| As a specialized membrane-associated coupling structure between mitochondria and endoplasmic reticulum,mitochondrial-associated endoplasmic reticulum membranes(MAMs)act an important role in the regulation of Ca2+transport and autophagy in neurocytes.As a heavy metal pollutant,the neurotoxicity of cadmium has been fully identified.Our previous investigations have confirmed that cadmium can induce endoplasmic reticulum stress,calcium homeostasis imbalance and autophagy.Exisiting studies have shown that Ca2+ transport regulates the autophagy process of neurocytes but the relationship between the changes of Ca2+transport capacity and autophagy mediated by MAMs of neurocytes exposed to cadmium has not been identified.In this study,rat pheochromocytoma cell line(PC 12)and primary rat cerebral cortical neurons(primary neurons)were used as models to investigate the role of MAMs and the Ca2+transport mediated by MAMs in cadmium induced autophagy of neurocytes in vitro.1 Effect of cadmium on MAMs and calcium transport in neurocytesTo investigate the effect of cadmium on MAMs and calcium transport in neurocytes,PC 12 cells and primary neurons were exposed to different concentrations(0 μM,2.5 μM,5μM and 10 μM)of CdCl2 for 6 hours or 5 μM CdCl2 for different periods(0 h,2 h,4 h,6 h and 12 h),or exposed to 5 μM CdCl2 for 6 hours and 12 hours.The following experiments were carried out:①Western blot was used to detect the expression levels of MAMs-related proteins-namely,Mfn2,Grp75 and VDAC1.②Laser scanning confocal microscopy was used to observe the change of mitochondria-endoplasmic reticulum coupling.The change of quantity of MAMs was verified by transmission electron microscopy(TEM).③Co-IP was used to detect the interaction of Grp75 and VDAC1,the scaffold proteins of Ca2+channel in MAMs.Laser scanning confocal microscopy was used to observe the co-localization of Grp75/VDAC1.④Flow cytometry was used to detect the change of Ca2+ levels of cytoplasm and mitochondria.The results were as follows:compared to the control group,①After exposed to different concentrations of cadmium for 6 h,the expression levels of Mfn2 and Grp75 in PC12 cells and Mfn2 in primary neurons increased significantly in the 2.5 μM cadmium treatment group(P<0.05).The expression levels of Mfn2,Grp75 and VDAC1 in two types of cells increased significantly in the 5 and 10 μM cadmium treatment group(P<0.05).After exposed to 5 μM cadmium for different periods,the expression levels of Mfn2,Grp75 and VDAC1 in PC 12 cells increased significantly(P<0.05)in 2 h,4 h,6 h and 12 h cadmium treatment groups,except for the VDAC1 in 2 h cadmium treatment group(P>0.05).In primary neurons,only the expression levels of Mfn2,Grp75 and VDAC1 in 6 h cadmium treatment group and Grp75 in 12 h cadmium treatment group increased significantly(P<0.05).②The level of correlation between mitochondria and endoplasmic reticulum in two types of cells and the quantity of MAMs in PC 12 cells increased in 6 h cadmium treatment group.The level of correlation between mitochondria and endoplasmic reticulum decreased in two types of cells treated with cadmium for 12 hours.③ After 6 hours of exposure,the co-localization of Grp75/VDAC1 in two types of cells increased,the protein interaction of Grp75/VDAC 1 in two types of cells increased significantly(P<0.05).After 12 hours of exposure,the co-localization of Grp75/VDAC1 in PC 12 cells increased,while the co-localization of Grp75/VDAC1 decreased in primary neurons.There was no significant change in the interaction of Grp75/VDAC 1 in two types of cells(P>0.05).④The levels of cytoplasmic Ca2+ and mitochondrial Ca2+ in two types of cells increased significantly in 6 h cadmium treatment group(P<0.05).The level of mitochondrial Ca2+in PC 12 cells and cytoplasmic Ca2+ in primary neurons increased significantly in 12 h cadmium treatment group(P<0.05).The results indicate that cadmium can induce the changes of MAMs and calcium transport capacity in neurocytes.2 The regulation of MAMs on autophagy and the change of calcium transport ability of neurocytes induced by cadmiumTo investigate the regulation of MAMs on autophagy and the change of calcium transport capacity in neurocytes induced by cadmium,PC 12 cells and primary neurons were exposed to different concentrations(0 μM,2.5 μM,5 μM and 10 μM)of CdCl2 for 6 hours or 5 μM CdCl2 for different periods(0 h,2 h,4 h,6 h and 12 h),or treated with 5 pM CdCl2 for 6 hours after the MAMs’ structural protein Mfn2 of the two types of cells was knocked down by Crispr-cas9 and RNA interference technology.The following experiments were carried out:①The expression levels of Mfn2 mRNA and protein were detected by qRT-PCR and Western blot respectively.②The change of quantity of MAMs was observed by TEM.The change of mitochondria-endoplasmic reticulum coupling was observed by laser scanning confocal microscopy.③The expression levels of autophagy-related proteins,namely,ATG7,ATG5,ATG14,Beclinl,p-ATG16L1s278 and LC3II were detected by Western blot.The co-localizaiton of LC3/Lamp2 was observed by laser scanning confocal microscopy.The interaction and co-localizaiton of Grp75/VDAC1 were detected by Co-IP and laser scanning confocal microscopy.⑤The change of the level of cytoplasmic Ca2+ and mitochondrial Ca2+were detected by flow cytometry.The results were as follows:①Compared to the control group(Con)or control group of negative siRNA(NCsiRNA),the transcription level of Mfn2 mRNA in primary neurons and the protein expression level of Mfn2 in two types of cells decreased significantly(P<0.05).②Knockdown of Mfn2 inhibited the increase of quantities of MAMs in PC 12 cells and the mitochondrial-endoplasmic reticulum coupling in two types of cells induced by cadmium.③After exposed to different concentrations of cadmium for 6 h,compared to the control group,only the expression levels of ATG7 and p-ATG16L1s278 in PC 12 cells increased significantly in 2.5 μM cadmium treatment group(P<0.05).The expression levels of autophagy-related proteins increased significantly in 5 μM cadmium treatment group(P<0.05).The expression levels of autophagy-related proteins in two types of cells increased significantly in 10 μM cadmium treatment group(P<0.05),except for the expression level of ATG16L1s278 and LC3II in primary neurons(P<0.05).After exposed to 5μM cadmium for different periods,only the expression level of ATG7 and p-ATG16L1s278 in primary neurons increased significantly in 2 h cadmium treatment group(P<0.05).The expression level of ATG5 and ATG14 in two types of cells and ATG7 in primary neurons increased significantly in 4 h cadmium treatment group(P<0.05).The expression level of ATG7,ATG5,ATG14 and LC3II in two types of cells increased significantly in 6 h cadmium treatment group(P<0.05).The expression level of ATG5,Beclin 1,p-ATG 16L1 s278 and LC3II in PC 12 cells and ATG7,ATG14,Beclin1 and LC3II in primary neurons increased significantly in 12 h cadmium treatment group(P<0.05).Knockdown of Mfn2 significantly inhibited the increase of expression level of ATG7,ATG5 and LC3II(P<0.05)and the co-localization of LC3/Lamp2 in two types of cells induced by cadmium.④Knockdown of Mfn2 significantly inhibited the increase of interaction and co-localization of Grp75/VDAC1 in two types of cells induced by cadmium(P<0.05).⑤Knockdown of Mfn2 significantly inhibited the increase of the level of cytoplasmic Ca2+ and mitochondrial Ca2+in two types of cells induced by cadmium(P<0.05).The results indicate that cadmium can induce autophagy and the changes of calcium transport capacity in neurocytes through MAMs.3 The regulation of calcium transport mediated by MAMs on autophagy induced by cadmium in neurocytesTo investigate the regulation of calcium transport mediated by MAMs on autophagy induced by cadmium in neurocytes,PC 12 cells and primary neurons were treated with RuR(mitochondrial MCU inhibitor)and cadmium alone or in combination for 6 h,or treated with 5μM cadmium for 6 h after the Ca3+ charmers protein Grp75 was knocked down by Crispr-Cas9 and RNA interference technology.The following experiments were carried out:①The cell viability was detected by CCK-8 assay.The change of mitochondrial Ca2+ level after RuR pretreatment was detected by flow cytometry.②The expression level of Grp75 mRNA and protein were detected by qRT-PCR and Western blot respectively.③The interaction of IP3R1/VDAC1 was detected by Co-IP.④The change of mitochondria Ca2+level was detected by flow cytometry.⑤The expression levels of autophagy-related proteins,ATG7,ATG5 and LC3II was detected by western blot.The co-localizaiton of LC3/Lamp2 and the changes of EGFP-RFP-LC3 fluorescence dots were observed by laser scanning confocal microscopy.The results were as follows:①Compared to the control group,different concentrations of RuR has no significant effect on the cell viability of the two types of cells.RuR pretreatment could significantly inhibit the increase of mitochondrial Ca2+ level caused by cadmium in two types of cells(P<0.05).②Compared to Con group or NCsiRNA group,the transcription level of Grp75 mRNA in primary neurons decreased significantly(P<0.05),and the expression level of Grp75 protein in two types of cells decreased significantly(P<0.05).③The interaction of IP3R1/VDAC1 was significantly inhibited by the knockdown of Grp75 in two types of cells(P<0.05).④Knockdown of Grp75 significantly inhibited the increase of mitochondrial Ca2+ level in two types of cells induced by cadmium(P<0.05).⑤The increase of the expression levels of ATG7,ATG5 and LC3II in two types of cells induced by cadmium was significantly inhibited by RuR pretreatment or knockdown of Grp75(P<0.05).Knockdown of Grp75 inhibited the increased level of co-localization of LC3/Lamp2 and autophagy fluorescence of EGFP-RFP-LC3 induced by cadmium.The results indicate that the Ca2+transport mediated by MAMs plays an important role in autophagy of neurocytes induced by cadmium. |
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