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Zebrafish as a model system to study
Pompe disease: new perspective for
therapeutic approaches
Rimini, 18 novembre 2017
Bragato Cinzia
Zebrafish as a model system to study
Pompe disease: new perspective for
therapeutic approaches
Rimini, 18 novembre 2017
Bragato Cinzia
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The species hailing from Southeast Asia,
where lives and proliferate in fresh water
reservoir (rivers, drains, basins, paddy fields,
etc.)0
Zebrafish is a Teleostei
(from Greek: teleios,
"complete" + osteon,
"bone”) fish, member of
the cyprinids family
Zebrafish (Danio rerio)
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Why zebrafish?
• It’s a vertebrate
• Rapid development
• Prolific• External fecundation
• Available in the lab year round
• Optically transparent
• Excellent embryology
• Easily injected
• Excellent genetics & genomics
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Rapid development
Zebrafish life cycle
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Zebrafish (Danio rerio)
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Zebrafish Facility
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Zebrafish (Danio rerio)
Despite the evolutionary distance between zebrafish and man, the morphogenetic
and molecular mechanisms are highly conserved. These mechanisms, which are
present at the beginning of the embryonic development and of the genetic
programming, are the main cause of diseases, if altered.
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Gene expression and function analysis
In situ hybridization on
whole-mount zebrafish embryo
Whole-mount immunofluorescence
of zebrafish embryo
Transgenic and mutants linesKnockdown and gene over-expression by RNA
and morpholino microinjection
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Alter normal developmental processes to identify the genes
that are important for thet process: from phenotype to gene
- Chemical mutagenesis: ENU (Ethyl Nitros Urea)
- Insertional mutagenesis
Modulation gene’s expression to understand is function:
from gene to phenotype specific gene inactivation
- Protein knock-down
- Gene knock-out
- Over expression
Forward genetics
Reverse genetics
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Knock-down zebrafish
Antisense oligonucleotides 25 bp long
Resistant to digestion Not RNase H mediated
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Loss of function analysis
Normal Splicing
Exon Skipping
Intron retention
MO against ATG
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Microinjection
• Different concentration of morpholino are injected in the yolk of one cell stage;
• Coinjection of viable tracker or GFP coding RNA are used to identify correctly
injected embryos;
• Morpholinos without target in zebrafish are used as negative control.
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Microinjection
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Knock-out zebrafish
Zinc-fingers
TALENs
CRISPR-Cas9
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CASPER zebrafish mutants
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Transgenic zebrafish lines
Tg(fli1:EGFP)The gene that codify
for EGFP (enhanced
green fluorescent
protein) is expressed
under control of the
endothelial fli1 (friend
leukemia integration 1)
gene promoter.
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Transgenic zebrafish lines
High-speed mSPIM video sequence
of a transgenic fish expressing
fluorescent proteins in the:
- endocardium Tg(flk1:GFP)s843,
- myocardium Tg(cmlc2:DsRed)s879
- blood Tg(gata1:DsRed)sd2
(Jan Huisken, Max-Planck-Institute of Molecular Cell Biology and Genetics )
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Cheaper than other animal models
Fast results advantageous for
pharmacological treatments and
pesonalized therapies
Advantages
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On-line tools
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Zebrafish model for Pompe DiseaseA model organism is a non-human species that is extensively studied to
understand particular biological phenomena.
Model organisms are amenable to experimental manipulation:
1) short life-cycle
2) techniques for genetic manipulation
3) special genome characteristics
characterization of zebrafish model with GAA deficiency, obtained by
knocking-down the GAA protein.
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Bioinformatic analysis
Zebrafish gaa
- 3166 bp
- 3203 bp
Expression panel
Gaa probe:
821 bp
Experimental design
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..from the beginning of
somitogenesis till 24 hpf
Preparation of gaa knock-down model
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(2-{N-[7-nitrobenz-2-oxa-1, 3-diazol 4-yl] amino}-2-deoxyglucose)
is a fluorescently-labeled deoxyglucose analog that
is used primarily to directly monitor glucose uptake
by living cells and tissues.
can be used:
• in real-time confocal,
• high-resolution
• wide-field fluorescence microscopy
• flow cytometry.
Experimental design
4 pMOL
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2-NBDG Results
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Pompe Disease’s phenotype
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Semi-thin sections
Scale bar = 40 um
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Transverse electron micrographs
STD-MO ATGgaa-MO I9E10gaa-MO
Scale bar = 2 um
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LysoTracker
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Ongoing experiments
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➢ Variable effects in late onset forms
➢ Low effectiveness on muscular fibers type II
➢ In animal models, high dosage (100 mg/kg/infusion)
is required in late stages of the disease.
➢ Expensive treatment
Enzymatic Replacement Therapy (ERT)
PROBLEMS
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Why couldn’t we try to find a new
strategy to improve the quality of life
of Pompe patients, especially late
onset patients?
…or try to improve ERT efficacy at
least?
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Chemical assays
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Schmidt et al. 2016 - Mixture toxicity of water contaminants-effect analysis using the zebrafish
embryo assay.
Chemical assays
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Preliminary Results
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Touch-evoked response test
Morphant MO treated with compound
Standard Control
3 dpf
embryos
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Future plans
Creation of a stable
Pompe disease
zebrafish line
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Single Plane Illumination Microscopy (SPIM)
Prof. Bassi Andrea
Tg(fli1:EGFP)y1 zebrafish embryo
showing the development of the
circulatory system in the yolk area.
Images were acquired every 5
minutes for 3 days
Vitale G, Gaudenzi G, Bassi A et al., 2017, Animal
models of medullary thyroid cancer: state of
the art and view to the future. Endocrin-Related
Cancer.
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Conclusions
• The new in vivo model for Pompe disease that we are
setting up will become a suitable tool for different scientific
fields.
• We will shed light on the relation
between glycogen storage and
autophagy that is still unclear,
taking advantage of the most
advanced imaging techniques
available, in order to directly
visualize glycogen formation
and accumulation in muscles.
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Dr. Mora Marina
Dr. Blasevich Flavia
Dr. Saredi Simona
Dr. Gibertini Sara
Dr. Zanotti Simona
Dr. Ruggieri Alessandra
Dr. Introna Clelia
Dr. Carra Silvia
Prof. Cotelli Franco
Dr. Beltrame Monica
Prof. Bassi Andrea
Acknowledgements
Dr. Mantegazza Renato
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..and thank you for your attention!