Mechanism Of Pathological Damage In Astrocytes After Alpha-synuclein Transferred To Astrocytes Promoted By METH

Backgroud:Methamphetamine(METH),a highly addictive psychostimulant drug,is commonly abused worldwide.The abuse of METH can cause neuron distortions and mitochondrial dysfunction.Alpha-synuclein(?-syn)is a neuronal protein.METH can cause ?-syn overexpression,which causes dopaminergic neuron damage.Astrocytes are the largest group of glial cells in the central nervous system(CNS).They are essential for maintaining the homeostasis of the brain and protecting neurons.After METH,astrocytes in the brain activate and proliferate.Studies have shown that ?-syn can spread prion-like protein between cells,and whether astrocytes play a role in?-syn-related pathologies,and the specific molecular mechanism needs further study.Objectives:To investigate whether ?-syn can be transmitted from neurons to astrocytes we used the model of METH poisoning in vivo and in vitro and examined the changes in the expression of ?-syn in astrocytes.Then,we explored the way of ?-syn transmission by detecting exosomes secreted by neuronal cells and the expression of?-syn in exosomes.To investigate pathological changes and molecular mechanisms of astrocytes after ?-syn enters astrocytes,we detected changes in the expression of inflammatory factors and Nurrl in astrocytes.Methods:Chapter 1.To explore the expression of ?-syn in astrocytes,and whether ?-syn can be transmitted from neurons to astrocytes,mouse models and cell models of METH chronic poisoning,and the co-culture model of SH-SY5Y cells and U87MG cells were established.WB,IHC and IF were used to detect the level of ?-syn in the hippocampal brain regions,neurons and astrocytesChapter 2.To explore the way of ?-syn transmitted from neurons to astrocytes,exosomes were isolated from cell culture media by ultracentrifugation and identified using TEM,NTA,and WB.Exosomes were added to astrocyte culture medium,and the changes of P-?-syn and inflammatory factors in astrocytes were detected by WB and IF.To investigate the mechanism of astrocyte inflammatory response,exosomes released from neurons of ?-syn knockout mice and recombinant ?-syn were,added to astrocyte culture medium.The expression of inflammatory factors and Nurrl in astrocytes were detected by WB and IF.Results:Chapter 1.(1)The expression of ?-syn and P-?-syn in the hippocampus were increased,astrocytes were activated,and P-?-syn was colocalized with astrocytes after METH exposure;(2)The expression of ?-syn in astrocytes is extremely low,and there is no significant change in the expression of ?-syn after METH exposure;(3)After co-culture of SH-SY5Y cells and U87MG cells,it was found that P-?-syn was colocalized with U87MG cells.Chapter 2.(1)The exosomes extracted from the cell culture medium have a higher purity;(2)the METH group released more exosomes,and the P-?-syn was increased in the exosomes;(3)The expression of P-?-syn and inflammatory factors in astrocytes was increased after exosomes in METH group were added to astrocyte culture medium;(4)After the ?-syn gene was knocked out,the inflammatory response in astrocytes was alleviated;(5)After recombinant ?-syn was added to astrocyte culture medium,the expression of inflammatory factors in astrocytes were increased and the expression of Nurrl was decreased.Conclusions:METH can increase the expression of ?-syn in neurons.Increased ?-syn can be transmitted from neurons to astrocytes through exosomes.The astrocytes will produce an inflammatory response after taking up ?-syn.Nurrl may be a key molecule in the inflammatory response of astrocytes.