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mRNA Regulation and Development
Martine Simonelig
Les organismes-modèles pour l’étude
des dystrophies musculaires
mRNA Regulation and Development
Martine Simonelig
Translational control of maternal mRNAs during early
development in Drosophila
Isabelle Busseau, Catherine Papin, Christel Rouget, Willy Joly, Bridlin Barckmann,
Anne-Cécile Meunier
Development of a Drosophila model of the human
oculopharyngeal muscular dystrophy or OPMD
Aymeric Chartier, Nicolas Barbezier, Cédric Soler
Les organismes-modèles pour l’étude des maladies
génétiques humaines
Les organismes-modèles:
levure
crible à grande échelle (molécules)
drosophile (Drosophila melanogaster)
rapidité
génétique/outils génétiques
très développés
nématode (Caenorhabditis elegans)
souris
génétique
mammifère/plus proche de l’homme
Quelles maladies génétiques humaines?
Cancer, retards mentaux, épilepsie, diabète, infection (immunité innée)...
Maladies neurodégénératives / Dystrophies musculaires
Conservation des génomes et des
fonctions moléculaires entre l’homme et
les organismes modèles
Gènes impliqués dans des maladies génétiques humaines
77% ont un homologue chez la drosophile
65% ont un homologue chez le nématode
Two ways to produce an animal model of a human
genetic disease
By loss of function of the gene homologous to the human gene mutated
in the disease
recessive genetic disease due to a loss of function mutation
requires that the animal model has the homologous gene affected in the disease
e.g.: - Fragile X syndrome (mutation in FMR1: fragile X mental retardation 1) (Drosophila model)
- Spinal muscular atrophy (mutation in SMN: survival motor neuron) (Drosophila model)
- Duchenne Muscular Dystrophy ( mutation in dystrophin) (C. elegans model)
By expressing or overexpressing the human mutant protein in the
animal
dominant genetic disease due to a gain of function mutation
does not require the homologous gene in the animal model
Neurodegenerative diseases / Oculopharyngeal Muscular Dystrophy
Inducible expression in Drosophila using the
UAS/Gal4 system (Brand & Perrimon 1993)
Gal4 Fly stock
GAL4
X
UAS Fly stock
cDNA-X
UAS
polyA
Genomic regulation
sequence
UAS/Gal4 embryos, larvae or flies
expression
GAL4
GAL4
UAS
cDNA-X
Genomic regulation
sequence
Gal4 drivers: ubiquitous, neurons, photoreceptor neurons, muscles ...
polyA
Drosophila models of human neurodegenerative diseases
Since 1998
Models of neurodegenerative diseases
By expressing in Drosophila the human mutant protein
Polyglutamine diseases:
at least 9 neurodegenerative diseases due to expansion of a polyglutamine tract in
different proteins: normal up to 35 glutamines / disease when 40 or more glutamines.
- Huntington's (huntingtin)
- Spinocerebellar ataxia type 3 (SCA3)
- Spinocerebellar ataxia type 1 (ataxin 1)
Non-polyglutamine diseases:
- Parkinson's (a-synuclein)
- Alzheimer's / Tauopathy (tau)
Pathology: neurodegeneration, late onset, progressive
memory loss, cognitive deficits, movement disorders
At the cellular level: the mutant protein forms insoluble aggregates
(protein conformation diseases)
Expression of polyglutamine expanded protein in the eye:
photoreceptor neurons (GMR-Gal4)
Marsh & Thompson, 2004
Features of the human diseases that are
conserved in Drosophila:
polyglutamine diseases:
- Neuronal degeneration
- Late onset
- Progressive
- Onset and severity correlate with polyglutamine repeat length
- Early death
- Cellular level:Nuclear inclusions (NI) containing the polyglutamine-expanded mutant
protein, or the polyglutamine alone with a tag
- Control: no NI with 20/27 glutamines.
Phenotypes reproduced in Drosophila with 127 glutamines out of the
context of a protein: The polyglutamine is intrinsically toxic
non-polyglutamine diseases:
- Parkinson's: adult onset loss of dopaminergic neurons, filamentous inclusions in neurons
(with a-synuclein), locomotor dysfunction.
- Alzheimer's / Tauopathy: adult onset, progressive neurodegeneration, accumulation of
abnormal tau.
Identify suppressor genes of these human diseases in Drosophila,
by a genetic approach
identify genes involved in the disease process without a priopri
of the molecular function
to understand molecular mechanisms of the disease
identify molecular pathways of the disease
to find targets for possible therapies
Identify suppressor genes of these human diseases in Drosophila,
by a genetic approach (genetic screens)
Mutagenesis, collections of mutants
loss of function mutants: chemical mutagenesis, P element inserted randomly in the genome
gain of fonction mutants:
P-UAS element inserted randomly in the genome
UAS
P-UAS element
gene X
expression
Screen
Suppressors or enhancers of the phenotype induced by expression of the polyglutamine
in the eye (by the UAS/Gal4 system)
suppressor
UAS-polyQ/+;
GMR-Gal4/+
phenotype
enhancer
UAS-polyQ/+;
GMR-Gal4/+
UAS-polyQ/+;
GMR-Gal4/+
Suppressor genes of neurodegenerative diseases in Drosophila
(by genetic screens)
1999/2007: genetic screens or test of candidates
Suppressors of polyglutamine diseases: increased expression of
- P35: viral anti-apoptotic apoptosis
- Chaperone proteins/or pathway:
HSP70 (human HSPA1L)
HSP40/ HDJ1 (chaperone-related J domain)
dTRP2 (chaperone-related J domain)
HS response factor
DnaJ1 64EF (chaperone)
Major pathways:
(N. Bonini, 2007)
Protein folding
Protein degradation
- ubiquitin/proteasome pathway
- proteins involved in autophagy (protein degradation by lysosomes)
- CBP: histone acetyltransferase
sequestration of transcription factors/or histone acetyltransferase by the poly-Q expanded protein
Suppressors of Parkinson’s: increased expression of HSP70
New approaches to find suppressors of these diseases
in the Drosophila model, based on the cellular pathways
identified by genetics
1) Prevention of aggregation
2) Protein folding: HSP70 pathway
3) Transcription regulation: inhibitors of histone deacetylase
New approaches to find suppressors of these diseases
in the Drosophila model, based on the cellular pathways
identified by genetics from 2002/2003
1) Prevention of aggregation
Design of suppressor peptides: for Huntington’s disease
25Q
17 aa
huntingtin
spacer ahelice
(54 to 67 aa)
25Q
myc
prevents polyglutamine aggregation
suppresses the phenotype in vivo in the Drosophila
model (L. Thompson, 2002)
Screen of suppressor peptides and test in vivo in the Drosophila model:
Polyglutamine Binding Peptide (QBP1) for polyglutamine diseases
suppresses neurodegeneration and early death (T. Toda, 2003)
Test of chemical or pharmacological compounds as suppressors
- Congo red and cystamine: suppressors in vivo for polyglutamine disease (L. Thompson, 2003)
New approaches to find suppressors of these diseases
in the Drosophila model 2002/2003
2) Protein folding: HSP70 pathway
Pharmacological compounds as suppressors
Geldanamycin: antibiotic, increases the level of HSP70
suppressor in vivo of Parkinson’s disease (N. Bonini, 2002)
3) Transcription regulation: inhibitors of histone deacetylase
Chemical compounds as suppressors
Butyrate and SAHA: inhibitors of histone deacetylases
suppressors in vivo of Huntington’s disease (L. Thompson 2001, Min 2003)
From Drosophila models to mouse models
Marsh &Thompson, 2004
New approaches to find suppressors: the Drosophila model
as an in vivo test.
High throughput test of molecules in Drosophila 2005/2006
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
2006: at least one molecule in clinical trials in patients, identified thanks
to a Drosophila model, in an academic lab (R. Cagan), for a cancer:
Multiple Endocrine Neoplasia Type 2. The molecule stops metastasis.
New approaches to find suppressors: the Drosophila model
as an in vivo test.
Test of intrabodies in Drosophila 2005
Ag
Intrabodies :
single chaine antibodies expressed within the cell
VH
VL
linker
Variable
Light
Variable
Heavy
IgG
In Drosophila (Messer 2005):
Cloning of intrabody DNA downstream of UAS:
expression with Gal4, whithin the cells
expressing polyQ-exon1Huntingtin:
- screen for intrabodies specific
to Huntingtin (yeast phage display)
-optimisation of the intrabody
(test in yeast model)
intrabody
UAS
expression
Reduction of neurodegeneration and formation of aggregates
Increase survival to adulthood (23% without intrabody/ 100% with intrabody)
Models for Muscular Dystrophies:
in Drosophila or C. elegans
Drosophila
C. elegans
Duchenne muscular
Dystrophy
dystrophin mutant
yes
yes
Spinal muscular atrophy
(SMA)
survival motor neuron
(SMN) mutant
yes
yes
yes
no
yes
yes
Myotonic Dystrophy
Oculopharyngeal
muscular Dystrophy
expression of noncoding CUG repeats
expression of the human
mutant protein
Duchenne Muscular Dystrophy
Loss of function mutation / recessive disease / X-linked
The most common of muscular dystrophies: 1 boy / 3500
Extremely severe: wheelchair-bound at 12, respiratory failure in early twenties
No treatment
Mutation in the gene encoding Dystrophin (very big gene: 2.9 megabases, 79
exons) (sporadic cases: 1/10 000 sperm or eggs)
Most DMD patients lack the Dystrophin
Dystrophin: 3685 AA protein present in skeletal and cardiac muscles
Duchenne Muscular Dystrophy
Dystrophin bridges extracellular matrix and cytoskeleton inside muscle cells
DGC:
dystrophin
glycoprotein
complex
Nowak & Davis 2004
Duchenne Muscular Dystrophy
Potential mechanisms contributing to muscle degeneration in DMD:
- Structural role of dystrophin:
degradation of proteins of the Dystrophin-Glycoprotein complex in the absence
of dystrophin: decrease in the amounts of the complex: Muscle fibers are more sensitive
to mechanical damage: leads to muscle degeneration, chronic inflammation, susceptibility to
oxidative stress
- inappropriate location of membrane components leading to alteration of ionic canals
- Role of the Dystrophin-Glycoprotein complex in the intracellular nitric oxide (NO) pathway:
loss of association between DGC and the nitric oxide synthase (nNOS) leads to impaired
nitric oxide production: role of NO in epigenetic regulations through the regulation of HDAC
(histone deacethylase).
Nitric oxide and HDAC have a role in DMD:
rescue of nNOS expression ameliorates the dystrophic phenotype in the mouse
model of DMD
deacethylase inhibitors are beneficial in the mouse model
Animal models for Duchenne Muscular Dystrophy
Mouse model: mutation in the gene encoding dystrophin (stop codon in exon 23):
mdx mice
mild myopathy
No cell model or in vitro model:need for a model useful for high-throughput screens
Possible models in Drosophila or C. elegans:
Dystrophin and the proteins of the DGC complex are conserved in
Drosophila and C. elegans / in smaller number
(dystrobrevin, sarcoglycans, syntrophins, dystroglycan, sarcospan)
muscles with a sarcomeric structure and protein composition similar to
mammalian striated muscles (but no satellite cells, and no fusion in C. elegans)
C. elegans model of Duchenne muscular dystrophy
(L. Ségalat, Lyon)
mutation in the Ce gene encoding dystrophin: dys-1 (null mutation)
phenotype: hyperactive locomotion, muscular hypercontraction, BUT...
no muscle degeneration
Double mutant in dys-1 and MyoD (myogenic factor)
dys-1-; CeMyoDts : locomotion defects / adult onset / progressive over time
protein homologous to a
rat protein interacting with
neural nitric oxide synthase
nNOS
WT
CeMyoDts
dys-1-
dys-1-;
CeMyoDts
dyc-1
dys-1-;
+
CeMyoDts overexpression
uncoordinated suppressor
C. elegans model of Duchenne muscular dystrophy:
analysis of muscle structure (optic microscopy)
active molecule
phalloidin staining:
visualization of actine fibers
WT
dys-1-; CeMyoDts
dys-1-; CeMyoDts
+ prednisone
(0.5 mg/ml / steroid)
Identification of prednisone from a test screen of 100 molecules (reduces muscle degeneration)
Prednisone is used as a treatment for DMD boys
Identification of this molecule in the C. elegans model in a blind screen indicates that
this model can be used for the search of active molecules
Suppressors of Duchenne muscular dystrophy
in the C. elegans model
Test of existing pharmaceutical compounds in the DMD C. elegans model :
Serotonin (neuro-hormone) is a suppressor of muscle degeneration
Reduction of serotonin levels leads to muscle degeneration in the CeMyoD mutant
A function of serotonin in muscles
Serotonin is beneficial to striated muscles
(Ségalat 2006)
- Mutation in the chn-1 gene decreases muscle degeneration in the DMD C. elegans model
CHN-1 is the homologue of human CHIP: interacts with E3 ubiquitin ligase and
E4 enzyme (ubiquitin-conjugating factor)
- A proteasome inhibitor has the same effect (MG132)
Implication of the ubiquitin/proteasome pathway in DMD
(Baumeister 2007)
Exon skipping therapy in Duchenne muscular dystrophy
(Garcia, Danos /Généthon 2004)
Knowledge of the disease in man, at the molecular level
Test in the mouse model, mdx mouse
Restoration of
dystrophin production
dystrophin
spectrin-like repeats
STOP
Muscle regeneration
Restoration of muscle
capacity
antisense RNA
Stable over time
possibly permanent
U7 snRNA
nonspliceosomal snRNA
modified to be incorporated in spliceosome
used to deliver antisense sequence
during splicing
in AAV vector: injected in mice, intramuscular or intra-arterial
(adeno-associated virus)
Drosophila model of oculopharyngeal muscular dystrophy:
OPMD
Aymeric Chartier, Béatrice Benoit & Martine Simonelig.
A Drosophila model of oculopharyngeal muscular dystrophy reveals intrinsic
toxicity of PABPN1. EMBO J. 2006, 25, 2253-2262.
Chartier Aymeric, Raz Vered, Sterrenburg Ellen, Verrips Theo C., van der Maarel Silvère
& Simonelig Martine. Prevention of oculopharyngeal muscular dystrophy by muscular
expression of Llama single-chain intrabodies in vivo.
Human Molecular Genetics 2009, 18, 1849-1859.
Contact: [email protected]
http://www.igh.cnrs.fr/equip/Martine.Simonelig/