Regulation Of PDE9A By CHIP And Its Use As A Serum Marker In PD Patients

BackgroundCarboxyl terminus of Hsc 70-interacting protein(CHIP)is a small molecule protein encoded by the STUB1 gene.It is widely involved in a variety of cellular physiological functions through its helper molecular chaperone function and ubiquitin E3 ligase activity.The pathological changes of neurodegenerative diseases,such as Alzheimer’s disease,Parkinson’s disease,Spinocerebellar ataxia,huntington’s disease and amyotrophic lateral sclerosis,mostly involve abnormal protein folding and aggregation,which leads to neuronal dysfunction and neuronal death.CHIP plays an important role in the occurrence and development of these diseases through its dual functions.Many studies have reported that mutations in STUB 1 cause ataxia.Phosphodiesterase(PDEs)is a super enzyme family responsible for the hydrolysis of cAMP and cGMP.By inhibiting PDE in the brain,the level of cAMP and cGMP response element binding proteins can be increased.Among PDE subtypes,phosphodiesterase 9A(PDE9A)has the highest expression in brain tissues,and has a high affinity with cGMP.It is widely involved in cell signaling pathways by hydrolyzing cGMP,which plays an important role in synaptic plasticity,phototransduction,learning,memory and stem cell differentiation.PDE9A inhibitors have protective effects on neurodegenerative diseases,such as Alzheimer’s disease.Studies have found that in the hereditary ataxia rat model of CHIP point mutations,the expression of PDE9A is significantly increased,suggesting that PDE9A may participate in pathogenicity by regulating cGMP.Regulating the expression of PDE9A may be a potential target for the treatment of this disease.Based on the above results,it is speculated that PDE9A and its regulated cGMP signaling pathway may be involved in the occurrence of CHIP-related hereditary ataxia.This study intends to further explore the regulatory effect of CHIP on PDE9A and the interaction between them in an in vitro cell model,in order to further study the potential neuroprotective effect of targeted PDE9A therapy on CHIP-related ataxia.Parkinson’s disease is the second most common neurodegenerative disease.Its motor symptoms include bradykinesia,resting tremor,muscle rigidity and postural disturbance.The main pathological features include the gradual loss of dopaminergic neurons in the substantia nigra and eosinophilic Lewy bodies in the cytoplasm of residual neurons.The symptoms of Parkinson’s disease are complex.The current clinical diagnosis mainly relies on motor symptoms,and early diagnosis is relatively difficult.The severity of Parkinson’s disease is mainly evaluated by scales,which is subjective by physicians.Therefore,it is of great significance to find biomarkers with high sensitivity and specificity related to Parkinson’s disease.CHIP is a key molecule in the protein quality control system,which can mediate the elimination of abnormal accumulation of proteins,which plays an important role in degenerative diseases.Studies have shown that CHIP protein interacts with α-synuclein,Parkin and LRRK2 respectively.PINK 1 gene is involved in the pathogenesis of Parkinson’s disease.CHIP protein can promote the ubiquitination of PINK1,thereby negatively regulating the stability of PINK1.Up-regulation of CHIP protein expression in PINK1 mutant drosophila model significantly improved movement disorders,loss of dopaminergic neurons,and mitochondrial defects.Therefore,CHIP protein may be involved in the pathogenesis of Parkinson’s disease.Serum is a biological sample that is relatively easy to obtain in clinical work.It has the advantages of being convenient to obtain and less harmful to the human body.In this part of the study,we collected serum from patients with Parkinson’s disease,detected the content of CHIP protein,and explored the feasibility of serum CHIP protein as a biomarker for Parkinson’s disease.Objective1.Through overexpression and inhibition of CHIP protein expression in cell models,explore the regulation of CHIP protein on PDE9A and the related mechanisms of their interaction.2.Serum CHIP levels of Parkinson’s patients and healthy control people were detected by ELISA to explore whether serum CHIP levels could be a potential biomarker for the clinical diagnosis of PD,and to provide new ideas for the diagnosis of PD.Methods1.Construct PDE9A overexpression vector,construct STUB 1 wild-type and mutant plasmids by cloning and recombination method.Cells were transfected with STUB1 and PDE9A overexpression vectors,and the levels of CHIP protein and PDE9A protein were detected by Western Blot to study the effect of CHIP expression on PDE9A protein.The interaction between CHIP and PDE9A protein was studied by immunoprecipitation.2.This study conducted an exploratory case-control study.A total of 65 Parkinson’s patients,age-and gender-matched normal people’s serum were collected,and the CHIP protein level was detected by ELISA.The t-test was used to analyze whether there was a difference in serum CHIP protein levels between Parkinson’s patients and healthy control,Spearman correlation analysis was used to evaluate the correlation between the level of serum protein CHIP and the course of the disease,MDS-UPDRS total score,MDS-UPDRS motor part score,Hoehn-Yahr grading and the Schwab score.The ROC curve was drawn to analyze the specificity and sensitivity of the diagnostic method.Results1.PDE9A overexpression vector and STUB1 wild-type and mutant plasmids were successfully constructed by recombination.2.The results of western blot analysis showed that the expression of PDE9A protein decreased when the expression of CHIP protein increased.When the expression of CHIP protein was inhibited,the expression of PDE9A protein increased.Compared with the wild type,when CHIP had H260Q mutation and K30A mutation,The expression of PDE9A increased.3.Co-immunoprecipitation proved that CHIP protein interacted with PDE9A protein.4.The serum CHIP protein levels of Parkinson’s patients and normal people were(52.47 ± 13.04)ng/mL and(43.07±12.45)ng/mL respectively,and statistical analysis showed P<0.01.The serum CHIP protein concentration in Parkinson’s disease group had no significant correlation with the course of disease,MDS-UPDRS total score,MDS-UPDRS motor part score,Hoehn-Yahr grade.The receiver operating characteristic(ROC)curve was established,and the results showed that the area under the curve was 0.698,P<0.01,and the 95%confidence interval was 0.608-0.787,indicating good specificity and sensitivity.Conclusion1.The PDE9A overexpression vector and STUB 1 wild-type and mutant plasmids were successfully constructed.2.CHIP expression level can regulate the expression level of PDE9A protein.CHIP protein interacts with PDE9A protein.3.Serum CHIP protein concentration is significantly increased in Parkinson’s disease patients,with good sensitivity and specificity,which can be used as a potential biomarker of Parkinson’s disease.