Proteomic Characterization of the Alzheimer's Disease Risk Factor BIN1 Interactome

The gene BIN1 is the second-largest genetic risk factor for late-onset Alzheimer's disease (LOAD). It is expressed in neurons and glia in the brain as cell-type-specific and ubiquitous isoforms. BIN1 is an adaptor protein that regulates membrane dynamics in many cell types. Previously, we reported that BIN1 predominantly localizes to presynaptic terminals in neurons and regulates presynaptic vesicular release. However, the function of neuronal BIN1 in relation to LOAD is not yet fully understood. A significant gap in the field is the unbiased characterization of neuronal BIN1-interacting proteins and proximal neighbors. To address this gap and help define the functions of neuronal BIN1 in the brain, we employed TurboID-based proximity labeling to identify proteins biotinylated by the neuronal BIN1 isoform 1-TurboID fusion protein (BIN1iso1-TID) in cultured mouse neuroblastoma (N2a) cells in vitro and in adult mouse brain neurons in vivo. Label-free quantification-based proteomic analysis of the BIN1iso1-TID biotinylated proteins led to the discovery of 360 proteins in N2a cells and 897 proteins in mouse brain neurons, identified as BIN1iso1-associated (proximal) or interacting proteins. A total of 92 proteins were common in both datasets, indicating that these are high-confidence BIN1-interacting or proximity proteins. SynapticGO analysis of the mouse brain dataset revealed that BIN1iso1-TurboID labeled 159 synaptic proteins, with 60 corresponding to the synaptic vesicle cycle. Based on phosphorylation site analysis of the neuronal BIN1iso1-TID interactome and related kinase prediction, we selected and validated AAK1, CDK16, SYNJ1, PP2BA, and RANG through immunostaining and proximity ligation assays as members of the BIN1 interactome in the mouse brain. This study establishes a foundation for further investigations into the function of neuronal BIN1 by identifying several previously unknown proximal and potential interacting proteins of BIN1.