PAK1 Kinase Regulates Parkinson’s Disease Through Microglial Activation

Objective:To study the role of p21-Activated Kinase on the neuroinflammation that triggered by activated microglia and explore the underline molecular mechanism.To study the effect of PAK1 on Parkinson’s disease.Methods:In vitro,BV2 cell line,primary microglia and dopaminergic neuron cell line MES23.5 were used in this study,PAK1 inhibitor IPA-3 and Pak1 knockout mice were used to inhibit PAK1 activity.Cell viability was detected by CCK-8.The mRNA levels of inflammatory cytokines on BV2 cells,such as inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2),interleukin-1 β(IL-1β),arginine 1(Arg1),CD206,interleukin-10(IL 10)were detected by Q-PCR.The protein levels of inflammatory cytokines(iNOS,COX-2,pro-IL-1β),IκBα protein expression,the distribution of p65 in nucleus and cytoplasm,the whole protein expressions and their phosphorylation levels of p65 and IKKα/β(p-p65/p65,p-IKKα/β/IKKα/β),the whole protein expressions and their phosphorylation levels of c-Fos and c-Jun(p-c-Fos/c-Fos,p-c-Jun/c-Jun),the whole protein expressions and their phosphorylation levels of JNK,p38 and ERK(p-JNK/JNK,p-p38/p38,p-ERK/ERK)were detected by Western blotting.The expression of Interleukin-6(IL-6),tumor necrosis factor a(TNFa)inflammatory cytokine secreted by microglial supernatant were detected by ELISA.Nuclear translocation of p65 was detected by immunofluorescence.Flow cytometry was used to detect the effect of inflammatory cytokines secreted from the supernatant medium of BV2 on the survival of MES23.5 dopaminergic neuron cell line.In vivo experiments,wild type and Pakl knockout mice were intraperitoneally injected with LPS or equivalent volume of saline.The protein levels of inflammatory cytokines and phosphorylated PAK1 in the brain were detected by Western blotting,and the activation of microglia were observed by immunofluorescence.A stereotaxic injection of 1.5 μL of LPS(5 mg/mL)into the left substantia nigra of wild-type mice was performed to obtain neruoiflammation-induced PD(Parkinson’s disease)mouse model.Intraperitoneal injection of IPA-3(5 mg/kg)or saline was given before and after modeling,LPS stereotaxic injection was also performed on wild type and Pak1 knockout mice.Immunofluorescence was used to detect the activation of microglia in the substantia nigra and the loss of dopamine neurons in the substantia nigra.Wild type and Pak1 knockout mice were intraperitoneally injected with MPTP to obtain MPTP subacute Parkinson’s disease animal model.The motor function of mice was evaluated by rotarod test.The protein levels of inflammatory cytokines,phosphorylated PAK1 and Tyrosine hydroxylase(TH)in the striatum were detected by Western blotting.The activation of microglia and the loss of dopamine neurons were detected by immunofluorescence assay.Results:After treatment with PAK1 inhibitor,the overexpression of pro-inflammatory factors such as iNOS,COX-2 and pro-IL-1β induced by LPS on BV2 cells were inhibited at both protein level and transcriptional level compared with LPS group alone,meanwhile the transcriptional level of anti-inflammatory factors remained unchanged.The same anti-inflammatory effect was observed on primary microglia.In vivo,Pak1 knockout mice also depressed LPS-induced proinflammatory overexpression.To explore the mechanism that PAK1 inhibited microglia activation.the nucleoplasmic separation kit was used to test that the increased protein of p65 tranferred to the nucleus that stimulated by LPS was suppressed by PAK1 inhibition,while the decline in cytoplasmic protein was alleviated.Immunofluorescence assay also exhibited nuclear translocation of p65 subunit.For further exploration of the molecular mechanism on the upstream of NF-κB,Western blotting results showed that compared with the LPS group,the up-regulation of p-p65 and p-IKKα/β was inhibited by PAK1 inhibitor,and the degradation of IκBα was rescued,indicating that NF-κB pathway is involved in PAK1 regulation.Previous studies have shown that AP-1 transcription factor is also involved in LPS-stimulated activation of microglia,so we detected the phosphorylation of c-Jun and c-Fos,and confirmed that inhibiting PAK1 activity can reduce LPS-stimulated phosphorylated proteins overexpression of c-jun and c-fos.Then,the upstream MAPK signaling pathway of AP-1 were concerned to be regulated by PAK1.We detected the total protein expression and their phosphorylated protein expression levels of p38,JNK and ERK which are the main components of MAPK signaling,and found that LPS-stimulated overexpression phosphorylation of these proteins were also regulated by PAK1 activity.In vitro flow cytometry staining showed that PAK1 inhibition alleviated microglia activation-mediated MES23.5 cell death.In vivo,LPS stereotactic injection model showed that pretreatment with IPA-3 or Pak1 knockout both attenuated the death of dopamine neurons by alleviating LPS induced microglial activation.In the MPTP subacute model of wild-type and Pak1 knockout mice,the rotarod experiment showed that the MPTP model could induce a decline in the motor ability of wild type mice,while not in Pak1 knockout mice.At the same time,Pak1 knockout mice also inhibited the activation of microglia cells and the resulting overexpression of neuroinflammation that caused by MPTP,and has a protective effect on the loss of dopaminergic neuron.Conclusion:PAK1 can regulate neuroinflammation mediated by microglial activation,PAK1 regulates microglial activation through NF-κB and AP-1 signaling pathways,Inhibition of PAK1 activity may alleviate PD-like phenotype in mice.