TY - JOUR T1 - Alzheimer’s disease BIN1 coding variants increase intracellular Aβ by interfering with BACE1 recycling JF - bioRxiv DO - 10.1101/2021.06.09.447716 SP - 2021.06.09.447716 AU - Catarina Perdigão AU - Mariana Barata AU - Tatiana Burrinha AU - Cláudia Guimas Almeida Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/06/10/2021.06.09.447716.abstract N2 - Genetics identified BIN1 as the second most important risk locus associated with late-onset Alzheimer’s disease, after APOE4. Here we show the consequences of two coding variants in BIN1 (rs754834233 and rs138047593), both in terms of intracellular beta-amyloid accumulation (iAbeta) and early endosome enlargement, two interrelated early cytopathological Alzheimer’s disease phenotypes, supporting their association with LOAD risk. We previously found that Bin1 deficiency potentiates beta-amyloid production by decreasing BACE1 recycling and enlarging early endosomes. Here, we demonstrate that the expression of the two LOAD mutant forms of Bin1 did not rescue the iAbeta accumulation and early endosome enlargement induced by Bin1 knockdown and recovered by wild-type Bin1. The LOAD coding variants reduced Bin1 interaction with BACE1 likely causing a dominant-negative effect since Bin1 mutants, but not wild-type Bin1, overexpression increased iAbeta42 due to defective BACE1 recycling and accumulation in early endosomes. Endocytic recycling of transferrin was similarly affected by Bin1 wild-type and mutants, indicating that Bin1 is a general regulator of endocytic recycling. These data show that the LOAD mutations in Bin1 lead to a loss of function, suggesting that endocytic recycling defects are an early causal mechanism of Alzheimer’s disease.ADAlzheimer’s diseaseAP-2adaptor protein complex 2APOE4Apolipoprotein E ε4APPamyloid precursor proteinARF6ADP-ribosylation factor 6Aβamyloid-betaBACE1 β-siteAPP-cleaving enzyme 1 or β-secretase 1BARBIN1/amphiphysin/RVS167 domainBIN1Bridging integrator 1 / MYC box-dependent-interacting protein 1BSAbovine serum albuminCTFcarboxyl-terminal fragmentcDNAcomplementary DNACLAPclathrin and AP2 binding domainDMEMDulbecco’s Modified Eagle MediumEEA1Early endosome antigen 1FADfamilial Alzheimer’s diseaseEGTAEthylene Glycol Tetra-acetic AcidFBSfetal bovine serumGFPGreen Fluorescent ProteinGWASgenome-wide association studiesHRPHorseradish peroxidaseIPImmunoprecipitationIgGImmunoglobulin GKDKnockdownLOADlate-onset Alzheimer’s diseaseN2aNeuro2amAbmonoclonal antibodyOEoverexpressionpAbpolyclonal antibodyPBSphosphate-buffered salinePICprotease Inhibitor CocktailRIPARadio-Immunoprecipitation AssayRVS167Reduced viability upon starvation protein 167SDS-PAGEsodium dodecyl sulfate-polyacrylamide gel electrophoresisSH3src homology 3 domainsiRNAsmall interfering RNASNX4sorting nexin-4 ER -