| Background: Parkinson disease(PD)is a neurodegenerative disease which is commonly in the middle-aged and older patients,with the main clinicopathological characteristics as the loss of dopamine neurons in the substantia nigra pars compacta and the formation of Lewy bodies(LBs).Some factors may participate in the occurrence of PD,such as Genetic factors,environmental factors,age factors and oxidative stress,and SNCA was the first discovered PD pathogenic gene,which contains 140 amino acids in the protein product α-Synuclein(α-S)encoded by it.α-Syn oligomerization was an intermediate step in the formation of LBs.The convertion of intracellular α-Syn monomer and oligomer to each other under normal physiologic states was maintaining the balance of dynamic.Therefore,the occurrence of PD may be due to the changes of environmental and genetic factors,causing the abnormal accumulationα-Syn of oligomers and the increasing of LBs.Currently,the specific mechanism of α-Syn oligomerization regulation is still unclear.Objective: The regulator α-Syn oligomers formation genes were screened out to reveal the specific mechanism of intracellular α-Syn oligomerization regulation,so as to provide targets for the diagnosis and treatment of α-Syn oligomerization abnormalities related PD.Methods:1.CRISPR-Cas9 whole-genome knockout library screening was performed for genes regulating α-Syn oligomerization in α-Syn Bi FC-GFP stable expression HEK293 cell line.After library lentivirus infection,the cells with high density GFP fluorescent was sorted by Flow cytometry for genome DNA isolation.PCR was used to amplification the fragments containing sg RNA sequences.Then,the amplicoms were sequenced by Next Generation Sequencing.2.The overlap genes of two independent experiments were analyzed by bioinformation technology for genes and pathways regulating α-Syn oligomerization.3.siRNA interference technology was used to confirm the top 10 candicate genes.The cross-linker DSG was used for oligomer cross-linking.After crosslink,Western blot was used to detect the changes of α-Syn oligomer level.The results showed that TSPAN3 were the most critical regulatory gene.4.With Brain EXP-NPD online database,the mRNA expression of TSPAN3 was analyzed in cerebellum,frontal cortex,medulla,striatum and midbrain/substantia nigra of PD patients and healthy population.5.Knockdown of TSPAN3 or exogenous expression of TSPAN3 wt and mutant in HEK293 cells stable expressing α-Syn,α-Syn oligomer content were detected by Western blot,immunofluorescence.The interaction between TSPAN3 and α-Syn was determinated by co-immunocoprecipitation and sub-cellular co-localization.6.Proteasome and lysosome inhibitors,as well as interfering endocytosis related genes Clathrin and AP2,respectively was used to study the role of Clathrin-AP2 mediated lysosome pathway in TSPAN3 regulating the content of α-Syn oligomer.7.The AAV9 serotype TSPAN3 adeno-associated virus with GFP label was injected into the primary motor cortex for overexpression of human α-Syn A53 T mutant transgene mouse.α-Syn aggreagate was detected by immunofluorescence in cortical neurons.Result:1.The amount of α-Syn oligomers was significantly increased in cells with interference of BACE1,TSPAN3 and STXBP5.However,the increase of α-Syn oligomers was the most significant when TSPAN3 knockdown.2.The expression of TSPAN3 in the frontal cortex,striatum and midbrain/substantia nigra of PD patients was significantly decreased as compared with the healthy population.3.The amount of α-Syn oligomer was significantly increased in cells with interference of TSPAN3,but not homologous genes TSPAN7.The content of α-Syn oligomer could be partially decreased by over expression of TSPAN3 wild-type(TSPAN3wt).While the inhibited endocytosis Y243 A TSPAN3 mutant could not decrease the content ofα-Syn oligomer as the TSPAN3 wt.4.The Immunofluorescence result showed the exogenous expression TSPAN3 was co-localization with α-Syn after treated by lysosomal inhibitor CQ,especially.exogenous expression TSPAN3 and endogenousα-Syn interaction was also determinated by co-immunocoprecipitation.5.The decreasing of α-Syn oligomers caused by overexpression of TSPAN3 wt can be blocked by the inhibition of lysosomal pathway.6.As a cell membrane protein,TSPAN3 could promote lysosomal degradation of cell membrane located α-Syn protein oligomers through Clathrin-AP2 mediated endocytosis pathway.7.The amount of α-Syn positive inclusions bodies in M1 region neurons of the mouse cerebral cortex could be decreased by overexpression of TSPAN3.Conclusion:1.TSPAN3,a novel regulator for α-Syn oligomer,was identified to regulate the content of α-Syn oligomer;2.TSPAN3 promotes the lysosomal degradation of cell membrane located α-syn oligomers through Clathrin-AP2 endocytosis pathway. |
