Drosophila: an alternative model for the modeling of human pathologies and the search for new therapies - FRANCOPA
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Drosophila: an alternative model for the modeling
of human pathologies and the search for new
therapies
Hervé TRICOIRE
Unité de Biologie Fonctionnelle et Adaptative
UMR8251 Université Paris Diderot/CNRS
Colloque OPAL & FRANCOPA 4 Novembre 2015
DISEASE
Associated genes CLINICAL STUDIES >100 M$
Disease models In vivo screening,
>0.5 M$
validation of compounds
Pathological mechanisms
« FDA approved » library
Putative therapeutic targets screening, high throughput
screening, other strategies
>80% of potential therapies fail in human clinical studies
Incorrect modelization
Genetic background S. Perrin, Nature 507 (2014) 423
Insufficient statistics
Why are animal models essential?
Multicellular organisms
• Cellular interactions
• Non autonomous cellular events Clemmons
• Long distance signalisation (hormones, doi:10.1038/nrd2359
neuropeptides, cytokines,...)
• Metabolic regulation
• ....
Cells or isolated tissues cannot reproduce the
complexity of an organism
DISEASE CLINICAL STUDIES
Associated genes Studies in mammals
Organoids
• Sophisticated molecular
Disease models In vivo screening, genetics models
validation of compounds
Pathological mechanisms • Ability to reproduce
« FDA approved » human disease features
Putative therapeutic targets library screening, • Short lifespan speed
other strategies
• Low cost of husbandry
and experiments good
statistics
• Controlled genetic
background
Drosophila and human pathologies
75% of genes involved in human diseases have a Drosophila
orthologue
700
Keyword: disease • Neurodegenerative diseases
•
600 Drosophila
Cancer
Number of papers per year
C. elegans
500
Zebrafish • Cardiac diseases
400
• Metabolic disorders
300 Genome • Gastro intestinal diseases
•
sequencing
200
SCA3
Sleep disorders
100
model
• Intellectual disability
• ….
0
1985 1990 1995 2000 2005 2010 2015 2020
Year
http://flystocks.bio.indiana.edu/Browse/HD/HDintro.htm
http://flydiseasemodels.blogspot.fr/
http://www.flyrnai.org/cgi-bin/DRSC_DG_query.pl/
http://flybase.org/lists/FBhh/
Modelization of a disease: the Fly toolbox
2 - Temporal control
3 - Control of expression level
Gal4GS RU486
Mutated
Gal4GS
protein
Promoter Gal4GS Mutated human gene GOF
UAS
1- Spatial
LOF
control c DNA
UAS
ds RNA
Phenotypic Caracterisation: a wide
range of assays
Human genetics
Epidemiology
Generation of a
Drosophila
model
TISSUE DEGENERATION
In vivo
phenotypic
caracterisation
BEHAVIOR
LONGEVITY
Example 1: Neurodegenerative diseases
A large number of NDs have been modelized in flies
McGurck et al.: Genetics. 2015 Oct;201(2):377-402
• Alzheimer disease Sentruck & Bellen: Curr Opin Neurobiol. 2018 Jun;50:24-32
• Parkinson disease
• Bouleau & Tricoire: J Alzheimers Dis. 2015;45(4):1015-38
• Huntington disease and SCAs • Hewitt & Whitworth Curr Top Dev Biol. 2017;121:173-200
• Krench & Littleton: Curr Top Dev Biol. 2017;121:201-223
• Amyotrophic lateral sclerosis • Casci & Pandey: Brain Res. 2015 May 14;1607:47-74
• Monnier et al.: Int J Mol Sci. 2018 doi: 10.3390/ijms19071989
• Freidreich ataxia
….
Sowa et al. PNAS 2018
115(11):E2624-E263
• Identifying modifier genes
• In vivo analysis of tissue specificities
• Identification of therapeutic compounds
• In vivo assays for putative disease variants
Validation of new susceptibility genes in
Parkinson disease
Nus1: an ER membrane protein
Whole exome sequencing of 39 EOPD families
39 Identification of de novo mutations
12 Filtering of variants Candidate genes
1 Screen gene variants on large PD cohorts
NUS1 as a new candidate gene for PD
Analysis in vivo in Drosophila models
Neuronal Tango14/dNUS1 KD
Locomotor activity Dopamine titer
• Impairment of locomotor activity
• Reduction of dopamine Neuronal
• Loss of dopaminergic neurons Loss
Guo et al.: Proc Natl Acad Sci U S A. 2018 Oct 22.
doi: 10.1073/pnas.1809969115
Example 2: Cancer
• Decoding oncogenic pathways
• In vivo analysis of cellular context
• In vivo analysis of the different phases of the disease
• Analysis of combinatorial treatment
Gonzales Nature Rev. Cancer 13, 172 (2013)A whole-animal Drosophila platform to identify highly
potent drugs against medullary thyroid carcinoma (MTC)
Assay of FDA anticancer kinase inhibitors First chemical optimisation
MTC model Sorafenib LS1-15
Assays in mouse Rationalized chemical optimisation Identification of pro-targets
(TT xenograft assay) and anti-targets for LS1-15
by genetic screening
LS1-15 APS6-45
Sorafenib
« more standard screening platforms … would have
led us toward different compounds” Sonoshita et al.: Nat Chem Biol. 2018 14(3):291-298Example 3: Friedreich ataxia (FA)
Most frequent recessive ataxia:
1/50000
Linked to GAA repeat expansion in
intron 1 of the frataxin (Fxn) gene
Frataxin is a mitochondrial
protein involved in :
Fxn down regulation (30%) • Fe/S complexes synthesis
• Resistance to oxydative
Neuronal degenerescence stress
Cardomyopathy (2/3) • Iron homeostasis
Diabetis (16%)How to do a cardiac model of
Friedreich ataxia in Drosophila
Gal4
Promoter Gal 4 c DNA
UAS
ds RNA
Constructions fhIR1, fhIR2
Mito-GFP
UASDilated cardiomyopathy model to screen
for FA therapeutic compounds
50 µm
+
WT
FA
fhRNAi
fhRNAi
MB 30µM
From Julio C. Rojas et al MB treatment protects again establishment of cardiac dilatation in
Progress in Neurobiology 2012
Drosophila FRDA model and has positive post symptomatic effects
Tricoire et al. HMG 23, 968 (2014)
METHYLENE BLUE (MB)
• Redox and antioxydant properties
• Already used in clinics (methemoglobinemia, ifosfamide-
induced encephalopathy…)
• Behaves as alternative electron transporter in mitochondria
• Protects in a rotenone model of PD
Wen et al; JBC 2011Screening of repositionnable compounds Prestwick Chemical library : 1280 repositionnable compounds Screening on the in vivo cardiac model of FA Fast screening with 8 flies (1 year) + secondary validation: +/- 30% de sauvetage de la dilatation cardiaque, p
The future of fly in human disease research
• Integrating CRISPR/CAS9 technology for better
disease model generation
Humanizing Drosophila gene
✓ Ensuring more relevant physiological
gene regulation
✓ Assaying the phenotype of human
variants in conserved proteins
• Implementing large scale collaborations between
clinic and biological researchIntegrating the CRISPR revolution for FA
New FA models A CRISPR induced Freidreich ataxia fly model
• Targeted mutations to
accurately mimic FA
• Physiological expression of fh
Reduced lifespan and Epigenetic repression of Heart
• Searching for new locomotor defects neighbouring genes dysfunction
pathological pathways and wild-type fh-GAAs
therapeutic drugs
• Assaying new therapeutic
strategies (CRISP mediated
repeat excision)Large scale multiple models consortium are
required for diseases research
Wangler et al. Genetics
(2017) 207, 9–27Rare diseases open the way
Wangler et al. Genetics
(2017) 207, 9–27H. Tricoire’s lab Véronique Monnier Élodie Martin Michael Rera Maria Russi Laura Tixier
Bilan des modèles Drosophile • Des succès indéniables dans un vaste spectre de pathologies (neurodégénératives, musculaires, oncogéniques, métaboliques,…) présentant des conservations évolutives • Un rapport « qualité/prix » élevé pour le criblage dans des organismes multicellulaires ✓ Faible taille, haute fertilité fortes statistiques (40 000 individus/m2) ✓ Nombreux phénotypes criblables et génétique moléculaire sophistiquée. Nouvelles possibilités avec CRISPR/CAS9. ✓ Pas (peu) de redondance génétique ✓ Cout d’exploitation limité ✓ Adaptés aux études sur des pathologies du vieillissement Un interfaçage encore insuffisant avec d’autres chaines de criblage (HTS en cellules, autres modèles animaux) Des capacités de flux de criblage in vivo parfois trop limitées pour certains paradigmes
Les modélisations chez la Drosophile
sont-elles pertinentes?
• Essentiellement les mêmes neurotransmetteurs sont utilisés
• Conservation fonctionnelle de certaines structures cérébrales
• De nombreuses interactions cellulaires critiques (ex: neurones-
astrocytes) sont aussi présentes
• De nombreux mécanismes physiopathologiques semblent conservésLimites possibles du criblage pharmacologique chez
la Drosophile
Administration des composés par ingestion
L’élimination des composés peut être très différente entre
une drosophile et des vertébrés
Des composés ciblant des structures très précises de
protéines de mammifères ont peu de chance d’être efficaces
chez la DrosophileWilloughby et al. Disease Models & Mechanisms 6, 521-529 (2013)
Recherche de modes d’actions
+
Stratégies génétiques par inactivation ou fhIR
surexpression de: AconIR
• Membres de la chaine respiratoire CG1970-IR
mitochondriale CG9172-IR
• Protéines antioxydantes CG4769-IR
• Régulateur du fer mitochondrial CG17856-IR
CG11015-IR
Analyse de composés apparentés:
• Effets sur le phénotype cardiaque 90
80
• Relations avec les caractéristiques 70
60
connues
ESD (µm)
50
40
30
20
Methylene Blue (MB) Toluidine Blue (TB) Neutral Red (NR) 10
0
- + + + + + + + + + + + + +
MB MB TB TB NR NR 2CP 2CP Pr Pr CP CP
10µM 30µM 10µM 30µM 10µM 30µM 10µM 30µM 10µM 30µM 10µM 30µM
2-chlorophenothiazine (2-CP) Promethazine (Pr) Chlorpromazine (CP)
Seule la restauration d’une activité respiratoire semble corrélée à l’effet
du BM et des phenothiazines testéesGénétique humaine
Epidémiologie
Génération d’un
modèle Drosophile
Caractérisation
phénotypique
in vivo
Autres modèles
Criblage haut débit
Criblage
Criblage génétique
pharmacologiqueAntiprion drugs 6-aminophenanthridine and guanabenz reduce PABPN1 toxicity and aggregation in oculopharyngeal muscular dystrophy. Barbezier N, Chartier A, Bidet Y, Buttstedt A, Voisset C, Galons H, Blondel M, Schwarz E, Simonelig M. EMBO Mol Med. 2011 Jan;3(1):35-49 Invertebrate models of lysosomal storage disease: what have we learned so far? Hindle S, Hebbar S, Sweeney ST. Invert Neurosci. 2011 Dec;11(2):59-71
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