Learning to swim: What fish can teach us about CMDs Jim Dowling, M.D., Ph.D. University of Michigan...
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Transcript of Learning to swim: What fish can teach us about CMDs Jim Dowling, M.D., Ph.D. University of Michigan...
![Page 1: Learning to swim: What fish can teach us about CMDs Jim Dowling, M.D., Ph.D. University of Michigan Congenital Muscular Dystrophy Family Conference August.](https://reader030.fdocuments.us/reader030/viewer/2022033104/56649dda5503460f94ad1123/html5/thumbnails/1.jpg)
Learning to swim:What fish can teach us about CMDs
Jim Dowling, M.D., Ph.D.
University of Michigan
Congenital Muscular Dystrophy Family Conference
August 14-15, 2010
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Meet the zebrafish
• Small, fresh water aquatic vertebrate• Lifespan 1-2 years• Independently swimming by day of life 3
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Why zebrafish??? (part 1)
Breeding age = 2-3 monthsAverage clutch size = 100-300
Time between clutches = 1 week
It’s a numbers game…Breeding age = 3
monthsAverage litter
size = 8-13Time between
litters = 20 days
Breeding age = 16+ years
Average litter size = 1
Time between litters = 9-12
months minimum
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Why zebrafish??? (part 2)
• Crystal clarity!–Zebrafish are optically translucent allowing for live imaging of muscle and heart
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Why zebrafish??? (part 3)
• Invertebrate style genetics– Large number of offspring
– Can easily introduce DNA/RNA
– Can do saturating mutagenesis screening
• Vertebrate style genome– Genome at least as complex as ours
– Genome sequenced as part of the NIH genome project
– All known muscular dystrophy genes are found in the zebrafish genome (Steffen et al., 2007)
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Zebrafish as a model for muscle disease
• Muscle development begins at 20 hpf and is completed by 48 hpf– i.e. very quickly!!!
• Fish muscle shares many features with human muscle– Slow and fast twitch fibers– Limb and trunk muscle– Contains the common muscle structures
• What’s different?– Slow and fast fibers in separate compartments
– Significantly more trunk than limb muscles
– Some reagents do not work in fish
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Zebrafish as a model for muscle disease (cont)
• Obvious phenotypic consequences from muscle dysfunction–Impaired swimming
• Abnormal muscle observable in live fish–Can see it with conventional microscopy
• Histopathologic changes that reflect human muscle pathologies –(dystrophic pattern in dystrophies, for example)Dowling et al. (2008) Circulation Resçearch
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Zebrafish as a model of muscle disease
Control embryos (3 days old) Myopathy embryos (3 days old)
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Zebrafish models of CMDs
• How do we make zf models of CMDs?
• What models currently exist?
• What have they been used for in the past?– In other words, what have we learned from them?
• What can we use these models for in the future?– How can they help patients with CMDs?
• Additional thoughts and future directions
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How do we make zebrafish CMD models?
• Two ways to make a model1. Transient models
– Morpholino knockdown– Mutant transgene
expression– Effect lasts 2-5 days
2. Stable models– Chemically induced
or spontaneous mutant
– Dominant transgenics– Direct gene
targeting– Gene trap– TILLING– ZF method
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What CMD models current exist?
• Transient Models (morpholino based)–LAMA2 (MDC1A)–FKRP–dystroglycan–COL6A1/A3 (UCMD)–SEPN1 (RSMD)
• Stable–LAMA2 (candyfloss)–RYR1 (relatively relaxed)
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Zebrafish model of MDC1A
• ENU induced mutation called candyfloss• Identified/characterized by Currie’s group
– (Hall et al., 2007)• Clinical
– Onset at 3 days– Progressive weakness– Death by 2 weeks
• Genetic– Point mutation in LAMA2 leading to premature stop codon– Absent LAMA2 staining
• Histopathologic– Progressive myofiber injury and degeneration– Fragility at the myotendinous junction
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Zebrafish model of UCMD• Experiment from my group
– (Telfer et al., 2010)• Clinical
– Severely reduced motor function at 24 and 48 hpf
– Obvious morphologic abnormalities
• Genetics– Morpholino mediated knockdown of COL6A1 (exon 9) and COL6A3 (exon 13)
– Models two common dominant mutations
• Histopathology– Myofiber disorganization and sarcolemmal breakdown
– mitochondrial swelling, increased apoptosis
– Reduced and disorganized collagen VI staining
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Zebrafish models of dystroglycanopathies
• Experiments from two groups– Thornhill et al., 2009– Kawahara et al., 2010
• Clinical– Reduced motor function – Morphologic changes at 24 and 48 hpf
• Genetic– Morpholino knockdown of FKRP (x2 groups: Straub, Kunkel)
• Histologic– Pathology in muscle, eye and brain
– Reduced laminin binding– Reduced glycosylated dystroglycan
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Zebrafish model of core myopathies (RSMD): ryr
• Identified at UM by John Kuwada and colleagues
• Spontaneous mutant with abnormal swimming (named relatively relaxed)
• Have impaired excitation-contraction coupling
• Found to have a homozygous recessive mutation in ryr1b
• Mutations cause significantly reduced levels of RYR1 in fast muscle
• Genetically and histologically a model for recessive core myopathies– RYR1 and SEPN1 related
myopathies
Hirata et al. 2007
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What did we learn from these models?
• Candyfloss (MDC1A)– LAMA2 important for the myotendinous junction– Like mice, LAMA2 zebrafish have an early onset, severe phenotype
• UCMD model– Confirmed mitochondrial proton pore hypothesis (and confirmed potential therapeutic role for molecules that act at the MPP)
– Unlike mice, UCMD zebrafish have an early onset, severe phenotype
• FKRP morphants– Demonstration of ability to model dystroglycanopathies in the zebrafish
– Defined key techniques for future experimentation
• Core myopathy models (SEPN1 and RYR1)– Confirmed key role of excitation-contraction coupling in muscle function
– Established/strengthened linked between SEPN1 and RYR1 related myopathies
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How can we use these models in the future?
• Identification of new/novel aspects of disease pathogenesis
• Identification of disease biomarkers
• Platform for rapid identification/development of novel therapeutics
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Therapy development in zebrafish models of CMDs
• Two stable genetic models of CMDs in zebrafish– Candyfloss (MDC1A)– Relatively Relaxed (ryr)
• Morpholino models recapitulate clinical, genetic, and pathologic features of CMDs
• No new therapies yet developed in these models– Verification of efficacy of CsA in UCMD model
• Zebrafish have excellent potential for MTS and HTS Telf er et al., 2010
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Drug screening in the zebrafish
• ZF and drug screening– Large number of offspring
– Frequent mating– Easily absorb drugs in media
– Translucent body plan plus many GFP markers
• Muscle specific phenotypes for drug screens– Birefringence– Motor function– Other targets? (for example, cardiac phenotypes)
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HTS and birefringence in zebrafish
• Birefringence– Property of muscle when placed under polarized light
– Abnormal birefringence is a measure of impaired myofiber integrity and/or organization
– Abnormal birefringence reported in all muscular dystrophy ZF yet described
• Birefringence and CMDs– Abnormal in candyfloss (LAMA2)
– Abnormal in MO knockdown of COL6A1 (UCMD model)
– Abnormal in MO knockdown of FKRP (dystroglycan model)
FKRP morphants (Kunkel)
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Embryos are fertilized and
collected
De-chorionate embryos
Place in 24 well dish with one drug
in each well (16 embryos per drug)
Allow exposure to drug for 3 day s
(change drug each day)
Examine birefringence at day
of life 4
Wash away durgEvaluate birefringence at days
of life 5-7
Positive hit = no fish with abn
birefringence at day of life 4
Re-test positive hits with a large sample
size
Secondary analysis of positive hits (dose response; post-sympt
Rx; survival; etc)
Drug screen of birefringence in zebrafish
Birefringence in UCMD model +/- cyclosporin A
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An example of a successful screen using birefringence with a DMD model
Drug 44: 2/16 with sap
Drug 50: 4/16 with sap
3 day washoutDrug 44: 0/16 with sap
Drug 50: 4/16 with sap
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Drug screening and motor function in zebrafish
• CMD models in zebrafish all have abnormal motor function–Impaired touch evoked escape response (starting at 3 dpf in candyfloss, earlier in MO models)
–Impaired swimming in all models
3 day old control
3 day old ryr
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Antioxidant treatment strategy
Identify ryr embryos (DOL3)
Place in 12 well dish with antioxidant or placebo (DMSO). Replace solution daily
Record daily swim speeds
Determine survival(lethality for ryr at 12-14 days)
Noldus Zebrafish System
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Proof of principle:Antioxidant treatment in ryr zebrafish
CTL CTL
ryr ryr
Untreated Antioxidant treated
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Future Directions- Zebrafish Models
• What do we have–Excellent model of MDC1A and RYR1–Transient models of UCMD and ZKRP dystroglycanopathy•Not really amenable to large scale drug screening
–Sound, quantitative assays for drug screening
• What do we need–Standardization of phenotypes and assays–“stable” models for UCMD, dystroglycanopathies, SEPN1, and laminopathies
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A few parting ideas…
• What validation is (or should be) required for novel concepts regarding pathogenesis that are developed in the zebrafish?
• What are the step(s) (or what should they be) between drug discovery in non-murine models and clinical trial?