The Role And Mechanism Of Calpain In The Inhibition Of Autophagy In SK-N-SH Human Neuroblastoma Cells By TOCP

Objective:In this project,the aim is to study the regulation and mechanism of calpain in autophagy disorder induced by Tri-ortho-cresyl phosphate（TOCP）by using different calcium channel inhibitors and constructing the cell model of CAPNS1 gene knockout SK-N-SH(CAPNS1^-/-).Methods:1.CAPNS1 gene knockout SK-N-SH cell line(CAPNS1^-/-)was constructed by using CRISPR/Cas9 technology2.MTT assay was used to detect the effects of TOCP（0,0.25,0.5,0.75,1 mmol/L）on the proliferation of wild-type SK-N-SH cells(CAPNS1^+/+)and mutant SK-N-SH cells(CAPNS1^-/-),and the dose of TOCP usage was determined.3.After pretreatment with Verapamil,2-APB and CsA,SK-N-SH cells were exposed to 0.75 mmol/L TOCP,and the levels of intracellular calcium and calpain activity were detected by fluorescence spectrophotometer.Calpain proteins and the Autophagy-related proteins were detected by Western-blotting.4.After wild-type SK-N-SH cells(CAPNS1^+/+)and mutant SK-N-SH cells(CAPNS1^-/-)were exposed to 0.75 mmol/L TOCP,the calpain activity and proteasome activity were detected by fluorescence spectrophotometer.The levels of calpain proteins,autophagy-related proteins,cytoskeletal proteins,phosphorylated proteins and Ub protein were detected by Western-blotting.Results:1.The CAPNS1^-/-SK-N-SH cells were constructed by CRISPR/Cas9 technology.The gene sequencing showed that the CAPNS1^-/-SK-N-SH cell line was successfully constructed,and CAPNS1,calpain1 and calpain2 protein levels were significantly decreased.2.The result of MTT showed that after treatment of TOCP at 48h,CAPNS1^+/+SK-N-SH cell and CAPNS1^-/-SK-N-SH cell proliferation was significantly inhibited.3.The intracellular calcium concentration and calpain activity were increased induced with TOCP.After pretreatment with Verapamil、2-APB and CsA,the promotion of intracellular calcium ion concentration and calpain activity induced by TOCP were inhibited.At the same time,Western-blotting results showed that the protein levels of calpain1,calpain2,LC3II and P62 were increased after exposed to TOCP,and after pretreatment with three inhibitors,the proteins of calpain1,calpain2,LC3II and P62 were decreased.In addition,compared with TOCP-treated CAPNS1^+/+SK-N-SH cells,the protein levels of calpain,calpain1 and calpain2 were decreased after exposed TOCP in CAPNS1^-/-SK-N-SH cells.Similarly,compared with TOCP-treated CAPNS1^+/+SK-N-SH cells,LC3II and P62 levels were decreased,while the level of beclin1 was increased after exposed TOCP in CAPNS1^-/-SK-N-SH cells.4.TOCP also decreased the levels of Tau,P-Tau,NF-H,and MAP2in both cell lines,while increased P-MAP2 and P-NF-H levels.Compared with CAPNS1^+/+SK-N-SH cells,The levels of NF-H and MAP2 were increased in CAPNS1^-/-SK-N-SH cells,the levels of P-MAP2 and P-NF-H were decreased,but the levels of Tau and P-Tau did not change significantly.In addition,TOCP treatment inhibited proteasome（PGPH-L）activity and increased ubiquitinated protein levels in both cells;however,CAPNS1^-/-SK-N-SH cells proteasome（PGPH-L）was increased activity and ubiquitinated protein levels was decreased compared to CAPNS1^+/+SK-N-SH cells after TOCP tretment.Conclusions:1.TOCP could increase the Ca²⁺concentration by activating L-type calcium channel,mitochondrial calcium channel and endoplasmic reticulum IP3R calcium channel in SK-N-SH cells.2.TOCP could increase calpain activity and decrease the level of beclin1,increase the levels of LC3-II and P62.And TOCP could decrease the level of autophagy receptor protein OPTN and increase the level of NBR1,and inhibit autophagy.3.TOCP could decrease the levels of skeletal-associated proteins Tau,MAP2 and NF-H in SK-N-SH cells by calpain activited.4.TOCP could reduce proteasome activity and increase the accumulation of ubiquitinated protein in SK-N-SH cells.