Ceramide Induces MIF-mediated Parthanatos In Primary Neurons By Increasing ROS Levels

BackgroundAs precursors of complex sphingolipids,ceramides can regulate intracellular signaling pathways and participate in cell growth,proliferation,motility,adhesion,differentiation,aging and apoptosis.The formation of ceramide occurs through three different pathways:de novo synthesis,sphingomyelinase pathway and salvage pathway,and the disturbance of its balance in the body may lead to the occurrence and development of neurodegenerative diseases.Alzheimer’s disease(AD)is a progressive neurodegenerative disease,clinically manifested as a slowly progressive global decline in cognitive function,accompanied by mental disorders and personality disorders.Its pathological feature is Extracellular deposition of Aβ and intracellular deposition of hyperphosphorylated tau protein.amyloid precursor protein(APP)abnormally cleaved under the action of β-secretase and y-secretase to release Aβ42 and Aβ40,of which Aβ42 oligomers are the most neurotoxic,deposit extracellularly to form senile inflammatory plaques leading to neuronal death.Same studies have found that ceramides can participate in the occurrence and development of AD by affecting the production of Aβ.In AD patients,elevated ceramide levels can directly increase Aβ production by stabilizing the key enzyme in APP production,beta-Secretase 1(BACE1),The generated Aβ oligomers in turn induce a further increase in the level of ceramide by activating sphingomyelinase,thereby forming a vicious circle that continuously promotes the occurrence and development of AD.Additionally,ceramides may affect disease progression in AD by increasing oxidative stress.Ceramides stimulate the production of reactive oxygen species(ROS)in a concentration-dependent manner,and ROS lead to changes in the structure and function of cellular macromolecules,including proteins,lipids,carbohydrates,and nucleic acids,leading to cell death,while neuronal the high oxygen consumption rate,limited antioxidant defense,and low activity of repair mechanisms of cells make them more susceptible to oxidative damage.Parthanatos,is a PARP-1-dependent cell death.Studies have found that ceramides mediate PARP-1 hyperactivation,PAR aggregation,and nuclear translocation of apoptosis-inducing factors(AIF)in SH-SY5Y cells and retinal neurons,resulting in large DNA fragmentation and chromatin aggregation,ultimately leading to cell death,these have been identified as common features of Parthanatos.However,AIF has no endonuclease activity and cannot cut DNA,and it is speculated that other molecules are involved.Kam TI et al.found that Parthanatos is involved in neuronal death in cerebral ischemia-reperfusion injury,and macrophage migration inhibitory factor(MIF)and AIF co-nuclear translocation,exerting endonuclease activity,mediating DNA Large fragmentation,induces cell death.Therefore,MIF may be the terminal molecule of Parthanatos,and it is necessary to further clarify its role in cell death.This study aimed to investigate the mechanism of ceramide-induced neuronal Parthanatos and the role of MIF in it.PurposeTo explore whether ceramide induces parthanatos in neurons and the related molecular mechanism,and to clarify the role of MIF in ceramide-mediated parthanatos in neurons.Thus,new ideas and possible therapeutic targets are provided for the occurrence,development and treatment of AD.MethodsThe primary cortical and hippocampal neurons of fetal mice(C57BL6J mice at 17-19 gestational days)were isolated and cultured.Cell viability was detected by CCK-8 assay,cell death was detected by Hoechst33342/PI double staining assay,and DNA damage was detected by comet assay.Western blot to detect the expression level of PAR polymer,nuclear translocation of AIF and MIF and the expression level of DNA damage marker y-H2AX,and cell immunofluorescence test to further verify the nuclear translocation of AIF and MIF;Virus transfection interfered with MIF and AIF gene expression in primary neuron cells.Real-time quantitative polymerase chain reaction(q-PCR)and Western blot were used to verify the transfection efficiency.Cellular immunofluorescence technique was used to further verify the role of MIF in ceramide-mediated neuronal Parthanatos;DCFH-DA probe was used to detect the level of intracellular reactive ROS,to verify that ROS in ceramide role in the induction of neuronal cell death.Results1.The results of CCK-8 and Hoechst33342/PI double-staining showed that compared with the control group,the neuronal viability of the ceramide treatment group was significantly reduced,and the cell death was significantly increased,and the difference was statistically significant(P<0.05).Compared with the ceramide-treated group,the PARP-1 inhibitor ABT888+ceramide group significantly increased neuronal activity and significantly decreased cell death(P<0.05);while the pan-caspase inhibitor Z-VAD-FMK+neuronal was no statistical difference in neuronal viability and cell death in the amide-treated group compared with the ceramide-treated group;Compared with the PARP-1 inhibitor ABT888+ceramide group,the neuron activity was significantly increased,and the cell death was significantly decreased,with statistical differences(P<0.05);However,there was no significant difference in neuronal activity and cell death between the pan-caspase inhibitor Z-VAD-FMK+ceramide-treated group and the ceramide-treated group.2.The comet test and Western blot results showed that compared with the control group,the ceramide treatment group had obvious tailing phenomenon,the DNA content in the tailing was significantly increased,and the expression level of the DNA damage marker γ-H2AX was significantly increased,and the difference was statistically significant.(P<0.05).3.Western blot results showed that the increase of ceramide concentration was positively correlated with the expression level of PAR polymer,and the difference was statistically significant(P<0.05).The results of Western blot and immunofluorescence test showed that compared with the control group,the expression levels of AIF and MIF in the nucleus of the ceramide treatment group were significantly increased,with a statistical difference(P<0.05).AIF and MIF co-localized in the nucleus,which proved that ceramide induces neuronal Parthanatos.4.The expression of AIF and MIF was interfered with the transfection of the lentivirus carrying the silenced AIF and MIF gene plasmids;the results of Western blot,comet test and Hoechst/PI double staining test showed that compared with the ceramide treatment group,the MIF KD+ceramide group Cell death was significantly reduced,comet tail length and tail DNA content were significantly reduced,and the expression level of y-H2AX was significantly reduced,with statistical significance(P<0.05).5.The results of immunofluorescence test showed that compared with the ceramide treatment group,the nuclear translocation of MIF in the AIF KD group was significantly reduced,while there was no significant difference in the nuclear translocation of AIF in the MIF KD group,indicating that MIF is the downstream of AIF.6.The results of DCFH-DA fluorescent probe showed that ceramide significantly increased the level of ROS in neurons compared with the control group,with a statistical difference(P<0.05).The results of Western blot,comet assay and Hoechst/PI double staining assay showed that compared with the ceramide-treated group,the ROS inhibitor NAC pretreatment+ceramide-treated group significantly decreased the levels of yH2AX and RAR,as well as the length of nuclear tail and DNA content,neuron death was significantly reduced,the difference was statistically significant(P<0.05).Conclusion1.Ceramide activates PARP-1,induces the formation of PAR multimers,and leads to DNA damage and cell death.2.Ceramide induces the interaction between AIF and MIF,and mutual nuclear translocation,MIF plays the function of endonuclease,and cleavage of DNA leads to the occurrence of Parthanatos in neurons.3.Ceramide induces Parthanatos in neurons through ROS/PARP-1/PAR/AIF/MIF pathway.