The Role And Mechanism Of Neuron-derived α-synuclein Transfer To Astrocytes In Blood-brain Barrier Damage Induced By Methamphetamine

Background:Methamphetamine(METH)is a widely used synthetic drug in the world today.Our previous studies found that a-synuclein plays an important role in neurotoxic effects induced by chronic METH abuse.The toxicity of a-syn is not limited within neurons,it can also enter astrocytes.Given the fact that astrocytes are the constituent elements of the blood-brain barrier,therefore,whether the accumulation of a-syn in astrocytes participate in METH-induced BBB injury?Nuclear receptor-related protein 1(Nurrl),a member of the nuclear receptor family,is widely expressed in the brain.Previous research found that there is a close relationship between Nurrl and a-syn,and Nurrl can regulate inflammatory response in astrocytes.However,it is not clear whether Nurrl-mediated inflammatory response in astrocytes engaged in the BBB injury elicited by METH.Objectives:To study whether METH can induce neuron-derived a-syn transferred to astrocytes,resulting in BBB injury,and to explore the role and mechanism of Nurrl in astrocytes during this process.Methods:By using Evans blue and fluorescein sodium vascular leakage test,immunoblotting,immunofluorescence staining,immunohistochemical staining,transmission electron microscope,enzyme-linked immunosorbent assay and other experimental methods to detect(1)whether METH can induce BBB injury and neuroinflammation(Chapter 1),(2)whether α-syn knockout or chemical inhibition can reduce neuroinflammation and BBB injury induced by METH(Chapter 2),and(3)whether astrocyte-specific overexpression of Nurrl can diminish the above mentioned inflammation and BBB damage(Chapter 3).Finally,in Chapter 4,primary cultured neurons,astrocytes,and brain microvascular endothelial cells were used to establish corresponding co-culture models to study(1)whether METH can induce neurogenic αsyn transferred to astrocytes and(2)whether α-syn induce inflammation and damage the BBB in vitro by downregulating the expression of Nurrl in astrocytes.Results:Chapter 1.(1)METH induces BBB injury and neuroinflammation characterized by the increase of permeability to Evans blue,the decrease of Claudin5,Occludin and PDGFRβ levels,the activation of astrocytes,and increase of IL-1β,IL-6,and TNF-αlevels.(2)METH treatment up-regulates the expression of α-syn(mainly located in hippocampus CA1 and DG area),and down-regulates the expression of Nurrl and GDNF.Chapter 2.(1)METH treatment can induce α-syn transferred to astrocytes.(2)Knock out α-syn can reduce METH-induced inflammation,apoptosis,BBB injury,and abnormal expression of Nurrl and GDNF.Specifically,decreased the number of astrocytes and the levels of various inflammatory factors;decreased Cleaved Caspase3 and Cleaved PARP levels;reduced the vascular leakage of Evans blue and fluorescein sodium;attenuated IgG deposition in brain parenchyma;increased Claudin5,Occludin,and PDGFRβ levels;improved BBB ultrastructural abnormities(abnormal cerebral microvascular diameter,astrocyte end-foot swelling,decreased pericyte coverage,and loss of tight junctions);and increased Nurrl and GDNF levels.(3)Chemical inhibition of α-syn can alleviate METH-induced inflammation,BBB injury,and abnormal expression of Nurrl and GDNF as proved by decreasing IL-1β,IL-6,and TNF-α levels;increasing Claudin5,Occludin,and PDGFRβ levels;and increasing Nurrl and GDNF levels.Chapter 3.Overexpression of Nurrl in hippocampal astrocytes alleviates inflammatory response,BBB injury,and abnormal expression of GDNF induced by METH.Specifically,decreased IL-1β,IL-6,and TNF-α levels;reduced the vascular leakage of Evans blue and fluorescein sodium;increased Claudin5,Occludin,and PDGFRβ levels;improved BBB ultrastructural abnormities;and increased GDNF levels.Chapter 4.(1)METH up-regulates α-syn expression in neurons and spreads it to astrocytes.(2)After the BBB cell model was treated with exogenous α-syn,the astrocytes(located in the lower chamber of the BBB cell model)could internalize αsyn and decrease its Nurrl and GDNF levels,while increase various inflammatory factors levels.Besides,there were also increased inflammatory factors expression and decreased GDNF expression in the culture medium of co-culture system.In addition,the permeability to fluorescein sodium increased,transendothelial electrical resistance decreased,and the levels of Claudin5 and Occludin decreased in BBB in vitro.Conclusion:METH up-regulates α-syn expression in neurons and spreads it to astrocytes.Theα-syn accumulates in astrocytes,induces astrocyte activation,increases and decreases inflammatory factors and GDNF levels by downregulating Nurrl expression,and eventually damages BBB.