Effects Of Atg7 Knockout On Injury Of Neuro-2a Cells Induced By Tri-ortho-cresyl Phosphate

ObjectiveOrganophosphorus(OPs)have a wide range of applications in agriculture and industry.Recent data suggested there are approximately three million individuals are exposed to OPs worldwide per year.Some OP compounds can cause toxic neuropathy known as organophosphorus-induced delayed neuropathy(OPIDN),which is pathologically characterized by Wallerian-like degeneration.Pathological changes consisted of loss of neurotubules and neurofilaments,accumulation of mitochondria and vesicle,and abnormal protein degradation process may occur.In previous research,TOCP treated hens and PC 12 cells,it was observed that autophagy activity in the cells changed,suggesting that autophagy plays an important role in OPIDN.So far,the research on the pathogenesis of OPIDN is still not thorough,and the specific role and mechanism of autophagy in OPIDN is not clear.Therefore,in this study,mouse neuroblastoma(Neuro-2a,N2a)wild-type cells and Atg7 knock-out cells were treated with TOCP after differentiation as an OPIDN in vitro model,and hens treated with TOCP as OPIDN in vivo model.Furthermore,it was investigated the role of autophagy in TOCP-induced axonal and cell body injure in Neuro-2a cells,in order to reveal the relationship of autophagy and TOCP-induced neuronal damage.Methods1.Establishment in-vitro model of TOCP-exposed N2a cellsThe wild-type and Atg7-/-Neuro-2a cells were incubated in culture plates.cells were induced in DMEM medium supplemented with 2%(v/v)FBS and 10 μM retinoic acid for 48 hours.Then,the differentiated N2a cells were treated with 0 μM,5μM,10 μM of TOCP respectively for 24 hours.2.Cellular morphologyAfter 24 h TOCP treatment,the axons of differentiated N2a cells were observed by microscope(200×).In each group,we have observed 20 scopes and recorded the length of 10 cells axons in one scope.Images of cellular morphology were conducted by TCapture.3.Measurement of Mitochondrial membrane potentialMitochondrial membrane potential assay kit with JC-1 was employed to measure the mitochondrial membrane potential.Briefly,at the end of treatment,cells were incubated at 37 0C with JC-1 staining solution(5 μg/mL)for 20 min.Then cells were washed with JC-1 staining buffer(1×).Then,the stained cells were observed by fluorescence microscope,and the fluorescence of JC-1 monomer was detected at 490/530 nm(excitation/emission wavelength)and the fluorescence of JC-1 aggregates was detected at 525/590 nm(excitation/emission wavelength)on Infinite 200 Pro multimode Plate readers.4.Western BlottingThe total proteins of TOCP-treated N2a cells were extracted by using ice-cold RIPA buffer for detecting the protein expression related to Wallerian degeneration,calpain degradative system,ER-stress and Necroptosis.The nuclear protein of TOCP-treated N2a cells were extracted by using NE-PER Nuclear and Cytoplasmic Extraction kit for detecting the protein expression related to ER stress signaling pathway.5.Quantitative real-time PCR analysisTotal RNA of N2a cells were extracted from cells using the TRIzol reagent and reverse-transcribed to cDNA.Furthermore,UPL fluorescent probe and quantitative real-time PCR were used to analyze the mRNA levels of anti-oxidation genes.6.Establishment in-vivo model of TOCP-exposed hensAfter three days adapt feeding,30 adult Roman hens were divided equally and randomly into control group,1-day group,5-day group,10-day group and 21-day group.All groups of hens were treated with TOCP at a single dose of 750 mg/kg by gavage,except of control group.Furthermore,the activitiy and gait changes of hens’limbs were observed every day,and the clinical symptoms of OPIDN was scored according to the eight point graded scale.After TOCP treated,the hens were sacrificed on the corresponding time of 1 day,5 days,10 days and 21 days.The spinal cord and tibial nerve were peeled off on ice.After three days adapt feeding,30 adult Roman hens were divided equally and randomly into control group,0-day group,1-day group,5-day group,10-day group and 21-day group.All groups of hens were pretreated with PMSF with a dose of 90 mg/kg by subcutaneous injection and treated with TOCP at a single dose of 750 mg/kg by gavage 24h later,except of control group.Furthermore,the activitiy and gait changes of hens’limbs were observed every day,and the clinical symptoms of OPIDN was scored according to the eight point graded scale.After TOCP treated,the hens were sacrificed on the corresponding time of 0 day,1 day,5 days,10 days and 21 days.The spinal cord and tibial nerve were peeled off on ice.The total proteins of spinal cord and tibial nerve were extracted for detecting the protein expression.Results1.Effects of Atg7 knockout on axon injury of N2a cells induced by TOCP(1)TOCP induced N2a cells axon injury:The length of N2a cells axons were decreased induced by TOCP in a dose-dependent manner.Knockout of Atg7 protected against TOCP induced N2a cells axon injury.(2)The changes of pro-survival and pro-degenerative factors:The level of NMNAT2 and SCG10 in N2a cells were significantly decreased after the treatment of TOCP,and the level of SARM1 and p-CRMP2 were increased in a dose-dependent manner.In the same setting of TOCP exposure,SCG10 and NMNAT2 were kept at a higher level in Atg7-/-cells than in wild-type cells,while SARM1 was lower.Knockout of Atg7 delayed TOCP induced increasing of pro-degenerative factors and decreasing of pro-survival factors.(3)The changes of DLK-MKK4-MAPK pathway:TOCP treatment resulted in a significant increase in DLK and p-MKK4 content of both wild-type and Atg7-/-N2a cells.It is the same with p-ERK1/2 and p-p38.The DLK-MAPK signaling was activated in N2a cells following TOCP treatment.(4)The changes of protein related to Wallerian degeneration in in-vivo model:Animals in the TOCP model experimental group began to show limbs weakkness,walking occasionally shook from the fifth day.The symptoms of OPIDN progressed rapidly,with the coordination of walking became worse and the shaking gradually increased or even fell.By the 15th day,all animals were completely paralyzed,and this state continued until the end of the experiment.The control animals did not show any abnormal changes during the experiment.Besides,animals in the PMSF intervention group were standing and walking normally during the experiment,and there was no OPIDN symptom occurred.p-MKK4(SARM1)and p-JNK in spinal cord and tibial nerve were higher than control group in result of TOCP treatment,representing a time-dependent manner.But the differences of p-MKK4 and p-JNK among groups in PMSF invention model had no sense.Take together,the result of animal models was consisting of N2a cells model.(5)The changes of calpain degradative system:The level of active m-calpain and p.-calpain in N2a cells showed a marked increase following 10μM TOCP,while the level of calpastatin was decreased in a dose-dependent manner.Calpain degradative system was activated in N2a cells following TOCP treatment.2.Effects of Atg7 knockout on cell body injury of N2a cells induced by TOCP(1)The change of mitochondrial membrane potential:The ratio of red fluorescence intensity and green fluorescence intensity in N2a cells were reduced apparently after TOCP treatment,for the mitochondrial membrane potential reduced.And knockout of Atg7 protected against TOCP induced N2a cells mitochondrial injury.(2)The changes of protein related to ER stress:Exposed to TOCP,wild-type and Atg7-/-N2a cells expressed much more ATF6 and CHOP,while only wild-type N2a cells expressed much more ATF4 and p-eIF2a because of 10μM TOCP.(3)The changes of anti-oxidation factors:TOCP induced the mRNA levels of phase II reaction genes in both kinds of cells.What’s more,In the same setting of TOCP exposure,the mRNA levels of anti-oxidation genes in Atg7-/-N2a cells are higher than wild-type N2a cells.(4)The changes of protein related to Necroptosis:The levels of RIP1 and p-MLKL were stimulated in by TOCP and they were higher in wild-type N2a cells.Necroptosis was activated in N2a cells following TOCP treatment.Knockout of Atg7 protected against TOCP induced N2a cells necroptosis.Conclusion1.TOCP treatment induced neuron cells axon injury.Knockout of Atg7 protected against TOCP induced N2a cells axon and cell body injury.2.Injury of N2a cells axon induced by TOCP was related to the consumption of axon pro-survival factors,the accumulation of axon pro-degenerative factors,and the activation of DLK-MAPK signaling and calpain degradative system.Knockout of Atg7 delayed TOCP induced increasing of pro-degenerative factors and decreasing of pro-survival factors.3.Injury of N2a cell body induced by TOCP was related to the activation of oxidative stress,ER stress,mitochondrial damage and necroptosis.Knockout of Atg7 protected against TOCP induced N2a cells oxidative stress,ER and mitochondrial damage.