Mitochondrial Dysfunctions And Intervention Strategy In Riboflavin-responsive Multiple Acyl-CoA Dehydrogenation Deficiency

BackgroundMultiple acyl-CoA dehydrogenation deficiency(MADD),also known as glutaricaceduria type Ⅱ,is an autosomal recessive fatty acid metabolism disorder.A considerable number of late onset MADD cases have been reported to be dramatically responsive to high dosage riboflavin supplementation.Such patients are referred to as riboflavin-responsive MADD(RR-MADD).The main cause of RR-MADD is the mutation of ETFDH gene,which leads to the deficiency of the electron transfer flavoprotein-ubiquinone oxidoreductase(ETF-QO)protein encoded by it.The deficiency of ETF-QO makes reduction equivalent that produced in the process of fatty acids β oxidation cannot be transferred downward,which in turn resulted in the accumulation of the substrate and the insufficient of ATP production.Previous case reports have indicated that patients with RR-MADD may experience mitochondrial dysfunction.This dysfunction is thought to be due to the instability structure of ETF-QO,which is caused by the ETFDH mutation in RR-MADD.As a result of this structural anomaly,electron leakage can easily occur during the process of electron transfer,ultimately leading to an increase in reactive oxygen species(ROS)within the mitochondria.Therefore,excessive ROS may be an important cause of mitochondrial function damage.Although riboflavin therapy can relieve the clinical symptoms of RR-MADD patients,it cannot completely correct the abnormal ROS levels and mitochondrial function in the patients,and some RR-MADD patients relapse in 3 weeks after high-dose riboflavin therapy alone.Previous studies on mitochondrial damage in RR-MADD patients were mostly case reports or making conclusions based on in vitro experiments,while there were numerous mutation sites of RR-MADD,and in vitro experiments could not simulate the complex metabolic environment and regulatory network in patients at the time of onset.Therefore,it is important to use model animals to study the mode and correction strategy of mitochondrial damage in RR-MADD in vivo to elucidate the complex regulatory network between mitochondria and lipid metabolism.In this study,we applied the RR-MADD mouse model with mutation site ETFDH c.250 G>A,which has been established previously,to explore the mitochondrial damage in RR-MADD patients.And we use XJB-5-131,which is a novel and safe mitochondrial ROS scavenger,to correct the mitochondrial damage in order to further clarify the relationship between ROS production and mitochondrial damage in RRMADD patients.Method1.Proteomics was used to explore the changes of mitochondria-related proteins in skeletal muscle of RR-MADD mice before induction,after onset and after treatment.2.Western Blot was used to determine the changes in mitochondrial complex protein expression levels in skeletal muscle of RR-MADD patients and mice,and mitochondrial enzyme complex activity was detected to determine the changes in mitochondrial complex activity in RR-MADD patients and mice.3.The changes of basic respiratory value,ATP production value,maximum respiratory value and electron leakage value of mitochondria in primary hepatocytes of RR-MADD mice(within 6h of extraction)before and after onset and treatment were analyzed by Seahorse cell energy analyzer.4.MitoSOX staining was used to observe the changes of mitochondrial ROS content in skin fibroblasts of RR-MADD patients and in primary hepatocytes of RR-MADD mice(within 6h of extraction)before and after onset,before and after treatment.And by comparing the changes of mitochondrial ROS content when XJB-5-131 was applied and not applied,to clear whether XJB-5-131 could effectively remove ROS or not.5.Changes of basic respiratory value,ATP production value,maximum respiratory value and electron leakage value in mitochondrial of RR-MADD mice before and after treatment combined with XJB-5-131 were detected by Seahorse cell energy analyzer to determine whether it can improve mitochondrial function of RR-MADD mice by clearing excessive ROS.Result1.RR-MADD patients had extensive damage of mitochondrial,and the protein contents and enzyme activities of mitochondrial complex Ⅰ,Ⅱ,Ⅲ and Ⅳ were significantly reduced.This condition does not vary with patients’ mutation site.2.RR-MADD mice had functional defects of mitochondrial enzyme complex before the onset of disease,and the protein contents and enzyme activities of mitochondrial complex Ⅰ,Ⅱ,Ⅲand Ⅳ were significantly decreased when the onset of disease.After riboflavin treatment,mitochondrial enzyme complex function can be partially restored.3.The basic respiratory value,ATP production and maximum respiratory value of mitochondria in primary hepatocytes of RR-MADD mice decreased at the onset of the disease.After riboflavin treatment,the mitochondrial basic respiratory value returned to the pre-onset level,but the ATP production was still lower than that of before the onset.And the Proton leak level was higher than that of before the onset.Even before onset and after treatment,the mitochondrial respiratory chain function of mice was different from that of the wild type.4.Increased ROS and antioxidant protein were present at the onset of RR-MADD,and ROS and antioxidant protein levels were still at a high level after riboflavin treatment reached remission,and mitochondrial function failed to recover to the pre-onset level.5.After the treatment of XJB-5-131,the antioxidant protein levels of mitochondrial ROS in RR-MADD mice and skin fibroblasts of RR-MADD patients were significantly decreased.The mitochondrial function of mice treated with riboflavin combined with XJB-5-131 was significantly improved compared with riboflavin alone.ConclusionExtensive mitochondrial damage was found in RR-MADD,and the decreased functions of mitochondrial enzyme complex Ⅰ,Ⅱ,Ⅲ and Ⅳ,the increased ROS production,and the defective function of mitochondrial enzyme respiratory chain were also found in RR-MADD.The mitochondrial dysfunction exists in the clinical compensatory period(when no disease occurs)and is significantly aggravated when the disease occurs in RR-MADD.Riboflavin therapy alone cannot restore the mitochondrial function to the state before the disease occurs.The mitochondrial function of RR-MADD mice could be partly restored when mitochondrial ROS scavenger XJB-5-131 was applied alone,while riboflavin combined with XJB-5-131 could further restore mitochondrial function.These results further demonstrated the important roles of ROS in mitochondrial damage of RR-MADD,and provided a basis for therapy strategy of riboflavin combined with mitochondrial antioxidants in patients with RR-MADD.