Fig. 3

Fenebrutinib blocks effects of FcγR activation in human brain tricultures and MCM-exposed neurons. A Immunocytochemistry of the human iPSC-derived brain triculture system showing GFAP⁺ astrocytes, pan-NF⁺ neurons and Iba1⁺ microglia. Scale bar is 50 µm. B TNF-α release from human iPSC-derived brain tricultures treated with fenebrutinib (1 μM) and stimulated with Agg IgG (1 mg/mL) for 24 h. Fenebrutinib treatment reduced Agg IgG-induced TNF-α release. C Representative live cell microscopy images of human iPSC-derived brain tricultures showing Agg IgG-induced clustering of GFP⁺ iMicroglia. Scale bar is 100 µm. D Quantification of GFP⁺ iMicroglia demonstrated a significant reduction in microglial clustering with fenebrutinib treatment. E Heatmap showing top differentially expressed genes from annotated gene sets in human iPSC-derived brain tricultures treated with fenebrutinib (1 μM) and stimulated with Agg IgG (1 mg/mL) for 72 h. F Box plots showing representative genes from the BSG pathway, which is the most significant gene set modulated by fenebrutinib treatment. G Representative confocal images of MCM-exposed iNGN2 showing reduced neurite staining with Agg IgG MCM, which is reversed by fenebrutinib treatment. Scale bar is 200 µm. H NfL levels in supernatants of MCM-exposed iNGN2. Fenebrutinib treatment significantly reduced NfL release compared with Agg IgG alone. Data are shown as mean ± SD; individual dots represent replicate wells. Results are representative of two to three independent experiments. Significance is indicated by ****P < 0.0001, ***P < 0.001 and *P < 0.05, determined by one‐way ANOVA and Tukey’s post hoc test. Agg IgG heat-aggregated immunoglobulin G, ANOVA analysis of variance, BSG basigin, DAPI 4′,6-diamidino-2-phenylindole, FcγR Fc gamma receptor, FEN fenebrutinib, GFAP glial fibrillary acidic protein, GFP green fluorescent protein, iMicroglia/iMGL induced pluripotent stem cell-derived microglia, iNGN2 neurogenin 2-inducible neuron, Iba1 ionized calcium-binding adaptor molecule 1, iPSC induced pluripotent stem cell, MCM microglia-conditioned media, NF neurofilament, NfL neurofilament light chain, ns not significant, SD standard deviation, TNF tumor necrosis factor, tpm transcripts per million