Peptide SS-31 Upregulates Frataxin Expression And Improves The Quality Of Mitochondria:implications In Treatment Of Friedreich Ataxia

Background:Friedreich ataxia(FRDA,Online Mendelian Inheritance in Man database#229300)is an autosomal recessive neurodegenerative disease.Age of onset is often in children early,the course of the process of development.Life expectancy averages between 40 and 50 years old.FRDA is characterized by a mixed spinocerebellar,sensory ataxia frequently associated with cardiomyopathy.The gene associated with the disease has been mapped to chromosome 9ql3-q21.1 and encodes a small mitochondrial protein called frataxin.The most common mutation is a GAA triplet-repeat expansion within the first intron of the frataxin gene,resulting in reduced expression of FXN protein.FXN is important in iron-sulfur cluster assembly where it likely functions as a chaperone that provides iron in a bioavailable form in the early steps of iron-sulfur cluster biosynthesis or as an allosteric factor that modulates the cysteine desulfurase activity.Iron-sulfur cluster is involved in the synthesis and activation of various enzymes in the mitochondria.Therefore,patients manifest mitochondrial iron-sulfur cluster assembly deficiency with decreased activities of iron-sulfur proteins such as mitochondrial aconitase and complex Ⅰ,Ⅱ,and Ⅲ,Moreover,the role of FXN in preventing formation of deleterious reactive oxygen species(ROS)has been well established,invoking an additional paradigm of FRDA pathology in which ROS toxicity leads to mitochondrial dysfunction with subsequent neuron death.No cure or effective treatment for FRDA has yet been reported though a few clinical trials have made some limited beneficial progress.Here,we report a potential therapeutic strategy for FRDA with a tetra-peptide SS-31.SS-31,a novel mitochondrion-targeted antioxidant,targets and concentrates in the inner mitochondrial membrane.SS-31 interacts with mitochondrial cardiolipin to maintain the mitochondrial structure and integrity.Previous studies have shown that SS-31 treatment improves ATP production,reduces mitochondrial ROS production,and decreases oxidative damage.SS-31 has been demonstrated to be highly effective in several animal models associated with mitochondrial dysfunction and oxidative stress.Objectives:In our study,we examined the effect of SS-31 treatment on lymphoblasts derived from FRDA patient to investigate whether SS-31 treatment can improve the mitochondrial function,and to determine whether SS-31 has the potential value on the treatment of FRDA.The main contents are as follows:(1)Clarify the effect of SS-31 on FXN expression.(2)Detect the mitochondrial function indexes to study the effect of SS-31 on the quality and quantity of mitochondria.(3)Detect the activity of iron-sulfur cluster-containing enzymes to study the effect of SS-31 on the iron-sulfur cluster biogenesis in FRDA patient-derived cells.(4)Detect the effect of SS-31 on expression of iron-related proteins IRP2,TfR1 and ferritin to study the iron metabolism in FRDA patient-derived cells.(5)Detect the effect of SS-31 on ROS levels and the antioxidative ability of FRDA patient-derived cells.(6)Reveal how SS-31 upregulates FXN expression.Methods:(1)Western Blotting analysis to optimise the condition,under which SS-31 treatment induced the expression of FXN in lymphoblasts(GM15850)derived from a FRDA patient.(2)The level of MMP,ATP,NADH/NAD+and mitochondrial copy number were detected.The mitochondrial morphology was observed by electron microscope.(3)The activity of aconitase,electron transport chain complexes and xanthine oxidase in SS-31 treated cells to study the effect of SS-31 treatment on the synthesis of iron-sulfur clusters.(4)The protein level of IRP2,TfRl and ferritin in cells were detected by western blotting,the bioavailable iron level was detected by:fluorescence probes.(5)Flow cytometry was used to detect the levels of ROS,the activity and protein level of super oxide dismutase,catalase were detected,and CCK8 was used to detect the ability of resisting hydrogen peroxide.(6)FXN mRNA levels detected by qPCR,newly synthesized FXN was detected by SILAC.Results:(1)SS-31 treatment upregulates FXN expression in FRDA patient-derived cells.(2)SS-31 improved the mitochondrial membrane potential,increased the content of ATP,and decreased the NADH/NAD+ratio in FRDA patient-derived cells.The ridge structure of the mitochondrial inner membrane was arranged in an orderly manner.SS-31 treatment improves the quality of mitochondria of FRDA patient-derived cells.(3)SS-31 treatment enhances the enzymatic activities of mitochondrial iron-sulfur-containing enzymes,such as mitochondrial aconitase and electron transport chain complex Ⅱ/Ⅲ,has no effect on the activity of oxidase XOD.(4)SS-31 treatment modulates the iron metabolism of cells derived from FRDA patient.SS-31 increases the bioavailable iron levels in the mitochondria of Patients.(5)SS-31 treatment decreases the ROS level and enhances the antioxidative ability of FRDA patient-derived cells.(6)SS-31 has no effect on FXN mRNA level and translationally upregulates FXN expression.Conclusion:SS-31 translationally upregulates FXN expression,improves the function of mitochondria,enhances the ability to resist oxidative stress in FRDA patient-derived cells.SS-31 is expected to be a potential drug for the treatment of FRDA.