| Parkinson’s disease (PD) is the secondary neurodegenerative diseases in the world,characterized by the impairment of motor function including tremor, muscle rigidity andslowness of movement. Patients with this disease are accompanied by the seriously reducedquality of life. As the exogenous neurotoxin MPTP has been discovered to causedegeneration of dopaminergic neurons, more and more researches aim to study therelationship between the pathogenesis and the endogenous structural analogues of MPTP,especially catecholamines isoquinolines (CAIQs) which are produced during dopaminemetabolism in the brain. On the basis of previous studies in our laboratory, a hypothesisabout CAIQs in PD is proposed, and the neurotoxicity could be significantly enhanced byN-methy-salsolinol (NMSal) which is the N-methylation product from salsolinol (Sal)catalyzed by N-methyltransferases (SNMT). Therefore, it is necessary to investigateneurological deficit induced by endogenerous CAIQs and develop biomarker candidate forearly diagnosis, which is helpful for diagnosis and pathogenesis of the disease.The dissertation focused on the neurological deficit protein and biomarker candidatefor the disease diagnosis. Based on the key deficit role of SNMT in the endogenerousneurotoxicity, an analytical method performed on high performance liquidchromatography-tandem mass spectrometry (HPLC-MS/MS) working in multiple reactionmonitoring (MRM) mode was established for determination of NMSal, providingmethodology for futher determination of biomarker. On the other side,18O labelingquantitative proteomics was applied to investigate protein changes, in order to interpretatethe profile of neurological deficit and analyze the correlation between the periphery andbrain, which is helpful for futher selection of biomarker candidate. The main contents andresults were as follows:1. A sensitive and specific analytical method was developed based on HPLC-MS/MSin MRM mode to determine NMSal. The precursor–product ion transitions were m/z194.1→145.1for NMSal. The methodology validation indicates the limits of detection ofNMSal was49pM and the limits of quantifcation was98pM. The quantitative range was0.098nM~100nM with the linear correlation coefficient greater than0.9995. Meanwhile,both the intra-day and inter-day precision was less than15.0%. This method also showedgood accuracy (94.0%~112.5%) as well as good recoveries (81.0%~91.4%). Theevaluation of the method indicated it was sensitive, specific and reproducible for futher determination.2. The SNMT activity in unilateral6-OHDA PD rat model was measured bydetermination of NMSal. Unilateral6-OHDA lesion surgery was performed in the striatum,behavioral evaluation results showed a success rate of60%for the model, andmorphological evaluation results showed that dopaminergic neurons in the PD group weresignificantly reduced compared with sham operated group (p<0.01). Results showed thatSNMT activity of striatum, substantia nigra and lymphocytes in PD model rats weresignificantly increased compared with the sham operated group (striatum: p<0.05;substanitia nigra and lymphocytes: p<0.01), and the the SNMT activity in lymphocyte wascoordinate with the tendency in the striatum and substantia nigra. The results not onlyindicated the SNMT and its activity was related with the production and metabolism ofCAIQs in brain, but also confirmed that the peripheral changes would reflect the changes inthe neuron system in the terms of SNMT activity which may be a promising earlydiagonosis biomarker.3.18O labeling with off-gel fractionation was developed based on ultrafiltration toremove trypsin for suppressing the back-exchange. Results on the peptides derived fromBSA (bovine serum albumin) showed that removal of soluble trypsin by ultrafiltrationprevented back-exchange effectively and enhanced the stability of18O-labeled peptidesunder off-gel separation even without trypsin inhibitor. After ultrafiltration, the18O labelingefficiency was95.8±2.3%under off-gel separation. In addition, these peptides had arelative high, approximately80%recovery.16O/18O ratio in peptides the mixture of1:1was0.99±0.17through ultrafiltration, and more than90%of the protein quantitative ratio was1.04±0.16in the biological sample, indicating no apparent effect on quantification. In hence,the useful and economical method presented here effectively inhibited back-exchange inoff-gel separation and might enable further applications in proteomics research.4. The comparative proteomics analysis based on18O labeling and2D-HPLC/MS/MSwas applied to investigate the protein changes in the unilateral6-OHDA PD rat model. Thepresent study explored the changes in protein abudance involved in the neurological deficitin the striatum and substania nigra. Results in striatum showed a total of1232peptide-spectrum and370proteins were identified with peptide FDR<1.0%, including76significantly differentially expressed proteins, and there were8439peptide-spectrum and1344proteins identified in substantia nigra with peptide FDR<1.0%. Furthermore, as the level of oxidative stress in unilateral lesioned PD rat models were both higer in striatumand substantia nigra, and data was interpreted by bioinformatics softwares and references,proteins with effect on neurological deficit included the following groups:(1) energymetabolism disorder was indicated by the up-regulated proteins in glycolysis whiledown-regulated mitochondrial proteins, in which seven mitochondiral proteins, threeglycolysis proteins in striatum and five mitochondrial proteins, three glycolysis proteins insubstantia nigra, and these changes were thought to compensate for a decrease in ATPproduction as a result of mitochondria dysfunction in PD;(2) there were twenty-twodown-regulated proteins found in composition of vesicle in striatum,10of which wereinvolved in neuronal transmission and recycling across synapses. These included SNAREproteins and clathrin-mediated endocytosis proteins. Moreover, mass spectrometry resultfor syntaxin-1A was confirmed by western blot analysis. These down-regulated proteins ofstriatum in lesioned rats indicated dyfunction in endocytosis and exocytosis;(3) differentialexpressed proteins in substantia nigra revealed thirteen proteins were significantly clusteredin immune response, including three proteins in microglia activation and proliferation, eightproteins in inflammation and immune function, and two proteins in calcium homeostasisand neuron protection. Further, western blot analysis of serum albumin was in consistentwith mass spectrum result. The differential expressed proteins in immune responseindicated abnormal immune function in the progress of disease.5. The comparative proteomics analysis based on18O labeling has revealed thatproteins participated in glycolysis and immune response, were up-regulated in lymphocyteof unilateral6-OHDA PD rat model. Moreover, the white blood cells and lymphocytes weresignificantly increased in PD rat model (p<0.01), indicating the inflammation appeared.And further results confirmed the abnormal immune function as lymphocyte cell subsetshowed CD4+/CD8+decreased (p<0.01) which was mainly induced by the decrease of Thelp cells CD4+. As immune function abnormalities were also involved in the substanianigra, correlation analysis of proteins between lymphocyte and substantia nigra showed atotal of seventeen proteins differentially expressed in both parts with a correlationcoefficient of0.45, indicating the positive correlation between the peripheral and brainsystem. Among the seventeen proteins, seven proteins were up-regulated both in thelymphocytes and substantia nigra with a correlation coefficient of0.89. Meanwhile, afterprincipal component analysis, the differential expressed proteins of fructose-bisphosphatealdolase A, glyceraldehyde-3-phosphate dehydrogenase,6-phosphofructokinase type C and 6-phosphogluconate dehydrogenase were found up-regulated in lymphocyte but nosignificant changes in substantia nigra, indicating lymphocytes were sensitive to energyrequirement, while fibrinogen, serum albumin, complement C3, transgelin-2andα-actinin-1with a coefficient of0.95were up-regulated both in lymphocyte and substantianigra. These changes and coefficiency indicated immune related proteins participated indisease, showing enhanced immune response. These proteins still need futher investigationbut may serve as diagnostic candidates because of the positive correltion.In summary, it was clear that not only sole target but also whole protein expressioncould be determined by mass spectrometry techniques, providing the protein profile withneurological deficits and biomarker candidates investigation in vivo, which could serve as areference for understanding the pathogesis of PD and was helpful for the characterization ofdisease, improving accuaracy of early diagnosis, rational subtyping, monitoring diseaseprogression, providing neuroprotective therapy and prevention of PD. |
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