Investigation Of The Pathological Mechanism Of DGUOK Mutant Mitochondrial DNA Depletion Syndrome Using An IPSC Disease Model

Mitochondrial DNA depletion syndrome(MDS)is a group of severe,phenotypically heterogeneous,recessively inherited disorders characterized by marked reduction of the mtDNA content in affected tissues and organs.deoxyguanosine kinase(DGUOK)is one of nine genes whicn are presently known to underlie the pathogenesis of MDS.Almost all DGUOK mutant patients developed iron overload progressing to liver failure,with some patients developing hepatocellular carcinoma in liver.Patients usually died before 2 years old without effective therapies except liver transplantation.However,the pathological mechanisms connecting mtDNA depletion and iron overload-induced hepatic damage remains uncovered.It has been difficult to acquire liver samples from DGUOK mutant patients for research,while fibroblasts or myoblasts from patients only showed some features observed in liver.Human induced pluripotent stem cells(iPSCs)can provide a renewable source of hepatocytes(iHep).Moreover,human iPSCs can be genetically edited by the CRISPR/Cas9 system to supply isogenic controls.Here,we establish an in vitro disease model using iPSCs from patients with DGUOK mutations,along with associated controls and isogenic cell lines corrected by the CRISPR/Cas9 system for studying hepatic pathology.At first,we found that mtDNA copy number and expression level were recovered in DGUOK corrected iHep.Furthermore,patient iHep displayed decreased mitochondrial membrane potential,oxygen consumption rate,ATP production as well as activities of respiratory chain complex ? and ? compare with control and corrected iHep.We also observed a significant increase of lactate/pyruvate ratio and cellular ROS in patient iHep compared to control and corrected iHep.Then,we found that DGUOK mutant iHep are more sensitive to iron overload-induced ferroptosis because of patient iHep displaying a reduction in glutathione(GSH)level,which can be rescued by the GSH precursor,N-Acetylcysteine(NAC).Mechanically,this ferroptosis is accompanied by degradation of ferritin in lysosomes but is not part of an autophagic process.We also found that nuclear receptor coactivator 4(NCOA4)was partially co-localizated with ferritin in DGUOK mutant iHep.It may suggest that NCOA4 plays a new role in autophagy-independent lysosomal ferritin degradation.Furthermore,iron-sulfur cluster is defect in DGUOK mutant iHep causing nuclear genome instability,which can be alleviated by NAC.Our study clarifies the underlying pathological mechanisms of this syndrome,and suggest that inhibiting ferroptosis and recovering iron-sulfur cluster could be viable therapeutic strategies.