Rôle de la microglie dans le

développement et les pathologies du SNC

Montpellier, Oct 2019

E. Audinat

Institut de Génomique Fonctionnelle

(etienne.audinat@igf.cnrs.fr)

Références:

Développement : Mosser et al., Prog Neurobiol 2017

Pathologie : Butovsky & Weiner, Nat Rev Neuro 2018

Epilepsie : Rassendren & Audinat, J Neurosci Res 2016

Bases d’électrophysiologie : le livre “Physiologie du Neurone”, Tritsch D,

Chesnoy-Marchais et Feltz A Eds. Douin

Microglia cells: the resident macrophages of the CNS

Resting

(dormant)

Activated

Healthy brain Pathology

• The professional phagocytes of the CNS

• Release different inflammatory mediators (cytokines, chemokines, growth

factors…) and orchestrate the inflammatory reaction occurring many CNS

disorders (TBI, Neuropathic pain, stroke, Parkinson, Alzheimer, ALS, epilepsy…)

Morphological changes

Translatome remodeling

Proliferation

Phagocytosis

Release mediators (trophic

factors, cytokines, ROS)

Microglia-synapse interactions

in physiological conditions : adult CNS

Active surveillance of the parenchyma

Nimmerjhan et al. 2005 (see also Davalos et al. 2005)

Tremblay et al. 2010

(see also Wake et al. 2009)

Neuron Microglia Direct contact with synapses

Control of synapse size and number

Parkhurst et al. 2013

control Microglia depletion

“Never resting” microglia monitor synapses and control their morphology/formation

Microglia-synapse interactions

in physiological conditions : adult CNS Microglia control of synaptic activity

1) specific activation of microglia receptors modulate synaptic transmission

Pascual et al.2012 Piccinin et al 2010

“Never resting” and “not just eating” microglia in the adult brain

2) Microglia bulbous endings contact active neurons and reduce their activity

Li et al.2012

In vivo time-lapse imaging of neuronal calcium and microglia morphology in the zebrafish optic tectum

Green: OGB-loaded neurons

Purple: microglia

Microglia-synapse interactions

In physiological conditions : Developing CNS

Synaptic pruning

Paolicelli et al. 2011 Schafer et al. 2012 Microglia PSD95 Microglia

Origin of microglia

Ginhoux et al. 2013

Microglial cells prune supernumerary synapses….

Microglia-synapse interactions

In physiological conditions : Developing CNS

… and help at forming new ones.

Miyamoto et al. 2016 Microglia/Neuron

Origin of microglia

Ginhoux et al. 2013

Microglia-synapse interactions

In physiological conditions : Developing CNS

Besides pruning and forming synapses, can microglia influence

synaptic activity during maturation of neuronal networks ?

Induction of developmental apoptosis (Marin-Teva et al., 2004 ; Bessis et al., 2006)

Neuronal survival (Ueno et al., 2013)

Migration of cortical inhibitory interneurons (Squarzoni et al., 2014)

Pruning of supernumerary synapses (Schafer et al., 2012 ; Paolicelli et al., 2011 ; Zhan et al., 2014)

Synaptogenesis (Miyamoto et al., 2016)

How can microglial cells be at the right place at the right time ?

200 µm

Heterogenous distribution of microglia

during early postnatal development of the brain

CX3CR1+/eGFP mice (Jung et al. 2000)

Thalamic axons

Spiny stellate neurons

Aronoff & Petersen 2008

Formation & maturation of the barrels :

P3

Clustering of Thalamocortical

axons (TCA)

The barrel cortex as a model of neuronal circuit development

• ↗ synaptic density

• Modification of glutamate receptor expression at TC synapses

• setting up of inhibitory network

Period of plasticity

P5

End of the critical window for LTP induction at TC synapses

P10

Inhib. Intern.

150 µm

Increased density of microglial cells in cortical

layers of the developing barrel cortex

CX3CR1+/eGFP mouse (Jung et al 2000)

Arnoux et al. 2013

P5 P5 P5

P7 P6

P5 P5

Scale bar:100µm

Distribution of microglial cells within layer 4 of the

barrel cortex during the first postnatal week

Hoshiko et al. 2012 Microglial cells invade the barrel centers after P5

Two microglia phenotypes in the developing barrel field C

urr

ent density

(pA

/pF

)

Vm (mV)

P5-P9 non-activated

P5-P9 “activated”

Adult controls

Kv1.3

Acute cortical slices from CX3CR1+/eGFP mice

Postnatal age

% o

f m

icro

glia

l cells

expre

ssin

g K

v1.3

What is the signaling mechanism responsible for the

recruitment of microglial cells at thalamo-cortical

synapses?

The fractalkine (CX3CL1) signaling pathway in the CNS

( From Richard M. Ransohoff; Immunity, Nov. 2009)

What is the signaling mechanism responsible for the

recruitment of microglial cells at thalamo-cortical

synapses?

Transgenic mice used (Jung and al, 2000):

- CX3CR1+/eGFP -> mice with functional CX3CR1

- CX3CR1 eGFP/eGFP -> mice lacking CX3CR1

Scale bar: 200 µm

Fractalkine (CX3CL1) immunoreactivity in the

developing somato-sensory cortex

Scale bar:100 µm

CX3CR1 deficiency delays the entry of microglia in the

barrel centers

Knockout mice

for CX3CR1

Heterozygous mice

for CX3CR1

Hoshiko et al. 2012

Scale bar: 100 µm

CX3CR1 deficiency does not impair the overall number

of microglial cells in layer 4 at P7 (but their distribution)

0

5000

10000

15000

20000

hetero homo

5000

10000

15000

CX3CR1+/eGFP

CX3CR1eGFP/eGFP

0

n=12 n=5

Hoshiko et al. 2012

Microglial cells enter the barrel centers during synaptic maturation

CX3CR1 deficiency delays microglia recruitment in the barrel centers

0

20

40

60

80

100

120

00-0

,2

0,2-0,4

0,4-0,6

0,6-0,8

0,8-1

1-1,2

1,2-1,4

1,4-1,6

1,6-1,8

1,8-2 >2

Mean velocity of soma (µm/min)

cum

ula

tive

pro

bab

ility

(%

)

CX3CR1+/-(n=9 exp)

CX3CR1 -/-(n=5 exp)

Mean Soma Velocity (µm/min)

CX3CR1+/-

CX3CR1-/-

Cu

mu

lative

Pro

ba

bili

ty

Reduced directional motility of microglia soma in

developing CX3CR1 knockout mice

Collaboration : Serge Charpak

Arnoux, Audinat 2015

Is there any consequence of CX3CR1 deficiency and of

the delayed recruitment of microglial cells on the

functional maturation of thalamo-cortical synapses?

Development : Conclusion # 1

Microglial cells enter the barrel centers during synaptic

maturation and fractalkine signaling contributes to the

timely recruitment of these immune cells at synaptic

sites

Recordings of spiny stellate cells

minimal stimulations of TC fibers

CX3CR1 deficiency impairs the development of

AMPAR/NMDAR ratio at TC synapses

Hoshiko, Arnoux et al. 2012

The delayed recruitment of microglia in the barrel centers in CX3CR1 knockout

mice is accompanied by an impaired functional maturation of TC synapses

P0 P3 P5 P8-10

Intra-cerebral

injection of liposomes Tests

Clodronate liposomes : another way to delay microglia

invasion of cortical layers

Free clodronate www.clodronateliposomes.org/

No sign of

astrogliosis @ P9

Depletion of microglia with clodronate liposomes also

impairs the functional maturation of TC synapses

The functional maturation of TC synapses is impaired

in the absence of microglia in the barrel centers

Do microglial cells also affect the maturation of

inhibitory circuitry in the barrel cortex ?

Development of TC feed-forward inhibition

in layer 4 of barrel cortex

The TC feed-forward inhibition matures mostly

after the end of the first postnatal week

The development of TC feed-forward inhibition decreases the

integration window that controls the timing and output of

spiny stellate neurons

Chittajallu & Isaac, 2010

P6

P11

P0 P3 P5 P10-11

Intra-cerebral

injection of liposomes Tests

Depletion of microglia impairs the development of

Thalamocortical feed-forward inhibition

The presence of microglia in the barrel cortex favors

the development of the TC feed-forward inhibition

By which mechanism microglial cells influence

the functional maturation of thalamocortical

synaptic circuits?

Development : Conclusion # 2

The presence of microglial cells in the barrel centers is

necessary for the timely maturation of excitatory and

inhibitory synapses of the thalamocortical circuit.

? Microglial activation Nerve injury

Microglial BDNF regulate adult synaptogenesis (Parkhurst et al. 2013)

In context of neuropathic pain, microglial BDNF induces

an increase of neuronal excitability (Coull et al., 2005 ; Ulmann et al., 2008)

An usual suspect : the Brain-Derived Neutrophic Factor

Conditional deletion of BDNF in microglia impairs the

development of the AMPAR/NMDAR ratio at TC synapses

CX3CR1CreER BDNFflox/flox X

P0 P3 P5 P10-11

Tamox Tests

P4

Microglial BDNF controls the functional maturation of TC synapses

Conditional deletion of BDNF in microglia impairs the

development of the TC feed-forward inhibition

CX3CR1CreER BDNFflox/flox X

P0 P3 P5 P10-11

Tamox Tests

P4

Microglial BDNF controls the development of TC feed-forward inhibition

Take home messages

Reciprocal interactions between neurons and microglial cells control the

functional maturation of the thalamocortical network

The presence of microglia in the barrel centers is necessary for the functional

maturation of thalamocortical excitatory synapses and the development of feed-

forward inhibition at early postnatal stages.

(Which of the 2 synapses is controlled by microglia during the development of the

feed-forward inhibition?)

BDNF is one of the mediators by which microglial cells influence the functional

maturation of synaptic networks in the barrel cortex.

(Direct or indirect effect of BDNF on pre- or postsynaptic elements?)

Microglia cells: the resident macrophages of the CNS

Resting

(dormant)

Activated

Healthy brain Pathology

• The professional phagocytes of the CNS

• Release different inflammatory mediators (cytokines, chemokines, growth

factors…) and orchestrate the inflammatory reaction occurring many CNS

disorders (TBI, Neuropathic pain, stroke, Parkinson, Alzheimer, ALS, epilepsy…)

Morphological changes

Translatome remodeling

Proliferation

Phagocytosis

Factor release (trophic factors,

cytokines, ROS)

Aronica, Gorter Neuroscientist 2007

Epilepsy, inflammation and glia

Choi, Koh Yonsei Medical Journal 2008

IL-1alpha, IL-1B, IL-6, NFkB, PLAT, TGFB, GFAP,

VIM, C1q, C3, C/EBPB, C/EBPd, FRD1…

Experimental TLE

- 600 000 patients suffering of different epilepsies in France

- Anti-epileptic drugs (neurocentric) refrain seizures but do not cure epilepsy

and have unwanted side effects

- New therapeutic targets are needed

Epilepsy:

Spontaneous

seizures

Acquired

hyperexcitability Evoked

hyper-excitability

Hours

Status

epilepticus

Release of

Mediators

Phagocytosis

Hypothesis: Purinergic receptors

which play central roles

in microglia functions

determine how these cells

influence hippocampus

remodeling

Days to weeks

“Latent period”

Neuronal death

Re-wiring of the network

Changes in activity

Plusieurs récepteurs purinergiques de types P2X et P2Y

sont exprimés dans la microglie

Fields et al.

Nat Rev Neurosci. 2006

P2X4

P2X7

P2Y6

P2Y12

Kainate

Microglia

phenotype

Status Epilepticus

CX3CR1+/eGFP mice

fluorescent microglia

Hippocampal microglia in a model of

Status epilepticus

Acute hippocampal slices

qPCR whole hippocampus

Immunohistochemistry

Increased expression of pro-inflammatory markers and

neuronal death after Status Epilepticus in CX3CR1+/eGFP

mice (C57bl6)

Control

48h

Post SE

Eosin hematoxylin Fluorojade B

mark

ed c

ells

(cells

/fie

ld)

CA1

CA3

0

10

20

30

40

Control 48h Post

SE

qPCR whole hippocampus

Avignone et al. 2008

Iba1 GFP Merged

PBS

24h

Post

SE

15 µm

Morphological changes of microglial cells in the CA1

region of the hippocampus after Status Epilepticus

Avignone et al. 2008

I density (

pA

/pF

)

Vm (mV)

PBS 3h 24h 48h

-100 0 100 -10

0

10

I d

en

sity (

pA

/pF

)

Vm (mV)

PBS3h24h48h

-100 0 100-10

0

10

I d

en

sity (

pA

/pF

)

Vm (mV)

PBS3h24h48h

-100 0 100-10

0

10

-100 0 100-10

0

10

PBS 3h 24h 48h

*

**

20

25

30

35

40

PBS 3h 24h 48h

Cm

(p

F)

*

**

20

25

30

35

40

PBS 3h 24h 48h

**

1

2

3

4

5

PBS 3h 24h 48h PBS 3h 24h 48h

**

1

2

3

4

5

1

2

3

4

5

Rin

(G

W)

Changes in intrinsic membrane properties of microglial

cells after Status Epilepticus

Acute hippocampal slices from 4-6

week old CX3CR1+/eGFP control

and epileptic mice.

200pA

20ms

PBS 48h after SE

200pA

20ms

200pA

20ms

PBS 48h after SE

Voltage steps from -140mV to +60mV

Holding potential -60 mV.

Avignone et al. 2008

200pA

20ms

Heterogeneity of hippocampal microglia cells

7 days after status epilepticus

P2X7

P2Y6

P2Y12

Production secretion

of, IL-1b, IL-6, TNFa

Phagocytosis

Polarisation

Process extension.

Purinergic receptors of microglial cells

Functions Receptor

P2X4 Production secretion

of bdnf; chemotaxis

Activation

Status Epilepticus induces an increase of purinergic

receptor mRNA expression in the hippocampus

0

1

2

48

12

P2X4P2X7P2Y6

**

PBS 48h3h 24h

P2Y12

**

**

**

**

*

* *

Fo

ldch

an

ge

B

*

0

1

2

48

12

P2X4P2X7P2Y6

**

PBS 48h3h 24h

P2Y12

**

**

**

**

*

* *

Fo

ldch

an

ge

B

*

Avignone et al. 2008

Larger P2X7 receptor-mediated currents in activated

microglia after Status Epilepticus

ATP (1mM)

10 pA

50 s

ATP (1mM)

A740003 (10 µM)

WT P2X7-/- WT+

A740003

0

2

4

6

I AT

P (

pA

/pF

)

PBS 48h after SE

Ca/Mg-free extracellular solution

CsGlu pipettes,

VH -60mV

P2X7

P2Y6

P2Y12

Production secretion

of IL-1b, IL-6, TNFa

Phagocytosis

Polarisation

Process extension.

Purinergic receptor remodeling

upon microglia activation

Function Receptor Activation

LPS

What about

P2X4?

Activation

SE

P2X4 is up-regulated in the hippocampus

48h after Status Epilepticus

Ulmann et al. 2013

P2X4 is up-regulated in microglia

48h after Status Epilepticus

Does P2X4 deficiency impact

microglia activation and neuronal remodeling?

Impaired microglia activation after Status Epilepticus in

P2X4-/- mice

Impaired increase in the number of microglial cells

Num

be

r o

f G

FP

ce

lls / m

m3 (

x1

03)

CX3CR1+/eGFP X P2X4-/-

CX3CR1+/eGFP

0

5

10

15

20

25

PBS Kainate

48h

***

**

*

*

PBS

Kainate

48h

CX3CR1+/eGFP

X P2X4-/- CX3CR1+/eGFP

Normal P2Y12 expression after Status Epilepticus in

P2X4-/- mice

P2Y12 immunohistochemistry (Ab D Julius) qPCR (whole hippocampus)

P2X4+/+ P2X4-/-

Normal P2Y12R-mediated motility of microglia

processes in hippocampal slices of P2X4-/- mice

Z-projection of 5 stacks of images (4 µm thick each)

taken every minute with dynamic speckle illumination

(movie by C Ventalon, V Emiliani, E Avignone) V

elo

city (

µm

/min

)

0

2

4

6

8

PBS 48h post SE

CX3CR1+/-

CX3CR1+/- ; P2X4 -/-

P2X4 deficiency does not impair P2X7 and P2Y6

receptor-mediated response in hippocampal microglia

No change in P2X7, P2Y6 and P2Y12 receptor-mediated

responses in microglia of control and epileptic mice

Impaired up-regulation of Kv1.3 mediated potassium currents

in microglia of P2X4-/- mice after Status Epilepticus

Voltage steps from -140mV to +60mV

Holding potential -60 mV.

Kv1.3

Decreased neuronal death in CA1 after Status

Epilepticus in P2X4-/- mice

CX3CR1+/eGFP CX3CR1+/eGFPxP2X4-/-

Fluoro-Jade B staining

48h post SE

Conclusions

- The reactivity of hippocampal microglia after a status epilepticus is characterized

by larger purinergic responses mediated by P2X7, P2Y6 and P2Y12

- P2X4 expression is also up-regulated in microglia

- P2X4 governs specific aspects of microglial cell reactivity (functional

expression of Kv1.3 and recruitment but not other purinergic responses)

- P2X4 promotes neuronal death after status epilepticus

>>> Specific pathways through which purinergic receptors control

neuronal survival and epileptogenesis remain to be identified

>>> Roles of the BBB and interactions with the peripheral immune

system (monocytes and T cells)