Sterol Side Chain Research Articles

Synthesis of a Side Chain Alkyne Analogue of Sitosterol as a Chemical Probe for Imaging in Plant Cells.

Clickable chemical tools are essential for studying the localization and role of biomolecules in living cells. For this purpose, alkyne-based close analogs of the respective biomolecules are of outstanding interest. Here, in the field of phytosterols, we present the first alkyne derivative of sitosterol, which fulfills the crucial requirements for such a chemical tool as follows: very similar in size and lipophilicity to the plant phytosterols, and correct absolute configuration at C-24. The alkyne sitosterol FB-DJ-1 was synthesized, starting from stigmasterol, which comprised nine steps, utilizing a novel alkyne activation method, a Johnson-Claisen rearrangement for the stereoselective construction of a branched sterol side chain, and a Bestmann-Ohira reaction for the generation of the alkyne moiety.

Cardiac glycosides protect wormseed wallflower (Erysimum cheiranthoides) against some, but not all, glucosinolate-adapted herbivores.

The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well-defended plant lineage impacts interactions with diverse herbivores. Erysimum cheiranthoides (Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model. We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes in E. cheiranthoides and characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. Using E. cheiranthoides cardiac glycoside-deficient lines, we conducted insect experiments in both the laboratory and field. EcCYP87A126 initiates cardiac glycoside biosynthesis via sterol side-chain cleavage, and EcCYP716A418 has a role in cardiac glycoside hydroxylation. In EcCYP87A126 knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate-tolerant specialist herbivores, but did not protect against all crucifer-feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores. These results begin elucidation of the E. cheiranthoides cardiac glycoside biosynthetic pathway and demonstrate in vivo that cardiac glycoside production allows Erysimum to escape from some, but not all, specialist herbivores.

Sterol methyltransferases in uncultured bacteria complicate eukaryotic biomarker interpretations

Sterane molecular fossils are broadly interpreted as eukaryotic biomarkers, although diverse bacteria also produce sterols. Steranes with side-chain methylations can act as more specific biomarkers if their sterol precursors are limited to particular eukaryotes and are absent in bacteria. One such sterane, 24-isopropylcholestane, has been attributed to demosponges and potentially represents the earliest evidence for animals on Earth, but enzymes that methylate sterols to give the 24-isopropyl side-chain remain undiscovered. Here, we show that sterol methyltransferases from both sponges and yet-uncultured bacteria function in vitro and identify three methyltransferases from symbiotic bacteria each capable of sequential methylations resulting in the 24-isopropyl sterol side-chain. We demonstrate that bacteria have the genomic capacity to synthesize side-chain alkylated sterols, and that bacterial symbionts may contribute to 24-isopropyl sterol biosynthesis in demosponges. Together, our results suggest bacteria should not be dismissed as potential contributing sources of side-chain alkylated sterane biomarkers in the rock record.

Structure-Activity Relationships of Ligand Binding to Oxysterol-Binding Protein (OSBP) and OSBP-Related Protein 4.

Oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4) have emerged as potentially druggable targets in antiviral and precision cancer drug development. Multiple structurally diverse small molecules function through targeting the OSBP/ORP family of proteins, including the antiviral steroidal compounds OSW-1 and T-00127-HEV2. Here, the structure-activity relationships of oxysterols and related compound binding to human OSBP and ORP4 are characterized. Oxysterols with hydroxylation at various side chain positions (i.e., C-20, C-24, C-25, and C-27)─but not C-22─confer high affinity interactions with OSBP and ORP4. A library of 20(S)-hydroxycholesterol analogues with varying sterol side chains reveal that side chain length modifications are not well tolerated for OSBP and ORP4 interactions. This side chain requirement is contradicted by the high affinity binding of T-00127-HEV2, a steroidal compound lacking the side chain. The binding results, in combination with docking studies using homology models of OSBP and ORP4, suggest multiple modes of steroidal ligand binding to OSBP and ORP4.

The effects of the nature of the sterol on the properties and stability of niosome bilayer vesicles

Niosome vesicles as potential nanocarriers for drug delivery systems have been in development since the mid-1980s. These vesicles form by self-assembly of cholesterol and nonionic surfactants in an aqueous phase. The aim of the present molecular dynamics (MD) simulation study is to determine how changes in the sterol side chain and ring affect the structure and stability of sorbitan monostearate (Span60) niosome bilayers. Five sterols are examined: cholesterol (CHL), β-sitosterol (BST), ergosterol (ERG), stigmasterol (STS), and lanosterol (LST). Different sets of simulations were performed by varying the number of molecules (30, 40, and 50 % sterol concentration). The interactions between Span60 and the different sterols are found to be strongly dependent on the sterol’s side chain and head group. BST is found to markedly reduce the effectiveness of the sterol on the ordering of the bilayers, a result that persists over the whole range of concentrations examined in this work.

Combined enhancement of the propionyl-CoA metabolic pathway for efficient androstenedione production in Mycolicibacterium neoaurum

BackgroundThe production of androstenedione (AD) from phytosterols by Mycolicibacterium neoaurum is a multi-step biotransformation process, which requires degradation of sterol side chains, accompanied by the production of propionyl-CoA. However, the transient production of large amounts of propionyl-CoA can accumulate intracellularly to produce toxic effects and severely inhibit AD production.ResultsIn the present study, the intracellular propionyl-CoA concentration was effectively reduced and the productivity of the strain was improved by enhancing the cytosolic methyl-branched lipid synthesis pathway and increasing the expression level of nat operator gene, respectively. Subsequently, the application of a pathway combination strategy, combined and the inducible regulation strategy, further improved AD productivity with a maximum AD conversion rate of 96.88%, an increase of 13.93% over the original strain.ConclusionsOverall, we provide a new strategy for reducing propionyl-CoA stress during biotransformation for the production of AD and other steroidal drugs using phytosterols.

Cardenolide Increase in Foxglove after 2,1,3-Benzothiadiazole Treatment Reveals a Potential Link between Cardenolide and Phytosterol Biosynthesis.

Cardenolides are steroidal metabolites in Digitalis lanata with potent cardioactive effects on animals. In plants, cardenolides are likely involved in various stress responses. However, the molecular mechanism of cardenolide increase during stresses is mostly unknown. Additionally, cardenolides are proposed to arise from cholesterol, but indirect results show that phytosterols may also be substrates for cardenolide biosynthesis. Here, we show that cardenolides increased after methyl jasmonate (MJ), sorbitol, potassium chloride (KCl) and salicylic acid analog [2,1,3-benzothiadiazole (BTH)] treatments. However, the expression of three known genes for cardenolide biosynthesis did not correlate well with these increases. Specifically, the expression of progesterone-5β-reductases (P5βR and P5βR2) did not correlate with the cardenolide increase. The expression of 3β-hydroxysteroid dehydrogenase (3βHSD) correlated with changes in cardenolide levels only during the BTH treatment. Mining the D. lanata transcriptome identified genes involved in cholesterol and phytosterol biosynthesis: C24 sterol sidechain reductase 1 (SSR1), C4 sterol methyl oxidase 1, and 3 (SMO1 and SMO3). Surprisingly, the expression of all three genes correlated well with the cardenolide increase after the BTH treatment. Phylogenetic analysis showed that SSR1 is likely involved in both cholesterol and phytosterol biosynthesis. In addition, SMO1 is likely specific to phytosterol biosynthesis, and SMO3 is specific to cholesterol biosynthesis. These results suggest that stress-induced increase of cardenolides in foxglove may correlate with cholesterol and phytosterol biosynthesis. In summary, this work shows that cardenolides are important for stress responses in D. lanata and reveals a potential link between phytosterol and cardenolide biosynthesis.

Characterization of Three Paris polyphylla Glycosyltransferases from Different UGT Families for Steroid Functionalization

Plant steroid glycosides, such as phytosterol glycosides, steroidal saponins, and steroidal glycoalkaloids, are natural products with great pharmaceutical values. In this study, we characterized three UDP-glycosyltransferases (UGTs) involved in the glycosylation of steroidal sapogenin from Paris polyphylla. Substrate specificity analysis revealed that UGT73CR1 could glycosylate steroidal sapogenins and steroidal alkaloids, with the highest affinity for diosgenin. The residues His27 and Asp129 of UGT73CR1 are conserved in corresponding positions of plant glycosyltransferases, which are crucial for activating the C-3 OH of the receptor substrates. In comparison, UGT80A33 and UGT80A34 exhibited a higher affinity for cholesterol than other steroids. UGT80s have a larger active pocket, which allows them to accommodate the side chain of sterols. In summary, we assessed three P. polyphylla glycosyltransferases from two UGT families for the functionalization of steroidal molecules, which will provide a basis for the future biomanufacturing of diverse bioactive steroid glycosides.

Comparative proteomic analysis for revealing the advantage mechanisms of salt-tolerant tomato (Solanum lycoperscium).

Salt stress causes the quality change and significant yield loss of tomato. However, the resources of salt-resistant tomato were still deficient and the mechanisms of tomato resistance to salt stress were still unclear. In this study, the proteomic profiles of two salt-tolerant and salt-sensitive tomato cultivars were investigated to decipher the salt-resistance mechanism of tomato and provide novel resources for tomato breeding. We found high abundance proteins related to nitrate and amino acids metabolismsin the salt-tolerant cultivars. The significant increase in abundance of proteins involved in Brassinolides and GABA biosynthesis were verified in salt-tolerant cultivars, strengthening the salt resistance of tomato. Meanwhile, salt-tolerant cultivars with higher abundance and activity of antioxidant-related proteins have more advantages in dealing with reactive oxygen species caused by salt stress. Moreover, the salt-tolerant cultivars had higher photosynthetic activity based on overexpression of proteins functioned in chloroplast, guaranteeing the sufficient nutrient for plant growth under salt stress. Furthermore, three key proteins were identified as important salt-resistant resources for breeding salt-tolerant cultivars, including sterol side chain reductase, gamma aminobutyrate transaminase and starch synthase. Our results provided series valuable strategies for salt-tolerant cultivars which can be used in future.

Synthesis and biological evaluation of cationic TopFluor cholesterol analogues

Cholesterol is not only a major component of the cell membrane, but also plays an important role in a wide range of biological processes and pathologies. It is therefore crucial to develop appropriate tools for visualizing intracellular cholesterol transport. Here, we describe new cationic analogues of BODIPY-Cholesterol (TopFluor-Cholesterol, TF-Chol), which combine a positive charge on the sterol side chain and a BODIPY group connected via a C-4 linker. In contrast to TF-Chol, the new analogues TF-1 and TF-3 possessing acetyl groups on the A ring (C-3 position on steroid) internalized much faster and displayed slightly different levels of intracellular localization. Their applicability for cholesterol monitoring was indicated by the fact that they strongly label compartments with accumulated cholesterol in cells carrying a mutation of the Niemann-Pick disease-associated cholesterol transporter, NPC1.

The Cerebrospinal Fluid Profile of Cholesterol Metabolites in Parkinson's Disease and Their Association With Disease State and Clinical Features.

Disordered cholesterol metabolism is linked to neurodegeneration. In this study we investigated the profile of cholesterol metabolites found in the cerebrospinal fluid (CSF) of Parkinson’s disease (PD) patients. When adjustments were made for confounding variables of age and sex, 7α,(25R)26-dihydroxycholesterol and a second oxysterol 7α,x,y-trihydroxycholest-4-en-3-one (7α,x,y-triHCO), whose exact structure is unknown, were found to be significantly elevated in PD CSF. The likely location of the additional hydroxy groups on the second oxysterol are on the sterol side-chain. We found that CSF 7α-hydroxycholesterol levels correlated positively with depression in PD patients, while two presumptively identified cholestenoic acids correlated negatively with depression.

Virtual screening of natural compounds for potential inhibitors of Sterol C-24 methyltransferase of Leishmania donovani to overcome leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by trypanosomatid parasite belonging to the genera Leishmania. Leishmaniasis is transmitted from one human to other through the bite of sandflies. It is endemic in around 98 countries including tropical and subtropical regions of Asia, Africa, Southern America, and the Mediterranean region. Sterol C-24 methyltransferase (LdSMT) of Leishmania donovani (L. donovani) mediates the transfer of CH3-group from S-adenosyl methionine to C-24 position of sterol side chain which makes the ergosterol different from cholesterol. Absence of ortholog in human made it potential druggable target. Here, we performed virtual screening of library of natural compounds against LdSMT to identify the potential inhibitor for it and to fight leishmaniasis. Gigantol, flavan-3-ol, and parthenolide showed the best binding affinity towards LdSMT. Further, based on absorption, distribution, metabolism, and excretion properties and biological activity prediction, gigantol showed the best lead-likeness and drug-likeness properties. Therefore, we further elucidated its antileishmanial properties. We found that gigantol inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes. Gigantol exerted its antileishmanial action through the induction of reactive oxygen species in dose-dependent manner. Our study, suggested the possible use of gigantol as antileishmanial drug after further validations to overcome leishmaniasis.

Editing sterol side chain reductase 2 gene (StSSR2) via CRISPR/Cas9 reduces the total steroidal glycoalkaloids in potato

Steroidal glycoalkaloids (SGAs) existing in most potato tissues are toxic to humans when the fresh weight is over 200 mg kg−1, and high SGAs content would also damage the quality of potato tubers. Sterol side chain reductase 2 (StSSR2) is a key enzyme in the biosynthetic of SGAs in potatoes. To reduce the concentration of SGAs in tetraploid cultivated potato (Solanum tuberosum L.), the gene StSSR2 was edited by the CRISPR/Cas9 system. A total number of 64 mutant lines were generated, and the mutation efficiency was 46%. The percentage of insertion was 6.25%, and the percentage of deletion was 93.75%. Compared to the wild-type (WT), the content of SGAs reduced significantly in the mutated potatoes. The lowest SGAs concentration was 54% of the WT level, which was recorded in the peels of line #360. In the tuber fleshes, the lowest SGAs were 66% of WT level, which was found in line #378. In the leaves, the lowest SGAs concentration was 44% of the WT level as found in lines #204 and #378. In a word, our results showed that the StSSR2 gene of tetraploid cultivated potato could be successfully edited by the CRISPR/Cas9 system to reduce toxic SGAs.

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D.

Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, Nocardioides simplex VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26-oic acid and 3-oxo-cholesta-1,4-dien-26-oic acid were identified as those that have not been earlier reported for N. simplex and related species. The transcriptomic approach revealed candidate genes of cholesterol and lithocholic acid (LCA) catabolism by the strain. A separate set of genes combined in cluster and additional 3-ketosteroid Δ1-dehydrogenase and 3-ketosteroid 9α-hydroxylases that might be involved in LCA catabolism were predicted. Bioinformatic calculations based on transcriptomic data showed the existence of a previously unknown transcription factor, which regulates cholate catabolism gene orthologs. The results contribute to the knowledge on diversity of steroid catabolism regulation in actinobacteria and might be used at the engineering of microbial catalysts for ecological and industrial biotechnology.

Targeted genome editing in tetraploid potato through transient TALEN expression by Agrobacterium infection.

Genome editing using site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9), is a powerful technology for crop breeding. For plant genome editing, the genome-editing reagents are usually expressed in plant cells from stably integrated transgenes within the genome. This requires crossing processes to remove foreign nucleotides from the genome to generate null segregants. However, in highly heterozygous plants such as potato, the progeny lines have different agronomic traits from the parent cultivar and do not necessarily become elite lines. Agrobacteria can transfer exogenous genes on T-DNA into plant cells. This has been used both to transform plants stably and to express the genes transiently in plant cells. Here, we infected potato, with Agrobacterium tumefaciens harboring TALEN-expression vector targeting sterol side chain reductase 2 (SSR2) gene and regenerated shoots without selection. We obtained regenerated lines with disrupted-SSR2 gene and without transgene of the TALEN gene, revealing that their disruption should be caused by transient gene expression. The strategy using transient gene expression by Agrobacterium that we call Agrobacterial mutagenesis, developed here should accelerate the use of genome-editing technology to modify heterozygous plant genomes.

Elevated expression of diosgenin-related genes and stimulation of the defense system in Trigonella foenum-graecum (Fenugreek) by cold plasma treatment

Atmospheric pressure plasma jets (APPJs) have been extensively studied for improving plant quality and yield. The effect of different working voltages (3500 V, 4000 V) as well as different exposure times (0.5, 1, 2 and 5 min) was evaluated using dielectric barrier discharge (DBD) plasma jet to determine the best combinations of treatment. The results revealed that there was a significant difference in all tested traits except for chlorophyll b and carotenoid content. The observed increasing activities of catalase (24%), glutathione peroxidase (53%), ascorbate peroxidase (86%) in comparison to control group, suggested that cold plasma treatment has a positive effect on the enhancement of Fenugreek defense system. The results also demonstrated that the total chlorophyll (88%) and protein (49%) content were significantly elevated. Further, the expression of Sterol side chain reductase, Squalene synthase, Squalene epoxidase, Cycloartenol synthase, and Sterol methyltransferase grew by 5.26, 3.42, 1.9, 4.19, and 3-fold, respectively, compared with the control group. This indicates cold plasma treatment as a promising approach to enhance the accumulation of diosgenin in Fenugreek. With respect to this fact that the classical breeding approach is a time-consuming and labor-intensive strategy, cold plasma could be an effective approach to improve plant traits.

Genome-wide response on phytosterol in 9-hydroxyandrostenedione-producing strain of Mycobacterium sp. VKM Ac-1817D

BackgroundAerobic side chain degradation of phytosterols by actinobacteria is the basis for the industrial production of androstane steroids which are the starting materials for the synthesis of steroid hormones. A native strain of Mycobacterium sp. VKM Ac-1817D effectively produces 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) from phytosterol, but also is capable of slow steroid core degradation. However, the set of the genes with products that are involved in phytosterol oxidation, their organisation and regulation remain poorly understood.ResultsHigh-throughput sequencing of the global transcriptomes of the Mycobacterium sp. VKM Ac-1817D cultures grown with or without phytosterol was carried out. In the presence of phytosterol, the expression of 260 genes including those related to steroid catabolism pathways significantly increased. Two of the five genes encoding the oxygenase unit of 3-ketosteroid-9α-hydroxylase (kshA) were highly up-regulated in response to phytosterol (55- and 25-fold, respectively) as well as one of the two genes encoding its reductase subunit (kshB) (40-fold). Only one of the five putative genes encoding 3-ketosteroid-∆1-dehydrogenase (KstD_1) was up-regulated in the presence of phytosterol (61-fold), but several substitutions in the conservative positions of its product were revealed.Among the genes over-expressed in the presence of phytosterol, several dozen genes did not possess binding sites for the known regulatory factors of steroid catabolism. In the promoter regions of these genes, a regularly occurring palindromic motif was revealed. The orthologue of TetR-family transcription regulator gene Rv0767c of M. tuberculosis was identified in Mycobacterium sp. VKM Ac-1817D as G155_05115.ConclusionsHigh expression levels of the genes related to the sterol side chain degradation and steroid 9α-hydroxylation in combination with possible defects in KstD_1 may contribute to effective 9α-hydroxyandrost-4-ene-3,17-dione accumulation from phytosterol provided by this biotechnologically relevant strain. The TetR-family transcription regulator gene G155_05115 presumably associated with the regulation of steroid catabolism. The results are of significance for the improvement of biocatalytic features of the microbial strains for the steroid industry.

The homoeologous genes encoding C24-sterol methyltransferase 1 in Triticum aestivum: structural characteristics and effects of cold stress

A unique structural feature of plant sterols is the presence of a 24-alkyl group in the sterol side chain, which is synthesized by C24-sterol methyltransferase (SMT). Here we report for the first time that the bread wheat genome (AABBDD) contains at least three homoeologous genes encoding C24-sterol methyltransferase 1. While these copies have similar coding regions, they differ markedly in the nucleotide sequences of their non-coding regions. Sequencing de novo of the promoter regions of the TaSMT1 homoeologs demonstrated the occurrence of common and specific stress-sensitive cis-elements such as LTR, the cis-element involved in low temperature response. These cis-elements, along with other factors, determine the differences in the effects of stress on the expression of homoeologous TaSMT1 genes. For example, TaSMT1-5A is constitutively expressed in the roots and leaves, while TaSMT1-4D gene is highly stress-responsive. Another important enzyme involved in sterol biosynthesis is C22-sterol desaturase, which converts β-sitosterol into stigmasterol. This enzyme is encoded by homoeologous TaCYP710A8 genes, which, in contrast to TaSMT1, are all up-regulated in response to stress. Cold-induced expression of TaCYP710A8 is greater in roots than in leaves. This may be due to the higher cold sensitivity of the roots and the necessity to increase the amount of stigmasterol known as a “stress sterol”. Our findings suggest that the existence of homoeologous genes of sterol biosynthesis in polyploid plants supports the diversity of genetic mechanisms of sterol-mediated response of plants to stresses.

Pushing the limits of concertedness. A waltz of wandering carbocations.

Among the array of complex terpene-forming carbocation cyclization/rearrangement reactions, the so-called "triple shift" reactions are among the most unexpected. Such reactions involve the asynchronous combination of three 1,n-shifts into a concerted process, e.g., a 1,2-alkyl shift followed by a 1,3-hydride shift followed by a second 1,2-alkyl shift. This type of reaction so far has been proposed to occur during the biosynthesis of diterpenes and the sidechains of sterols. Here we describe efforts to push the limits of concertedness in this type of carbocation reaction by designing, and characterizing with quantum chemical computations, systems that could couple additional 1,n-shift events to a triple shift leading, in principle to quadruple, pentuple, etc. shifts. While our designs did not lead to clear-cut examples of quadruple, etc. shifts, they did lead to reactions with surprisingly flat energy surfaces where more than five chemical events connect reactants and plausible products. Ab initio molecular dynamics simulations demonstrate that the formal minima on these surfaces interchange on short timescales, both with each other and with additional unexpected structures, allowing us a glimpse into a very complex manifold that allows ready access to great structural diversity.

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.