Effects Of Treadmill Exercise On Endoplasmic Reticulum Proteostasis In Hippocampus Of APP/PS1 Transgenic AD Mice

ObjectiveAlzheimer’s disease（AD）is a neurodegenerative disease associated with proteostasis impairment,which is characterized by increased production of amyloidβ-protein（Aβ）and phosphorylated tau protein（p-Tau）,decreased synthesis of synapse related proteins,and impaired clearance of abnormal proteins.The endoplasmic reticulum regulates proteostasis,a process that monitors the protein biogenesis,folding,trafficking and degradation.From the perspective of endoplasmic reticulum proteostasis,this study aimed to investigate the effects of three months treadmill exercise on the pathological and behavioral symptoms of AD in APPswe/PS1d E9（APP/PS1）mice,the expression of endoplasmic reticulum protein folding related genes,endoplasmic reticulum unfolded protein response（UPR）signal,and endoplasmic reticulum stress（ERS）autophagy coupling,so as to provide molecular and behavioral basis for exercise prevention and alleviation of AD.MethodsExperimental grouping:Three month old male APP/PS1 transgenic mice and wild type mice were randomly divided into four groups:Wild type control group（WT-Sed,n=11）,Wild type exercise group（WT-Exe,n=11）,APP/PS1 control group（APP/PS1-Sed,n=11）and APP/PS1 exercise group（APP/PS1-Exe,n=11）.Exercise program:Mice in APP/PS1-Exe group and WT-Exe group were subjected to treadmill exercise for 3 months,45 minutes a day,5 days a week.Behavior experiment:After 3 months of exercise intervention,Morris water maze test was used to detect the spatial learning and memory ability of mice in each group.Tissue preparation:24 hours after the end of the behavioral experiment,the mice were taken for tissue sampling.Five mice from each group were selected to deep anesthesia and transcardially perfused with phosphate buffered saline（PBS）,and then the brain tissue was taken out.The right hemisphere was made into paraffin sections for immunohistochemistry;the left hippocampus was collected,and total RNA was extracted for Real-time PCR.The other six mice of each group were sacrificed after deep anesthesia.The left and right hippocampi were collected and the total protein was extracted for ELISA,Western blotting and Simple western analysis.Molecular biology experiment:（1）Detection of hippocampal Aβand p-Tau production and expression of synapse related proteins:Immunohistochemistry was used to detect hippocampal Aβplaque;ELISA was used to detect the content of hippocampal Aβ40 and Aβ42;Western blotting was used to detect the protein levels of BACE1,PS1,Tau,p-Tau,GSK3β,p-GSK3β^S9,p-GSK3β^Y216;Real time PCR was used to detect the m RNA level of KAL7;and the protein levels of SYN,PSD95,BDNF,CREB and p-CREB^S133 were detected by Simple western.（2）Detection of endoplasmic reticulum protein folding and endoplasmic reticulum stress related molecules in hippocampus:Real time PCR was used to detect the m RNA levels of ERO1L,PDIA2,PDIA3,PDIA4,PDIA5,PDIA6,HSPA5,HYOU1,HSPA1A,HSPA1B,HSPH1,HSPA8,HSP90B1,HSP90AB1,DNAJB1,DNAJB2,CRYAB,CNX and CRT;The protein levels of CNX and CRT were detected by Simple Western;The co-localization level of APP and GRP78 in hippocampus was detected by immunofluorescence double labeling.（3）Detection of endoplasmic reticulum UPR signal pathway in hippocampal:Western blotting and Simple western were used to detect the protein levels of PERK,p-PERK,e IF2α,p-e IF2α,ATF4,ATF6,IRE1α,p-IRE1α,XBP1,CHOP,BAX,BCL2 and CASPASE12;Immunohistochemistry was used to detect the protein levels of p-PERK,p-e IF2α,ATF6 and p-IRE1α;Real time PCR was used to detect the m RNA level of XBP1s;The nuclear ATF4 level was detected by immunofluorescence;TUNEL immunofluorescence was used to detect the apoptosis level in hippocampus.（4）Detection of hippocampal ERAD and ERS-autophagy coupling:Simple western was used to detect the protein levels of HERPUD1,SYVN1,VCP and DERL2.Real time PCR was used to detect the m RNA levels of autophagy related genes ATG5,ATG7,ATG12 and BECN1;Western blotting was used to detect the protein levels of LC3 and P62;Immunofluorescence double labeling was used to detect the co-localization levels of CRT and LC3 in hippocampus.Results（1）In the navigation test,compared with WT-Sed group,the latency of APP/PS1-Sed group on day 4 and 5 was significantly increased,and the percentage of platform quadrant time on day 3,4 and 5 was significantly decreased.Compared with WT-Exe group,the latency of APP/PS1-Exe group on day 3 and 4 was significantly increased,and the percentage of platform quadrant time on day 3 and 4 was significantly decreased.Compared with the APP/PS1-Sed group,the latency of the APP/PS1-Exe group on day 5 was significantly decreased.In the probe test,compared with WT-Sed group,the number of platform crossing in APP/PS1-Sed group was significantly reduced.Compared with APP/PS1-Sed group,the number of platform crossing in APP/PS1-Exe group was significantly increased.Compared with WT-Sed group,the levels of Aβplaque,Aβ40 and Aβ42,and the protein levels of BACE1,PS1,p-Tau^S396 and p-GSK3β^Y216 were significantly increased,and the protein levels of SYN,BDNF,p-CREB^S133 as well as the m RNA level of KAL7were significantly decreased in hippocampus of APP/PS1-Sed group.Compared with the APP/PS1-Sed group,the levels of Aβplaque,Aβ40 and Aβ42,and the protein levels of BACE1,PS1,p-Tau^S396 and p-GSK3β^Y216 were significantly decreased,and the protein levels of p-GSK3β^S9,SYN,PSD95,BDNF and p-CREB^S133 as well as the m RNA level of KAL7 were significantly increased in the hippocampus of APP/PS1-Exe group.（2）Compared with WT-Sed group,the m RNA levels of PDIA4,PDIA5 and HSPA1B in hippocampus of WT-Exe mice were significantly decreased;the m RNA levels of PDIA2,PDIA3,PDIA4,PDIA5,PDIA6,HSPA1B,HSPA8,HSP90B1,DNAJB2,CRYAB,CNX and CRT,and the protein level of CRT in hippocampus of APP/PS1-Sed mice were significantly decreased;the m RNA levels of ERO1L in hippocampus of APP/PS1-Exe mice was significantly increased,while the m RNA levels of PDIA3,PDIA4,PDIA5 and HSPA1B were significantly decreased.Compared with WT-Exe group,the m RNA levels of ERO1L and the protein level of CRT in hippocampus of APP/PS1-Exe mice were significantly increased,while the m RNA levels of PDIA3,PDIA6 and CRT were significantly decreased.Compared with APP/PS1-Sed group,the m RNA levels of ERO1L,PDIA2,PDIA4,PDIA6,HSPA1A,HSPA8HSP90AB1 and DNAJB2,and the protein levels of CRT in hippocampus of APP/PS1-Exe mice were significantly increased.Compared with WT-Sed group,the co-localization level of APP/GRP78 and the average fluorescence intensity of APP and GRP78 were significantly increased in hippocampus of APP/PS1-Sed mice;the co localization level of APP/GRP78 was significantly increased in hippocampus of APP/PS1-Exe mice.Compared with the APP/PS1-Sed group,the co-localization level of APP/GRP78 and the average fluorescence intensity of GRP78 in the DG region of hippocampus in APP/PS1-Exe mice were significantly decreased.（3）Compared with WT-Sed group,the protein levels of p-PERK,p-e IF2α,ATF4,p-IRE1,XBP1u,XBP1s,CHOP,CASPASE12 and BAX,the ratio of BAX/BCL2,the average fluorescence intensity of ATF4,the m RNA level of XBP1s and the number of TUNEL positive cells in hippocampus of APP/PS1-Sed mice were significantly increased.Compared with APP/PS1-Sed group,the protein levels of p-PERK,p-e IF2α,ATF4,p-IRE1,XBP1s,CHOP,CASPASE12 and BAX,the ratio of BAX/BCL2,the m RNA level of XBP1s and the number of TUNEL positive cells in hippocampus of APP/PS1-Exe mice were significantly decreased.（4）Compared with WT-Sed group,the protein level of VCP in hippocampus of APP/PS1-Sed mice was significantly decreased,and that of HERPUD1 was significantly increased;the protein level of DERL2 in hippocampus of APP/PS1-Exe mice was significantly increased.Compared with APP/PS1-Sed group,the protein levels of VCP and DERL2 in hippocampus of APP/PS1-Exe mice were significantly increased,while the protein level of HERPUD1 was significantly decreased.Compared with WT-Sed group,the m RNA level of ATG5 in hippocampus of WT-Exe mice was significantly increased,while the m RNA level of ATG7 was significantly decreased;the m RNA levels of BECN1 and ATG7 in hippocampus of APP/PS1-Sed mice was significantly decreased,the m RNA level of ATG12 and the protein levels of LC3Ⅱand P62 were significantly increased,the average fluorescence intensity of CRT in DG region was significantly decreased,and the average fluorescence intensity of LC3 in DG region was significantly increased;The m RNA level of ATG7 in hippocampus of APP/PS1-Exe mice was significantly decreased,the m RNA level of ATG12 was significantly increased,and the average fluorescence intensity of LC3in DG region was significantly increased.Compared with WT-Exe group,the m RNA levels of BECN1 and ATG5 in hippocampus of APP/PS1-Exe mice were significantly decreased,while the m RNA levels of ATG12 and the average fluorescence intensity of LC3 in DG region were significantly increased.Compared with APP/PS1-Sed group,the m RNA level of ATG12 in hippocampus of APP/PS1-Exe mice was significantly increased,the protein level of P62 was significantly decreased,and the average fluorescence intensity of CRT in DG area was significantly increased.ConclusionsThree months of treadmill exercise could prevent APP/PS1 transgenic induced learning and memory deficits,the increase of Aβand p-Tau production,and the down-regulation of synaptic associated protein expression.The biological mechanism may be related to the maintenance of endoplasmic reticulum proteostasis,specifically as follows:（1）In regulating protein synthesis,treadmill exercise can not only promote endoplasmic reticulum protein folding and quality monitoring in the hippocampus,thus reducing APP accumulation in the endoplasmic reticulum and alleviating ERS,but also prevent the over activation of ERS by down regulating IRE1α/XBP1 signal;Furthermore,by down regulating PERK/e IF2αsignal,the expression of BACE1 and PS1,the activity of GSK3βand the apoptotic signal of CHOP were decreased,and then the production of Aβ,p-Tau and the apoptosis of hippocampal neurons were decreased;In addition,the expression of synapse related proteins may be up-regulated by restoring the de novo synthesis of proteins.（2）In regulating abnormal protein degradation,treadmill exercise can promote the reverse cytoplasmic transport of misfolded proteins and proteasome degradation pathway of ERAD,improve autophagy activity,and up-regulate the coupling of ERS and autophagy mediated by CRT,thus reducing the aggregation of misfolded proteins in brain of AD.