A Study On The Exosomic Mechanism In The Pathogenesis Of Parkinson's Disease

Part 1 The characteristic analysis of serum derived exosomes from Parkinson's disease patients and neurological control,besides exosomes from various experimental animal modelsObjective:To compare the difference of serum exosomes from PD patients and neurological controls,normal and experimental PD animal models.Methods:PD patients(duration>5 year)and neurological controls were enrolled,and approximately 10 ml blood were acquired,then exosomes were isolated with ultracentrifugation or with exosomal isolation kit.Transmission electron microscope(TEM)was used to define the morphology and Nanosight was used to characterize the diameter and concerntration of the extract,besides,western blotting was used to evaluate the exosomic mareker CD63 and TSG101.Toxicants such as rotenone and MPTP were intraperitoneally injected and 6-OHDA was intrastratally injected to induce parkinsonian animal model on mouse or rat,Nanosigt was also performed to compare the characteriscs of serum exosomes of toxicant treated group and untreated group.Results:Ultrastructurally cup-shaped or spherical particles of these exosomes could be visualized under TEM both in exosmes acquired by ultracentrifugation or exosomic isolation kit.Exosomic marker CD63 and TSG101 were detected by western blotting and more abundant in exosomic isolation kit.The diameter results revealed by Nanosight was located in the theoretical range 30-120nm.The exosomic concentration result indicated by Nanosight showed that PD patients contain more serum exosomes than neurological controls,as was the same trend for PD animal models such as rotenone or MPTP or 6-OHDA.Conclusion:The concerntration of serum exosomes is increased in PD patients as well as several PD animal models,which may act as potential disease marker.Part 2 The pathogenesis of PD patients derived exosomes(cell model)Objective:To observe the pathogenic effect of PD patients derived exosomesMethod:Blood was extracted from PD patients(duration>5 years)and neurological control,PCR reaction was performed to eclude the SNCA mutation in PD.Then exosomes were isolated by ultracentrifugation,western blotting was performed to evaluate the a-syn expression level between PD group and neulogical group.Then two groups of exosomes and rotenone were added to SH-SY5Y cells,CCK 8 assay was used to evaluate the cell viability,then the protein level of cellular a-syn was evaluated by western blotting and immunofluorescence.Results:Exosomes from PD patients and neurological control both express ?-syn while PD exosomes contain more oligomers,besides,neither of them contain a-syn oligomer.Exosomes from PD also trigger more cellular a-syn formation as revealed by western blotting,and more obviously,the a-syn filaments formation observed by IF.Conclusion:PD serum derived exosomes contain more a-syn oligomers and could trigger a-syn aggregation in recipient cells.Part 3 The pathogenesis of PD patients derived exosomes(animal model)Objective:To observe the pathogenic effect of PD exosomes on the mouse model with different genetic background.Method:To examine the blood-brain barrier permeability of exosomes by in vivo imaging system(IVIS).Then PD exosomes and neurological exosomes were tail injected into the balb/c and NOD/SCID mouse,weight change was recorded.Besides,the expression level of TH,?-syn?p-syn(ser129)?ubiquitin and P62 was evaluated by western blotting and immunohistochemistry or IF,HE staining was used to observe the Lewy body formation.The behavioral change was evaluated by pole test and rotarod test.Results:IVIS showed that exosomes could cross the blood brain barrier.The body weight decreased after exposure to PD exosomes.The level of TH decreased while the level of a-syn,p-syn(serl29),P62 and ubiquitin increased as revealed by western blotting,IF or immunohistochemistry.HE staining showed that PD exosomes induced morphological change and nuclear hyperchromatism of neurons in the substantia nigra.Pole test and rotarod test indicated that both balb/c and NOD/SCID mouse showed slight bradykinesia.Conclusion:Tail vein injected PD exosomes could initiate PD-like pathological and behavioral changes in both balb/c mouse and T-cell and B cell deficient mouse-NOD/SCID mouse.Part 4 The pathogenesis of PD patients derived exosomes(stereotactic animal model)Objective:To observe the effect of stereotactic injected PD derived exosomes on the mouse model.Methods:Cannulas were implanted in the striatum of the balb/c mouse to achieve intermittent injection of PD and neurological exosomes for 2 months.The expression level of TH,?-syn?p-syn(ser129)?ubiquitin?P62?IL-1? and IBA-1 was evaluated by western blotting and immunohistochemistry or IF,HE staining was used to observe the Lewy body formation.The behavioral change was evaluated by pole test,rotarod test and apomorphine induced rotation.Results:The level of TH decreased while the level of a-syn,p-syn(ser129),P62,ubiquitin,IL-1? and IBA-1 increased as revealed by western blotting,IF or immunohistochemistry.HE staining showed that PD exosomes induced morphological change and nuclear hyperchromatism of neurons in the substantia nigra.Pole test and rotarod test indicated that both balb/c and NOD/SCID mouse showed slight bradykinesia.Besides,apomorphine induced rotation can be induced by PD exosomes injection.Conclusion:Striatal injection of PD derived exosomes could induce PD-like pathological and behavioral change in balb/c mouse.