| Background and Aim Parkinson's disease(PD)is a common degenerative disease of the central nervous system and it tends to occur in elderly people over 65 years old.With the growth of the aging population,the prevalence and incidence of PD in the global are increasing.China is in the stage of rapid increase of PD patients.The number of PD patients will account for about half of the global patient numbers at present and in the future.As a disease with low fatality rate and high disability rate,PD not only seriously threatens the physical and mental health of PD patients,but also aggravates the familial and social burden.At present,the pathogenesis of PD is still unclear,and further studies are urgently needed.The disease progresses of PD is tardy.In the early stage of the disease,non-motor symptoms such as olfaction dysfunction and REM sleep behavior disorder have occured.With the aggravation of the disease,some typical clinical symptoms,such as static tremor and myotonia,will gradually appear.Now,the clinical evaluation and diagnosis of PD mainly depend on its motor symptoms.It is noteworthy that about 30~70% of dopaminergic neurons in the substantia nigra are already lost when the patient is diagnosed as a PD patient.Therefore,it is crucial to diagnose PD in the early stage of the disease for delaying or early intervene the development of PD.In order to realize the early diagnosis of PD,many researchers focus on the biomarkers in the early stage of disease.The main pathological features of PD are the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the formations of Lewy bodies(LBs)and Lewy neurites(LNs).The main components of LBs and LNs are ?-synuclein(?-syn).?-syn is a monomer protein composed of 140 amino acids,which mainly enriches in the presynaptic terminal.The Post-translational modifications of ?-syn,such as phosphorylation,can promote its monomer aggregating to gradually develop into oligomers and fibrils,and eventually accumulate into LBs and other inclusions,which seriously damages the normal function of cells.It was found that only less than 4% ?-syn was phosphorylated at Ser129 in normal brains,but at least 90% ?-syn was phosphorylated in LBs of PD brains,suggesting that the ?-syn phosphorylated at Ser129(P-?-syn)was prone to aggregation.Under physiological conditions,the aggregated ?-syn can be cleared by various protein degradation pathways,such as the autophagy-lysosomal pathway and the ubiquitin-proteasome system.However,when the intracellular protein degradation pathways are impaired,their ability to degrade abnormal ?-syn is limited,which may activate other non-classical secretory pathways,such as exosome-related secretory pathways,to release abnormal ?-syn into the extracellular environment.Therefore,some non-classical vesicle release pathway in the cell may be the compensatory mechanism of protein degradation pathway,which plays a protective role on themselves.More and more evidences showd that exosomes can not only promote the aggregation of ?-syn,but also played a role in the propagation of pathological ?-syn from cell to cell,resulting in the acceleration of PD progression.Exosomes are 30~150 nm extracellular vesicles found in cerebrospinal fluid,blood and urine.In order to find effective biomarkers for PD,many researchers focus on exosomes.Currently,some body fluids such as cerebrospinal fluid,blood and urine are concerned,which are regarded as the sources of PD biomarkers.It was reported that the total level of ?-syn in cerebrospinal fluid of PD patients was significantly lower than that of control group,but the level of oligomeric ?-syn,P-?-syn and exosomal ?-syn in cerebrospinal fluid were significantly higher.The levels of exosomal ?-syn and Tau derived from the central nervous system in plasma of PD patients were both significantly higher than control group.In addition,phosphorylated ?-syn in cerebrospinal fluid of PD patients was demonstrated that might play a key role in the pathogenesis and the early diagnosis of PD.These results imply that the exosomal ?-syn and P-?-syn in plasma may be potential biomarkers for PD.In this study,Western Blot,mass spectrometry and other techniques are used to detect the expression levels of exosomal ?-syn and P-?-syn in plasma between PD patients and healthy controls so that looks for PD biomarkers in plasmic exosomes.It will provide the oretical basis for the early diagnosis of PD,and futher improve the accuracy of the diagnosis,gain better prognosis and obtain potential disease intervention.In addition,we also investigated the effect of ?-syn overexpression on protein degradation pathway and vesicle release pathway to explore the pathogenesis of PD.Materials and MethodsPart 1 Detection of the level of exosomal ?-syn and P-?-syn in plasma1.Plasma samples were collected from 36 healthy controls and 36 PD patients,and then exosome precipitation kit and differential centrifugation were used to isolate plasma exosomes.2.The morphology,particle size and surface marker protein of exosomes in plasma were identified by transmission electron microscopy,nanoparticle tracing analysis and Western Blot,respectively.3.Protease K was used to detect the degradation characteristics of exosomal ?-syn and P-?-syn in plasma.4.Expression levels of exosomal ?-syn and P-?-syn in plasma were detected by Western Blot.And the differentially expression levels of proteins in plasma was preliminarily detected by mass spectrometry.Part 2 Effects of ?-syn overexpression on protein degradation systems in PC12 cells1.PC12 cells were transfected with pc DNA3.1(-)and SNCA-coding plasmids,respectively,and then the m RNA levels of proteins related to the autophagy-lysosomal pathway and the ubiquitin-proteasome system were detected.2.PC12 cells were transfected with pc DNA3.1(-)and SNCA-coding plasmids,and the co-localization of proteins related to the autophagy-lysosomal pathway and the ubiquitin-proteasome pathway with P-?-syn aggregates in PC12 cells was detected by immunofluorescence.Results1.Both differential centrifugation method and exosome precipitation method could isolate exosomes from the plasma of PD patients and healthy controls,and some exosomes were derived from the central nervous system.Exosome precipitation kit produced higher yields of exosomes than differential centrifugation method(P < 0.05).2.?-syn and P-?-syn were existed on the surface and inside of exosomes isolated from the plasma of both PD patients and healthy controls.And the exosomal ?-syn and P-?-syn in PD patient plasma were more difficult to be degraded by protease K(P < 0.05).3.The ratio of ?-syn monomer(17 KDa)to total ?-syn level in plasma exosomes of PD patients was significantly lower than that of healthy controls(P < 0.05),but the ratio ?-syn oligomers(26~42 KDa)to total ?-syn was higher(P < 0.05).4.The ratio of P-?-syn oligomers to total P-?-syn in plasma exosomes of PD patients was significantly higher than that of healthy controls(P < 0.05).5.The level of exosomal ?-syn oligomers and P-?-syn oligomers derived from the central nerve system in plasma of PD patients were higher than healthy controls,but there was no significant difference(P > 0.05).6.The ROC analysis of the ratio of ?-syn oligomers to total ?-syn in plasma exosomes of PD patients and healthy controls was moderate(AUC = 0.7054,sensitivity = 60.5%,specificity = 59.4%)and the ratio of P-?-syn oligomers to total P-?-syn was also moderate(AUC = 0.6895,sensitivity = 60.0%,specificity = 59.5%).7.?-syn could be detected in the plasma of PD patients(n = 4),while ?-syn could not be detected in healthy control plasma(n = 3)in this study.8.Compared with PC12 cells transfected with pc DNA3.1(-)plasmids,the m RNA level of ?-syn in ?-syn-overexpressed cells was significantly higher(P < 0.0001).The m RNA levels of proteins in the autophagy lysosomal pathway(LC3,ATG5,ATG16L2,LAMP2)and ubiquitin-proteasome system(ubiquitin)showed an increased trend,only the m RNA level of ATG12 was significant increase(P < 0.05).The m RNA level of UCHL1 which associated with PD pathology was significantly increased(P < 0.001).9.Compared with PC12 cells transfected with pc DNA3.1(-)plasmid,the co-localization of P-?-syn with LC3 B was significantly increased,but no significant change with ubiquitin.10.Compared with PC12 cells transfected with pc DNA3.1(-)plasmid,the m RNA levels of CD63 and RAB11 A,proteins related to vesicle release pathway,were significantly higher in ?-syn-overexpressed PC12 cells(P < 0.05).Conclusions1.The aggregates of ?-syn and P-?-syn in plasmic exosomes of PD patients are more difficult to be degraded by protease K than those in healthy controls,and the enhanced levels of ?-syn oligomers/total ?-syn and P-?-syn oligomers/total P-?-syn in plasmic exosomes that derived from PD patients may be the potential marker of PD that can be used to diagnose PD.2.The autophagy-lysosome pathway in ?-syn overexpressed PC12 cells plays a main role in clearing ?-syn and ?-syn overexpression can induce the activation of the non-classical secretory pathway,including exosomes and RAB11 A related secretory pathway. |
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