Fig 2: P2RY13 is upregulated in UC by Bioinformatics Analysis. (A) The DEGs were obtained from GSE9689 and GSE10191, 219 DEGs were upregulated and 86 DEGs were downregulated. (B) Cytoscape software (v3.8.0) was used to screen the core DEGs according to the analysis of DEGs. (C) KEGG enrichment analysis was performed for the DEGs from (A). (D) KEGG enrichment analysis was performed for 21 core DEGs. (E) Go enrichment analysis of core DEGs, was performed using Omicshare online tool. (F-G) Verification by two GEO datasets: GSE87466 and GSE38713. P2RY13 is upregulated in UC with significance. (H-I) ROC curve of the P2RY13 genes in UC in GSE87466 (AUC=0.859) and GSE38713 (AUC=0.731). AUC area under the ROC curve. KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; DEGs, differentially expressed genes.

Fig 3: Effect of P2Y13 receptor knock-out on microglial surveillance in hippocampal slices. (a) Specimen images taken 5 min apart of P85 WT and P88 P2Y13 KO Iba1-eGFP microglia, showing process extensions and retractions (red = retracted, green = extended processes) and the less ramified shape of microglia in the KO. (b) Quantification of surveillance for microglia from 3 WT and 3 KO mice, showing less surveillance in the KO than in their WT littermates. (c) Time course of increase of the number of surveyed pixels in maximum intensity projections of images of microglia in WT and KO Iba1-eGFP mice. Initial value is the area of the cell in the first image frame. (d) Initial slope of graphs in c (measured over the first 2 min, when assessment of surveillance is least confounded by pixel overlap in the maximum intensity projection. (e) Time course of surveillance indices with no drugs (no PSB-0739) in 3 WT mice or with application after 20 min of PSB-0739 (PSB) to block P2Y12 receptors in 3 WT and 3 KO Iba1-GFP hippocampal slices. Data show less surveillance in the KO and no significant effect of PSB. Data are normalized to the mean baseline values of the WT mice (averaged over the period from 1 to 19 min). (f) Quantification of the normalized surveillance index in the presence of PSB-0739, calculated as the mean surveillance index in PSB-0739 (averaged over the last 5 min in the drug) relative to the mean baseline surveillance index (averaged over the last 5 min of the baseline). The value shown for the Control bar is obtained from data at the same times as for the slices to which PSB-0739 was applied, but for the WT slices to which no PSB was applied. Age for b–f was P85–P119 for WT and P88–P131 for KO. Number of microglia shown on bars; p values were from t tests [Color figure can be viewed at http://wileyonlinelibrary.com]

Fig 4: P2Y13 KO increases baseline release of interleukin 1ß in brain slices, however, the microglia in the intact P2Y13 KO brains are not detectably activated. (a) ELISA-measured IL-1ß levels released from hippocampal slices from 8 WT (at P56–P93) and 8 P2Y13 KO mice (at P58–P105) exposed to no drugs, LPS (50 µg/ml), ATP (1 mM), or ATP + LPS, showing an enhanced resting level of IL-1ß release in the KO. (b) ELISA-measured TNFa levels released from hippocampal slices from 3 WT and 3 P2Y13 KO mice exposed to the same protocol as (a), showing a lack of effect of knocking out P2Y13 receptors on TNFa release. (c-d) mRNA levels of selected “activation” (il6, il-1ß, iNos), “priming” (tlr2, csf1, lgals3), and “homeostasis” (p2ry12) marker genes in whole brain samples from 5 WT (at P64-P115, 4 males, 1 female) and P2Y13 KO mice (at P62–P115, 4 males, 2 females), relative to Gapdh, as measured by quantitative RT-PCR. (e) Transcript levels of the selected markers shown in c and d in P2Y13 KO brains expressed as n-fold change relative to their WT aged-matched controls. The number of mice is shown on bars

Fig 5: Effect of P2Y13 receptor knock-out on microglial directed motility in hippocampal slices. (a-b) Directed motility to an ATP-filled pipette. (a) Time course of directed motility quantified as reduction of the “clear area” not occupied by microglia around the 1 mM ADP-filled pipette tip in 6 WT and 5 KO mice. (b) Time course of directed motility quantified as reduction of the mean distance (µm, averaged over sectors) from the microglial processes to the ADP-filled pipette tip in 6 WT and 5 KO mice. (c,d) Directed motility to a laser ablation. (c) Time course of directed motility quantified as reduction of the “clear area” not occupied by microglia around the ablation site in 3 WT and 3 KO mice. (d) Time course of directed motility quantified as reduction of the mean distance (µm, averaged over sectors) from the microglial processes to the ablation site in three WT and three KO mice. (e) Time course of directed motility quantified as reduction of the normalized “clear area” around the ADP-filled pipette in 6 WT and 5 KO mice and around the laser ablation site in 3 WT and 3 KO mice. The values were normalized to the initial “clear area” values of each recording. (f) Quantification of the time needed for the plots in (e) to drop to the 1/e value, that is, the time needed for the “clear area” to drop 36.7% of the initial area. (g) Quantification of the speed of convergence of the WT and KO microglial processes onto the tip of the pipette and the laser ablation site, calculated as the distance (µm) moved per 30 sec, averaged over the period needed to converge on the pipette tip or the ablation site, and then averaged over all the slices. Number of slices are shown on bars. Age for pipette experiments (a, b, e–g) was P55–P121 for WT and P56–P136 for KO and for laser ablation experiments (c, d, e–g) was around P60