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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
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
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
Prolifera/on Survival / Matura/on
Mature neurone
Immature neurone
Differencia/on
Mito/c stage Post-‐mito/c stage
Neuroblaste
0 21 28 > 42 7 Days 3 5 100%
Neo-‐neurones rate 25% 100%
Quiescent glial cell
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
Mito/c stage Post-‐mito/c stage
Neuroblaste
Days
Prolifera/on Survival / Matura/on
Quiescent glial cell
Ac/vated glial cell
Introduc/on
Quiescent glial cell
Ac/vated glial cell
Progenitor cell
Mature neurone
Immature neurone
Mito/c stage Post-‐mito/c stage
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
Quiescent glial cell
Ac/vated glial cell
Progenitor cell
Mature neurone
Immature neurone
Mito/c stage Post-‐mito/c stage
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
Quiescent glial cell
Ac/vated glial cell
Progenitor cell
Mito/c stage Post-‐mito/c stage
GR = GlucocorAcoid Receptors
Introduc/on
Neuroblaste
GR+
GR-‐
?
GR = GlucocorAcoid Receptors
• 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
Mito/c stage Post-‐mito/c stage
Ac/vated glial cell
Progenitor cell
Mature neurone
Immature neurone
Quiescent glial cell
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
I. a) Neuroblas/c GR expression in rat
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
I. a) Neuroblas/c GR expression in rat
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
Brain slides (cryostat)
• 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
Brain slides (cryostat)
• NesAne • DoublecorAne • CalreAnine • GR • TUNEL
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
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
Brain slides (cryostat)
• NesAne • DoublecorAne • CalreAnine • GR • TUNEL
Quan/fica/on
Confocal Microscope
-‐5 0 Days
Immunohistochimistry GROUPE 1: Chronically stressed
GROUPE 2: Control 12
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
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
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
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
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
III.a) Chronic stress effect on s/mulated neurogenesis
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
III.a) Chronic stress effect on s/mulated neurogenesis
?
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
III.a) Chronic stress effect on s/mulated neurogenesis
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
• Deap brain sAmulaAon (DBS) induice cell proliféraAon
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
• Deap brain sAmulaAon (DBS) induice cell proliféraAon
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