cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain

Abstract

The turnover of cholesterol in the brain is thought to occur via conversion of excess cholesterol into 24S-hydroxycholesterol, an oxysterol that is readily secreted from the central nervous system into the plasma. To gain molecular insight into this pathway of cholesterol metabolism, we used expression cloning to isolate cDNAs that encode murine and human cholesterol 24-hydroxylases. DNA sequence analysis indicates that both proteins are localized to the endoplasmic reticulum, share 95% identity, and represent a new cytochrome P450 subfamily (CYP46). When transfected into cultured cells, the cDNAs produce an enzymatic activity that converts cholesterol into 24S-hydroxycholesterol, and to a lesser extent, 25-hydroxycholesterol. The cholesterol 24-hydroxylase gene contains 15 exons and is located on human chromosome 14q32.1. Cholesterol 24-hydroxylase is expressed predominantly in the brain as judged by RNA and protein blotting. In situ mRNA hybridization and immunohistochemistry localize the expression of this P450 to neurons in multiple subregions of the brain. The concentrations of 24S-hydroxycholesterol in serum are low in newborn mice, reach a peak between postnatal days 12 and 15, and thereafter decline to baseline levels. In contrast, cholesterol 24-hydroxylase protein is first detected in the brain of mice at birth and continues to accumulate with age. We conclude that the cloned cDNAs encode cholesterol 24-hydroxylases that synthesize oxysterols in neurons of the brain and that secretion of 24S-hydroxycholesterol from this tissue in the mouse is developmentally regulated.

Figures

Figure 1

Alignment of murine and human cholesterol 24-hydroxylase protein sequences. The cDNA-deduced sequences of the enzymes are shown in single-letter code with identical residues highlighted in black. Amino acids are numbered on the right. An asterisk above the alignment marks a cysteine residue (position 437) conserved in P450 enzymes. The GenBank accession numbers for the murine and human cDNAs are AF094479 and AF094480, respectively.

Figure 2

Expression of cholesterol 24-hydroxylase cDNAs in cultured cells. (A) The indicated expression plasmid was transfected into 293 cells by lipofection. Some cells (+) were treated before substrate addition for 1 h with 20 mg/ml 2-hydroxypropyl-β-cyclodextrin to remove cholesterol from membranes. Thereafter, fresh medium containing radio-labeled substrate was added and the incubation continued for 4, 8, or 24 h. Sterols were extracted from the media and separated by TLC, and the plates were subjected to PhosphorImage analysis to determine the % conversion of substrate into products. A photograph of a typical experiment is shown in the center. Sterol standards are marked on the left. The calculated % conversion for each lane is shown at the bottom. (B) Chemical analyses of 24-hydroxylase products. Cultured 293 cells were transfected with vector alone or a murine 24-hydroxylase expression plasmid (pCMV-m24) as described above except that the posttransfection media was supplemented with 10 μg/ml cholesterol. After 48 h, sterols were extracted from the media, purified by silica chromatography, derivatized to trimethylsilyl ethers, and subjected to GC-MS. (Left) GC profiles of sterols extracted from the media of mock-transfected cells (Upper) or expression vector-transfected cells (Lower). (Right) Ionization spectra of authentic 24R/S-hydroxycholesterol standard (Upper) and the sterol product eluting at 22.33 min from the GC obtained with transfected cell medium (Lower).

Figure 3

Structure of cholesterol 24-hydroxylase gene. The positions of introns and exons in the gene are indicated on a schematic of the protein. A conserved amino acid (Cys-437) that is postulated to be the sixth ligand of the heme cofactor is shown below the drawing. Introns interrupt codons of the murine and human 24-hydroxylase genes at the same positions.

Figure 4

Tissue distribution of cholesterol 24-hydroxylase mRNAs in mouse and human. Multiple tissue blots containing 2 μg of polyadenylated RNA from the indicated organs were subjected to hybridization using radio-labeled 24-hydroxylase cDNA probes. The positions of RNA standards are shown on the left. The murine and human RNA blots were exposed to film for 5 and 7 days, respectively.

Figure 5

Cell type specific expression patterns of cholesterol 24-hydroxylase in murine brain. Coronal sections were subjected to in situ mRNA hybridization analysis using a 33P-labeled RNA probe (A) or immunohistochemical analyses using an antipeptide antibody probe (B-D). (A) Light pink mRNA hybridization signals reveal an expression pattern consistent with neurons in the cortex (C), hippocampus (HC), dentate gyrus (DG), and thalamus (T). Magnification = ×0.5. (B) A similar pattern of cholesterol 24-hydroxylase protein is revealed at the same magnification by dark purple signal. (C) A photomicrograph of the cortical region of the brain taken at higher magnification (×10). Individual pyramidal neurons in layers I, II, III, and V contain dark red signal indicative of 24-hydroxylase protein. Smaller nonpyramidal cells concentrated in cortical layer IV contain little or no detectable signal. (D) Staining of 24-hydroxylase protein in Purkinje cells of the cerebellum (arrowheads). Signal is also present in the dendritic trees of Purkinje cells. Magnification = ×2.

Figure 6

Expression of cholesterol 24-hydroxylase as a function of age. (A) Ontogeny of serum and brain 24S-hydroxycholesterol in mice. Sterol levels were measured by isotope dilution GC-MS in sera and tissues from animals of the indicated ages. For days 3–30, each point represents a separate measurement in pooled sera from multiple animals (n = 3–10). For days 42–300, each point represents a measurement in a single animal. Serum levels (●), brain levels (○). (B) Ontogeny of 24-hydroxylase protein expression in murine brain. Aliquots (30 μg) of membrane protein prepared from animals of the indicated ages were immunoblotted with an antipeptide antibody directed against 24-hydroxylase. Antigen-antibody complexes were detected by enhanced chemiluminescence. Size standards are indicated on the left. The lane marked + is a control containing lysate (5 μg protein) from 293 cells transiently transfected with a murine 24-hydroxylase expression plasmid (pCMV-m24). (C) Ontogeny of 24-hydroxylase protein expression in human brain. Aliquots of total cell protein (50 μg) were prepared from individuals of the indicated ages and analyzed by immunoblotting as described in B. Size standards are indicated on the left. The lane marked + is a control containing lysate (1 μg protein) from 293 cells transiently transfected with a human 24-hydroxylase expression plasmid (pCMV-h24) mixed with 50 μg of total cell protein from a frontal lobe specimen of a 24-year-old individual.