| ObjectivesResearches have confirmed that gene mutations in the adaptor protein 4 complex are associated with autosomal recessive cerebral palsy syndrome. Genetic mutation in the μ subunit (AP4M1) can cause developmental deficiency of neural system, resulted in congenital spastic tetraplegia (CST), which is similar to the perinatal leukomalcia caused by glutamate excitotoxicity. The possible mechanism is distributional abnormality of AMPA receptors mediated by AP4M1. Whether AP4M1 is changed in hypoxic-ischemic encephalopathy (HIE) remains uncertain. The purposes of this study are to investigate whether and how AP4M1 expression is changed in hypoxic injured neurons, and the mechanism of AMPA receptor trafficking mediated by adaptor protein 4 complex. This research may elucidate the mechanism of HIE, provide a new clue for HIE treatment strategy.Materials and MethodsPrimary cultured hippocampal neurons were prepared for this experiment. They were subjected to oxygen-glucose deprivation (OGD), mimicking brain ischemia. Neuron-specific enolase (NSE) was labeled immunofluorescently to confirm that the purity of neuron was higher than 90%. Neurons were divided into two groups:sham group and OGD group. The testing time points are 0,12 and 24 hours after the OGD procedure for real-time PCR, wertern blot and immunofluorescent colorimetric assay. Double immunofluorescent labeling, immunofluorescent colorimetric assay and co-immunoprecipitation assay were performed immediately after the OGD.Real-time PCR and were performed to measure the mRNA expression of AP4M1. Western blotting was exploited to measure the protein expression of AP4M1 and AMPARs. AP4M1 and AMPARs was labeled immunofluorescently with MAP2 or Tau-1 to observe the distribution. Immunofluorescent colorimetric analysis was used to test the surface expression of AP4M1, G1uR1 and G1uR2. AP4M1 is labeled with G1uR1, GluR2 and GluR4 to see if their distributions were consistent with each other. The interactions between AP4M1 and G1uR1 or G1uR2 were detected with co-immunoprecipitation assay.Results1. Primary culture and identification of hippocampal neurons:Primary hippocampal neurons were well developed and became mature on the on the 13th day in vitro. By immunofluorescent labeling NSE, we found out approximately 95% of the cells were neurons.24 hours after the OGD procedure, More than 90% of was dead, some neurons showed apoptotic morphological changes, the others showed necrotic changes.2. Real-time PCR analysis:The AP4M1 mRNA expression of oxygen-glucose deprived hippocampal neurons is much lower than normal neurons. The AP4M1 mRNA expression of oxygen- glucose deprived neurons is 77.9 percent (p=0.009, n=8) of normal neurons immediately after the OGD, then this figure decreased to 34.8 percent (p<0.001, n=8)after 12 hours and 34.2 percent (p<0.001, n=8) after 24 hours. The mRNA expression of GluRl-4 remained consistent across the various time points in the sham group. At the time point of 0 hours, none of these four genes showed expression difference between the sham group and the OGD group. At the time point of 12 hours after the OGD, G1uR4 is mildly down-regulated while the other genes are not. By the time of 24 hours after the OGD, G1uR1 and G1uR2 showed notable down-regulation in oxygen-glucose deprived neurons, the mRNA expressions are 20.2 percent (p<0.001, n=8) and 20.8 percent (p<0.001, n=8) of normal neurons. But GluR4 still showed mild down-regulation at this time point, the percentage is 62.0 percent (p=0.009, n=8). G1uR3 remained to be similar between the two groups.3. Western blotting analysis:The AP4M1 protein levels were similar immediately after the procedure between the OGD group and the sham group(p=0.835, n=8). However, down-regulation was observed 12 hours after the reperfusion, and became more notable at 24 hours. The protein expressions of OGD group are 40.87 percent (p<0.001, n=8) at 12 hours and 19.79 percent (p<0.001, n=8) at 24 hours. There is no difference between the OGD group and the sham group in the quantitavely expression of AMPARs.4. Immunofluorescent labeling:AP4M1 is distributed in the dendrites of normal neurons, but it is redistributed to the axons after the OGD procedure. The distribution of G1uR2 is mainly focused in the dendrites of normal neurons. But it is less distributed in this area after the OGD procedure. G1uR1 and G1uR4 are mainly expressed in the cell bodies both in the sham group and the OGD group, showed no difference after the procedure.5. Immunofluorescent colorimetric assay:The total expression of AP4M1 in oxygen-glucose deprived neurons did not down-regulated until 24 hours after the OGD, it is 79.8 percent of normal neurons (p<0.001, n=6). The surface expression and the ratio of surface/total expression in the OGD group are similar to the sham group immediately after the OGD, and then they became lower at the time point of 12 hours, which is 62.8 percent (p<0.001, n=6) and 58.7 percent (p<0.001, n=6) of normal neurons, By the time of 24 hours after the OGD, these percentages decreased to 37.3 percent (p<0.001, n=6) and 46.6 percent (p<0.001, n=6). Neither of total expression, surface expression and the ratio of G1uR1 showed difference between the sham and OGD group. The total expression of GluR2 in oxygen-glucose deprived neurons showed no difference from normal neurons at all these three time points. The surface expression and the ratio of surface/total expression in the OGD group are similar to the sham group immediately after the OGD procedure. But both of them are down-regulated at 12 hours and 24 hours later. The surface expression and the ratio of surface/total expression in the OGD group are similar to the sham group immediately after the OGD, and then they became lower at the time point of 12 hours, which is 61.9 percent (p<0.001, n=6) and 61.2 percent (p<0.001, n=6) of normal neurons, By the time of 24 hours after the OGD, these percentages decreased to 28.4 percent (p <0.001, n=6) and 29.2 percent (p<0.001, n=6).6. Double immunofluorescent labeling: AP4M1 can be merged with G1uR2 in both sham group and OGD group. However, the mergence of AP4M1 with G1uR1 or G1uR4 is much lower. 7. Co-immunoprecipitation assay:AP4M1 can not be co-immunoprecipited with either G1uR1 or G1uR2.ConclusionsAP4M1 is not only quantitatively down-regulated in oxygen-glucose deprived hippocampal neurons, both at mRNA and protein levels, but also showed distributional alterations. This subunit is redistributed from dendrites to axons and is less distributed on cell surface.The mRNA expression of G1uR2 is down-regulated in oxygen-glucose deprived hippocampal neurons. The distributions of G1uR2 are also changed; it is less distributed both in the dendrites and on the surface of neurons.AP4M1 may regulate G1uR2 trafficking indirectly in oxygen-glucose deprived hippocampal neurons. |
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