Metformin Mitigates Craniofacial Abnormalities and Reduces Oxidative Stress in a Zebrafish Model of Treacher Collins Syndrome

TORRES, MERCEDES; GRAZIATI, SEBASTIÁN; COUX, GABRIELA

Rosario

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2023

SAIB

Treacher Collins Syndrome (TCS) is a hereditary mandibulofacial dysostosis resulting primarily frommutations in the TCOF1 (treacle ribosome biogenesis factor 1) gene. TCS is characterized by distinctivecraniofacial abnormalities ranging from almost unnoticeable to severe even among individuals withidentical mutations. However, the molecular mechanisms underlying the variable expressivity of TCSremain unclear. Previous research has indicated increased levels of reactive oxygen species (ROS)together with reduced CNBP protein expression in a zebrafish model of TCS. CNBP (CCHC-type zincfinger nucleic acid binding protein) is a ROS-sensitive protein involved in the regulation of neural crestcells (NCC) proliferation and apoptosis. Phosphorylation of CNBP by AMP-activated protein kinase(AMPK) plays a role in its stability. The anti-diabetic biguanide metformin is an AMPK activator withantioxidant properties. We propose that metformin may ameliorate TCS-related manifestations bymodulating cellular metabolism.In this study, we employed a zebrafish TCS model generated via specific Morpholino (designed to inhibitthe translation of tcof1 transcript) injection into 1-cell staged zebrafish embryos. Negative controls wereinjected with the standard universal Morpholino, which does not bind to any zebrafish transcript.Incubations with varying concentrations of metformin diluted in embryo medium were carried out from6 to 24 hours post-fertilization (hpf). Craniofacial cartilage morphology of the embryos was analyzed by Alcian Blue staining of 120 hpf larvae. Remarkably, treatment with 1 mM metformin resulted instatistically significant improvements in craniofacial cartilage morphology of TCS-like larvae. Analysisof ROS levels (using DCFH-DA) revealed a normalization of these in TCS-like embryos treated with 1 mMmetformin. The expression of redox-response genes (sod2, cat, nfe2l2α), assessed by qPCR,corroborated these findings. Additionally, metformin-treated embryos displayed a decrease inneuro-epithelial cell death (detected by Acridine Orange staining) and a restoration of NCCpopulations (evaluated by flow cytometry analyses with GFP-labelled NCC from Tg(-4.7sox10:GFP)embryos). Furthermore, metformin treatment led to the restoration of CNBP protein levels.This study sheds light on the mechanism of action underlying metformin´s protective effects,emphasizing its role in mitigating oxidative stress and preserving craniofacial development in TCS.Furthermore, thanks to the conservation of proteins and processes involved in vertebrate development,and although more in-depth studies are required, our findings suggest that metformin could be atherapeutic alternative to moderate the TCS phenotype. Finally, our results highlight the role of externalfactors (as oxidative stress) in TCS manifestations.