The Mechanistic Study Of C-Abl Involved In The Apoptosis Of Embryonic Neural Progenitor Cells Induced By High Glucose

Maternal diabetes significantly increases the risk of congenital malformations in humans and in animal models. Neural tube defects (NTDs) are among the most common of the malformations associated with diabetic embryopathy and are the major sources of neonatal mortality, leading to place an enormous emotional and economic burden on the population. Understanding the underlying molecular basis for diabetic neural tube defects is important for devising strategies aimed at reducing their occurrence.Previous studies from several laboratories have shown that neuroepithelial apoptosis is responsible for maternal diabetes-induced neural tube defects and high glucose can result in neuroepithelial apoptosis. Recently, there is considerable evidence that hyperglycemia-induced oxidative stress is one of main causes of maternal diabetes-induced neural tube defects. Hyperglycemia effectively enhanced oxygen free radical formation. Supplemental antioxidants can prevent neural tube defects caused by hyperglycemia in rodent embryos. Therefore, hyperglycemia -induced oxidative stress plays a key role in diabetes-induced neural tube defects. Although a clear link has been established between oxidative stress and neuroepithelial apoptosis, a question that may be raised by these observations is how oxidative stress induced by hyperglycemia contributes to neuroepithelial apoptosis and which cellular proteins are involved in oxidative stress and neuroepithelial apoptosis. We screened many genes (c-Abl, Atm, Atr and PKC-δetc.) which are associated with oxidative stress and apoptosis and found that the expression of c-Abl was increased in neuroepithelium exposed to hyperglycemia.The present study is aimed to investigate the mechanisms that c-Abl regulates the apoptosis of neural progenitor cells (NPCs) induced by high glucose, which may be involved in oxidative stress and the NPCs apoptosis. For the aim, we established the cellular model of high glucose-induced NPCs apoptosis. Through immunohistochemistry, Co-IP, siRNA and Western blot techniques, we investigated the relationship between c-Abl and NPCs apoptosis.一,The in vitro cellular model of the apoptosis of neural progenitor cells induced by high glucoseNeuroepithelial cells from neural tube are the most primitive neural progenitor cells. These neuroepithelial stem cells are a population of undifferentiated cells that are capable of extended self-renewal and multipotential ability to generate multilineage cell types. At mice E12.5d, Neural progenitor cells suspension was successfully isolated by stereoscopic microscope and mechanical blowing, and neural progenitor cells gained in serum-free medium. BrdU labeling technique and Nestin immunofluorescence staining technique were used to detect their self-duplication and self-renewal. Microtubule associated protein 2 ( MAP2) and glial fibrillary acidic protein (GFAP) immunofluorescence staining technique were used to identify their differentiation. The present results suggest that neural progenitor cells from brain cortex of embryonic mice are easily extracted and naturally differentiate into neurocyte and neuroglial cells and they are the preferred cell model of study on high glucose and apoptosis.Primary NPCs were used as a model system to investigate the mechanisms by which HG induces apoptosis. First, cell viability was determined by MTT assay. Cells were treated with glucose (D-glucose) and mannitol at 5,25,45,60 and 100 mM in cultures for 12 h,24 h and 48 h. Mannitol was used as a control to exclude a possible effect of osmolarity on apoptosis. The cell viability in 25mM glucose is best among five groups. There was a significant decrease in cell viability in the cultures exposed to 45 mM glucose compared to 25 mM glucose (P<0.01). This was not observed in NPCs cultured with mannitol at the same osmolality. On the basis of this, 45 mM HG was used in the following experimental groups. The similar glucose concentration was commonly used by others. To assess the degree of NPCs injury induced by high glucose (HG), the percentage of apoptotic cells was determined using fluorescein isothiocyanate (FITC)-conjugated annexin V. In the control NPCs, there were 8% apoptotic cells at 24 h, which was expected in this system; however, the frequency of apoptotic cells increased to 40% at 24 h, and 57% at 48 h treatment with 45 mM HG. These indicated that high glucose (45 mM) could induce the apoptosis of neural progenitor cells.二,c-Abl is involved in the apoptosis of neural progenitor cell induced by high glucoseTo ascertain a possible role for c-Abl in HG induced NPCs apoptosis, we evaluated the c-Abl mRNA and protein levels of NPCs exposed to HG. In the presence of HG, there was a significant increase in the levels of c-Abl mRNA after 12,24 and 48 h incubation. Quantification of results from three experiments confirmed that the upregulation is statsitically significant at 24 h (P<0.01) and 48 h (P<0.01). Furthermore, the protein levels of c-Abl at 24 h and 48 h were markedly greater than that of the control. Because the nuclear pool of c-Abl has been implicated in proapoptotic functions, the subcellular distribution of c-Abl protein in NPCs exposed to HG was analyzed. By immunofluorescence labeling, c-Abl was present in both the cytoplasm and the nucleus of the control NPCs. The level of c-Abl in the nucleus increased greatly in NPCs after 24 h of HG treatment. Subcellular fractionation analysis confirmed these results showing that the c-Abl protein level was significantly increased in the nuclear fraction of the NPCs exposed to HG; however, the cytoplasmic c-Abl levels did not show a significant change. To determine whether c-Abl alters high glucose-induced apoptosis, c-Abl siRNA was used to specially inhibit the c-Abl expression. Non-silencing siRNA was also transfected as control. The protein levels of c-Abl at 24 h were efficiently reduced in NPCs transfected with c-Abl siRNA. The relative increase of viable cells in c-Abl siRNA group was significantly higher compared to the control groups exposed to HG (P<0.05).These suggested that c-Abl could regulate the apoptosis of neural progenitor cell induced by high glucose.三,The molecular mechanism of c-Abl involved in NPCs apoptosis induced by high glucosePrevious studies have demonstrated that oxidative stress mediates HG-induced NPCs apoptosis. In order to investigate the relationship between reavtive oxygen species (ROS) and c-Abl, we first tested whether HG led to an increase of ROS in NPCs. The levels of ROS formation were determined by fluorimeter from 0 h to 24 h after HG treatment. It was found that within the critical initial period, 6 h, after HG treatment, DCF levels peaked; thereafter, the levels of DCF declined. We then tested whether c-Abl was regulated by ROS levels.α-lipoic acid (LA), a potent antioxidant, was used to block ROS production in NPCs. The induction of c-Abl by HG was attenuated by LA (P<0.05). Pretreatment the NPCs with STI571, an inhibitor of c-Abl that has exceptionally high affinity and selectivity, did not alter the intracellular level of ROS in the presence of HG These suggest that ROS can up-regulate the expression of c-Abl protein and c-Abl was a downstream of ROS.Many studies have shown that p53-mediated apoptosis is responsible for neural abnormalities during embryogenesis. Inhibition of p53 accumulation in neuroepithelium could prevent cell death. In the light of this, we tested whether c-Abl would also play a role in the accumulation of p53 in NPCs exposed to HG. First, the expression of p53 was analysed. While the levels of p53 mRNA did not show a significant change, there was a significant increase in the protein levels of p53 in the nucleus (P<0.01). The c-Abl-p53 interaction in NPCs exposed to HG was then evaluated. This association was observed in both directions when either c-Abl or p53 proteins were immunoprecipitated. The c-Abl-p53 complex increased in NPCs after 24 h of treatment with HG. To determine the involvement of p53 in c-Abl mediated NPCs apoptosis, we tested whether inhibition of ROS and c-Abl affected the protein levels of p53 in the presence of HG. Nuclear fractions were prepared, and the accumulation of p53 in the presence of STI571 and LA was markedly lower than the control group.To determine whether ROS and c-Abl have influence on NPCs apoptosis, we evaluated the effect of ROS scavengers (LA) and c-Abl inhibition by STI571 on NPCs apoptosis induced by HG. The results showed that treatment with LA and STI571 inhibited NPCs apoptosis markedly when compared to NPCs exposed to high glucose (P<0.01). This showed that both ROS and c-Abl can mediate NPCs apoptosis exposed to HG.Conclusion:We report here that high glucose could induce the NPCs apoptosis, 45 mM glucose was optimal concentration for the cellular modle of NPCs apoptosis induced by HG. The expression of c-Abl is increased in NPCs apoptosis exposed to high glucose in vitro, which is an important cellular protein involved in NPCs apoptosis induced by high glucose. High glucose up-regulates the level of ROS, leading to increasing the expression of c-Abl, especially in the nucleus, which subsequently mediates nuclear p53 protein levels, likely through physical interaction. Our results support that ROS→c-Abl signaling pathway plays an important role in NPCs apoptosis induced by high glucose. These researches, not only illuminated the molecular biological mechanism of NPCs apoptosis induced by high glucose, but also provided a new experimental evidence for the precaution and therapy for neural tube defects.