Molecular Regulatory Mechanism Of Neurodegenerative Processes In MPTP/probenecid-induced Progressive Parkinson's Disease Mice Revealed By Transcriptome

Objective Parkinson's disease(PD)is a neurodegenerative disease caused by a variety of complex pathogenic factors.The progressive PD mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid(MPTP/p)is recognized as a classic PD model,but the neurodegenerative process of this model is still unclear.This study was designed to explore the progressive neurodegeneration process in MPTP/p-induced progressive PD mice by RNA-seq and bioinformatics analysis,and to explore the molecular dynamic changes of neurodegeneration process in substantia nigra of progressive PD model mice at the transcription level,so as to provide scientific basis for exploring potential drug targets and treatment strategies of PD.Methods 1.The MPTP/p-induced progressive mouse PD model was established according to precious research[1].12 week-old C57BL/6J mice were randomly divided into the model group and the control group.The model group was intraperitoneally injected with MPTP(25 mg/kg)and probenecid(250 mg/kg),while the control group was intraperitoneally injected with saline,twice a week for 5 weeks.After 3,6 and 10 times of intraperitoneal injection,the olfactory test,the beam traversal test and the pole test were used as behavioral indicators to detect the changes of non-motor and motor function in mice.Meanwhile,the changes of dopaminergic neurons were detected by TH immunofluorescence to identify the preparation of the model.2.Total RNA was extracted from the substantia nigra of the model group and the control group at specific time points(after 3rd,6th and 10th injections),and also the normal group.The total RNA was used for RNA-seq by BGI-500 platform.The differentially expressed genes between the model group and the control group at different time points during MPTP/p-induced progressive PD model mice were identified.Next,a soft clustering strategy using Mfuzz was employed to overview expression patterns of DEGs in normal and model groups,and then we identified the top 20 KEGG pathway enrichment and GO enrichiment of DEGs between the group and the control group at different time points.The analysis of KEGG pathway and GO enrichment illustrated that the neurodegenerative process of MPTP/p-induced progressive PD model mice was divided into three transcriptional reaction stages;after that gene set enrichment analysis(GSEA)was used to explore the relevant correlation among "Parkinson's disease" gene set,MPTP/p injections and identify leading-edge subsets that accounts for the enrichment signal;Finally,qRT-PCR was used to verify the quality of RNA sequencing data.Result(1)Behavioral experiments showed that the olfactory and the motor functions of MPTP/p-induced progressive PD mice were gradually damaged with the increase of the injection times,and the behavior decreased significantly after the 10th injection(p<0.01);(2)TH immunofluorescence results showed that with the increase of MPTP/p administrations,the number of TH positive cells in MPTP/p-induced progressive PD mice decreased gradually,and reached the minimum after the 10th injection(p<0.01);(3)KEGG pathway ang GO enrichment analysis showed that the transcription response of MPTP/p-induced progressive PD mice could be divided into three stages:"stress response phase" maintained stable microenvironment,"prophase of neurodegeneration" showed significant MPTP/p cytotoxicity and gradual degeneration of dopaminergic neurons,and "neurodegenerative phase" reflected obvious damage of dopaminergic neurons;(4)glial cells can protect dopaminergic neurons induced by MPTP/p after 3rd and 6th MPTP/p injections,but after 10th MPTP/p injection,glial cells play a promoting role in oxidative stress-induced PD and tissue damage;(5)this study also showed that the substantia nigra of PD mice presents a unique change pattern in each stage.The enhancement of the function through neurotropic signal pathway,ECM-receptor interaction,oxidative phosphorylation,apoptosis and necrosis may be related to the occurrence and development of PD.Conclusion The neurodegerative process of MPTP/p-induced progressive PD model mice were preliminarily explored by RNA-seq,and the regularity and network relationship between molecular regulatory mechanism and biological phenotype of the model were revealed.RNA-seq analysis showed that the transcriptional response in MPTP/p-induced progressive PD mice could reflect three stages:stress response stage,neurodegenerative phase and neurodegenerative phase,and eventually lead to the loss of dopaminergic neurons.In the early stage,the level of neurotrophic related genes increased,which may explain to some extent that DA neurons did not decrease significantly in this stage.The dynamic changes of genes involve neurotrophic signaling pathway,ECM-receptor interaction,oxidative phosphorylation,apoptosis,necrosis and dopaminergic synapses.This study provides a wider range of dynamic molecular changes at the transcriptional level for understanding the disease process of Parkinson's disease,and provides more comprehensive theoretical knowledge for the study of potential therapeutic targets of Parkinson's disease.