Bridging integrator 1 (BIN1) is the second most prevalent genetic risk factor identified by genome-wide association studies (GWAS) for late-onset Alzheimer's disease. BIN1 encodes an adaptor protein that regulates membrane dynamics in the context of endocytosis and neurotransmitter vesicle release. In vitro evidence suggests that BIN1 can directly bind to tau in the cytosol. In addition, BIN1's function limits extracellular tau seed uptake by endocytosis and subsequent propagation as well as influences tau release through exosomes. However, the in vivo roles of BIN1 in tau pathogenesis and tauopathy-mediated neurodegeneration remain uncharacterized. We generated conditional knockout mice with a selective loss of Bin1 expression in the forebrain excitatory neurons and oligodendrocytes in P301S human tau transgenic background (line PS19). PS19 mice develop age-dependent tau neuropathology and motor deficits and are commonly used to study Alzheimer's disease tau pathophysiology. The severity of motor deficits and neuropathology was compared between experimental and control mice that differ with respect to forebrain BIN1 expression. BIN1's involvement in tau pathology and neuroinflammation was quantified by biochemical methods and immunostaining. Transcriptome changes were profiled by RNA-sequencing analysis to gain molecular insights. The loss of forebrain BIN1 expression in PS19 mice exacerbated tau pathology in the somatosensory cortex, thalamus, spinal cord and sciatic nerve, accelerated disease progression and caused early death. Intriguingly, the loss of BIN1 also mitigated tau neuropathology in select regions, including the hippocampus, entorhinal/piriform cortex, and amygdala, thus attenuating hippocampal synapse loss, neuronal death, neuroinflammation and brain atrophy. At the molecular level, the loss of forebrain BIN1 elicited complex neuronal and non-neuronal transcriptomic changes, including altered neuroinflammatory gene expression, concomitant with an impaired microglial transition towards the disease-associated microglial phenotype. These results provide crucial new information on in vivo BIN1 function in the context of tau pathogenesis. We conclude that forebrain neuronal BIN1 expression promotes hippocampal tau pathogenesis and neuroinflammation. Our findings highlight an exciting region specificity in neuronal BIN1 regulation of tau pathogenesis and reveal cell-autonomous and non-cell-autonomous mechanisms involved in BIN1 modulation of tau neuropathology.

Background The BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease. BIN1 undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth. Methods Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells. Bin1 expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches. Results Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencing Bin1 expression in primary microglial cultures, we demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specific Bin1 conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss of Bin1 impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene, Ifitm3 . Conclusions Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship between Bin1 and Ifitm3 , two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulate Ifitm3 upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases. Graphical Abstract

BIN1, a member of the BAR adaptor protein family, is a significant late-onset Alzheimer disease risk factor. Here, we investigate BIN1 function in the brain using conditional knockout (cKO) models. Loss of neuronal Bin1 expression results in the select impairment of spatial learning and memory. Examination of hippocampal CA1 excitatory synapses reveals a deficit in presynaptic release probability and slower depletion of neurotransmitters during repetitive stimulation, suggesting altered vesicle dynamics in Bin1 cKO mice. Super-resolution and immunoelectron microscopy localizes BIN1 to presynaptic sites in excitatory synapses. Bin1 cKO significantly reduces synapse density and alters presynaptic active zone protein cluster formation. Finally, 3D electron microscopy reconstruction analysis uncovers a significant increase in docked and reserve pools of synaptic vesicles at hippocampal synapses in Bin1 cKO mice. Our results demonstrate a non-redundant role for BIN1 in presynaptic regulation, thus providing significant insights into the fundamental function of BIN1 in synaptic physiology relevant to Alzheimer disease.

BIN1, a member of the BAR adaptor protein family, is a significant late-onset Alzheimer’s disease risk factor. Here, we investigated BIN1 function in the brain using conditional knockout (cKO) models. The loss of neuronal Bin1 expression resulted in select impairment of spatial learning and memory. Examination of hippocampal CA1 excitatory synapses revealed a deficit in presynaptic release probability and slower depletion of neurotransmitter during repetitive stimulation, suggesting altered vesicle dynamics in Bin1 cKO mice. Super-resolution and immunoelectron microscopy localized BIN1 to presynaptic sites in excitatory synapses. Bin1 cKO significantly reduced synapse density and altered presynaptic active zone protein cluster formation. Finally, 3D-electron microscopy reconstruction analysis uncovered a significant increase of docked and reserve pool of synaptic vesicles at hippocampal synapses in Bin1 cKO mice. Our results demonstrate a non-redundant role for BIN1 in presynaptic regulation, thus providing novel insights into BIN1's fundamental function in synaptic physiology relevant to Alzheimer’s disease.