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.

Bile acid biosynthesis in Smith-Lemli-Opitz syndrome bypassing cholesterol: Potential importance of pathway intermediates.

Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ5-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography - mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7ß-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3ß-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7ß-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7ß-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway.

Formation and metabolism of oxysterols and cholestenoic acids found in the mouse circulation: Lessons learnt from deuterium-enrichment experiments and the CYP46A1 transgenic mouse.

While the presence and abundance of the major oxysterols and cholestenoic acids in the circulation is well established, minor cholesterol metabolites may also have biological importance and be of value to investigate. In this study by observing the metabolism of deuterium-labelled cholesterol in the pdgfbret/ret mouse, a mouse model with increased vascular permeability in brain, and by studying the sterol content of plasma from the CYP46A1 transgenic mouse overexpressing the human cholesterol 24S-hydroxylase enzyme we have been able to identify a number of minor cholesterol metabolites found in the circulation, make approximate-quantitative measurements and postulate pathways for their formation. These "proof of principle" data may have relevance when using mouse models to mimic human disease and in respect of the increasing possibility of treating human neurodegenerative diseases with pharmaceuticals designed to enhance the activity of CYP46A1 or by adeno-associated virus delivery of CYP46A1.

Mining for Oxysterols in Cyp7b1-/- Mouse Brain and Plasma: Relevance to Spastic Paraplegia Type 5.

Deficiency in cytochrome P450 (CYP) 7B1, also known as oxysterol 7α-hydroxylase, in humans leads to hereditary spastic paraplegia type 5 (SPG5) and in some cases in infants to liver disease. SPG5 is medically characterized by loss of motor neurons in the corticospinal tract. In an effort to gain a better understanding of the fundamental biochemistry of this disorder, we have extended our previous profiling of the oxysterol content of brain and plasma of Cyp7b1 knockout (-/-) mice to include, amongst other sterols, 25-hydroxylated cholesterol metabolites. Although brain cholesterol levels do not differ between wild-type (wt) and knockout mice, we find, using a charge-tagging methodology in combination with liquid chromatography-mass spectrometry (LC-MS) and multistage fragmentation (MSn), that there is a build-up of the CYP7B1 substrate 25-hydroxycholesterol (25-HC) in Cyp7b1-/- mouse brain and plasma. As reported earlier, levels of (25R)26-hydroxycholesterol (26-HC), 3ß-hydroxycholest-5-en-(25R)26-oic acid and 24S,25-epoxycholesterol (24S,25-EC) are similarly elevated in brain and plasma. Side-chain oxysterols including 25-HC, 26-HC and 24S,25-EC are known to bind to INSIG (insulin-induced gene) and inhibit the processing of SREBP-2 (sterol regulatory element-binding protein-2) to its active form as a master regulator of cholesterol biosynthesis. We suggest the concentration of cholesterol in brain of the Cyp7b1-/- mouse is maintained by balancing reduced metabolism, as a consequence of a loss in CYP7B1, with reduced biosynthesis. The Cyp7b1-/- mouse does not show a motor defect; whether the defect in humans is a consequence of less efficient homeostasis of cholesterol in brain has yet to be uncovered.

Metabolism of Non-Enzymatically Derived Oxysterols: Clues from sterol metabolic disorders.

Cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol) is formed from cholestan-5,6-epoxide (5,6-EC) in a reaction catalysed by cholesterol epoxide hydrolase, following formation of 5,6-EC through free radical oxidation of cholesterol. 7-Oxocholesterol (7-OC) and 7ß-hydroxycholesterol (7ß-HC) can also be formed by free radical oxidation of cholesterol. Here we investigate how 3ß,5α,6ß-triol, 7-OC and 7ß-HC are metabolised to bile acids. We show, by monitoring oxysterol metabolites in plasma samples rich in 3ß,5α,6ß-triol, 7-OC and 7ß-HC, that these three oxysterols fall into novel branches of the acidic pathway of bile acid biosynthesis becoming (25R)26-hydroxylated then carboxylated, 24-hydroxylated and side-chain shortened to give the final products 3ß,5α,6ß-trihydroxycholanoic, 3ß-hydroxy-7-oxochol-5-enoic and 3ß,7ß-dihydroxychol-5-enoic acids, respectively. The intermediates in these pathways may be causative of some phenotypical features of, and/or have diagnostic value for, the lysosomal storage diseases, Niemann Pick types C and B and lysosomal acid lipase deficiency. Free radical derived oxysterols are metabolised in human to unusual bile acids via novel branches of the acidic pathway, intermediates in these pathways are observed in plasma.

First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry.

Enzyme-assisted derivatization for sterol analysis (EADSA) is a technology designed to enhance sensitivity and specificity for sterol analysis using electrospray ionization⁻mass spectrometry. To date it has only been exploited on sterols with a 3ß-hydroxy-5-ene or 3ß-hydroxy-5α-hydrogen structure, using bacterial cholesterol oxidase enzyme to convert the 3ß-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for cholesterol oxidase, making the method applicable to sterols with a 3α-hydroxy-5ß-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C27 acids give an intense molecular ion ([M]⁺) upon electrospray ionization and informative fragmentation spectra. The method shows promise for analysis of bile alcohols and 3α-hydroxy-5ß-C27-acids, enhancing the range of sterols that can be analyzed at high sensitivity in sterolomic studies.

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.

International descriptive and interventional survey for oxycholesterol determination by gas- and liquid-chromatographic methods.

Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7α-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5-73.3% (survey 1), 56.8-60.3% (survey 2); 36.2-35.8% (survey 1), 56.6-59.8, (survey 2); 61.1-197.7% (survey 1), 47.2-74.2% (survey 2) for 24(S)-, 27-, and 7α-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed.

Identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in human cerebrospinal fluid and plasma.

Dihydroxyoxocholestenoic acids are intermediates in bile acid biosynthesis. Here, using liquid chromatography - mass spectrometry, we confirm the identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in cerebrospinal fluid (CSF) based on comparisons to authentic standards and of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic and 7α,x-dihydroxy-3-oxocholest-4-en-26-oic (where hydroxylation is likely on C-22 or C-23) based on exact mass measurement and multistage fragmentation. Surprisingly, patients suffering from the inborn error of metabolism cerebrotendinous xanthomatosis, where the enzyme CYP27A1, which normally introduces the (25 R)26-carboxylic acid group to the sterol side-chain, is defective still synthesise 7α,24-dihydroxy-3-oxocholest-4-en-26-oic acid and also both 25 R- and 25 S-epimers of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic acid. We speculate that the enzymes CYP46A1 and CYP3A4 may have C-26 carboxylase activity to generate these acids. In patients suffering from hereditary spastic paraplegia type 5 the CSF concentrations of the 7α,24- and 7α,25-dihydroxy acids are reduced, suggesting an involvement of CYP7B1 in their biosynthesis in brain.

Identification of unusual oxysterols and bile acids with 7-oxo or 3ß,5α,6ß-trihydroxy functions in human plasma by charge-tagging mass spectrometry with multistage fragmentation.

7-Oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC), and its hydrolysis product cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol) are normally minor oxysterols in human samples; however, in disease, their levels may be greatly elevated. This is the case in plasma from patients suffering from some lysosomal storage disorders, e.g., Niemann-Pick disease type C, or the inborn errors of sterol metabolism, e.g., Smith-Lemli-Opitz syndrome and cerebrotendinous xanthomatosis. A complication in the analysis of 7-OC and 5,6-EC is that they can also be formed ex vivo from cholesterol during sample handling in air, causing confusion with molecules formed in vivo. When formed endogenously, 7-OC, 5,6-EC, and 3ß,5α,6ß-triol can be converted to bile acids. Here, we describe methodology based on chemical derivatization and LC/MS with multistage fragmentation (MSn) to identify the necessary intermediates in the conversion of 7-OC to 3ß-hydroxy-7-oxochol-5-enoic acid and 5,6-EC and 3ß,5α,6ß-triol to 3ß,5α,6ß-trihydroxycholanoic acid. Identification of intermediate metabolites is facilitated by their unusual MSn fragmentation patterns. Semiquantitative measurements are possible, but absolute values await the synthesis of isotope-labeled standards.

Comparison of the composition of bile acids in bile of patients with adenocarcinoma of the pancreas and benign disease.

Bile acids have been implicated in the development of gastrointestinal malignancies. Both the specific nature of individual bile acids and their concentration appear key factors in the carcinogenic potency of bile. Using liquid chromatography mass spectrometry (LC-MS) we performed quantitative profiling of bile extracted directly from the common bile duct in 30 patients (15 patients with pancreatic cancer and 15 patients with benign disease). Separation and detection of bile acids was performed using a 1.7µm particle size reversed-phase C18 LC column at a flow rate of 200µL/min with negative electrospray ionization MS. A significant difference (p=0.018) was seen in the concentration of unconjugated cholic acid in the malignant group (0.643mmol/L) compared to the benign group (0.022mmol/L), with an overall significant difference (p=0.04) seen in the level of total unconjugated bile acids in the malignant group (1.816mmol/L) compared to the benign group (0.069mmol/L). This finding may offer the possibility of both understanding the biology of cancer development in the pancreas, as well as offering a potential diagnostic avenue to explore. However, a larger study is necessary to confirm the alterations in bile acid profiles reported here and explore factors such as diet and microbial populations on the bile acid profiles of these patient groups.

Charge-tagging liquid chromatography-mass spectrometry methodology targeting oxysterol diastereoisomers.

The introduction of a hydroxy group to the cholesterol skeleton introduces not only the possibility for positional isomers but also diastereoisomers, where two or more isomers have different configurations at one or more of the stereocentres but are not mirror images. The differentiation of diastereoisomers is important as differing isomers can have differing biochemical properties and are formed via different biochemical pathways. Separation of diasterioisomers is not always easy by chromatographic methods Here we demonstrate, by application of charge-tagging and derivatisation with the Girard P reagent, the separation and detection of biologically relevant diastereoisomers using liquid chromatography - mass spectrometry with multistage fragmentation.

Sterols and oxysterols in plasma from Smith-Lemli-Opitz syndrome patients.

Smith-Lemli-Opitz syndrome (SLOS) is a severe autosomal recessive disorder resulting from defects in the cholesterol synthesising enzyme 7-dehydrocholesterol reductase (Δ7-sterol reductase, DHCR7, EC 1.3.1.21) leading to a build-up of the cholesterol precursor 7-dehydrocholesterol (7-DHC) in tissues and blood plasma. Although the underling enzyme deficiency associated with SLOS is clear there are likely to be multiple mechanisms responsible for SLOS pathology. In an effort to learn more of the aetiology of SLOS we have analysed plasma from SLOS patients to search for metabolites derived from 7-DHC which may be responsible for some of the pathology. We have identified a novel hydroxy-8-dehydrocholesterol, which is either 24- or 25-hydroxy-8-dehydrocholesterol and also the known metabolites 26-hydroxy-8-dehydrocholesterol, 4-hydroxy-7-dehydrocholesterol, 3ß,5α-dihydroxycholest-7-en-6-one and 7α,8α-epoxycholesterol. None of these metabolites are detected in control plasma at quantifiable levels (0.5ng/mL).

Reduced Plasma Levels of 25-Hydroxycholesterol and Increased Cerebrospinal Fluid Levels of Bile Acid Precursors in Multiple Sclerosis Patients.

Multiple sclerosis (MS) is an autoimmune, inflammatory disease of the central nervous system (CNS). We have measured the levels of over 20 non-esterified sterols in plasma and cerebrospinal fluid (CSF) from patients suffering from MS, inflammatory CNS disease, neurodegenerative disease and control patients. Analysis was performed following enzyme-assisted derivatisation by liquid chromatography-mass spectrometry (LC-MS) exploiting multistage fragmentation (MS n ). We found increased concentrations of bile acid precursors in CSF from each of the disease states and that patients with inflammatory CNS disease classified as suspected autoimmune disease or of unknown aetiology also showed elevated concentrations of 25-hydroxycholestertol (25-HC, P < 0.05) in CSF. Cholesterol concentrations in CSF were not changed except for patients diagnosed with amyotrophic lateral sclerosis (P < 0.01) or pathogen-based infections of the CNS (P < 0.05) where they were elevated. In plasma, we found that 25-HC (P < 0.01), (25R)26-hydroxycholesterol ((25R)26-HC, P < 0.05) and 7α-hydroxy-3-oxocholest-4-enoic acid (7αH,3O-CA, P < 0.05) were reduced in relapsing-remitting MS (RRMS) patients compared to controls. The pattern of reduced plasma levels of 25-HC, (25R)26-HC and 7αH,3O-CA was unique to RRMS. In summary, in plasma, we find that the concentration of 25-HC in RRMS patients is significantly lower than in controls. This is consistent with the hypothesis that a lower propensity of macrophages to synthesise 25-HC will result in reduced negative feedback by 25-HC on IL-1 family cytokine production and exacerbated MS. In CSF, we find that the dominating metabolites reflect the acidic pathway of bile acid biosynthesis and the elevated levels of these in CNS disease is likely to reflect cholesterol release as a result of demyelination or neuronal death. 25-HC is elevated in patients with inflammatory CNS disease probably as a consequence of up-regulation of the type 1 interferon-stimulated gene cholesterol 25-hydroxylase in macrophages.

The role of microRNA-155/liver X receptor pathway in experimental and idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is progressive and rapidly fatal. Improved understanding of pathogenesis is required to prosper novel therapeutics. Epigenetic changes contribute to IPF; therefore, microRNAs may reveal novel pathogenic pathways. OBJECTIVES: We sought to determine the regulatory role of microRNA (miR)-155 in the profibrotic function of murine lung macrophages and fibroblasts, IPF lung fibroblasts, and its contribution to experimental pulmonary fibrosis. METHODS: Bleomycin-induced lung fibrosis in wild-type and miR-155-/- mice was analyzed by histology, collagen, and profibrotic gene expression. Mechanisms were identified by in silico and molecular approaches and validated in mouse lung fibroblasts and macrophages, and in IPF lung fibroblasts, using loss-and-gain of function assays, and in vivo using specific inhibitors. RESULTS: miR-155-/- mice developed exacerbated lung fibrosis, increased collagen deposition, collagen 1 and 3 mRNA expression, TGF-ß production, and activation of alternatively activated macrophages, contributed by deregulation of the miR-155 target gene the liver X receptor (LXR)α in lung fibroblasts and macrophages. Inhibition of LXRα in experimental lung fibrosis and in IPF lung fibroblasts reduced the exacerbated fibrotic response. Similarly, enforced expression of miR-155 reduced the profibrotic phenotype of IPF and miR-155-/- fibroblasts. CONCLUSIONS: We describe herein a molecular pathway comprising miR-155 and its epigenetic LXRα target that when deregulated enables pathogenic pulmonary fibrosis. Manipulation of the miR-155/LXR pathway may have therapeutic potential for IPF.

Defective cholesterol metabolism in amyotrophic lateral sclerosis.

As neurons die, cholesterol is released in the central nervous system (CNS); hence, this sterol and its metabolites may represent a biomarker of neurodegeneration, including in amyotrophic lateral sclerosis (ALS), in which altered cholesterol levels have been linked to prognosis. More than 40 different sterols were quantified in serum and cerebrospinal fluid (CSF) from ALS patients and healthy controls. In CSF, the concentration of cholesterol was found to be elevated in ALS samples. When CSF metabolite levels were normalized to cholesterol, the cholesterol metabolite 3ß,7α-dihydroxycholest-5-en-26-oic acid, along with its precursor 3ß-hydroxycholest-5-en-26-oic acid and product 7α-hydroxy-3-oxocholest-4-en-26-oic acid, were reduced in concentration, whereas metabolites known to be imported from the circulation into the CNS were not found to differ in concentration between groups. Analysis of serum revealed that (25R)26-hydroxycholesterol, the immediate precursor of 3ß-hydroxycholest-5-en-26-oic acid, was reduced in concentration in ALS patients compared with controls. We conclude that the acidic branch of bile acid biosynthesis, known to be operative in-part in the brain, is defective in ALS, leading to a failure of the CNS to remove excess cholesterol, which may be toxic to neuronal cells, compounded by a reduction in neuroprotective 3ß,7α-dihydroxycholest-5-en-26-oic acid.

An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway.

In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway.

New methods for analysis of oxysterols and related compounds by LC-MS.

Oxysterols are oxygenated forms of cholesterol or its precursors. They are formed enzymatically and via reactive oxygen species. Oxysterols are intermediates in bile acid and steroid hormone biosynthetic pathways and are also bioactive molecules in their own right, being ligands to nuclear receptors and also regulators of the processing of steroid regulatory element-binding proteins (SREBPs) to their active forms as transcription factors regulating cholesterol and fatty acid biosynthesis. Oxysterols are implicated in the pathogenesis of multiple disease states ranging from atherosclerosis and cancer to multiple sclerosis and other neurodegenerative diseases including Alzheimer's and Parkinson's disease. Analysis of oxysterols is challenging on account of their low abundance in biological systems in comparison to cholesterol, and due to the propensity of cholesterol to undergo oxidation in air to generate oxysterols with the same structures as those present endogenously. In this article we review the mass spectrometry-based methods for oxysterol analysis paying particular attention to analysis by liquid chromatography-mass spectrometry (LC-MS).

Lipid metabolism in mycobacteria--Insights using mass spectrometry-based lipidomics.

Diseases including tuberculosis and leprosy are caused by species of the Mycobacterium genus and are a huge burden on global health, aggravated by the emergence of drug resistant strains. Mycobacteria have a high lipid content and complex lipid profile including several unique classes of lipid. Recent years have seen a growth in research focused on lipid structures, metabolism and biological functions driven by advances in mass spectrometry techniques and instrumentation, particularly the use of electrospray ionization. Here we review the contributions of lipidomics towards the advancement of our knowledge of lipid metabolism in mycobacterial species.