Regulatory Effect Of G Protein/?-arrestin2 Signaling Pathway Downstream Of Astrocyte Drd2 On Neurotrophic Factor's Production

Astrocytes are the most widely distributed cells in the central nervous system of the brain,and are the largest cells in glial cells,accounting for about 20%-40%of glial cells.Astrocytes play an important role in many central nervous system diseases,such as Parkinson's disease,depression and other neurodegenerative diseases.It has many functions,including the biochemical support of the endothelial cells to support the blood-brain barrier,providing nutrients to nerve tissue,maintaining extracellular ion balance,and the role of brain and spinal cord repair and scar formation after traumatic injury.Dopamine physiology is mediated by five distinct but closely related G protein-coupled receptors(GPCRs).Among them,D2 receptor is essential for learning and memory.The D2 receptor is the major receptor for most antipsychotics and is coupled to the Gi subtype of GPCR.Consistent with most GPCR receptors,the D2 receptor activates downstream signals after interaction with the G protein through the classical pathway,and also activates downstream signals through the non-canonical pathway,interacting with the scaffold protein ?-arrestin2,and is independent of In the G protein pathway.At present,a large number of clinical drugs play a role in the treatment of neuropsychiatric diseases by stimulating DRD2,and the importance thereof is further expanded.At present,the importance of neurotrophic factors in neuropsychiatric diseases is gradually recognized.It is a kind of biomolecules that support the growth,survival and differentiation of developing and mature neurons.Neurotrophic factors also promote the initial growth and development of neurons in the central and peripheral nervous systems,which can regenerate damaged neurons in test tubes and animal models.At present,brain-derived neurotrophic factor(BDNF),glial-derived neurotrophic factor(GDNF),nerve growth factor(NGF),vascular endothelial growth factor(VEGF),basic fibroblast growth factor(FGF-2),etc.,which play a key role in the central nervous system.Among them,the important role of neurotrophic factors such as BDNF and GDNF in depression has been reported in many literatures,and the hypotrophic hypotrophy has become one of the five hypotheses of the cause of depression.At the same time,in Alzheimer's disease(AD),BDNF can also down-regulate abnormal A? accumulation to alleviate the AD process,showing the importance of its neuroprotective effects in neuropsychiatric diseases.Previous research by our research team found that mRNA expression of neurotrophic factors decreased in all groups after chronic mild stress(CMS)modeling.However,after ?-arrestin2-/-mice were treated with fluoxetine,the regulation of vasopressin on neurotrophic factors was abolished,and after CMS modeling,wild-type mice were given ?-arrestin2-preferred DRD2 agonist UNC9995 and antidepressant fluoxetine,we found that in the animal behavioral test,the results of Forced swimming test(FST),Social interaction test(SI),Novelty-Suppressed Feeding Test(NST),and Tail suspension test(TST)were all improved,suggesting that the non-canonical pathway of ?-arrestin2 may be related to trophic factors after activation of DRD2.At present,no studies have reported that the G protein classical pathway of Drd2 and the non-canonical pathway of ?-arrestin2 regulate the release of astrocyte neurotrophic factor.In this study,we studied the regulation of different non-selective DRD2 agonists on different neurotrophic factors by in vitro culture of astrocytes;exploring the activation of DRD2 on astrocytes by blocking the G protein pathway and the ?-arrestin2 pathway,respectively.Whether the regulation of trophic factor release has pathway selectivity and explores specific possible regulatory mechanisms.The results showed that different DRD2 agonists 1y171555,Quinelorane and Bromocriptine can increase the levels of BDNF,GDNF and NGF and decrease the level of VEGF.Further studies have shown that the activation of DRD2 on neurotrophic factors depends on G protein classics.The pathway also relies on the non-canonical pathway of ?-arrestin2,which together have a common regulatory effect on the production of neurotrophic factors.This study reveals the role of the D2 receptor downstream classical G protein pathway and the non-canonical?-arrestin2 pathway in the release of astrocyte trophic factor.This study also provides the new ideas and experimental basis for the subsequent selection of targeted and preferred DRD2 agonists for neuropsychiatric disorders.AIM:To elucidate the regulation and mechanism of Drd2 agonists on astrocyte neurotrophic factor productionMETHODS:(1)Primary astrocytes were incubated with 1y171555(40 ?M),Quinelorane(100 ?M),Bromocriptine(100 ?M),and Western blot(WB)was used to detect the protein expression levels of BDNF,GDNF,NGF and VEGF;(2)Real-time Polymerase Chain Reaction(RT-PCR)was used to detect the mRNA levels of BDNF,GDNF,NGF and VEGF under different concentrations of DRD2 agonists;(3)using the original Drd2 CKO mice's primary astrocyte or transfection of primary astrocytes with Drd2 siRNA,1y171555(or Quinelorane,Bromocriptine)was incubated,western blot or enzyme-linked immunosorbent assay(Elisa)was used to detect the protein level of BDNF,GDNF,NGF,test whether 1y171555(or Quinelorane,Bromocriptine)depended on Drd2 for the action of neurotrophic factors,and screen out the trophic factors with consistent changed under the action of different Drd2 agonists for further study;(4)Primary astrocytes were given pertussis toxin PTX(100 ng/ml)for 3 h,inhibiting the G protein pathway,and 1y171555 was incubated to detect the level of neurotrophic factor.It varied whether the changes depending on the G protein pathway;(5)In ?-arrestin2-/-primary astrocytes or after transfection of ?-arrestin2 siRNA to primary astrocytes,1y171555 was incubated,and neurotrophic factor(BDNF,GDNF,NGF)was detected whether the changes in the levels depending on the ?-arrestin2 pathway by western blot or elisa;(6)In ?-arrestin2-/-mice's primary astrocytes or transfection of primary astrocytes with ?-arrestin2 siRNA,pertussis toxin PTX(100 ng/ml)was given for 3 h to inhibit the G protein pathway.Incubate with lyl 71555(40 ?M)to verify whether changes in neurotrophin levels were abolished;(7)Western blot analysis of downstream p-CaMKII,CaMKII,p-CREB and CREB proteins after incubation with 1y171555(40 ?M)for the classical G protein pathway Level;(8)For the non-canonical p-arrestin2 pathway,1y171555(40 ?M)was incubated,and the expression levels of downstream p-ERK1/2,ERK1/2,CREB and other proteins were detected by western blot.RESULTS:(1)Different DRD2 agonists can up-regulate the protein expression of BDNF,GDNF and NGF in astrocytes and down-regulate the protein expression of VEGF.Different DRD2 agonists,Quinpirole(1y171555)(40 ?M),Quinelorane(100 ?M)and Bromocriptine(100 ?M),were able to up-regulate the protein levels of BDNF,GDNF and NGF in cultured mouse primary astrocytes(p<0.05),down-regulated the protein level of VEGF(p<0.05).(2)Different concentrations of DRD2 agonists could up-regulate the mRNA levels of BDNF,GDNF and NGF in astrocytes and down-regulate the mRNA levels of VEGF.Different concentrations of DRD2 agonists 1y171555(10 ?M,20 ?M and 40 p?M),Quinelorane(10 ?M,50 ?M and 100 p,M),Bromocriptine(10?M,50?M and 100?M)were able to up-regulate the mRNA levels of BDNF,GDNF and NGF(p<0.05).,VEGF mRNA levels were down-regulated(p<0.05).(3)The regulation of astrocyte neurotrophic factor by DRD2 agonists is dependent on DRD2.After the astrocyte was transfected with Drd2 siRNA and interfered with the expression of astrocyte Drd2,the regulation of DRD2 agonist 1y171555(40 ?M)on the production of neurotrophic factors BDNF,GDNF and NGF was abolished.(4)The regulation of astrocyte neurotrophic factors by DRD2 agonists is incompletely dependent on the classical pathway of the G protein.Primary astrocytes were pretreated with pertussis toxin(PTX)(100 ng/ml)for 3 h and then stimulated with DRD2 agonist 1y171555(40 ?M)for 12 h.Astrocyte neurotrophic factors BDNF,GDNF and NGF were detected.The up-regulation effect still exists(p<0.05).It indicates that the regulation of DRD2 activation on astrocyte neurotrophic factor still exists after PTX blocks the G protein pathway,suggesting that the regulation of DRD2 activation on astrocyte neurotrophic factor is incompletely dependent on the classical G protein pathway.(5)The regulation of astrocyte neurotrophic factor by DRD2 agonists is incompletely dependent on the non-canonical pathway of ?-arrestin2.After culturing the primary astrocytes of ?-arrestin2-/-mice or transfecting astrocytes with ?-arrestin2 siRNA,the cytokine neurotrophic factors were detected after stimulation with Drd2 agonist 1y171555(40 ?M)for 12 h.The up-regulation of cytokine neurotrophic factors BDNF,GDNF and NGF was still present(p<0.05).It is shown that after knocking out ?-arrestin2 and blocking the non-canonical pathway of ?-arrestin2,the regulation of DRD2 activation on astrocyte neurotrophic factor still exists,suggesting that the regulation of DRD2 activation is incompletely dependent on the ?-arrestin2 non-canonical pathway.(6)The regulation of astrocyte neurotrophic factor by DRD2 agonists depends both on the classical pathway of G protein and the non-canonical pathway of P-arrestin2.Afiter culturing the primary astrocytes of ?-arrestin2-/-mice or transfecting astrocytes with ?-arrestin2 siRNA,pretreatment with PTX(100 ng/ml)for 3 h followed by 1y171555(40 ?M)incubation.After 12 h,WB detected that the regulation of astrocyte cytotrophic factors BDNF,GDNF and NGF by Drd2 agonists was abolished,suggesting that DRD2 agonists regulate astrocyte neurotrophic factor,which the role depends both on the classical pathway of G protein and the non-canonical pathway of ?-arrestin2.(7)DRD2 agonists increase the gene expression of trophic factors by activating the level of astrocyte p-CaMKII to regulate the level of p-CREB.After culturing the primary astrocytes of ?-arrestin2-/-mice or transfecting astrocytes with ?-arrestin2 siRNA,they were incubated with 1y171555(40 ?M)for 12 h,and WB detected The protein level of p-CaMKII,CaMKII downstream of the G protein pathway and p-CREB in the upstream of the trophic factor was increased(p<0.05).(8)DRD2 agonists induce an increase in the expression of trophic factors by activating astrocyte p-ERK1/2 to regulate the level of p-CREB.Pretreatment of primary astrocytes with pertussis toxin(PTX)(100 ng/ml)for 3 h followed by DRD2 agonist 1y171555(40?M)for 12 h,WB detected ?-arrestin2 downstream signal ERK1/2,p-ERK1/2 and trophic factor upstream p-CREB Protein levels were elevated(p<0.05).CONCLUSIONS:(1)Agitated DRD2 can regulate the level of astrocyte neurotrophic factor.(2)agonistic DRD2 induces the expression of trophic factors by regulating the level of p-CREB through the classical pathway of G protein and the non-canonical pathway of ?-arrestin2.The major contributions of the present study lie in:(1)The specific regulation of neurotrophic factors by the activation of DRD2 in astrocytes was found.(2)To elucidate the mechanism of DRD2 downstream G protein/?-arrestin2 signaling pathway in the role of DRD2 in regulating astrocyte trophic factors.Expanded the role of DRD2 agonists on astrocytes,providing new ideas for the development of clinical application and function of DRD2 agonists.