The Expression In Astrocytes And The Effects On Neurons Of Beta-synuclein Protein Linked To Dementia With Lewy Bodies

Astrocytes(Astrocytes,As)are the largest types of the glial cells and the role in Parkinson's disease(Parkinson's Disease,PD)is increasing.In PD,there is not only the loss of dopaminergic neurons in substantia nigra,but also the significant activation and proliferation of astrocytes.Aggregation of beta-synuclein(beta-synuclein,beta-syn)mutants V70 M and P123 H induced apoptosis and accelerated neurodegeneration in neurons.However,the mechanisms of beta-syn mutants V70 M and P123 H on the degradation of neurons expressed in astrocytes in PD pathological process is not clear.In this study,we will use U251 cells stably expressed of beta-syn V70 M and P123 H to study the effects on the neurons when beta-syn mutants expressed in astrocytes,and to explore the molecular mechanisms of astrocyte-derived beta-syn mutants in the pathological process of PD.In this experiment,we constructed wild type and two kinds of mutant beta-syn plasmids,which were transiently transfected into U251 cells.Western blotting and immunofluorescence were used to identify that the beta-syn could be expressed in the U251 cells.WST-1 and LDH experiments showed that the survival rate of astrocytes transfected with beta-syn V70 M and P123 H was significantly decreased and the cytotoxicity was increased.Next,we constructed the stable cell lines of U251 cells expressed beta-syn wild-type and mutants with flag tags.Immunofluorescence and western blot analysis to investigate the effect of beta-syn mutants on astrocytes.The normal and transfected vector U251 as the control groups,we found that beta-syn WT in the cell is a diffuse distribution state.Beta-syn mutants V70 M and P123 H in U251 cells aggregated,and promoted the accumulation of alpha-syn in U251 cells.To some extent,the expression of GFAP and S100? as increased,and the expression of apoptosis related proteins were also changed in U251 cells expressed beta-syn mutants.The conditional medium of U251 cells expressed beta-syn wild type and V70 M and P123 H was collected.Western blot analysis was used to detect whether beta-syn in astrocytes could be released into extracellular.The results showed that either wild-type or mutants beta-syn could be released into the extracellular.We used the conditional medium of U251 cells expressed beta-syn wild type and mutant to culture SH-SY5 Y,through WST-1 assay,immunofluorescence,western blotting and TUNEL assay to detect the effect of mutants beta-syn expressed in astrocytes on neurons.The results showed that the cell viability of SH-SY5 Y significantly reduced,which was treated with the conditional medium of U251 cells expressed beta-syn V70 M and P123 H,the expression of alpha-syn and caspase3,as well as p53 increased.On the other hand,the apoptosis rate of SH-SY5 Y increased,and the length of the axons was shortened.Finally,we co-cultured SH-SY5 Y cells and astrocytes with recombinant flag-beta-syn WT or mutants to measure whether the astrocytes-derived beta-syn could be transferred into neurons.Immunofluorescence showed that astrocytes-derived beta-syn can be taken up into neurons.Through homologous recombination,we used Cre and Loxp systems to constructe the transgenic mice with the beta-syn V70 M gene specific knocked into astrocytes.Weight,footsteps,tail suspension test,and a series of behavioral experiments found that the behavior of transgenic mice changed,accompanied with depression and anxious symptoms.Western blot analysis showed that the expression of alpha-syn and S100? increased in the different brain regions of transgenic mice.In conclusion,the beta-syn mutations V70 M and P123 H accumulation in astrocytes,which promoted the aggregation of alpha-syn,led to astrocytes activate,the cell survival rate decrease,cytotoxicity increase,and promoted the expression of S100?,then caused to alpha-syn aggregated in neurons,induced neuronal apoptosis and promoted neurodegeneration.We also found that neuronal cells can uptake astrocytes-derived beta-syn.Astrocytes specific expression of beta-syn V70 M gene mice appeared behavioral disorders.These evidence suggest that astrocyte-derived beta-syn mutants can promote the neurodegeneration,which may contribute to the neurodegeneration mechanisms of Parkinson's disease.