Abstract BackgroundTheBIN1locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease.BIN1undergoes 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. MethodsMicroglial 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.Bin1expression 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. ResultsHere, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencingBin1expression 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-specificBin1conditional 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 ofBin1impaired 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. ConclusionsOur 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 betweenBin1andIfitm3, two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulateIfitm3upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases. Graphical Abstract
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This content will become publicly available on August 1, 2026
A programmable microfluidic platform to monitor calcium dynamics in microglia during inflammation
Neuroinflammation is characterized by the elevation of cytokines and adenosine triphosphate (ATP), which in turn activates microglia. These immunoregulatory molecules typically form gradients in vivo, which significantly influence microglial behaviors such as increasing calcium signaling, migration, phagocytosis, and cytokine secretion. Quantifying microglial calcium signaling in the context of inflammation holds the potential for developing precise therapeutic strategies for neurological diseases. However, the current calcium imaging systems are technically challenging to operate, necessitate large volumes of expensive reagents and cells, and model immunoregulatory molecules as uniform concentrations, failing to accurately replicate the in vivo microenvironment. In this study, we introduce a novel calcium monitoring micro-total analysis system (CAM-μTAS) designed to quantify calcium dynamics in microglia (BV2 cells) within defined cytokine gradients. Leveraging programmable pneumatically actuated lifting gate microvalve arrays and a Quake valve, CAM-μTAS delivers cytokine gradients to microglia, mimicking neuroinflammation. Our device automates sample handling and cell culture, enabling rapid media changes in just 1.5 s, thus streamlining the experimental workflow. By analyzing BV2 calcium transient latency to peak, we demonstrate location-dependent microglial activation patterns based on cytokine and ATP gradients, offering insights contrasting those of non-gradient-based perfusion systems. By harnessing advancements in microsystem technology to quantify calcium dynamics, we can construct simplified human models of neurological disorders, unravel the intricate mechanisms of cell-cell signaling, and conduct robust evaluations of novel therapeutics.
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- Award ID(s):
- 2240094
- PAR ID:
- 10533749
- Publisher / Repository:
- Nature
- Date Published:
- Journal Name:
- Microsystems & Nanoengineering
- Volume:
- 10
- Issue:
- 1
- ISSN:
- 2055-7434
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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