Establishment Of Rat Model Of Synucleinopathy Disease By Transfection With Human α-synuclein Mediated By Adeno-associated Virus In Vivo

Part I Construction of a recombinant AAV vector encoding humanα-synuclein gene with myelin basic protein PromoterObjective To construct recombinant adeno-associated virus carrying humanα-synuclein gene under the control of cytomegalovirus (CMV) or murine myelin basic protein (MBP) promoter and to measure the virus titer and verify the recombination.Methods Humanα-Synuclein and MBP cDNA were amplified by polymerase chain reaction (PCR), digested and subcloned into shuttle plasmid pSNAV2.0 to obtain recombinant plasmid pSNAV2-CMV-α-Synuclein and pSNAV2-MBP-α-Synuclein. The resulting recombinant plasmids were co-transfected into the 293T cells by calcium-phosphate precipitation method to complete rAAV2-α-Synuclein packaging. The titer of the recombinant rAAV2-α-Synuclein was determined by dot-blot assay. The recombinant rAAV-α-Synuclein was verified by PCR of the exogenous interest genes.Results The recombinant AAV vector containingα-Synuclein genes under the control of CMV or MBP promoter were successfully constructed. The physical particle titer of rAAV2-CMV-α-Synuclein and rAAV2-MBP-α-Synuclein were respectively 5.6×1011 vg/ml and 3.1×1011 vg/ml. The recombinant virus was confirmed by PCR of exogenous a-Synuclein and MBP promoter gene.Conclusion rAAV2-α-Synuclein was successfully constructed with a high virus titer, which may offer foundation for in vitro and in vivo experiments and provide a new method for the study of pathogenetic mechanisms and exploration of new therapeutic targets of particular relevance to human neurodegenerative disorders. Keywordsα-synuclein;myelin basic protein;recombinant adeno-associated virus; plasmid. Part II Behavioral and Pathological study of establishment in a-synucleinopathy rat modelObjective To establish overexpression of a-synuclein rat model and investigate the influence of overexpression of a-synuclein on motor function and pathology in a-synucleinopathy rat model.Methods We used a high-titer recombinant adeno-associated virus (rAAV) vector to express human a-synuclein in the substantia nigra of adult rats. After four months, Behavior was evaluated with narrow-beam tests and pole tests. The amount and morphology of dopaminergic neuron in mesencephalic nigra were studied with HE staining and Nissl staining.Results The rAAV-a-synuclein-treated rats showed obviously progressive decline in motor performance, compatible with this magnitude of nigrostriatal damage. Whereas brain-injured group were significantly impaired in their performance on the beam balance compared with control group(P<0.05). compared with the control group, the pole climbing time of brain-injured group was obviously lasting(P<0.05), and those treated displayed markedly hypoactive behavior, the control group didn't display parkinsonism behavior. Pathological examination demonstrated that the number and volume of neurons and nissl body in SN were decreased,and the tissue structure of SN became indistinct in brain-injured groups.Conclusion rAAV vector-mediated a-synuclein gene transfer in nigrostriatal provides a rat model of a-synucleinopathy that is of particular interest because it develops slowly over time and expresses neuropathological features that are similar to those seen in idiopathic PD. This model offers new opportunities for the study of pathogenetic mechanisms of particular relevance to human PD. PartⅢprimary verification of overexpression ofα-synuclein inα-synucleinopathy rat modelObjective To investigate the effect of overexpression ofα-synuclein on the expression of tyrosine hydroxylase (TH) in dopaminergic neurons. Furthermore, we were going to study the overexpression ofα-synuclein and the pathologic basements of rat brain.Methods The amount and morphology of dopaminergic neuron in mesencephalic nigra were detected expression of TH andα-synuclein with immunohistochemistry. double immunofluorescent labeling was used to analyze the expression of TH andα-synuclein and their relationship in rAAV-α-Synuclein treated rat.α-synuclein protein expression in rat brain were detected by western-blot assay.Resultsα-synuclein protein expression enhanced in global brain but not in an even lever, especially mostly increased in corpus striatum, substantia nigra, and brain stem reticular formation. the density ofα-synuclein positive staining was increased significantly (p<0.05) andα-synuclein positive inclusions was revealed in substantia nigra of rat. both the immune positive neurons of TH was significantly decreased in the substantia nigra of the rats. Double staining showed thatα-synuclein and TH protein coexpressed with in some neurons in the substantia nigra of rats.Conclusion These results indicate that overexpression of humanα-synuclein may inhibit TH synthesis in dopaminergic neurons, which may eventually lead to the degeneration of nigral neurons. we have suecessfully established overexpression of humanα-synuclein of rat model. Animal models based on overexpression of humanα-synuclein which can offer new opportunities for the study of pathogenetic mechanisms and exploration of new therapeutic targets of particular relevance to human neurodegenerative diseases.