A Study On Exosomes Derived From Brain Tissue Of VMAT2 Knockout Mouse Models In The Pathogenesis Parkinson’s Disease

Part 1 The characteristic analysis of exosomes originated from brain tissue of VMIAT2 knockdown mouse PD modelsObjective:To constructing PD model by VMAT2 knockdown combined with subacute exposure of MPTP,and to verify the reliability of the model by behavioral,histopathological,and molecular biology methods,and to compare the differences of exosomes originated from brain tissue of different groups.Methods:Six-month-old male HT(50%expression level of VMAT2)and wild-type(WT)mice were randomly divided into MPTP intervention group(MPTP 30 mg/kg,continuous intraperitoneal injection for 1 week)and untreated group.Rotarod and pole test to evaluate animal behavior,Immunofluorescence staining to detect the expression of TH and abnormal aggregation of a-syn;psyn(S129),ubiquitin and P62 in the substantia nigra and striatum,Western blot analysis to detect the expression of TH and VMAT2.Exosomes of different groups were isolated by ultracentrifugation;detecting the exosomal marker CD63 and TSG101 by Western blot analysis;defining the morphology by transmission electron microscope(TEM);measuring diameter by Naosight;determining the concentration based on the activity of AChE.Results:MPTP-treated HT mice displayed more obvious locomotor retardation;more significant decrease in the expression level of TH revealed by Western blot analysis and Immunofluorescence staining;more prominent aggregation of a-syn,psyn(S129),ubiquitin and P62 by Immunofluorescence staining in the substantia nigra compared to MPTP-treated WT mice.The exosomes isolated by ultracentrifugation were cup-shaped under TEM;Western blotting confirmed that the extract contained exosomal marker CD63 and TSG101,the diameter of the extract by Naosight were located in 62 nm,and there was no significant difference in the diameter of each group;the concentration result determined by AChE enzymatic activity revealed that HT(50%expression level of VMAT2)mice with MPTP exposure secreted more exosomes than other mice.Conlusions:The concentration of exosomes originated from brain tissue is increased in the HT mice with MPTP exposure which is a very good PD animal model.Part 2 Analysis of exosome secretion and release in VMAT2 knockdown mouse models and explore possible regulatory mechanismsObjective:To compare the expression level of protein related to exosome biogenesis and release of different groups and the possible mechanisms involved.Methods:The expression level of proteins related to biogenesis and release of exosomes were evaluated by Western blotting、immunohistochemistry and immunofluorescence;the co-localization of proteins was observed by cell immunofluorescence and co-immunoprecipitation(Co-IP);determining the concentration based on the activity of AChE.Results:The results of Western blotting、immunohistochemistry and immunofluorescence indicated that there was no difference in the expression level of exosome biogenesis related proteins such as CD63 and TSG101 in different groups,while the expression level of release related proteins Rab35 were significantly increased in the HT mice with MPTP exposure;cell immunofluorescence and Co-IP results suggested the co-localization and interaction between VMAT2 and Rab35.There was more secretion of exosomes derived from brain tissue in LO mice and HT mice with exposure.Conclusion:VMAT2 with very low expression level can increase the secretion of exosomes in brain tissue by affecting Rab35.Part 3 Pathogenicity of exosomes derived from brain tissue of VMAT2 knockdown mouse models(cell model)Objective:To survey the pathogenicity of exosomes derived from brain tissues of VMAT2 knockdown mouse models on cells.Methods:Exosomes from different groups were superinduced into SH-SY5Y cells,then,CCK-8 assay was used for determining cell viability;exosomes were labeled with PKH26 and then were added to SH-SY5Y;Western blotting and immunofluorescence were used to detemine the expression of a-syn,psyn,a-syn filament,P62,ubiquitin in different groups.Results:SH-SY5Y could absorb exosomes,There was no significant difference in absorption among the groups;the results of Western blotting and immunofluorescence indicated that the expression of α-syn,psyn,a-syn filament,P62 and ubiquitin was increased after exosomes derived from brain tissues of HT with MPTP exposure mice added to the cells.Conclusion:Exosomes derived from brain tissues of HT with MPTP exposure mice can cause PD-like damage in SH-SY5Y cells.Part 4 Pathogenicity of exosomes derived from brain tissue of VMAT2 knockdown mouse models(animal model)Objective:To observe the effect of striatal stereotactic injected exosomes derived from brain tissue of PD animal models on wild-type mice.Methods:Intracranial catheterization was performed in the striatum of wild-type mice to carry out intermittent stereotactic injection of exosomes derived from VMAT2 knockdown mouse models for 3 months;apomorphine induced rotation,rotarod test,pole test were used to evaluate motor function;the expression level of TH,α-syn,psyn(S129),p62,ubiquitin and IBA1 in midbrain and striatum were observed by Western blotting,immunofluorescence and immunohistochemistry;the mRNA level of IL6,TNF-a and IL-1β was determined by RT-qPCR.Results:Apomorphine induced lateral rotation in mice receiving exosomes derived from brain tissue of HT with MPTP exposure mice injection,and impaired motor function was further confirmed by rotarod test and pole test;the expression level of TH decreased,while the expression level of a-syn,ubiquitin,psyn(S129),p62 and IBA1 augmented by Western blotting,immunofluorescence and immunohistochemistry in mice receiving exosomes derived from brain tissue of HT with MPTP exposure mice injection;the mRNA level of IL6,TNF-a and IL-1β elevated determined by RT-qPCR in mice receiving exosomes derived from brain tissue of HT with MPTP exposure mice injection.Conlusion:Striatlal stereotactic injection of exosomes derived from brain tissue of HT with MPTP exposure can induce PD-like behavioral and pathological damage in wild-type(WT)mice.Part 5 Possible pathogenic mechanism of exosomes derived from brain tissue of VMAT2 knockdown mouse modelsObjective:To explore the possible pathogenic mechanism of exosomes derived from brain tissue of VMAT2 knockdown mouse models.Methods:Western blotting were used to analyze the protein components of exosomes from brain tissues of different groups;exosomes were treated with DNAase,RNAase,Proteinase K and high temperature,and then added into SH-SY5Y cells,at last,western blotting were used to detect the expression ofα-syn in SH-SY5Y cells;exosome were added into BV2 cells,ELISA and western blotting were used to evaluate the expression level of IL6,TNF-a,IL-1β,NFκB;TNF-neutralizing antibody was co-incubated with exosomes and then added to BV2 cells,the expression level of NFκB in different treatment groups were evaluated by Western blotting.Results:The results of Western blotting indicated that the amount of iNOS、IL6,TNF-a,IBA1 increased,while,NeuN、GFAP declined contained in exosomes from HT mice with MPTP exposure;the expression level of a-syn decreased in SH-SY5Y cel s co-incubated with exosomes treated with Proteinase K and high temperature compared to treated with DNAase,RNAase,it indicated that the protein carried by exosomes was closely linked to its pathogenicity;the resu ts of ELISA showed that the exosomes derived from the brain tissue of HT mice with MPTP exposure could significantly increase the contents of IL-6,TNF-a and IL-1β in BV2 cells;the results of Western blotting indicated that the exosomes from the brain tissue of HT mice with MPTP exposure could activate the NFκB pathway in BV2 cells in a TNFa dependent way.Conclusion:The pathogenicity of exosomes is mainly related to the proteins they carry,the pathogenic mechanism may be propagating pathogenic a-syn,activating microglia to induce exaggerated inflammatory response,the NFκB pathway is involved.