Objectives: Osteogenesis imperfecta (OI) is a bone disease mainly caused by collagen type I mutations and characterized by bone fragility. No definitive cure is available and the search for novel treatments is necessary. The small teleost D. rerio is particularly appealing for drug screening approaches. A zebrafish OI model (Chihuahua) carrying in heterozygosis the G574D substitution in the α1 chain of collagen type I is available. To use this model for drug tests it is mandatory to understand the composition of collagen type I, since three genes coding for three different α(I) chains have been described, but their stoichiometry is still unknown.
Methods: Collagen I was extracted from skin, scales and bone of WT and Chihuahua adult fishes and from embryos, and separated by 1D and 2D SDS-PAGE followed by mass spectrometry. Amino acid analysis and Differential Scanning Calorimetry were performed, such as RT-PCR for collagen genes. Skeleton was analyzed by X-ray, microCT, alizarin red/alcian blue and calcein staining. Electron microscopy of osteoblasts was performed.
Results: Electrophoretic analysis followed by mass spectrometry revealed in adults the comigration of α1 and α3 chains in a unique band/spot, while in embryos it was possible to distinguish an α1 and an α1/ α3 comigrating band. The α2 band was always separated and in the classical 1:2 ratio respect with α1 and α3 bands. These data and the amino acid analysis suggest the existence of [α1(I)]2α2(I) and α1(I) α2(I) α3(I) heterotrimers in embryos and of α1(I)α2(I)α3(I) in adults. The Tm for bone, scales and skin collagen was similar. SDS-PAGE showed overmodification of the mutant collagen I compared to WT. X-ray and microCT revealed severe skeletal deformities, multiple bone fractures and reduced mineralization in adult Chihuahua fishes, while skeletal staining demonstrated a delayed mineralization in embryos. The presence of ER cisternae enlargement in osteoblasts was evident by electron microscopy analysis.
Conclusion: The collagen composition in embryos and adult fishes is different. The collagen overmodification and ER retention, the skeletal deformities and abnormal mineralization found in Chihuahua reproduce the main features reported in OI patients, validating this model for the study of the disease and to evaluate novel possible target for OI treatment.
Fondazione Cariplo 2013-0612, Telethon GGP13098 and the European Community n. 602300.
Disclosure: The authors declared no competing interests.
27 Jun 2015 - 30 Jun 2015