Fig. 4: AXL activation promotes uptake of synaptic material in OPCs.
From: Human oligodendrocyte progenitor cells mediate synapse elimination through TAM receptor activation
a Representative images of OPC monocultures displaying typical OPC morphology and high yield of PDGFRα + OLIG2+ cells (mean ± s.e.m, n = 3 biological lines). b Immunostaining of neuron-derived synaptosomes expressing the ligand GAS6 and synaptic markers SYN1 and GEPH. c Live-cell imaging (IncuCyte) showing internalisation of synaptosomes into acidic phagolysosomes of OPCs, indicated by red fluorescence from pHrodo. d Quantification of pHrodo-labelled synaptosome uptake in cytochalasin D (10uM)-treated and untreated OPCs (phagocytic index = mean pHrodo+ area per OPC; mean ± s.e.m, n = 3 lines; two-way repeated-measures ANOVA with Sidak’s tests). e Confocal images and Imaris-based volumetric reconstructions confirming engulfment of pHrodo-labeled synaptosomes by OPCs (magenta = complete internalisation, left), and co-localisation with PSD-95+ puncta (right) indicating internalised synaptic material. f Relative mRNA levels of TAM receptors MERTK and AXL normalised to GAPDH. g Phagocytic indices in OPCs treated with 50, 150, or 200 nM UNC2025 or vehicle (DMSO) at 24 h (mean ± s.e.m., n = 3 lines represented by colour; Kruskal-Wallis with Dunn’s post hoc). h Quantification of AXL-expressing cells normalised to total nuclei (DAPI) showing ~50% knockdown following AXL siRNA treatment versus non-targeting control (NT; unpaired two-sided t-test, no multiple-comparison adjustment). i Comparison of phagocytic indices in NT- versus AXL siRNA-treated OPCs at 24 h (median: 170.2 vs 19.5; two-tailed Mann-Whitney U test; n = 3 lines represented by colour). j Representative immunostaining and volumetric reconstructions of PDGFRα + OPCs showing SYN1+ internalisation in vehicle- and UNC2025-treated neuron-OPC co-cultures at day 26 (magenta = complete internalisation). k Quantification of total internalised SYN1+ volume overlapping ≥90% with OPC surface, normalised by cell volume, showing reduced uptake with 150 nM UNC2025 (median: 0.56 vs 0.17; n = 25 OPCs per condition). l Representative immunostaining and volumetric reconstructions of AXL+ OPCs showing SYN1+ internalisation in vehicle- and UNC2025-treated forebrain organoids. m Quantification of total internalised SYN1+ volume, normalised by cell volume, showing decreased uptake with 150 nM UNC2025 (medians: 0.83 vs 0.35; n = 30 cells per condition; two-tailed Mann-Whitney U test). Scale bars: 20 μm (a–c), 10 μm (j), 5 μm (e), 3 μm (l).
