C. elegans models in polygutamine disorders

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Criblage sur modèle nématode C. elegans une
composante du processus de découverte de
médicaments pour les maladies dégénératives
Nicolas Offner Laboratory of Neuronal Cell
Biology Pathology Center for Psychiatry
Neuroscience UMR 894 Ste Anne Hospital, Paris,
France neri_at_broca.inserm.fr
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• Neurodegenerative diseases
• Needs neuroprotective drugs, markers
• Rationale
• promote neuronal cell adaptation to
  proteotoxicity
• Focus
• apply a top-down approach to identify the key
  modifiers of
• proteotoxicity as promising targets disease
  modifiers/markers
• find drugs able to slow-down the early stages
  of several diseases
• Interest
• role of longevity/cell maintenance modulators
  and developmental
• genes in diseased cell survival

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Huntingtons disease and huntingtin (htt)
Phosphorylation regulates clivage by caspases
Ubiquitination, sumoylation acetylation
regulate stability
Nuclear export
hydrophobic core
3145
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polyP
36 Glns
Protein Interaction (WW, SH3, CH2, CH3)
A cytoskeletal scaffold involved in development
and neurogenesis

• Complex disease mechanisms
• Loss of normal htt function
• gain of toxic properties soluble N-ter
  fragments
• abnormal transcription protein trafficking
• abnormal neurotransmission

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Clinical variability in HD

• What are the modifier genes ?
• Biomedical potential ?

Htt belongs to a large protein network covering
several cellular processes focus on partner
proteins HD onset age spans late development
and adult lifetime focus on pathways involved
in development and/or longevity
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C. elegans as a genetically-tractable and
well-characterized model system to study the
neuronal effects of expanded polyQs at the
genomic level
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Life cycle
2-3 week mean lifespan
1 generation 3.5 days At 20?C
4 larval stages (L1-L4) punctuated by molts
Low food, high pheromone enters a long-lived,
stress resistant, non-reproductive stage, the
dauer
Mango and Jorgensen, Nat Rev Gen (2003)
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Good things about C. elegans

• ANATOMICAL INVARIANCE , SMALL CELL NUMBER
  allow IDENTIFICATION of every cell in the animal
• TRANSPARENCY, SIMPLICITY, SPEED and INVARIANCE
  OF DEVELOPMENT allowed description of COMPLETE
  CELL LINEAGE reproducible series of cell
  divisions by which the fertilized egg produces an
  adult
• CELL LINEAGE high-resolution marker of cell
  fate, entire nervous system reconstructed by
  serial-section electron microscopy.
• GENETICS small size, rapid development,
  reproduction by self-fertilization and
  facultative crossing all facilitate genetic
  analysis
• MOLECULAR ANALYSIS genome sequence well aligned
  with genetic map permits rapid progress to
  molecular genetic analysis

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Touch receptor neurons (P mec-3 targets)

• Response to light touch controlled by one pair of
  neurons
• Easily-assayable and quantitative read-out
• Non essential neurons
• Reproducible responses

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-
Q19
Q128
Parker et al., PNAS 2001

• Early phases of expanded polyQ toxicity neuronal
  dysfunction

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Chemical synapse of ALMR/L neurons
httRFP SNB1GFP

Number of SNB1GFP signals / neuron
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Diseased neuron survival
Development Longevity
FoxO network FoxONet

• Mechanisms for protection of HD neurons ?
• Therapeutic implications ?
• 3) Contribution to phenotypic variability in HD ?
  

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FoxO and the Insulin/IGF pathway

• Metabolism (glucose production,
• protection against diabetes)
• Cell differenciation/cell
• cycle arrest (tumor suppression
• if repressed)
• Stress resistance/longevity
• (long life-span if activated)

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Regulation of FOXO activity
Deacetylation (Sir2)
Phosphorylation (IGF path, AMPK) Ubiquitination
(skp2)
Fine tuning intracellular distribution
interaction w/ promoters
Nucleocytoplasmic distribution, degradation
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• Effect of Increased dosage of sir-2.1/SIRT1

Lifespan

• (Tissenbaum Guarente, Nature 2001)

Touch assay
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128Q
19Q
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rescue of touch response at the tail
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0
-10
-20
(Parker et al., Nature Genetics 2005)
sir-2.1 SIRT1
age-1 PI3 kinase
daf-16 FOXO

• sir-2.1
• SIRT1

• sir-2.1
• daf-16

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FoxONet
Lifespan
Development
IGF1/insulin signaling
Jnk signaling
Developmental (Smad) and stress response (hsf-1)
TFs
Sir2.1/SIRT1 aak-2/AMPK
Daf-16/FoxO
About 700 putative or established
targets Antioxydative activity, mitochondrial
function, protein folding, protein degradation,
cell cycle, others (Cynthia Kenyon coll., Gary
Ruvkun coll., Heidi Tissenbaum coll.)
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Downstream to FoxO
Hundreds of targets
Genome-wide analysis in polyQ nematodes Genetics
RNAi screen microarray analysis
Data integration Comparisons with other models
Unbiased selection of genes of high interest
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BIOGEMIX/HD database and data analysis package
RNAi screen (modifiers of response to light
touch) 5000 genes tested
including 446 FoxO targets Microarray analysis of
FACS-purified P mec-3 target cells
2000 genes dysregulated by mutant polyQs
PolyQ worms
HD
Published and unpublished datasets relevant to
HD Htt partner proteins (RE Hughes, E.
Wanker) Genes dysregulated in mouse and human
brains (R. Luthi-Carter) Modifiers in flies and
fly cells (J. Botas, N. Perrimon) Modifiers
(ShRNA scren) in cellular models (RE Hughes,
Biofocus) HD modifier loci (N. Wexler genome
scan data) Other large datasets of
interest longevity (C. Kenyon, G.
Ruvkun) synaptic structure and activity (J.
Kaplan) neural outgrowth (N. Perrimon)
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FoxO targets are significantly enriched in
modifiers of neuronal dysfunction produced by
mutant polyQs More largely, there is a role for
modulators of mitochondrial function including
FoxO targets
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daf-16/FoxO
Putative target (Ruvkun lab)
ucp-4 / UCP4
Mitochondria uncoupling ATP production
respiration
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Neuroprotection by Sir2 requires ß-catenin and
ucp-4
UCP-4 is neuroprotective
Ucp-4 LOF
Increased Sir2 dosage bar-1 or ucp-4 LOF
UCP2/4 siRNAs modulate the survival of HdhQ111
striatal cells
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No effect
Protective
JNK
PDK
Detrimental
GSK3?
ftt-2
Validated in striatal cells
AMPK
Lit-1/NLK
Effector mechanisms
UCP2/4
SOD-3
CTL-2
Mitochondrial function
Antioxidant enzymes
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Diseased neuron survival
Development Longevity
FoxO network FoxONet

• Mechanisms for protection of HD neurons ?
• Therapeutic implications ?
• 3) Contribution to phenotypic variability in HD ?
  

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David Sinclair, Nature Genetics 2005
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Incubation
mix
Lecture
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Parker et al. Nature Genetics (2005)
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Insulin/IGF
Gene activity
DAF-2
No effect
Weak allele
PI3K
Protective
JNK
SGK
PDK
Detrimental
AKT
GSK3?
Validated in striatal cells
FOXO
Sir-2.1
FOXO
SIRT1
FOXO
AMPK
UCP2
SOD-3
Mitochondrial uncoupling
Antioxydant
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ALMR/L neurons N-ter httRFP SNB1GFP

Number of SNB1GFP signals/neuron
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General validity of Sirtuin-FoxO protection
Sir2 activation and resveratrol protect worm and
mouse neurons from mutant PrP toxicity
(collaboration w/JL Laplanche)
Would Sir2-FoxO be protective in OPMD, a late
onset muscular dystrophy caused by polyAla
expansion in the nuclear protein PABPN1
(collaboration w/G. Rouleau) ?
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Target cells body wall muscles PolyAla tract
13, 10 or 0 Alas
Abnormal motility Loss of nuclear GFP
signals Muscle cell loss Phenotypes primarily
caused by soluble PABPN1
Catoire, Pasco et
al. Hum Mol Genet 2008
Suppressing Sir2, FoxO and AMPK is protective
motility
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Conclusions
Key developmental genes and longevity modulators
in FoxONet modulate diseased cell survival and
they have therapeutic potential Working model
cross-talk between several FoxONet members in
promoting HD neuron survival through specific
effectors like mitochondrial uncoupling Drug
treatment efficient neuroprotection by using
targets that signal onto the same effectors
Sir2-FoxO modulators GSK3ß inhibitors Several
of these notions may be of general validity for
several diseases
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Past Alex Parker, PhD Margarita Arango,
PhD Hélène Catoire, PhD Céline Lefebvre,
PhD Sébastien Holbert, PhD Emmanuel
Lambert Present Nicolas Offner, PhD Rafael
Vasquez, PhD Cendrine Tourette Matthieu
Pasco Sophie Menet Juliette Pascaud Aurélie
Darbois Cedric Bicep Hazare Nouari David Bragado

• Working Group in Biomathematics, Paris 5
• Antoine Chambaz, CNRS, Paris
• Jean-Philippe Vert, Ecole des Mines, Paris

• And with
• Alexis Brice, Inserm
• Nicole Déglon, CEA
• Elena Cattaneo, Univ. of Milano
• Robert Ferrante, Boston Univ.
• Michael Hayden, Univ. British Columbia
• Norbert Perrimon, Harvard Med School
• Christopher Ross, Johns Hopkins Univ.
• Guy Rouleau, Univ. of Montreal
• David Sinclair, Harvard Med School
• Heidi Tissenbaum, MIT
• The French HD Group
• The European HD Network
• Transcriptomics platform, ENS
• Cytometry platform, Pasteur Institute

INSERM, ANR, FRM, AFM, Univ. of Montreal,
Hereditary Disease Foundation, CHDI
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