Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis

The sterol storage disorder cerebrotendinous xanthomatosis (CTX) is characterized by abnormal deposition of cholesterol and cholestanol in multiple tissues. Deposition in the central nervous system leads to neurological dysfunction marked by dementia, spinal cord paresis, and cerebellar ataxia. Deposition in other tissues causes tendon xanthomas, premature atherosclerosis, and cataracts. In two unrelated patients with CTX, we have identified different point mutations in the gene (CYP27) encoding sterol 27-hydroxylase, a key enzyme in the bile acid biosynthesis pathway. Transfection of mutant cDNAs into cultured cells results in the synthesis of immunoreactive sterol 27-hydroxylase protein with greatly diminished enzyme activity. We have localized the CYP27 gene to the q33-qter interval of human chromosome 2, and to mouse chromosome 1, in agreement with the autosomal recessive inheritance pattern of CTX. These findings underscore the essential role played by sterols in the central nervous system and suggest that mutations in other sterol metabolizing enzymes may contribute to diseases with neurological manifestations.     

Molecular basis of familial chylomicronemia: mutations in the lipoprotein lipase and apolipoprotein C-II genes.

The molecular basis of familial chylomicronemia (type I hyperlipoproteinemia), a rare autosomal recessive trait, was investigated in six unrelated individuals (five of Spanish descent and one of Northern European extraction). DNA amplification by polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) analysis allowed rapid identification of the underlying mutations. Six different mutant alleles (three of which are previously undescribed) of the gene encoding lipoprotein lipase (LPL) were discovered in the five LPL-deficient patients. These included an 11 bp deletion in exon 2, and five missense mutations: Trp 86 Arg (exon 3), His 136 Arg (exon 4), Gly 188 Glu (exon 5), Ile 194 Thr (exon 5), and Ile 205 Ser (exon 5). The Trp 86 Arg mutation is the only known missense mutation in exon 3. The other missense mutations lie in the highly conserved "central homology region" in close proximity with the catalytic site of LPL. These and other previously reported missense mutations provide insight into structure/function relationships in the lipase family. The missense mutations point to the important role of particular highly conserved helices and beta-strands in proper folding of the LPL molecule, and of certain connecting loops in the catalytic process. A nonsense mutation (Arg 19 Term) in the gene encoding apolipoprotein C-II (apoC-II), the cofactor of LPL, was found to underlie chylomicronemia in the sixth patient who had normal LPL but was apoC-II-deficient.     

Mutations in the gene encoding 21-hydroxylase detected by solid-phase minisequencing

We have developed an assay based on solid-phase minisequencing to screen for the following seven point mutations in the gene CYP21 encoding 21-hydroxylase: Pro30Leu, I2-splice, Ile172Asn, Cluster-E6, Val281Leu, Gln318Stop, and Arg356Trp. 5′-Biotinylated PCR products of CYP21 are bound to streptavidin-coated microtiter wells, where the minisequencing reaction takes place after denaturation of DNA. Depending on the sequence investigated, one specific ³H-labelled deoxyribonucleotide is incorporated to extend a detection primer. By using an appropriate set of detection primers, it is possible to screen the gene for several mutations within the same PCR amplificate. This fast and reliable method very clearly distinguishes between DNA from homozygous mutant, heterozygous, and normal individuals and is well suited for routine diagnosis of patients with 21-hydroxylase deficiency and for carrier detection. Received: 19 August 1996     

LDL-R and Apo-B-100 gene mutations in Polish familial hypercholesterolemias

A group of 30 Polish families with clinical signs of familial hypercholesterolemia was studied for the presence of germ-line mutations in the LDL-R and ApoB-100 genes. Screening of the LDL-R gene was performed at the genomic DNA level by single-strand conformation polymorphism analysis of all 18 exons and extended by sequencing of polymerase chain reaction (PCR) products showing abnormalities. The occurrence of large LDL-R gene alterations was evaluated by analysis of restriction enzyme patterns on Southern blots and using the long-PCR technique. The ApoB-100 gene was studied by combined allele-specific and asymmetric PCR for the occurrence of the common B-3500 missense mutation G to A at nucleotide position 10,708. Germ-line mutations were found in 17 families. In 12 of them LDL-R gene mutations were detected. Three of 11 different mutations had previously been described in other populations (3-bp deletion of codon 197; Ser156Leu; Gly571Glu). Of the mutations not previously recognized and identified in Polish families, there were three small deletions (2-bp deletion AG at codon 291; 4-bp deletion CCCT at codons 661–662; 1-bp deletion A at codon 830), and four point mutations (Arg239Stop, Cys331Stop, Asn543Ser, Gln665Stop). Additionally, one large (∼1-kb) LDL-R gene deletion between exons 6 and 9 was identified. In five families, the B-3500 mutation within the ApoB-100 gene was revealed. Received: 15 September 1997 / Accepted: 10 February 1998     

Genetic polymorphisms and haplotype structures of the CYP4A22 gene in a Japanese population

The CYP4A fatty acid monooxygenases oxidize endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid that acts as a regulator of blood pressure. Among the isoforms of the CYP4A subfamily, the human CYP4A22 was recently identified. In this study, we report the comprehensive investigation of polymorphisms in the CYP4A22 gene. To investigate genetic variation in CYP4A22 in 191 Japanese subjects, we used denaturing HPLC (DHPLC) and direct sequencing. Our investigation has enabled the identification of 13 sequence variations in the CYP4A22 coding region, thereby demonstrating for the first time that this gene is subject to polymorphism. Two of these sequence variations correspond to silent mutations located in exons 8 (His323His) and 9 (Gly390Gly). Nine of these sequence variations correspond to missense mutations located in exons 1 (Arg11Cys), 3 (Arg126Trp), 4 (Gly130Ser and Asn152Tyr), 5 (Val185Phe), 6 (Cys231Arg), 7 (Lys276Thr), 10 (Leu428Pro), and 12 (Leu509Phe). One of these sequence variations corresponds to nonsense mutations located in exon 9 (Gln368stop). The 13th mutation corresponds to a nucleotide deletion (G7067del) that causes a frameshift and consequently results in a stop codon 80 nucleotides downstream. In addition to the wild-type CYP4A22*1 allele, 20 variants, namely CYP4A22*2-15, were characterized by haplotype analysis. Based on these data, we concluded that allelic variants of the human CYP4A22 gene exist and speculated that some of these variants may be functionally relevant.     

Glucocerebrosidase mutations in Gaucher disease.

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.     

New insights into the pathological mechanisms of cerebrotendinous xanthomatosis in the Taiwanese using genomic and proteomic tools

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid-storage disorder caused by a deficiency of the mitochondrial sterol 27-hydroxylase. Genetic analysis utilizing SSCP and direct DNA sequencing identified a new mutation. One base-pair of cytosine was deleted at codon 326 on exon 2 of CYP27 in all CTX patients while their father was heterozygotic. This novel point deletion predicts a frameshift in mRNA (Pro(102) -->Leu) and results in the appearance of a premature termination codon (TGA) to substitute for Val(106) (GTG). To characterize the pathological mechanism of CTX patients, the protein profiles of serum and leukocytes extracted from these subjects were presented by means of proteomic technologies including 2-DE and MALDI-TOF analysis. According to the results, the amount of vinculin, ABP-280, talin and vimentin in leukocytes of CTX patients had changed significantly, reflecting the changes in membrane dynamics concerning cholestanol accumulation. The expression of target proteins in CTX patients and control was further verified by western blotting which indicated the same tendency as 2-DE data. This is the first paper to integrate both genomic and proteomic concepts for analyzing the possible mechanism of CTX and provides more information for related study in the future.     

Premature termination codon at the sterol 27-hydroxylase gene causes cerebrotendinous xanthomatosis in a French family

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid-storage disease caused by mutations in the sterol 27-hydroxylase gene (CYP27). So far several mutations causing CTX have been identified and characterized. A new mutation creating an insertion of cytosine at position 6 in the cDNA, which is expected to result in a frameshift and a premature termination codon at codon 179, has been identified in a French family. The mutation creates a new site for the restriction endonuclease HaeIII.     

Identification of a novel splicing mutation and 1-bp deletion in the 17α-hydroxylase gene of Japanese patients with 17α-hydroxylase deficiency

We report studies of two unrelated Japanese patients with 17α-hydroxylase deficiency caused by mutations of the 17α-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17α-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient’s mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17α-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation. Received: 15 November 1997 / Accepted: 15 March 1998     

Two new mutations in the sterol 27-hydroxylase gene in two families lead to cerebrotendinous xanthomatosis

This report concerns two new mutations in the sterol 27-hydroxylase gene in two patients with cerebrotendinous xanthomatosis (CTX). In a Surinam-Creole patient (patient A), a G deletion on position cDNA 546/547 in exon 3 led to a frameshift and the introduction of a premature termination codon. In a Dutch patient (patient B), a C→T transition at position 496 in exon 3 also led to a premature termination codon. Patient A was homozygous for the mutation, whereas patient B was compound heterozygous, a C→T transition also being found in exon 6 at position 1204. The two new mutations were confirmed by restriction analysis with the restriction enzymes FokI and MaeI, respectively. Received: 24 July 1996 / Revised: 9 August 1996     

Analysis of naturally occurring and site-directed mutations in the argininosuccinate lyase gene.

Argininosuccinic aciduria is an inborn error of metabolism due to the genetic deficiency of argininosuccinate lyase. In order to determine the molecular basis for the disease, RNA isolated from cultured skin fibroblasts derived from four unrelated patients was reverse-transcribed and amplified using the polymerase chain reaction and the products were cloned and sequenced. Three single base missense mutations were identified: Arg111----Trp, Gln286----Arg, and Arg193----Gln. One single base amber mutation was identified at Gln454. One mutation involved a 13-base pair deletion within exon 13, and it was noted that the majority of the mature RNA derived from this allele was deleted for the entire exon rather than containing the exon with the 13 bases deleted. A final mutation was observed in which exon 2 was deleted from the mature RNA. The molecular basis for this deletion was not determined. Of the eight potential mutations present in the four cell lines studied, six mutations were identified and further data indicate that the remaining two unidentified mutations were different from those identified. Two site-directed mutations were created in the cDNA, Lys51----Asn and His89----Gln, and these were expressed in yeast. The Lys51 mutation caused an approximate 2-fold reduction in activity and the His89 mutation resulted in an approximate 10-fold reduction in activity. The combination of determination of naturally occurring mutations and the study of the effect of site-directed mutations on the activity of argininosuccinate lyase provide insight into the amino acid residues critical to the function of the enzyme.     

2 Novel deletions of the sterol 27-hydroxylase gene in a Chinese Family with Cerebrotendinous Xanthomatosis

Background  

Cerebrotendinous xanthomatosis (CTX) is a rare lipid-storage disease. We investigated the clinic manifestation, histopathology and sterol 27-hydroxylase gene (CYP27A1) in a Chinese family with Cerebrotendinous Xanthomatosis (CTX).     

On the substrate specificity of human CYP27A1: implications for bile acid and cholestanol formation

The mitochondrial sterol 27-hydroxylase (CYP27A1) is required for degradation of the C27-sterol side chain in bile acid biosynthesis. CYP27A1 seems, however, to have roles beyond this, as illustrated by patients with a deficient sterol 27-hydroxylase due to mutations of the CYP27A1 gene [cerebrotendinous xanthomatosis (CTX)]. These subjects have symptoms ranging from accumulation of bile alcohols and cholestanol to accelerated atherosclerosis and progressive neurologic impairment. The present work describes a detailed investigation on the substrate specificity of recombinant human CYP27A1. In accordance with some previous work with rat liver mitochondria, the activity in general increased with the polarity of the substrate. An obvious example was the finding that cholesterol was 27-hydroxylated more efficiently than cholesterol oleate but less efficiently than cholesterol sulfate. The oxysterols 24S-hydroxycholesterol and 25-hydroxycholesterol were 27-hydroxylated less efficiently than cholesterol, possibly due to steric hindrance. Surprisingly, sterols with a 3-oxo-Delta4 structure were found to be hydroxylated at a much higher rate than the corresponding sterols with a 3beta-hydroxy-Delta5 structure. The rates of hydroxylation of the sterols were: 7alpha-hydroxy-4-cholesten-3-one>4-cholesten-3-one>7alpha-hydroxycholesterol>24-hydroxy-4-cholesten-3-one> cholesterol>25-hydroxy-4-cholesten-3-one>24-hydroxycholesterol>or=25-hydroxycholesterol. The possibility is discussed that the findings may have implications for oxysterol-mediated regulation of gene expression. The very high activity of CYP27A1 towards the cholestanol precursor 4-cholesten-3-one may be of importance in connection with the accumulation of cholestanol in patients with CTX.     

Rifampicin-induced CYP3A4 activation in CTX patients cannot replace chenodeoxycholic acid treatment

Cerebrotendinous xanthomatosis (CTX) is a rare neurodegenerative disorder with cholestanol accumulation resulting from mutations in the sterol 27-hydroxylase gene (CYP27A). Conventional treatment includes chenodeoxycholic acid and HMG-CoA reductase inhibitors. Mice with disrupted Cyp27A (Cyp27 KO) do not show elevated cholestanol levels nor develop CTX manifestations. This phenomenon was proposed to be due to murine CYP3A overexpression leading to an alternative pathway for degradation of bile alcohols including cholestanol. Our objective was to examine the influence of CYP3A4 induction on cholestanol elimination in CTX patients. Rifampicin (600 mg/day x 7 days), known to induce the PXR, and thereby to increase CYP3A activity, was used. The degree of CYP3A4 induction was assessed by comparing midazolam pharmacokinetics before and after rifampicin treatment. Cholestanol levels and cholestanol/cholesterol ratios were assayed during the experimental period and compared to a 3 weeks period without treatment. The results show that despite 60% increase in CYP3A4 activity following rifampicin treatment, there is no significant change in cholestanol levels. We conclude that up-regulated expression of CYP3A affects cholestanol elimination in mice differently as compared to its effect in CTX patients. Therefore, CYP3A4 inducers cannot replace chenodeoxycholic acid for the treatment of CTX.     

Cerebrotendinous xanthomatosis in the Israeli Druze: molecular genetics and phenotypic characteristics.

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid-storage disease caused by mutations in the sterol 27 hydroxylase gene (CYP27). Clinically, a multitude of neurological, skeletal, and vascular manifestations are usually present. Premature atherosclerosis has been reported in CTX and may be related to the metabolic derangement caused by the deficiency of the enzyme. A CYP27 nonsense mutation created by the deletion of cytosine376 has been identified in four Israeli Druze CTX patients residing in the same village. Molecular screening for this mutation in families of two probands revealed a total of 10 homozygotes and 28 heterozygotes whose clinical and biochemical characteristics are described. Overall, except for tendon xanthomas, most of the clinical manifestations progress with age. The CYP27 mutation was associated with modest differences in the levels of plasma total cholesterol (TC) and LDL cholesterol (LDL-C). The distribution of plasma concentrations of TC and LDL-C in the CTX families was consistent with a polygenic model. A similar model that includes also the effects of the CYP27 genotypes was not better supported by the data. It may be concluded that, in CTX, the presence of a CYP27 mutation does not significantly affect the plasma concentrations of lipids and lipoproteins. Therefore, the reported increased prevalence of atherosclerosis in this disease must be related to other factors.     

Mutations in argininosuccinate synthetase mRNA of Japanese patients,causing classical citrullinemia.

Citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase. In order to characterize mutations in Japanese patients with classical citrullinemia, RNA isolated from 10 unrelated patients was reverse-transcribed, and cDNA amplified by PCR was cloned and sequenced. The 10 mutations identified included 6 missense mutations (A118T, A192V, R272C, G280R, R304W, and R363L), 2 mutations associated with an absence of an exon 7 or exon 13, 1 mutation with a deletion of the first 7 bp in exon 16 (which might be caused by abnormal splicing), and 1 mutation with an insertion of 37 bp within exons 15 and 16 in cDNA. The insertion mutation and the five missense mutations (R304W being excluded) are new mutations described in the present paper. These are in addition to 14 mutations (9 missense mutations, 4 mutations associated with an absence of an exon in mRNA, and 1 splicing mutation) that we identified previously in mainly American patients with neonatal citrullinemia. Two of these 20 mutations, a deletion of exon 13 sequence and a 7-bp deletion in exon 16, were common to Japanese and American populations from different ethnic backgrounds; however, other mutations were unique to each population. Furthermore, the presence of a frequent mutation--the exon 7 deletion mutation in mRNA, which accounts for 10 of 23 affected alleles--was demonstrated in Japanese citrullinemia. This differs from the situation in the United States, where there was far greater heterogeneity of mutations.     

Molecular analysis of CYP-21 mutations for congenital adrenal hyperplasia in Singapore

BACKGROUND: Congenital adrenal hyperplasia arising from 21-hydroxylase deficiency is associated with mutations in the CYP21 gene on chromosome 6p. This is the first report on the mutational spectrum of the CYP21 gene in Singapore. METHODS: To catalogue the mutations, ten exons of the CYP21 gene from 28 Singaporean patients were analyzed by PCR amplification and direct sequencing. RESULTS: Common mutations in descending order were the intron 2 splice site mutation (32.7% of the alleles), the I172N mutation (23.1% of the alleles), and the R356W mutation (19.2% of the alleles). Two potentially novel mutations were discovered: (1) duplication of 111 bp from codon 21 to codon 57 (exon 1) and (2) missense mutation (L261P, exon 7). There was generally a good genotype-phenotype correlation, allowing accurate prediction of the disease severity.     

Novel splice-affecting variants in CYP27A1 gene in two Chilean patients with Cerebrotendinous Xanthomatosis

Cerebrotendinous Xanthomatosis (CTX), a rare lipid storage disorder, is caused by recessive loss-of-function mutations of the 27-sterol hydroxylase (CYP27A1), producing an alteration of the synthesis of bile acids, with an accumulation of cholestanol. Clinical characteristics include juvenile cataracts, diarrhea, tendon xanthomas, cognitive impairment and other neurological manifestations. Early diagnosis is critical, because treatment with chenodeoxycholic acid may prevent neurological damage. We studied the CYP27A1 gene in two Chilean CTX patients by sequencing its nine exons, exon-intron boundaries, and cDNA from peripheral blood mononuclear cells. Patient 1 is a compound heterozygote for the novel substitution c.256-1G > T that causes exon 2 skipping, leading to a premature stop codon in exon 3, and for the previously-known pathogenic mutation c.1183C > T (p.Arg395Cys). Patient 2 is homozygous for the novel mutation c.1185-1G > A that causes exon 7 skipping and the generation of a premature stop codon in exon 8, leading to the loss of the crucial adrenoxin binding domain of CYP27A1.     

The carrier rate of the phenylalanine hydoxylase gene (<Emphasis Type="Italic">PAH</Emphasis>) mutations p.Arg408Trp,pArg261Gln,and p.Arg261X in the populations of Eurasia

Analysis of the carrier frequency of p.Arg408Trp, p.Arg261Gln, and p.Arg261X mutations in the PAH gene was carried out in different unrelated indigenous individuals representing 58 populations of Eurasia taking into account their linguistic identity and territorial location. Mutation p.Arg408Trp in the PAH gene was found in 14 studied populations with the highest average carrier frequency of 0.0127 in the Volga-Ural region and 0.0134 in the representatives of the Slavic language group. Mutation p.Arg261Gln in the PAH gene was detected only in two populations with average carrier frequency rate of 0.0012 in the Volga-Ural region. Mutation p.Arg261X in the PAH gene was identified in four North Caucasus populations with highest carrier frequency in Karachays—at 0.0526. All PAH gene mutations in populations of Eurasia were identified in the heterozygous state.     

Additional pathways of sterol metabolism: Evidence from analysis of Cyp27a1−/− mouse brain and plasma

Cytochrome P450 (CYP) 27A1 is a key enzyme in both the acidic and neutral pathways of bile acid biosynthesis accepting cholesterol and ring-hydroxylated sterols as substrates introducing a (25R)26-hydroxy and ultimately a (25R)26-acid group to the sterol side-chain. In human, mutations in the CYP27A1 gene are the cause of the autosomal recessive disease cerebrotendinous xanthomatosis (CTX). Surprisingly, Cyp27a1 knockout mice (Cyp27a1−/−) do not present a CTX phenotype despite generating a similar global pattern of sterols. Using liquid chromatography – mass spectrometry and exploiting a charge-tagging approach for oxysterol analysis we identified over 50 cholesterol metabolites and precursors in the brain and circulation of Cyp27a1−/− mice. Notably, we identified (25R)26,7α- and (25S)26,7α-dihydroxy epimers of oxysterols and cholestenoic acids, indicating the presence of an additional sterol 26-hydroxylase in mouse. Importantly, our analysis also revealed elevated levels of 7α-hydroxycholest-4-en-3-one, which we found increased the number of oculomotor neurons in primary mouse brain cultures. 7α-Hydroxycholest-4-en-3-one is a ligand for the pregnane X receptor (PXR), activation of which is known to up-regulate the expression of CYP3A11, which we confirm has sterol 26-hydroxylase activity. This can explain the formation of (25R)26,7α- and (25S)26,7α-dihydroxy epimers of oxysterols and cholestenoic acids; the acid with the former stereochemistry is a liver X receptor (LXR) ligand that increases the number of oculomotor neurons in primary brain cultures. We hereby suggest that a lack of a motor neuron phenotype in some CTX patients and Cyp27a1−/− mice may involve increased levels of 7α-hydroxycholest-4-en-3-one and activation PXR, as well as increased levels of sterol 26-hydroxylase and the production of neuroprotective sterols capable of activating LXR.