β-secretase 1 Down-regulates β-dystroglycan And Modulates The Phenotypic Switching Of Vascular Smooth Muscle Cells

β-secretase 1(BACE1)is a key enzyme for amyloid precursor protein cleaving and amyloid-β production.The protein level and enzyme activity of BACE1 were significantly increased in neurons and cerebral vascular smooth muscle cells(vSMCs)of Alzheimer’s disease(AD)and cerebral amyloid angiopathy(CAA)patients.This suggests that BACE1 is a key target for interfering with the pathological development of AD and CAA.However,clinical trials of BACE1 inhibitors for AD treatment have failed.The reason may lie that BACE1 has a variety of substrates and binding proteins and is involved in many important physiological functions,and current BACE1 inhibitors cannot specifically inhibit the cleavage of amyloid precursor protein.Therefore,the further study of BACE1 substrates or binding proteins will help to understand the physiological and pathological functions of BACE1 and provide a theoretical background for AD and CAA drug development.A proteomics analysis of Bace1-/-mice found that the protein level of dystroglycan(DG,gene symbol DAG1)decreased significantly in the cerebrospinal fluid of Bace1-/mice compared with wild-type mice,suggesting that BACE1 may down-regulate the protein level of DG.DG contains two non-covalently connected subunits:α-DG and βDG,α-DG is located outside the plasma membrane and is a receptor of the extracellular matrix,β-DG is a single transmembrane integrated protein that connects α-DG with cytoskeletal proteins and multiple signaling molecules.In the central nervous system,DG is highly expressed in vSMCs.Vascular SMCs undergo phenotypic switching in the face of vascular injury stimulation,which is characterized by the down-regulation of contractile protein levels and enhanced cell proliferation and mobility.Studies have found that β-DG can regulate the proliferation and regeneration of cardiomyocytes.Therefore,we propose that BACE1 can regulate the phenotypic switching of vSMCs by down-regulating the protein level of β-DG.The scientific question of this study focuses on:Can BACE1 regulate the protein level of β-DG,and what is the mechanism?Can BACE1 affect the phenotypic switching of vSMCs?Does BACE1 regulate the phenotypic switching of vSMCs through DG?First,we used cell transfection to overexpress BACE1 and DG in HEK-293T cells.Western blotting showed that BACE1 significantly reduced the protein level of β-DG.When the endogenous BACE1 in HEK-293T cells was knocked out by gene editing,the protein level of β-DG was significantly increased.As for the mechanism,this study found that BACE1 did not change the transcription level of the DAG1 gene by real-time quantitative PCR analysis.Protein co-immunoprecipitation assay showed that there was an interaction between BACE1 and β-DG.The treatment of the BACE1 inhibitor revealed that the down-regulation of β-DG by BACE1 was dependent on the enzymatic activity of BACE 1.However,the carboxyl-terminal fragment of BACE1-related β-DG was not detected in the experiment,and current results cannot prove that β-DG is a substrate of BACE1.In summary,these results showed that the overexpression of BACE1 could down-regulate the protein level of β-DG in HEK-293T cells.To further study the regulatory effect of BACE1 on the endogenous β-DG,we selected vSMCs and primary astrocytes with a high level of endogenous DG.After overexpression of BACE1 in these two cells,the protein levels of endogenous β-DG were significantly decreased by western blotting analysis.This result indicates that BACE1 can downregulate the protein level of endogenous β-DG.Second,to study the effect of BACE1 on vSMCs phenotype,we performed RNAsequencing of BACE1-overexpressing vSMCs.The results showed that cell proliferation and migration signaling pathways were significantly enriched in BACE1overexpressing vSMCs.Real-time quantitative PCR analysis showed that BACE1 overexpression increased the transcription levels of synthetic marker genes(Mmp9,Mcp1)and decreased the transcription levels of contractile marker genes(Myh11,Acta2,Tagln).Immunofluorescence staining of Ki67(a cell proliferation marker)and cell scratch assay showed that BACE1-overexpressing vSMCs exhibited enhanced proliferation and migration capabilities compared to control cells.These findings suggest that the overexpression of BACE1 promotes the phenotypic switching of vSMCs to synthetic phenotype.Finally,to study whether BACE1 affects the phenotypic switching of vSMCs through DG.We transferred exogenous DG into BACE1-overexpressing vSMCs.Cell scratch assay showed that the migration of vSMCs was inhibited by DG transfection.Western blotting showed that the overexpression of BACE1 down-regulated the levels of three contractile marker proteins,namely smooth muscle myosin heavy chain,smooth muscle actin α,and smooth muscle protein 22α(encoded by Myh11,Acta2,Tagln genes).However,in the presence of BACE1,the addition of DG significantly increased the levels of three contractile phenotype marker proteins.The above results indicate that BACE1 affects the phenotypic switching of vSMCs through DG.In summary,this study determined that BACE1 reduces the protein level of β-DG,and this regulation depends on the enzyme activity of BACE1.Secondly,BACE1 may promote the transformation of vSMCs to a synthetic phenotype by reducing the protein level of endogenous β-DG.This is the first time find that BACE1 has been implicated in the phenotypic switching of vSMCs.This study provides a new mechanism for explaining vascular functional defects in AD and CAA patients.