Comparative Proteomic Studies On Parkinson's Disease-Related Mice Model And Sera From Patients.

Parkinson's disease(PD) is the second most common neurodegenerative disorder, with a prevalence of about 2%among people over the age of 65 years.PD is typified clinically by motor symptoms and less frequent non-motor complications such as dementia.The neuropathological characters of PD include loss of dopaminergic neurons from the Substantia nigra(SN),depletion of striatal dopamine(DA) and presence of intraneuronal inclusions called Lewy body(LB).The pathogenesis of PD is still unknown, but it appears to be induced by genetic and environmental risk factors.In this work,the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine(MPTP)-treated mice model andα-synuclein(A53T) transgenic mice model were employed to represent the environmental toxin-induced and the genetic factor-induced PD-like models,respectively.The comparative proteomic researches were performed in these two mice models as well as the sera samples from PD patients,and the disease-related proteins were yielded and further investigated.PartⅠ.Comparative Proteomic Research of Striatal Proteins in MPTP-induced Mice Model and the Functional Investigation of Differential Proteins.It has been proved that exposure to environmental toxins(such as MPTP,Rotenone, Maneb and Paraquat) can produce Parkinson's disease through specific inhibiting the mitochondrial respiratory chain complexⅠorⅢin dopaminergic neurons.MPTP is a potent and selective nigrostriatal dopaminergic neurotoxin that can induce parkinsonism when administered to primates and rodents,particularly in the C57BL/6 strain.In this part of work,MPTP was used to treat mouse for 7 d.The behaviors and neuropathological studies showed PD-like changes.The mitochondrial protein profiles in the striatum(STR) were compared between MPTP-treated mice and control by employing two-dimensional electrophoresis(2DE).After MPTP treatment,five proteins were predominantly changed abundance.Four of them were significantly decreased in MPTP-treated mice and identified as Zinc finger A20 domain containing 1,Lectin-related NK cell receptor LY49S,Protein similar to sodium channel associated protein 1 isoform 2 and Proteasome 19S ATPase Rpt6;and the other protein was highly increased in MPTP mice and identified asα-synuclein(α-Syn).The following investigations were focused on the potential relationships between 19S ATPase Rpt6(Rpt6) reduction andα-Syn elevation in MPTP mice STR.It is well-known that a full-functional 26S proteasome in mammaline cells is characterized by a catalytic 20S core particle and one or two 19S regulator particles.The 19S ATPase Rpt6 is one of the subunits involved in the 19S regulator particle,and whose deficit implied an insufficient role of 26S proteasome which might respond to the increased level ofα-Syn. Western blot analysis revealed that proteins expression of Rpt6 and Rpt4 were decreased by 85.0%and 30.5%,repectively;meanwhile,α-Syn level was increased by 164.8%in STR of MPTP-treated mice,however the protein level of 20Sα6 was not changed. Interestingly,none of the above protein subunits were altered in SN after MPTP treatment. In addition,the MPTP-induced reduction of ATP concentration(36.4%) was found in STR but not in SN.Because of the 19S ATPases function depends on the ATP level,the decline of ATP and deficit of subunits expression might lead to a dysfunction of 19S proteasome particle.To gain insight into the role of Rpt6 inα-Syn accumulation,RNA interference was performed in cultured striatal neurons.It showed that,compared with control,α-Syn was increased by 50%when Rpt6 was knocked down by siRNA.To further elucidate whetherα-Syn augment might partially be due to the Rpt6 deficit-induced suppression of 20S activities,the proteolytic activities of 20S were tested.However,the results combined with the Western blot analysis showed that Rpt6 deficit neither suppressed the striatal 20S activities nor effected on the striatal 20Sα6 subunit expression in MPTP-treated mice. Thus,the above data revealed that Rpt6-relatedα-Syn increase in STR might through a distinct mechanism which was not associated with 20S activities. Alpha-synuclein-induced neuronal toxicity was considered as one of the harmful risk factors contribute to PD pathogenesis.Three PD-linked missense mutations including A53T,A30P and E46K inα-Syn gene were discovered in familial PD.In sporadic PD, aggregatedα-Syn is the primary fibrillar component of LBs,which are hallmarks of PD and Lewy body dementia.To demonstrate the correlations betweenα-Syn and PD pathology,in the recent years,the wild-type and mutantα-Syn transgenic mice were wildly used in PD researches.In this part of work,the behavior,TH and TUNEL immunohistochemistry and the level of DA-related neurotransmitters were evaluated in a-Syn transgenic mice and control in different age.The behavioral tests revealed that no signigicant difference was found between 3-month-old transgenic mice and control,however,9-month-old transgenic mice presented a declined movement ability.Meanwhile,the TH and TUNEL immunostainning assays implied a decreased tendency of TH-positive dopaminergic neurons in SN and an increased trend of TUNEL-positive cells in both SN and STR of 9-month-old transgenic mice compared with control.In addition,the striatal level of HVA were decreased in 3-month-old transgenic mice,furthermore,DA and its metabolites,DOPAC and HVA, were consistently reduced in 9-month-old transgenic mice.These data implied that the 9-month-old transgenic mice gradually appeared the PD-like pathological changes.To further elucidate the correlation between the status ofα-Syn overexpression and phosphorylation and the proteasome function,we examed the proteins expression ofα-Syn, CKⅡand 19S ATPase Rpt6 and the enzymatic activity of 20S proteasome in SN and STR of transgenic mice and control.Data showed thatα-Syn was continously overexpressed in both SN and STR of 3-and 9-month-old transgenic mice compared with control. Meanwhile,the level of CKⅡwas significantly increased in STR of 3-and 9-month-old mice,whereas was attenuated in SN of 9-month-old mice.The distinct changes of CKII in SN and STR might lead to different status ofα-Syn phosphorylation,which would produce various physiopathological alterations.Moreover,the level of 19S ATPase Rpt6 was only decreased in SN of 9-month-old transgenic mice,while the 20S activity was not changed in both 3-and 9-month-old transgenic mice.The results indicated that high level ofα-Syn would prefer to affect the expression of 19S proteasome subunit rather than changed 20S activity.To gain insight into the protein targets involved inα-Syn(A53T) toxicity,the comparative proteomics investigation was carried out for analyzing the protein profiles in SN and STR between transgenic mice and age-matched control.The proteomics study of SN showed that 9 differential spots were viewed in 3-month-old transgenic mice,2 of which were dramatically elevated and the other 7 were newly appeared spots.Besides these 9 differential spots,in 9-month-old transgenic mice, another 4 fresh differential spots were yielded and presented declined abundances or almostly disappeared.The proteomics study of STR indicated that 9 differential spots were presented in 3-month-old transgenic mice,1 of which was significantly increased,1 of which was predominantly deficit and the other 7 were newly appeared spots.Besides these 9 differential spots,in 9-month-old transgenic mice,another 2 differential spots were discovered,and one spot was decreased while the other one was increased.In addition,we compared 13 differential spots in SN with 11 differential spots in STR of 9-month-old mice and found an overlap of 8 spots.The 8 spots were altered in 3-month-old transgenic mice without regional specificity.Furthermore,those differential spots which only presented in SN or STR of 9-month-old transgenic mice were thought to be more valuable for PD investigation.PartⅢ.Comparative Proteomic Study on Serum of Parkinson's Disease Patients and Age-Matched Normal PeopleThe life quality of people suffered with PD is from bad to worse.However,no means exist to evaluate or predict the PD in the early-stage betbre the motor symptom onset. Moreover,the only current method for diagnosis of this disease is through physical examination and L-Dopa test,and no serum biochemical markers exist.Hence,to make efforts for PD prognosis and further advance a comprehensive understanding of PD pathology,we conducted a sera based comparative proteome study of healthy and diseased human in this part of work.The presence of high-abundant proteins(such as albumin,IgG,antitrypsin,etc.) in human serum always brings difficulty in separating and visualizing the meaningful low-abundant protein markers by 2DE.To address this problem,the Agilent Human 14 Multiple Affinity Removal System combinding with HPLC system were employed. Specfic removal of 14 high-abundant proteins depletes approximantly 94%of total protein mass of human serum.The low-abundant proteins in the flow-through fractions can be studied.Then,a comparative analysis of serum proteomes with low-abundant proteins was performed to discover proteins associated with sporadic PD.2DE patterns of human sera from 10 PD patients and 10 normal subjects were analyzed.The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-TOF-MS).In total,more than 1000 spots were separated in low-abundant protein fraction of human serum.The gross number of differentially regulated proteins in PD was 16,2 of which were significantly increased in PD samples and 14 of which were decreased in different levels.Among them,7 spots were successfully identified as five known proteins, which included thrombin,Apolipoprotein A-I,Apolipoprotein A-IV,Apolipoprotein L-! and Complement 2.These proteins were consistently decreased in PD patients.The results point towards a heterogeneous aetiopathogenesis of the disease,including alterations of Apolipoprotein-related lipid metabolism,alterations of the classical pathway involved in complement system as well as alterations of proteins involved in thrombin-induced coagulation system.