Il modello Zebrafish di Osteogenesis Imperfecta: uno strumento per lo screening farmacologico

INTRODUCTION Osteogenesis imperfecta (OI) is a heterogeneous group of inherited skeletal dysplasias characterized by growth deficiency, bone deformity and fragility. OI was traditionally known as a disease caused by dominant mutations in COL1A1 and COL1A2 genes, encoding the α1 and α2 chains of collagen type I. OI type III is the most severe non-lethal form of dominant OI and patients’ osteoblasts are characterized by endoplasmic reticulum (ER) stress, caused by the mutant collagen type I retention, resulting in ER cisternae enlargement and cellular malfunction. The use of zebrafish (Danio Rerio) as an animal model to investigate skeletal disorders have increased in the last years, due to its embryos transparency and the conserved bone cells, ossification process and gene expression profiles with mammals. During my Master thesis I evaluated the use of the dominant OI zebrafish model Chihuahua (Chi/+), carrying a Gly574Asp substitution in the α1 chain of collagen type I, as a pharmacological screening tool. Based on the previously obtained positive results of the chemical chaperone 4-phenylbutyrate (4-PBA) on Chi/+ bone phenotype, I evaluated the effect of the 4-PBA administration combined with the well known antiresorptive drug Alendronate. I also tested on Chi/+ embryos and adult fish the effect of two novel 4-PBA drugs, modified to increase their half-life. RESULTS A delay in mineralization was demonstrated in Chi/+ compared to WT. The administration of 4-PBA combined to Alendronate improved the mineralization of Chi/+ compared to controls and only 4-PBA treated fish. WT and Chi/+embryos were then treated with the modified 4-PBA drug X1. In this case the amelioration in bone mineralization was observed mostly in WT treated compared to controls. The effect of X1 was also tested on 4-months WT and Chi/+ fish during caudal fin regeneration experiments. No positive effects were detected after the administration of X2 in both WT and Chi/+. Finally, an optimized micro-computed tomography (μCT) protocol was developed to analyze bone quality parameters in controls and X1-injected WT fish. No difference was found between treated and untreated fish and the optimized protocol will be applied on Chi/+ zebrafish in the future. CONCLUSION The impaired bone mineralization and regeneration confirmed Chi/+ as valid model for dominant OI and the experiments on bone formation in embryos, on tail regeneration and on bone remodeling in adult fish confirm the zebrafish as a suitable tool for pharmacological screening analysis. The combination of 4-PBA to bisphosphonates will increase the chemical chaperone effect specifically on bone tissue.