The Investigation Of Endogenous Neurotoxin In Pathogenesis Based On The α-synuclein Transgenic Rat Model Of Parkinson’s Disease

Parkinson’s disease(PD) is the second most common neurodegenerative disorder. PD occurs predominantly in the aging with the movement symptoms consist of resting tremor, bradykinesia, rigidity and postural instability. The patients of PD are increasing in China every year. Loss of dopaminergic neuron in substantia nigra pars compacta(SNpc) and protein aggregation named Lewy Bodies(LBs) are two important characters of PD. There are many hypotheses about PD such as oxidative stress hypothesis and free radical hypothesis, but the pathogenesis is not clear. The genetic factor and environmental factor are the two fields investigated most and environmental factor play dominant roles in pathogenesis. Since MPTP was discovered, neurotoxins as causative factors of PD have been widespread concern, and catechol tetrahydroisoquinolines(CTIQs) is a class of endogenous neurotoxins among them. Our group generated a new hypothesis about the biosynthesis and neurotoxicity of CTIQs neurotoxin based on the long-period studies of CTIQs formation and metabolism in vivo.When the brain is attacked by external stimuli(physical combat or environmental pollution, etc.), aldehydes were produced as the final product through lipid peroxidation(LPO) in the process of oxidative stress. These aldehydes condense with dopamine(DA) to form CTIQs such as Salsolinol(Sal). CTIQs then process N-methylation and oxidation to form ion compounds which can combine with the mitochondrion complex Ⅰto make the cell dysfunctions in energy metabolism and exacerbate oxidative stress. On the other hand, α-synuclein in the neuron may be aggregation under the CTIQs. The aggregated α-synuclein can also exacerbate oxidative stress. All of the processes described above can be combined to form a cycle and induce death in neurons.In this thesis, α-synuclein overexpressed rat models were used to investigate the hypothesis. The investigation was focused on the effect of N-methyl-Salsolinol(NMSal) to α-synuclein aggregation. The aim is explain the mechanism of α-synuclein aggregation in brain and the. The aim of this subject is to investigate if NMSal can induce α-synuclein aggregation in the brain and neurotoxicity of NMSal. Through this study, it can accumulate data to support the hypothesis described above.The achievements acquired in this subject are listed as follow:(1) α-synuclein overexpressed rat model of PD was established using Recombinant Adeno-Associated Viral(rAAV). The method of rAAV package and purification was optimizeand and the final titer was more than 1013gc/m L. The rAAV was injected to the substantia nigra pars compacta(SNpc) by cerebral stereotaxic technique. After a series of time node behavior test, the rats showed apomorphine induced rotation to uninjected side which is characteristic behavior to loss of dopaminergic neurons. The rotation speed of α-synuclein A53 T group was 3.91±1.62 rpm whereas WT group was 2.68±1.74 rpm. α-synuclein expression was detected in brain slices using immunohistochemistry(IHC) showed that α-synuclein was expressed in the dopaminergic neurons, and the express level was increasing from the 3rd week to the12 th week in α-synuclein WT group whereas the level reached the highest point in the 9th week in the A53 T group. IHC of tyrosine hydroxylase(TH) showed that loss of dopaminergic neurons with positive correlation to the expression of α-synuclein. The two rat models of PD consist of α-synuclein WT and A53 T were established successfully.(2) NMSal can enhance the toxicity of α-synuclein in rat model of PD. 100 nmol NMSal was injected to the same place after 12 weeks from r AAV injection. After 4 weeks, the behavior test showed that the NMSal injection accelerated apomorphine induced rotation in rAAV-α-synuclein groups. The IHC results showed that NMSal can enhance the loss and apoptosis of dopaminergic neurons in the PD models, and the apoptosis ratio was increase 3.7% and 7% in α-synuclein WT group and A53 T group respectively. These results NMSal can increase the loss of dopaminergic neurons by α-synuclein in rat model of PD. The content of malondialdehyde(MDA) and reactive oxygen species(ROS) was dectected by ELISA, and the data showed the level was increased 50% by NMSal induction. The result showed that the oxidative stress level was increased by NMSal in the brain of PD model. The HPLC-MS/MS results showed the DA level in striatum was lower in the rAAV-α-synuclein groups with the content of WT and A53 T group is 44% and 27% of control respectively, and NMSal can decrease the level to 24% and 15%. The content of endogenous neurotoxin Sal and NMSal was also elevated after NMSal induction. These results showed that NMSal can enhance the toxicity of α-synuclein in rat model of PD, and the effect is even significant in the α-synuclein A53 T group.(3) NMSal enhances the LBs like aggregation of α-synuclein in rat model of PD. The western bolt results showed that insoluble α-synuclein was increased after NMSal induction, and the trimer of α-synuclein is 2 fold and 3 fold of control in α-synuclein WT group and A53 T group respectively. IHC results showed that NMSal can increase the Proteinase K-resistant inclusions in striatum, especially in the A53 T group. These results showed that NMSal can enhance α-synuclein aggregation. The IHC of S129 phosphorylated α-synuclein indicated that only α-synuclein A53 T group showed phosphorylation significantly. There are inclusions distribute in the striatum, and NMSal increased the number of inclusions from 4.5±1.7 to 8.8±3.3. The IF result of ubiquitin and α-synuclein double-stain showed co-localization of the two proteins, and this indicated ubiquitination of α-synuclein. NMSal induction can enhance ubiquitination. These results showed that the inclusions of α-synuclein were phosphorylation and ubiquitination as the LBs,and NMSal can elevate the level in the α-synuclein A53 T group especially.(4) NMSal may inhibit the degradation of α-synuclein through autophagy-lysosomal pathway(ALP). ALP and ubiquitin-proteasome system(UPS) are two pathways involved in the degradation of α-synuclein. The western bolt results showed that NMSal induction can repress expression of Beclin-1, Atg5 and Atg3 in ALP, whereas Parkin and UCHL1 in UPS was not affected by NMSal. These results showed that NMSal induced α-synuclein aggregation maybe thought the inhibition of ALP.In this subject, PD model base on α-synuclein over-expression was combined with NMSal injection. It was the first time to investigate NMSal reacts with α-synuclein in vivo directly. Thought these experiments, the results showed that NMSal can enhance α-synuclein aggregation and increased its toxicity. The oxidative stress level and the content of endogenous neurotoxin were also elevated after NMSal induction. These results provided some proofs to the hypothesis of biosynthesis and neurotoxicity of CTIQs neurotoxin, and supply a useful animal model to investigate the hypothesis in the future.