Effects Of Mitochondrial Calcium Uniporter (MCU) On Lead-induced Oxidative Stress In Neuronal Cells

BackgroundThe lead poisoning is a serious public health problem atpresent, has a significant threat to human health.Lead is a kind of poison with a broud affinity to numerous organs, such as brain,heart,kidney and liver as well as central nervous system. Leadneurotoxicity is able to cause neural function disorder that characterized by lead encephalopathy, peripheral neuropathy, hearing impairmentand cognitive impairment. What’s more, lead exposure can lead to an irreversible toxic effect on neural function, especially for developing children. Recently, toxicological and epidemiological studies have indicated that Pb can cause brain damage, neurochemical dysfunction, and severe behavioral problems, especially in children.The key mechanisms of lead neurotoxicity include the competition effect with calcium ion, cell apoptosisand the lipid peroxidation damage of neurons. Althought the lead neurotoxicity mechanismsare not yet clarified, but there are lots of literature point out that the lead induced oxidative stress may plays an important part in it. These articles clearly stated that the mechanisms of Pb-induced oxidative stress include the effects of Pb on cell membranes, DNA, and the cellular antioxidant defense system. It is noteworthy that some studies point out that the developing brain is more vulnerable to Pb neurotoxicity because it can spontaneously release several neurotransmitters even in low Pb levels. Therefore, studying the mechanism of lead neurotoxicity is a subject with profound significance, since clarifyingthe mechanism of lead induced oxidative stress and adjusting related pathophysiological process of huge importance, which can further elucidate the pathogenesis of related diseases and help to find some new therapeutic targets to provide basic experiment basis.Mitochondria plays an important role in maintaining ionic balance and energy metabolism and the stress of external factors of cells. What’s more, mitochondria is the main site to produce reactive oxygen free radicals, an excess of active oxygen free radicals (ROS) and reactive nitrogen production is the necessary condition to oxidative stress[12].It’s reported that lead can induce the generation of reactive oxygen free radicals and finally cause oxidative damage to the body[13], by reducing the defense capability of plasma membrane to the active oxygen free radicals and weaken the antioxidant defense capability of cells,such as consumption the intracellular glutathione, inhibit the activity of superoxide dismutase (SOD) and change the integrity of plasma membrane[14]. Mitochondrial calcium uniporter (MCU) is located in the mitochondrial membrane, is an important way to transfer calcium ions from outside into the mitochondrial matrix, it regulating mitochondrial calcium dynamic balance, and closely related to cell energy metabolism and survival conditional With literature points out that active oxygen free radical adjust the calcium signal network, as a prospective to regulates the active oxygen free radicals.As we all know, as a second messenger, Calcium plays an irreplaceable role in the physiology and pathology function of cells, and there are reports that the intracellular calcium ion release induced by fluoridecan lead to the activation of active oxygen free radicals, thus play a role of its cytotoxicity. Mitochondrial calcium uniporter (MCU) is a selective calcium ion channels, plays an key rolein the intracellular calcium homeostasis. A survey of the literature available shows that lead is the competitive antagonist of calcium ion,and lead can suppress alpha7acetylcholine receptor activity by blocking the calcium concentration,and play a role of lead neurotoxicity. Therefore, although the study of mechanisms of lead neurotoxicity have made great achievements in many aspects, But whether mitochondrial calcium uniporter play a a role in the neuronal oxidative stress induced by lead is unclear,and has not been reported that lead can interfere the mitochondria calcium balance through mitochondrial calcium uniporter,,so as to break the intracellular calcium homeostasis and play the role of lead neurotoxicity.ObjectivesIn the current study, the mechanism underlying oxidative stress induced by Pb2+was assessed by examining MCU expression and function, including mitochondrial Ca2+influx, in human neuroblastoma SH-SY5Y cells and primitive rat hippocampal neurons andrat pups exposed to low levels of Pb2+, using the MCU activator spermine and blocker Ru360, and by MCU overexpression and knockdown.Methods1. Human neuroblastoma SH-SY5Y cellswere maintained in RPMI1640containing10%FBS. Cultures were maintained at37℃in a humidified atmosphere of5%CO2.seting in different concentrations groups,the SH-SY5Y cells were incubated with lead acetate for48h which observing the cell morphology change by microscope and determining cell vitality by MTT experiment.2. ROS fluorescent probe reagents and Reduced Glutathione Detection Kit are used to detect redox status indicator-intracellular ROS and GSH after SH-SY5Y cells incubating with lead acetate for48h. 3. After incubating with the ROS fluorescent probe, the SH-SY5Y cells were observing under laser confocal microscope by differentLead groups4. Western blot is used to detect content of mitochonddrialcalcium uniporter (MCU) and neuronal nitric oxide synthase (nNOS) protein.5. After using the MCU activator spermine and blocker Ru360and by MCU overexpression and knockdown,ROS and GSH were detected by ROS fluorescent probe reagents and Reduced Glutathione Detection Kit.6. Incubating with mitochondrial calcium sensitivity indicator Frod-2cell,we use laser confocal microscope real-time record mitochondrial calcium ion in different lead groups.7. primary hippocampal neuronswere maintained in Neurobasal containing2%B27. Cultures were maintained at37℃in a humidified atmosphere of5%CO2.seting in different concentrations groups,the primary hippocampal neurons were incubated with lead acetate for24h which observing the cell morphology change by microscope and determining cell vitality by MTT experiment.8. ROS fluorescent probe reagents and Reduced Glutathione Detection Kit are used to detect redox status indicator-intracellular ROS and GSH after primary hippocampal neurons incubating with lead acetate for24h.9. Western blot is used to detect content of mitochonddrialcalcium uniporter (MCU) protein after primary hippocampal neurons incubating with lead acetate for24h.10. After using the MCU activator spermine and blocker Ru360,ROS and GSH were detected by ROS fluorescent probe reagents and Reduced Glutathione Detection Kit.11. Animals were maintained under12:12h light/dark conditions at25℃and40-60%humidity. Dams were provided with food and water ad libitum prior to breeding. Litters were culled to four pups per group on the day after birth (day1) and divided into control and Pb2+exposure groups. In the latter, pups were nursed by dams receiving deionized drinking water with2mmol/1Pb acetate. Blood Pb levels in pups were evaluated at the time of weaning on day21, and were euthanized immediately afterward and the hippocampus was dissected for measurement of protein expression,including MCU and nNOS protein.12. Statistical analysis was performed using SPSS13.0software. Results were expressed as mean±SEM. Data were analyzed by one-way analysis of variance. Differences Group compared with one-way ANOVA and inter-group comparision was significant using LSD when the homogeneity of variances was meet, while, Group compared with Welch and inter-group comparision was significant using Dunnett T3when the homogeneity of variances was not meet. P<0.05means the significance.Results1. Cell viability was assessed in SH-S Y5Y neuroblastoma cells after treatment with a range of Pb2+concentrations.No effects were observed24h after treatment (data not shown); however, at10and50μM Pb2+, cells with abnormal morphology were observed at48h after treatment. This was accompanied by a dose-dependent decrease in viability of25.5%and47.0%, respectively, at48h compared to control cells (P<0.01).2. a dose-dependent increase in ROS and decrease in GSH levels were noted in cells treated with10μM Pb2+for48h, which was confirmed by an increase in intensity of the C400signal representing ROS production (P<0.01).3. ROS fluorescence intensity increased with a range of Pb2+concentrations.4. MCU protein levels decreased as a function of Pb2+concentration by27.2%and70.0%at10and50μM, respectively. nNOS protein increased with a range of Pb2+concentrations compared with control group(P<0.01). 5. When cells were treated with spermine, a MCU agonist, Pb2+-induced ROS generation was abrogated, and GSH levels were restored to control levels; application of Ru360, an MCU inhibitor, produced the opposite effect, enhancing the oxidative stress response. The Pb2+-induced upregulation of ROS levels was also abolished, as was the decrease in GSH (P<0.05).Furthermore, siRNA-1knockdown of MCU enhanced the Pb2+-induced increase in ROS levels and further suppressed GSH levels compared to Pb2+-treated cells transfected with the scrambled control siRNA (P<0.01)6. In the presence of10μM Pb2+,the mitochondrial Ca2+influx from the addition of extracellular Ca2+decreased by50%; however, the Ca2+influx was partially restored when cells were pretreated with spermine, while pretreatment with Ru360resulted in a significant reduction in mitochondrial Ca2+uptake by the cells. The overexpression of MCU likewise rescued the Pb2+-induced suppression of Ca2+influx compared to cells transfected with a control vector (P<0.01). In contrast, knocking down MCU expression led to a greater decrease in Ca2+uptake in Pb2+-treated cells (P<0.01), while cells transfected with the scrambled siRNA construct had comparable mitochondrial Ca2+levels.7. The purity of primary hippocampal neurons were detected by MAP-2antibody, which up to90%. cultured hippocampal neurons can do follow-up experiments. primary hippocampal neuronswith abnormal morphology were observed at24h after treatment. Cell viability was dramaticlly decreased witha range of Pb2+concentrations(P<0.01).8. a dose-dependent increase in ROS and decrease in GSH levels were noted in cells treated with10μM Pb2+for24h in primary hippocampal neurons which incubated with a range of Pb2+concentrations.9. MCU protein levels decreased as a function of Pb2+concentration at10μM. 10. When primary hippocampal neuronswere treated with spermine, a MCU agonist, Pb2+-induced ROS generation was abrogated, and GSH levels were restored to control levels; application of Ru360, an MCU inhibitor, produced the opposite effect, enhancing the oxidative stress response.11. In rat pups exposed postnatally to Pb2+, nNOS protein expression was increased in the hippocampus compared to untreated rats (P<0.05).Similarly, MCU protein expression in the hippocampus of Pb2+-exposed postnatal rats decreased by48.9%compared to control rats (P<0.01).ConclusionsTaken together, these results indicate that the oxidative stress induced by Pb2+are mediated via Ca2+uptake by the MCU.In this study, Pb2+decreased mitochondrial Ca2+concentration by inhibiting the activity of the MCU, but the precise mechanism of action is still an open question to be addressed by future studies.