The Mechanism And Regulation Effect Of MiR-30a-5p On The Expression Of GLT-1 In A Mouse Model Of Parkinson’s Disease

Parkinson’s disease(PD)is the second common chronic neurodegenerative disease in middle-aged and aged people,which is characterized by the progressive degeneration of dopaminergic(DA)neurons in the pars compacta of substantia nigra(SNpc)and the formation of Lewy bodies containing alpha-synuclein(α-Syn),leading to dopamine neurons degeneration.Thus,the clinical symptoms such as bradykinesia,resting tremor,rigidity,postural instability appeared.So far,there are multiple mechanisms which are believed to contribute to dopaminergic neurons death in PD,including age,environmental factors,genetic factor,neuroinflammation,glutamate excitotoxicity and so on.The effect of glutamate excitotoxicity in the development of PD becomes more and more important,dysfunctional glutamate transporters in astrocytes and neurons will lead to abnormal intake of the intercellular space of glutamic acid causing DA neurons damage,especially glutamate transporter-1(GLT-1,also known as the EAAT2)expressed on astrocytes.Improvement of GLT-1 can keep the extracellular glutamate concentration at a low level in the synaptic cleft,avoiding the excitatory toxicity effect of high glutamate in DA neurons.Therefore,it is of great significance to study the regulation of glutamate transporter.In recent years,microRNA(miRNA)has become a hot spot,a great deal of researches have indicated that dysregulated miRNAs are related to PD,including miR-133b,miR-7,miR-7116-5p,miR-124,miR-126,miR-155 and so on.Some target genes of the miRNAs have been confirmed.Although there have been many reports about the role of miRNAs in PD,there are still little reports about whether MiRNAs regulate GLT-1 in PD,except for the research in our lab.Therefore,what are the miRNAs which regulate glutamate transporter GLT-1 in the pathogenesis of PD?What are the regulatory effects and mechanisms?What does it mean for PD?Can regulating miRNA targeting glutamate transporter GLT-1 achieve the goal of alleviating or treating PD?Can it be a new therapeutic target for the treatment of PD?Solving these problems has a very important clinical significance for the prevention and treatment of PD.This study mainly identified the miRNAs that play a regulatory role in glutamate transporter GLT-1 in PD,clarified its regulation mechanism and its significance in the pathogenesis of PD.Through the intervention of specific miRNA to improve the expression and function of GLT-1 can reduce the neurotoxic effect of glutamate and alleviate the condition of PD,which can provide a new therapeutic target for clinical treatment of PD.Recently we found that miR-30a-5p is a very promising candidate which can regulate GLT-1 based on the results of high-throughput sequencing and bioinformatics analysis in the early stage.So we choose miR-30a-5p for further study.In the previous experiments,our research group used the neurotoxin MPTP to prepare the PD mouse model,then we detected the expression profiles of miRNAs in different regions of the brain(cerebral cortex,substantia nigra,hippocampus,cerebellum)at different time points after using MPTP.The miRNAs differentially expressed were screened out and bioinformatics analysis of miRNAs differentially expressed showed that Slc1a2(the gene name of GLT-1)might be a target of miR-30a-5p.We investigated the effects of miR-30a-5p on the expression and function of GLT-1 in astrocytes.Our results show that MPP+can decrease the expression of GLT-1 in both total and membrane protein.Nevertheless,when treated with miR-30a-5p inhibitor,the expression of GLT-1 increased significantly in astrocytes compared with those exposed to MPP+,and treated with miR-30a-5p mimic could aggravate the reduction of GLT-1 caused by MPP+in both total and membrane protein.We further to explore whether miR-30a-5p affect the function of GLT-1 in MPP+-treated astrocytes,we found that miR-30a-5p inhibitor could improve the abnormal function of GLT-1 caused by MPP+,and miR-30a-5p mimic aggravated abnormal function of GLT-1 caused by MPP+.Finally,we tested if miR-30a-5p could affect the Slc1a2 mRNA,we found MPP+could decrease Slcla2 mRNA but miR-30a-5p inhibitor could reverse this change.Thus,our findings suggest that miR-30a-5p could regulate the expression and function of GLT-1.We further verified the regulatory effect of miR-30a-5p on GLT-1 in MPTP-treated mice.We first constructed a lentivirus vector(LV)containing the miR-30a-5p inhibition sequence labeled the green fluorescent protein markers,and the lentivirus was injected into the pars compacta of substantia nigra by using brain stereotaxic instrument and MPTP administration.We found that the total and membrane protein levels,mRNA levels,function of GLT-1 and the number of tyrosine hydroxylase positive cell were increased significantly in MPTP-treated mice with miR-30a-5p lentivirus compared with those exposed to MPTP.And we found that α-Syn and apoptotic cells were significantly decreased.Taken together,these results confirmed that the miR-30a-5p could regulate GLT-1 in MPTP-treated mice,and revealed that the expression and function of GLT-1 could be enhanced by miR-30a-5p knockdown in MPTP-treated mice.Based on our results that miR-30a-5p could regulate the expression and function of GLT-1 in vitro and in vivo.So we postulated that Slc1a2 might be is one of target genes of miR-30a-5p.Here,we used dual luciferase reporter gene experiments to test this hypothesis.However,the results showed that there was no direct binding site between miR-30a-5p and Slc1a2,which suggested that miR-30a-5p might regulate GLT-1 through an indirect pathway.So we concluded that miR-30a-5p might play an indirect role in regulating GLT-1 in MPTP-treated mice.Due to Slc1a2 was not a target gene of miR-30a-5p,but miR-30a-5p could regulate the expression and function of GLT-1 indeedly,we speculated that miR-30a-5p regulated GLT-1 through an indirect pathway,so we chose some pathway signal molecules which might be related to PD,like PI3K,PKCα,STAT3,mTOR and so on.We found that PI3K,PKCα,STAT3 were activated and mTOR was suppressed in MPTP-treated mice.When MPTP-treated mice were injected with miR-30a-5p lentivirus,PI3K,PKCα,STAT3 and mTOR were rescued to normal.It is indicated that the function of miR-30a-5p on regulating GLT-1 might be combined with one or several pathways through PI3K,PKCα,STAT3,mTOR,and the exact pathway mechanism remains to be further studied.Meantime,we used immunofluorescent double staining to detect ubiquitin degradation pathway and endocytosis degradation pathway in the SNpc of MPTP-treated mice.We found that those two degradation pathways of GLT-1 were upregulated in MPTP-treated mice,and miR-30a-5p lentivirus could reverse the upregulated ubiquitination but not endocytosis degradation pathway.Thus,we confirmed miR-30a-5p can regulate the expression of GLT-1 by ubiquitin degradation pathway.Based on the above experimental results,we used PI3K antagonist(LY294002),PKCa antagonist(Staurosporin),STAT3 antagonist(SH-4-54)and mTOR agonist(MHY1485)to deal with astrocytes to test whether these pathway signal molecules affected the function of GLT-1.We found that PI3K antagonist(LY294002)and STAT3 antagonist(SH-4-54)couldn’t change the abnormal function and expression of GLT-1 caused by MPP+and PKCa antagonist(staurosporin),mTOR agonist(MHY1485)could improve the abnormal function of GLT-1 caused by MPP+.And staurosporin could reverse the decrement of GLT-1 in membrane protein caused by MPP+,rather than total protein,while MHY1485 couldn’t affect the decrement of GLT-1 in both total and membrane protein.Taken together,these results suggested that miR-30a-5p could regulate the function and expression of GLT-1 through PKCa pathway,at least partly.Based on the results that miR-30a-5p could regulate the expression of GLT-1 through ubiquitin degradation pathway,we want to test whether miR-30a-5p regulate ubiquitination through PKCa or mTOR.We used immunofluorescent double staining to detect the effect of PKCa antagonist(Staurosporin)or mTOR agonist(MHY1485)on ubiquitin degradation pathway.The results showed staurosporin could reverse the increase of ubiquitin degradation pathway caused by MPP+,rather than MHY1485.Based on those results,we speculated that miR-30a-5p could regulate ubiquitin degradation of GLT-1 through PKCa pathway,at least partly.Finally,in order to assess the functional outcome of miR-30-5p knockdown in MPTP-treated mice,behavioral experiments were performed before we sacrificed the mice.MiR-30a-5p knockdown shortened the climbing time and prolonged the holding time of MPTP-treated mice.These results suggested that miR-30a-5p knockdown could alleviate the motor disorder in MPTP-treated mice.Then we tested whether miR-30a-5p knockdown affected the pathological changes in MPTP-treated mice.We found the expression of α-Syn was reduced significantly in the SNpc of MPTP-treated mice injected with miR-30a-5p shRNA compared with MPTP-treated mice.Furthermore,miR-30a-5p knockdown reversed the reduced number of TH-positive neurons in the SNpc and TH density in the striatum after using MPTP.As we all know,astrocytic activation and microgliosis are the characteristics of PD.We further explored the role of miR-30a-5p knockdown in astrocytic activation and microgliosis,we examined the astroglial and microglial expression in the SNpc.Here,we found that MPTP-induced astrocytic activation and microgliosis could be ameliorated by miR-30a-5p shRNA.Based on our results,we conclude that miR-30a-5p knockdown has a neuroprotective effect in MPTP-treated mice.It could be miR-30a-5p knockdown can improve the expression and function of GLT-1 so that alleviate glutamate excitotoxicity in MPTP-treated mice.Therefore,miR-30a-5p may be a key therapeutic factor for PD.