Molecular Mechanism Of Exercise Training Improving Neural Function In Key Brain Regions In Parkinson’s Disease

Parkinson’s disease（PD）is a neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra and Lewy bodies.A number of clinical trials and animal experiments have shown that exercise training can effectively improve motor balance disorders in PD patients,and the molecular mechanism may involve exercise promoting dopamine secretion,protecting mitochondrial function,improving inflammation,etc.However,different brain regions have different degrees of involvement in the occurrence and development of PD,and exercise has different effects on each brain region.At present,it is not clear the difference of the effect of exercise training on the brain regions related to motor regulation,nor the mechanism of the influence of the body’s extracerebral differences on the brain regions related to the brain.Therefore,it is necessary to more systematically and comprehensively evaluate the differences in gene expression in the motor cortex,striatum,substantia nigra and cerebellum before and after PD training and the possible non-central regulatory effects,so as to elucidate the molecular mechanism of exercise training correcting balance disorders in PD.PartⅠStudy on the pathological and motor symptoms in PD mice by exercise training ObjectiveTo investigate the changes of gene expression levels in related brain regions（cortex,cerebellum,substantia nigra and striatum）of PD mice before and after exercise training.To investigate the most important brain regions in response to exercise and elucidate the molecular mechanism of training improving motor symptoms in PD mice.Methods1.After 5 days of training,4 mice were randomly assigned to each group,including Saline group,MPTP group and MPTP +EX group.After anesthesia,PBS heart perfusion was used to remove the whole brain as soon as possible,and the cerebellum was removed from the ice disc.Striatum,substantia nigra and motor cortex of the brain were separated under anatomical microscope,and total RNA was extracted.2.Gene expression profile detection: 500 ng Total RNA was extracted from each sample as experimental material,and Ion Total RNA-SEQ Kit V2 was used for c DNA library construction.Library quality and concentration were measured using Qubit Fluorometer（Invitrogen,USA）.Then diluted 10 p M samples were taken from each sample c DNA library and sequenced using the Ion Proton^TM System.3.Analysis of gene expression profile: Bioinformatics analysis software was used to conduct in-depth analysis of gene coverage and sequencing,analysis of gene expression differences（cluster analysis,GO analysis,Pathway analysis）,transcription factor analysis,RHHO analysis,and screening of key regulatory sites and molecular regulatory mechanisms.4.Another 5 mice from each group were anesthetized,their hearts were perfused with PBS,their brain tissues were removed,fixed and embedded,and their potential regulatory mechanisms were further verified by immunofluorescence experiments.Results1.The results of behavioral experiment showed that the motor ability of MPTP group was significantly decreased in the three indexes of rod rotation,rod climbing and open field,and significantly improved after exercise training.2.Compared with the Saline group,the number of TH positive dopaminergic neurons in the MPTP group and the MPTP+EX group was significantly reduced by 70%,suggesting that exercise training could not restore the number of lost DA neurons in the substantia nigra area of PD mice.Conclusion The number of DA neurons in the substantia nigra decreased and motor behavior was impaired in PD mice.And exercise improved the impaired behavior of PD mice,but did not increase the number of DA neurons in the substantia nigra of PD mice.PartⅡStudy on the effection and mechanism of exercise training on gene expression in related brain regions ObjectiveTo explore the mechanism of exercise training on the expression of genes in motor and PD-related brain regions such as cortex,cerebellum,substantia nigra and striatum in PD mice,find the most important brain regions responding to exercise and clarify the molecular mechanism of improvement of PD motor symptoms after exercise training.Methods 1.5 days after the training,5 mice from the Saline group,MPTP group and MPTP+EX group were randomly selected and anesthetized.After heart perfusioned with PBS,the whole brain was separated as soon as possible,and the cerebellum were separated on the icing disk.2.Gene expression profile: 500 ng Total RNA was extracted from each sample as experimental material,and Ion Total RNA-SEQ Kit V2 was used for c DNA library construction.Library quality and concentration were measured using Qubit Fluorometer（Invitrogen,USA）.Then diluted 10 p M samples were taken from each sample c DNA library and sequenced using the Ion Proton^TM System.3.Gene expression profile analysis: Bioinformatics analysis software was used to conduct in depth analysis of data,gene coverage and sequencing in depth analysis,gene expression difference analysis（cluster analysis,GO analysis）,transcription factor analysis,RHHO analysis,and screening of key regulatory regions and mechanisms.4.After anesthesia,the remaining 4-5 anesthetized mice in each group were with perfusioned with PBS and PFA,and the brain tissues were separated for fixed and embedded sections to further verify the potential mechanism.ResultsAfter PD exercise training,cortex,cerebellum,substantia nigra and striatum were analyzed,for instance:1.The correlation analysis of the four related brain regions found that the substantia nigra and striatum were the most prominent in response to PD and exercise training in PD,suggesting that the substantia nigra and striatum system may be the key brain for the improvement of motor symptoms after exercise training in PD.2.The four brain regions were analyzed respectively.Phosphoinositide 3-kinase signaling and Vascular associated smooth muscle cells were observed in the cortex.Up-regulation of migration,positive regulation of cell motility,epithelial cell proliferation,endothelial cell development and other metabolic and angiogenesis pathways.In cerebellum region showed little response to PD and exercise training.and circadian regulation of gene expression was differentially expressed pathway,which mainly reflected the downregulation of circadian rhythm-related rhythmic process after exercise training.The differentially expressed genes in striatum were mainly concentrated in response to nerve growth factor,regulation of translation at synapse and modulating synaptic.Transmission and other pathways associated with synaptic increase and improvement in synaptic function are upregulated.In addition,we also found downregulation of pathways associated with ubiquitination.The variation pattern of differential genes in the substantia nigra was mainly up-regulated and down-regulated after modeling.Synaptic transmission glutamatergic,signal release and regulation of postsynaptic neurotransmitter receptor were identified by pathway enrichment analysis,glutamate receptor signaling pathway and other glutamate neurotransmitter related pathways.At the same time,we found that response to growth factor,vasculature development and other pathways of angiogenesis and neuronutrition were up-regulated.Further,transcription factor analysis showed that genes related to neurongenesis（Rtn4rl2,Neurod1,Neurod6,etc.）and inflammation（Cd5,F7）were enriched.3.Analysis of inflammation-related genes in the substantia nigra showed that inflammation-related factors IL-16,IL-22 and Cd5 were significantly down-regulated after exercise training,and Socs3,which regulates the transformation of microglia from M1 to M2,was up-regulated after exercise.4.Correlation analysis of PD and four brain regions after exercise training was conducted in pairs,and the results showed that the substantia nigra striatum pathway plays an important role in PD and exercise training.ConclusionBy analyzing the differences of genes in cortex,cerebellum,substantia nigra and striatum before and after PD exercise training,it was revealed that after exercise training:cerebellum area changes mainly limited in circadian rhythm related pathways.In cortex,pathway change embodied in the new angiogenesis of active.Genetic changes in the striatum of nutritional factors reaction,neurogenesis,synaptogenesis and transmission way raised after exercise.And in the substantia nigra,most response exercise intervention PD differences in gene number,Inflammation in the substantia nigra was inhibited,glutamate-related pathways were inhibited,and pathways related to memory,axon guidance,and synaptic assembly were upregulated.The correlation analysis of RRHO expression pattern successfully identified that the gene expression pattern between the substantia nigra and striatum was highly similar in PD with exercise.Part III Study on the mechanism of exercise training improving intracerebral and extracerebral inflammation in PD mice ObjectiveTo explore the relationship between exercise training and PD inflammation,lay a foundation for regulatory targets.Methods1.Through the analysis of peripheral blood transcriptome data of mice in the Saline group,MPTP group and MPTP+EX group,the changes of peripheral inflammatory pathways and molecules were revealed.2.Immunofluorescence staining was used to observe the changes of microglia in the substantia nigra of mice in the Saline group,MPTP group and MPTP+EX group.3.Intestinal flora of Salien group,MPTP group and MPTP+EX group were analyzed by 16 S r RNA sequencing.4.MPTP-induced subacute PD model was constructed.After modeling,feces of mice in this group were collected and transplanted to normal mice.Feces,peripheral blood and brain tissue were collected after completion.5.16 S r RNA sequencing was performed on MPTP-induced subacute model mice,fecal microbiota transplantation（FMT）group and fecal bacteria control group mice to explore the changes of intestinal flora of mice after PD modeling and verify whether intestinal bacteria transplantation was successful in FMT group mice.6.Changes of inflammatory pathways and molecules in peripheral blood of MPTP group,FMT group and Saline group were analyzed by peripheral blood transcriptome data.7.Immunofluorescence staining was used to determine the loss of DA neurons and the changes of microglia in the substantia nigra of MPTP-induced subacute model mice,FMT group mice and Saline group.Results1.Exercise training can inhibit peripheral inflammation in PD mice and PD patients.RNA-Sequencing analysis of PD expression profile in peripheral blood after exercise training showed that Th17 cell differentiation（T cell receptor signaling Pathway）was down-regulated in peripheral blood after exercise training.Further,analysis found that the expression of inflammation-related genes in the pathway was decreased in MPTP+EX group.2.Immunofluorescence staining of microglia in the substantia nigra further confirmed that the inflammation in the substantia nigra was corrected after exercise intervention,and that the inflammatory changes in the substantia nigra were closely related to the improvement of PD exercise symptoms.3.Exercise training can change the intestinal flora of PD mice.After exercise training,the β diversity and OTU levels of intestinal flora in mice were more similar to those in Saline group.Further analysis of genus level proportion of intestinal bacteria showed that Lactobaxillus and Akerrmansia increased while Lachnospiraceae decreased after exercise training.The function prediction analysis of intestinal bacteria showed that the function of intestinal bacteria after exercise was more similar to that of the Saline group.HIF-1signaling Pathway,AMPK signaling Pathway,Alzheimer’s disease,Huntington’s disease,Parkinson’s disease,Insulin signaling Pathway,IL-17 signaling Pathway /Th17 cell differentiation and other pathways are reversed after exercise.4.After MPTP modeling,the diversity of intestinal flora in mice decreased,and intestinal type and principal component analysis were changed.At the same time,normal mice were gacaged facal from MPTP mice.It was found that the diversity,intestinal type and principal component analysis of intestinal bacteria were similar to those of MPTP group.In the Saline group,bacteroidia was the main intestinal type,while bacili was the main intestinal type in PD group and FMT group.Principal component analysis also showed that the intestinal bacteria in PD group and FMT group were more similar.Further analysis on the genus level proportion of intestinal bacteria showed that Lactobacillus,Turicibacter,Lachnospiracea and other intestinal bacteria with specific changes in PD patients were uniformly up-regulated in PD group and FMT group mice.5.Transcriptome analysis of peripheral blood showed that inflammatory pathways in peripheral blood were upregulated after PD modeling and after fecal transplantation from PD model mice to normal mice.6.The number of DAergic neurons and microglia in the substantia nigra was analyzed.The results showed that the number of DAergic neurons in MPTP group decreased by about 30%,but there was no significant change in FMT group,and the number of microglia in MPTP group and FMT group increased significantly.ConclusionExercise improved inflammation in brain and peripheral blood,and the abundance and structure of intestinal microflora in PD mice were partially corrected after exercise.We observed changes in fecal bacteria,peripheral blood and intracerebral inflammation in the group of mice（FMT）by transplantation of intestinal bacteria from PD mice to normal mice.It is suggested that peripheral inflammation（Th17 and other T cell pathways）may be the target of microbial-gut-brain axis mediated PD motor intervention.