Effect Of Microglial MyD88 Signaling On Neuronal Ferroptosis After Spinal Cord Injury

BackgroundSpinal cord injury（SCI）is a serious traumatic disorder of the central nervous system（CNS）.The annual incidence of SCI in China is about 37 per million,which causes a heavy medical and economic burden on individuals and society.Unfortunately,due to the complexity of the pathological mechanism of SCI and the non-regenerative nature of the neurons in the CNS,there has not been breakthrough in clinic therapy to SCI so far.Therefore,it is still a great task for neuroscience to explore novel and effective therapeutic strategies for SCI,and fulfilling it relies on intensive study about the pathological mechanism.Neuronal death is the direct cause of neurological dysfunction after SCI.In addition to the primary injury,mechanisms of the secondary injury including tissue edema,inflammation,ischemia and hypoxia are important factors that aggravate neuronal death.To save damaged neurons by preventing or mitigating secondary injury is one of the important principles of SCI therapy.Ferroptosis is a programmed cell death（PCD）discovered in recent years.The feature of ferroptosis is programmed cell lipid peroxidation induced by iron overload and accumulation of reactive oxygen species（ROS）.Ferroptosis has been involved in various diseases.According to existing studies,ferroptosis occurs after SCI,due to bleeding and hemolysis led to iron overload ROS prodution and stress response in the lesion area.Ferroptosis inhibitor deferoxamine can inhibit ferroptosis and improve functional recovery after SCI.During the secondary injury stage,microglia-mediated pro-inflammatory response is one of the critical mechanisms,which is mainly initiated by damage associated molecular patterns（DAMPs）binding to innate immune receptors especially Toll-like receptors（TLRs）,and trigger TLRs/myeloid differentiation factor 88（My D88）signaling cascade.The occurrence of ferroptosis is usually accompanied by proinflammatory activation of microglia.However,the role of microglia mediated inflammation in regulating neuronal ferroptosis after SCI and its underlying mechanism remain unclear.Therefore,to explore the role of microglial My D88 signaling in regulation neuronal ferroptosis after SCI and the possible underlying mechanism may help better understand the mechanisms of secondary injury and provide research basis for new therapeutic strategies and potential targets to SCI.PurposeThrough SCI model of mice and the cell culture experiments,this study was designed to investigate regulatory role of microglial My D88 signaling in the occurrence neuronal ferroptosis and the underlying molecular mechanism after SCI,so as to find out new target for rescue of neurons undergoing ferroptosis after SCI.1.To investigate the possible relationship between ferroptosis of neurons and pro-inflammatory activation of microglia after SCI through in vivo experiments on mice.2.To explore the regulatory role and mechanism of pro-inflammatory activated microglia in regulating ferroptosis of neurons through cell cultures as in vitro experiments as well as the in vivo experiments with mouse SCI model.Methods:1.Spinal cord crush injury in mice as the animal model was used for study in vivo.Oxygen and glucose deprivation/reoxygenation（OGD/R）was used to neuron cell line SH-SY5Y and primary cortical neuron culture as the object in vitro.2.Basso Mouse Scale（BMS）and gate analysis were used to estimate the changes in motor function of the mice after SCI surgery.3.Hematoxylin eosin（HE）staining was used to detect the size of the lesion cavity caused by SCI in the mouse.4.Iron accumulation and antioxidant levels in the spinal cord were determined by measurement of iron concentration and GSH content in the tissues.5.The expression levels of related protein molecules in tissues and cells were observed by Western Blot（WB）and immunofluorescence staining.6.Transwell chambers were used to co-culture microglia and neurons to observe the cellular interaction in between.7.The death rate of neuronal cells was analyzed by propidium iodide（PI）staining and flowcytometry.8.Use Cell Counting Kit-8（CCK8）to detect changes in cell vitality of neurons.9.Use detection of malondialdehyde（MDA）and 4-hydroxynonenal（4HNE）level to reflect lipid peroxidation in neurons.10.The production of lipid ROS in neurons was detected by BODIPY C11 581/591（lipid peroxidation sensor）.11.The morphology of mitochondria in the spinal cord neurons was observed by transmission electron microscopy.12.Changes in mitochondrial membrane potential of the neurons were indicated by mitochondrial membrane potential probe JC-1.13.Introduce My D88^f/f;TMEM119-Cre/ERT transgenic mice to observe the effect of microglia-specific My D88 knockout on ferroptosis of neurons after SCI.14.Fluorescent in situ hybridization was used for My D88 RNA scope in the spinal cord of the mice with My D88 conditional knock out.Results:Part I.Neuronal ferroptosis and microglial My D88 signaling activation were involved in the lesion area of the spinal cordThree days after the SCI model was established in wild-type mice,the iron concentration was increased and GSH level was reduced significantly.In addition,immunofluorescence staining showed increased expression of acyl-Co A synthetase long-chain family member 4（ACSL4）in the neurons near to the injury center.At the same time point,it was found obviously activated microglia were adjacent to the neurons with lipid peroxidation accumulation through immuno-fluorescence staining to the sections of the injured spinal cord.Results from Western Blot showed that the ferroptosis related signaling was upregulated after SCI,and the My D88/NF-κB signaling pathway was significantly activated as well.Part II.Pro-inflammatory activated microglia promoted ferroptosis in neurons by upregulation of My D88The results of CCK8 detection showed that the viability of SH-SY5Y cells could be further decreased obviously by the conditional medium（CM）induced by lipopolysaccharide（LPS）stimulating microglia culture on the base of OGD/R treatment.Flowcytometry after PI staining also showed that CM derived from proinflammatory microglia increased the mortality of OGD/R treated SH-SY5Y neurons.Consistent with Western Blotting assay,glutathione peroxidase 4（GPX4）,an antioxidative molecule,was significantly decreased in CM cultured SH-SY5Y cells.In addition,the expressions of My D88,NF-κB p65 and i NOS were significantly increased in microglia stimulated by LPS,indicating the pro-inflammatory activation of these microglia;while My D88 inhibitor ST2825 distinctly inhibited LPS induced microglia activation and the CM derived from such microglia culture did not reduce the cell viability or increase the cell mortality of OGD/R treated SH-SY5Y cells.Similarly,CCK8 and flow cytometry showed further decreased cell viability and further increased mortality of the OGD/R treated primary neurons that were co-cultured neurons with LPS stimulated microglia.Western Blot analysis indicated that ST2825 significantly inhibited the expression levels of My D88/NF-κB,IL-1βand i NOS in microglia cells stimulated by LPS.And the decreased cell viability and increased mortality of neurons were reversed when microglial My D88 was inhibited by ST2825 prior to co-culture.By detecting the expression of neuronal Lipid ROS with immunofluorescence staining,it is found that inhibition of My D88 expression in microglia significantly reduced neuronal lipid peroxidation.The detection of malondialdehyde（MDA）concentration in neurons further demonstrated that LPS stimulated microglia promoted lipid peroxidation through up-regulation of My D88.Western Blot showed that inhibition of My D88 expression in microglia significantly suppressed the activation of ferroptosis signal in co-cultured neurons.The immunofluorescence staining of neuron mitochondrial membrane potential JC-1proved that inflammatory activated microglia exacerbated the decrease of neuronal mitochondrial membrane potential.Part III.Microglia-specific My D88 deletion inhibited neuronal ferroptosis and promote motor function recovery after SCIThe conditional knockout（CKO）of My D88 in microglia was induced in TMEM119-Cre ERT;My D88^flox/flox transgenic mice by tamoxifen oral gavage.The success of microglia specific knockout of My D88 was verified by fluorescence in situ hybridization and Western Blot.Then SCI model was established with this CKO mice,and BMS score and gait analysis showed that microglia-specific My D88 knockout mice exhibited earlier hind limb standing,faster and better recovery of motor function,compared with the WT group.HE staining also showed that the tissue cavities in the spinal cord of CKO mice were smaller than that of WT animals on the third day after SCI.Transmission electron microscopy showed that the abnormal mitochondria were fewer and the shrinkage of mitochondria decreased on the third day post SCI in CKO mice,compared with WT mice.Western Blot analysis to spinal cord tissue further confirmed that the My D88/NF-κB pathway in the spinal cord of CKO mice was significantly inhibited on the 3rd day after SCI,and the signaling of ferroptosis was also significantly weakened,compared with the sham operation group.Consistently,the lipid peroxidation and GSH of neurons near the lesion center reduced in the CKO mice 3 d after SCI,compared with WT mice.Part IV.The potential molecular mechanism of microglial My D88 involved in ferroptosis of neurons after spinal cord injuryIn the co-culture system between microglia and OGD/R-treated neurons,Western Blot and cell immunofluorescence showed that LPS stimulation increased the expression of hepcidin in microglia,while the expression of ferroportin（FPN）in neurons was significantly decreased and the transferrin receptor（Tf R）in neurons was significantly increased.However,when microglial My D88 was inhibited,the expression of hepcidin was also significantly decreased,in the meantime,the decrease in FPN and increase in Tf R of neurons were reversed.Western Blot results of spinal cord also showed that hepcidin in My D88 f/f group andMy D88 CKO group were significantly up-regulated after SCI.However,compared with My D88 f/f SCI group,hepcidin expression in spinal cord of My D88 CKO SCI group was significantly inhibited.Correspondingly,FPN decreased and Tf R increased in My D88 f/f mice after SCI.However,in My D88 CKO SCI mice,the changes in FPN and Tf R were significantly reversed also,compared with My D88 f/f mice.Conclusion:The above findings in the injured spinal cord during the acute phase that neuronal ferroptosis occurred accompanied by activation of adjacent microglia cells and activation of My D88 signaling pathway,suggest close relationship between the activation state of microglia and the ferroptosis in neurons induced by SCI.Cell culture experiments have demonstrated that inflammatory activation of microglia can aggravate ferroptosis in the neurons based on OGD/R treatment.Inhibition of My D88 expression in microglia can significantly inhibit the incidence of neuronal ferroptosis,suggesting that microglial My D88 signaling play a key role in regulating neuron ferroptosis.The specific knockout of microglial My D88 in transgenic mice TMEM119-Cre ERT;My D88^flox/floxcan significantly down-regulate the ferroptosis gene of neurons in the acute phase of SCI,thus significantly promoting the recovery of motor function after SCI in vivo.It is proved that the up-regulation of microglial My D88 can promote the ferroptosis of neurons after SCI,while the down-regulation of microglia My D88 signal can alleviate the ferroptosis of neurons.The cell interaction is mainly regulated by IL-1β/hepcidin/FPN axis.This study provides a new understanding of the regulatory mechanism of neuronal ferroptosis in SCI,and is expected to provide new ideas and targets for future treatment of SCI.