Marine Biotechnology

Student name: Hagay Livne

I.D: 300171113

03.06.2013

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Duchenne muscular dystrophy (DMD) Giovanni Semmola in 1834 and Gaetano

Conte in 1836 named after the French neurologist

Guillaume Benjamin Amand Duchenne (1806–1875)

The first who did a biopsy to obtain tissue from a living patient for microscopic examination

at first the muscle forms normally but then degenerates faster than it can be repaired.

Fatal ,life expectancy is around 25 years (a frequency of about 1 in 3,500 new-born males)

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Duchenne muscular dystrophy (DMD)

caused by a mutation of the dystrophin gene at locus Xp21 (nonsense or frame shift mutations)[2]

DMD is inherited in an X-linked recessive pattern

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Deutekom et al, 2003.

Dystrophin

The largest gene found in nature (2.4 MB) Highly complex Large, rod-like cytoskeletal protein Found at the inner surface of muscle fibers Part of the dystrophin-glycoprotein complex (DGC) Bridges the inner cytoskeleton (F-actin) and the

extra-cellular matrix

http://www.ncbi.nlm.nih.gov/gene/1756

www.humgen.nl

The zebra-fish (Danio Rerio)

Small, cheap and easy to grow Penetrable for small compounds suitable for chemical screens Easily mutated in a large scale Orthologs for the Dystrophin

gene (DGC) Follow the Formation of the

muscle fibers

The research goal is to perform a chemical

screen in zebra fish dystrophin mutants

That might correct the pathology of the

Muscle structure

Materials and Methods Fish Cultures – zebrafish mutants

sapje (stop codon in exon 4) sapje-like (splice site mutation in exon 62)

The Prestwick chemical library (Harvard Institute of Chemistry and Cell Biology)

Birefringence Assay Genotyping Histology and Immunohistochemistry Antisense MO Injection Western Blotting PKA Assay

methodology

First-Round Screen (Pooled Compounds)

Second-Round Screen Using Individual Compounds

Kawahara et al, 2011.

Kawahara et al, 2011.

Genotyping and Birefringence Assay

some fish show normal birefringence despite genotyping results

Kawahara et al, 2011.

Immunostaining - an anti-dystrophin antibody.

restored muscle structure

no expression of dystrophin

Kawahara et al, 2011.

Dystrophin Morphants (MO)

(A and B) Normal light image (C and D) Birefringence image (E and G) Immunostaining - anti-dystrophin antibody (F and H) Immunostaining - anti-laminin antibody

WT

WT

WT

WT

Dys-mut

Dys-mut

Dys-mut

Dys-mut

Kawahara et al, 2011.

one to two cell stage WT embryos

4 dpf

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Testing 7 Candidate Chemicals (MO)

For each chemical treatment, the percentage of affected fish is reduced (4 dpf)

Kawahara et al, 2011.

Long-Term Culture Fish with 7 Candidate Chemicals (4-30 dpf)

chemicals 2,3,7 proved toxic to zebrafish the average number of surviving fish is greater in

chemical 4 Kawahara et al, 2011.

Long-Term Culture Fish with 7 Candidate Chemicals (1-30 dpf)

Red – survivorsLight Blue – controlBlue – WTGreen - untreated fish

Kawahara et al, 2011.

Chemical 4 - Aminophylline Surviving fish (30 dpf) were sectioned

skeletal muscle structure restored

Kawahara et al, 2011.

Chemical 4 - Aminophylline

Kawahara et al, 2011.

Treatment with chemical 4 restored the muscle structure of these

dystrophin-null fish

Aminophylline A nonselective PDE5 inhibitor Increases the levels of intercellular cAMP Activation of cAMP-dependant PKA Anti-inflammatory effects:

inhibition of inflammatory mediators activation of NF-κB

PDE5 inhibitor restores mdx mouse muscle to normal

The expression, phosphorylation, and activation of PKA were examined

PKA Expression and Activity

Immunoblot (C) pPKA/PKA Ratio (D) pProteins/proteins (E)

Kawahara et al, 2011.

Activated phosphorylated PKA and the activity

of PKA were increased in aminophylline-treated

fish

Intracellular cAMP is increased with

Aminophylline treatment

Treatment of sapje Fish with a Series of PDE Inhibitors (1-4 dpf)

Kawahara et al, 2011.

birefringence assay

sildenafil citrate and aminophylline decreased the percentage of fish showing abnormal birefringence

Kawahara et al, 2011.

Discussion

The muscle structure of aminophylline-treated dystrophin-null fish appeared normal

The activity of PKA is clearly up-regulated in aminophylline-treated dystrophin-null fish

Each of the seven chemicals increased the percentage of fish with normal birefringence

The chemical treatment did not restore dystrophin expression

Discussion

A two-tiered screening strategy PDE inhibitors cause an increase in intracellular

cAMP and/or cGMP Mutations in the zebrafish dystrophin gene (sapje

and sapje-like mutants): good models for studies of DMD ideally suited for use in chemical screens easily detectable by a highly accurate birefringence assay

Discussion

The zebrafish: small enough to be permeable to small molecules can be assayed in large numbers

Sildenafil (viagra ©) and Tadalafil (Cialis ©) have been independently identified by others

Thousands more compounds are now available for further screening

Thank you for listening!

Questions?

References1. Kawahara, Genri, et al. "Drug screening in a zebrafish model of

Duchenne muscular dystrophy." Proceedings of the National Academy of Sciences 108.13 (2011): 5331-5336.

2. Koenig, Michel, et al. "Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals." Cell 50.3 (1987): 509-517.

3. Bassett, David I., and Peter D. Currie. "The zebrafish as a model for muscular dystrophy and congenital myopathy." Human molecular genetics 12.suppl 2 (2003): R265-R270.

4. Van Deutekom, Judith CT, and Gert-Jan B. Van Ommen. "Advances in Duchenne muscular dystrophy gene therapy." Nature Reviews Genetics 4.10 (2003): 774-783.