The Role Of D1R/Shp-2 Complex In D1R/Shp-2/ERK1/2 Signal Pathway Of Dyskinesia Rats

Parkinson's disease(PD)is a debilitating neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra.This disease is the second major neurodegenerative disorder after Alzheimer's disease.When the dopaminergic neurons are damaged more than 70-80 percentage,the clinical traits of PD are expressed of bradykinesia,resting tremor,muscle rigidity and postural instability.Exogenous levodopa can improve the clinical symptoms of most patients at first,but it can not prevent the progress of the disease.And long-term use of exogenous levodopa will lead to the decline or disappearance of the efficacy.To the best of our knowledge,l-3,4-dihydroxyphenylalanine(L-DOPA)continues to be the gold standard therapy for PD.However,the long-term use of L-DOPA leads to a severe motor side effect known as levodopa-induced dyskinesia(LID),which depletes its efficacy gradually.Despite great progress in basic research on LID in recent years,the mechanisms of LID are still not fully understood.As we known,there are direct and indirect pathways from striatum to internal segment of the globus pallidus(Gpi)and substantia nigra pars reticulata(SNr)in the basal ganglia.In PD patients,the degeneration of DA neurons leads to excessive inhibition of basal ganglia's feedback to the motor cortex,resulting in reduced motor symptoms.Previous studies have shown that D1 receptor hypersensitivity mediates the overactivation of ERK1/2 in striatum and abnormal downstream mTORC1 signaling pathway,which is one of the main mechanisms of LID.Moreover,this hypersensitivity is attributed to the abnormal activation of signal transduction.Accumulating evidence indicates that LID develops in response to activation of sensitized D1 Rs and modifications of ERK signaling pathway.One study has recently identified a new mechanism for D1R-mediated activation of ERK1/2 in striatal neurons involving the tyrosine phosphatase Shp-2.In particular,in the striatum,Shp-2 directly interacts with the D1 R and is activated by D1 R stimulation which is essential for downstream signaling pathway.In this study,we aim to investigate the role of the D1R/Shp-2 complex in L-Dopa-induced aberrant activaion of ERK1/2 signaling and development of dyskinesia in the 6-OHDA rat model of PD,using D1 receptor agonists and antagonists specifically disrupting striatal D1R/Shp-2 interaction.We aim to define the role of the D1R/Shp-2 complex in the development of LID since it could represent a promising new drug target for LID.The mechanism of LID is the pulsative dopamine stimulation in the striatum.Studies showed that continuous dopamine stimulation was helpful in reducing LID.Thus,in the present study,levodopa and benserazide were microencapsulated into poly(lactide-co-glycolide)(PLGA)microspheres which can sustain release levodopa and benserazide.Then we will observe the effect of the microspheres on the D1R/Shp-2 complex and phosphorylation of Shp-2,and provide experimental basis for further clarifying the mechanism of LID.The present study was divided into three parts: Part ?: The interaction of D1R/Shp-2 complexAfter the treatment with L-DOPA/benserazide for 22 days,PD rats presented with dyskinesia.In order to explore the relationship between D1 R and Shp-2 in the pathogenesis of LID,we confirm the co-localization of D1 R and Shp-2 in striatum cells by immunofluorescence firstly.Then the interaction between D1 R and Shp-2 was confirmed in the sham and LID rats through the methods of coimmunoprecipitation.The interaction between D1 R and Shp-2 in the normal rats was kept stable after the long-term use of L-DOPA.All the above,these data verified the existence of D1R/Shp-2 complex.Part ?: The effect of D1R/Shp-2 complex involved in LID in PD ratsTo further confirm the role of D1R/Shp-2 complex,we designed this study with the purpose of exploring the role of D1R/Shp-2 complex in the D1R-mediated signaling pathway in the occurrence of LID.The 6-hydroxydopamine(6-OHDA)was injected unilaterally to produce the rat models of PD.Successful PD rat models were randomly divided into three groups to receive the treatment with L-DOPA/benserazide,L-DOPA /benserazide + D1 R antagonist(SCH23390)or D1 R agonist(SKF38393).Abnormal involuntary movements were assessed in different groups during the treatment.In addition,the levels of p-Shp-2,p-ERK1/2 and p-mTOR were determined by Western blot in different groups.D1 R agonist,SKF38393,induced similar involuntary movements in PD rats.In contrast,the dyskinetic movements were not induced by coadministration of L-DOPA + D1 R antagonist(SCH23390).Moreover,we found that the pulsatile levodopa administration induced hyperphosphorylation of Shp-2,ERK1/2 and mTOR,while the co-administration of L-DOPA and D1 R antagonist,SCH23390,did not induce the hyperphosphorylation of these proteins.These data verified the crucial role of D1R/Shp-2 complex in the D1R-mediated signaling pathway in dyskinetic rats.Focus on the D1R/Shp-2 complex might be a potential treatment of LID in the future.Part ?: Levodopa/Benserazide loaded microspheres alleviate L-dopa induced dyskinesia through preventing the over-expression of D1R/Shp-2/ERK1/2 signaling pathway in a rat model of parkinson's diseaseTo observe the effect of levodopa/benserazide-loaded PLGA microspheres on D1R/Shp-2/ERK1/2 signal pathway of LID and to explore the underlying mechanisms.Here we designed the study in which two different methods of L-dopa delivery(continuous dopamine stimulation vs.intermittent dopamine stimulation)were used to further identify.6-OHDA-lesioned rat models of PD were randomly divided into two groups to receive intermittent L-dopa stimulation(L-dopa/benserazide standard group,LS group)or CDS(L-dopa/benserazide loaded microspheres,LBM group)for 21 days.Dyskinesia and anti-parkinsonian effect were compared between the two groups through the AIMs assessment and cylinder test.The critical protein changes in the D1R/Shp-2/ERK1/2 signaling pathway were compared between the two groups through Western blotting.We found that intermittent L-dopa administration induced serious dyskinetic movements in the 6-OHDA-lesioned rats,and the anti-parkinsonian effect of L-dopa was gradually counteracted by the occurrence of dyskinesia.Intermittent L-dopa administration enhanced the expression of membrane D1 R,and induced a robust increase of phosphorylation of Shp-2,Src,DARPP-32,and ERK1/2 in the 6-OHDA-lesioned striatum.In contrast,CDS played a dose-dependent antiparkinsonian role,without inducing such apparent dyskinetic movements.Moreover,CDS induced no change of membrane D1 R expression or phosphorylation of Shp-2,Src,DARPP-32,and ERK1/2 in the 6-OHDA-lesioned striatum.The aberrant activation of D1R/Shp-2 complex was evidenced to be required for the D1 R mediating ERK1/2 phosphorylation and the occurrence of LID.CDS effectively prevented the overexpression of D1R/Shp-2/ERK1/2 signaling pathway,resulting in the reduction of LID in 6-OHDA-lesioned rats model of PD.