?-Arrestins-orchestrated Microglia Polarization And Their Potential Roles In The Pathogenesis Of Parkinson's Disease

As the second-most common neurodegenerative disorder in the world,Parkinson's disease?PD?affects the physical and mental health of numerous patients.It contains three neuropathological hallmarks:the progressive loss of dopaminergic neurons,the presence of Lewy bodies?LBs?and chronic neuroinflammation in the substantia nigra pars compacta?SNc?.Correspondingly,it leads to a movement disorder characterised by classical motor symptoms,including bradykinesia,muscular rigidity,and rest tremor.Parkinson's disease is also associated with a multitude of non-motor symptoms,such as olfactory dysfunction,sleep disorders,psychiatric symptoms,and cognitive impairment.Emerging evidence indicates that PD is caused by a variety of genetic factors and environmental exposures.However,the etiology of PD has not yet been fully understood.PD remains an incurable neurological disorder.In clinical treatment,Levodopa?L-Dopa?compensating for the loss of dopamine is a mainstay drug to alleviate the motor symptoms of PD.However,this treatment can not cure or delay the progress of PD.Simultaneously,long-term use will lead to a series of medication?related complications,such as dyskinesias?L-Dopa?induced dyskinesias,LIDs?and motor fluctuations.Therefore,it is necessary to further explore the mechanisms of PD pathogenesis to provide direction for disease-modifying therapies.Chronic neuroinflammation is one of the characteristic features of PD and plays a crucial role in the development of PD.Neuroinflammation is closely associated with the degeneration of dopaminergic neurons.Under neuroinflammatory conditions,activated glial cells release pro-inflammatory and neurotoxic factors,such as cytokines,reactive oxygen species?ROS?and complement proteins,which induce neuron damage and degeneration.As the disease progresses,degenerating neurons release adenosine triphosphate?ATP?and?-synuclein??-syn?to further activate glial cells,forming a vicious cycle of chronic inflammatory state.Moreover,dopaminergic neurons are more vulnerable to glial cell-induced neurotoxicity,leading to the activation and proliferation of glial cells in the SNc.Microglia,the main resident immune cells of the brain,play an important role in neuroinflammation.Activated microglia have been classified into two phenotypes:the M1 pro-inflammatory phenotype and the M2 anti-inflammatory phenotype.M1microglia produce pro-inflammatory factors,chemokines,ROS,and nitric oxide?NO?,which are neurotoxic to dopaminergic neurons.M2 microglia produce anti-inflammatory factors and neurotrophic factors,which promote neuron survival and tissue repair.The ratio of M1 and M2 microglia is altered in the pathological process of PD,which show a decrease in M2 microglia and an increase in M1 microglia.Therefore,in-depth investigations are urgently needed to fine-tune the transitions of M1/M2 microglia by suppressing pro-inflammatory phenotype and enhancing anti-inflammatory phenotype,and this appears a valid therapeutic approach for PD treatment.?-Arrestins?ARRBs?were first identified as proteins involved in mediating G-protein-coupled receptors?GPCRs?desensitization and were,hence,called?-arrestin1and 2.Then it was discovered that ARRBs can mediate receptors internalization,ubiquitination,degradation and also serve as multifunctional scaffold proteins that recruit various molecules to trigger ARRB-dependent signaling pathways and play a critical role in inflammation and inflammatory diseases.Our previous study revealed that ARRB2 inhibits the assembly and activation of NOD-like receptor protein-3?NLRP3?by binding to NLRP3,which accounts for the anti-inflammatory effects of dopamine D2 receptor?DRD2?and?2-adrenoreceptor??2AR?.It has been reported that ARRB2 is involved in LIDs.ARRB2 deficiency aggravates LIDs,and overexpression of ARRB2 attenuates LIDs.It is suggested that ARRBs might be involved in the pathogenesis of PD.However,there has no document reporting a role for ARRBs in PD.Based on the above research,in the first part of this work we used Arrb1^-/-and Arrb2^-/-mice to induce acute 1-methyl,4-phenyl-1,2,3,6-tetrahydropyridine?MPTP?PD mouse model to investigate the relationship between ARRBs and PD in vivo.The results showed that ARRB1 deficiency reduces loss of dopaminergic neurons and glial cells activation,and also decreases M1 while increases M2 markers in the midbrain tissues.Deletion of ARRB2 exacerbates loss of dopaminergic neurons and glial cell activation,and also enhances the expression of M1 markers but reduces the expression of M2 markers in the midbrain tissues.In the second part of the work we further studied the roles of ARRBs in microglia/macrophage polarization and the effects on the survival of dopaminergic neurons in vitro.The results showed that ARRB1 promotes M1 and inhibits M2 microglia polarization,knockdown of ARRB1 alleviates the damage of dopaminergic neurons induced by M1 microglial conditioned medium?CM?.While ARRB2 suppresses M1 and boosts M2 microglia polarization,knockout of ARRB2 aggravates dopaminergic neuron damage caused by M1 microglial CMs.In the third part of this paper we elucidated the molecular mechanisms for ARRBs regulating the shift of microglia phenotype.The data revealed that ARRB1downregulation inhibits NF-?B,STAT1 signaling pathways and promotes STAT6pathway,and ARRB2 downregulation enhances the activation of NF-?B,STAT1signaling pathways and reduces the activation of STAT6 pathway.We then used RNA-seq to find the key molecules accounting for the opposite roles of ARRB1 and ARRB2in regulating microglia phenotype.The results indicated that Nprl3 is the key molecule accounting for the opposite functions of ARRB1 and ARRB2 in regulating M1microglia polarization and Samd4 is the core protein responsible for the distinct roles of ARRB1 and ARRB2 in mediating M2 microglia polarization.In summary,ARRB1and ARRB2 play important but opposite roles in the pathogenesis of PD.ARRBs regulate the transformation of microglia phenotype by acting on Nprl3 and Samd4,thereby affecting the survival of dopaminergic neurons and participating in the pathogenesis of PD.Part ? The roles of?-arrestins in the pathogenesis of Parkinson's diseaseAIM:To investigate the roles of ARRBs in the pathogenesis of PD.METHOD:WT mice were used to prepare acute MPTP model.The expression of ARRB1 and ARRB2 in midbrain tissues were measured by Western Blot.We detected the expression of ARRB1 and ARRB2 and the colocalization with ionized calcium binding adaptor molecule-1?Iba-1?positive microglia by immunofluorescence.WT,Arrb1^-/-and Arrb2^-/-mice were used to establish acute MPTP model,we used Tyrosine hydroxylase?TH?,Glial fibrillary acid protein?GFAP?and Iba-1immunohistochemical staining to investigate the effects of ARRB1 and ARRB2deletion on the loss of dopaminergic neurons,the proliferation and activation of astrocytes and microglia.Moreover,we extracted total RNA or protein of the midbrain tissues,and detected the mRNA or protein levels of M1/M2 microglia markers by RT-PCR or Western Blot.RESULTS:1)The expression of ARRB1 was upregulated and ARRB2 was downregulated in the midbrain tissues of MPTP model mice,and expression of ARRBs on Iba-1⁺microglia had the same changes.2)Knockout of ARRB1 alleviated the loss of dopaminergic neurons and the activation of astrocytes and microglia in the SNc of MPTP model mice,and inhibited the expression of M1 markers and promoted the expression of M2 markers in the midbrain tissues.3)ARRB2 deficiency aggravated the loss of dopaminergic neurons and the activation of astrocytes and microglia in the SNc of MPTP model mice,and augmented the expression of M1 markers and attenuated the expression of M2 markers in the midbrain tissues of MPTP model mice.CONCLUTION:ARRBs might be involved in the pathogenesis of Parkinson's disease by modulating microglia phenotypes,and the two ARRB isoforms display functional specialization.Part ? ?-Arrestin1/2-mediated microglia polarization and their effects on neuronsAIM:To investigate the regulation of ARRBs on microglia polarization and their effects on neuron survival.METHOD:The primary microglia from neonatal WT mice were cultured and stimulated with lipopolysaccharide?LPS,100 ng/ml?+interferon-??IFN-?,20 ng/ml?or interleukin-4?IL-4,20 ng/ml?,the expression of ARRBs were measured by Western Blot.Downregulating the expression of ARRB1 in microglia by ARRB1 siRNA.After48 h,Western Blot was used to detect the interference efficiency.Microglia transfected with ARRB1 siRNA were stimulated with LPS+IFN-?or IL-4.After 6 h,RT-PCR was used to measure the mRNA levels of M1 markers?IL-6,IL-1?,TNF-?,iNOS?or M2 markers?Arg1,Ym1,CD206?to observe the effect of ARRB1 on microglia polarization.The primary microglia from neonatal WT and Arrb2^-/-mice were cultured and stimulated with LPS+IFN-?or IL-4 for 6 h,RT-PCR was used to detect the mRNA levels of M1 markers or M2 markers to observe the effect of ARRB2 on the transformation of microglia phenotype.The primary bone marrow-derived macrophages?BMDMs?from WT mice were cultured and transfected with ARRB1siRNA for 48 h and then stimulated with LPS+IFN-?or IL-4.After 6 h,the mRNA levels of M1 markers or M2 markers were detected by RT-PCR.After 24 h,cell supernatants were collected,and the levels of pro-inflammatory cytokines?IL-6,IL-1?,TNF-??or anti-inflammatory factors?TGF-?,IL-10?were measured by ELISA,or cell proteins were extracted and the expression of iNOS or CD206 were observed by Western Blot.Moreover,we analyzed the expression of CD16 or CD206 by immunofluorescence to investigate the effect of ARRB1 on macrophage polarization.The primary BMDMs from WT and Arrb2^-/-mice were cultured,and the same stimulations and methods were applied to observe the regulation of ARRB2 in macrophage polarization.The primary BMDMs from WT mice were cultured and transfected with pcDNA-Arrb1 or pcDNA-Arrb2 plasmid.After 24 h,the overexpression efficiency was detected by Western Blot.BMDMs transfected with the corresponding plasmid were stimulated with LPS+IFN-?or IL-4 for 6 h,the mRNA levels of M1 markers or M2 markers were detected by RT-PCR.We collected the CMs from M1 microglia transfected with ARRB1 siRNA or deleted ARRB2 to stimulate the primary neurons.After 24 h,CCK8,Hoechst 33342 staining,Western Blot and TH immunocytochemical staining were used to observe neuron damage.RESULTS:1)Under LPS+IFN-?treatment,the expression of ARRB1 was upregulated,while ARRB2 was downregulated in microglia.On the contrary,under IL-4 stimulation,the expression of ARRB1 was reduced,while ARRB2 was elevated in microglia.2)ARRB1 knockdown inhibited the mRNA levels?Il-6?Il-1b?Tnf?Nos2?of M1 markers elicited by LPS+IFN-?treatment,and promoted the mRNA levels?Arg1?Ym1?Mrc1?of M2 markers by IL-4 stimulation in microglia.3)ARRB2deficiency enhanced the mRNA levels of M1 markers by LPS+IFN-?treatment,and reduced the mRNA levels of M2 markers using IL-4 stimulation in microglia.4)Downregulation of ARRB1 suppressed the mRNA and protein levels?IL-6,IL-1?,TNF-?,iNOS,CD16?of M1 markers in macrophage with LPS+IFN-?stimulation,and augmented the mRNA and protein levels?CD206,TGF-?,IL-10?of M2 markers by IL-4 treatment.5)Overexpression of ARRB1 increased the mRNA levels of M1markers using LPS+IFN-?treatment,and attenuated the mRNA levels of M2 markers by IL-4 stimulation in macrophage.6)Knockout of ARRB2 exaggerated the mRNA and protein levels of M1 markers by LPS+IFN-?stimulation,and diminished the mRNA and protein levels of M2 markers in macrophage with IL-4 treatment.7)Overexpression of ARRB2 suppressed M1 marker genes by LPS+IFN-?treatment,and upregulated M2 marker genes using IL-4 stimulation in macrophage.8)Knockdown of ARRB1 in microglia attenuated ventral mesencephalic neuron damage induced by M1 microglial CM.9)ARRB2 deletion in microglia aggravated ventral mesencephalic neuron damage caused by M1 microglial CM.CONCLUTION:ARRB1 and ARRB2 play vital but distinct roles in microglia activation,which further affect the survival of dopaminergic neurons.Part ? The mechanism and relationship of?-arrestin1/2 in modulating microglia/macrophage polarizationAIM:To investigate the molecular mechanisms by which ARRBs regulate microglia/macrophage polarization and display opposite roles.METHOD:The primary BMDMs from WT mice were cultured and transfected with ARRB1 siRNA or ARRB2 siRNA for 48 h,then stimulated with LPS?100 ng/ml?+IFN-??20 ng/ml?or IL-4?20 ng/ml?for 2 h.We detect the phosphorylation and total protein expression of IKK?,p65,STAT1 or STAT6 by Western Blot,to observe the effect of ARRB1 or ARRB2 knockdown on NF-?B,STAT1 and STAT6 pathways.The primary BMDMs from WT mice were cultured and stimulated with LPS+IFN-?or IL-4.Cell proteins were extracted after 1 h,we used co-immunoprecipitation?Co-IP?to detect the interaction between ARRB1?ARRB2?with IKK?,p65,STAT1 or STAT6.The primary BMDMs from WT mice were cultured and transfected with ARRB1siRNA or ARRB2 siRNA for 48 h,then stimulated with LPS+IFN-?.After 1 h,cell proteins were extracted and the binding of ARRB2 or ARRB1 to p65 was detected by Co-IP.The primary microglia from neonatal WT and Arrb2^-/-mice were cultured and stimulated with LPS+IFN-?or IL-4.After 6 h,total RNA was extracted for RNA-seq.Through screening analysis,RT-PCR verification and interference of protein expression,we found the key molecules accounting for the distinct functions of ARRB1 and ARRB2 in microglia/macrophage polarization.RESULTS:1)Downregulation of ARRB1 inhibited the phosphorylation protein expression of IKK?,p65,and STAT1 induced by LPS+IFN-?stimulation,and promoted the phosphorylation protein expression of STAT6 induced by IL-4stimulation.2)ARRB2 knockdown promoted the phosphorylation protein expression of IKK?,p65,and STAT1 induced by LPS+IFN-?stimulation,and inhibited the phosphorylation protein expression of STAT6 induced by IL-4 stimulation.3)Both ARRB1 and ARRB2 interacted with p65 upon LPS+IFN-?stimulation,but ARRB1and ARRB2 were not associated with IKK?,STAT1 or STAT6.4)Under LPS+IFN-?treatment,downregulation of ARRB1 enhanced the association of ARRB2 with p65,and ARRB2 knockdown also increased the interaction of ARRB1 with p65.5)In ARRB1-knockdown or ARRB2-knockout M1 microglia,Il12rb1,Lpar1 and Nprl3 had opposite changes,knockdown of ARRB1 reduced the mRNA levels of these genes,while ARRB2 deletion enhanced the mRNA levels of these genes.6)The change of Samd4 was opposite in ARRB1-knockdown and ARRB2-knockout M2 microglia,ARRB1 downregulation increased the mRNA level of Samd4,while knockout of ARRB2 decreased the mRNA level of Samd4.7)Knockdown of Nprl3 augmented the inhibition of M1 microglia polarization by ARRB1 downregulation and partially abolished the promotion of M1 microglia polarization by ARRB2 deficiency.8)Knockdown of Samd4 partially abrogated facilitation of M2 microglia polarization by ARRB1 knockdown and enhanced the suppression of M2 microglia polarization by ARRB2 deletion.CONCLUTION:ARRB1 and ARRB2 differently regulate the expression of Nprl3 and Samd4 and the activation of NF-?B,STAT1 and STAT6 signaling pathways,thus exerting distinct functions in microglia/macrophage activation.The major contributions of the present study lie in:1.ARRB1 andARRB2 play crucial but opposite roles in PD.ARRB1 knockout attenuates the loss of dopaminergic neurons and the proliferation and activation of glial cells in the SNc,while ARRB2 knockout exacerbates the loss of dopaminergic neurons and the proliferation and activation of glial cells in the SNc,suggesting that both ARRB1 and ARRB2 are involved in the pathogenesis of PD,but exert opposite effects,which provide a theoretical basis for the development of PD drugs targeting GPCRs or ARRBs.2.ARRBs modulate microglia polarization then affect the survival of dopaminergic neurons.ARRB1 knockdown inhibits M1 and promotes M2 microglia polarization,thus alleviating the damage of dopaminergic neuron induced by M1microglial CM;knockout of ARRB2 augments M1 and attenuates M2 microglia polarization,thus aggravating the damage of dopaminergic neuron caused by M1microglial CM,which indicate ARRBs are involved in the pathogenesis of PD by regulating microglia polarization to affect the survival of dopaminergic neurons.These results provide promising experimental evidence for developing neuroprotective agents that modulate microglia phenotypes.3.ARRBs regulate the phenotype shift of microglia via mediating Nprl3 and Samd4 signaling.Nprl3 and Samd4 are the key molecules for ARRB1 and ARRB2regulating the phenotype shift of microglia,providing important targets for the development of drugs switching microglia phenotypes.