The Investigation Of The Biological Function Of α-synuclein And Its Phosphorylation

Parkinson’s disease（PD）is the second most common neuronal degenerative disorder in the world.The pathological hallmarks of PD are the progressive loss of dopaminergic neurons in the substantia nigra pars compacta（SNc）,and the formation of Lewy bodies（LBs）.Even though numerous studies have demonstrated the mechanism and a variety of hypotheses been proposed,unfortunately,the detailed mechanism of PD remains unclear.α-synuclein（α-syn）is one of the key proteins in PD pathogenesis as it’s the main component of Lewy bodies（LBs）,one of the main hallmarks of PD.Aggregatedα-syn can be detected both in sporadic and familial PD,particularly,the duplication and triplication of SNCA and three site mutations（A53T,A30P,and E46K）inα-syn lead to PD.Mechanical studies of the phosphorylatedα-syn in PD pathogenesis has attracted widespread attention due to the fact that approximately 90%ofα-syn deposition in LBs is phosphorylated at Ser129,whereas in normal brains,only 4%or less ofα-syn is phosphorylated at this residue.The great disparity suggests that post-translational modification ofα-syn,especially phosphorylation,may be worthy of further investigation.This study can be divided into three branches:1.Firstly,we detected the effects ofα-syn PD-linked mutations and Ser129phosphorylation on PD pathogenesis.Using these plasmids pcDNA3.1::-syn、pcDNA3.1::-syn A53T、pcDNA3.1::-syn A30P as templates,we have successfully constructed 9 plasmids containing double-mutations,such as pcDNA3.1::-synS129A,pcDNA3.1::-synS129D,pcDNA3.1::-synA53T S129A,pcDNA3.1::-synA53TS129D et al.These newconstructed plasmids were identified by restriction enzyme digestion and nucleotide sequencing.Next,the plasmids were transfected in the PC12 cells 48h and their expression levels were detected and proved by Western Blot.Then we investigated the apoptosis of cell and theα-syn distribution in the cell after over-expression of the plasmids.We also detected the function of possible degradation pathways,like Ubiquitin-protesasome system（UPS）and autophagy-lysosome pathway（ALP）on regulation ofα-syn PD-linked mutation and Ser129 phosphorylation by detecting the key protein expressions in vitro.The results indicated that differentα-syn PD-linked mutations and Ser129 phosphorylation interaction may contribute to PD pathogenesis via different mechanisms.In vivo,the immunohistochemical experiment was performed with SNCA transgenic mice and the control mice,and the results proved that the degree ofα-syn Ser129phosphorylation is higher in theα-syn overexpression mice group;and subsequently,Tyrosine Hydroxylase（TH）expression was deleted and the level of ubiquitin and LC3B all became higher both in substantia nigra and the striatum.The results indicated that the increase ofα-syn Ser129 phosphorylation may lead to the deletion of dopaminergic neurons,and may promote the UPS and ALP pathway.This study may provide novel insights into the mechanism underlying theα-syn PD-linked mutation and Ser129 phosphorylation on PD pathogenesis,as well as the interplay between PD-linked mutations and the pathogenic phosphorylation.2.Secondly,we explored the function of a small molecule inhibitor Epigallocatechin gallate（EGCG）onα-syn and phosphorylatedα-syn aggregation.EGCG has been considered as a potential agent for PD.In this study,the inhibitory mechanism of EGCG onα-syn,α-syn A53T and their phosphorylated form aggregation was detected.We first extracted,purified theα-syn andα-syn A53T protein.We used the Thioflavine T（ThT）staining method and the electron microscope（EM）method to detect theα-syn aggregation pattern in vitro and the effect of EGCG inhibition.The results indicated that EGCGcouldeffectivelyinhibittheα-synaggregationina concentration-dependent manner.The ED₅₀ of EGCG inhibition was 250 nM.The binding sites ofα-syn that potentially interactes with EGCG,were detected on peptide membranes GKTKEGVLY,GVLYVGSKT,and AAATGFVK.In the PD tissue based assay,α-syn aggregates were also blocked by EGCG in a concentration dependent manner.And it was implicated that EGCG binds toα-syn by instable hydrophobic interactions.We also found that,EGCG could effectively inhibit the aggregation of phosphorylatedα-syn,however,for theα-syn A53T mutation and its phosphorylated form,EGCG could only inhibit the aggretation in a short period,as time goes,EGCG may promote the protein aggregation,which presented the opposite effect.In this study,we suggested that EGCG could be a potent small molecular remodeling agent ofα-syn aggregates and a potential disease modifying drug for the treatment of PD and otherα-synucleinopathies.3.In this part of study,we did theα-syn quantitation in human plasma based on liquid chromatography stable isotope dilution tandem mass spectrometry（HPLC-SID-MS）:the quantitation of PD biomarker.First,we used the quadrupole-time of flight mass spectrometer（Q-TOF）to detect theα-syn specific peptides GVVAAAE（GAE）and GVLYVGSKTKE（GKE）,and optimized the multiple reaction monitoring（MRM）parameters,then we chose 5 ion transitions as the qualitative peptides,and 1 ion transitions as the quantitative peptide（590.6->648.8）.Theα-syn SH-SY5Y stable cell strain was used to identify the specificity of the peptides.The ¹⁸O labeling efficiency was 96.44%,and the labeling was stable.We used the plasma of healthy controls as the matrix,the methodology validation indicated the limits of detection ofα-syn was 0.5nM,and the limits of quantification was 1nM.The quantitative range was 1nM-80nM,and the linear correlation coefficient was 0.9973.The inter-day precision was less than 15%,and the intra-day procision was less than 20%.The method also showed good accuracy 71.65%¹01.51%,and this method can meet the needs of complex clinical samples.Finally,we used the diagnosed PD patients and healthy controls to detectα-syn in plasma with the method.The results indicated that totalα-syn content in PD patient plasma was higher than the control（p=0.058）.For the ROC curve,The specificity was 37.5%and the sensitivity was 87.5%,AUC was 0.7813,which indicated that the method accuracy was good.The establishment of the method may provide the theoretical foundation and experiment evidence for PD biomarker quantitation.This article is focused on the effect ofα-syn PD-linked mutation and Ser129phosphorylation on PD pathogenesis;α-syn and phosphorylatedα-syn aggregation small molecule inhibitor Epigallocatechin gallate（EGCG）function and the quantitation ofα-syn in human plasma based on the mass spectrum ¹⁸O stable isotope label.Based on these three parts of the molecular mechanism,drug development and PD biomarker quantitation,using molecular biology tools,cell model,experimental animal,and human samples,we studied theα-syn protein and its phosphorylation.This study may provide theoretical foundation and experiment evidence for PD pathogenesis and translational medicine study.