An Experimental Study On Glyceraldehyde-3-phosphate Dehydrogenase Cytoplasmic Aggregation And Nuclear Translocation In The Pathogenesis Of Parkinson’s Disease

Part1The Establishment of Rotenone-induced SH-SY5Y Cell PD ModelSection1The neurotoxicity of rotenone on SH-SY5Y cellsObjective To investigate the neurotoxicity of rotenone on human neuroblastoma SH-SY5Y cells.Methods To confirm the morphological change of SH-SY5Y cell after giving rotenone. MTT assay was used to measure the activity of retonone-induced cells, and to decide the intervention concentration of rotenone. Meanwhile, AO/EB fluorescent staining technology was used to check the way of cell death under fluorescence microscope and to calculate the cell apoptotic rate.Results Rotenone-induced SH-SY5Y cells have great morphological change, their growth was suppressed, neurites were shortened, part of cells turned round and contracted, and their adherence ability reduced. MTT assay pointed out that rotenone could reduce the SH-SY5Y cell activity concentration-and time-dependently. AO/EB fluorescent staining showed that rotenone-induced SH-SY5Y cell death was mainly time-and concentration-dependent cell apoptosis.Conclusion Rotenone induced the apoptosis of dopaminergic SH-SY5Y cells with a time-and concentration-dependent manner. Section2The establishment of rotenone-induced undifferentiated SH-SY5Y cell PD modelObjective To investigate the formation of rotenone-induced intracellular inclusions, the expression level and the localization of α-synuclein.Methods HE staining was used to confirm the morphological change of SH-SY5Y cells and the formation of intracellular inclusions at24hours and48hours after giving retonone. Laser confocal fluorescence microscopy was used to observe the changes of distribution and properties of a-synuclein in retonone induced cells. Meanwhile, western blot was used to measure the protein level of a-synuclein in rotenone-induced and uninduced SH-SY5Y cells.Results HE staining showed that rotenone-treated SH-SY5Y cell and its nucleus enlarged, neurites shortened, cytoplasm loosened and vesiculated, cell nucleus appeared karyopycnosis, and cytoplasmic Lewy body-like inclusions which was eosinophil and had clear boundary could be observed. Laser confocal fluorescence microscopy showed granulated positive staining of a-synuclein aggregated in SH-SY5Y cells, and was costained by Thioflavin S. The up-regulation of a-synuclein protein was detected by western blot.Conclusion The induction of rotenone on SH-SY5Y cells can lead to the formation of eosinophil inclusions, the aggregation and up-regulation of a-synuclein protein. It can be concluded that rotenone induced SH-SY5Y cells can simulate the pathological characteristics of PD very well, and rotenone as a neurotoxin can successfully establish PD cell model. Part2The Expression and Distribution of GAPDH in Rotenone-induced SH-SY5Y Cell PD ModelSection1The influence of rotenone on the subcellular location and expression of GAPDH in SH-SY5Y cellsObjective To investigate the influence of rotenone on the expression and subcellular location of GAPDH in SH-SY5Y cells.Methods Laser confocal fluorescence microscopy followed immunostaining was used to show the distribution, subcellular localization and characteristic change of GAPDH. Western blot and real-time PCR technologies were used to measure GAPDH protein and mRNA expression levels in retonone-induced SH-SY5Y cells. Results Laser confocal fluorescence microscopy showed that GAPDH protein distributed uniformly in the normal SH-SY5Y cell cytoplasm, with a small amount appeared in the nucleus. The exposure to rotenone leaded to the enhanced GAPDH staining and obvious granulated positive aggregation in the cytoplasm and increased nuclear GAPDH distribution, and brought the progressive increase of GAPDH protein and mRNA levels followed the time extension.Conclusion In PD cell model, rotenone could induce the overexpression and abnormal aggregation of GAPDH, and lead to the abnormal subcellular localization and characteristics of GAPDH. This might be one of the most important mechanism in dopaminergic neuron apoptosis induced by rotenone. Section2Effect of rotenone on GAPDH location and expression in the nucleus of SH-SY5Y cellsObjective To explore the effect of rotenone on GAPDH location and expression in the nucleus of SH-SY5Y cells.Methods Immunofluorescence staining and confocal microscopy were used to examine the location and expression of GAPDH in the nucleus of SH-SY5Y cells; Immunohistochemical staining was used to explore the effect of rotenone on GAPDH; Western blot was used to examine the expression of GAPDH.Results Results from immunofluorescence staining showed that GAPDH uniformly distributed in the cytoplasma in normal condition, with a small amount in the nucleus. Rotenone treatment induced enhancement and concentration of GAPDH fluorescence in the nucleus. Immunohistochemical staining also showed GAPDH staining was obviously increased by rotenone treatment, reflecting by some nuclei were stained to dense brown. Analysis from western blot also revealed that rotenone dose-dependently increased the expression of GAPDH in the nucleus, suggesting that nuclear translocation of GAPDH occured. Conclusion Rotenone can induce nuclear translocation of GAPDH in a time-and concentration-dependent manner, suggesting that nuclear translocation of GAPDH may underlie rotenone-induced neuron apoptosis. Part3GAPDH Cytoplasmic Aggregation and Nuclear Translocation in the Pathogenesis of Rotenone-induced PD Cell ModelSection1The effect of GAPDH abnormal degradation in rotenone-induced SH-SY5Y cellsObjective To investigate the role of ubiquitin-proteasomal pathway and autophagy-lysosomal pathway in GAPDH degradation.Methods MTT assay was used to measure the effects of different drugs on SH-SY5Y cell activity, and to decide the intervention concentrations. Protein immunoblot analysis (western blot) was used to detect GAPDH protein levels in cells induced by rotenone after selective inhibition or activation of ubiquitin-proteasomal pathway and autophagy-lysosomal pathway.Results Overexpression of GAPDH was found in rotenone-induced PD cell model. GAPDH protein levels of proteasomal inhibitor MG132treated cells and lysosomal inhibitor3-MA treated cells raised significantly than that of the normal control group and the sole rotenone-induced group (P<0.05). Activation of ubiquitin-proteasomal pathway by Mg2+or autophagy-lysosomal pathway by rapamycin decreased rotenone-induced GAPDH expression. And GAPDH level of Mg2+treated group declined more pronouned than that of rapamycin treated group.Conclusion We found that not only is GAPDH degraded by proteasome, but it is also degraded by autophagy. And proteasomal pathway may play a more important role in GAPDH degradation. Increasing clearance of GAPDH merits consideration as a potential therapeutic for Parkinson’s disease. Section2Involvement of Siahl in rotenone-induced GAPDH nuclear translocation and the downstream mechanismObjective To investigate the effects of Siahl on rotenone-induced GAPDH nuclear translocation and dopaminergic cell apoptosis.Methods Immunofluorescence staining and confocal microscopy were used to examine the location and expression of GAPDH and Siahl in the nucleus of SH-SY5Y cells. Western blot was used to examine the expression of Siahl in the nucleus, and to explore the correlation of Siahl and GAPDH, as well as the role of Siahl and GAPDH in rotenone-induced cell death.Results Results from immunofluorescence staining showed rotenone treatment induced enhancement and concentration of GAPDH and Siahl fluorescence in both the cytoplasm and nucleus. Confocal microscopy showed bright yellow fluorescence in the cytoplasm and nucleus of rotenone-induced cells. Results from western blot revealed that Siahl was concentration-dependently increased in the cell nucleus after rotenone treatment, which was consistent with the changing trend of GAPDH. And we also found apoptosis-related proteins p53and Bax were concentration-dependently increased.Conclusion Siahl participates in rotenone-induced GAPDH nuclear translocation. They are postulated to combind in the cytoplasm and then to co-transfer to the nucleus, promoting apoptotic cascades. Section3The effects of RNA interference on Siahl expression and rotenone-induced GAPDH nuclear translocationObjective To investigate the effects of siRNA on Siahl expression and rotenone-induced GAPDH nuclear translocation. Methods The small interfering RNA (siRNA) that was specific targeted to Siahl gene was transfected into SH-SY5Y cells using Lipofectamine2000. Interfering efficiency was identified by western blot analysis. Effects of rotenone on GAPDH nuclear location and expression in non-interfering and interfering SH-SY5Y cells were detected using western blot analysis.Results Results from western blot analysis showed that Siahl siRNA inhibited Siahl expression efficiently. GAPDH nuclear translocation was found in rotenone-treated SH-SY5Y cells; while Siahl siRNA-treated cells had lesser GAPDH translocation. Down-regulation of Siahl expression significantly decreased rotenone-induced GAPDH nuclear translocation.Conclusion Down-regulation of Siahl decreases rotenone-induced GAPDH nuclear translocation, which may play a role in protection cells from rotenone-induced apoptosis.