The Effects Of SIRT3 In Neural Stem Cells On Dysfunction Induced By Microglia Activation

BackgroundThe discovery of neural stem cell (NSC), especially adult neural stem cells in the mammalian central nervous system (CNS) brings new hope for nerve regeneration and repair. The process of neurogenesis continues throughout life, with thousands of new neurons generate every day in the mammalian brain. NSC proliferation, differentiation, apoptosis, and other life activities are affected by the around complex microenvironment. Impairment of hippocampal neurogenesis has been suggested to be involved in neurodegenerative conditions, including the cognitive decline associated with aging, Alzheimer’s disease. These neurodegenerative conditions are all characterized by proinflammatory changes and increased numbers of activated microglia. Activated microglia produce a variety of proinflammatory factors, including tumor necrosis factor-α and nitric oxide. Reactive oxygen species (ROS) induce inflammatory reaction and oxidative stress. They destroy the normal microenvironment in which neural stem cells can grow, inhibiting nerve regeneration and mitochondrial function. High ROS level also improves intracellular Aβ concentration, causing AD neurons damaged. Neurons are particularly sensitive to mitochondrial dysfunction, elevated ROS promotes cell death, protein deposition, thus accelerating the progress of neurodegenerative diseases, such as AD. Mitochondria plays a crucial role in neurogenesis. Mitochondrial protection and overexpression of antiapoptotic proteins can promote neural differentiation in vitro, and enhance neural precursor cells viability in the animal’s brain damaged area.Sirtuin3 (SIRT3) is a member of the Sirtuin gene family, mainly located in mitochondria, having deacetylation enzyme activity. SIRT3 has many biological functions, regulating oxidative stress, energy metabolism, ATP production, ROS removal, mitochondrial function, etc., and plays an important role in the development of neurodegenerative, diabetes, aging, cancer and other diseases. The central auditory cortex SIRT3 and SOD2 expression in aging rat model reduced, SIRT3 expression level in AD rat cortical neurons also declined obviously. SIRT3 deletion of neurons increases oxidative stress, which can promote H2O2 induced cell death, and also exacerbates dopaminergic neuron degeneration in PD model mice nigra striatum. SIRT3 plays a role in development of nerve diseases, but the researches about SIRT3 in neural stem cells have not seen to be reported. Early studies in our research team confirmed that microglia activation induced NSC apoptosis, and inhibited NSCs to differentiate into neurons, which associated with energy metabolism. We speculate that, SIRT3 has a role of neural protection in microglia activation induced NSC dysfunction by reducing oxidative stress and inflammation damage, improve mitochondrial function, and so on, and promotes neurogenesis. Research results can give a new view for revealing mechanisms of nerve inflammation mediated NSC dysfuction, and provide new targets for clinical treatment and drug intervention for neurodegenerative diseases such as AD.ObjectiveTo study the effects of SIRT3 in neural stem cells on microglia activation induced their dysfunction. The effects of NSC gene SIRT3 overexpression and interference on cell apoptosis, proliferation and differentiation, mitochondrial function, ROS generation, and its related molecular mechanisms were explored.MethodsThis study used tanswell system to co-culture mice neural stem cell line C17.2 (NSC) and microglia BV-2 (MG), C17.2 cells culture in the down department adherently, and BV-2 cells culture in the up department adherently.10 μM Aβ was added into co-culture system, which resulted in BV-2 cells activation. The co-cultured cell model of activated microglia induced NSC dysfunction was established.Establishment of SIRT3 gene overexpression and interference model:before C17.2 cells co-culture with BV-2 cells, C17.2 cells were inoculated on the bottom of 6 holes cell culture plate. SIRT3 overexpression adenovirus was added into SIRT3 gene overexpression co-culture model, SIRT3-siRNA was added into SIRT3 gene interference co-culture model, respectively, when the confluence value was about 40 %～50%. The duration of serum-free culture transfection was 6 h. At the same time, BV-2 cells was inoculated on the bottom of up chamber of transwell devices, which were located in another 6 wells cell culture plate. The BV-2 cell confluence reached about 50%～60% when adenoviruses or siRNA cell transfection ended. After C17.2 transfected, BV-2 transwell nested devices were migrated into the 6 wells cell culture plate of C 17.2 cells.10 μM Aβ was added into the co-culture system.NSC marker nestin and SIRT3 protein expression of C17.2 were identified by cell immunofluorescence. The concentration of inflammatory factors (TNF-a, IL-1β, IL-6) and NO in cell culture medium of co-culture system were detected by ELISA and spectrophotometry. DHE fluorescent probe was used to detect the level of superoxide anion in NSC cell lysis liquid. Cell apoptosis and cell cycle distribution were examined by flow cytometry. JC-1 was used to detect the change of mitochondrial membrane potential, the Calcein-AM incubation with CoCl2 were used to test the open of mitochondrial permeability transition pore. The related genes mRNA expression levels were examined by qRT-PCR, PCR products were detected by agarose gel electrophoresis. ELISA was also used to detect the activity of NSC intracellular caspase-3 and caspase-9. The related genes protein expression levels were detected by Western blotting, semi-quantitative analysis was carried out with gray scan.Results1. Effects of microglia activation on NSC function and SIRT3 expressionUp to now, there is no literature reported regarding about SIRT3 function in NSC and neurogenesis. The experiment was divided into C17.2 cultured group separately (NSC), microglia BV-2 and C17.2 co-cultured group (MG+NSC group), C17.2 cultured group treated by Aβ (Aβ+NSC group), BV-2 and C17.2 co-cultured model group treated by Aβ (Aβ+MG+NSC group).Results showed that the NSC-C17.2 cells could express SIRT3 protein, and mainly located in the extranuclear. The activated microglia down-regulated NSC SIRT3, MnSOD expression level. NSC SIRT3, Mn-SOD protein expression levels in Aβ+MG+NSC group obviously lower than that in Aβ+NSC group, SIRT3, Mn-SOD mRNA expression levels also significantly decreased (P=0.000, P=0.000).Aβ activated microglia could secrete inflammatory factors and NO, up-regulated NSC intracellular ROS levels, induced oxidative stress injury. Compared with that in Aβ+NSC group, the concentration of inflammatory factors (TNF-a, IL-1β, IL-6) and NO in cell-culture medium of Aβ+MG+NSC group increased significantly (P=0.000, P=0.000, P=0.012, P=0.000), and superoxide anion, hydrogen peroxide level in NSC cell lysis liquid increased significantly (P=0.000, P=0.000). Compared with that in Aβ+NSC group, the NSC apoptosis rate in Aβ+MG+NSC group increased obviously (P=0.000), the propotion of NSC G0/G1 cell increased significantly (P=0.000), the propotion of S phase and G2/M phase cells dramatically reduced (P=0.000, P=0.000). The NSC JC-1 red/green fluorescence intensity ratio in Aβ+MG+NSC group significantly became smaller (P=0.003), and calcein-AM green fluorescence intensity obviously reduced by comparison to that in Aβ+NSC group (P=0.034).2. Effects of NSC SIRT3 on the NSC damage induced by activated microgliaThe influences of SIRT3 on NSC cell survival and the related molecular mechanisms of mitochondrial apoptosis pathway were investigated from positive and negative aspects. Tests included SIRT3 overexpressed part and interference part. Overexpressed group were divided into blank control group, empty vector adenovirus transfection group, SIRT3 overexpressed adenovirus transfection group. The experiments in interference group were divided into blank control group, scrambled siRNA negative control group, siSIRT3 interference group.Results in overexpressed group showed that SIRT3, Mn-SOD mRNA expression levels of NSC in SIRT3 overexpressed group were significantly about seven times higher than that in control group (P=0.000, P=0.000), SIRT3, Mn-SOD protein expression levels also significantly raised. Compared with control group, the NSC DHE red fluorescence intensity in SIRT3 overexpressed group weakened, super oxygen anion obviously reduced (P=0.009), and the concentration of hydrogen peroxide dramatically decreased (P=0.008). Compared with control group, the NSC apoptosis rate in SIRT3 overexpressed group significantly decreased (P=0.003), the proportion of G0/G1 phase cells significantly decreased (P=0.005), S, G2/M phase cells proportion increased significantly (P=0.004, P=0.013). NSC JC-1 red/green fluorescence intensity ratio in SIRT3 overexpressed group was obviously greater than that in control group (P=0.004). Compared with the control group, NSC mitochondria CypD protein expression significantly reduced, calcein-AM green fluorescence intensity in SIRT3 overexpressed group dramatically enhanced (P=0.006). Compared with control group, NSC Cyt C released into the cytoplasm in SIRT3 overexpressed group decreased significantly, mitochondria pro-apoptosis factors cleaved caspase-3, Bax expression dramatically lowered, anti-apoptosis factor Bcl-2 protein expression obviously increased, caspase-3, caspase-9 activity also significantly declined (P=0.012, P=0.022).Results in interference group indicated that SIRT3, Mn-SOD mRNA expression levels of NSC in siSIRT3 interference group were obviously lower than that in control group (P=0.000, P=0.000), SIRT3, Mn-SOD protein expression also significantly dropped. Compared with control group, the NSC DHE red fluorescence intensity in siSIRT3 interference group enhanced, the concentration of super oxygen anion and hydrogen peroxide dramatically increased (P=0.003, P=0.006). Compared with control group, the NSC apoptosis rate in siSIRT3 interference group significantly increased (P=0.001), the proportion of G0/G1 phase cells significantly increased (P=0.012), the proportion of S phase cells obviously decreased (P=0.003). NSC JC-1 red/green fluorescence intensity ratio in siSIRT3 interference group was significantly less than that in control group (P=0.001). Compared with the control group, NSC mitochondria CypD protein expression significantly up-regulated, calcein-AM green fluorescence intensity in siSIRT3 interference group dramatically reduced (P=0.013). Compared with control group, NSC Cyt C released into the cytoplasm in siSIRT3 interference group raised significantly, mitochondria pro-apoptosis factors cleaved caspase-3, Bax expression dramatically up-regulated, anti-apoptosis factor Bcl-2 protein expression obviously down-regulated, caspase-3, caspase-9 activity also significantly increased (P=0.001, P=0.011).The results of pros and cons demonstrated that NSC SIRT3 expression up-regulated the level of MnSOD expression, reduced ROS level significantly, inhibited the NSC apoptosis, promoted cell division. SIRT3 gene expression rose the mitochondrial membrane potential, decreased the open of mitochondrial permeability transition pore and CypD protein expression. The molecular mechanisms of SIRT3 expression improved NSC dysfunction induced by microglia activation included the reduction of mitochondrial Cyt C released into the cytoplasm, decrease of Bax expression and caspase-3, caspase-9 activity, the increase of Mn-SOD, Bcl-2 expression levels.3. Effects of SIRT3 expression on NSC differentiation and its relative molecular mechanismMicroglia activation impairs brain microenvironment through oxidative stress and inflammation injury, producing negative effects on adult neurogenesis. SIRT3 can alleviate microglia activation induced NSC damage, but whether the impact of SIRT3 on NSC differentiation potential remains to be studied. Adenovirus and siRNA were used to transfect NSC in co-culture model, the role of SIRT3 in all-trans retinoic acid induced C17.2-NSC differentiation and its related molecular mechanisms were investigated. Test was divided into the NSC group, control model group, SIRT3 overexpressed group, siSIRT3 interference group, all-trans retinoic acid (1.0 μM) was added into each group to induced NSC differentiation for 48 h.Results demonstrated that, compared with control model group, NSC NeuN, MAP-2 mRNA expression levels in SIRT3 overexpressed group significantly rised (P=0.002, P=0.001), while the nestin, GFAP mRNA expression levels decreased obviously (P=0.001,P=0.000). However, in siSIRT3 interference group, NSC NeuN, MAP-2 mRNA expression levels significantly reduced (P=0.001, P=0.000), nestin and GFAP mRNA expression levels increased (P=0.003, P=0.005). The proteins expression followed the same varying trend as mRNA expression in every group. Compared with the control model group, NSC β-catenin, p-GSK-3β protein expression in SIRT3 overexpressed group increased significantly (P=0.001, P=0.001), p-P-catenin, GSK-3β, Axin2 protein expression obviously decreased (P=0.001, P=0.026, P=0.000), However, in siSIRT3 interference group, NSC p-catenin, p-GSK-3β protein expression significantly reduced (P=0.006, P=0.000), p-β-catenin, GSK-3β, Axin2 protein expression dramatically up-regulated (P=0.020, P=0.000, P=0.000).The above results showed that SIRT3 overexpression induced differentiation of NSC into neurons in co-cultured system. After SIRT3 interfered, the number of NSC differentiated to neurons became smaller, on the contrary, more NSC differentiated into glial cells. SIRT3 promoted NSC to differentiate to neurons through Wnt/p-catenin signaling pathway activation, with β-catenin, p-GSK-3β expression levels up-regulation, and p-β-catenin, GSK-3β, Axin2 expression levels down-regulation.Conclusion(1) Activated microglia caused oxidative stress and inflammatory injury to co-cultured NSC, and reduced the SIRT3 gene expression of NSC;(2) NSC SIRT3 could alleviate the activated microglia induced damages on NSC, inhibit cell apoptosis, promote cell cycle progression, and improve mitochondrial function;(3) The mechanisms of NSC SIRT3 attenuated activated microglia induced NSC dysfunction included decreasing the number of mitochondrial permeability transition pore opening and CypD protein expression, inhibition of mitochondrial Cyt C release to cytoplasm, declining the Bax/Bcl-2 ratio, reducing caspase-3/caspase-9 activity;(4) SIRT3 promoted the differentiation of NSC into neurons, Wnt/β-catenin signal transduction pathway activation mediated the process;(5) Results could provide a new insights on microcircumstance improvement for NSC survival, and give a novel intervention targets for neurodegenerative diseases such as AD.