Mechanistic Study Of Central Nervous System Disease Based On CXCR2 And DYRK1A Targets

Multiple sclerosis(MS)is a degenerative and inflammatory disease of the central nervous system(CNS)characterized by perivascular inflammation,demyelination,axonal damage,and neuronal loss.Studies have identified that environmental,lifestyle,and genetic risk factors are relevant to the development of MS.However,the underlying pathophysiological mechanisms involved in this disorder are not fully understood.In the early stages of demyelinating injury,Oligodendrocyte precursor cells(OPCs)are recruited into the demyelinating areas.During the disease progression,remyelination stops attribute to the differentiation disability of OPCs into oligodendrocytes(OLs),which are the main myelin sheath-forming cells.Therefore,stimulating OPC differentiation and maturation is an important therapeutic strategy for promoting the regeneration of myelin sheaths and ameliorating persistent demyelination in the CNS.CXC chemokine receptor 2(CXCR2)is a typical G-protein-coupled receptor that is highly expressed in peripheral neutrophils and central oligodendrocytes.Numerous researches have illustrated that CXCR2 could interact with its ligand to modulate multiple cellular processes,which include migration,proliferation,and differentiation.However,the underlying mechanisms of CXCR2 inhibition in promoting OPC maturation and improving myeline repair were not yet clarified.In the present study,we utilized the primary cultured OPCs and ethidium bromide(EB)-induced rat demyelinating model to mainly investigate the effects of CXCR2 on myelin regeneration.Specific markers in different cell stages of oligodendrocyte differentiation were detected to represent OPC differentiation and maturation.Western blot results demonstrated that the expression of the immature OPCs marker,platelet derived growth factor receptor alpha(PDGFRα),was significantly decreased under the stimulation of SB225002 in primary cultured OPCs.Consistently,the expression of differential and mature markers of pre-OLs and OLs,including myelin basic protein(MBP),proteolipid protein 1(PLP1),myelin-associated glycoprotein(MAG),myelin oligodendrocyte glycoprotein(MOG),and contactin associated Protein(Caspr),were markedly increased in dose-dependent manners when OPCs were incubated with SB225002.These results revealed that CXCR2 inhibition could facilitate the differentiation and maturation of OPCs to OLs.Luxol fast bule(LFB)staining were further applied to evaluate the change of myelin sheath.The corpus callosum of rats exhibited obvious demyelination,and the structure as well as completeness of myelin sheath suffered significant damage and even broke under the stimulation of EB.While suppressing CXCR2 could alleviate the injury of myelin sheath and promote the biosynthesis of myelin proteins,resulting in OPC differentiation and maturation,in addition to enhance the CNS remyelination and alleviate motor dysfunction of demyelinating rat model.In our previous study of CPZ-intoxicated demyelinated mice,the transcriptome data revealed that PDE10A was one of the important proteins that was affected in CXCR2 signaling.We then verified the PDE10A expression both in vitro and in vivo using Western blot.Consistent with the transcriptome profiling results,CXCR2 antagonism by SB225002 significantly down-regulated PDE10A expression both in primary cultured OPCs and in the corpus callosum of EB-intoxicated rats.Additionally,the co-immunoprecipitation and immunofluorescence assays were used to detect the interactions between CXCR2 and PDE10A in OPCs.The co-staining results showed that the PDE10A protein was co-localized and co-expressed with surface-bound CXCR2,indicating direct binding and specifical interaction between CXCR2 and PDE10A in primary cultured OPCs.It is also important to note that CXCR2 expression was not affected when PDE10A was inhibited by TAK-063 or overexpressed by transfecting the PDE10A gene with plasmid in OPCs.Moreover,PDE10A expression was down-regulated when CXCR2 was inhibited by SB225002,in comparison with PDE10A overexpressed OPCs.These data collectively suggested that PDE10A was an important downstream protein of CXCR2,and suppressing CXCR2 could facilitate the myelin repair and regeneration.To further address the critical role of PDE10A on OPC differentiation and maturation,we detected the different markers of oligodendrocyte lineage cells when PDE10A expression was inhibited by TAK-063.The immunofluorescence assay and western blot indicated that PDE10A antagonism significantly reduced the intensity of A2B5,a marker of OPCs in the precursor stage,and markedly up-regulated the accumulation of cell-stage specific markers that are characteristic of differentiated OLs,indicating the constant conversion of immature OPCs to mature OLs,while overexpression of PDE10A significantly restrained the formation of mature OLs.Furthermore,treatment with TAK-063 could promoted the expression of myelin sheath associated markers and facilitated the regeneration of corpus callosum in EB-induced demyelinating rats.In order to further verify the molecular mechanism that PDE10A regulates OPC differentiation and remyelination,qPCR assay was employed to measure the levels of transcription factors.Additionally,ELISA and Western blot were utilized to assess the signaling pathway and protein phosphorylation both in vitro and in vivo.These results strongly demonstrated that inhibition of CXCR2/PDE10A activated the cAMP/ERX1/2 signaling pathway,and up-regulated the expression of key transcription factors for myelination regulated proteins,including SRY-box transcription factor 10(SOX10),oligodendrocyte Lineage Transcription Factor 2(OLIG2),Myelin regulatory factor(MYRF),and Zinc finger protein 24(ZFP24),that ultimately promoted remyelination and myelin protein biosynthesis.In conclusion,our findings suggested that inhibition of CXCR2 promoted OPC differentiation and enhanced remyelination by regulating PDE10A/cAMP/ERK1/2 signaling pathway.The present data also highlighted that CXCR2 might serve as a potential target for the treatment of MS,and modulating the CXCR2/PDE10A/cAMP signal might serve as a promising therapeutic strategy for demy elination and other neurological diseases that involve white matter pathologies.Parkinson’s disease(PD)is the second largest prevalent neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the formation of Lewy bodies.The pathogenesis of PD is not yet fully understood,but it is believed to be related to the genetic factors,age,gender,environmental factors,and behavioral habits.Numerous studies have demonstrated that the onset of PD is associated with notable neuroinflammation and immune dysfunction,which have been implicated in multiple non-motor symptoms including depression,sleep dysfunction and gastrointestinal disorders.Moreover,a marked elevation in the levels of proinflammatory factors was observed in the serum and cerebrospinal fluid of PD patients.There was also a noticeable augmentation in inflammatory cell infiltration and activation of microglia within the brain.Histological and imaging studies also support the presence of sustained neuroinflammatory response in the brains of PD patients,which facilitate the damage of dopaminergic neurons and significantly contribute to the neurodegenerative process.Therefore,it is crucial to prevent the degeneration of dopaminergic neurons and restore the normal level of dopamine transmitter as a key strategy for treating PD.Dual-specificity tyrosine-phosphorylation-regulated kinase 1A(DYRK1A)is a protein kinase that can be activated by auto-phosphorylated tyrosine residues.And it has been implicated in the progression of multiple neurodegenerative diseases by regulating the neuroinflammation and biological processes,such as chromatin transcription,cell cycle regulation and neuronal development.In the progression of PD,DYRK1A could mediate the neurotoxicity by phosphorylating α-synuclein(α-syn)and PARKIN protein.However,its specific role and mechanism in the regulation of neuroinflammation remain unclear.Therefore,this research aims to investigate the effects and mechanism of DYRK1A in mediating neuroinflammation and protecting dopaminergic neurons in Lipopolysacchride(LPS)-induced inflammatory microglia cells and rotenone-challenged PD mouse model.This study firstly utilized LPS-induced BV2 microglia to investigate the role of DYRK1A in neuroinflammation.The results revealed that both the expression and kinase activity were upregulated in LPS-mediated neuroinflammatory reaction.Inhibition of DYRK1A using Harmine significantly reduced the activation of microglia,as detected by increased expression of ionized calcium binding adaptor molecule 1(Iba1),Cyclooxygenase 2(COX2)and Interleukin-1β(IL-1β).In order to explore the role of DYRK1A in neuroinflammation by regulating microglial polarization,Western blot assays were then used to evaluate the expression of microglial markers.Harmine could reversed the LPS-induced the upregulation of M1 markers,such as nitric oxide synthase(iNOS)and CD86,as well as the decrease of M2 markers,including Arginase 1(Arg1)and macrophage mannose receptor 1(CD206),demonstrating that suppressing DYRK1A promoted the transformation of Ml-phenotype to M2-phenotype in BV2 cells.Additionally,SH-SY5Y cells were incubated with BV2 conditional medium to elucidate the protective effects of DYRK1A on neurons.LPS stimulated the over-production of inflammatory factors,resulting in abnormal morphology and death of SH-SY5Y cells.While inhibition of DYRK1A markedly alleviated neuronal injury and improved the numbers of SH-SY5Y cells by modulating the expression of inflammatory proteins and release of cytokines.In the rotenone-challenged PD mice,inhibition of DYRK1A improved the motor and coordination ability,and reduced the neuroinflammation by modulating the microglial polarization.Immunohistochemical staining showed that rotenone induced a significant loss and death of dopaminergic neurons in the substantia nigra.While it was observed that Harmine could mitigate the injury and enhance the cell viability of dopaminergic neurons in PD mice.In order to further validate the regulatory role of DYRK1A in PD,an adenovirus vector containing DYRK1A shRNA was injected into the substantia nigra region of α-syn(A53T)transgenic PD mice.We also analyzed the expression of neuroinflammatory proteins and tyrosine hydroxylase(TH)-positive neurons using Western blot and immunohistochemical staining.The data revealed a significant increase of DYRK1A expression in the midbrain of A53T transgenic mice.After silencing DYRK1A with shRNA,the expression of inflammatory proteins,iNOS and COX2,was remarkably reduced,which also led to a decrease in the activation of microglial cells,as well as mitigation on the damage of TH-positive neurons in the substantia nigra.Further mechanism studies demonstrated that apoptosis signal regulating kinase-1(ASK1)acted as a substrate of DYRK1A and involved in the neuroinflammatory reactions.Suppression of DYRK1A modulated the polarization of microglia both in vitro and in vivo by reducing the activation of ASK1/JNK/P38 signaling pathway,thereby inhibiting the neuroinflammation and protecting dopaminergic neurons in PD model mice.Additionally,inhibition of ASK 1 was found to regulate M1/M2-phenotype polarization by decreasing the activation of JNK/P38 proteins in LPS-mediated BV2 microglial inflammatory cells.In conclusion,our data demonstrated that suppression of DYRK1A effectively reduced the neuroinflammation by promoting the microglia from M1 to M2 phenotype.This ultimately led to the upregulation of dopaminergic neurons in the substantia nigra and alleviation of motor dysfunction in PD mice.Mechanistic study had identified ASK1/JNK/P38 as an important signaling pathway for DYRK1A in the regulation of microglial polarization and neuroinflammation.This study highlighted the important role and underlying mechanism of DYRK1A in regulating neuroinflammation of PD,and it provided a theoretical foundation for the development of drugs targeting DYRK1A.