Disruption of the astrocytic TNFR1-GDNF axis accelerates motor neuron degeneration and disease progression in amyotrophic lateral sclerosis

Transcription

1 Disruption of the astrocytic TNFR1-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, 216

2 Amyotrophic Lateral Sclerosis Prevalence: 2-8 per, people Incidence: 1-2 per, people each year Clinical features: Muscle weakness Atrophy Spasticity Pathological features: degeneration and death of corticospinal and spinal motor neurons Death due to respiratory failure within 2-5 years of clinical onset

3 ALS gene frequencies Alsultan et al, Degener Neurol Neuromusc Disease, 216

4 ALS gene frequencies Alsultan et al, Degener Neurol Neuromusc Disease, 216

5 Human SOD1 mutant sites Sites of the mutant residues depicted are derived from the ALS Online Genetic Database

6 Transgenic mutant SOD1 mouse models Turner & Talbot, Prog Neurobiol., 8

7 SOD1 G93A transgenic mice Wild type SOD1 G93A presymptomatic SOD1 G93A end stage Chiu et al, Mol Cell Neurosci, 1995 Turner & Talbot, Prog Neurobiol., 8

8 Is motor neuron degeneration independent on the microenvironment?

9 Clement et al, Science, 3

10 Astrocyte involvement in ALS Yamanaka et al, Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis, Nat. Neurosci., 8 Wang et al, Astrocytes loss of mutant SOD1 delays ALS disease onset and progression in G85R transgenic mice, Hum. Mol. Genet., 211 Papadeas et al, Astrocytes carrying the superoxide dismutase 1 (SOD1G93A) mutation induce, Proc. Natl. Acad. Sci. USA, 211 Ilieva et al, Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond, J Cell Biol., 9 Ferraiuolo et al, Dysregulation of astrocytemotoneuron cross-talk in mutant SOD1-related amyotrophic lateral sclerosis, Brain, 211 Van Damme et al, Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity, Proc. Natl. Acad. Sci. USA, 7 Nagai et al, Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons, Nat. Neurosci., 7 Di Giorgio et al, Non-cell autonomous effect of glia on motor neurons in an embryonic stem cellbased ALS model, Nat. Neurosci., 7 Di Giorgio et al, Human embryonic stem cellderived motor neurons are sensitive to the toxic effect of glial cells..., Cell Stem Cell, 8 Marchetto et al, Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived., Cell Stem Cell, 8 Cassina et al, Mytochondrial dysfunction in SOD1G93A-bearing astrocytes promotes motor neuron degeneration, Hum. Mol. Genet., 211 Yang et al, Presynaptic regulation of astroglial excitatory neurotransmitter transport GLT1, Neuron, 9 Aebischer et al, IFNgamma triggers a LIGHTdependent selective death of motoneurons contributing., Cell Death Differ., 211 Haidet-Phillips et al, Astrocytes from familial and sporadic ALS patients are toxic to motor neurons, Nat. Neurosci., 211 Diaz-Amarilla et al, Phenotypically aberrant astrocytes that promote motoneuron damage in a model.., Proc. Natl. Acad. Sci. USA, 211 Rossi et al, Focal degeneration of astrocytes in amyotrophic lateral sclerosis, Cell Death Differ., 8

11 Deleterious interplay between motor neurons and astrocytes Gain of function Loss of function Cytokines (IL-1, IL-6, IL-1 INFg, GM-CSF, TGFb, TNFa Chemokines (CCL2, MCP-1, CCL5, CCL2, CXCL1, CXCL12, CXCL1, CXCL2, CX3CL1) Eicosanoids Reactive oxygen species Nitric Oxide Excitatory aminoacids Glutamate uptake Aberrant [Ca 2+ ] i GluR2 subunit of AMPAR modulation Metabolic substrates Trophic factors (GDNF, BDNF, NT3, CNTF, VEGF, bfgf, IGF1)

12 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)

13 TNFa and its receptors TNF TNF

14 Regulatory Neurotoxic/neuroprotective Physiological processes Pathological processes Synaptic plasticity Learning and memory Sleep Glial transmission Food/water intake Ischemic and traumatic injuries Epilepsy Chronic pain Multiple sclerosis Amyotrophic Lateral Sclerosis Infectious diseases Alzheimer s disease Parkinson s disease

15 Relative GDNF mrna expression (% of ctrl) Relative GDNF protein expression (% of ctrl) Relative GDNF protein release (% of ctrl) TNFa /TNFR1 signalling controls GDNF expression and secretion from human spinal astrocytes ctrl TNFa ctrl TNFa *** 15 *** *** ***p<.1 vs. ctrl Brambilla et al, Hum Mol Genet, 216

16 Relative GDNF mrna expression (% of ctrl) Relative GDNF protein expression (% of ctrl) Relative GDNF protein release (% of ctrl) TNFa/TNFR1 signalling controls GDNF expression and secretion from mouse spinal astrocytes *** ctrl TNFa 15 *** ctrl TNFa *** Wild type Wild type tnfr1 -/- Wild type ***p<.1 vs. ctrl Brambilla et al, Hum Mol Genet, 216

17 Relative GDNF mrna expression (% of ctrl) Relative GDNF protein expression (% of ctrl) Relative GDNF protein release (% of ctrl) TNFa/TNFR1 signalling controls GDNF expression and secretion from mouse spinal astrocytes *** ctrl TNFa 15 *** ctrl TNFa *** Wild type tnfr1 -/- Wild type tnfr1 -/- Wild type tnfr1 -/- ***p<.1 vs. ctrl Brambilla et al, Hum Mol Genet, 216

18 The TNFa/TNFR1 pathway controls GDNF expression in vivo i.c.v. injection 6-7 wks (age) 48 h vehicle TNFa Wild type tnfr1 -/- Wild type tnfr1 -/- RNA Protein cdna ELISA RT-qPCR Brambilla et al, Hum Mol Genet, 216

19 Relative GDNF mrna expression (% of vehicle) Relative GDNF protein expression (% of vehicle) The TNFa/TNFR1 pathway controls GDNF expression in vivo i.c.v. injection 6-7 wks (age) 48 h vehicle TNFa Wild type tnfr1 -/- Wild type tnfr1 -/- RNA cdna RT-qPCR Protein ELISA ** vehicle TNFa * vehicle TNFa Wild type tnfr1 -/- Wild type tnfr1 -/- **p<.1, *p<.5 vs. vehicle Brambilla et al, Hum Mol Genet, 216

20 GDNF is a potent pro-survival agent for spinal motor neurons

21 GDNF delivery to spinal motor neurons by muscle cells Retrograde viral delivery of GDNF SOD1 G93A ALS model Murine myoblasts or hmscs genetically modified to secrete GDNF before disease onset Delayed degeneration of motor neurons and decline in motor function Prolonged survival Acsadi et al, Hum Gene Ther, 2; Kaspar et al, Science, 3; Manabe et al, Apoptosis, 2; Wang et al, J Neurosci, 2; Mohajeri et al, Hum Gene Ther, 1999; Suzuki et al, Mol Ther, 8

22 Direct GDNF delivery into the CNS Viral delivery of GDNF or hnpc GDNF SOD1 G93A spinal cord Protection of motor neurons Guillot et al, Neurobiol Dis, 4 Klein et al, Hum Gene Ther, 5 Suzuki et al, Plos One, 7

23 Neurotrophic support 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. Bohn, Experimental Neurology 4

24 Relative mrna expression (%of 3 days) Correlation between the endogenous TNFa/TNFR1 system and GDNF expression in SOD1 G93A mouse spinal cord 3 days days 13 days TNFa TNFR1 GDNF #### **** *** * #### **** *** #### **** 3 * *** *p<.5 and **p<.1 vs. -day-old Wild-type and SOD1 WT mice ***p<.1 vs. 3-day-old SOD1 G93A mice ****p<.1 vs. 13-day-old Wilde-type and SOD1 WT mice #### p<.1 vs. 3- and -day-old SOD1 G93A mice Wild type SOD1 WT SOD1 G93A Brambilla et al, Hum Mol Genet, 216

25 MAC-1 SMI32 GFAP Expression of GDNF in SOD1 G93A mouse spinal cords is predominantly in the astrocytes GDNF merge Brambilla et al, Hum Mol Genet, 216

26 Relative mrna expression (%of controls ) Correlation between the endogenous TNFa/TNFR1 system and GDNF expression in sporadic ALS spinal cords Controls sals ** TNFa Patients Age of death (Years) Sex Onset Duration of illness (Months) Cause of death 18 ** TNFR1 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 ** GDNF **p<.1 vs. Controls Brambilla et al, Hum Mol Genet, 216

27 Breeding scheme for the generation of mutant SOD1-expressing mice that are either wild-type or knockout for the tnfr1 gene SOD1 G93A X tnfr1 -/- SOD1 G93A ;tnfr1 +/- X SOD1 G93A ;tnfr1 +/+ tnfr1 +/- SOD1 G93A ;tnfr1 -/- Brambilla et al, Hum Mol Genet, 216

28 Relative mrna expression (% of 3 days) Disruption of endogenous TNFa/TNFR1 signalling abrogates GDNF expression 6 5 **** TNFa 4 3 #### ** 3 days days 13 days 5 **** GDNF 4 3 #### *** SOD1 G93A ;tnfr1 +/+ SOD1 G93A ;tnfr1 -/- **p<.1 and ***p<.1 vs. 13-day-old SOD1 G93A ;tnfr1 +/+ mice ****p<.1 vs. 3- and -day-old SOD1 G93A ;tnfr1 +/+ mice #### p<.1 vs. 3-day-old SOD1 G93A ;tnfr1 +/+ mice Brambilla et al, Hum Mol Genet, 216

29 Relative mrna expression (% of 3 days) Expression of neuroprotective/ neurotoxic factors is unchange in spinal cord of tnfr1 -/- ALS mice 15 5 VEGF-B *** *** IL-1b *** *** *** *** 3 COX-2 ** ** Bcl-X L 25 ### ### 15 5 SOD1 G93A ;tnfr1 +/+ SOD1 G93A ;tnfr1 -/- 3 days days 13 days *** ### *** MMP-9 ***### *** SOD1 G93A ;tnfr1 +/+ SOD1 G93A ;tnfr1 -/- ***p<.1 and **p<.1 vs 3-day-old genotype-matched mice ### p<.1 vs 3- and -day-old day-old genotype-matched mice Brambilla et al, Hum Mol Genet, 216

30 Probability of onset (peak of body weight) Probability of onset (rotarod deficits) Ablation of the tnfr1 gene has no impact on the onset of ALS disease.. A B SOD1 G93A ;tnfr1 +/+ SOD1 G93A ;tnfr1 -/- SOD1 G93A ;tnfr1 +/+ SOD1 G93A ;tnfr1 -/ Age (days) Age (days) Brambilla et al, Hum Mol Genet, 216

31 Probability of survival Duration...but accelerates its progression in SOD1 G93A mice C SOD1 G93A ;tnfr1 +/+ D SOD1 G93A ;tnfr1 -/- tnfr1 -/ Early phase Late phase # Age (days) SOD1 G93A ;tnfr1 +/+ SOD1 G93A ;tnfr1 -/- # p<.5 vs SOD1 G93A ; tnfr1 +/+ in the Late phase Brambilla et al., Hum Mol Genet, 216

32 Ablation of the tnfr1 gene causes a significant decrease in the number of motor neurons in symptomatic SOD1 G93A mice Motor neuron counts SOD1 G93A ;tnfr1 +/+ A B C D SOD1 G93A ;tnfr1 +/+ SOD1 G93A ;tnfr1 -/- SOD1 G93A ;tnfr1 -/- E F G H 3 3 days ** * *** Late phase 3 days Late phase **p<.1, ***p>.1 vs 3-day-old genotype-matched mice *p<.5 vs SOD1 G93A ; tnfr1 +/+ at the Late phase Brambilla et al, Hum Mol Genet, 216

33 Relative GDNF mrna expression (% of ctrl) SOD1 G93A astrocytes respond to TNFa/TNFR1 signalling by producing normal GDNF levels 3 25 ** ** *** ctrl TNFa 15 **** 5 Wild type SOD1 WT SOD1 G93A ;tnfr1 +/+ SOD1 G93A ;tnfr1 -/- **p<.1, ***p>.1 vs. ctrl astrocytes ****p<.1 vs. TNFα in Wild-type, SOD1 WT and SOD1 G93A ; tnfr1 +/+ astrocytes Brambilla et al, Hum Mol Genet, 216

34 asymptomatic stage onset of motor deficits Microglia symptomatic stage Astrocytes Motor Neuron X X Muscle TNFa TNFR1 GDNF Brambilla et al, Hum Mol Genet, 216

35 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 SOD1 G93A ALS mice Motor neuron loss is more pronounced in SOD1 G93A 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 SOD1 G93A astrocytes

36 Istituti Clinici Scientifici Maugeri SpA SB Liliana Brambilla Giulia Guidotti Chiara Malatino Federica Sorrentino Former members of the lab Chiara Valori Francesca Martorana Chiara Bergamaschi University of Lausanne, Switzerland Paola Bezzi Academisch Medisch Centrum, Amsterdam, The Netherlands Anand M. Iyer Eleonora Aronica