| OBJECTIVE: Parkinson’s disease(PD)is a common neurodegenerative disease that occurs in middle-aged and elderly people.Movement disorder is the main evidence for the clinical diagnosis of PD.Most PD patients show digestive tract symptoms such as malnutrition and constipation over a course of decades that precedes the onset of typical motor symptoms.The abnormal expression ofα-Synuclein(α-Syn)in neurons and the aggregation of Lewy bodies are the specific pathological changes of PD.The Braak hypothesis suggests that α-Syn pathological changes may originate earlier in the gastrointestinal and then propagate from the bottom up to the brain.In the early stage,our team established PD model of mice with abnormal increase of α-Syn by injectingα-Syn pre-formed fibrils(PFF)into the intracerebral striatum,which proved that the expression of Dopamine receptor D1(DRD1)in substantia nigra and striatum decreased.Studies have shown that the level of dopamine in the smooth muscle of the colon in PD patients is low,and whether the abnormal increase of α-Syn affects the expression of DRD1 in colon and its mechanism has not been confirmed.In addition,there are studies to prove that α-Syn can reduce the expression of N-methyl-D-aspartic acid receptor 1(NMDAR1)on the cell surface in PD,while DRD1 and N-methyl-D-aspartic acid(NMDA)receptor are assembled in the form of constitutive heterogeneous complexes and transferred to functional sites.This study intends to explore the potential relationship between the abnormally increased α-Syn and the changes in DRD1 in the substantia nigra and the colon,and whether α-Syn PFF may affect the expression of DRD1 through NMDAR1,in order to provide new evidence for elucidating the pathogenesis of PD.METHODS: C57BL/6J mice were randomly divided into a control group and a model group.In the model group,a stereotactic injection of α-Syn PFF in the striatum was used to prepare a mouse PD model,and the control group was injected with an equal volume of PBS.Balance beam,climbing-pole and open field tests were performed to detect the behavioral differences of mice at 6months after the operation.Western blot,immunohistochemistry,and immunofluorescence were used to detect the expression of DRD1,α-Syn,and tyrosine hydroxylation(TH)of the substantia nigra and colon.Take Sprague-Dawley fetal rat cerebral cortex to culture primary neurons,add α-Syn monomer or α-Syn PFF respectively,and detect the expression changes of DRD1 and NMDAR1 by Western Blot and immunofluorescence.B104 cells were infected with lentivirus to overexpress NMDAR1 and supplemented withα-Syn PFF,real-time quantitative PCR detection included NMDAR1 and DRD1 m RNA levels,immunofluorescence detection of NMDAR1 and DRD1 expression changes.RESULTS: Compared with the control group,the mice in the model group exhibited prolonged time in balance beam and climbing-pole,and slower average moving speed in open field test.The results of Western Blot,immunohistochemistry and immunofluorescence showed that the number of DRD1,TH positive cells was significantly reduced(P<0.01,0.01,0.05)and the expression of DRD1 and TH in the substantia nigra and colon of the model mice were lower than those in the control group(P<0.01,0.05,0.01,0.01);The expression in the substantia nigra and colon is positively correlated(r = 0.8943,0.9026,P<0.05).In cell experiments,Western Blot and immunofluorescence results showed that compared with the control group,the expression of DRD1 and NMDAR1 was not significantly differentin in the group with α-Syn monomer.The expression of DRD1 and NMDAR1 in the group with α-Syn PFF was significantly reduced.In the cells overexpressing NMDAR1 through lentivirus infection,real-time quantitative PCR and immunofluorescence results showed that compared with the control group,the expression levels of NMDAR1 and DRD1 in the overexpression group increased.Afterwards,the expression levels of NMDAR1 and DRD1 in the α-Syn PFF-added group were lower than the overexpression group;compared with the α-Syn PFF-added group,the expression levels increased.The expression of DRD1 and NMDAR1 decreased with the abnormal increase of α-Syn,and the expression of DRD1 was same with the overexpression of NMDAR1.Therefore,the above experimental results suggest that α-Syn PFF may reduce DRD1 expression through NMDAR1.CONCLUSION: The abnormal increase of α-Syn may decrease the expression of DRD1 by down regulating NMDAR1. |
