View
8
Download
0
Category
Preview:
Citation preview
Brain endogenous Liver X Receptor ligands selectively promote midbrain
neurogenesis
Spyridon Theofilopoulos1, Yuqin Wang2, Satish Srinivas Kitambi1, Paola Sacchetti1,7, Kyle M
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-
Epoxycholesterol
C-4-en-3,6-dione 532.3898 0.160 9.79 C-4-en-3,6-dionee 6-Oxocholestenonee Autoxidation, Rt, MSn C-4-en-22R-ol-3-one 534.4054 0.004 6.19 C-5-en-3β,22R-diol 22R-Hydroxycholesterol Rt, MSn C-4-en-24S-ol-3-one 534.4054 0.028 7.18/
7.58 C-5-en-3β,24S-diol 24S-Hydroxycholesterol
Appear as syn and anti conformers, Rt, MSn
C-4-en-25-ol-3-one 534.4054 0.011 7.43 C-5-en-3β,25-diol 25-Hydroxycholesterol Rt, MSn C-4-en-26-ol-3-one 534.4054 0.012 8.46/
8.88 C-5-en-3β,26-diolf 27-Hydroxycholesterolf
Appear as syn and anti conformers, Rt, MSn
C-4-en-7β-ol-3-one
534.4054
0.064 9.14/ 9.53
C-5-en-3β,7β-diol
7β-Hydroxycholesterol
Autoxidation, appears as syn and anti conformers, Rt, MSn
C-4-en-7α-ol-3-one 534.4054 0.045 9.62 C-5-en-3β,7α-diol 7α-Hydroxycholesterolg Rt, MSn C-7-en-x-ol-3-one
534.4054
0.123 9.29/ 9.81
C-7-en-3β,x-diol
Hydroxylathosterol
No reference, MSn
Nature Chemical Biology doi:10/1038/nchembio.1156
C-4-en-6-ol-3-one
534.4054
1.234
9.95
C-4-en-6-ol-3-one/C-3β,5α,6β-triol/C-3β-ol-5,6-epoxideh
6-hydroxycholest-4-en-3-one/cholestane-3β,5α,6β-triol/5,6-epoxycholesterol
Autoxidation, Rt, MSn
C-4-en-24,26-diol-3-one
550.4003
0.012 4.82
C-5-en-3β,24,26-triol
24,27-Dihydroxycholesterol No reference, MSn
Aldol 552.4160 0.096 9.62 Aldoli Aldoli MSn
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
m/z 407.28 ± 0.02 (upper trace, biologically active fraction 21_33; lower trace, authentic [M-
H]- ion of cholic acid). (b, right) Mass spectra of the components eluting at 7.98 (upper trace,
biologically active fraction 21_33) and 7.97 (lower trace, authentic cholic acid) min, showing
similar mass and isotopic pattern. (c, d) Comparison of the transcriptional activation capacity
of 22-HC and several bile acids (10 µM for 24 h) on Lxrα (c) and Fxr (d) in luciferase
reporter assays. Firefly luciferase activity was normalized to Renilla luciferase activity, and
the values are expressed as fold activation over the normalised basal LXRE-Luc activity set
to 1. (e) CA did not regulate Lxr reporters in liver HepG2 cells, while 22-HC did. (f) CA
activated Fxr reporters in HepG2 cells in a similar manner as other Fxr ligands such as CDCA
and LCA. (g) Dose-response curves of the activational capacity of CA and 22-HC on Lxrα.
(h) While CA increased the expression of the Lxr target gene, abca1, equimolar
concentrations of two closely related bile acids, CDCA and LCA, had the opposite effect.
Data represent mean ± SEM (n=3), *P<0,05, **P<0,01 compared to vehicle treatment.
Nature Chemical Biology doi:10/1038/nchembio.1156
Supplementary Figure 2: Characterization of the activity of endogenous Lxr ligands and
GGPP on Lxrs and other nuclear receptor reporters
(a) (i) Reconstructed ion-chromatogram (RIC) for GP-derivatised 24S,25-epoxycholesterol
(m/z 532.3898 ± 5 ppm). (ii) MS2 (532�) of the component eluting at 6.68 min in RIC of m/z
532.3898. (iii) MS3 (532�453�) spectrum of the component eluting at 6.68 min in RIC of
m/z 532.3898. The peak at 6.89 min gives identical spectra indicating that these are the syn
and anti conformers of the derivative. The peak at 7.39 min is 24-oxocholesterol, an
isomerisation product of 24S,25-epoxycholesterol formed during the derivatisation process.
(b) Comparison of the transcriptional activation capacity of several oxysterols and sterols (10
µM) identified by LC-MSn on Lxrα. Cells were stimulated for 24h with vehicle, 22-HC, CA
or 24,25-EC (10 µM). For all luciferase assays, firefly luciferase activity was normalized to
Renilla luciferase activity, and the values are expressed as fold activation over the normalised
basal LXRE-Luc activity set to 1. (c) Dose-response curves of the activational capacity of
24,25-EC and 22-HC on Lxrα. (d,e) Analysis of luciferase activity of LXRE-luciferase
constructs in SN4741 cells transfected with wild-type Lxrα, Lxrβ or the respective deletion
mutants Lxrα∆AF-2 and Lxrβ∆AF-2. (f) GGPP blocked Lxrβ-dependent CA- or 24,25-EC-
induced luciferase activity. (g) The Lxr-specific inhibitor geranylgeranyl pyrophosphate
(GGPP) did not affect Fxr- or Rxrα- or Nurr1-dependent transcriptional activity. Data
represent mean ± SEM (n=3), *P<0,05, **P<0,01 compared to vehicle treatment.
Supplementary Figure 3: Regulation of CA- or 24,25-EC-induced Lxr activation by N-
CoR, Src-1 and Rxr.
(a, b) Analysis of luciferase activity of LXRE-luciferase constructs in SN4741 cells
transfected with Lxrα (a) or Lxrβ (b), and treated with the Rxr ligand 9-cis-RA, as indicated.
(c, d) Analysis of luciferase activity of LXRE-luciferase constructs in SN4741 cells
Nature Chemical Biology doi:10/1038/nchembio.1156
transfected with Lxrα (c) or Lxrβ (d), and in the presence or absence of the co-activator Src-1,
as indicated. (e, f) Analysis of luciferase activity of LXRE-luciferase constructs in SN4741
cells transfected with Lxrα (e) or Lxrβ (f), and in the presence or absence of the co-repressor
N-CoR, as indicated. For all assays, cells were stimulated for 24h with vehicle, 22-HC, CA,
24,25-EC, or 9-cis-RA (10µM). Data represent mean ± SEM (n=3), *P<0,05, **P<0,01
compared to vehicle treatment.
Supplementary Figure 4: siRNAs against Lxrα or Lxr β specifically downregulate the
expression of each receptor.
SiRNAs against Lxrα (a) or Lxrβ (b) specifically blocked the mRNA expression of their
respective receptors by 60-70% compared to scramble siRNA in either control or CA-treated
cells.
Supplementary Figure 5: 24,25-EC and CA require Lxrs to regulate transcript levels of
DA neuronal markers, and diencephalic TH+ cells in zebrafish.
(a) Lxr morpholinos (lxrMO) abolished the CA- or 24,25-EC-induced increases in th1, nurr1,
and dat at 3 dpf. (b) LxrMO did not decrease the number of TH+ cells but abolished the
ligand-induced increase in TH+ cell numbers. Representative pictures are shown in (c). Data
represent mean ± SEM (n=3), *P<0,05, **P<0,01 compared to each respective vehicle
treatment.
Supplementary Figure 6: Effect of CA and 24,25-EC on TH+ cell numbers in mouse
organotypic ‘open-book’ cultures.
(a) Image of an E11.5 ‘open-book’ culture after 2 days in vitro. The ventral part where
individual TH+ cells were identified and quantified is marked. (b) Representative pictures of
Nature Chemical Biology doi:10/1038/nchembio.1156
the ventral part of ‘open-book’ cultures [outlined in (a)] treated with vehicle, CA or 24,25-
EC. (c) Quantitation of the TH+ cells shows that only 24,25-EC increased the number of TH+
neurons. Higher magnification segments of the cultures are shown in (d). Data represent
mean ± SEM (n=3), *P<0,05, compared to vehicle treatment.
Supplementary Figure 7: CA increases the number of Nkx6.1+ red nucleus progenitor
cells.
(a) CA increased the number of Nkx6.1+ cells in E11.5 VM cultures, whereas 24,25-EC had
no effect. Representative pictures shown in (b). It should be noted that Nkx6.1 antibodies
label both red nucleus progenitors and Islet1+ neurons (trochlear and oculomotor neurons).
Since the number of Islet1+ cells was not regulated by CA, changes in the number of Nkx6.1
directly reflect changes in the number of red nucleus progenitors. (c) Sumary of the different
activities of cholic acid (CA) and 24,25-epoxycholesterol (24,25-EC) on midbrain progenitor
cells. CA specifically increases the number of Red Nucleus neurons by decreasing cell death
and increasing neurogenesis. 24,25-EC specifically increases the number of midbrain DA
neurons by selectively inducing DA neurogenesis (“S”: survival, “N”: neurogenesis).
Nature Chemical Biology doi:10/1038/nchembio.1156
Supplementary Figure 1
f
0
2
4
6
8
10
12
-2 -1 0 1
Fol
d ac
tivat
ion
concentration (10ˆn µM) log scale
LXRα
22-HCCA
a b
e
g
50 VMs HPLC fractions pools
Acidic fraction pools (7)
Neutral fraction
+ +56 individual acidic HPLC samples
0
2
4
6
8
10
12
veh veh 22-HC CA veh 22-HC CA
- LXRα LXRβ
Fol
d ac
tivat
ion
HepG2 cells*
*
0
2
4
6
8
10
12
14
veh veh 22-HC CA CDCA LCA
- FXR
Fol
d ac
tivat
ion
HepG2 cells
*
** **
d
0
2
4
6
8
10
12
vehicle vehicle 22-HC CA CDCA LCA
- LXRα
Fol
d ac
tivat
ion
*
*
SN4741 cells
SN4741 cells
c
0
0.5
1
1.5
2
2.5
vehicle vehicle 22-HC CA CDCA LCA
- FXR
*F
old
activ
atio
n
**SN4741 cells
0
0.5
1
1.5
2
vehicle CA 50 µM
LCA 50 µM
CDCA 50 µM
AB
CA
1 ex
pres
sion
(fol
d in
crea
se)
*
*
*
vehicle
veh CA50 µM
LCA50 µM
CDCA50 µM
h SN4741 cells
Nature Chemical Biology doi:10/1038/nchembio.1156
Supplementary Figure 2
f
a
c
02468
10121416
vehicle 22-HC CA 24,25-EC
Fol
d ac
tivat
ion
SN4741 cells-
LXRα WT
LXRα∆AF2
**
**
**
**
0
2
4
6
8
10
12
14
16
-4 -3 -2 -1 0 1
Fol
d ac
tivat
ion
concentration (10^n µM) log scale
LXRα
22-HC24,25-EC
SN4741 cells
0
2
4
6
8
10
12
14
16
vehi
cle
vehi
cle
5,6-
24,2
5-di
EC
22-H
C
24(S
)-H
C
25-H
C
27-H
C
24,2
5-E
C
desm
oste
rol
- LXRα
Fol
d ac
tivat
ion *
**
****
* *
SN4741 cellsb
d
0123456789
10
vehicle 22-HC CA 24,25-EC
Fol
d ac
tivat
ion
-
LXRβ WT
LXRβ∆AF2
**
****
*
eSN4741 cells
0123456789
10
vehicle vehicle 22-HC CA 24,25-EC
- LXRβ
Fol
d ac
tivat
ion
- GGPP
+ GGPP
**
*
****
LXRβ
0
1
2
3
vehicle vehicle CDCA
- FXR
Fol
d ac
tivat
ion
-GGPP
+GGPP
0
1
2
3
4
5
vehicle vehicle 9-cis-RA
- RXRα
Fol
d ac
tivat
ion
-GGPP
+GGPP
f g
iiii ii
0
1
2
3
4
vehicle vehicle 9-cis-RA
- Nurr1
Fol
d ac
tvat
ion
-GGPP
+GGPP
Nature Chemical Biology doi:10/1038/nchembio.1156
0
5
10
15
20
vehicle vehicle 22-HC CA 24,25-EC
- LXRa
Fol
d ac
tivat
ion
-
+ 9-cis-RA
LXRα
0
2
4
6
8
10
12
14
16
18
vehicle vehicle 22-HC CA 24,25-EC
- LXRb
Fol
d ac
tivat
ion
-
+ 9-cis-RA
LXRβ
*
*
* **
*
**
*
Supplementary Figure 3
02468
101214161820
vehicle vehicle 22-HC CA 24,25-EC
- LXRβ
Fol
d ac
tivat
ion
- SRC-1
+ SCR-1
**
**
**
**
0
5
10
15
20
25
30
35
vehicle vehicle 22-HC CA 24,25-EC
- LXRα
Fol
d ac
tivat
ion
- SRC-1
+ SRC-1**
**
**
**
0123456789
vehicle vehicle vehicle 22-HC CA 24,25-EC
- + LXRα + LXRα + N-CoR
Fol
d ac
tivat
ion **
0
1
2
3
4
5
vehicle vehicle vehicle 22-HC CA 24,25-EC
- + LXRβ + LXRβ + N-CoR
Fol
d ac
tivat
ion
**
a b
c d
e f
Nature Chemical Biology doi:10/1038/nchembio.1156
Supplementary Figure 4
a b
0
0.2
0.4
0.6
0.8
1
1.2
1.4
src
siR
NA
siLX
Rα
siLX
Rβ
siLX
Rα
+β
src
siR
NA
siLX
Rα
siLX
Rβ
siLX
Rα
+β
vehicle 50 µM CA
LXRα
expr
essi
on
(fol
d in
crea
se)
****
****
scr
siR
NA
scr
siR
NA
0
0.2
0.4
0.6
0.8
1
1.2
src
siR
NA
siLX
Rα
siLX
Rβ
siLX
Rα
+β
src
siR
NA
siLX
Rα
siLX
Rβ
siLX
Rα
+β
vehicle 50 µM CA
LXRβ
expr
essi
on
(fol
d in
crea
se)
** **** **
scr
siR
NA
scr
siR
NA
Nature Chemical Biology doi:10/1038/nchembio.1156
Supplementary Figure 5
cMO+veh lxrMO+veh
lxrMO+24,25-ECcMO+24,25-EC
lxrMO+CAcMO+CA
b
c
0
5
10
15
20
Num
ber
of T
H+
neu
rons * *
a
Fol
d ch
ange
in e
xpre
ssio
n
0
0.5
1
1.5
2
2.5
3
cMO
lxrM
O
cMO
lxrM
O
cMO
lxrM
O
cMO
lxrM
O
cMO
lxrM
O
cMO
lxrM
O
cMO
lxrM
O
cMO
lxrM
O
cMO
lxrM
O
veh CA 24,25-EC
veh CA 24,25-EC
veh CA 24,25-EC
th1 nurr1 dat
***
*****
**
*
*
*
**
*
*
*
*
Nature Chemical Biology doi:10/1038/nchembio.1156
vehicle CA 24,25-ECa
Supplementary Figure 6
b
0
200
400
600
800
vehicle CA 24,25-EC
TH
+ c
ells
(per
ope
n-bo
ok c
ultu
re)
*
TH/Tuj1c d
TH
Nature Chemical Biology doi:10/1038/nchembio.1156
Supplementary Figure 7
b
a
vehicle CA 24,25-EC
Nkx6.1
% N
kx6.
1+ c
ells
/Hoe
chst
0
5
10
15
veh CA 24,25-EC
cRed Nucleus neurons
Dopaminergic neurons
VM progenitor cell
CA
24,25-EC
Nature Chemical Biology doi:10/1038/nchembio.1156
Recommended