Fig. 4: Microglial MMP14 promoted brevican digestion in human cells.
From: Extracellular matrix proteolysis maintains synapse plasticity during brain development
a, Scatter plot of gene expression (sorted by rank) of iPS-cell-derived microglia and human fetal microglia. MMPs, ADAMs and ADAMTSs families that have transmembrane domain in dark green; MMPs, ADAMs and ADAMTSs without transmembrane domains in light green; microglial marker genes in violet. Data were reanalyzed from the previous study55. b, Schematic of the iPS-cell-derived astrocyte–neuron–microglia tri-culture system. c, Schematic of the proteomics analysis with iPS-cell-derived cells. Cell supernatants were collected from co-culture (neurons and astrocytes; no microglia) and tri-culture (neurons, astrocytes and microglia; with microglia) and analyzed. MS, mass spectrometry. d, Venn diagram of proteins detected in cell culture supernatants with or without microglia. Proteins detected only in the absence of microglia are listed on the left and proteins detected only in the presence of microglia are listed on the right. ECM-related proteins as defined by the matrisome database MatrisomeDB2.080 are labeled in red. e, Volcano plot of proteins in the intersection of Venn diagram in d. The fold change is calculated by comparing the ‘with microglia’ condition with the ‘no microglia’ condition. Thresholds: adjusted P < 0.05 (horizontal dashed line), and average log2(fold change) > 1 (vertical dashed gray lines). ECM-related proteins as defined by the matrisome database MatrisomeDB2.080 in red. Significant matrisome proteins are outlined in blue. f, Strategy for MMP14 knockdown (KD) by shRNA (shRNA interference) with a scrambled shRNA control, and MMP14 rescue by expression of MMP14 fused to GFP, versus a GFP-only control in microglia. The rescue construct has silent mutations in the MMP-GFP to prevent knockdown by shMMP14. All constructs were delivered by lentivirus at the iPS cell stage and used the ubiquitous promoter EF1a. g, Representative images of the tri-culture system with MMP14 KD and rescue in microglia. Iba1 (microglia), S100β (astrocyte) and neuron (MAP2) stainings are shown. Scale bar, 100 µm. h, Quantification of the process length of microglia in the tri-culture system with MMP14 KD and rescue. Dots show mean microglial process length per field of view from 18 fields of view over 3 independent experiments. Box-and-whiskers plot: box shows 25th to 75th percentile; whiskers show minimum to maximum. Statistics performed on means per field of view (Kruskal–Wallis test; asterisks in the figure show results of Dunn’s multiple comparisons). i, Representative image of western blotting for brevican in cell supernatant and MAP2 loading control in cell lysate without microglia or with microglia after MMP14 KD and rescue. j, Quantification of brevican in the cell supernatant without microglia or with microglia after MMP14 KD and rescue. Each dot represents mean brevican intensity normalized to no microglia control from n = 4 independent experiments (one-way ANOVA; asterisks in the figure show results of Tukey’s multiple comparisons). Values were plotted as mean ± s.e.m. ****P < 0.0001; *P < 0.05. Illustrations in b, c and f created using BioRender.com.
