Fig 1: Presynaptic caspase-3 activation promotes complement-dependent synaptic phagocytosis by microglia.a Images displaying colocalization between C1qA-647 and a presynaptic bouton (arrowheads) at 6 hours after CNO application. b Proportion of presynapses colocalized with C1qA-647 6 hours after drug application. DEVD refers to the caspase-3 inhibitor Z-DEVD-FMK. Mean ± SD, n = 20 regions from 2 independent experiments, Dunn’s test after one-way ANOVA on ranks. Box plots are defined as follows: the central line within the box represents the median (50th percentile), while the bounds of the box represent the 25th and 75th percentiles. The whiskers extend to the minimum and maximum values. All individual data points are plotted. c C1qA-647 signals in H2B-GFP-positive soma, βIII-tubulin-immunostained axons and synaptophysin-mCherry-positive presynapses at 6 hours after drug application. Plots from the same region are connected with a line. n = 7, 14 regions from 2 independent experiments, Tukey’s test after two-way repeated-measures ANOVA. d Images displaying a time-lapse assay monitoring synaptic phagocytosis by microglia. Arrows indicate phagocytosed synaptophysin-mCherry. The inset image is an orthogonal view of the yellow-line square. e Volumetric assay of synaptophysin-mCherry signals within microglia before and after drug application. Mean ± SD, DMSO: n = 170, 193 microglia from 4 independent experiments; CNO: n = 155, 172 microglia from 4 independent experiments; C1qA + DMSO: n = 92, 96 microglia from 3 independent experiments; C1qA + CNO: n = 163, 173 microglia from 4 independent experiments; C1qA + CNO + DEVD: n = 79, 82 microglia from 3 independent experiments, two-sided Mann‒Whitney U test. Box plots are defined as follows: the central line within the box represents the median (50th percentile), while the bounds of the box represent the 25th and 75th percentiles. The whiskers extend to the minimum and maximum values. All individual data points are plotted. f A representative image of microglia, presynapses, and axons in the coculture system. The experiment was independently repeated 30 times with similar results. g An orthogonal view showing the phagocytosis of a presynapse by microglia 24 minutes after starting live imaging. h Timeline showing drug application and live imaging of microglial synaptic phagocytosis. i Snapshots of live imaging representing microglial synaptic phagocytosis in control (DMSO) and corresponding 3D reconstructed images of presynapses and axons. Arrows indicate synaptophysin-mCherry-positive puncta phagocytosed by microglia. Arrowheads indicate the axon where phagocytosis occurred. The elapse time from the start of live imaging is shown in the upper left corner of each image. j Number of presynaptic puncta phagocytosed by microglia during the continuous 2-hour live imaging session. Mean ± SD, n = 7 (DMSO), 7 (CNO), 8 (C1qA + DMSO), 8 (C1qA + CNO) independent experiments, Dunnett’s test after one-way ANOVA. Source data are provided as a Source Data file.
Fig 2: Graphical abstract of the present research.Increased neuronal activity induces nonapoptotic caspase activation at presynapses, probably via mitochondrial accumulation. Caspase activation causes synaptic tagging by C1q, leading to C3 localization at presynapses. Microglial CR3 recognizes C3, inducing synapse-specific phagocytosis by microglia.
Fig 3: Optogenetic caspase-3 activation induces complement-dependent synaptic phagocytosis.a Diagram illustrating optic induction of caspase activation. b Images showing cleaved caspase-3 signals at presynapses (arrows) following blue light stimulation of a single presynaptic bouton (arrowhead). The experiment was independently repeated 4 times with similar results. c Timeline showing caspase activation and synaptic complement tagging identification by immunocytochemistry (ICC). d Images showing C1qA and presynapses. The stimulated points are outside the ROI. e Proportion of C1qA area in a presynaptic bouton. LEHD refers to the caspase-9 inhibitor Z-LEHD-FMK TFA. n = 119, 63, 78, 74 presynapses from 3 independent experiments, Dunn’s test after one-way ANOVA on ranks. Box plots are defined as follows: the central line within the box represents the median (50th percentile), while the bounds of the box represent the 25th and 75th percentiles. The whiskers extend to the minimum and maximum values. All individual data points are plotted. f Timeline showing caspase activation, live imaging of microglial synaptic phagocytosis, and ICC detection of complement receptors. g A representative image of microglia‒neuron interactions. h Magnified images from g (dashed-line square). Top: live images of microglial synaptic phagocytosis. The elapsed time from the start of live imaging is shown in the upper left corner of each image. Bottom: ICC evidence of CR3 surrounding phagocytosed synapses. i Magnified images from h detailing phagocytosed presynapses and CR3. j Comparison of CR3 intensity around phagocytosed synapses (phagocytic site) and at other sites in microglial processes (nonphagocytic site). Plots from the same region are connected with a line. n = 12 presynapses from 7 independent experiments, two-sided paired t test. Source data are provided as a Source Data file.
Fig 4: Inhibitory synaptic caspase-3 activation leads to complement tagging.a, c Immunohistochemical images of C1q and C3 in the middle molecular layer 6 hours after febrile seizures (FSs). b, d Density of C1q and C3 puncta in the middle molecular layer 6 hours after FSs. Mean ± SD, n = 5 mice, two-sided unpaired t test. e–g Images displaying colocalized inhibitory presynapses with PSVue (PS) and cleaved caspase-3 or C1q or C3 6 hours after FSs (arrows). DEVD refers to the caspase-3 inhibitor Z-DEVD-FMK. h–j Density of inhibitory presynapses colocalized with PSVue and cleaved caspase-3 or C1q or C3 6 hours after FSs. Mean ± SD, n = 5 mice, Tukey’s test after one-way ANOVA. Source data are provided as a Source Data file.
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