Effect of GSK-3β-mediated DRP1 on inhibition of primary hippocampal neuronal growth induced by aluminum
取出生24 h以内的乳鼠海马提取神经元进行原代培养。培养至第6天，采用免疫荧光鉴定神经元纯度；培养至第10天，选择生长状态良好的神经元进行Al染毒以及GSK-3β抑制剂SB216763（SB）干预，实验分组为空白对照组、二甲基亚砜（DMSO）组、Al组（20 μmol·L−1）、SB组（1 μmol·L−1）、SB（1 μmol·L−1）+Al（20 μmol·L−1）组。在干预原代海马神经元48 h后，采用CCK-8法检测神经元细胞活力，透射电子显微镜观察原代海马神经元线粒体形态，激光共聚焦成像对原代海马神经元进行扫描并分析其突起总长度，Sholl分析其复杂程度。采用蛋白印迹法检测原代海马神经元磷酸化GSK-3β、GSK-3β、DRP1蛋白表达水平。
P>0.05），Al组明显降低（ P=0.006）；SB+Al组细胞存活率与Al组相比没有明显差异（ P>0.05）。与空白对照组相比，DMSO组和SB组神经元平均突起总长无明显差异（ P>0.05），Al组明显减少（ P<0.001）；SB+Al组神经元平均突起总长度明显高于Al组（ P=0.001）。在距胞体130 μm以内，各组神经元的交叉点个数均随突起距离的增加而增加。在距胞体130 μm以上，各组神经元的交叉点个数均随突起距离的增加而逐渐减少。在距胞体130、310 μm处，与空白对照组相比，DMSO组与SB组神经元交叉点个数无明显差异（ P>0.05），Al组明显减少（ P<0.05）；SB+Al组神经元交叉点个数与Al组无明显差异（ P>0.05）。空白对照组线粒体结构完整，嵴清晰可见；DMSO组与SB组线粒体结构未见明显变化；Al组线粒体有明显破裂甚至成空泡化，嵴结构消失；SB+Al组与Al组相比嵴结构更清晰。各组间GSK-3β磷酸化水平差异具有统计学意义（ F=45.841， P<0.001）。与空白对照组相比，DMSO组GSK-3β磷酸化水平无明显差异（ P>0.05），SB组明显增加（ P=0.022），Al组明显减少（ P<0.001）；SB+Al组GSK-3β磷酸化水平明显高于Al组（ P<0.001）。各组间DRP1蛋白水平差异具有统计学意义（ F=8.389， P=0.003）。与空白对照组相比，DMSO组和SB组DRP1蛋白水平无明显差异（ P>0.05），Al组升高（ P=0.001）；SB+Al组DRP1蛋白水平明显低于Al组（ P=0.029）。 结论
Aluminum (Al) can cause irreversible damage to neurons and synapses function, and the mechanism may be connected to mitochondrial damage caused by glycogen synthase kinase-3β (GSK-3β) regulating dynamin-related protein 1 (DRP1), resulting in inhibition of the growth of neuronal protrusions.
To investigate the role of GSK-3β regulating DRP1 in the inhibition of primary hippocampal neurite growth induced by Al.
Neurons were extracted from the hippocampus of newborn mice (≤24 h old) for primary culture. On day 6, the purity of neurons was detected by immunofluorescence. On day 10, neurons with good growth state were selected for Al exposure and GSK-3β inhibitor SB216763 (SB) intervention. The experiment design included a blank control group, a dimethyl sulfoxide (DMSO) group, an Al (20 μmol·L−1) group, a SB (1 μmol·L−1) group, and a SB (1 μmol·L−1) + Al (20 μmol·L−1) group. After primary hippocampal neurons were treated with Al or SB for 48 h, cell viability was detected by CCK-8 assay, the mitochondrial morphology of primary hippocampal neurons was observed by transmission electron microscopy, the total protrusion length of primary hippocampal neurons was scanned and analyzed by laser confocal imaging, and their complexity was analyzed by Sholl analysis. The expression levels of phospho-GSK-3β, GSK-3β, and DRP1 were detected by Western blotting.
The immunofluorescent results showed that the purity of primary neurons was higher than 90%. After the Al exposure and the SB intervention for 48 h, compared with the blank control group, there was no obvious difference in cell viability in the DMSO group and the SB group (
P>0.05), and the Al group showed reduced cell viability ( P=0.006); there was no obvious difference in cell viability between the SB+Al group and the Al group ( P>0.05). Compared with the blank control group, there was no obvious difference in the average total length of protrusion in the DMSO group and the SB group ( P>0.05), and the Al group showed reduced average total length of neurite ( P<0.001); the average total neurite length in the SB+Al group was significantly increased compared with that in the Al group ( P=0.001). The results of Sholl analysis revealed that, within 130 μm from the cytosol, the number of intersections of neurons in each group increased with the increase of distance. Above 130 μm from the cytosol, the number of intersections of neurons in each group decreased gradually with increase of distance. At 130 μm and 310 μm from the cytosol, compared with the blank control group, the number of neuronal intersections in the DMSO group and the SB group had no obvious difference ( P>0.05), and that in the Al group was significantly reduced ( P<0.05); there was no obvious difference in the number of neuronal intersections between the SB+Al group and the Al group ( P>0.05). The mitochondrial structure of the blank control group was complete and the crest was clearly visible; there was no apparent variation in the mitochondrial structure in the DMSO group and the SB group; the mitochondria in the Al group were vacuolated and the crista disappeared; the SB+Al group showed clearer crista than the Al group. The difference in GSK-3β phosphorylation level among groups was statistically significant ( F=45.841, P<0.001). Compared with the blank control group, the GSK-3β phosphorylation level showed not significantly different in the DMSO group ( P>0.05), increased in the SB group ( P=0.022), and significantly reduced in the Al group ( P<0.001); the GSK-3β phosphorylation level was significantly higher in the SB+Al group than in the Al group ( P<0.001). The difference in DRP1 protein level among groups was statistically significant ( F=8.389, P=0.003). Compared with the blank control group, the DRP1 protein levels in the DMSO group and the SB group were not significantly different ( P>0.05), and significantly increased in the Al group ( P=0.001); the DRP1 protein level in the SB+Al group was significantly lower than that in the Al group ( P=0.029). Conclusion
Al may increase the level of DRP1 protein by activating GSK-3β, causing mitochondrial damage and inhibiting neuronal protrusions growth.