Immune checkpoint molecule Tim-3 regulates microglial function and the development of Alzheimer's disease pathology [scRNA-Seq]

Microglia, resident immune cells in the brain, play a critical role in neurodevelopment and neurological diseases. We investigated the role of the immune checkpoint molecule Tim-3 (Havcr2), which was recently identified as a genetic risk factor for late-onset Alzheimer's disease (LOAD) in microglia. While Tim-3 has been shown to play an important role in inducing T cell exhaustion, it shows high and specific expression in the microglia where its role is unknown. Here we show that Tim-3 expression in microglia was induced by TGFß signaling. Mechanistically, Tim-3 binds both Smad2 and Tgfbr2 by its C-terminus tail and enhances TGFß signaling by promoting the phosphorylation of Smad2 by Tgfbr, thereby contributing to microglial homeostasis. Genetic deletion of Tim-3 in microglia resulted in increased phagocytic activity with a gene expression profile skewed towards neurodegenerative microglia (MGnD) phenotype, also known as disease-associated microglia (DAM). Moreover, microglia-targeted deletion of Tim-3 ameliorated AD pathology in 5xFAD mice. Single-nucleus RNA sequencing (snRNA-seq) identified a subpopulation among MGnD/DAM microglia in Havcr2-deficient 5xFAD mice, characterized by increased pro-phagocytic and anti-inflammatory gene expression together with decreased proinflammatory gene expression. Additional single-cell RNAseq confirmed these transcriptomic shifts in Havcr2-deficient 5xFAD mice in most microglial clusters. Our study shows a Tim-3-mediated regulatory mechanism of homeostatic microglia through its interaction with TGFß signaling and the beneficial role of targeting microglial Tim-3 in AD mice. Our findings promise a potential therapeutic strategy targeting checkpoint molecule Tim-3 in currently intractable AD. Overall design: Female Tim3 f/f, 5XFAD and Tim3 f/f, CX3CR1 CreERT2, 5XFAD mice were prepared. Tamoxifen (150 mg/kg body weight) was injected intraperitoneally in two consecutive days at the age of 6 weeks. Microglia were isolated from the brain cortex at the age of 5 months. Mice were deeply anesthetized with CO2 and transcardially perfused with cold HBSS (Gibco). The brains were quickly removed from the skull into cold HBSS. The brain cortices were gently separated with forceps on ice under the microscope. The prepared cortices were homogenized gently with Dounce Homogenizer (Bellco Glass) on ice. After centrifugation, the pellet was dissolved in 70% Percoll Plus (GE Healthcare) solution, with 37% Percoll Plus put gently on top of it. After gentle centrifugation, the cell layer in the middle was collected into cold PBS-based flow cytometry (FCM) buffer, including 0.5% BSA and 2mM EDTA. The sorted microglia were then collected into PBS with 1% BSA.