ROS-mediated LncRNA NR₀30777 And Non-Coding RNA M⁶A Modification In Paraquat-induced Neuron Mitophagy

Objective:To explore the role of mitochondrial fission in PQ-induced mitophagy and the underlying molecular mechanism in neuron.To clarify the role and mechanism of NR?030777 in PQ-induced mitophagy,in the hope that it can become a new prevention and treatment target for PD.To explore the possible mechanism of PQ-induced neuron damage by detect the changes profiles of m~6A in lnc RNAs and circ RNAs mediated by ROS.Methods:(1).CCK8 was used to determine the neurotoxicity of PQ in N2a cells.Flow cytometry was used to detect the ROS and mitochondrial membrane potential caused by PQ.Confocal laser scanning microscopy and transmission electron microscopy was used to confirm the role of mitochondrial fission in mitophagy in PQ-exposed neuron.The autophagy proteins were detected by western blot,and the autophagy inhibitor chloroquine(CQ),bafilomycin(Bfa1)were added to analyze the autophagy flux of PQ.The role of ROS in the process of mitophagy and mitochondrial fission caused by PQ was confirmed by the ROS scavenger NAC.The DRP1 inhibitor mdivi-1 was used to investigate the role of mitochondrial fission in the process of mitophagy caused by PQ.(2).Through the establishment of PQ-infected mouse dopamine neuron model,to study the mitophagy on in the substantia nigra of mice and the long non-coding RNA NR?030777.By establishing the N2a and primary neuronal cell injury models treated with different concentrations and times of PQ,the change trend of long non-coding RNA NR?030777 was studied.NAC was used to verify the role of ROS in NR?030777changes caused by PQ.The role of NR?030777 in the changes of mitophagy induced by PQ was determined by NR?030777 stably overexpressed.The target protein of NR?030777 was determined by RIP and RNA pulldown experiments.The region of interaction between NR?030777 and the target protein was determined by truncated RNA pulldown experiment.By establishing a PQ-infected NR?030777 brain conditional overexpression C57BL/6 mouse model,the role of NR?030777 in PQ-induced changes in mitophagy in the substantia nigra of mice was verified.(3).Construct the N2a cell model of the combined effect of NAC and PQ,detect the changes in the expression profile of m~6A lnc RNAs and circ RNAs through me RIP-seq,and explore the possible mechanism of PQ nerve cell toxicity through bioinformatics analysis.Results:(1).N2a cells activity was decreased in a dose-dependent manner(100,200 and 300?M PQ).Mitophagy was increases in a dose-dependent manner(100,200 and 300?M PQ)in neuronal cells.Mitochondrial fission-related proteins were increased.The mitochondrial membrane potential(MMP)was decreased and the ROS was increased in N2a cells treated by PQ.In particular,mitophagy was promoted by DRP1-mediated mitochondrial fission induced by PQ.(2).NR?030777 participates in the regulation of mitophagy in PQ-treated neuron cells through DRP1 mediated mitochondrial fission.The results further revealed that during the enhancement of mitophagy caused by PQ exposure,NR?030777,activates the CDK1 signaling pathway,causing the phosphorylation of serine at position 616 of the DRP1 protein,thereby promoting damaged mitochondria fission.On the other hand,NR?030777 promotes the formation of ATG12-ATG5complex by acting on ATG12,accelerating the process of autophagy membrane formation,and quickly enveloping the short round mitochondria split from damaged mitochondria,making them rapidly degraded by autophagy.(3).When N2a cells were exposed to PQ,the level of m~6A in lnc RNAs increases.After PQ-treated N2a cells,the differentially changed m~6A lnc RNAs mainly came from chromosomes 2,4,5,and 11.In ROS-mediated PQ neurotoxicity,m~6A is only enriched around CDS and at the beginning of 3?UTR.Differentially methylated lnc RNAs participate in many biological processes(such as:ubiquitinated protein degradation,autophagy,RNA splicing,etc.).In addition,m~6A perturbed the gene network related to lnc RNAs in ROS-mediated PQ neurotoxicity.4.Our research results show that NAC pretreatment can partially reverse the changes in circ RNA s driven by m~6A methylation after PQ exposure.In addition,gene ontology(GO)and pathway analysis showed that differentially methylated circ RNAs can regulate target genes involved in neurotoxic pathways(UBC and PPP2CA).Conclusions:1.PQ exposure causes mitochondria damage in neuron cell,increased reactive oxygen species,and increased damaged mitochondria fission to short round mitochondria,which is conducive to autophagy clearance and mitophagy were enhanced.2.Long non-coding RNA NR?030777 is a key regulator of the enhancement of mitophagy caused by PQ exposure.It promotes DRP1-mediated mitochondrial division through CDK1 and at the same time promotes autophagy through ATG12,thereby eliminating the damaged mitochondria caused by PQ exposure.3.In N2a cells,ROS mediates the changes of lnc RNA and circ RNA m~6A caused by PQ.The methylation of lnc RNA and circ RNA is involved in many important biological processes,which need to be further studied.