TRH,p-ERK1/2 And FosB-△FosB Are Involved In A Positive Feedback Loop Influencing The Development Of Levodopa-induced Dyskinesia

Sectin 1:The relationship between LID and TRH,p-ERK1/2,FosB-△FosB and other relevant signal moleculesObjective To clarify the alterations of the whole transcripts and the major signal molecules in the dopamine-depleted striatum associated with LID.Methods 6-OHDA(2pg/μl,4μl)were injected into the medial forebrain bundle of SD rats.After two weeks,the model was validated using the apomorphine-induced rotation test.For the successful PD models,levodopa and benserazide were intraperitoneally administered 3 days later,once daily for 21 days to replicate the lateral LID rat model.Axial,orofacial,forelimb and locomotive abnormal involuntary movements(AIMs)were assessed every other day.Lesions of dopaminergic neurons in SN were verified using immunohistochemistry.The alterations of the whole transcripts in the striatum of lesioned side were detected using microarray.The expression of TRH mRNA and FosB mRNA was further validated by qRT-PCR.The co-expression of TRH and FosB-△FosB was detected by immunofluorescence double-labeling methods.The protein expression levels of TRH,FosB-AFosB and p-ERK1/2 were quantified using Western blot.Results Compared with PD group,TRH mRNA levels in the dopamine-depleted striatum of rats in the LID group were significantly increased(P<0.001).And TRH mRNA was in a sustained high level after L-DOPA administration.Double immunolabeling images exhibited complete colocalization between TRH and FosB-△FosB in rats with dyskinesia.No expression of the two molecules was observed in the intact side of striatum.In addition,the protein levels of TRH,FosB-△FosB and p-ERK1/2 of rats in LID group were higher than those in PD group.Conclusion The occurrence and development of LID is associated with persistent high expression of TRH in the striatum,and the abnonnal expression of TRH could be related to FosB-△AFosB,an established molecular hallmark of LID.Section 2:The effects of TRH on LID and p-ERK1/2 and other relative signal moleculesObjective The major purposes of this section were to investigate the effects of TRH on LID and relative signal molecules,determine whether TRH could directly regulate the phosphorylation of p-ERK1/2.Methods TRH tripeptide or TRH analogue(Taltirelin)was injected into the dorsolateral striatum of rats with unilateral 6-OHDA lesions of the nigrostriatal pathway through intracranial catheterization once daily 30 minutes before intraperitoneal injection of levodopa and benserazide for 1 week.Axial,orofacial and forelimb abnormal involuntary movements(ALO AIMs)were assessed every day.Rats were sacrificed on the 7th day,and the expression levels of p-ERK1/2 and FosB-AFosB in the striatum were detected.Lentivirus LV-TRH-shRNA-GFP was packaged,and then infected PC 12 cells.The best sequence was screened out which could effectively interfere with TRH mRNA.The TRH-specific interference sequence was packaged into adeno-associated virus.And AAV-TRH-shRNA-GFP was injected into the dorsolateral striatum.After 4 weeks,Rats received daily L-DOPA treatment for 1 week,and axial,orofacial and forelimb abnormal involuntary movements(ALO AIMs)were assessed every day.Rats were sacrificed on the 7th day,and the expression levels of TRH,p-ERK1/2 and FosB-△FosB in the striatum were detected.PC 12 Cells were incubated with vehicle,MEK inhibitor(U0126)and TRH receptor TRH-R1 antibody(Anti-TR1).After 2 hours,TRH tripeptide and TRH analogue Taltirelin were employed to treat cells.Western Blot technique was used to detect the phosphorylation levels of p-ERK1/2 under different treatment conditions.Results TRH tripeptide and TRH analogue Taltirelin could significantly aggravate LID,increase the expression of p-ERK1/2 and AFosB.AAV-TRH-shRNA could significantly alleviate LID,down-regulate TRH mRNA and decrease the expression of p-ERK1/2 and AFosB.TRH tripeptide,TRH analogue Taltirelin could significantly increase the phosphorylation level of p-ERK1/2,and this elevated effect could be inhibited by MEK inhibitor U0126 and TRH-R1 antibody.Conclusion TRH could be involved in the progression of LID and positively regulate ERK1/2 phosphorylation and AFosB expression.Section 3:The regulation of p-ERK1/2 on FosB-AFosB expression and the regulation of FosB-AFosB on TRH expressionObjective The major purposes of this section were to observe the effects of MEK inhibitor U0126 on LID and FosB-△FosB expression,clarify the regulation of FosB gene on TRH expression.Methods Rats with unilateral 6-OHDA lesions of the nigrostriatal pathway received chronic daily L-DOPA treatment for three weeks,and axial,orofacial and forelimb abnormal involuntary movements(ALO AIMs)were assessed every other day.U0126 was injected into the dorsolateral striatum for 12 days starting from day 10 of L-DOPA treatment.The protein levels of p-ERK1/2,△FosB and other signal molecules were quantified by Western Blot.Lentivirus LV-FosB-GFP was packaged,and then infected PC 12 cells.TRH mRNA and FosB mRNA were detected by qRT-PCR.Results Administration of MEK inhibitor U0126 could significantly alleviate the severity of fully-established AIMs and reverse the overexpression of FosB-△FosB and TRH.Overexpression of FosB could significantly increase the expression of TRH.Conclusion P-ERK1/2 could positively regulate the expression of FosB-△FosB and TRH.FosB-AFosB could positively regulate the expression of TRH.