Study On The Mechanism Of Hippocampal Neuron Apoptosis Mediated By NR1D1-OPA1 Pathway Under Hypoxia

Research Backgroud:Various aerospace environmental factors will have varying degrees of impact on the brain function of astronauts.The most significant influencing factor is microgravity,also known as weightlessness.The head distribution of blood is one of the most important changes that organisms face under microgravity conditions.Studies have shown that spaceborne microgravity can cause a decrease in blood volume,a slowdown in blood flow,and a decrease in the number of red blood cells and hemoglobin content.These changes in the blood circulatory system will affect its ability to bind oxygen and transport oxygen,resulting in brain tissue hypoxic state.Hypoxia is one of the serious cellular stresses,which can cause cell damage or even death.Hypoxia can cause damage to hippocampal neurons and other cells,and play an important role in the progression of the disease as the main pathological mechanism in stroke,ischemic heart disease and other diseases.The studies indicate that hypoxia can induce mitochondrial damage,calcium overload,increased oxygen free radicals and hypoxia-inducible factors（HIF）expression to induce apoptosis.Our previous studies have shown that 28-day tail suspension simulated microgravity can cause significant upregulation of HIF-1αm RNA and protein,increased number of mitochondria,swollen and vacuolated mitochondria,and increased apoptosis of hippocampal neurons.Meanwhile,the apoptosis-related proteins Cleaved Caspase-3 and Bax were significantly up-regulated.The dynamic change of mitochondrial morphology is the basis of mitochondrial function.Studies have shown that the dynamic change of mitochondrial morphology is related to apoptosis.Therefore,we hypothesize that simulated microgravity effect may cause hypoxia in hippocampus and induce apoptosis of hippocampal neurons by affecting the morphology and function of mitochondria,but the molecular mechanism remains unclear.Many genes are regulated by HIF.Nuclear receptor subfamily 1 group D member 1（NR1D1）is a nuclear receptor,and acts as a transcription inhibitor.Mitochondrial endomembrane fusion protein Optic Atrophy 1（OPA1）is a kind of GTPase similar to dynein.It has been reported that OPA1 can inhibit apoptosis by regulating the release of mitochondrial cytochrome c.We predicted that there were 17 binding sites between HIF-1αand NR1D1 promoter,and two binding sites between NR1D1 and OPA1promoter.Objectives:1.To elucidate the effects of simulated microgravity and hypoxia on mitochondrial morphology and apoptosis of hippocampal neurons.2.To disclose the regulatory relationship between HIF-1αand NR1D1.3.To elucidate the mechanism of NR1D1 on hypoxia induced mitochondrial morphological changes and apoptosis of hippocampal neurons by inhibiting OPA1transcription.Methods:1.C57BL/6 mice were suspended at 30°to simulate the microgravity effect,and the hippocampus tissues were taken 28 days later.The mouse hippocampal neuron HT22 cells were rotated for 72 hours to simulate the microgravity effec.Co Cl₂（0,75,150,300μM）treated HT22 cells to simulate cell hypoxia for 48 hours.2.The mice hippocampal mitochondrial morphology was observed under the transmission electron microscope.Nissl staining was to show the hippocampal neuron damage.The cell apoptosis was detected by the flow cytometry and the cell proliferation was detected by the CCK-8 assay.Mito Tracker Red fluorescent staining was to label mitochondria,and laser confocal microscope was used to observe the changes in the number and morphology of mitochondria.The mitochondrial DNA（mt DNA）quantitative analysis was to detect the number of mitochondria.3.NR1D1 or OPA1 small interfering RNA（si RNA）was transfected with RNAi MAX respectively.Lipofectamine LTX and PLUS transfected HIF-1αand NR1D1 plasmid respectively or NR1D1 and OPA1 reporter gene plasmid respectively.4.Western Blot examined the expressions of HIF-1α,NR1D1,OPA1,Bax,Caspase-3and Cleaved Caspase-3 protein.The SYBR Green dye q RT-PCR method detected the HIF-1αand NR1D1 m RNA levels.5.Bioinformatics JASPAR software predicted the binding sites between HIF-1αand NR1D1 promoter,and between NR1D1 and OPA1 promoter.The dual luciferase reporter gene experiment detected the interaction between HIF-1αand NR1D1 promoter,and between NR1D1 and OPA1 promoter.Results:1.Transmission electron microscopy showed that the number of mitochondria in the hippocampus of the mice after 28 days of tail suspension increased significantly,the swelling was obvious,the cristae was loose and dissolved,and vacuoles appeared.The number of hippocampal neurons decreased in the number of Nissl bodies and the neuron arrangement was scattered in the mouse hippocampus tissues.The expressions of HIF-1αand NR1D1 proteins increased significantly,the expression of OPA1 protein decreased,and the expression of Bax protein increased in HT22 cells.2.The apoptosis of HT22 cells treated with Co Cl₂showed a significant increase with the increase of Co Cl₂concentration（P<0.001）,the cell proliferation showed a significant decline（P<0.001）.The expression of Cleaved Caspase-3 protein increased significantly and the number of mitochondria increased,whereas OPA1 protein expression was down-regulated.3.Co-transfection of HIF-1αexpression plasmid and NR1D1 reporter gene plasmid did not significantly increase the luciferase activity（P>0.05）.The luciferase activity was significantly increased after NR1D1 reporter gene plasmid transfection together with Co Cl₂treatment（P<0.01）.In NR1D1 knockdown HT22 cells,the expression of OPA1protein increased,and the expression of Cleaved Caspase-3 protein decreased.However,the expression of OPA1 protein decreased,and the expression of Cleaved Caspase-3protein increased in the HT22 cells transfected with NR1D1 expression plasmid.The luciferase activity was significantly reduced when NR1D1 expression plasmid co-transfected with OPA1 reporter gene plasmid（P<0.05）,and the expression of Cleaved Caspase-3 protein increased in OPA1 knockdown HT22 cells.4.NR1D1 antagonist SR8278 or NR1D1 knockdown can partially reversed HT22 cell apoptosis after Co Cl₂treatment（P<0.01 or P<0.05 respectively）,and the cell viability was restored to some degree（P<0.01）.The mt DNA analysis and Mito Tracker Red staining showed that SR8278 could reduce the number and volume of mitochondria,the expression of OPA1 protein was down-regulated,and the expression of Cleaved Caspase-3 protein was up-regulated.Conclusions:1.The simulated microgravity effect can lead to hypoxia in brain tissue and increased apoptosis of hippocampal neurons,which may be related to the morphological changes of mitochondria caused by the increased expression of HIF-1αand NR1D1.2.NR1D1 can cause changes in mitochondrial morphology under hypoxic conditions by inhibiting the transcription of mitochondrial fusion protein OPA1,and promote hippocampal neuronal apoptosis.In summary,the NR1D1-OPA1 pathway can mediate hippocampal neuron apoptosis under hypoxic conditions,and may serve as a potential therapeutic target for hypoxic-ischemic diseases.