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

daniela.rossi@icsmaugeri.it

Turin, December 1-3, 2016

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

ALS gene frequencies

Alsultan et al, Degener Neurol Neuromusc Disease, 2016

ALS gene frequencies

Alsultan et al, Degener Neurol Neuromusc Disease, 2016

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

Transgenic mutant SOD1 mouse models

Turner & Talbot , Prog Neurobiol., 2008

Wild type SOD1G93A

presymptomaticSOD1G93A

end stage

SOD1G93A transgenic mice

Chiu et al, Mol Cell Neurosci, 1995

Turner & Talbot , Prog Neurobiol., 2008

Is motor neuron degeneration

independent on the

microenvironment?

Clement et al, Science, 2003

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

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)

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)

TNFa and its receptors

TNF TNF

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

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

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

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

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

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

GDNF is a potent pro-survival agent for

spinal motor neurons

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

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

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.

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

****

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

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

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

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

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+/+

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

....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

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

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

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

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

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

daniela.rossi@icsmaugeri.it