The Role And Mechanism Of Lactylation Mediated By Microglial Glycolysis In Parkinson’s Disease

Objective: To observe the effects of glycolysis inhibiting on microglial activation,dopaminergic(DA)neurons damage and and motor function in the substantia nigra(SN)of a mouse model of Parkinson’s disease(PD).And to explore the role and mechanism of lysine lactylation(Kla)in the activation of microglia.Methods: In this study,a subacute PD mouse model was established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)and an inflammatory PD mouse model was established by unilateral stereotactic injection of lipopolysaccharide(LPS)into SN.Real-time quantitative PCR(q PCR),western blotting(WB)and immunofluorescence(IF)staining were employed to investigate the effects of intraperitoneal injection of 2-deoxyd-glucose(2-DG)on microglial activation,DA neurons injury and motor function in the PD models.Furthermore,WB and IF staining were used to investigate the levels and cellular localization of pan-Kla and histone 3 lysine 9lactylation(H3K9la)in the SN of MPTP and LPS-induced PD mouse models and the A53 T transgenic PD mouse model.The inflammatory model of primary microglia was also used to validate these findings.Then,p300 and Cbp genes were knocked down to observe their impact on pan-Kla and H3K9 la levels and microglial activation.Subsequently,the target genes of H3K9 la were screened through CUT&Tag and RNA-seq and were validated using Chip-q PCR.Finally,pharmacological inhibition of target gene was utilized to explore its effects on microglial activation and DA neuron damage in the PD mouse model and the impact of primary microglia-conditioned medium on SH-SY5 Y neuronal apoptosis.Results: Compared with control group,the levels of the key glycolytic enzymes hexokinase 2(HK2)and lactate dehydrogenase A(LDHA)proteins and lactate concentration increased in the SN in MPTP mouse model.Intraperitoneal injection of2-DG to inhibit glycolysis relieved microglial activation and DA neurons damage,and improved motor dysfunction of PD mice.WB showed that pan-Kla and H3K9 la were increased in the SN of MPTP model mice,LPS model mice,and A53 T transgenic mice.IF staining showed that H3K9 la co-labeled with microglia,and the fluorescence intensity of H3K9 la was significantly higher in PD model group than in the control group.Intraperitoneal injection of 2-DG decreased the fluorescence intensity of pan-Kla and H3K9 la in microglia.In vitro experiments showed that the levels of p300 and CBP proteins were increased in LPS-stimulated primary microglia.Knockdown the expression of p300 and Cbp gene using si RNA not only reduced the levels of pan-Kla and H3K9 la proteins,but also inhibited the expression of pro-inflammatory cytokines.CUT&Tag,RNA-seq,and Chip-q PCR experiments showed that H3K9 la enriched at the promoter of Slc7a11 and activated its transcription.Pharmacological inhibition of SLC7A11 relieved microglial activation and DA neurons damage in the SN and improved motor dysfunction of PD mice.Moreover,inhibition of SLCA11 reduced the damage of primary microglia cultured medium to SH-SY5 Y neurons.Conclusions: This study suggests that inhibition of glycolysis reduces neuroinflammation-mediated DA neurons damage by suppressing microglial activation.We report for the first time the role of enhanced glycolysis in promoting microglial activation through H3K9la/SLC7A11 pathway in the PD pathogenesis,which provides a new target for metabolic immunotherapy of PD.Part Ⅰ: Inhibition of glycolysis alleviates microglial activation and dopaminergic neuron damage in PD miceObjective: This study investigates the effects of intraperitoneal injection of 2-DG to inhibit glycolysis on microglial activation,neuroinflammation,and DA neurons damage in the SN in PD mouse model.Methods: The MPTP mouse model and LPS mouse model were constructed.The motor function of mice was tested by Rotarod test,climbing pole test and open field test.Lactate concentrations in the SN and striatum were measured by colorimetric assays.q PCR and WB were used to measure the expression of glycolytic key enzymes HK2 and LDHA,as well as pro-inflammatory cytokines i NOS,IL-1β,IL-6,TNF-α and NLRP3,and anti-inflammatory cytokines Arg1,CD206 and YM1.IF staining was used to evaluate the activation of microglia and the damage to DA neurons.Changes in the above indicators were also detected after inhibition of glycolysis by intraperitoneal injection of 2-DG.In addition,a cell model of inflammation was constructed by stimulating primary microglia with LPS,and the effects of pretreatment with 2-DG or sodium oxalate,which inhibit glycolysis,on the above indicators were evaluated.Finally,primary microglia were stimulated with different concentrations of exogenous lactate to observe their effects on the expression of pro-inflammatory and anti-inflammatory cytokines.Results: Compared with the control group,lactate concentrations in the SN and striatum of MPTP model mice was significantly increased.The levels of HK2 and LDHA proteins in the SN were increased.Moreover,the number of microglia increased and showed an ameboid state,while the number of DA neurons decreased.Intraperitoneal injection of2-DG alleviated microglia activation and DA neuron damage,decreased the m RNA and protein of TNF-α and increased the protein level of ARG1,and finally improved motor dysfunction in MPTP mice.In LPS model mice,intraperitoneal injection of 2-DG alleviated the proliferation and activation of microgliaand loss of DA neurons in the SN.In vitro experiments showed that the expression of Glut1,Hk2 and Ldha were upregulated in primary microglia stimulation by LPS,and lactate concentration in the medium was increased.The inhibition of glycolysis by 2-DG and sodium oxalate not only inhibited the expression of pro-inflammatory cytokines Il1β,Nos2,Il6,Tnf and Nlrp3,but also promoted the expression of anti-inflammatory cytokines Cd206 and Ym1.At the same time,the primary microglia changed from amoebia-like form to resting state.Additionally,exogenous lactate promoted the expression of Il-6,Il-1β and Tnf-α in primary microglia in a concentration-dependent manner.Conclusions: Inhibition of glycolysis alleviates neuroinflammation-induced DA neurons damage,and improves motor dysfunction in PD mice by suppressing microglia activation and the expression of pro-inflammatory cytokines.Part Ⅱ: Glycolysis promotes microglial activation by H3K9 lactylation in PD miceObjective: To investigate the expression and cellular localization of histone lactylation and its modification sites in SN of MPTP model mice,LPS model mice and A53 T transgenic mice,and to study the effect of glycolysis inhibition on histone lactylation.Methods: MPTP mouse model,LPS mouse model and 10~12 months old A53 T transgenic mice were constructed.The expression of pan-Kla and potential sites(H3K9la,H3K18 la,H4K12la and H4K16la)in the SN of PD mice were detected by WB.The localization of H3K9 la in microglia(IBA1+),neuronal(Neu N+),DA neurons(TH+)and astrocytes(GFAP+)were determined by IF.The changes of pan-Kla and H3K9 la levels were observed after 2-DG intraperitoneal injection.In addition,mouse primary microglia were stimulated with LPS,IL-4 and different concentrations of exogenous lactate.WB and IF were used to detect the changes in pan-Kla and H3K9 la before and after inhibition of glycolysis and lactate production by 2-DG,sodium oxalate and Ldha-si RNA.Results: WB results showed that pan-Kla and H3K9 la protein levels were significantly higher in the SN of MPTP model mice than in control mice.IF staining showed that H3K9 la was expressed in neurons,DA neurons,microglia and astrocytes.However,compared to control group,the fluorescence intensity of H3K9 la was significantly increased in microglia in the SN of MPTP mice.In addition,compared with wild-type mice,the protein levels of HK2 and LDHA and lactate concentration were significantly increased,and the fluorescence intensity of pan-Kla and H3K9 la co-labeled with microglia was significantly enhanced in the SN of A53 T transgenic mice.Intraperitoneal injection of2-DG significantly reduced the fluorescence intensity of pan-Kla and H3K9 la in microglia in the SN of MPTP and LPS models.In vitro experiments showed that exogenous lactate upregulated pan-Kla protein levels in a concentration-dependent manner.pan-Kla and H3K9 la protein levels were significantly increased in primary microglia stimulated with LPS but were not altered in response to IL-4 stimulation.Inhibition of glycolysis and lactate production by 2-DG,sodium oxalate and Ldha-si RNA reduced the levels of pan-Kla and H3K9 la protein.Conclusions: Lactate produced by enhanced glycolysis promotes the activation of microglia in PD model mice through histone lactylation.Part ⅡI: p300/CBP mediates H3K9 lactylation in microglia activationObjective: To explore the role of p300 and its complex CREB-binding protein(CBP)in histone lactylation and microglial activation.Methods: LPS was used to stimulate primary microglia to construct an inflammatory cell model.C646 was used to inhibit p300 and CBP activity,or si RNA was transfected to knock down the expression of p300 and Cbp.The changes of pan-Kla and H3K9 la protein levels were detected by WB,and the expression of pro-inflammatory and anti-inflammatory cytokines was detected by q PCR and WB.Results: WB results showed that the levels of p300 and CBP proteins in primary microglia were significantly increased after LPS stimulation.Inhibition of p300 and CBP activity with C646 significantly reduced the levels of pan-Kla and H3K9 la proteins,and inhibited the levels of TNF-α,NLRP3 and i NOS proteins.Knockdown the expression of p300 and Cbp not only attenuated protein levels of pan-Kla and H3K9 la,but also down-regulated the expression of Il6,Il1 b,and Nos2 and up-regulated expression of Arg1 in primary microglia stimulated by LPS.Conclusions: p300/CBP is a potential lactyltransferase involved in histone lactylation during microglial activation.Targeting p300/CBP may alleviate microglial activation by inhibiting histone lactylation.Part IV: H3K9 lactylation promotes microglial activation in PD mice by activating the transcription of Slc7a11Objective: To screen and verify the target genes of H3K9 la,and to explore the effect of target gene inhibiting on microglial activation and DA neurons damage in the SN of PD model mice.Methods: LPS-stimulated primary microglia were collected for CUT&Tag and RNA-seq to screen the target genes of H3K9 la,and the target genes were verified by Chip-q PCR.In vitro,Slc7a11 gene expression was knocked down by Erastin or si RNA,and the expression of pro-inflammatory and anti-inflammatory cytokines was detected by q PCR and WB.CCK8 was used to detect the effect of erastin-inhibiting SLC7A11 activity conditioned medium on apoptosis of SH-SY5 Y neurons.In vivo,SLC7A11 activity was inhibited by intraperitoneal injection of sulfasalazine(SAS),and the motor function of LPS model mice was evaluated by climbing pole test,Rotarod test and open field test.q PCR,WB and IF staining were used to detect the effects of SAS on microglial proliferation and activation and DA neurons injury.Results: CUT&Tag and RNA-seq analyse showed that Slc7a11 was the potential target gene for H3K9 la.Chip-q PCR showed that H3K9 la was significantly enriched at the promoter of Slc7a11 in LPS-stimulated primary microglia,and inhibition of glycolysis with 2-DG and sodium oxalate significantly reduced its enrichment.q PCR showed that the Slc7a11 expression was up-regulated in LPS-stimulated primary microglia and in the SN of LPS model mice.Using erastin to inhibit SLC7A11 activity or si RNA to knock down Slc7a11 expression suppressed the activation of primary microglia and the expression of pro-inflammatory cytokines,and alleviated conditioned medium injury to SH-SY5 Y neurons.In addition,ntraperitoneal injection of SAS inhibited microglial activation and neuroinflammation,alleviated DA neurons damage and improved motor dysfunction in PD mice.Conclusions: Slc7a11 is the target gene for H3K9 la.Inhibition of Slc7a11 improves the motor dysfunction in PD mice by alleviating microglial activation and DA neurons damage.