Serine Protease HtrA2/Omi Regulates Adaptive Mitochondrial Reprogramming In Neurocyte Via UCP2-SIRT3-PGC1? Pathway

It has been found that mitochondria play an important role in the development of cerebral ischemia-reperfusion injury?IRI?in patients with acute ischemic stroke.By the following researches,people have a deeper understanding of the function and regulatory network of mitochondria,which provides a new theoretical and experimental basis for exploring the role of mitochondria in IRI.Because mitochondrial damage is a gradual process,there may be different levels of quality control,electron chain activity,ROS production and antioxidant capacity in different stages of mitochondrial damage.SIRT3 as a deacetylase and PGC1?as a transcription coactivator,are considered to be one of the important protein molecules that regulate mitochondrial quality and biogenesis,and play a role in protecting mitochondria.This effect can be reflected in the molecular chaperones such as HSP10,HSP60 and LONP,which can degrade damaged misfolded proteins through mtUPR and maintain the normal functions of mitochondria.Therefore,this adaptive response to mitochondrial damage is called adaptive mitochondrial recombination?AMR?.Our previous study found that UCP2can be a potential regulatory pathway by increasing the ratio of reduction equivalent NAD⁺/NADH to activate SIRT3 and regulate PGC1?when IRI occurs.When C57BL/6J mice were intraperitoneally injected with genipin,an inhibitor of UCP2,the IRI could be reduced by UCP2-SIRT3 regulation.These results suggest that UCP2 may be related to mtUPR,but the specific mechanism is not clear.By observing the changes of UCP2 and SIRT3-PGC1?signal pathway during mitochondrial injury,it may be possible to explore the mechanism of this pathway in mitochondrial adaptive recombination.HtrA2/Omi is a serine protease located in the mitochondrial membrane space,which has been found to be related to Parkinson's disease,Alzheimer's disease and other diseases.HtrA2/Omi mutant homozygote(HtrA2^mnd2)mice would show neurodegenerative disease symptoms in early stage.Therefore,using HtrA2/Omi mutant mice,we first observed the relationship between mitochondrial function and neurocyte damage.Secondly,by using WT and HtrA2/Omi mutant heterozygotes to replicate IRI model,we explore the changes of UCP2-SIRT3-PGC1?activity and its impact on mitochondrial quality and function.Finally,the mechanism of UCP2regulating AMR through SIRT3-PGC1?pathway was described by using genipin of intraperitoneal injection pretreatment before replicating IRI model.By targeting UCP2,the changes of SIRT3-PGC1?signal pathway were observed,and the role of protease HtrA2/Omi in neurocytes was further explored.Methods:1.Western blot,qPCR,PCR array,immunohistochemistry,transmission electron microscopy and enzyme activity kit were used to evaluate the quality control level and mitochondrial function of HtrA2/Omi mutant mice.2.WT and HtrA2^hetero were replicated by MCAO and TTC staining,Western blot,PCR array,immunohistochemistry and multiple kits were used to evaluate the anti injury ability of HtrA2^hetero and the activity of UCP2-SIRT3-PGC1?pathway.3.WT and HtrA2^hetero with intraperitoneal injection of Genipin,an inhibitor of UCP2,were replicated by MCAO.TTC staining,immunohistochemistry,Western blot,transmission electron microscopy,enzyme activity kit and other techniques were used to analyze the possibility mechanism of HtrA/Omi regulating AMR by UCP2-SIRT3-PGC1?pathway.Results:1.HtrA2^mnd2 growth stagnated and died in the early stage,HtrA2^hetero appeared hair shedding earlier than WT.The expression of HtrA2/Omi protein and mRNA in cortex of HtrA2^hetero increased.HE staining and TUNEL staining showed that the number of HtrA2^mnd2 nneurocyte was significantly reduced,shrunk,arranged in disorder and the number of apoptotic nuclei was significantly increased,but HtrA2^hetero was the second.2.HtrA2^hetero cerebral cortex showed SOD activity increased,MDA content decreased,ATP content increased,mitochondrial UPR related protein expression increased,mtDNA copy number and mitochondrial number increased,UCP2 protein expression increased,NAD⁺/NADH ratio and SIRT3 activity significantly increased,while HtrA2^mnd2 significantly decreased.3.After 1.5h ischemia and 24h reperfusion in HtrA2^hetero,the area of cerebral infarction stained by TTC increased significantly.HE staining showed the aggravation of cell damage and TUNEL staining suggested that the number of apoptotic nuclei increased.HtrA2/Omi protein expression decreased,SOD activity decreased,MDA content increased,ATP content decreased,hSP10,HSP60,LONP quality control related proteins expression were significantly down-reguregulated.The increased expression of UCP2 did not increase the activity of SIRT3-PGC1?.4.After pretreatment with Genipin,TTC staining showed that the infarct area decreased.In the HtrA2^Hetero model group,the expression of LOPN,ATP content,mtDNA copies number and activity of respiratory volume complex I increased significantly.At the same time the expression of UCP2 was not significantly increased,while the expression of HtrA2/Omi was maintained.The ratio of NAD⁺/NADH and the activity of SIRT3were significantly increased.Conclusions:1.The decrease of HtrA2/Omi protein function is closely related to brain injury.2.Compared with homozygous mice,HtrA2^hetero may induce adaptive mitochondrial reorganization through SIRT3-PGC1?pathway,and partially antagonize the mitochondrial damage of neurocytes caused by the decrease of HtrA2/Omi function.3.When HtrA2/Omi function decreased,ischemia-reperfusion injury could cause SIRT3-PGC1?pathway down-regulated,resulting in the decline of mitochondrial adaptive recombination.4.Genipin may activate SIRT3-PGC1?through UCP2 and/or protease HtrA2/Omi,thus reducing the degree of ischemia-reperfusion injury caused by the decrease of HtrA2/Omi function.