Study Of Mechanisms Of Acrylamide-induced Learning And Memory Impairment

Part ? Effect of acrylamide on learning and memory ability in ratsObjectives:In the present study,sub-chronic ACR exposure model was established by oral gavage.The neurobehavioral and pathology methods were used to investigate whether ACR exposure can cause learning and memory impairment in rat.Methods:Thirty-six Male Sprague-Dawley rats,weight 140 g to 160 g,were randomly assigned to three groups:control?received vehicle for ACR by oral gavage?,ACR1?received 5 mg/kg ACR by oral gavage?,and ACR2?received 10 mg/kg ACR by oral gavage?.The study was conducted for a period of 14 weeks.The body weights and gait scores were recorded once per week to assess the locomotion.The landing foot splay and grip strength tests were conducted at the terminal stage of ACR exposure to assess motor coordination and muscle strength,respectively.The Morris water maze test was used to assess the learning and memory of the rats at the 13^th weeks.The neuronal morphological characteristic in the hippocampus was identified by the H&E staining.The number of NeuN positive neurons was explored by immunohistochemistry.Immunofluorescence staining was used to assess the activation of glial cells in the hippocampus.Morphological changes of neuronal ultrastructure were evaluated by transmission electron microscopy.Results:?1?General toxicity:The body weight and the organ coefficient of the rats displayed no statistical difference in the three groups?P>0.05?.?2?The motor function changes:The gait score in the ACR1 group showed a statistical difference at the 10^th to13^th weeks?P<0.05?.While in the ACR2 group,the gait score revealed a markedly difference at the 6^th to 13^th weeks?P<0.05?.The landing foot splay and the grip strength of rats in the ACR1 group showed no difference.However,in the ACR2 group,the grip strength of rats were significantly reduced,and the landing foot splay of rats were increased at the end of ACR exposure?P<0.05?.?3?Learning and memory changes:The average swim speed indicated no significant difference in the three groups?P>0.05?.The rats in ACR1 group revealed no change in escape latency,while the rats in ACR2 group demonstrated a prolonged latency to find the hidden platform?P<0.05?.The time and swimming distance spent in ACR1 and ACR2 groups were markedly decreased?P<0.05?.?4?General pathological changes:?1?H&E staining:In ACR1and ACR2 groups,the cell membrane and the nuclear boundary became unclear because of nucleus condensation and pycnosis.?2?NeuN staining:The immunoreactivities of NeuN in ACR1 group were slightly decreased?P<0.05?,while,it was severely reduces in ACR2 group?P<0.001?.?3?Glial cell activation:5 mg/kg ACR exposure for 14 weeks significantly enhanced GFAP and Iba-1 fluorescence intensity in hippocampus and CA3 region?P<0.05?.By contrast,the fluorescence intensity of GFAP and Iba-1 in the ACR2 group were much stronger than that in the control and ACR1groups?P<0.05?.?5?The ultrastructure changes:?1?Synapses:The pre-synaptic terminals were slightly swollen,and the synaptic cleft were faintly visible in ACR1 group.The post-synaptic densities became thicker and electron denser,the synaptic cleft became unclear,and the presynaptic membrane was incomplete in ACR2group.?2?Nerve filaments:The lamellae splitting of myelin sheath were obviously seen in ACR1 and ACR2 group.?3?Myelin sheaths:The aggregation of the neurofilaments were obviously seen in ACR1 and ACR2 group.Conclusion:The results obtained here indicated that ACR exposure at 5 and10mg/kg/d for 14 weeks disrupted the ability of learning and memory abilities.In addition,the hippocampal neurons in the ACR treated rats were lost and they demonstrated obvious astrocytosis and microglial activation.Moreover,ACR induces ultrastructural changes of synapses,myelin and neurofilament in the hippocampal CA1region.Part ? Molecular mechanisms of acrylamide-induced learning and memory impairmentObjective: Abnormal phosphorylated tau in neurons can damage the synaptic structure and affect the learning and memory ability.Synaptic vesicular proteins play an important role in synaptic plasticity and memory formation.PERK-e IF2? signaling and ALP have an important influence on p-tau formation and synaptic vesicle protein expression.In this part,the molecular biology techniques were adopted to explore the roles of p-tau,abnormal expression of synaptic vesicle protein,PERK-e IF2? and ALP signaling in ACR-induced learning and memory impairment.Methods: Experimental groups and brain processing were same as part I.Immunohistochemistry was applied for AT8 distribution.Abnormal tau expression,the related protein kinases,and synaptic vesicle proteins were examined through Western blot analyses,as well as the proteins involved in PERK-e IF2? and ALP signaling pathway.Results:?1?Abnormal tau detection:?1?AT8 distribution: Notable increase in AT8 staining could be seen in hippocampal sub-regions of rats in ACR1 and ACR2 groups with respect to the control?all P < 0.001?.?2?Expression of p-tau: The levels of p S262 and AT8 were markedly increased in ACR1 group.While in ACR2 group,notable increase in p S262,p S396 and AT8 levels could be seen with respect to the control and ACR1 groups?all P < 0.05?.?3?Key protein kinases GSK-3? and cdk5: The inactive p-GSK-3? Ser9 was significantly reduced and p25:p35 ratio was significantly increased in ACR-treated rats.?4?Protein phosphatase 2A: The inactivated form p-PP2Ac?Y307?was markedly increased in ACR-treated groups,indicating the PP2 A activity was down-regulated by ACR?all P < 0.05?.?2?Synaptic vesicle proteins: In ACR1 group,only a 19% decrease in the amount of synaptophysin was seen?P < 0.05?.In ACR2 group,the amount of synapsin-1 and synaptophysin decreased to 51% and 62% respectively?both P < 0.05?.Moreover,the expression of BDNF and P-CREB were attenuated considerably in a dose-dependent way in ACR exposure rats?P < 0.05?.?3?PERK-e IF2?-ATF4 signaling pathway: The protein levels of GRP78,P-PERK,P-e IF2?,ATF4 and CHOP were up-regulated considerably in the hippocampus of ACR-treated rats?P < 0.05?.?4?ALP signaling pathway: p62 was dramatically up-regulated and mature Cathepsin-D was markedly decreased in ACR1 group?P < 0.05?.In ACR2 group,the ratio of L3-II/I and p62 were significantly increased and mature CathepsinD was significantly reduced in ACR2 group compared with control?P < 0.05?.Conclusion: The increased p-tau and decreased CREB-BDNF,synapsin-1 and synaptophysin are involved in ACR-induced learning and memory impairment in rats.The activation of PERK-e IF2? signaling pathway and inhibition of ALP signaling pathway may be involved in ACR-induced learning and memory impairment in rats.Part ? Roles of PERK and ALP in acrylamide-induced learning and memory impairmentObjective:Specific inhibitor and agonist were used to explore the roles of PERKe IF2? and ALP signaling pathways in ACR-induced tau phosphorylation and synaptic vesicle protein reduction in SH-SY5 Y cells.Methods: Cell treatment: SH-SY5 Y cells were treated with 1.25,2.5,and 5 m M ACR for 24 h.Cell viability were determined by CCK8 assay.The morphological changes of cells were displayed under an optical microscopy.Specific inhibitor and agonist application: Cells were pretreated with 200 n M Rapamycin or 0.5 ?M GSK2606414 for 2 h and then incubated with 2.5 m M for 24 h.Abnormal tau expression and synaptic vesicle proteins were examined through Western blot analyses,as well as the proteins involved in PERK-e IF2? and ALP signaling pathway.Immunofluorescence staining was used to assess the distribution of p-tau in SH-SY5 Y cells.LC3 immunofluorescence staining was applied to label autophagosome.The formation of acidic vesicular organelles accompanied with lysosome accumulation were seen through acridine orange staining and Lyso Tracker Red staining.Results:?1?Cell viability: Cell viability was reduced significantly in a dosedependent manner in cells treated with 2.5 ¹0 m M ACR?P < 0.001?.After exposure to 2.5 or 5m M of ACR for 24 hours,most cells shrank and the cell body became round.?2?Expression of p-tau and synaptic vesicle proteins:?1?p-tau: The levels of p S262,p S396 and AT8 were markedly increased and the inactive p-GSK-3? Ser9 was significantly reduced in 2.5 and 5 m M ACR treated group?P < 0.05?.?2?Synaptic vesicle proteins: The expression of synapsin-1,synaptophysin,BDNF and P-CREB were attenuated considerably in a dose-dependent way after ACR exposure compared with control?all P < 0.05?.?3?PERK and ALP signaling pathway:?1?PERK: The protein levels of GRP78,P-PERK,P-e IF2?,ATF4 and CHOP were up-regulated considerably after ACR exposure?all P < 0.05?.?2?ALP: The ratio of L3-II/I and p62 were significantly increased and mature Cathepsin-D was significantly reduced in ACR treated group compared with control?P < 0.05?.?4?PERK inhibition: GSK2606414 pretreatment significantly reduced levels of P-PERK,P-e IF2?,ATF4 and CHOP in SHSY5 Y cells?P < 0.05?,suggesting the blockage of the PERK-e IF2? signaling pathway by GSK2606414.The inhibition of P-PERK with GSK2606414 considerably attenuated the expression of p S262 and AT8 and enhanced the inactive?p-Ser9?forms of GSK3? was in SH-SY5 Y cells compared with ACR-treated group?P < 0.05?.Moreover,the GSK2606414 pretreatment markedly increased the levels of P-CREB and BDNF,but failed to rescue synapsin-1 or synaptophysin.?5?ALP induction: The activation of ALP with Rapamycin considerably attenuated the ratio of L3-II/I and p62 and enhanced the mature Cathepsin-D in SH-SY5 Y cells compared with ACR-treated group?P < 0.05?.Moreover,confocal microscopy imaging displayed that the lysosomes accumulation was more obvious and the formation of acidic vesicles accumulation was markedly increased compared with that in ACR-treated group.The activation of ALP with Rapamycin considerably attenuated the expression of p S262,p S396 and AT8 in SH-SY5 Y cells compared with ACR-treated group?P < 0.05?.Moreover,the GSK2606414 pretreatment markedly increased the levels of BDNF,but failed to rescue P-CREB,synapsin-1 or synaptophysin.Conclusion: ACR-induced tau phosphorylation and BDNF reduction are mediated by PERK-e IF2? activation and ALP inhibition.The reduction of synapsin-1 and synaptophysin is independent of PERK-e IF2? or ALP signaling pathway.