From cattle Pseudomyotonia to Zebrafish: alternative animal models for studying Brody disease and exploring innovative therapeutic approaches for this human pathology

Brody disease (BD) is an ultrarare inherited disorder that impairs skeletal muscle function in humans. BD is a myopathy characterized by the deficiency of SERCA1 protein, resulting from mutations or in-frame deletions of the ATP2A1 gene. SERCA1 deficiency is responsible for impaired muscle relaxation after contraction due to a prolonged increase of Ca2+ concentration in the cytoplasm of skeletal muscle fibers. Noteworthy, BD represents an orphan disease since a specific therapy does not exist to date. Indeed, BD patients are treated with generic muscle relaxant drugs that prevent Ca2+release from the sarcoplasmic reticulum. However, these molecules are unsuitable for long-term treatments and the therapy is often suspended due to side effects. A muscular disorder named “congenital pseudomyotonia” (PMT) has been described in bovine species. Based on clinical, genetic (i.e. mutations in bovine ATP2A1 gene) and biochemical analyses, PMT represents the real counterpart of BD. Interestingly, missense mutations in the ATP2A1 gene are responsible for the pseudomyotonia phenotype and the pathogenic mechanism underlying pseudomyotonia involves ubiquitin-proteasome system (UPS) degradation of the mutated bovine SERCA1. The mutation in the ATP2A1 gene generates a SERCA1 protein most likely corrupted in proper folding which is prematurely degraded by the UPS, but still retains its catalytic activity. Starting from the knowledge that in bovine pseudomyotonia SERCA1 mutation does not abolish pump activity, leads us to hypothesize that the retention of the functional properties might represent a prerequisite for the development of a potential innovative pharmacological therapy for Brody disease. Based on different studies in this thesis we are proposing a novel pharmacological approach to repair the defective SERCA1 protein by using small molecules. This thesis starts with the investigation of the G211/286V mutation in a muscle biopsy of Romagnola cattle semimembranosus muscle, HeLa and HEK293 cells and their protein activity. Further ex-vivo and in-vivo experiments were made to test our novel pharmaceutical approach based on the CFTR correctors with the calves carrying compound heterozygous mutations, R164H mutation in one allele and the two-point mutations (G211/286V) in the other allele. The results obtained from treatment were quite encouraging. Although bovine PMT is an alternative animal model of Brody disease it demonstrated its difficulties and complications to study drug administrations. To overcome difficulties faced with the bovine, as recommended by Hirata et al., and Gleason et al., an animal model of Brody disease commercially available zebrafish accordion is deeply characterized and treated with CFTR correctors. However, during the characterization, the accordion zebrafish line demonstrated different catalytic activity in SERCA1 protein than bovine PMT, although was a carrier of the S766F SERCA1 mutation. To overcome this problem new knock-in zebrafish models, carrying both Chianina and Romagnola mutations were generated with CRISPR/Cas9 genome editing technology.