The Role And Mechanism Of IST1 In Acrylamide-Induced Learning And Memory Impairment

Acrylamide（ACR）is a widely used environmental chemical with neurotoxicity.However,the molecular mechanism and intervention targets of ACR have not been fully elucidated.IST1 factor associated with ESCRT-Ⅲ（IST1）is an important regulatory subunit of endosomal sorting and transport complex Ⅲ,which can promote autophagy flux and accelerate autophagic clearance of the phosphorylated Tau protein（p-Tau）andα-synuclein（α-Syn）.In this study,IST1 overexpression(IST1^oe)cell model and mouse model were constructed to explore the role and mechanism of IST1 in learning and memory decline induced by ACR,and to provide theoretical basis for elucidating the neurotoxic mechanism of ACR and investigating molecular targets for prevention and treatment.Part Ⅰ Cytotoxicity of acrylamide on SH-SY5Y cells and IST1 protein expressionObjective:To investigate the dose-effect and time-effect relationships of ACR toxicity on SH-SY5Y cells,and to lay the foundation for subsequent mechanistic studies.Methods:⑴Cell grouping and treatment:SH-SY5Y cells were exposed to 0.00,0.50,0.75 and 1.00 mmol/L ACR for 72 h（control group was treated with distilled water）,and the dose-effect relationship was observed.SH-SY5Y cells were treated with 0.75 mmol/L ACR for 0,24,48 and 72 h,and the time-effect relationship was observed.（2）CCK8 was used to detect cell viability.Mitochondrial membrane potential（ΔΨm）,reactive oxygen species（ROS）and early apoptosis rate were measured by flow cytometry.Kits were used to detect glutathione（GSH）and malondialdehyde（MDA）contents and caspase-3 enzyme activity.Apoptotic cells were observed by Tunel assay.Lenti-GFP-LC3B and Lenti-m Cherry-GFP-LC3B were transfected to observe LC3 puncta and m Cherry⁺GFP⁺,m Cherry⁺GFP^-puncta,respectively.Western blot analysis was used to test the expression levels of p-PERK,t-PERK,GRP78,ATF4 pathway proteins of Endoplasmic reticulum stress（ERS）,learning and memory related proteins p-CREB（Ser133）,t-CREB,BDNF,AT8（p-Tau Ser202&Thr205）,autophagy and autophagy flux regulator proteins Beclin,LC3-Ⅱ,P62,IST1,STX17,VAMP8,apoptosis-related proteins Bcl-2,Bax,CHOP.Results:⑴Dose-response relationship:（1）0.75 and 1.00 mmol/L ACR significantly reduced cell survival rate（P<0.001）;（2）0.50 and 0.75 mmol/L ACR significantly increased the content of ROS and MDA（P<0.05）,decreased GSH content（P<0.05）;0.75 mmol/L ACR significantly increased expression levels of P-PERK/t-PERK,GRP78 and ATF4 in ERS pathway（P<0.05）;（3）0.75 mmol/L ACR significantly reduced the protein levels of p-CREB（Ser133）and BDNF（P<0.01）,increased AT8 protein level（P<0.05）;（4）0.75mmol/L ACR significantly increased the rate of early apoptosis（P<0.05）,Tunel⁺cell number（P<0.001）,caspase-3 enzyme activity（P<0.001）,ane the expression levels of pro-apoptotic proteins Bax and CHOP（P<0.01）,decreased the expression levels ofΔΨm and anti-apoptotic protein Bcl-2（P<0.001）;（5）0.75 mmol/L ACR significantly increased LC3-Ⅱ and p62 protein levels（P<0.01）;0.75 mmol/L ACR significantly increased the puncta of LC3 and m Cherry⁺GFP⁺（P<0.001）,while had no significant effect on m Cherry⁺GFP^-puncta（P>0.05）;（6）0.75 mmol/L ACR could significantly reduce the level of IST1 protein（P<0.01）and had no significant effect on STX17 and VAMP8 expression（P>0.05）.⑵Time-effect relationship:0.75 mmol/L ACR decreased IST1 significantly for 48 h and lasted for 72h（P<0.01）,while lc3-Ⅱ,Bax and Bcl-2 protein expression levels did not change significantly until 72 h after exposure（P<0.01）.Conclusion:The treatment with 0.50 mmol/L ACR for 72 h induced oxidative stress in SH-SY5Y cells.After 0.75 mmol/L ACR treatment for 72 h,oxidative stress,ERS,apoptosis,autophagy flux blockage and autophagy regulatory protein IST1 levels were decreased,and the expressions of learning and memory related proteins were abnormal.The effect of ACR on IST1 protein was earlier than that of apoptosis and autophagy protein.Part Ⅱ The role and mechanism of IST1 in acrylamide-induced cytotoxicity in SH-SY5Y cellsObjective:The IST1^oe cell model was constructed and the autophagy flux blocker bafilomycin A1（Baf A1）was used to study the role and mechanism of IST1 in ACR cytotoxicity.Methods:⑴Lipofectamine 3000 kit was used to transfect e GFP-N1-IST1 plasmid into SH-SY5Y cells to construct IST1^oe cell model.Meanwhile,vector control was set up.⑵Cell grouping and treatment:cells were divided into Vector group,IST1^oe group,Vector+ACR group and IST1^oe+ACR group.The final ACR concentration was 0.75 mmol/L and the exposure time was 72 h.⑶The cells in intervention study of Baf A1,an autophagolysosomal fusion inhibitor,were divided into IST1^oe+ACR group and IST1^oe+Baf A1+ACR group.ACR alone group treated IST1^oe cells with 0.75 mmol/L ACR for 72h,Baf A1 combined group treated IST1^oe cells with 0.75 mmol/L ACR and 7.5 nmol/L Baf A1for 72h.⑷Test indicators:the same as part 1.Results:⑴The expression of IST1 in the IST1^oe group was significantly higher than that in the Vector group（P<0.05）,indicating IST1^oe cell model was successfully constructed;⑵IST1^oe can significantly promote autophagy flux,there were more autophagosomes in the Vector+ACR group,but a few autophagosomes in IST1^oe+ACR group.The protein levels of LC3-Ⅱ and p62 in IST1^oe+ACR group were significantly lower than those in Vector+ACR group（P<0.05）,LC3 and m Cherry⁺GFP⁺puncta significantly decreased（P<0.001）,m Cherry⁺GFP^-puncta significantly increased（P<0.05）;⑶IST1^oesignificantly inhibited the decrease of p-CREB（Ser133）and BDNF protein levels and the increase of AT8 protein levels（P<0.05）induced by ACR;⑷IST1^oe had no significant effect on the content of GSH,MDA and ERS-related protein induced by ACR（P<0.05）;⑸The early apoptosis rate（P<0.01）,Tunel⁺cell number（P<0.001）,caspase-3 enzyme activity（P<0.05）and Bax protein（P<0.05）in IST1^oe+ACR group were significantly lower than that in Vector+ACR group,and Bcl-2 protein was significantly higher than that in Vector+ACR group（P<0.05）,CHOP protein level had no significant change（P>0.05）;⑹Baf A1 significantly inhibited the alleviating effect of IST1^oe on ACR cytotoxicity.Compared with IST1^oe+ACR group,the number of autophagosomes in IST1^oe+Baf A1+ACR group was more under transmission electron microscope.LC3-Ⅱ,p62,AT8,Bax protein levels and early apoptosis rate were significantly increased（P<0.01）,Bcl-2significantly decreased（P<0.05）.Conclusion:IST1^oe alleviated ACR-induced autophagy flux blockage by promoting the fusion process of autophagosome and lysosome,thereby reducing the changes of learning and memory-related proteins and cell apoptosis,but had no significant effect on ACR-induced oxidative stress and ERS.Part Ⅲ The role and mechanism of IST1 in acrylamide-induced learning memory impairment in miceObjective:AAV9-CMV-IST1-GFP was stereotaxically injected into the lateral ventricle of C57BL/6J mice to construct the hippocampal IST1^oe mouse model,to study the role and mechanism of IST1 in ACR-induced learning and memory impairment in mice.Methods:Forty healthy male 7-week-old SPF C57BL/6J mice,twenty of them were injected with AAV9-CMV-IST1-GFP in bilateral ventricles to construct the animal model of hippocampal IST1^oe,and twenty of them were injected with AAV9-CMV-GFP in bilateral ventricles as vector control.Three weeks after injection,animals were randomly divided into Vector group,IST1^oe group,Vector+ACR group and IST1^oe+ACR group,with 10animals in each group.Mice in the Vector+ACR and IST1^oe+ACR groups were exposed to 10 mg/kg bw/d ACR by gavage for 7 weeks,and mice in the Vector and IST1^oe groups were treated with distilled water.The weight,water intake and food intake of the mice were weighed and recorded weekly.In the last week of exposure,the rotation acceleration test was performed to detect motor coordination,the open field test and the elevated cross maze test to detect autonomous movement and anxiety-like behavior,and the water maze test to detect learning and memory ability.24 h after the last treatment,all of the mice were sacrificed,the serum was collected,and the hippocampus was separated.H&E staining,Niss staining,Golgi staining,Tunel staining and ultrastructural pathological examination were performed.The kits were used to detecte GSH,MDA,catalase（CAT）,total superoxide dismutase（T-SOD）and caspase-3 enzyme activity.Western blot was performed to analyze the levels of IST1 and the Histone 4 Acetylation at Lysine 12（Ac H4K12）,autophagy-related proteins Beclin-1,LC3-Ⅱ and p62,learning and memory related proteins p-CREB（Ser133）,t-CREB,BDNF,AT8 p-α-Syn（Ser129）,ERS pathway proteins,and apoptosis proteins.Results:⑴There was a large amount of green fluorescence in the hippocampus of mice,and the level of IST1 protein in the IST1^oe group was significantly higher than that in the Vector group（P<0.01）,indicating the hippocampal IST1^oe model is constructed successfully.⑵ACR significantly reduced the level of IST1 in hippocampus（P<0.05）,Ac H4K12 had no significant change（P>0.05）,hippocampal IST1^oe reduced the inhibitory effect of ACR on IST1（P<0.05）.⑶Hippocampal IST1^oe could significantly inhibit autophagy flus blockage,by electron microscopy,a large number of autophagosomes were observed in the CA1 region of the Vector+ACR group,and the levels of LC3-Ⅱ and p62 in hippocampus of Vector+ACR group were significantly higher than those in the Vector group（P<0.001）,hippocampal IST1^oe significantly alleviated the accumulation of autophagosomes,and the elevation of LC3-Ⅱ,p62 protein levels（P<0.05）.⑷There were no significant differences in body weight,water intake,food intake and main organ coefficients among all groups（P>0.05）,ACR significantly reduced the stay time of mice on the stick（P<0.001）,hippocampal IST1^oe did not significantly prolong the stay time on the rotating stick（P>0.05）.⑸The results of water maze test showed the escape latency and swimming distance of ACR group on the fourth and fifth days were significantly higher than those of Vector group（P<0.05）,the residence time in the original target quadrant was significantly lower than that in the Vector group（P<0.05）,hippocampal IST1^oesignificantly improved the effects of ACR on escape latency,swimming distance and residence time in primary target quadrant（P<0.05）.Open field test and elevated cross maze test showed ACR had no significant effect on autonomic movement and anxiety-like behavior of mice（P>0.05）;⑹The number of Nissl body in Dentate Gyrus（DG）,CA3,CA1 regions of ACR group was decreased,the arrangement of neurons was disorganized,the extra-cellular space was enlarged,the number of glial cells was increased,and TNF-αlevel in hippocampus was significantly increased（P<0.05）,Golgi staining showed that the density of dendritic spine in DG and CA1 region decreased significantly in Vector+ACR group（P<0.001）.The ACR induced changes of dendrite spine density and TNF-αwere significantly reversed by IST1^oe（P<0.01）,and also reduced the proliferation of glial cells and neuron structure damage;⑺The serum GSH,hippocampal GSH,CAT and T-SOD in the Vector+ACR group were significantly lower than those in Vector group（P<0.05）,MDA content in serum and hippocampus of Vector+ACR group were higher than that of Vector group（P<0.05）,the levels of ERS-related proteins p-PERK/t-PERK,GRP78 and ATF4 in the hippocampus of Vector+ACR group were significantly higher than Vector group（P<0.05）,hippocampal IST1^oe had no significant effect on ACR-induced oxidative stress and ERS（P>0.05）.⑻ACR significantly decreased the protein levels of p-CREB（Ser133）and BDNF（P<0.001）,increased AT8 and p-α-Syn（Ser129）levels（P<0.001）,hippocampal IST1^oe significantly reversed the effect caused by ACR on p-CREB/t-CREB,BDNF（P<0.05）and AT8,p-α-Syn（Ser129）（P<0.01）.⑼Compared to the Vector group,the number of Tunel⁺cells in CA1,CA3 and DG region of hippocampus in Vector+ACR group was significantly higher（P<0.01）,Bax protein level higher significantly（P<0.001）,Bcl-2 significantly lower（P<0.001）,IST1^oe significantly decreased Tunel⁺cell numbers in CA1,CA3 and DG regions（P<0.05）,and alleviated the changes of Bax and Bcl-2 caused by ACR（P<0.01）.Conclusion:Hippocampal IST1^oe ameliorated the damage of hippocampal neurons and dendrites and improved the learning and memory ability of mice by alleviating of ACR-induced autophagy flux blockage,apoptosis and learning and the abnormal expression of learning and memory-related proteins.Hippocampal IST1^oe did not alleviate oxidative stress and ERS induced by ACR.