Cortisol +RU486 (50nM)

-5

0

5

10

15

20

25

n=3

Brd

U+

cel

ls (

% c

hang

e fr

om v

eh)

MAP2+,

NeuN+,

tuj1+

Dcx+, PSA-

NCAM+

Lie et al. 2004

�MR activation mediates the effects of low cortisol concentrations, while high

concentrations of cortisol activate the GR in human hippocampal progenitor cells.

� Cortisol decreases human hippocampal neurogenesis via GR-dependent

upregulation of SGK1.

� SGK1 regulates GR function by phosphorylation.

Cortisol - 1nM 10nM 100nM 1µM 10µM 100µM

Cortisol +Spironolactone (1µM)

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*Cortisol - 1nM 10nM 100nM 1µM 10µM 100µM

*

CORTISOL DECREASES HIPPOCAMPAL NEUROGENESIS BY

REGULATING THE ENZYME SGK1C. Anacker1, P.A. Zunszain1, A. Cattaneo2, K. Musaelyan1, S. Thuret1, J. Price1, C.M. Pariante1.

1Centre for the Cellular Basis of Behaviour, Institute of Psychiatry, King’s College London, United Kingdom

2 IRCCS San Giovanni di Dio, Genetics Unit, Brescia, Italy.

BACKGROUND

• Glucocorticoids and adult hippocampal neurogenesis in depression

� Glucocorticoids (cortisol in humans) can activate

two intracellular receptors:

� Mineralocorticoid Receptor (MR), high affinity

� Glucocorticoid Receptor (GR), low affinity

HYPOTHESIS

RESULTS

• Bimodal effects of cortisol on cell proliferation

CONCLUSIONS� MR activation by low concentrations of cortisol increases proliferation, while GR activation by high concentrations decreases proliferation

� GR-dependent activation of SGK1 expression mediates the cortisol-induced reduction in neurogenesis

� Cortisol-induced SGK1 expression activates the GR by phosphorylation at the GR serine residues S203 and S211

DISCLOSURE: This poster is financially supported by the European Union Framework7, the Medical Research Council UK and an NIHR BRC studentship in Biomedical and Mental Health to C. Anacker. J. Price acts as a consultant for ReNeuron, UK.

• Cortisol decreases neuronal differentiation

� The enzyme, serum- and glucocorticoid-regulated kinase 1 (SGK1) mediates some

effects of glucocorticoids on working memory, oligodendrocyte morphology and

glucocorticoid responsiveness. (Yuen et al., 2011; Miyata et al., 2011, Luca et al., 2009)

� GR function is critically regulated by phosphorylation at the serine residues S203,

S211 and S226.

a. b.

Figure 2. (a) The MR-antagonist, spironolactone, blocks the increase in proliferation. (b) The GR-

antagonist, RU486, blocks the decrease in proliferation. *p<0.05

• GR activation by cortisol increases SGK1 expression

Figure 4. (a) High cortisol concentrations increase SGK1 expression. (b) The effect at 12hrs is blocked

by RU486 (50nM). *p<0.05, **p<0.01

• SGK1 mediates the cortisol-induced reduction in neurogenesis

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cells

(%

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from

veh

)

Cortisol - 1nM 10nM 100nM 1uM 10uM 100uM

**

**

Hoechst BrdU

MRGR

SGK1 inhibitor SGK1 inhibitor

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n=3

100µMCortisol 100µM

100nM_

MA

P2+

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rons

(% c

hang

e)

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-5

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n=4

_ 10nM 50nM 100nM

Cortisol 100µM 100µM 100µM100µM

Brd

U+ c

ells

(%

cha

nge)

** *

1h 3h 12h 72h1.01.11.21.31.41.51.61.71.81.92.0

Cort100µM

* ** *

n=6

Cort100nM

fold

cha

nge

gene

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

RU486 (50nM)

Spironolactone (1µM)

_ _

_

_

__ +

+

Cort 100nM

Cort 100µM

***

n=3fold

cha

nge

gene

exp

ress

ion

Dcx & MAP2

Immunostaining

&

Cell counting

BrdU

Immunostaining

Quantitative

Real-Time PCR

Western Blot

METHODS

• Proliferation assay

• Differentiation assay

7 days Differentiation

1.

2.

3 days Proliferation

Treatment

Treatment

4hrs

BrdU

Treatment

(Cortisol, Spironolactone 1µM [MR antagonist],

RU486 50nM [GR antagonist], GSK650394 [SGK1 inhibitor])

3days Proliferation

� Human embryonic hippocampal progenitor cell line HPC03A/07 (ReNeuron, UK)

Treatment

� Adult hippocampal neurogenesis has recently

been demonstrated to contribute to the

development of depressive symptoms in

situations of stress (Snyder et al., 2011)

Figure 5. The SGK1-inhibitor, GSK650394, counteracts the effects of cortisol (100µM) on

(a) proliferation and (b) neuronal differentiation.*p<0.05, **p<0.01

a. b.

a. b.

• SGK1 phosphorylates the GR

0.0

0.5

1.0

1.5

2.0

_ + + _

_ + +_

n=5

fold

indu

ctio

n

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**

_ + + _

_ + +_

*

fold

indu

ctio

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4

*

_ + + _

_ + +_

*

Cort (100uM)

SGK1 inhibitor(100nM)

fold

indu

ctio

n

S203

GR

S211GR

0

1

2

3

4

*

_ + + _

_ + +_

fold

indu

ctio

n

S226GR

GR

ACTB

Figure 6. The SGK1-inhibitor, GSK650394, counteracts the cortisol-induced GR phosphorylation at S203

and S211, but not at S226. *p<0.05, **p<0.01

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low Cortisol(100nM)

RU486 (50nM)

Spironolactone (1 µM)

_ _

_

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high Cortisol(100µM)

** n=3**

MA

P2+

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ls (

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e)

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low Cortisol(100nM)

high Cortisol(100µM)

n=3

MA

P2+

cel

ls (

% c

hang

e)

a. b.

Hoechst

MAP2

2.1.

Figure 3. (a) Cortisol treatment during proliferation and differentiation (paradigm 1 ) decreases

neuronal differentiation at both 100nM and 100µM via MR and GR-dependent effects, respectively.

(b) Treatment only during the differentiation phase (paradigm 2 ) has no effect. **p<0.01

� Increased levels of glucocorticoid hormones are commonly observed in situations

of chronic stress and in depression.

� High levels of glucocorticoid hormones decrease adult hippocampal neurogenesis

Figure 1. Low concentrations of cortisol (100nM) increase proliferation, while high

concentrations (100µM) decrease proliferation.**p<0.01

• MR- and GR-dependent effects of cortisol on proliferation