Brain endogenous Liver X Receptor ligands selectively promote midbrain neurogenesis Spyridon Theofilopoulos 1 , Yuqin Wang 2 , Satish Srinivas Kitambi 1 , Paola Sacchetti 1,7 , Kyle M Sousa 1,8 , Karl Bodin 1 , Jayne Kirk 3 , Carmen Saltó 1 , Magnus Gustafsson 1 , Enrique M Toledo 1 , Kersti Karu 4 , Jan-Åke Gustafsson 5 , Knut R. Steffensen 6 , Patrik Ernfors 1 , Jan Sjövall 1 , William J Griffiths 2 & Ernest Arenas 1 1 Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, 17177, Sweden. 2 Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, U.K. 3 Waters UK Ltd, Atlas Park, Simonsway, Manchester, M22 5PP, U.K. 4 The School of Pharmacy, 29-39 Brunswick Square, London WCN 1AX, U.K. 5 Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX 77204, U.S.A. 6 Department of Biosciences and Nutrition, Center for Biosciences, Novum, 14186 Stockholm, Sweden 7 Current address: Department of Biological Sciences, Mount Holyoke College, 50 College Street, South Hadley, Massachusetts, 01075, U.S.A. 8 Current address: Department of Gene Regulation & Drug Discovery, City of Hope – Beckman Research Institute, Duarte, CA 91010 U.S.A. Nature Chemical Biology doi:10/1038/nchembio.1156
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Brain endogenous Liver X Receptor ligands selectively promote midbrain
Sousa1,8, Karl Bodin1, Jayne Kirk3, Carmen Saltó1, Magnus Gustafsson1, Enrique M Toledo1,
Kersti Karu4 , Jan-Åke Gustafsson5, Knut R. Steffensen6, Patrik Ernfors1, Jan Sjövall1,
William J Griffiths2 & Ernest Arenas1
1 Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and
Biophysics, Karolinska Institute, Stockholm, 17177, Sweden.
2Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park,
Swansea SA2 8PP, U.K.
3Waters UK Ltd, Atlas Park, Simonsway, Manchester, M22 5PP, U.K.
4The School of Pharmacy, 29-39 Brunswick Square, London WCN 1AX, U.K.
5Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX
77204, U.S.A.
6Department of Biosciences and Nutrition, Center for Biosciences, Novum, 14186 Stockholm,
Sweden
7Current address: Department of Biological Sciences, Mount Holyoke College, 50 College
Street, South Hadley, Massachusetts, 01075, U.S.A.
8Current address: Department of Gene Regulation & Drug Discovery, City of Hope –
Beckman Research Institute, Duarte, CA 91010 U.S.A.
Nature Chemical Biology doi:10/1038/nchembio.1156
Supplementary Results: Supplementary Table 1: Oxysterols identified by LC-MSn in Ventral Midbrain (VM) of embryonic mouse (E11.5). Vma Identified Structure after Treatment with Cholesterol Oxidase
[M]+ of GPa
m/z
µµµµg/g Rt/min Inferred Structure prior to Treatment with Cholesterol Oxidase
Inferred Compound Trivial Name
Comment/ Parameters for Identification
C-4-en-24S,25-epoxide-3-one
532.3898
0.036 6.68/ 6.89
C-5-en-3β-ol-24S,25-epoxide
24S,25-Epoxycholesterol
Appear as syn and anti conformers, Rt, MSn
C-4-en-3,24-dioneb
532.3898
0.123 7.39
C-5-en-3β-ol-24S,25-epoxide
24S,25-Epoxycholesterol
Rt, MSn
C-4-en-24,25-diol-3-onec
550.4003
0.081 4.42/ 4.97
C-5-en-3β-ol-24S,25-epoxide
24S,25-Epoxycholesterol
Appear as syn and anti conformers, Rt, MSn
C-4-en-24-ol,25-OMe-3-oned
564.4160
0.146
6.06/ 6.43
C-5-en-3β-ol-24S,25-epoxide
24S,25-Epoxycholesterol
Appear as syn and anti conformers, Rt, MSn
0.386 C-5-en-3β-ol-
24S,25-epoxide 24S,25-Epoxycholesterol Total 24S,25-
Experimental methods are fully described in reference 27. Systematic nomenclature adopted according to Lipid Maps http://www.lipidmaps.org/ C = cholestane, a number preceding “en” indicates the location of carbon-carbon double bonds, a number(s) preceding “ol(diol, etc)” or “one” indicates the location of hydroxy and oxo groups, respectively (see C-5-en-3β-ol below). Rt = retention time; No reference = no authentic standard available. a Data for GP derivatives. b Isomerisation product of C-4-en-24S,25-epoxide-3-one. c Hydrolysis product of C-4-en-24S,25-epoxide-3-one. d Alternatively C-4-en-25-ol,24-OMe-3-one, methanolysis product of C-4-en-24S,25-epoxide-3-one. e C-4-en-3,6-dione reacts with GP reagent without oxidation by cholesterol oxidase. f According to systematic nomenclature recommended by the Lipid Maps consortium, hydroxylation of the terminal carbon of cholesterol introducing 25R stereochemistry is at C-26 leading to C-5-en-3β,26-diol. However, the common name is 27-hydroxycholesterol. g Formed enzymatically by CYP7A1 and/or by autoxidation. h C-4-en-6-ol-3-one can be formed from C-3β,5α,6β-triol and C-3β-ol-5,6-epoxides during the cholesterol oxidase/GP derivatisation reaction. i 3β,5β-dihydroxy-B-norcholestane-6β-carboxyaldehyde (aldol) reacts with GP reagent without oxidation by cholesterol oxidase. n MS/MS or MS/MS/MS
HO
123 4 5 6 7
8910
19 1211 1314 15
1617
18 20
21 22
2324 25
26
27
C-5-en-3β-ol
Nature Chemical Biology doi:10/1038/nchembio.1156
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1: Characterization of Lxr-dependent transcriptional activity of
bile acids.
(a) Overview of the chromatographic and fractionation procedure followed in order to
identify endogenous acidic ligands that activate Lxrα and/or Lxrβ in the developing mouse
VM. 56 samples were pooled to 7 groups, analyzed and individual samples of positive pools
were tested for Lxr reporting activity. (b, left) Reconstructed ion-chromatograms (RICs) for