Effects Of Mitochondrial Calcium Uniporter On Apoptosis Induced By 1-methyl-4-phenylpyridinium Ion In PC12 Cells

BackgroundParkinson’s disease (PD) is a common neurodegenerative disease among middle aged and elderly people. It is characterized by a selective loss of dopaminergic neurons, which results in motor impairments involving resting tremor, bradykinesia, postural instability, gait difficulty and rigidity. PD which leads to disability is progressive and uncurable; it’s a serious threat to health and quality of life. So far, the pathogenesis of PD has not been fully understood, and there is also no exact and reliable drug to cure PD. A growing number of experimental research in vitro or in vivo showed that PD is closely related to apoptosis. Therefore, clarifying the link between PD and apoptosis, preventing neuronal apoptosis, and thus slowing or preventing PD process is particularly important.The posibility of the link between PD and apoptosis was first proposed in the 1950s. The resercher observed nucleus aggregation characteristics of high-density which was similar to apoptosis in the substantia nigra of the brain of a PD patients through electron microscopy. Later Mochizuki et al. found that 0.6% to 4.8% of dopaminergic neuron was undergoing apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay.1-methyl-4-phenylpyridinium (MPP+) which can cause PD-like symptoms in vivo has been extensively used in cultured neurons to estabilish a cell model of PD for the purpose of exploring the pathomechanism of PD. The apoptosis in dopaminergic neurons caused by MPP+, an exogenous poison, is due to the induction of oxidative stress and inhibition of mitochondrial complex I.As a second messenger Ca2+ plays an important role in signal transduction. Accumulating evidence supports the notion that increased cytoplasmic Ca2+ could cause cell apoptosis both at early and late stages of apoptotic processes. Normally, cytoplasmic Ca2+ concentration is low, while it’s much higher in endoplasmic reticulum, mitochondria and extracellular fluid. Thus, a slight Ca2+ release will cause a greatly improvement in the concentration of cytoplasmic Ca2+, thereby activating of Ca2+-dependent enzymes and causing a series of biochemical reactions, leading to intracellular calcium overload. The homeostasis of intracellular calcium is important for neurons to normal founction. Calcium dyshomeostasis will lead to cell injury or death. Transient variation of its intracellular concentration controls a multitude of biological processes ranging from proliferation, muscle contraction, secretion, gene expression, ATP production to cell death. Therefore, the concentration of intracellular calcium should be strictly controled to maintain the normal physiological functions.In the physiological condition, cellular Ca+ homeostasis occurs through repetitive bursts of rising intracellular Ca2+ that, sometimes are referred to as Ca2+ oscillations. Mitochondrial Ca2+ buffering through MCU shapes the amplitude and spatio-temporalpatterns of cytosolic Ca2+ concentration ([Ca2+]cyt) increases.It has been known for almost 50 years that Ca2+ uptake across the ion-impermeable inner mitochondrial membrane (IMM) is mediated by low-affinity mitochondrial Ca2+ uniporter (MCU), that has the properties of a highly selective ion channel. Indeed, it was known that the driving force for Ca2+ accumulation in the mitochondrial matrix is the steep membrane potential (negative inside) (△Ψm) across the inner membrane and mitochondrial Ca2+ uptake occurs by a uniport mechanism driven by the negative-inside membrane potential without direct coupling to ATP hydroslysis or transport of other ions, but its molecular identity, however, has remained elusive. Most traditional approaches in this molecular search could not be applied or failed because no highly specific inhibitor can be utilized in biochemical purifications. Although ruthenium red (RuR) and the related compound Ru360 can inhibit the activity of the uniporter, it shows some major drawbacks because it binds a broad array of glycoproteins. Until 2011, researchers of Harvard Medical School and Massachusetts General Hospital found the key protein of mitochondrial calcium uniporter CCDC109A (MCU) by refering to the human genome project database combined with experimental analysis.MCU, localized to the inner membrane, is composed of MICU1 (regulatory partner) and MCU (poreformingsubunit). A characteristic feature of the MCU is its low affinity for Ca2+. But the mitochondrial Ca2+ concentrations can increase quickly by cell stimulation, as mitochondria are exposed to microdomains of high [Ca2+]. Mitochondria localized at close proximity to intracellular Ca2+ stores or plasma membrane Ca2+ channels sense and respond to the Ca2+ transients by taking up Ca2+.Whether MCU participates in MPP+-induced apoptosis in PC12 cells, and the specific mechanism has not been reported. Therefore, this study focuses on the effect of MCU on apoptosis induced by MPP+ in PC12 cells and what role it plays in it, to provide a theoretical basis for the future development of drugs for PD. PurposeTo explore the mechanism of apoptosis induced by MPP+ in PC12 cells.To clarify weather MCU is involved in the apoptosis induced by MPP+ in PC12 cells.To explore the mechanism of MCU participate in the apoptosis induced by MPP+ in PC12 cells.To clarify weather PGC-1α is involved in the apoptosis induced by MPP+ in PC12 cells.To explore the mechanism of ROS participate in the apoptosis induced by MPP+ in PC12 cells.Methods1 PC12 cells were incubated in a 37℃ incubator (CO2,5%;air,95%). After treatment of MPP+ with different concentrations, we measured cell viability by MTT, then chose appropriate time and concentrations as the basic condition of establishing PD cell model.2. Hoechst method, flow cytometry instrument technology were used to detect apoptosis of PC12 cells3. We measured the changes of PGC-1α and mitochondrial calcium uniporter (MCU) expression after the treatment of MPP+ with certain concentrations in western blot.4. We observed the effect of pretreated with inhibitor Ru360, stimulator Spermine and downregulate or upregulate the expression of MCU on apoptosis induced by MPP+ in PC12 cells.5. We observed the effect of downregulate or upregulate the expression of MCU, downregulate the expression of PGC-1α on ROS induced by MPP+ in PC12 cells.6. We measured the changes of mitochondrial calcium uniporter (MCU) expression after downregulating the expression of PGC-1α in western blot.Results1. MPP+ caused a decrease in viability of PC12 cells with a dose and time-dependent.The viability decreasing of PC 12 cells was correlated with the concentration exposure of MPP+ within 0-4mM after treatment of 24h or 48h. Compared with the control, the viability of PC 12 cells in each treatment group decreased with statistically significant difference.2. MPP+ induced the apoptosis of PC12 cellsThe apoptosis of PC 12 cells induced by MPP+was confirmed by hoechst 33342 staining, where we observed condensation and fragmentation of chromatin in PC 12 cells with 1mM MPP+ incubated for 24h. The mitochondrial transmembrane potential significantly decreased and the cleavage of PARP which is a marker of the apoptosic response was increased with 1mM MPP+ incubated for 24h. The apoptosic rate of PC12 cells was increased from 2.14%± 0.15% to 11.07%± 1.07% with statistically significant difference.3. The effect of MPP+ at different dose on the expression of MCUWe measured the expression of MCU by western blot at different dose of MPP+. The expression of MCU was gradually decreased with the increaseing dose of MPP+ which showed that the expression of MCU in PC 12 cells was affected by MPP+.4. The effect of MCU inhibitor and agoniston on the apoptosis induced by MPP+ in PC12 cellsWe know that the expression of MCU in PC 12 cells was affected by MPP+ through the foregoing experiment, then weather MCU is involved in the apoptosis induced by MPP+ in PC12 cells? PC12 cells were preincubated with 10μM Ru360 and 20μM Spermine, which were non-specificity inhibitor and stimulator of MCU for 1h, then we applied 1mM MPP+ for 24h. The apoptosis of PC12 cells in the group of Ru360+ MPP+ were decreased significantly by annexinV+ PI assay compared with those of control cells, while there was no significant difference between the group of Sper+ MPP+ and the control.5. The effect of MCU inhibitor and overexpression on the apoptosis induced by MPP+ in PC12 cellsTo identify the effect of MCU on the apoptosis induced by MPP+ in PC12 cells, we downregulated and upregulated the MCU by siRNA and transfecting MCU into PC12 cells respectively. Western blot method was used to detect the interference efficiency of knockdown after siRNA interference MCU expression, showed that the interference efficiency of MCU is about 90% and the MCU transfection efficiency was 143%. The change of apoptosis rate with MCU overexpression or siRNA was detected by flow cytometry, the results showed that overexpression ofMCU decreased the apoptosis rate, contrary to the results of downregulate MCU. All the above confirmed that MCU was invloved in the apoptosis induced by MPP+ in PC12 cells.6. The effect of MCU interference and overexpression on the Oxidative stress induced by MPP+ in PC12 cellsTo determine the role of MCU on the oxidative stress induced by MPP+ in PC12 cells, we downregulated and upregulated the expression of MCU separately by siRNA and transfecting MCU into PC12 cells. ROS detection kits were used to detect the interference efficiency of knockdown after siRNA interference MCU expression showed that the interference of MCU induced the ROS rate.The change of ROS rate with MCU overexpression or siRNA was detected by ROS detection kits, the results showed that overexpression of MCU decreased the ROS rate, contrary to the results of downregulate MCU. All the above confirmed that MCU was invloved in the Oxidative stress induced by MPP+ in PC12 cells.7. The effect of PGC-lα interference on the apoptosis induced by MPP+in PC12 cellsTo identify the effect of PGC-1α on the apoptosis and oxidative stress induced by MPP+ in PC 12 cells, we downregulated the expression of PGC-1α by siRNA in PC12 cells. Western blot method was used to detect the PGC-la rate after interference of knockdown after siRNA interference MCU expression. The results showed that downregulated and upregulate the expression of PGC-la decreased the rare of MCU.The change of ROS rate with PGC-la siRNA was detected by ROS detection kits, the results showed that downregulated the expression of PGC-la induced the ROS rate. All the above confirmed that PGC-1α was invloved in the apoptosis and oxidative stress induced by MPP+in PC 12 cells.8. The effect of ROS on the apoptosis induced by MPP+in PC12 cellsThe apoptosis of PC 12 cells induced by MPP+ was confirmed by ROS detection kits. We applied 1mM MPP+ for 24h, the ROS rate was increased.The change of ROS rate with NAC was detected by ROS detection kits, the results showed that with NAC treatment, the ROS rate was decreased.Western blot method was used to detect the PGC-la rate and the MCU rate after pretreatment with NAC. The results showed that after the pretreatment with NAC decreased the PGC-1 rate, and increased the MCU rate.ConclusionIn conclusion, this study shows for the first time that MCU is involved in the apoptosis induced by MPP+in PC 12 cells. MPP+treatment increases the expression of PGC-la and decreases the expression of MCU. Interference of MCU will promote the apoptosis induced by MPP+ in PC 12 cells, while overexpression of MCU will inhibit the apoptosis. MCU participates and inhibit the apoptosis induced by MPP+ in PC12 cells. Interference the expression of MCU increases the expression of PGC-1α. The mechanism may be that PGC-1α, as an important transcriptional regulation factor for the mitochondria, control the important circulation within the calcium on mitochondrial membrane protein MCU, Participate in and can suppress the apoptosis of PC12 cells induced by MPP+.