| 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 possibility of the link between PD and apoptosis was first proposed in the 1950s.The researcher 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.As an, 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 Ca2+ 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 there’s 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 SH-SY5Y cells, and the specific mechanism has not been reported. Therefore, this study focuses on the effect of MCU on apoptosis induced by MPP+ in SH-SY5Y 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 SH-SY5Y cells.To clarify weather MCU is involved in the apoptosis induced by MPP+ in SH-SY5Y cells.To explore the mechanism of MCU participate in the apoptosis induced by MPP+ in SH-SY5Y cells.MethodsSH-SY5Y 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. We measured the changes of MCU expression after the treatment of MPP+ with different concentrations and observed the effect of pretreated with non-specificity inhibitor Ruthenium Red, stimulator Spennine and downregulate or upregulate the expression of MCU on apoptosis induced by MPP+ in SH-SY5Y cells by western blot.We detected the effect of downregulate or upregulate the expression of MCU on calcium concentration in endoplasmic reticulum ([Ca2+]ER) and mitochondrial Ca2+concentration in matrix([Ca2+]mt) induced by MPP+ through laser scanning confocal microscope(LSCM). We measured the effect of changes of MCU expression on the expression of GRP78 in SH-SY5 Y cells by western blot.Results1. MPP+ caused a decrease in viability of SH-SY5Y cells with a dose and time-dependent.The viability decreasing of SH-SY5Y 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 SH-SY5Y cells in each treatment group decreased with statistically significant difference. A dose-response study established that lmmol/L MPP+ produced 50% death of SH-SY5Y cells after 24h under our conditions.2. MPP+ induced the apoptosis of SH-SY5Y cellsThe apoptosis of SH-SH5Y cells induced by MPP+ was confirmed by hoechst 33342 staining, where we observed condensation and fragmentation of chromatin in SH-SY5Y cells with 1mM MPP+ incubated for 24h.The mitochondrial transmembrane potential significantly decreased with 1mM MPP+ incubated for 24h.The apoptosic rate of SH-SY5Y cells was increased from 4.6%±0.43% to 11.3%±1.66% 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 SH-SY5Y cells was affected by MPP+.4. The effect of MCU inhibitor/ interference and agoniston/overexpression on the apoptosis induced by MPP+in SH-SY5Y cellsWe know that the expression of MCU in SH-SY5Y cells was affected by MPP+ through the foregoing experiment, then weather MCU is involved in the apoptosis induced by MPP+ in SH-SY5Y cells? SH-SY5Y cells were preincubated with 50 μM Ruthenium Red and 100μM Spermine,which were non-specificity inhibitor and stimulator of MCU for 30min,then we applied 1mM MPP+ for 24h.The apoptosis of SH-SY5Y cells in the group of RuR+MPP+ were decreased signifificantly by annexin V+PI assay compared with those of control cells,while there was no significant difference between the group of Sper+MPP+ and the control.To identify the effect of MCU on the apoptosis induced by MPP+ in SH-SY5Y cells, we downregulated and upregulated the expression of MCU separately by siRNA and transfecting MCU into SH-SY5Y cells.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 50% andthe MCU transfection efficiency was 175%. The change of apoptosis rate with MCU overexpression or siRNA was detected by flow cytometry, the results showed that overexpression of MCU 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 SH-SY5Y cells.5. The effect of interference and overexpression of MCU on [Ca2+]mt and [Ca2+]ER changes induced by MPP+in SH-SY5YcellsWe detected [Ca2+]ER after treated with lmM MPP+by LSCM. It showed that MPP+ caused [Ca2+]ER decline. And interference of MCU in the cells would promote the effect, while overexpression of MCU can reverse this inhibition.[Ca2+]mt decreased first and then increased after treated with 1mM MPP+. Interference of MCU results in decrease of [Ca2+]mt, while [Ca2+]mt temporary decline, then rebounded more than the original concentration levels and remained at that level after overexpression of MCU.6. The effect of changes of MCU expression on unfolded protein response (UPR) caused by MPP+ in SH-SY5Y cellsMPP+can cause UPR. We interferenced and overexpressed MCU in SH-SY5Y cells, then treated the cells with 1mM MPP+ for 24h. The expression level of GRP78/Bip, a marker protein of UPR, was detected by immunoblotting. The results show that the interference caould promote the upregulating of GRP78 protein induced by MPP+, whereas overexpression MCU is able to reduce the expression level of GRP78 protein. MCU could reduce the UPR.ConclusionIn conclusion, this study shows for the first time that MCU is involved in the apoptosis induced by MPP+ in SH-SY5Y cells. MPP+ treatment decreases the expression of MCU. Interference of MCU will promote the apoptosis induced by MPP+ in SH-SY5Y cells, while overexpression of MCU will inhibit the apoptosis. MCU participates and inhibit the apoptosis induced by MPP+ in SH-SY5Y cells.The mechanism may be that MCU can alleviate [Ca2+]ER decrease caused by MPP+,thereby maintain the caicium in ER and reduce the UPR.The maintenance of homeostasis of [Ca2+]mt for normal bioenergetic energy may be another mechanism. |
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