disruption of the astrocytic tnfr1-gdnf axis accelerates ...ferraiuolo et al, dysregulation of...
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Disruption of the astrocyticTNFR1-GDNF axis accelerates
motor neuron degeneration and disease progression in
amyotrophic lateral sclerosis
Daniela Rossi
Laboratory of Research on Neurodegenerative Disorders
Turin, December 1-3, 2016
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Prevalence: 2-8 per 100,000 people
Incidence: 1-2 per 100,000 people each year
Amyotrophic Lateral Sclerosis
Clinical features:
Muscle weakness
Atrophy
Spasticity
Death due to respiratory
failure within 2-5 years
of clinical onsetPathological features:
degeneration and death of
corticospinal and spinal
motor neurons
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ALS gene frequencies
Alsultan et al, Degener Neurol Neuromusc Disease, 2016
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ALS gene frequencies
Alsultan et al, Degener Neurol Neuromusc Disease, 2016
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Human SOD1 mutant sites
Sites of the mutant residues depicted are derived from the ALS Online Genetic Database
http://alsod.iop.kcl.ac.uk/Overview/gene.aspx?gene_id=SOD1
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Transgenic mutant SOD1 mouse models
Turner & Talbot , Prog Neurobiol., 2008
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Wild type SOD1G93A
presymptomaticSOD1G93A
end stage
SOD1G93A transgenic mice
Chiu et al, Mol Cell Neurosci, 1995
Turner & Talbot , Prog Neurobiol., 2008
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Is motor neuron degeneration
independent on the
microenvironment?
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Clement et al, Science, 2003
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Astrocyte involvement in ALSYamanaka et al, Astrocytes as determinants of
disease progression in inherited amyotrophic
lateral sclerosis, Nat. Neurosci., 2008
Wang et al, Astrocytes loss of mutant SOD1
delays ALS disease onset and progression in
G85R transgenic mice, Hum. Mol. Genet., 2011
Papadeas et al, Astrocytes carrying the
superoxide dismutase 1 (SOD1G93A) mutation
induce……, Proc. Natl. Acad. Sci. USA, 2011
Ilieva et al, Non-cell autonomous toxicity in
neurodegenerative disorders: ALS and beyond,
J Cell Biol., 2009
Ferraiuolo et al, Dysregulation of astrocyte-
motoneuron cross-talk in mutant SOD1-related
amyotrophic lateral sclerosis, Brain, 2011
Van Damme et al, Astrocytes regulate GluR2
expression in motor neurons and their
vulnerability to excitotoxicity, Proc. Natl. Acad.
Sci. USA, 2007
Nagai et al, Astrocytes expressing ALS-linked
mutated SOD1 release factors selectively toxic to
motor neurons, Nat. Neurosci., 2007
Di Giorgio et al, Non-cell autonomous effect of
glia on motor neurons in an embryonic stem cell-
based ALS model, Nat. Neurosci., 2007
Di Giorgio et al, Human embryonic stem cell-
derived motor neurons are sensitive to the toxic
effect of glial cells ……... , Cell Stem Cell, 2008
Marchetto et al, Non-cell-autonomous effect of
human SOD1 G37R astrocytes on motor neurons
derived……. , Cell Stem Cell, 2008
Cassina et al, Mytochondrial dysfunction in
SOD1G93A-bearing astrocytes promotes motor
neuron degeneration……, Hum. Mol. Genet., 2011
Yang et al, Presynaptic regulation of astroglial
excitatory neurotransmitter transport GLT1,
Neuron, 2009
Aebischer et al, IFNgamma triggers a LIGHT-
dependent selective death of motoneurons
contributing……., Cell Death Differ., 2011
Haidet-Phillips et al, Astrocytes from familial and
sporadic ALS patients are toxic to motor neurons,
Nat. Neurosci., 2011
Diaz-Amarilla et al, Phenotypically aberrant
astrocytes that promote motoneuron damage in a
model ….., Proc. Natl. Acad. Sci. USA, 2011
Rossi et al, Focal degeneration of astrocytes in
amyotrophic lateral sclerosis,
Cell Death Differ., 2008
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Deleterious interplay between motor neurons and astrocytes
Gain of function Loss of function
Cytokines (IL-1, IL-6, IL-10
INFg, GM-CSF, TGFb, TNFa
Chemokines (CCL2, MCP-1, CCL5, CCL20,
CXCL10, CXCL12, CXCL1, CXCL2, CX3CL1)
Eicosanoids
Reactive oxygen species
Nitric Oxide
Excitatory aminoacids
Glutamate uptake
Aberrant [Ca2+]i
GluR2 subunit of AMPAR modulation
Metabolic substrates
Trophic factors (GDNF, BDNF,
NT3, CNTF, VEGF, bFGF, IGF1)
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Tumor Necrosis Factor α is released as a soluble cytokine
(a homotrimer of 17 kDa monomers) after being enzymatically cleved from
its cell surface bound precursor by the TNFa converting enzyme (TACE)
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TNFa and its receptors
TNF TNF
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Neurotoxic/neuroprotective
Pathological processesPhysiological processes
Regulatory
Synaptic plasticity
Learning and
memory
Glial transmission
Food/water
intake
Sleep Infectious
diseases
Alzheimer’s
disease
Parkinson’s
diseaseMultiple
sclerosis
Chronic
pain
Epilepsy
Ischemic and
traumatic injuries
Amyotrophic Lateral
Sclerosis
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TNFa /TNFR1 signalling controls GDNF
expression and secretion from
human spinal astrocytes
0
100
200
300
400
500
ctrl TNFa
Re
lati
ve
GD
NF
mR
NA
ex
pre
ssio
n (
% o
f c
trl) ***
Rela
tive
GD
NF
pro
tein
ex
pre
ssio
n (
% o
f c
trl)
0
50
100
150
200
0
50
100
150
200
Rela
tive
GD
NF
pro
tein
rele
ase
(%
of
ctr
l)
ctrl TNFa
***
***
***p<0.001 vs. ctrl
Brambilla et al, Hum Mol Genet, 2016
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Rela
tive
GD
NF
mR
NA
ex
pre
ssio
n (
% o
f c
trl)
Wild type
ctrl
TNFa
0
50
100
150
200
250
***
Wild type Wild type
Rela
tive
GD
NF
pro
tein
ex
pre
ssio
n (
% o
f c
trl)
Rela
tive
GD
NF
pro
tein
rele
ase
(%
of
ctr
l)
ctrl TNFa
0
50
100
150
200
tnfr1-/-
0
50
100
150
200***
***
TNFa/TNFR1 signalling controls
GDNF expression and secretion
from mouse spinal astrocytes
***p<0.001 vs. ctrl
Brambilla et al, Hum Mol Genet, 2016
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Rela
tive
GD
NF
mR
NA
ex
pre
ssio
n (
% o
f c
trl)
Wild type
ctrl
TNFa
tnfr1-/-
0
50
100
150
200
250
***
Wild type Wild type
Rela
tive
GD
NF
pro
tein
ex
pre
ssio
n (
% o
f c
trl)
Rela
tive
GD
NF
pro
tein
rele
ase
(%
of
ctr
l)
ctrl TNFa
0
50
100
150
200
tnfr1-/- tnfr1-/-
0
50
100
150
200***
***
TNFa/TNFR1 signalling controls
GDNF expression and secretion
from mouse spinal astrocytes
***p<0.001 vs. ctrl
Brambilla et al, Hum Mol Genet, 2016
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The TNFa/TNFR1 pathway controls GDNF expression in vivo
TNFa
vehicle Wild type
tnfr1-/-
Wild type
tnfr1-/-
6-7 wks (age)
48 h
i.c.v.
injection
RNA RT-qPCRcDNA
Protein ELISA
Brambilla et al, Hum Mol Genet, 2016
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The TNFa/TNFR1 pathway controls GDNF expression in vivo
TNFa
vehicle Wild type
tnfr1-/-
Wild type
tnfr1-/-
6-7 wks (age)
48 h
i.c.v.
injection
RNA RT-qPCRcDNA
Protein ELISA
0
20
40
60
80
100
120
140
Re
lati
ve
GD
NF
pro
tein
ex
pre
ss
ion
(% o
f ve
hic
le)
Wild type tnfr1-/-
vehicle
TNFa*vehicle
TNFa
Re
lati
ve
GD
NF
mR
NA
exp
ressio
n(%
of
veh
icle
)
tnfr1-/-0
20
40
60
80
100
120
140
Wild type
**
**p<0.01, *p<0.05 vs. vehicle
Brambilla et al, Hum Mol Genet, 2016
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GDNF is a potent pro-survival agent for
spinal motor neurons
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GDNF delivery to spinal motor neurons by muscle cells
Retrograde viral delivery of GDNF
Murine myoblasts or hMSCs
genetically modified to secrete GDNF
Delayed degeneration of motor neurons
and decline in motor function
Prolonged survival
Acsadi et al, Hum Gene Ther, 2002; Kaspar et al, Science, 2003;
Manabe et al, Apoptosis, 2002; Wang et al, J Neurosci, 2002;
Mohajeri et al, Hum Gene Ther, 1999; Suzuki et al, Mol Ther, 2008
SOD1G93A ALS model
before disease onset
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Direct GDNF delivery into the CNS
Viral delivery of GDNF
or hNPCGDNF
SOD1G93A spinal cord
Protection of motor neurons
Guillot et al, Neurobiol Dis, 2004
Klein et al, Hum Gene Ther, 2005
Suzuki et al, Plos One, 2007
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Neurotrophic support
Bohn, Experimental Neurology 2004
Cartoon of potential sources of GDNF for motor neurons. GDNF can be secreted from muscle (M, red arrows)
and Schwann cells (SC, yellow arrows) at the motor neuron (MN) terminal, as well as from astrocytes (A, green
arrows) surrounding motor neuron perikarya, dendrites, and proximal axons. GDNF might also be secreted
from motor neurons themselves and affect neighbouring motor neurons in a paracrine manner.
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Correlation between the
endogenous TNFa/TNFR1
system and GDNF
expression in SOD1G93A
mouse spinal cord
100 days
130 days
30 days
TNFa
0
1000
2000
3000
4000
5000
6000
*
####
****
***
Wild type SOD1G93ASOD1WT
0
GDNF####
****Rela
tive
mR
NA
ex
pre
ssio
n
(%o
f 3
0 d
ays
)
TNFR1
0
100
200
300
400
500
600
700####
****
*****
Brambilla et al, Hum Mol Genet, 2016
*p<0.05 and **p<0.001 vs. 100-day-old Wild-type and SOD1WT mice
***p<0.001 vs. 30-day-old SOD1G93A mice
****p<0.001 vs. 130-day-old Wilde-type and SOD1WT mice####p<0.001 vs. 30- and 100-day-old SOD1G93A mice
100
200
300
400
500
****
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GFA
PM
AC
-1S
MI3
2GDNF merge
Expression of GDNF in SOD1G93A mouse spinal cords is
predominantly in the astrocytes
Brambilla et al, Hum Mol Genet, 2016
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Controls
sALS
Rela
tive
mR
NA
ex
pre
ssio
n
(%o
f c
on
tro
ls)
0
50
100
150
200
250
300 TNFa**
0
50
100
150
200
250
300 ** GDNF
TNFR1
0
20
60
100
140
180 **
Patients Age of
death
(Years)
Sex Onset Duration of
illness
(Months)
Cause of
death
ALS1 56 F Limb 7 ALS
ALS2 52 M Limb 49 ALS
ALS3 58 M Bulbar 11 ALS
ALS4 64 F Leg 15 ALS
ALS5 43 M Arm 32 ALS
Control1 42 M NA NA Myocardial
infarction
Control2 54 M NA NA Myocardial
infarction
Control3 57 F NA NA Myocardial
infarction
Control4 59 F NA NA Pneumonia
Control5 55 M NA NA Myocardial
infarction
Brambilla et al, Hum Mol Genet, 2016
**p<0.01 vs. Controls
Correlation between the
endogenous TNFa/TNFR1 system
and GDNF expression in sporadic
ALS spinal cords
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SOD1G93A
tnfr1-/-
SOD1G93A;tnfr1+/-
tnfr1+/-
SOD1G93A;tnfr1+/+
SOD1G93A;tnfr1-/-
X
X
Breeding scheme for the generation of mutant
SOD1-expressing mice that are either wild-type or
knockout for the tnfr1 gene
Brambilla et al, Hum Mol Genet, 2016
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Disruption of endogenous TNFa/TNFR1 signalling
abrogates GDNF expression
SOD1G93A;tnfr1-/-
0
100
200
300
400
500
GDNF
***
SOD1G93A;tnfr1+/+
****
####
**p<0.01 and ***p<0.001 vs. 130-day-old SOD1G93A;tnfr1+/+ mice
****p<0.001 vs. 30- and 100-day-old SOD1G93A;tnfr1+/+ mice####p<0.001 vs. 30-day-old SOD1G93A;tnfr1+/+ mice
Brambilla et al, Hum Mol Genet, 2016
Re
lati
ve
mR
NA
ex
pre
ss
ion
(% o
f 3
0 d
ays
)TNFa
0
1000
2000
3000
4000
5000
6000
**
****
####
100 days
130 days
30 days
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Expression of neuroprotective/
neurotoxic factors is unchange
in spinal cord of tnfr1-/- ALS mice
0
50
100
150VEGF-B
*** ***
0
50
100
150
200
250Bcl-XL
SOD1G93A;tnfr1-/-SOD1G93A;tnfr1+/+
Re
lati
ve
mR
NA
exp
ress
ion
(% o
f 3
0 d
ays
)
######
100 days 130 days30 days
Brambilla et al, Hum Mol Genet, 2016
***p<0.001 and **p<0.01 vs 30-day-old genotype-matched mice###p<0.001 vs 30- and 100-day-old day-old genotype-matched mice
MMP-9
***
***
******######
0
200
400
600
800
1000IL-1b
****** ***
***
0
100
200
300 COX-2
****
0
40
80
120
SOD1G93A;tnfr1-/-SOD1G93A;tnfr1+/+
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Ablation of the tnfr1 gene has no impact
on the onset of ALS disease…..P
rob
ab
ilit
y o
f o
ns
et
(pea
k o
f b
od
y w
eig
ht)
90 110 130 150
0
25
50
75
100
Age (days)
SOD1G93A;tnfr1-/-
SOD1G93A;tnfr1+/+
A B
Pro
bab
ilit
y o
f o
ns
et
(ro
taro
dd
efi
cit
s)
Age (days)
90 110 130 150
0
25
50
75
100SOD1G93A;tnfr1-/-
SOD1G93A;tnfr1+/+
Brambilla et al, Hum Mol Genet, 2016
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....but accelerates its progression
in SOD1G93A mice
C D
Early phase
Du
rati
on
Late phase
SOD1G93A;tnfr1-/-
0
5
10
15
20
25
0
2
4
6
8
10
12
#
SOD1G93A;tnfr1+/+
Pro
bab
ilit
y o
f s
urv
iva
l
90 110 130 150 170
0
25
50
75
100
Age (days)
SOD1G93A;tnfr1-/-
SOD1G93A;tnfr1+/+
tnfr1-/-
Brambilla et al., Hum Mol Genet, 2016
#p<0.05 vs SOD1G93A; tnfr1+/+ in the Late phase
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Ablation of the tnfr1 gene causes a significant
decrease in the number of motor neurons in
symptomatic SOD1G93A mice
A
E
C
G
SO
D1
G93A;t
nfr
1-/
-
Late phase30 days
SO
D1
G93A;t
nfr
1+
/+ B D
F H
SOD1G93A;tnfr1-/-
Mo
tor
ne
uro
n c
ou
nts
0
100
200
300
400
500
600
Late phase30 days
*
SOD1G93A;tnfr1+/+
*****
Brambilla et al, Hum Mol Genet, 2016
**p<0.01, ***p>0.001 vs 30-day-old genotype-matched mice
*p<0.05 vs SOD1G93A; tnfr1+/+ at the Late phase
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ctrl
SOD1G93A;tnfr1-/-
Rela
tive
GD
NF
mR
NA
ex
pre
ssio
n (
% o
f c
trl)
****
SOD1WTWild type
0
50
100
150
200
250
300
SOD1G93A;tnfr1+/+
TNFa** **
***
SOD1G93A astrocytes respond to TNFa/TNFR1
signalling by producing normal GDNF levels
Brambilla et al, Hum Mol Genet, 2016
**p<0.01, ***p>0.001 vs. ctrl astrocytes
****p<0.001 vs. TNFα in Wild-type, SOD1WT and SOD1G93A; tnfr1+/+ astrocytes
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asymptomatic stage
TNFa TNFR1 GDNF
Astrocytes
Motor Neuron
Muscle
onset of motor deficits
X
Microglia
symptomatic stage
X
Brambilla et al, Hum Mol Genet, 2016
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Conclusions
• Stimulation of astroglial TNFR1 can potentiate the production and
delivery of GDNF by human and mouse spinal cord astrocytes in vitro
• Activation of TNFR1 signal transduction by intracerebroventricular
injection of exogenous TNFa can strengthen the production of GDNF
also within the CNS in vivo
• Ablation of the cytokine receptor 1 significantly affects GDNF levels,
thus confirming a role for endogenous TNFa/TNFR1 signalling in
modulating the expression of GDNF in SOD1G93A ALS mice
• Motor neuron loss is more pronounced in SOD1G93A mice lacking
TNFR1 during the late phase of the disease, i.e. when ALS progression
is significantly accelerated
• The capacity to release GDNF upon TNFα stimulation is not impaired
in SOD1G93A astrocytes
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Chiara Valori
Francesca Martorana
Chiara Bergamaschi
Former members of the lab
Istituti Clinici Scientifici Maugeri SpA SB
Paola Bezzi
University of Lausanne, Switzerland
Anand M. Iyer
Eleonora Aronica
Academisch Medisch Centrum,
Amsterdam, The Netherlands
Liliana Brambilla
Giulia Guidotti
Chiara Malatino
Federica Sorrentino