Mechanism Of Mitochondrial UCP4 Protection Of Cerebellar Purkinje Cells Under Hyperbaric Hypoxia

Objective:The plateau is an important area for Chinese economic development and national defense construction.With the progress of social development,the number of activities in the plateau gradually increases,but its low-pressure and oxygen-deficient environmental characteristics make the brain-body operational capacity and labor efficiency of people in the plateau decrease significantly,which seriously affects people’s activity capacity,economic construction and military operations in the plateau.At present,there is a lack of new technical means to improve the brain-body working ability in the plateau.The aim of this study is to investigate the relationship between mitochondrial UCP4 and locomotion related to cerebellar Purkinje cells under hyperbaric hypoxia,which is expected to shed light on the direction of improving our military operational capacity and medical research development in the plateau,and is likely to provide strategies for improving human locomotor performance under low-pressure hypoxic environment.Methods:UCP4-specific knockout mice in cerebellar Purkinje cells,Pcp2-Cre::Ucp4^flox/flox mice,were constructed using Cre-Loxp technology,and mouse genotypes were identified by murine tail PCR,and molecule changes of UCP4 in cerebellar Purkinje cells were determined by Western blot,q PCR with RNAscope.The changes in locomotor ability of Pcp2-Cre::Ucp4^flox/flox mice were assessed by the open field test,elevated plus-maze test,and rotarod test.The hyperbaric hypoxia model was constructed to simulate condition on the plateau with the altitude of 5000 m,and mice were put in a low-pressure oxygen chamber for five consecutive days for 16 h per day.Then the molecular changes of Ucp4,Ucp2,Ucp5,Drp1,Fis1,Mff,Mfn1,Mfn2,Opa1and Tfam were detected by q PCR,immunofluorescence staining and RNAscope;the emotion or motor ability of Pcp2-Cre::Ucp4^flox/flox mice were evaluated by the open field test,elevated plus-maze test,and rotarod test;Changes in mitochondrial morphology of cerebellar Purkinje cells from Pcp2-Cre::Ucp4^flox/flox mouse before or after hyperbaric hypoxia were observed by electron microscopy.Constructed UCP4 overexpression virus:p AAV-CMV-MTS-UCP4-EGFP-3x FLAG-WPRE;The virus were injected at the normal mouse cerebellum to overexpresse UCP4 protein in cerebellar cortex,after which the changes of motor ability before and after hyperbaric hypoxia in mice were assessed by open field test,elevated plus-maze test,and rotarod test.Results:The results of the tail identification experiments showed that:The mice with UCP4-specific knockout in cerebellar Purkinje cells were successfully constructed,Pcp2-Cre::Ucp4^flox/flox mice.q PCR,Western blot and RNAscope experiments showed that the expression of UCP4 in Purkinje cells was reduced.The results of the open field test showed that Pcp2-Cre::Ucp4^flox/flox mice showed a decrease in locomotor performance.The results of elevated plus maze test and rotarod test showed no significant changes in emotion,coordination and endurance of Pcp2-Cre::Ucp4^flox/floxmice.（2）Compared with the Ucp4^flox/flox mice,the results of the open field test showed that the motor ability of Pcp2-Cre::Ucp4^flox/floxmice was significantly reduced after simulated hyperbaric hypoxia.q PCR,immunofluorescence staining and RNAscope experiments showed that UCP4 and UCP5 were increased,while UCP2 was decreased in the cerebellar cortex of the control or experimental groups after simulated hyperbaric hypoxia.The q PCR results showed that the expression of Drp1 and Fis1 was increased in the cerebellar cortex of the both groups after simulated hyperbaric hypoxia.the expression of Mff and Tfam was increased in the cerebellar cortex of Ucp4^flox/flox mice,and the expression of Mfn1 was increased in the cerebellar cortex of Pcp2-Cre::Ucp4^flox/flox mice.Electron microscopic results showed that the area occupied by mitochondria in the cytoplasm of cerebellar Purkinje cells was significantly increased and the number of mitochondria per unit area was significantly increased after hyperbaric hypoxia in mice.（3）The results of the open field test,elevated plus-maze test,and rotarod test showed that hyperbaric hypoxia did not affect locomotion,mood,endurance and motor coordination in the cerebellar cortex from normal mouse after overexpression of UCP4.Conclusion:Knockdown of the Ucp4 gene in cerebellar Purkinje cells under hyperbaric hypoxia attenuates locomotor activity in mice,possibly through a mechanism that affects mitochondrial biosynthesis and functions.