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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
Transcript
Page 1: Brain endogenous Liver X Receptor ligands selectively promote midbrain ... · Brain endogenous Liver X Receptor ligands selectively promote midbrain neurogenesis ... the values are

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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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Supplementary Figure 4

a b

0

0.2

0.4

0.6

0.8

1

1.2

1.4

src

siR

NA

siLX

siLX

siLX

src

siR

NA

siLX

siLX

siLX

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

siLX

siLX

src

siR

NA

siLX

siLX

siLX

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

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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

***

*****

**

*

*

*

**

*

*

*

*

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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

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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


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