Neurogenesis & stress (EN)

Laura TZEPLAEFF

Neurogenesis and Stress

Ini/a/on à la Démarche Scien/fique en Neurosciences

Eléonore GOVILAS Laura TZEPLAEFF Lucas PHAM-‐VAN QuenAn FUCHS M1 NCI

Supervisors : Alexandre CHARLET José-‐Luis GONZALEZ DE AGUILAR SylveKe CHASSEROT-‐GOLAZ

Project Presenta/on

Introduc/on

Spa/al memory

Stone et al., 2011

Neurogenesis in the dentate gyrus

Chronic stress

2

-‐

Introduc/on

Quiescent glial cell

Ac/vated glial cell

Cellule progenitor

Prolifera/on Survival / Matura/on

Mature neurone

Immature neurone

Differencia/on

Mito/c stage Post-‐mito/c stage

Chronic stress ?

Neuroblaste

-‐

3

0 21 28 > 42 7 Days 3 5

Chronic stress

-‐ -‐ -‐

Introduc/on

10

30

50

70

90

0 1j 3j 1sem 2sem 4 sem 3 mois 6 mois 12 mois

25 %

Temps après division Pourcentage de

néo

-‐neu

rone

s


Ac/vated glial cell

Cellule progenitor


Mature neurone

Immature neurone

Differencia/on


Neuroblaste

3

0 21 28 > 42 7 Days 3 5 100%

Neo-‐neurones rate 25% 100%

Adapté de Kempermann et al., 2003 Snyder et al., 2009

CorrelaAon between neurogenesis and :

•  Survival rate : 85 %

•  Proliféra/on rate : 19 %

100%

Introduc/on

0 10 20 30 40 50 60 70 80 90 100

10% 0 -‐ 10% 0%

Glucocor/coid receptor expression levels during neurogenesis

50%

Garcia et al., 2004

Pourcentage of GR+ neu

rone

s

4

Cellule progenitor


Mature neurone

Immature neurone

Differencia/on


Neuroblaste

0 21 28 > 42 7 Days 3 5 100%

Neo-‐neurones rate 25% 100%


Ac/vated glial cell

Introduc/on

0 21 28 > 42 7 3 5

0 10 20 30 40 50 60 70 80 90 100

10% 0 -‐ 10% 0%

50%

Garcia et al., 2004

25% 100%

4

Pourcentage of GR+ neu

rone

s

Glucocor/coid receptor expression levels during neurogenesis

Neo-‐neurones rate

Cellule progenitor

Mature neurone

Immature neurone

Differencia/on


Neuroblaste

Days



Ac/vated glial cell

Introduc/on


Ac/vated glial cell

Progenitor cell

Mature neurone

Immature neurone


Neuroblaste

During the neurogenesis ? 5

Prolonged stress

Glucocor/coides

Hippocampal neurons death Stein-Behrens et al. 1994 Lupien et al. 1998

Introduc/on

•  Does GR reseptors play a role in neo-‐neurones viability ?

50% GR+

6

GR = GlucocorAcoid Receptors


Ac/vated glial cell

Progenitor cell

Mature neurone

Immature neurone


Neuroblaste

Introduc/on

Neuroblaste

GR+

GR-‐

?

•  Does GR reseptors play a role in neo-‐neurones viability ? •  Are GR+ cells tag for death ?

6

Mature neurone

Immature neurone


Ac/vated glial cell

Progenitor cell



Introduc/on

Neuroblaste

GR+

GR-‐

?


•  Does GR reseptors play a role in neo-‐neurones viability ? •  Are GR+ cells tag for death ? •  Can chronic stress increase cell death via the GR receptor during survival satge ?

Stress chronique

?

Surval / Matura/on

6


Ac/vated glial cell

Progenitor cell

Mature neurone

Immature neurone


Impact of chronic stress on adult hippocampal neurogenesis:

Regula/on of neuroblas/c survival by glucocor/coids

7

Project

Overview

I.   Does GR expresion influence the neuroblas/c survival rate in chronic stress condi/on? a)  NeuroblasAc GR expression in rats b)  GR expression and neuroblasAc survival rate → in-‐vivo experimenta/ons

II.   Is the neuroblas/c pro-‐appopto/c effect caused by direct ac/on of glucocor/coides on GR ? a)  GR acAvaAon and neuroblasAc appoptosis b)  Traitement using a GR antagonist → in-‐vitro experimenta/ons

III.   Does chronic stress have an impact on cogni/ve process related to hippocampal neurogenesis : Spacial memory a)  Chronic stress effect on a sAmulated neurogenesis b)  Consequences on spaAal memory → in-‐vivo experimenta/ons

8

Social defeat chronic stress protocol

Cage with perforated separa/on

9

Dominant

24 hours before the experimentaAon

1 10 0

Social defeat

-‐1

Days Dominanted

5 minutes in the same compartment

Every day :

Blood sample of dominated rat

Separeted the rest of the day

I. a) Neuroblas/c GR expression in rat

Mesurement of glucocor/coide level in plasma using HPLC

ConcentraAon plasmaAque totale en corAcostérone (CORT) chez les rats stressés et non-‐stressés

0 1 2 4 6 8 10

Concen

tra/

on en CO

RT

Etat Basal

Stress Chronique

Jours

GROUPE 2: Control

Fixa/on

Coupe au cryostat

Quan/fica/on

-‐5 0 12 jours

GROUPE 1: Chronically stressed

Immunofluorescence

•  NesAn •  DoublecorAne •  CalréAnine •  GR

Microscope confocal

10


GR + NesAne – DoublecorAne + CalréAnine –

Neuroblaste

GR –

10

Fixa/on

Brain slides (cryostat)

•  NesAne •  DoublecorAne •  CalreAnine •  GR

Quan/fica/on

Confocal Microscope

-‐5 0 Days

Immunohistochimistry

12

GROUPE 1: Chronically stressed

GROUPE 2: Control


Garcia et al., 2004

Modified from Garcia et al., 2004 & Guidoj et al., 2013

Neuroblas/c GR+/GR-‐ rate :

0% 20% 40% 60% 80%

100%

GR-‐

GR+

20μm Quiescente glial cell

AcAvated glial cell

Progenitor cell

Neuroblaste

10

Fixa/on


•  NesAne •  DoublecorAne •  CalreAnine •  GR

Quan/fica/on

Confocal Microscope

-‐5 0 Days

Immunohistochimistry GROUPE 1: Chronically stressed

GROUPE 2: Control 12

I. b) GR expression and neuroblas/c survival

NeuroblasAc TUNEL labeling in the rat dental gyrus

200μm 80μm

Reisi et al. 2015 Etat basal Stress Chronique

Taux de ne

urob

lastes TUNEL+

0

Neuroblas/c apoptosis rate:

11

Fixa/on


•  NesAne •  DoublecorAne •  CalreAnine •  GR •  TUNEL

Quan/fica/on

Confocal Microscope

-‐5 0 Days



I. b) GR expression and neuroblas/c survival

Etat Basal Stress Chronique

GR-‐ GR+

Neuroblas/c apoptosis rate: GR+ & GR-‐ cells

Etat basal Stress Chronique

Taux de ne

urob

lastes TUNEL+

Taux de ne

urob

lastes TUNEL +

0 0

11

Neuroblas/c apoptosis rate:

Fixa/on


•  NesAne •  DoublecorAne •  CalreAnine •  GR •  TUNEL

Quan/fica/on

Confocal Microscope

-‐5 0 Days



I.   Does GR expresion influence the neuroblas/c survival rate in chronic stress condi/on?

→ Expression of GR on 50% neuroblasts at baseline state → Increasing of this rate in chronic stress condiAon → Increasing of GR+ neuroblasAc apoptosis

12

Overview

13




II. a) GR ac/va/on and neuroblas/c apoptosis

Basline concentra/on

Control

Solvant (ethanol)

Chronic stress concentra/on

Neu

roblastomes

cultu

re

( SH

-‐SY5Y)

Labeling

•  GR •  TUNEL (apoptosis)

Cell count fluorescence microscope

Taux de neuroblastomes GR +

Contrôle EtOH CORT [+] CORT [+++]

Taux de neuroblastomes en apoptose

Nom

bre de

neu

roblastomes GR +

Nom

bre de

neu

roblastomes TUNEL +

Contrôle EtOH CORT [+] CORT [+++] 0 0

Lactate dehydrogenase

ac/vity (LDH)

(necrosis)

14

II. b) Traitement using a GR antagonist

Neuroblastomes apoptosis rate:

CORT [+] CORT [+++] CORT [+++] +RU-‐38486

TUNEL+ ne

urob

lastom

es ra

te

0

Chronic stress concentra/on + RU-‐38486

(GR antagoniste)

Chronic stress concentra/on

Baseline concentra/on

Neu

roblastomes

cultu

re

( SH

-‐SY5Y)

15

Lactate dehydrogenase

ac/vity (LDH)

(necrosis)

Labeling

•  GR •  TUNEL (apoptosis)

Cell count fluorescence microscope

II.   Is the neuroblas/c pro-‐appopto/c effect caused by direct ac/on of glucocor/coides on GR ?

è  Direct acAon of glucocorAcoids on neuroblasts by binding to the GR.

è Chronic stress concentraAon of glucocorAcoid:

•  Increase the GR + neuroblasAc rate •  Increase neuroblasAc apoptoAque rate

16

Overview

17




III.a) Chronic stress effect on s/mulated neurogenesis

-‐8

+ -‐ + -‐

DBS

BrdU 2 1 0 -‐3

Mini pump ac/va/on

+5

Lot 1 (+10)

+10 +42

Morris water maze

Lot 2 (+49)

+49

18

Electrodes

Mini pump


Confocale microscope

Not S/mulated Vehicule

S/mulated Vehicule

Not S/mulated Glucocor/coid

S/mulated Glucocor/coid

1

2

3

4

-‐8

+ -‐ + -‐

DBS

BrdU 2 1 0 -‐3

Mini pump ac/va/on

+5

Lot 1 (+10)

+10 +42

Lot 2 (+49)

+49

19

Morris water maze


?

Cor/co

Cellules B

rdU+

CORT

Cellules B

rdU+

S

S

NS NS

CORT V

?

20

-‐8

+ -‐ + -‐

DBS

BrdU 2 1 0 -‐3

Mini pump ac/va/on

+5

Lot 1 (+10)

+10 +42 +49

Morris water maze

•  Deap brain sAmulaAon (DBS) induice cell proliféraAon


20

-‐8

+ -‐ + -‐

DBS

BrdU 2 1 0 -‐3

Mini pump ac/va/on

+5

Lot 1 (+10)

+10 +42 +49

Morris water maze

Cellules B

rdU+

CORT

BrdU

+ cells

S

S

NS NS

CORT V

HP1


Ø  Hypothesis 1 : GlucocorAcosterone inhibit DBS effect

III. b) Consequences on spa/al memory

V CORT

BrdU

+ cells

S

S

NS NS AcAvity

in th

e target area

HP1 HP1

CORT

21

-‐8

+ -‐ + -‐

DBS

BrdU 2 1 0 -‐3

Mini pump ac/va/on

+5 +10 +42 +49

Lot 2 (+49)

NS-‐V

S-‐V

NS-‐CorAco S-‐CorAco

III. b) Consequences on spa/al memory BrdU

+ cells

S

S

NS NS

S

NS

HP1

CORT CORT V

21

HP2


Ø  Hypothesis 1 : GlucocorAcosterone inhibit DBS effect

Ø  Hypothesis 2 : GlucocorAcosteronehas a negaAve effect on BrdU+ cells

-‐8

+ -‐ + -‐

DBS

BrdU 2 1 0 -‐3

Mini pump ac/va/on

+5

Lot 1 (+10)

+10 +42 +49

III. b) Consequences on spa/al memory BrdU

+ cells

S

S

NS NS

S

NS

AcAvity

in th

e target area

NS-‐V

S-‐V

NS-‐CorAco S-‐CorAco

HP1 HP1 HP2 HP2

CORT CORT V

22

-‐8

+ -‐ + -‐

DBS

BrdU 2 1 0 -‐3

Mini pump ac/va/on

+5 +10 +42 +49

Lot 2 (+49)

III.  Does chronic stress have an impact on cogni/ve process related to hippocampal neurogenesis : Spacial memory

è  Hypothesis 1 : GlucocorAcoid inhibite DBS induiced neurogenesis but do not decrease the basal neurogenesis and spacial memory

è Hypothèse 2 : GlucocorAcoid have a negaAv effect on neurogenesis during the survival stage of neuroblasts and so impaire the spacial memory.

23

Bibliographie Ø  Ar/cles

•  Egeland M. et al. (2015) Molecular mechanisms in the regulaAon of adult neurogenesis during stress. Nature Reviews Volume 16 189-‐200

•  Garcia et al. (2004) Age-‐dependent expression of glucocorAcoids-‐and mineralocorAcoid recepteurs […] adult murine hippocampus. ASGBI 00130.x

•  Gould et al. (2010) Stress, stress hormones and adult neurogenesis. Exp Neurol 233(1): 12-‐21

•  Kuhn H.G (2015) Control of cell survival in adult mammalian neurogenesis. CSH Perspect Biol a018895

•  Kempermann G (2006) Natural variaAon and geneAc covariance in adult hippocampal neurogenesis. Epub Jan 9. 103(3): 780-‐5

•  Stone S. et al. (2011) SAmulaAon of entorhinal cortex promotes adult neurogenesis and facilitates spaAal memory. J. Neurosci 31(38):13469-‐13484

•  Kirby E. et al. (2013) Acute stress enchances adult rat hippocampal neurogenesis and acAvaAon of newborn neurons via secreted astrocyAc FGF2. eLife 2:e00362

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Neurogenesis & stress (EN)