Marine Biotechnology Student name: Hagay Livne I.D:

 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)

 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

Deutekom et al, 2003.

 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

 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

 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

 First-Round Screen (Pooled Compounds)  Second-Round Screen Using Individual Compounds Kawahara et al, 2011.

 some fish show normal birefringence despite genotyping results Kawahara et al, 2011.

 restored muscle structure  no expression of dystrophin Kawahara et al, 2011.

 (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 Dys-mut Kawahara et al,  one to two cell stage WT embryos  4 dpf

 For each chemical treatment, the percentage of affected fish is reduced (4 dpf) Kawahara et al, 2011.

 chemicals 2,3,7 proved toxic to zebrafish  the average number of surviving fish is greater in chemical 4 Kawahara et al, 2011.

Red – survivors Light Blue – control Blue – WT Green - untreated fish Kawahara et al, 2011.

 Surviving fish (30 dpf) were sectioned  skeletal muscle structure restored Kawahara et al, 2011.

Treatment with chemical 4 restored the muscle structure of these dystrophin-null fish

 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

 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

Kawahara et al, 2011.

 sildenafil citrate and aminophylline decreased the percentage of fish showing abnormal birefringence Kawahara et al, 2011.

 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

 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

 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

Questions?

1. Kawahara, Genri, et al. "Drug screening in a zebrafish model of Duchenne muscular dystrophy." Proceedings of the National Academy of Sciences (2011): 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): 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-R Van Deutekom, Judith CT, and Gert-Jan B. Van Ommen. "Advances in Duchenne muscular dystrophy gene therapy." Nature Reviews Genetics 4.10 (2003):