Study Of The Role Of Metalloproteinase ADAMTS18 In Central Nerves System

The ADAMTS(A disintegrin and metalloproteinase with thrombospondin motifs)enzymes are secreted,multi-domain matrix-associated zinc metalloendopeptidases.Now there are 19 members found in human in this family,which can be synthesized and secreted by macrophage,vascular endothelial cell,smooth muscle cell and fibroblast.They can interact with cell surface ligands or extracellular matrixs(ECM)by their special protein region,and thus play crucial roles in development,tissue morphogenesis,inflammation,fertility,tumor,angiogenesis and central nervous system(CNS)damage by modifying ECM.ADAMTS18,an orphan ADAMTS,remains to be characterized with regard to its functions and substrates.It was first found in 2002 using an extensive computer screening of human genome databases.It has been shown that ADAMTS18 expression was detected in human fetal lung,liver and kidney,whereas it was found mainly in adult brain,prostate,submaxillary gland,endothelium,and retina.In mice,Adamts18 expression was detected mainly in brain,eye,kidney,lung and testicle,especially in brain with the hightest expression level.A genome-wide association study(GWAS)of 542050 single-nucleotide polymorphisms(SNPs)in healthy people of 72-74 years old in 2012 showed that ADAMTS18 is related to white matter integrity in the brain,which suggests that ADAMTS18 is relevant to brain development,but the potential role of ADAMTS18 in CNS remain largely unclear.Murine ADAMTS18 is 88%homologous with human ADAMTS18(PubMed blast),and share the similar protein expression profile with human.In this study,Adamts18knockout(KO)mice were used as a model to identify the potential role of ADAMTS18 in CNS by a combination of molecular biological method,cell biological method,pathologic histology study and animal behavior tests.The research methods included are as following:1)Determination of Adamts18 expression profile in mouse brains at different developmental stages by using reverse transcription-polymerase chain reaction(RT-PCR)and In situ hybridization(ISH)methods.2)Determination of the changes of anatomic form of mouse brains caused by Adamts18 deficiency using Hematoxylin and Eosin(H&E)staining or Nissl staining.3)Immunofluorescence(IF)staining and Golgi staining were performed to further examine morphological alterations in neurons from wild-type mice(WT)and Adamts18 KO mice.4)The influence of Adamts18 deficiency on mouse brain function was detected by using a battery of animal behavioral tests.5)Quantitative Real-Time-PCR(qRT-PCR),Enzyme linked immunosorbent assay(ELISA)and Western blotting were used to investigate the underlying mechanisms.The results showed:1)Adamts18 mRNA was highly expressed in developing brains,including cerebellum,dentate gyrus(DG),cerebral cortex,thalamus and superior colliculus,especially in cerebellum granular cell layer and the hippocampus DG inner granular cell layer.2)Adamts18 KO mice exhibited higher dendritic branching complexity and spine density on hippocampal DG granular cells compared with WT littermates[avearge total dendritic length:1110±91.02?m(KO)vs.870.3±65.64?m(WT),~*P<0.05;Dendrite spine numbers:9.919±0.285 per 10?m(KO)vs.8.811±0.372 per 10?m(WT),~*P<0.05].3)Adamts18 KO mice showed decreased immobility time in both Tail suspension test(TST)and Forced swim test(FST)[TST:201±14.9 s(KO)vs.242±7.3 s(WT),~*P<0.05;FST:66±8.4 s(KO)vs.93±8.6 s(WT),~*P<0.05],and higher sucrose preference(%)[68±3.1%(KO)vs.57±2.8%(WT),~*P<0.05]compared with WT littermates.4)Laminin(LN)levels in Adamts18 KO mouse embryonic brains were significantly increased compared with WT littermates[LN:458.4±27.1 ng/mg(KO)vs.402±39.9 ng/mg(WT),~*P<0.05].Western blotting results further showed that phosphatidylinositol3-kinase(PI3-kinase)/AKT/glycogensynthasekinase-3?(GSK-3?)/collapsing response mediator protein-2(CRMP2)signaling pathway which can regulate neuron polarization mediated by LN was activated in Adamts18 KO mouse embryonic brains.In summary,this study revealed for the first time that ADAMTS18 plays a critical role in CNS.ADAMTS18 deficiency could give rise to the increased LN in mouse embryonic brains,which activates the PI3K/AKT/GSK-3?/CRMP2 signaling pathway mediated by LN;The activation of this pathway may be responsible for the increased average total dendrite length and dendrite spine numbers of dendrites of hippocampal DG granular cells;The alteration of dendrite structural plasticity could partially contribute to the decreased depression-like behavior in Adamts18 KO mice.This study not only supplements the functional spectrum of ADAMTS18,but also provides new evidences for the roles of ADAMTS family members in CNS.In addition,the association between ADAMTS18 and depression-like behavior provides a potential target for the treatment of depression-related disorders.