| Parkinson's disease?PD?is the second most common neurodegenerative disease after Alzheimer's disease?AD?.PD is characterized by bradypragia,tremble,numbness in limbs,at the same with advanced nerve dysfunction complication such as dementia,depression,and petients even lost life self-care ability.This not only impact on the patient's physical and mental health seriously,but also bring heavy burden to patients' families and the society.Currently the pathogenesis of PD is recognized as the degeneration and death of dopamine?DA?neurons in the substantia nigra pars compacta and the formation of Lewy bodies which contain alpha-synuclein?a-Syn?as the main ingredients of eosinophilic inclusions.Glutamic acid?Glu?is the main neurotransmitter of central nervous system excitatory synaptic transmission.In order to end up neurotransmitter of glutamate and keep the extracellular glutamate concentration at low levels,glutamic acid released by nerve endings is recycle back into the cytoplasm by the glutamate transporte of glial and rneurons cells.As a consequence the excitability neurons could avoid suffering from high levels toxicity of excitatory effect.Among the numerous pathology theories of PD,the theory of excitatory amino acids toxicity has been the hot topic all over the world,and glutamate transporter plays a key role in this theory.There are many studies have shown that the abnormal function and the descending uptake ability of glutamate transporter are closely related to the pathogenesis of PD.Eukaryotes high affinity glutamate transporter?excitatory amino acid from,EAAT?mainly divides into five subtypes,and among the five subtypes,GLAST?EAAT1?and GLT-1?EAAT2?play an important role.Eliminating the glutamate that accumulate in the intercellular spaces and preventing glutamate excitotoxicity are mainly accomplished by GLT-1 and GLAST.GLT-1 is widely distributed in the central nervous system,mainly expressed on the membrane of astrocytes in the cerebral cortex,hippocampus,forebrain,striatum and so on.In addition,about 90%of glutamate can be cleared by GLT-1.Although the change of glutamate transporter in protein level plays an important role on its function,the phase regulation of mRNA translation to generate the glutamate transporter protein plays a vital effect on the expression and function of glutamate transporter,and microRNA?miRNA?is one of the important regulatory factors in the phase.microRNA is a kind of endogenous noncoding RNAs,which size is among 20-25nt.miRNAs play the role of regulation by degrading target mRNA or inhibiting target mRNA translation with the 3' end of the target genes the translation section?3' untranslated regions,3'UTR?complete complement or incomplete matching.At present,a lot of microRNAs have been revealed that are relate to neurodegenerative diseases,some of which have been found involved in Parkinson's disease process.Though the microRNAs associated with Parkinson's disease have been found and some have identified the target genes,some reported that miR-124 may be involved in regulation of glutamate transporter GLT-1 expression,but the microRNA that play to the role of regulation of glutamate transporter in Parkinson's disease has not been reported,it is worth us to explore further.Therefore,this study mainly research the change of expression and function of glutamate transporter GLT-1,and find the microRNA which is play a part in the change.We try to improve the expression and function of GLT-1 by intervening in specific microRNA to reduce the neurotoxic effect of glutamic acid and alleviate Parkinson's disease.It may provide new targets for clinical treatment of Parkinson's disease.Part ? The change of expression and function of GLT-1 in the course of PDFirst,We created PD mice model with MPTP and the model was considered to be acute model of Parkinson's disease,which were verified by immunehistochemical and behavioral experiments.Previous studies had reported that EAATs function abnormalities and decline of glutamate uptake were closely related to the occur of Parkinson's disease.With declining of EAATs expression in PD,its ability of glutamate uptake was decline.As a consequence,it leaded to the rise of extracellular glutamate levels and increase the toxic effect of neurons.In our study,at the three days after the MPTP injection,one day and three days after the created PD mice models,we separated the midbrain,striatum hippocampus and cerebral cortex from certain number of mice,and examined the expression of GLT-1 and the activity of synaptosome.The protein expression of GLT-1 was reduced in the striatum and midbrain in three days after the created PD mice model compared with control by Western blotting.Comparing with the control,the GLT-1 mRNA expression of PD mice models in different brain tissues were reduced at different time points by RT-PCR.The results showed that GLT-1 protein and mRNA expression level in the midbrain changed most significantly at 3 days after the created of PD mice models.Although the change of protein lever is important for GLT-1 expression and function,the mRNA translation phase plays an important role for its expression and function and microRNAs?miRNA?is one of the important regulatory factors.In addition,there were a large number of GLT-1 on postsynaptic membrane,we tested the synaptic activity from different part of brain at the different time by specific substrate of glutamate transporter with radioactive element tag.It can indicate the changing of GLT-1 transport function.The results showed that the retake activity of glutamate transporter of each part of the brain detection was reduced in PD mice compared with control.In this part of our study,the expression and function of GLT-1 were significantly decreased in the process of PD,and this implied that GLT-1 may play a important role in the pathogenesis of PD.In addition,the midbrain GLT-1 protein and mRNA expression level changed most significantly at the three days after created the PD mice,and the mRNA translation phase plays an important role for GLT-1 expression and function.Consequencely,the expression and function of GLT-1 can be improved by intervening in the post-transcription regulation.microRNA is an important factor,and specific miRNA also may be a new target for the treatment of PD.Part ? Filter miRNAs differential expression in PD mice by High throughput sequencing and function analysis for these miRNAsmiRNA different expression was a hot focus in the study of miRNA,and abnormal expression of miRNA was associated with many diseases,including Parkinson's disease.miRNA was closely related with the occurrence and development of PD,miRNA often can present different expression patterns in the process of the occurrence of diseases.In this part of the study,we screened different expression of miRNAs by high-throughput sequencing between the PD mice?n=3?and control mice?n=3?.The results showed that 12 miRNAs expression were abnormal in the midbrain after the created PD mice model compared with control,6 of them were up-regulated and others were down-regulated.Then,6 miRNAs of the abnormal expression miRNAs?mmu-miR-30a-5p,mmu-miR-543-3p,mmu-miR-1197-3p,mmu-miR-6538,Cluster141,Cluster59?was verified by RT-PCR,the results were basically consistent with high-throughput sequencing,and that can indicate the reliability of high-throughput sequencing.All above results showed that there were differentially expressed miRNAs in the midbrain between PD mice and control mice,that may play an important role in the development of PD.We obtained 12 differentially expressed miRNAs in the midbrain of PD mice by high-throughput sequencing and analyzed the miRNAs by bioinformatics.The target genes of differentially expressed miRNAs were predicted with TargetScan databases,miRanda database,miRDB database and so on.What delighted was that we found glutamate transporter GLT-1 was one of target genes of mmu-miR-30a-5p and mmu-miR-543-3p.At the same,We predicted the miRNA which target genes was GLT-1 by TargetScan databases,miRanda,miRDB databases and so on,the results showed that the mmu-miR-30a-5p and mmu-miR-543-3p were involved in the miRNAs.Not only the miRNA but also gene were verified the the relationship of glutamate transporter GLT-1 with mmu-miR-30a-5p and mmu-miR-543-3p,so we get mmu-miR-30a-5p and mmu-miR-543-3p as an importantly alternative miRNA in the follow-up study.Part ? Research on miRNAs which play the regulatory role of GLT-1Through forecasting of the miRNA target genes in the second part,we found that glutamate transporter GLT-1 was the target gene of mmu-miR-30a-5p and mmu-miR-543-3p.So we study the regulatory effect on GLT-1 of mmu-miR-30a-5p and mmu-miR-543-3p in this part.Overexpression or inhibits of mmu-miR-30a-5p and mmu-miR-543-3p had an influence on expression and function of GLT-1 by cell experiment.The miR-30a-5p inhibitors?reverse inhibit sequence of miR-30a-5p?,miR-30a-5p mimics?simulate sequence of miR-30a-5p?and the miRNA-543-3p inhibitors?reverse inhibit sequence of miRNA-543-3p?,miR-543-3p mimics?imulate sequence of miR-543-3p?were added into the MPP+ infected astrocytes respectively.The total protein and membrane protein expression of GLT-1 were detected by Western blotting and the results showed that the GLT-1 protein expression was significantly increased after miR-30a-5p inhibitors treatment compared with the MPP+ infected astrocytes.In contrast,protein expression was significantly lower with miR-30a-5p mimics treatment.miR-543-3p inhibitors and mimics had the same influence on the protein expression of GLT-1 as miR-30a-5p inhibitors and mimics.The mRNA expression of GLT-1 was significantly increased after miR-30a-5p inhibitors treatment compared with the MPP+ infected astrocytes by RT-PCR.In contrast,the mRNA expression was significantly lower after miR-30a-5p mimics treatment.miR-543-3p inhibitors and mimics had the same influence on the mRNA expression of GLT-1 as miR-30a-5p inhibitors and mimics.In addition,we also tested the GLT-1 transport activity of different treatment cells,and the results showed that GLT-1 transport activity was significantly increased after miR-30a-5p inhibitors treatment compared with the MPPr infected astrocytes.In contrast,the transport activity was significantly after miR-30a-5p mimics treatment.miR-543-3p inhibitors and mimics had the same influence on GLT-1 transfer activity as miR-30a-5p inhibitors and mimics.Above all,cell experiments results showed that the mmu-miR-30a-5p and mmu-miR-543-3p had influence on expression and function of GLT 1.Whether the two miRNAs effects on GLT-1 was direct or not?In order to confirm this problem,we had the dual-luciferase reporter gene experiment to confirm if there was a direct binding site between the two miRNAs and GLT-1.The results showed that there was no direct binding site between miR-30a-5p and GLT-1,which indicated that GLT-1 was not the target gene of GLT-1 and miR-30a-5p regulated the expression of GLT-1 indirectly.However,there was a direct binding site between miR-543-3p and GLT-1,which indicated that GLT-1 was the target gene of miR-543-3p and GLT-1 can be regulated by miR-543-3p directly.In other word,miR-543-3p is the miRNA we're looking for that play a role of regulation to GLT-1 directly in PD.Then we study the changing of glutamate transporter GLT-1 that regulated by mmu-miR-543-3p in vivo.In this part,we contrusted the green fluorescent protein labeld lentivirus containing mmu-miR-543-3p reverse inhibit sequence,then the lentivirus was injected unilateraliy into the substantia nigra of mice by the brain stereotaxic instrument positioning.After the lentivirus injection,we isolated the midbrain and extracted the total protein,membrane protein,total RNA and complete synaptosome to detect the GLT-1 protein,mRNA expression level and synaptic activity respectively.The total and membrane protein expression of GLT-1 were significantly increased in the midbrain after the lentivirus injection compared with PD mice by Western blotting,and the mRNA expression of GLT-1 was elevated significantly in the midbrain after the lentivirus injection compared with PD mice by RT-PCR,Similarly,synaptosome activity was increased significantly in the midbrain after the lentivirus injection compared with PD mice.Above all,the experiments confirmed that the mmu-miR-543-3p had an important regulatory influence on glutamate transporter GLT-1 in PD mice.In conclusion,our research firstly proved that the expression and function of GLT-1 were reduced in the process of occurrence and development of PD.Then we screened out 12 differentially expressed miRNAs and analyzed them by bioinformatics.We predicted that mmu-miR-30a-5p and mmu-miR-543-3p perhaps regulate GLT-1.Finally,we confirmed that mmu-miR-30a-5p could regulate GLT-1 indirectly,mmu-miR-543-3p could regulate GLT-1 directly and GLT-1 was the target gene of mmu-miR-543-3p.In addition,the results showed that the expression and function of GLT-1 were increased after inhibiting mmu-miR-543-3p.Our studies provide theoretical and experimental basis for the miRNA that can regulate GLT-1 and be a new therapy of PD. |
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