The Expression And Mechanism Of CIP2A In Parkinson’s Disease

Part 1 Patients with Parkinson’s disease have decreased CIP2 A concentration in their peripheral plasmaObjective: To compare peripheral plasma CIP2 A levels in patients with Parkinson’s disease(PD)and healthy controls.Methods: Peripheral blood was collected from patients with idiopathic PD and healthy controls,and plasma was obtained by centrifugation.Demographic information about PD patients and healthy controls,including name,age,gender and duration of disease,was collected.Scale information for PD patients is also needed,including the Unified Parkinson’s Disease Rating Scale Part III(UPDRS-III),the Mini-Mental State Examination(MMSE),the Montreal Cognitive Assessment(Mo CA),the Hamilton Depression Scale(HAMD),and the Hamilton Anxiety Scale(HAMA).Plasma CIP2 A concentrations in idiopathic PD patients and age-and gender-matched healthy controls were measured by ELISA test.The Mann-Whitney U test was utilized to statistically analyze the plasma CIP2 A concentration in the PD group and the control group.The ROC curve was made,and the area under the curve was calculated to obtain the specificity and sensitivity of the index.Correlation analysis evaluated whether there was a correlation between CIP2 A concentration with motor and non-motor symptoms.Results: The plasma CIP2 A concentration in PD patient group was significantly lower than that in the age-and sex-matched control group,1.721(1.435-2.428)ng/ml vs 3.051(2.36-5.475)ng/ml,respectively;P <0.0001.The area under the plasma CIP2 A curve that distinguished PD patients from normal controls was 0.776,indicating that CIP2 A plasma concentration can be used as an indicator for diagnosing PD.Its cutoff is 2.528 ng/ml,sensitivity is 80.65%,and specificity is 69.84%.Correlation analysis was performed between plasma CIP2 A concentration with UPDRS-III,MMSE,Mo CA,HAMD,and HAMA scale score.The results indicated that plasma CIP2 A concentration of PD patients had no significant correlation with these scale scores.Conclusion: Compared with healthy controls,the plasma CIP2 A concentration in PD patients is significantly lower,it may be a potential diagnostic biomarker for PD patients.There is almost no correlation between plasma CIP2 A concentration in PD patients with disease duration,motor impairment,non-motor impairment,and cognitive level.So it has no value in monitoring the progression of the disease.Part 2 Increased expression of CIP2 A in PD cell modelObjective: To observe the expression of CIP2 A molecule and pathological changes in PD cell models.Methods: The appropriate concentration of rotenone was tested by CCK8 on SH-SY5 Y cell line.To establish rotenone induced cell model and then verified the success of the model.Cellular immunofluorescence and Western Blot were used to compare the differences of CIP2 A expression before and after modeling,and explore the changes of α-syn,phosphorylated α-syn and PP2 A expression in rotenone model.Results: After treatment with rotenone,PD-like changes were manifested at the cellular level,such as increased expression of α-syn and phosphorylated α-syn,which indicate that rotenone cell model was successfully established.And it was found that the expression level of CIP2 A increased and the level of PP2 A decreased after rotenone treatment.Conclusion: After rotenone treatment of SH-SY5 Y cells,the level of CIP2 A increased and the downstream PP2 A protein decreased in the cell model.This change may be the cause of increased expression of α-syn and phosphorylated α-syn.Whether the pathological changes in PD are affected by CIP2 A,which through reduced activity of PP2 A,remains to be further investigated.Part 3 Increased CIP2 A expression in mouse model of PDObjective: To establish a mouse model of PD and observe the expression of CIP2 A in the substantia nigra of the mouse brain.Methods: Adult C57 / B6 J male mice were randomly divided into the MPTP subacute intervention group(MPTP 30mg/kg/d,continuous intraperitoneal injection for 1 week)and the wild type(WT)control group(continuous intraperitoneal injection of equal volume of normal saline for 1 week).Rotarod test and Pole test were used to evaluate the motor coordination and balance ability of mice.The expression of TH positive neurons in the substantia nigra of mice was observed by immunofluorescence and Western Blot,which confirmed the success of the model.The expression of CIP2 A in the substantia nigra region of mice was observed by immunofluorescence,immunohistochemical staining and Western Blot.Results: Motor coordination ability of the mice in the MPTP group was significantly decreased,TH positive staining neurons in the substantia nigra were significantly reduced,indicating that the model was successfully constructed.The expression of CIP2 A in the substantia nigra is increased in MPTP induced model mice.Conclusion: The mouse model of PD was successfully established.In the mouse model of PD,the level of CIP2 A protein was also increased,which was consistent with that in the cell model.It is necessary to explore the role of CIP2 A in PD.Part 4 Protective effect of knockdown of CIP2 A on a cell model of PD Objective: To construct a small interfering RNA(si RNA),transfecting SH-SY5 Y cells and knocking down CIP2 A protein level,and then observe the effect of knockdown CIP2 A on the pathology of PD.Methods: Three si RNAs were designed and synthesized based on previous literature and published sequence data.SH-SY5 Y cells were transiently infected,Western Blot was used to evaluate the interference effects of the three si RNAs.Screening a si RNA with good interference effect for the subsequent experiment.SH-SY5 Y cells are divided into four groups: control group,si RNA intervention group,rotenone treatment group,si RNA intervention + rotenone treatment group.Western Blot and cell immunofluorescence used to detect possible changes in the related downstream proteins,including CIP2 A,α-syn,phosphorylated α-syn and PP2 A.Results: All three constructed si RNAs have good interference effects.After knocking down the CIP2 A protein level,the expression of PP2 A protein increased,which improve the PD-like pathology caused by rotenone.Conclusion: Knocking down the CIP2 A protein level has a protective effect on PD cell models induced by rotenone.This protective effect may be caused by an increase in PP2 A activity through knocking down CIP2 A.