The Role Of MCU And MICU1 In Brain Infarction

Objective:Brain infarction is a common disease in neurology with high morbidity and mortality. Although the ultra-early thrombolytic treatment is effective, but receiving treatment within a specified time window is difficult for most patients. The physiological and pathological mechanisms of neuron injury after infarction is more complex cascade, including the excitatory amino acid toxicity, oxidative stress, inflammation and apoptosis. One of the key mechanism of glutamate excitotoxicity after infarction is calcium overload caused by calcium influx, which causing mitochondria damage, lipid peroxidation, ATP deprivation, ROS generation, nucleic acid fracture, eventually leading to neuronal death. Mitochondrial calcium concentration closely related with mitochondrial energy metabolism and membrane permeability, but also affect cell function by modifing cytosolic calcium signals. Mitochondrial calcium uniporter(MCU) is an recently identified protein which is an important structure located on mitochondrial inner membrane that involved in mitochondrial calcium uptake. Mitochondrial calcium uptake 1(MICU1)is regulatory protein of MCU, which role is controversial about activated or inhibited. In view of this, this highly complex and precise regulatory role has still need to be further studied. Research has shown that MCU involved in glutamate excitotoxicity of neuron cell, inhibition of MCU can reduce the mitochondrial calcium uptake, protect mitochondrial function thereby reducing neuronal death. So we hypothesize MCU, MICU1 are relevant with cerebral infarction, inhibiting MCU and MICU1 expression may confer the neuroprotective effect via curb calcium overload. We expect this new approach provide a new potential target for cerebral infarction treatment.1 Male, healthy Sprague-Dawley rats were used and randomly assigned to eight groups: control(normal control) group, sham(sham operated) group, MCAO groups(Divided into 3h, 6h, 12 h, 24 h, 48 h, 72 h six subgroups). MCAO model was performed by using intraluminal filament technique in rats. Neurological behavior was evaluated after anesthetic sober. The animals were sacrificed at different time points, Infarct volume was analyzed with 2, 3, 5-triphenyl tetrazolium chloride(TTC) staining, MCU, MICU1, cleaved-caspase3 protein and m RNA expression were measured by Western Blot, realtime-q PCR and immunohistochemistry. Cell and mitochondrial ultrastructure was observed by Transmission Electron Microscope(TEM), Flow cytometry technique was used to measure mitochondrial function.2 Male, healthy Sprague-Dawley rats were used and randomly assigned to eight groups: control(normal control) group, sham(sham operated) group, MCAO group(Vehicle), Kallikrein+MCAO group(Kallikrein0.00875PAN/kg + MCAO). MCAO model was performed in rats according to Longa method. Kallikrein 0.00875PAN/kg was administrated by intravenously injected after cerebral ischemia. For MCAO, sham and control group, equal volume saline was administered in the same manner. Neurological behavior was evaluated after anesthetic sober. the animals were sacrificed after 12 h of ischemia. Infarct volume was analyzed with TTC staining, MCU and cleaved-caspase3 expression were measured by western blot.Results:1 Rats in MCAO groups at various time points performed a left palsy. Neurological deficit score was increased significantly(P<0.05). No infarction was observed in sham and control group, MCAO group showed a large area of infarcts in right hemisphere by TTC staining.2 The protein and m RNA expression of MCU, MICU1 in brain tissue after MCAO were measured by western blot, realtime-q PCR and immunohistochemistry. Western blot shows that MCU, MICU1 protein in MCAO group were significantly upregulated compared with sham and control group(P<0.05), beginning at 6h and peaking at 12 h. The m RNA expression Method: of MCU and MICU1 in MCAO group were significantly increased compared with sham and control group(P<0.05), cleaved-caspase3 was also dramatically upregulated at 3-12 h after MCAO(P<0.05).3 Ultrastructural changes of neuron cells. Control and sham group show a structured cortical cells with normal morphology, evident nucleolus, nuclear membrane integrity, cytoplasm mitochondria, endoplasmic reticulum complete visible. Blurred nuclear membrane, dilated endoplasmic reticulum and unswollen mitochondria were found in the neurons of cortex at 3h after MCAO. Cytolymph edema, blurred nuclear membrane, liquified nuclear, dilated endoplasmic reticulum and balloon-like mitochondria with crista on the rim were observed in neurons at 12 h after MCAO. Interstitial edema, cell pyknosis and peripheral edema were found around edema at thalamencephon-basal nucleus at 12 h after MCAO.4 Mitochondrial membrane potential decreased significantly at 12-24 h after infarction compared with control and sham group by flow cytometry(P<0.05).5 Neurological deficit score was significantly decreased in Kallikrein+MCAO group compared with MCAO group(P<0.05), the infarct volume was also significantly reduced in Kallikrein+MCAO group compared with MCAO group(P<0.05), MCU protein expression in MCAO group was upregulated compared with sham group(P<0.05), Kallikrein significantly inhibited the MCU protein expression compared with MCAO group(P<0.05), Cleaved-caspase3 was also significantly reduced by Kallikrein compared with MCAO group.Conclusion:Our studies showed that the expression of MCU, MICU1 were up-regulated after cerebral infarction, the expression of cleaved-caspase3 was increased simultaneously, and mitochondrial membrane potential was decreased, which indicated MCU and MICU1 play an important role in the pathophysiology of cerebral infarction by promoting mitochondrial calcium uptake, reducing mitochondrial membrane potential, damaging mitochondrial function, causing calcium overload, leading to apoptosis ultimately.Kallikrein improve neurological deficit situation, reduce infarct volume,which via to inhibit protein expression of MCU, reduce calcium influx of mitochondria, protecte mitochondrial function and curb calcium overload, eventually inhibit apoptosis.