The Role And Mechanism Of RNA Demethylase FTO Regulating Ephrin-b2 In Manganese-induced Parkinsonism

Objective:Excessive manganese（Mn）exposure may cause persistent and progressive extrapyramidal dysfunction by damaging the dopaminergic neuron projection of nigro-striatal projection system.Its symptoms are similar to the clinical manifestations of parkinson’s disease（PD）.Although a series of studies have been carried out on the neurotoxic mechanism of Mn,the molecular mechanism of abnormal dopaminergic neurons projection in nigro-striatal projection system and motor dysfunction induced by Mn is still unclear.In addition,there is no specific drug for the treatment of patients with Mn poisoning.Therefore,in-depth study the mechanism of dopaminergic neurons projection injury and motor dysfunction induced by Mn has great practical significance to protect the health of people exposed to Mn.The accurate establishment of neural circuits involves axons navigating neurons to their target areas.The process of neuronal axonal guidance is essential for the release of DA from dopaminergic neurons in the substantia nigra to the striatum.The researchers found that axon guidance molecules are highly expressed in the striatum.Axon guidance molecules modulate dopaminergic neuron projections that affect brain motor function.N6-methyladenosine（m⁶A）is one of the most common methylation modifications in RNA,which can regulate gene expression at the post-transcriptional level.m⁶A modification can regulate RNA splicing,translation and degradation.It is reported that m⁶A modification can regulate different neuronal functions,such as motor function,axonal guidance function and synaptic function.The newly reported m⁶A demethylase Fat mass and obesity-associated protein（FTO）shows that m⁶A modification can be dynamically regulated.Studies have shown that YT521-B homeodomain family protein 2（YTHDF2）is identified as the first protein to recognize m⁶A preferentially,which can promote mRNA degradation after recognizing m⁶A.In addition,the researchers found that inhibition of demethylase FTO impaired neuronal activity and motor function.However,up to now,whether FTO and YTHDF2 can regulate nigra-striatal dopaminergic neurons projection through axon guidance molecules and then participate in Mn-induced motor dysfunction remains to be further studied.In this study,the over-expression animal and cell models of FTO and key target axon guidance molecules ephrin-B2 were constructed,using molecular biology,behavioral,morphological and bioinformatics methods to explore the important mechanism of FTO-mediated mRNA m⁶A methylation of ephrin-B2 and YTHDF2-dependent degradation in Mn-induced abnormal projection of dopaminergic neurons,and to further clarify the mechanism of Mn-induced motor dysfunction.Methods:1.According to the principle of body weight balance,C57BL/6 mice were divided into 8 groups（n=16）,including control group,12.5,25 and 50 mg/kg Mn Cl₂group,and AAV5 negative control（NC）group,AAV5-FTO control group,AAV5-NC+50 mg/kg Mn Cl₂group and AAV5-FTO+50 mg/kg Mn Cl₂group.The mice in the control group were given intraperitoneal injection of 0.9%Na Cl.Mice in Mn Cl₂group received intraperitoneal injection of 12.5,25 and 50 mg/kg Mn Cl₂.The concentration of treatment was 5 m L/kg.Once a day for two weeks.For AAV5 virus injection,0.2μL AAV5-FTO or AAV5-GFP virus was injected into the striatum via stereotaxic injection.At the end of the behavior experiment,2 of the 16 mice were used in FG retrograde tracing experiment.Then all the mice were decapitated under anesthesia,and the striatum of the other 14 mice in each group was separated on ice for morphological observation,immunofluorescence detection,Mn and DA content detection,and to determine the target mRNA expression,protein and other index levels.2.After SH-SY5Y cells were exposed to Mn,the protein levels and mRNA expression of m⁶A demethylase FTO were detected by Western blotting and RT-qPCR.The effect of Mn on the viability of SH-SY5Y cells was detected by CCK-8 method.Immunofluorescence staining was used to observe the changes of FTO expression location and fluorescence intensity.Construction of FTO over-expression cell model:FTO over-expression lentivirus or GFP control（FTO-NC）lentivirus was transfected into SH-SY5Y cells,and SH-SY5Y cells with stable FTO over-expression were obtained after puromycin selection.FTO-NC cells were divided into control group and Mn group,and FTO over-expression cells were divided into control group and Mn group.The RNA of each group was collected for RNA-seq experiment to screen the potential axon guidance molecular of FTO.Then RT-qPCR assay was used to detect potential target genes mRNA expression of FTO.Finally,Me RIP-qPCR assay was used to detect the mRNA m⁶A level of potential target genes in order to screen the key axon guidance molecular of FTO.3.The over-expression cell model of FTO and/or YTHDF2 was constructed:FTO over-expression lentivirus and/or YTHDF2 over-expression lentivirus and GFP control lentivirus were transfected into SH-SY5Y cells.Stable FTO over-expression and/or YTHDF2over-expression cells were screened and used in the follow-up experiment.FTO-NC and/or YTHDF2-NC cells were divided into control group and Mn group,FTO and/or YTHDF2over-expression cells were divided into control group and Mn group.Actinomycin D experiment was used to detect the mRNA stability of ephrin-B2.The protein levels and mRNA expression of YTHDF2 were detected.Then isolate total RNA from SH-SY5Y cells and perform RIP-seq experiments to identify the target transcripts of YTHDF2 and whether YTHDF2 binds to ephrin-B2.The protein levels of ephrin-B2 were detected by Western blotting assay.The expression of ephrin-B2 mRNA was detected by RT-qPCR assay.C57BL/6 mice were divided into 4 groups according to their body weight,with 16 mice in each group,including ephrin-B2-NC control group,ephrin-B2-NC+50 mg/kg Mn Cl₂group,ephrin-B2-NC+20 mg/kg MA2+50 mg/kg Mn Cl₂group and AAV5-ephrin-B2+20 mg/kg MA2+50 mg/kg Mn Cl₂group.Mice in the control group were given intraperitoneal injection of 0.9%Na Cl after subcutaneous injection of 0.9%Na Cl 2 h.Mice in the Mn Cl₂group received subcutaneous injection of 0.9%Na Cl for 2 h followed by intraperitoneal injection of50 mg/kg Mn Cl₂.The mice in the MA2 group were given intraperitoneal injection of 0.9%Na Cl after subcutaneous injection of 20 mg/kg MA2 for 2 h.The mice in 20 mg/kg MA2+50mg/kg Mn Cl₂group received subcutaneous injection of 20 mg/kg MA2 for 2 h and then intraperitoneal injection of 50 mg/kg Mn Cl₂.The concentration of treatment was 5 m L/kg.Once a day for two weeks.AAV5 virus injection and follow-up detection indicators refer to the first part of animal experiments.Results:1.The content of Mn in the striatum of mice was increased with increasing dose of Mn Cl₂,while the expression of FTO in the striatum was significantly decreased.The difference was statistically significant compared with the control group.The results of behavioral experiment observed that the fatigue running distance,endurance time,moving distance and moving speed of mice in 50 mg/kg Mn Cl₂group were significantly decreased.Over-expression of FTO can reverse the symptoms of motor function injury caused by Mn.The results of HE staining and Nissl staining found that the pathological damage of striatum in the 50 mg/kg Mn Cl₂group was serious,and the over-expression of FTO could improve the injury symptoms caused by Mn exposure.The results of retrograde labeling with FG and double staining of TH and DARPP32 found that the function of dopaminergic neurons in nigro-striatal projection system was significantly impaired in the 50 mg/kg Mn Cl₂group.The dopaminergic neurons projection damage was recovered after FTO over-expression.In addition,the content of DA in striatum was significantly decreased in the 50 mg/kg Mn Cl₂group,and the content of DA increased after over-expression of FTO.2.The results of Western blotting and RT-qPCR showed that Mn exposure significantly decreased the expression of FTO in SH-SY5Y cells.CCK-8 assay found that Mn exposure was significantly decreased the cell viability of SH-SY5Y,and the cell viability increased after over-expression of FTO.In addition,it was found that the fluorescence intensity of FTO in SH-SY5Y cells decreased significantly after Mn exposure,and there was no significant change in the location of FTO expression.RNA-seq and RT-qPCR results found that ephrin-B2,NTN4,SLITRK5,SEMA3A and SEMA7A may be potential target genes of FTO in Mn exposed cell model.Subsequently,we used the Me RIP-seq data after specific knockout of mouse mesencephalic dopaminergic neurons FTO published by HESS et al for pathway analysis.The results showed that the axon guidance signaling pathway was significantly changed.Finally,through the Me RIP-qPCR experiment,it was found that Mn exposure could up-regulate the mRNA m⁶A levels of ephrin-B2,while the mRNA m⁶A levels decreased after FTO over-expression,suggesting that ephrin-B2 may be the key target gene of FTO in Mn exposure model.3.The results of actinomycin D experiment showed that Mn exposure decreased the stability of ephrin-B2 mRNA,while the over-expression of FTO significantly increased the stability of ephrin-B2 mRNA.Mn exposure did not affect YTHDF2 expression.The results of FTO RNA-seq and YTHDF2 RIP-seq analysis found that ephrin-B2 was an axon guidance molecule which could be regulated by FTO and bound to YTHDF2.The results of RT-qPCR and Western blotting observed that the over-expression of FTO could antagonize the decrease of ephrin-B2 expression induced by Mn exposure.It is worth noting that the over-expression of YTHDF2 significantly decreased the level of ephrin-B2 in FTO over-expression cells.In addition,the over-expression of YTHDF2 further reduced the stability of ephrin-B2 mRNA on the basis of Mn exposure.Further analysis found that the intervention of MA2 aggravated the damage of dopaminergic neurons projection and the decrease of DA level after exposed to Mn.In addition over-expression of ephrin-B2 in FTO inhibition model significantly improved the damage of dopaminergic neurons projection and the decrease of DA content induced by Mn.Behavioral experiments observed that Mn and MA2 exposure significantly reduced the fatigue running distance and endurance time.The over-expression of ephrin-B2 in FTO gene suppressed model mice significantly improved the performance of mice in rotating rod assay and fatigue assay.Conclusion:1.Mn exposure can reduce the expression of FTO in the striatum of mice,thereby causing dopaminergic neurons projection injury and motor dysfunction of mice.2.Mn can regulate the mRNA m⁶A modification of ephrin-B2 through FTO,thus affecting its expression level.3.FTO/m⁶A/ephrin-B2/YTHDF2 signaling pathway plays a key role in Mn-induced dopaminergic neurons projection injury and motor dysfunction.