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mRNA Regulation and Development
Martine Simonelig
Validation d'outils thérapeutiques dans le
modèle drosophile
exemple de la dystrophie musculaire
oculo-pharyngée (OPMD)
Aymeric Chartier, Nicolas Barbezier, Yannick Bidet
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Drosophila as a model to study human genetic
diseases
Why does it work?
Genomes and molecular functions are conserved
between man and Drosophila 77 of genes involved
in human genetic diseases have a homologue in
Drosophila
Advantages of Drosophila
Rapid analysis (new generation in two weeks)
Drosophila is genetically tractable highly
sophisticated genetic tools
Possibility of large scale screens genetics or
molecules - to understand molecular mechanisms
of the disease - to find targets for possible
therapies
Disease models available in Drosophila Cancer,
mental retardation, diabetes, innate immunity,
neurodegenerative diseases, muscular
dystrophies, etc....
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Drosophila models of human neurodegenerative
diseases
Models of neurodegenerative diseases by
expressing in Drosophila the human mutant protein
(since 1998)
Polyglutamine diseases neurodegenerative
diseases due to expansion of a polyglutamine
tract in different proteins, e.g. -
Huntington's - Spinocerebellar ataxia type 3
(SCA3) Non-polyglutamine diseases - Parkinson's
(a-synuclein) - Alzheimer's / Tauopathy (tau)
Features of the disease conserved in Drosophila
Pathology neurodegeneration, late onset,
progressive, onset and severity correlate with
polyglutamine repeat length At the cellular
level the mutant protein forms insoluble
aggregates (protein aggregation diseases)
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The Drosophila model as an in vivo test to
identify or validate therapeutic tools
Validation or design of therapeutic tools
Anti-aggregation peptides Huntingtons
disease 2006 a molecule in clinical trials in
patients, identified in the Drosophila model, in
an academic lab (R. Cagan), for a cancer
Multiple Endocrine Neoplasia Type 2. The
molecule stops metastasis
High throughput test of molecules in Drosophila

• Companies that test large collections of
  molecules (EnVivo Pharmaceuticals)
• molecules are delivered in the food from the
  embryonic stage/ change of the food
• every day
• - e.g. 20 000 flies per week of a disease model
• possible test of collections up to 30 000
  molecules
• Hits (or positive) their effects are analysed at
  cellular and molecular levels, for validation
• Test of the hits in mouse models

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OPMD oculopharyngeal muscular dystrophy
Autosomal dominant muscular dystrophy Late onset
(fifth decade) and progressive weakening of
muscles that hold eyelids, leading to
ptosis involved in swallowing, leading to
dysphagia limb muscles Characterized at the
ultrastructural level by nuclear inclusions of
tubular filaments (8.5 nm diameter), found in
muscle fiber nuclei only in 2 to 9 of the
muscle nuclei
Hino et al., HMG 2004
OPMD is a rare disease, with a worldwide
distribution 1/100 000 in France But more common
in Quebec 1/ 1000
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OPMD is due to mutations in the PABPN1 gene
B. Brais, Nature Genetics 1998 In 144 OPMD
families, from 15 different countries all present
a GCG triplet expansion in the PABPN1
gene leading to a polyalanine tract extension in
the PABPN1, Nuclear Poly(A) Binding Protein I
Wild-type PABPN1
PABPN1
10 alanines
PABPN1 in OPMD patients
PABPN1
12 to 17 alanines
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Molecular mechanisms leading to OPMD?
OPMD is a protein aggregation disorder
Nuclear inclusions in muscles of OPMD patients
contain mutant PABPN1, HSP70, ubiquitin,
proteasome subunits, poly(A) RNA
PABPN1
DNA
poly(A) RNA
(M. Carmo-Fonseca, 2000)
ubiquitin
Extension of the alanine tract in PABPN1 leads
to the formation of insoluble PABPN1 aggregates
in muscle nuclei
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Function of PABPN1 role in nuclear
polyadenylation of mRNA
(E. Wahle)
poly(A) site
CPSF
AAA
PAP
AAUAAA
5'

• In Drosophila
• PABP2 has the same function as its mammalian
  homologue, PABPN1 in nuclear polyadenylation in
  vitro and in vivo
• - Identification of another cytoplasmic function

(Benoit al. Developmental Cell 2005)
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The Drosophila model of OPMD
Expression of mammalian PABPN1 in Drosophila
using the UAS/Gal4 system,
an inducible expression system (Brand Perrimon
1993)
X
Gal4 driver trangenic lines Mhc-Gal4 muscle
(Myosin heavy chain) 24B muscle
precursors (early)
UAS transgenic lines UAS-PABPN1 wild-type10
alanines UAS-PABPN1-17ala mutant
protein UAS-PABPN1-Dala (no alanine)
muscle defects
lethality
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Drosophila adult musculature
thoracic adult musculature
Indirect flight muscles (IFM)
involved in wing posture and flight
DLM dorso-longitudinal muscles
DVM dorso-ventral muscles
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Phenotypes in adults wing posture defects (25C
and 18C)
control
These visible phenotypes occur with
Mhc-Gal4/
Mhc-Gal4/ UAS-PABPN1 UAS-PABPN1-17ala UAS-PABPN1-
Dala
wings up
wings down
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The wing posture phenotype is progressive and
increases with the number of alanines in the
tract

• phenotype progressive
• its strength depends on the number of alanines

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Muscle degeneration is progressive
UAS-PABPN1-17ala/ Mhc-Gal4/
day 1 no muscle defect
day 6 50 of thorax with degenerating IFM (n26)
DLM dorso-longitudinal muscles
day 16 100 of thorax with degenerating
IFM (n35)
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Nuclear inclusions in adult flight muscles
contain HSP70 and ubiquitinylated proteins
Nuclear Inclusions in adult flight muscles (at
25C)
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Nuclear inclusions vizualised by EM presence of
fibrils
UAS-PABPN1-17ala/ Mhc-Gal4/
OPMD patient
(X80 000)
Diameter of fibrils 8.5 nm in OPMD patients
8.3 to 10 nm in Drosophila
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Role of PABPN1 domains in the OPMD-like phenotype
abnormal wing position
self interaction
Day 6
Day 11
coiled coil
17ala
PABPN1-17ala
87
Arg rich
96
Y215A
F175A
0-3
0-3
PABPN1-17ala-dm
OPMD-like phenotypes in Drosophila requires
the RRM and the function of PABPN1 in RNA
binding OPMD depends on intrinsic toxicity of
PABPN1, not on toxicity of the alanine tract per
se
Chartier et al. EMBO J. 2006
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In vivo validation of therapeutic tools using the
Drosophila model
Identification of drugs as suppressors of OPMD in
Drosophila
Anti-PABPN1 intrabodies as suppressors of OPMD
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Assay to screen for drugs
Medium Drosophila Instant Medium 1 yeast
molecule (mg/ml ou µM)
Test of toxicity 40 wild-type 1st instar larvae
per vial at 25C on medium molecule
toxicity
Score of adults of viability
90
70
Doxycycline
of adult viability
50
30
10
-
mg/ml
0.5
0.25
1
2
3
6
working range

• Acitivity of the molecule on OPMD
• - 40 OPMD 1st instar larvae per vial at 18C, on
  medium molecule
• Adults in a new vial at 18C, on medium
  molecule
• Score of wing position phenotypes

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Identification of drugs as suppressors of OPMD
in Drosophila
Test of candidate drugs
Ibuprofen
Trehalose
Doxycyline
90
90
90
70
70
70
flies with abnormal wing position
flies with abnormal wing position
flies with abnormal wing position
50
50
50
30
30
30
10
10
10
0
0.25
mg/ml
0.5
1
0
2

5
10
mg/ml
0
0.1
0.25
0.5
0.6
0.8
1
(active in mouse model of OPMD)
(active in cell model of OPMD)
(active in mouse model of OPMD)
Drugs inactive in the Drosophila model of
OPMD Doxycycline, Trehalose, Ibuprofen, Congo
red (250 µM to 400 µM)
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PABPN1 intrabodies as suppressors of the
OPMD-like phenotype in Drosophila
P. Verheesen, S. van der Maarel T.
Verrips Development of anti-PABPN1 antibodies as
tools in OPMD Camelidae have a repertoire of
antibodies devoid of light chains the heavy
chain antibodies The variable domain of these
antibodies, called VHH efficiently binds the
antigen The variable domain VHH are single
chain antibodies they have a small size (14
kDa), they can be cloned and expressed within the
cells they can efficiently bind the antigen
within the cell
Llama VHH phage-display library screened against
PABPN1 6 antibodies against PABPN1 One of them
reduces PABPN1 aggregation when expressed
intracellularly in a cell model of OPMD
P. Verheesen et al., HMG, 2006
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Collaborations Marc BLONDEL, Université de
Brest Hervé GALONS Fabienne GUG, Université
Descartes, Paris Silvère van der MAAREL, Leiden
University, Leiden
Institut de Génétique Humaine Montpellier
Martine SIMONELIG Isabelle BUSSEAU Cathy
PAPIN Christel ROUGET Perrine BENOIT Bénédicte
FRANCO Yannick BIDET Aymeric CHARTIER Nicolas
BARBEZIER
The muscle team