The Underlying Mechanisms For Tri-ortho-cresylphosphate-Induced Mitophagy Alteration In Neuro-2a Cells

ObjectiveTri-ortho-cresyl phosphate(TOCP)is an ordinary organophosphorus compound that can cause one type of toxic neuropathy known as organophosphate-induced delayed neuropathy(OPIDN),which is pathologically characterized by axonal degeneration.Nowadays,the mechanisms leading to axonal degradation after TOCP exposure is unclear.The disturbance in elimination of toxic materials including damaged mitochondria and aggregated proteins may contribute to the development of TOCP-induced axonal degeneration.In previous research,it was observed that autophagy was induced in TOCP treated animals and cells.Currently,the conditions of mitophagy in TOCP are unclear.Also,the mechanisms underlying autophagy induction under TOCP exposure remains unknown.Therefore,this research was designed to study how mitophagy changed under TOCP treatment by using N2a cells.Furthermore,it was investigated that how TOCP accounted for autophagy induction in TOCP-dosed N2a cells.Methods1.In vitro model establishment by TOCP-exposed N2a cellsN2a cells were incubated in culture plates.Ra were used to induce N2a cell differenciation.Then,CCK-8 was used to assess TOCP cytotoxicity to differentiated N2a cells for dose determination.According to the results of cytotoxicity assays,four groups of differenciated N2a cells were dosed with 0,5,10 and 20 ?M TOCP respectively for 24 h.After TOCP treatment,N2a cells were observed by microscope.Meanwhile,images of cellular morphology were conducted by TCapture.2.MorphologyMeasurement of Intracellular Ca2+ levels:After TOCP treatment,intracellular Ca2+in N2a cells was stained by Fluo-3 AM and images were conducted by Metaporph.In addition,another batch of cells stained by Fluo-3 AM were harvested for measuring fluorescence intensity.Finally,intracellular Ca2+ concentration was calculated.Measurement of reactive oxygen species(ROS)levels:Intracellular ROS generation in TOCP-dosed N2a cells was monitored by using 2'7'-dichlorofluorescin diacetate(DCFH-DA).Stained cells were conducted by Metaporph or harvested for fluorescence intensity measurement.Mitochondria staining by Mito-tracker:TOCP-treated N2a cells were stained by MitoTracker probe.Subsequently,cells were observed by microscope or were collected.Stained mitochondria in harvested cells can be measured by fluorescence intensity.Immunofluorescence staining:LC3 and Parkin expression in TOCP-exposed N2a cells were detected via immunofluorescence.3.Detection of expression in relative proteinsWestern Blotting:TOCP-treated N2a cells proteins were extracted.The expression of LC3,P62,p-P62(S351),kinases in Ca2+-controled pathway and proteins in PINK1-Parkin mediated mitophagy was tested.4.Statistical analysis:Data are reported as mean values ± standard error of the mean(SEM).The statistical significance of differences was calculated by One-way ANOVA where P<0.05 was considered to be significant.Results1.The effects posed on N2a cells by TOCP administration:The results of cytotoxicity assay showed that TOCP-dosed N2a cell viability decreseased in a dose-dependent manner when TOCP concentration reached over 20 ?M.All cells would lose viability under 1000 ?M TOCP administration.According to the results of cytotoxicity assay,the in vitro model was established under 0,5,10 and 20 ?M TOCP treatment where N2a viability was little affected.It was observed that the length of axons decreased significantly in TOCP-dosed N2a cells in a dose-dependent manner.2.The changes of mitophagy in TOCP-treated N2a cells:TOCP induced significant ROS enhancement in N2a cells(P<0.05),which implied that mitochondria were damaged under TOCP administration.The results of Western Blotting illustrated that the contents of LC3-II,P62 and p-P62(S351)rose dramatically in N2a cells after TOCP administration(P<0.05).It could also be found that mitochondrial Drp1,PINK1,Parkin,LC3-?,P62 and p-P62(S351)contents rose dramatically in N2a cells after TOCP exposure(P<0.05).In contrast,mitochondrial OPA1 and inner mitochondria membrane protein Tim23 declined significantly in TOCP-treated N2a cells(P<0.05).These results implied that TOCP could impair mitochondrial structure and,thus,trigger mitophagy in N2a cells.3.The detection of Ca2+-dependent upstream pathway of autophagy:First,it were observed that TOCP induced significant intracellular Ca2+ rise in N2a cells(P<0.05).Subsequently,It could be found that expression of CAMKK ?,p-AMPK(T172),ULK1 and p-ULK1(S317)was enhanced significant by a dose-dependent manner in TOCP-dosed N2a cells(P<0.05).Besides,the results demonstrated that BAPTA-AM intervention could attenuate TOCP-induced p-AMPK(T172)and p-ULK1(S317)increase(P<0.05).Also,BAPTA-AM treatment could alleviate TOCP-triggered LC3-?,p62 and p-p62(S351)accumulation in N2a cells.These results suggested that Ca2+mediated signal pathway was involved in TOCP-induced autophagy.4.The expression of transport proteins in TOCP-exposed N2a cells:Western Blotting results showed that dynactin and kinesin levels decreased significantly in N2a cells after TOCP administration by dose-dependent manner(P<0.05).Conclusion1.TOCP could induce autophagy in N2a cells.2.TOCP exposure could cause mitochondrial damage in N2a cells and,subsequently,stimulate PINK 1-Parkin mediated mitophagy.3.Ca2+ mediated signal pathway was involved in autophagy induction in TOCP-treated N2a cells.Increased Ca2+ by TOCP exposure would enhance the initiation of autophagy via activating CAMKK ?-AMPK-mTOR-ULK1 pathway.BAPTA-AM could inhibit the activation of this pathway in TOCP-treated N2a cells.4.The expression of dynactin and kinesin decreased dramatically in TOCP-dosed N2a cells.