Журнал гликомики и липидомики
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ISSN: 2153-0637
ISSN: 2153-0637
Анна Мельон
Дефицит цитохрома P450 (CYP) 7B1, также известного как оксистерол 7α-гидроксилаза, у людей приводит к наследственной спастической параплегии типа 5 (SPG5) и в некоторых случаях у младенцев к заболеванию печени. SPG5 с медицинской точки зрения характеризуется потерей двигательных нейронов в кортикоспинальном тракте. В попытке лучше понять фундаментальную биохимию этого расстройства мы расширили наше предыдущее профилирование содержания оксистеролов в мозге и плазме мышей с нокаутом Cyp7b1 (-/-), включив, среди прочих стеролов, 25-гидроксилированные метаболиты холестерина. Хотя уровни холестерина в мозге не различаются между мышами дикого типа (wt) и нокаутированными мышами, мы обнаружили, используя методологию маркировки заряда в сочетании с жидкостной хроматографией-масс-спектрометрией (ЖХ-МС) и многоступенчатой фрагментацией (МСn), что в мозге и плазме мышей Cyp7b1-/- происходит накопление субстрата CYP7B1 25-гидроксихолестерина (25-HC). Как сообщалось ранее, уровни (25R)26-гидроксихолестерина (26-HC), 3β-гидроксихолест-5-ен-(25R)26-овой кислоты и 24S,25-эпоксихолестерина (24S,25-EC) также повышены в мозге и плазме. Известно, что оксистеролы боковой цепи, включая 25-HC, 26-HC и 24S,25-EC, связываются с INSIG (индуцированный инсулином ген) и ингибируют обработку SREBP-2 (белок-2, связывающий регуляторный элемент стерола) в его активную форму как главный регулятор биосинтеза холестерина. Мы предполагаем, что концентрация холестерина в мозге мыши Cyp7b1-/- поддерживается за счет баланса сниженного метаболизма, как следствие потери CYP7B1, со сниженным биосинтезом. Мышь Cyp7b1-/- не показывает двигательного дефекта; является ли дефект у людей следствием менее эффективного гомеостаза холестерина в мозге, еще предстоит выяснить. Цитохром P450 (CYP) 7B1 (семейство цитохромов P450 7 подсемейство B член 1) был впервые идентифицирован в 1995 году и, как было обнаружено, в первую очередь экспрессируется в мозге грызунов. CYP7B1 — это оксистерол- и стероид- 7α-гидроксилаза, принимающая в качестве субстратов множество оксистеролов и холестериновых кислот, а также стероиды, включая дегидроэпиандростерон (ДГЭА). У людей дефицит фермента впервые был выявлен у десятинедельного мальчика presenting with severe liver disease. In more recent studies, the treatment of another infant with this enzyme deficiency with chenodeoxycholic acid has proved successful in resolving liver disease [6]. CYP7B1 is expressed in human hippocampus and interestingly CYP7B1 mRNA is significantly reduced in dentate neurons from Alzheimer’s disease subjects. In mice, deletion of Cyp7b1 results in a mild phenotype despite elevation of tissue and plasma levels of its oxysterol substrates 25-hydroxycholesterol (25-HC) and 26-hydroxycholesterol, presumably the 25R-epimer, (26-HC, also known as 27-hydroxycholesterol, see Supplementary Materials, Table S1 for abbreviations, common and systematic names) [8]. In light of this mouse data, it was surprising when Tsaousidou et al. found in 2008 that sequence alterations in CYP7B1 were associated with hereditary spastic paraplegia type 5 (SPG5) in humans [9]. Subsequent studies have confirmed patients suffering from SPG5 have a metabolic phenotype characteristic of inactive CYP7B1. In an effort to understand the differences between mouse and human with respect to defective CYP7B1, we embarked on a sterolomic investigation of mouse brain and plasma exploiting enzyme-assisted derivatization for sterol analysis (EADSA) and liquid chromatography–mass spectrometry (LC–MS) with multistage fragmentation (MSn). We previously found that in Cyp7b1 knockout (Cyp7b1-/-) mouse brain the concentration of cholesterol was similar to that of the wild-type (wt, Cyp7b1+/+), as were the levels of 24S-hydroxycholesterol (24S-HC) [14]. On the other hand, concentrations of (25R)26-hydroxycholesterol (26-HC), 3β-hydroxycholest-5-en-(25R)26-oic acid (3β-HCA) and 24S,25-epoxycholesterol (24S,25-EC) were elevated, presumably being CYP7B1 substrates. Now, delving deeper into the sterolome, we reveal that 25-HC and 26-hydroxydesmosterol (26-HD) are also elevated in Cyp7b1-/- mouse brain, whereas 7α,25-dihydroxysterols are reduced in abundance, but other oxysterols 24R-hydroxycholesterol (24R-HC), 7α- and 7β-hydroxycholesterol (7α-HC, 7β-HC) do not change in concentration between the two genotypes. We suggest that elevated levels of 25-HC, 26-HC and 24S,25-EC in brain reduce the expression of cholesterol biosynthetic genes by inhibiting the processing of SREBP-2 (sterol regulatory element-binding protein 2) to its active form as a master transcription for the mevalonate pathway, thereby reducing cholesterol synthesis and compensating for its reduced metabolism (via the CYP7B1 pathway), thus maintaining cholesterol levels in Cyp7b1-/- mouse brain at wild-type levels. 25-HC, 24S,25-HC, 3β-HCA, and in some studies 26-HC, have all been found to be ligands to the liver X receptors (LXRα, NR1H3; LXRβ, NR1H2), activation of which increases the expression of ATP-binding cassette transporter A1 (ABCA1) and apolipoprotein E (APOE), transporter and carrier proteins important for maintaining correct sterol levels in neurons [19] and avoiding overload by potentially toxic oxysterols. Brain and plasma samples were from male mice of 13 and 23 months of age. Cyp7b1-/- and Cyp7b1+/+ mice were littermates generated from Cyp7b1+/- crosses at the University of Edinburgh animal facilities. All mice were housed under standard conditions (7:00 am to 7:00 pm light/dark cycle, 21 °C) with food and water available ad libitum. Tissue sampling was performed under the aegis of the UK Scientific Procedures (Animals) Act, 1986, amended in 2012 to comply with the European Directive 2010/63/EU. The study was conducted under PPL No.70/7870 with prior approval from the University of Edinburgh Animal Welfare and Ethical Review Body. All mice were sacrificed in the morning by cervical dislocation, trunk blood collected and brains removed, frozen on powdered dry ice and stored at −80 °C. Sterols including oxysterols were extracted from brain as described in. In brief, mouse brain was homogenised in ethanol containing isotope-labelled internal standards and oxysterol- and cholesterol-rich fractions separated by solid phase extraction (SPE) on a reversed phase C18 column. The oxysterol-rich fraction was then divided into two aliquots (a and b) and the first treated with cholesterol oxidase enzyme from Streptomyces sp. to oxidise 3β-hydroxy-5-ene groups to 3-oxo-4-ene equivalents, suitable for subsequent derivatisation with the Girard P (GP) reagent (i.e., fraction a). The second aliquot of the oxysterol fraction was treated with GP reagent directly, in the absence of cholesterol oxidase (i.e., fraction b). This allowed the differentiation of oxysterols with a native 3-oxo-4-ene structure (fraction b) from those with a 3β-hydroxy-5-ene structure (i.e., (fraction a)–(fraction b)). The cholesterol-rich fraction was treated separately, but in the same way as the oxysterol-rich fraction. Plasma samples were prepared as described in Autio et al. and Crick et al. by extraction into ethanol followed by SPE to separate cholesterol- and oxysterol-rich fractions . The oxysterols were then derivatised with GP reagent with (fraction a), or without (fraction b), prior oxidation by cholesterol oxidase. For plasma analysis, two GP reagents were used [2H0]GP and [2H5]GP with either fraction a or with fraction b, respectively. This allowed the duplex LC-MS analysis of oxysterol fractions prepared with (fraction a) or without (fraction b) treatment with cholesterol oxidase The oxidised/derivatised oxysterol-rich fractions were analysed by LC-MS (MSn) using an Ultimate 3000 LC system (Thermo Fisher Scientific, Loughborough, UK) and LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Loughborough, UK) as described in Meljon et al. and Crick et al. . In brief, GP-derivatised oxysterols were separated on a reversed phase Hypersil Gold C18 column (Thermo Fisher Scientific) using a methanol/acetonitrile/0.1% formic acid gradient. The eluent was directed to an electrospray ionisation source (ESI) and analysed by high-resolution (60,000 at m/z 400) MS and MS3 ([M]+→[M-Py]+→, where “-Py” corresponds to the loss of the pyridine group from the molecular ion M+) scans performed in parallel in the Orbitrap and LTQ linear ion-trap, respectively. Quantification was performed using the isotope dilution method.