| Physical inactivity or sedentary behavior is closely associated with the occurrence and development of various chronic non-communicable diseases,seriously endangering the population’s health.Exercise,on the contrary,can improve the metabolism and promote the health of multiple systems,such as the heart,lungs,and brain.Specifically,exercise can promote or improve hippocampus-dependent learning and memory functions via inducing adult neurogenesis,accelerating the maturation of new neurons,increasing the volume of(human)hippocampus,promoting angiogenesis,and producing neurotrophin.However,the underlying molecular mechanisms still remain unclear.Objective:1.To explore the temporal characteristics of the mRNA expression of histone acetyltransferases(HATs)and deacetylases(HDACs)in mouse hippocampus during the 24 hours’ recovery phase after a single bout of high-intensity exercise.2.To explore the role of lysine acetylation modification in the remodeling of hippocampal functions after moderate-intensity chronic exercise.3.To explore the difference of high-intensity exercise at different time in one day(rest phase in the early morning and active phase in the early evening)upon hippocampal molecular functions through protein phosphorylation.Methods:1.Groups of exercise mouse model with different recovery periods after a single bout of high-intensity exercise(0h,0.5h,1h,4h,7h and 24h)were constructed,as well as a group of sham-exercise mouse model.The mRNA levels of HATs and HDACs were detected by real-time polymerase chain reaction(RT-qPCR).Western blot was used to detect the lysine acetylation on the whole proteins and some specific locations of histone,as well as various acylation modifications on the total proteins.2.A moderate-intensity chronic exercise mouse model and a sedentary control group were constructed.Novel object recognition test was used to assess short-term memory of recognizing new objects,and Y-maze test was used to assess short-term spatial memory.Multiplex immunofluorescence staining was used to detect markers of adult neurogenesis(BrdU,NeuN and DCX).The hippocampal lysine acetylome and proteome were performed by affinity enrichment and liquid chromatography-tandem mass spectrometry(LC-MS/MS).Thereafter,a series of bioinformatic analysis was applied,including posttranslational modification(PTM)correlation analysis,subcellular distribution and protein domain analysis,functional enrichment analysis and protein-protein interaction(PPI)network,etc.3.At both the rest phase in the early morning(ZT3)and the active phase in the early evening(ZT15),the acute high-intensity exercise mouse model and the sham-exercise group were established.The hippocampal phosphoproteome and proteome were detected by affinity enrichment and LC-MS/MS.Aspects of differentially regulated phosphorylated sites and proteins between the rest and active phase were compared,including functional enrichment analysis,PPI network analysis,motif analysis and kinase prediction,etc.Results:1.(1)Except the four unaffected genes(Hdac4,Ncoa1,Ncoa2,Sirtl),acute highintensity exercise changed the mRNA expression trajectories of other 21 HATs or HDACs genes:genes in Cluster 1 increased quickly following exercise with a peak at 0.5h and/or 1h,and remained at a high expression level till 24h post exercise;Cluster 2 genes presented a gradual increase with a delayed peak at 4h or 7h post-exercise before returning to baseline;Cluster 3 genes decreased in response to exercise at 0.5h and/or 1h with some rebounding to overexpression(Hdac1 and Sirt3).(2)Acute exercise neither affected the global nor residue-specific histone acetylation levels at 0.5h post-exercise.(3)Immediately after exercise,metabolism related acylation on the total proteins,including acetylation,succinylation,2-hydroxyisobutyryllysine,β-hydroxybutyryllysine and lactylation displayed a decreased trend and mainly occurred on non-histones.2.(1)Chronic moderate-intensity exercise significantly improved novel object recognition memory and spatial working memory,and strengthened the expression of NeuN,BrdU and DCX.(2)A total of 3,876 acetyl sites on 1,764 proteins were identified,which were predicted to distribute in extensive subcellular compartments,with cytoplasm and mitochondria the most dominant.(3)PTM correlation analysis showed that 2,423 novel acetylation sites were identified in our data,and that various PTMs might occur at or within six residues of our identified lysine sites.(4)Acetylation sites that were upregulated by chronic exercise tended to be near acidic amino acids while the other tended to be near basic amino acids.(5)272 acetyl sites on 252 proteins were significantly regulated,and they were predicted to be significantly enriched in coil compared with the non-modified lysine residues.(6)Functional enrichment analysis showed that differentially acetylated proteins were highly enriched in carbon-related metabolism(carbohydrate digestion and absorption,glycolysis/gluconeogenesis,fructose and mannose metabolism,and pyruvate metabolism),Hippo signaling pathways,ribosomes,and protein processing.(7)18.58%differentially acetylated proteins were annotated in mitochondria,and enriched in pathways of citrate cycle,butanoate metabolism,fatty acid metabolism,amino acid catabolism,and oxidative phosphorylation.(8)Only 21 proteins were significantly expressed and enriched in the pathway of complement and coagulation cascades.3.(1)Acute high-intensity exercise at ZT3 and ZT15 significantly changed the modification level with 7.8%(932/11970)and 6.9%(828/12,007)of phosphorylation sites,respectively,corresponding to 24.9%(648/2,607)and 22.3%(585/2,619)of proteins.There were only 49 overlapping differentially regulated sites on 44 proteins.The proportion of up-regulated phosphorylation sites was similar with that of down-regulated sites in both phases.(2)Functional enrichment analysis displayed that ZT3 exercise exerted a broader impact on the biological function of hippocampus.Enriched pathways in ZT3 phase were dominated by upregulated phosphorylation proteins while pathways in ZT15 were dominated by downregulated phosphorylation proteins.(3)Proteins with upor down-regulated phosphorylation sites in both phases were enriched in some identical pathways,but their sites were different(such as glutamatergic synapses,dopaminergic synapses,and Ras signaling pathways);The phosphorylation states of proteins enriched in some identical pathways were diametrically opposite between ZT3 and ZT15(such as calcium signaling pathways,Wnt signaling pathways,insulin and gastric acid secretion).(4)There were 29%differentially phosphorylated proteins related to the structure or function of synapses.Exercise at both ZT3 and ZT15 caused extensive changes in synaptic pathways like glutamate synapse and dopamine synapse.(5)Kinase-substrate predictions showed that exercise at ZT3 and ZT15 activated multiple kinases,including PKC and CAMK2.Meanwhile,13 kinases were predicted to have converse activity.(6)Both exercise at ZT3 and ZT15 might significantly affect the pathway of the glutamate synapse-calcium signaling pathway-long-term potential in hippocampus.(7)Exercise at ZT3 and ZT15 induced expression changes of 27 and 15 proteins,respectively,and no core proteins related to circadian rhythm were detected.(8)Blood glucose decreased only after ZT15 exercise,when the phosphorylation of proteins enriched in the insulin secretion pathway was downregulated,and the expression of proteins enriched in the cellular response to insulin stimulus was also downregulated.Conclusions:1.During the 24 hours’ recovery phase after acute high-intensity exercise,the transcriptional trajectory of HATs or the same class of HDACs in hippocampus were distinct.Though acute exercise didn’t affect the selected sites on histone lysine,it possibly incurred acetylation as well as other acylation on non-histone proteins.2.Moderate-intensity chronic exercise enhanced both memory and adult neurogenesis.Chronic exercise also induced changes in the lysine acetylation modification and related biological function,which partially reveals the molecular mechanism of the benefits underlying exercise upon on the hippocampal functions.3.The changes in the phosphorylation and expression of hippocampal proteins at different time of a day were similar,but acute exercise at ZT3 exerted a broader impact on the biological functions.They both significantly affected pathways related to synaptic function but with distinct phosphorylation sites or states.This reveals the characteristic changes of circadian rhythm on the exercise physiology of hippocampus,and enriches the current exercise chronobiology database. |
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