Cystatin F Derived From Blood-borne Monocyte Ameliorates Cognitive Impairment Of APP/PS1 Transgenic Mice

Objective:Alzheimer's disease(AD)is a neurodegenerative disorder,having a negative impact on human health.Oligomers of intracellular amyloid?protein(A?)are strongly cytotoxic and play crucial roles in synaptic transmission and cognitive function in Alzheimer's disease(AD).It's meaningful for the prevention and cure of AD to understand AD pathogenesis and find effective treatments.the core pathogenic substances of AD,and the most important targets for the prevention and treatment of AD.Brain microglial cells can be replenished by blood-derived monocytes,but many aspects of this repopulation remain unclear,known as"bone marrow-derived microglia"(BMDM).BMDM can effectively prevent the deposition of A?and promote the clearance of A?plaques in the brain.In previous study,we detected that the expression and secretion of cystatin F was up-regulation in AD plasma groups compared with that of aged-matched controls.Cystatin F overexpressed could effectively degrade the oligomers of intracellular amyloid?protein and significantly ameliorates cognitive impairment of APP/PS1 transgenic mice.In this paper,we will explore the molecular mechanism of cystatin F derived from blood-borne monocyte ameliorates cognitive impairment of APP/PS1 transgenic miceMethods:1.A transgenic mouse line expressing cystatin F in monocyte was established;2.A?expression was detected in peripheral blood and brain with cystatin F~+transgenic mice and cystatin F~+/APP~+hybrid mice that was produced by crossing mice of two different inbred strains;3.Through the water maze test,learning and memory was detected in four groups transgenic mice,which was including WT mice,cystatin F~+transgenic mice,APP~+transgenic mice,cystatin F~+/APP~+transgenic mice;4.A?binding assay.ELISA binding plates was coated with A?1-42,A?1-40,and A?42-1,then adding cystatin F recombinant protein at different concentrations,detecting with anti-cystatin F antibody,reading the OD450 absorbance value;5.Using commercial GST 1-42,GST 1-40,and GST-C99proteins and then co-incubating with U937 cells,GST pull down assay was used to analyze the interaction between A?and cystatin F;Using GST-cystatin F co-incubating with the human brain microvascular endothelial cells(HBMEC),GST pull down technology was used to detect the interaction between cystatin F and A?;6.Add fluorescent A?1-42,A?1-40 to U937 cells incubated with labeled cystatin F antibody.Immunofluorescence staining image showed that the labeled cystatin F with A?1-42 and A?1-40 was observed with a confocal laser scanning microscope.And then fluorescent A?1-42 was incubated with labeled cystatin F and lysosome.Results:1.Cystatin F~+transgenic mice expressed human cystatin F;2.A?content of cystatin F~+/APP~+transgenic mice was significantly reduced in the peripheral blood and brain;3.Cystatin F~+/APP~+transgenic mice ameliorated cognitive impairment of APP/PS1transgenic mice;4.Cystatin F binding with A?1-42 and A?1-40 in vitro was concentration dependently;5.After co-incubation of GST-cystatin F with HBMEC,APP was detected.In contrast,co-incubation of GST-A?1-42,GST-A?1-40,and GST-C99 with U937 cells,cystatin F was detected;6.The co-localization of cystatin F and A?was observed with a confocal laser scanning microscope.Conclusion:Cystatin F derived from blood-borne monocyte participated in?-amyloid degradation and ameliorated cognitive impairment of APP/PS1 transgenic mice;Cystatin F could directly interact with A?,and effectively degrade the oligomers of amyloid?protein.