged between mice of different Cyp46a1 genotypes (Fig. 5, middle panel), and the total excretion of sterols in these mice (acidic plus neutral sterols) was the same (Fig. 5, bottom panel). Bile acid pool sizes in 3-month-old knockout males were no different from those of wild type controls (Cyp46a1+/+, 19 ± 1.0 µmol/mouse; Cyp46a1/, 19.5 ± 1.5 µmol/mouse), and the ratios of cholic acid to muricholic acid were 1.3 and 1.4 in the wild type and knockout mice, respectively, indicating no major differences in bile acid composition.



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FIG. 5.
Effect of gender on external sterol balance in wild type and Cyp46a1/ mice. All animals used in this experiment were 7 weeks of age. 72-h fecal samples were collected, and acidic (panel A) and neutral (panel B) sterols were determined by enzymatic assay and gas chromatography, respectively. The acidic and neutral sterol weights were added together to give values for total fecal sterol excretion (panel C). Means ± S.E. are shown for 10–12 animals of each genotype.

 

Plasma cholesterol levels as determined by an enzymatic method were not significantly different between wild type and Cyp46a1/ mice, although a trend toward lower values was detected in the mutant animals (Fig. 6, inset). Analysis of plasma lipoproteins by fast protein liquid chromatography showed that the distribution of cholesterol (Fig. 6), and of triglycerides (data not shown), among the very low density lipoprotein, LDL, and HDL fractions also were not different between mice of different Cyp46a1 genotypes. A slightly reduced level of HDL was detected in the mutant mice, but the difference was not statistically significant. For unknown reasons, the plasma cholesterol pool was significantly lower in male but not female knockout mice when this parameter was calculated and normalized to a constant body weight of 1 kg (Table I).



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FIG. 6.
Distribution of plasma cholesterol in the major lipoprotein fractions of male Cyp46a1+/+ and Cyp46a1/ mice. All animals used in these experiments were 7 weeks of age. Plasma was pooled from five to six animals of each genotype and lipoproteins were separated by fast protein liquid chromatography on a Superose 6 column. The inset shows total plasma cholesterol concentrations expressed as milligrams/dl; means ± S.E. are shown for 10–12 animals of each genotype.

 

The cholesterol contents of ten different tissues and the remaining carcass were determined by gas chromatography and found to be similar in wild type and 24-hydroxylase knockout mice (Fig. 7). Whole body cholesterol, calculated as the sum of the tissue cholesterol concentrations, also was unchanged (Fig. 7, inset, and Table I). These data indicated that steady-state levels of cholesterol were maintained in all tissues of the mutant mice, including the brain, despite the elimination of cholesterol 24-hydroxylase.



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FIG. 7.
Tissue cholesterol concentrations and whole animal cholesterol pools in male Cyp46a1+/+ and Cyp46a1/ mice. All animals used in these experiments were 7 weeks of age. The major organs and remaining carcass were saponified. Cholesterol concentrations were measured by gas chromatography and expressed as milligrams of sterol per gram of wet weight of tissue. The inset shows the cholesterol pools in whole animals expressed as milligrams of sterol per kilogram of body weight. Means ± S.E. are shown for 6–8 animals of each genotype.

 

The synthesis of cholesterol in 13 tissues was examined next. The only significant difference in de novo synthesis between control and knockout mice was in the brain, where the rate of production per gram of tissue was reduced by 40%, from 171 ± 6 to 105 ± 4 nmol/h/g of tissue. Whole body cholesterol synthesis was not significantly different between the two groups (Fig. 8, inset), because brain cholesterol synthesis typically accounts for only ~1% of the total.



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FIG. 8.
Tissue and whole animal cholesterol synthesis rates in Cyp46a1+/+ and Cyp46a1/ mice. All animals used in these experiments were 7 weeks of age. One hour after each animal was injected with 50 mCi of 3H2O, the major organs and remaining carcass were saponified. The rates of cholesterol synthesis were determined and expressed as nanomoles of 3H2O incorporated into sterols per hour per gram of tissue. The inset shows whole animal cholesterol synthesis rates calculated as mg of sterol synthesized per day per kilogram of body weight. Means ± S.E. are shown for 6–8 animals of each genotype. The asterisk identifies the value in Cyp46a1/ animals that was significantly different (p < 0.05, Student's t test) from that in the Cyp46a1+/+ animals.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of inactivation of the mouse cholesterol 24-hydroxylase gene (Cyp46a1) on sterol metabolism are reported in this paper. The major finding of the study is that inactivation of Cyp46a1 results in a selective reduction in cholesterol synthesis in the brain. Despite cholesterol synthesis being reduced by ~40% in the knockout mice, the steady-state level of cholesterol in the brain was unchanged relative to wild type mice. These results indicate that cholesterol 24-hydroxylase is responsible for the turnover of at least 40% of brain cholesterol, and that in the enzyme's absence, homeostasis is maintained by decreasing the synthesis of new cholesterol by an equivalent amount.

The synthesis of 24(S)-hydroxycholesterol in the brain and its potential role in the turnover of cholesterol in this tissue were initially reported by Bjorkhem and colleagues in 1996 (3), and an increasing body of evidence supports these original findings (45). The experiments in Cyp46a1/ mice extend these observations and provide a quantitative measure of the contribution of sterol 24-hydroxylase enzyme to cholesterol catabolism in the brain.

Cholesterol biosynthesis is decreased but not eliminated in cholesterol 24-hydroxylase-deficient mice. Inasmuch as a decrease in de novo cholesterol biosynthesis reflects a decrease in turnover of the sterol, this result indicates that there must be other mechanisms that allow removal of cholesterol from the brain. Several candidate proteins have been proposed to play a role in the turnover of cholesterol in the central nervous system, including members of the low density lipoprotein (LDL) receptor family (46), the common ligand for these receptors, apolipoprotein E (13), and other oxysterol biosynthetic enzymes like sterol 27-hydroxylase. Studies in knockout mice demonstrate that inactivation of the LDL receptor or the apolipoprotein E gene does not alter cholesterol biosynthetic rates in the central nervous system (12). Similarly, sterol 27-hydroxylase-deficient mice,2 and cholesterol 25-hydroxylase-deficient mice,3 have no alterations in brain cholesterol metabolism. Thus, additional enzymes involved in cholesterol excretion from the brain remain to be found.

Although steady-state levels of cholesterol in the whole brain were unchanged in the mutant mice (Fig. 7), localized concentrations of cholesterol may differ between Cyp46a1/ and wild type animals. Membrane cholesterol is concentrated in specialized sub-domains, including coated pits, lipid rafts, and caveolae (47), which serve as organization centers for receptors, intracellular signaling molecules, and glycosylphosphatidylinositol-anchored proteins. Whether the levels of cholesterol in these domains differ between the mutant and wild type mice remains to be determined, but the absence of apparent developmental or postnatal defects in the cholesterol 24-hydroxylase-deficient mice suggests a non-essential role for the enzyme in subcellular cholesterol targeting.

Cholesterol 24-hydroxylase is expressed in some but not all neurons of the developing embryo (Figs. 3 and 4) and adult mouse brain (26) and does not appear to be present in the much more plentiful support cells. Neurons constitute just 2–10% of cells in the human central nervous system (48). Assuming that a similar proportion of cells in the adult mouse brain are neurons, and estimating from the immunohistochemical data that 10% of these cells express cholesterol 24-hydroxylase, then it can be calculated that 0.2–1% of cells in the brain are responsible for ~40% of cholesterol turnover in this organ. This outcome is unexpected based on the distribution of cholesterol in the brain, which is approximately 10 times more abundant in the myelin sheaths elaborated by oligodendrocytes than in neuronal and other support cell membranes (12), and it raises interesting questions concerning the role of cholesterol turnover in this subset of cells and the consequences of its absence in the Cyp46a1/ mice.

Despite apparent robust expression of cholesterol 24-hydroxylase in early embryos as judged by {beta}-galactosidase staining (Figs. 3 and 4), the knockout mice appear to develop normally. Preliminary experiments in which movement and balance were examined in open field and rotorod tests, respectively, failed to reveal a difference in these parameters between adult wild type and knockout mice. When these negative data are considered with the abundant expression of the cholesterol 24-hydroxylase in pyramidal neurons of the cortex and Purkinje cells of the cerebellum (26), which are involved in regulating motor skills (49), it seems unlikely that cholesterol turnover plays an essential role in the establishment of the motor system. A maintenance role also is unlikely in that some of the Cyp46a1/ mice in our colony are 2 years of age and do not show obvious motor problems. Similar arguments can be made for a large number of essential functions controlled by neurons that express cholesterol 24-hydroxylase. Cholesterol turnover in the developing and adult brain may have a more subtle functional role in the mouse, perhaps in remodeling neurons for higher order tasks like learning and memory, which may involve dynamic changes in sterol content or distribution within neuronal membranes. Alternatively, other metabolic pathways may compensate for loss of cholesterol 24-hydroxylase in the mutant mice.

A slight reduction in circulating HDL levels and the total plasma pool of cholesterol was detected in the Cyp46a1/ mice (Table I and Fig. 5), but the mechanism underlying this modest lowering effect of the induced mutation was not determined. The product of the enzyme, 24(S)-hydroxycholesterol is a ligand for the liver X receptor (25), a nuclear hormone receptor that regulates the expression of many genes involved in lipid metabolism. It thus is possible that the reduction of plasma cholesterol in the knockout mice may be due in part to the absence of 24(S)-hydroxycholesterol and a corresponding reduction in LXR activity. In agreement with this possibility, LXR{alpha} knockout mice have slightly reduced levels of circulating HDL (50, 51). Old mice (>1 year) that lack both LXR{alpha} and LXR{beta} genes exhibit altered brain morphologies, including closed lateral ventricles, enlarged blood vessels, abnormal lipid deposition, and disordered myelin sheaths (52). This histopathology was not observed in younger mice of this genotype (52). In preliminary studies, we have not detected any brain abnormalities in young (3–4 months) or old (14.5 months) cholesterol 24-hydroxylase-deficient mice.4 The apparent normal architecture of the brains of 24-hydroxylase-deficient animals agrees with the absence of obvious behavioral abnormalities in these mice.

In summary, the current studies show that the synthesis of 24(S)-hydroxycholesterol and its secretion from the brain represent a quantitatively important mechanism of cholesterol turnover in this organ. Mice with an introduced mutation in the biosynthetic enzyme, cholesterol 24-hydroxylase, maintain cholesterol homeostasis in the central nervous system by decreasing the de novo synthesis of cholesterol. As such, these animals may represent a useful model to study the effects of altered rates of cholesterol synthesis on certain neuropathological conditions in which disruptions in cholesterol metabolism are thought to play a permissive or causative role. Our future studies will focus on how loss of cholesterol 24-hydroxylase affects the progression of Neiman Pick type C and Alzheimer disease and in deducing the role of cholesterol turnover in mouse behavior.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grants P01 HL20948 (to D. W. R.) and R37 HL09610 (to J. M. D.), the Moss Heart Fund (to J. M. D.), the Keck Foundation (to D. W. R.), and Academy of Finland Fellowship 80540 (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Merck Research Laboratories, Rahway, NJ 07090. Back

|| To whom correspondence should be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9046. Tel.: 214-648-2007; Fax: 214-648-6899; E-mail: david.russell{at}utsouthwestern.edu.

1 The abbreviations and trivial names used are: cholesterol, 5-cholesten-3{beta}-ol; 24(S)-hydroxycholesterol, 5-cholesten-3{beta},24(S)-diol; 25-hydroxycholesterol, 5-cholesten-3{beta},25-diol; 27-hydroxycholesterol, 5-cholesten-3{beta},27-diol; cholic acid, 5{beta}-cholanic acid-3{alpha},7{alpha},12{alpha}-triol; muricholic acid, 5{beta}-cholanic acid-3,6,7-triol; LDL, low density lipoprotein; HDL, high density lipoprotein; LXR, liver X receptor. Back

2 C. Xie, E. G. Lund, S. D. Turley, D. W. Russell, and J. M. Dietschy, unpublished observations. Back

3 G. Liang and D. W. Russell, unpublished observations. Back

4 T. Kotti and E. G. Lund, unpublished observations. Back


    ACKNOWLEDGMENTS
 
We thank Kevin Anderson, Daphne Davis, Amanda Fletcher, Brian Jefferson, Elizabeth Moore, Stephen Ostermann, and Monti Schneiderman for excellent technical assistance, Bob Hammer for construction of knockout mice, Brian Luikart for a discussion regarding {beta}-galactosidase-staining patterns, the Pathology Core laboratory at UT Southwestern for histological sections, and Helen Hobbs and Jay Horton for critical reading of the manuscript.



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