The Role And Mechanism Of Mitochondrial Depolarization In Secondary Brain Injury After Intracerebral Hemorrhage

Intracerebral hemorrhage（ICH）is a common and serious disease.The prevalence of ICH is approximately 120/100,000 and 58%of ICH patients die within one year.Two-thirds of survivors remain disabled.The high morbidity and mortality of ICH are based on both the primary injury and secondary injury.The current lack of evidence-based strategies for surgery has prompted researchers to search for possible intervention targets of secondary injury after ICH.Secondary brain injury of ICH includes neuronal cell death,ROS burst and DNA damage.Evidence from both animal models and human samples have shown that the major type of neuronal cell death after ICH is necrosis,which was thought to be a non-programmed type of cell death accompanied by inflammation and tissue damage.Since it was first reported in2005 that necrotic cell death occurs through a programmed pathway,the spectrum of different forms of regulated cellular necrosis has expanded dramatically and many of these types of necrosis are related to central nervous system（CNS）diseases.Currently,regulated necrosis（RN）is a major executioner of cell death in the central nervous system.Acute and severe iron overload is observed in brain tissues from patients and experimental animals after ICH and iron overload is closely related to secondary brain injury.Accumulating evidence from animal studies also indicate that removing the overloaded iron improves the prognosis of intracerebral hemorrhage.Some evidence indicates that iron may trigger neuronal death,while other studies have shown that iron accumulation does not directly cause neuronal death.The mechanism and effect on neuronal death of iron accumulation are still not fully understood.Free iron can cause oxidative damage to lipid,proteins and DNA of different cell types due to producing highly reactive hydroxyl radicals.Therefore,the attention to the treatment of iron-induced brain injury after ICH is mainly focused on iron chelation and antioxidants（10）.However,the recent clinical trials failed to show any improvement on the prognosis of ICH patients who had been treated with iron chelators or antioxidants.Iron participates in normal physiological functions of cells,such as cytochrome oxidase,and synaptic formation in the central nervous system（13,14）.Reactive oxygen species are also involved in normal immune responses like NOXes（nicotinamide adenine dinucleotide phosphate[NADPH]oxidases）-mediated phagocytic function.Therefore,the challenge of iron chelation and ROS clearance is to reduce brain injury induced by pathological overloaded iron and ROS accumulation without disturbing of iron and ROS-related functions.Searching for specific and accurate iron-related intervention targets to neuronal damage is urgently needed to alleviate secondary brain injury after ICH.In this study,we focus on three aspects:1)We firstly confirmed the iron valence in the brain parenchyma around hematoma in ICH mice model.Then,RNA sequencing was used to find the targets and main characteristics of neuronal cell damage induced by ferrous iron;2)The mechanism of neuron damage caused by ferrous iron overload was determined in mice after ICH;3)The therapeutic effect of the drug was explored in experimental mice after ICH.Part one:Mitochondrial dysfunction is the main feature of neuronal injury caused by ferrous iron overload after intracerebral hemorrhage.Objective:To find out and confirm the main targets of ferrous iron overload after intracerebral hemorrhage.Methods:To verify the valence of iron around hematoma after ICH,cortical and internal capsule tissues were extracted after ICH and an iron detection method that can separately measure ferrous and ferric iron was used.RNA was extracted from PC-12 neuronal-like cells treated with ferrous iron and were sequenced to explore the potential mechanism.Next,the effect of ferrous iron on the function of mitochondrial complex I was examined.And the oxygen consumption rate（OCR）and adenosine triphosphate（ATP）level were also examined.Results:1.The concentration of ferrous iron,but not ferric iron,increased acutely after 24 hours and the concentration of ferrous iron in the cortex and internal capsule remained high at 28days after ICH.2.PC-12 cells were treated with 50 or 250μM FeCl₂ and the differentially expressed genes were detected by RNA sequencing.Compared with untreated PC-12 cells,the expressions of 110 genes changed significantly after 50μM FeCl₂ treatment,while 129 genes changed significantly after 250μM FeCl₂ treatment.In addition,the percentage of mitochondrial related genes was 42%and 19%of differentially expressed genes after 50 or250μM FeCl₂ treatment.Further analysis of mitochondrial related genes showed that the expression of mitochondrial respiratory complex I related genes changed significantly in PC-12 cells exposed to 50μM and 250μM FeCl₂.3.The oxygen consumption rate（OCR）and adenosine triphosphate（ATP）level were decreased by ferrous iron in a dose-dependent manner.Moreover,ferrous iron overload significantly reduced the mitochondrial NAD⁺/NADH ratio（Fig.2E）.And it was also found that the fluorescence intensity of TMRM decreased after treatment with 250μM ferrous iron for 12 hours.Conclusions:Mitochondrial dysfunction is an important biomarker of neurons with ferrous iron overload,which is characterized by the decrease of NAD⁺/NADH ratio,OCR and ATP content,and depolarization of mitochondrial membrane potential.Part two:Mitochondrial Fe²⁺influx/CypD acetylation positive feedback loop mediates mitochondrial membrane depolarization and promotes neuronal necrosis after intracerebral hemorrhage.Objective:To confirm the positive feedback loop of mitochondrial Fe²⁺influx/CypD acetylation and its role in mitochondrial membrane depolarization dependent neuronal death after intracerebral hemorrhage.Methods:RPA was used to detect labile Fe²⁺in mitochondria.CypD acetylation level was detected by immunoprecipitation method.MPTP opening was evaluated in situ by calcein release test.Gene of CypD in PC-12 cells was knocked out by using CRISPR-cas9technology and RNAi was used to knock down the gene ABCB10 of PC-12 cells.Results:1.Mitochondrial ferrous iron overload induced the decrease of the mitochondrial membrane potential in neurons.2.Increased acetylation of CypD in response to mitochondrial ferrous iron overload opened the mPTP in neurons.3.The opened mPTP facilitated the mitochondrial Fe²⁺overloads in neurons.4.Ferrous iron overload-induced mitochondrial dysfunction and neuronal cell death were alleviated by an inhibitor of CypD acetylation or CypD deletion.Conclusion:Mitochondrial labile Fe²⁺overload decreased mitochondrial membrane potential and increased the acetylation of CypD in neuronal cells.The increased CypD acetylation opened the mPTP and consequently facilitated the entrance of ferrous iron(mitochondrial Fe²⁺influx)through the opened mPTP,resulting in the neuronal death.Inhibition of CypD acetylation or CypD deletion decreased Fe²⁺overload-induced mitochondrial dysfunction and neuronal death.In brief,the positive feedback molecular loop of mitochondrial Fe²⁺influx/CypD acetylation mediates mitochondrial membrane depolarization leading to neuronal necrosis after intracerebral hemorrhage.Part third:The neuroprotective effect of inhibiting mitochondrial depolarization after intracerebral hemorrhage.Objective:To explore the neuroprotective effect of inhibiting mitochondrial depolarization after intracerebral hemorrhage.Methods:PI staining was used to detect the death of primary neurons under the condition of ferrous iron and excitatory amino acids.The neurological function of mice after was evaluated by BMS,pole test and open field test.Results:1.Excitatory amino acids aggravated the neuronal necrosis induced by ferrous iron overload.2.Cyclosporin A（CSA）treatment or CypD deletion inhibited neuronal necrosis in vivo and in vitro and improved neurological function after ICH.3.The selective mitochondrial ROS scavenger MitoQ may improve the efficacy of antioxidants treatment of ICH by white matter injury alleviation.Conclusion:The positive feedback molecular loop of mitochondrial Fe²⁺influx/CypD acetylation is the therapeutic target of ICH.CSA,MitoQ or CypD deletion can significantly reduce the neurological deficit in the acute and chronic stages after intracerebral hemorrhage.