The Effect Of ADAM10 Deletion On Expression Of Synaptophysin,PSD-95 And Synaptic Ultrastructure In Adult Murine Brain

Aims:A disintegrin and metalloprotease 10(ADAM10)is an important member of ADAM family.ADAM10 mediates cleavage of more than 30 transmembrane proteins,such as APP,Notch,cell adhesion molecule,and transmembrane inducing factor.The recent studies showed that ADAM10 resides in the postsynaptic densities(PSDs)of excitatory synapses,interacts with the synapse-associated protein-97(SAP97),controls the shedding of different cell adhesion molecules(CAMs)at the postsynaptic site thereby contributing to the development and functioning of synapses,and promotes signal transduction.In order to investigate ADAM10 function in the brain,we used adult ADAM10 conditional knockout mice(cKO)to explore the effects of ADAM10 deletion on expression of synaptophysin,PSD-95 and synaptic ultrastructure in the murine brain,.Methods:Western blotting was first used to detect brain samples.Proteins were taken from the different brain areas of ADAM10 cKO mice(n=3)and C57BL/6J mice(n=3)at 12-month-old and quantified using standard BCA assay.Levels of synaptophysin in the cortex and hippocampus and PSD-95 in the cortex,hippocampus and cerebellum were detected respectively.Software Image j was used to analyze the gray value of images.Then standard immunohistochemical procedures were used to analyze the expression of synaptophysin and PSD-95 in the cortex and hippocampus of ADAM10 cKO mice(n=3)and C57BL/6J mice(n=3)at 12-month-old,respectively.Software image pro-plus 6.0 were used to analyze the image mean density.Finally,we used both transmission electron microscopy and software image pro-plus 6.0 to analyze the synaptic interface curvature,PSD and synaptic cleft in the hippocampal CA1 area of 12-month-old ADAM10 cKO mice(n=3)and C57BL/6J mice(n=3).Results:The results indicated that the expression levels of synaptophysin proteins decreased by 24.75%(P < 0.05)and 40.10%(P < 0.01)in the cortex and hippocampus of ADAM10 cKO mice compared with controls.There was no significant difference at PSD-95 levels between ADAM10 cKO and control cortex.But PSD-95 levels decreased by 23.37%(P< 0.05)in the hippocampus of ADAM10 cKO mice.The immunohistochemical results showed that synaptophysin expression decreased by 24.59%(P < 0.05),18.78%(P < 0.001),14.00%(P < 0.01)and 21.17%(P < 0.01)in the cortex,hippocampus CA1,CA3,and dentate gyrus of ADAM10 cKO mice compared with controls,respectively.There were no significant differences at PSD-95 expression in the cortex and hippocampus CA3 region of both genotypes.However,PSD-95 expression decreased by 26.23%(P < 0.05)in the CA1 area of ADAM10 cKO mice.In addition,the thickness of PSD and the curvature of synaptic interface were decreased,and the width of synaptic space was increased in the hippocampal CA1 area of ADAM10 cKO mice compared with controls.Conclusion:Taken together,these findings demonstrated that ADAM10 deletion resulted in downregulation of the synaptophysin and PSD-95 expression to different content,abnormal synaptic ultrastructure,and impaired synaptic plasticity.Therefore,ADAM10 plays a crucial role in synaptic function.