Microglial P2Y6 calcium signaling promotes phagocytosis and shapes neuroimmune responses in epileptogenesis

Abstract

Microglial calcium signaling is rare in a baseline state but shows strong engagement during early epilepsy development. The mechanism and purpose behind microglial calcium signaling is not known. By developing an in vivo UDP fluorescent sensor, GRAB_UDP1.0, we discovered that UDP release is a conserved response to seizures and excitotoxicity across brain regions. UDP signals to the microglial P2Y₆ receptor for broad increases in calcium signaling during epileptogenesis. UDP-P2Y₆ signaling is necessary for lysosome upregulation across limbic brain regions and enhances production of pro-inflammatory cytokines-TNFα and IL-1β. Failures in lysosome upregulation, observed in P2Y₆ KO mice, can also be phenocopied by attenuating microglial calcium signaling in Calcium Extruder ("CalEx") mice. In the hippocampus, only microglia with P2Y₆ expression can perform full neuronal engulfment, which substantially reduces CA3 neuron survival and impairs cognition. Our results demonstrate that calcium activity, driven by UDP-P2Y₆ signaling, is a signature of phagocytic and pro-inflammatory function in microglia during epileptogenesis.

Figure 1:. Development of a fluorescent sensor for in vivo imaging of extracellular UDP

(A) To engineer the UDP biosensor, a circularly permutated enhanced GFP (cpEGFP) molecule with linker sequences was inserted into the extracellular loop between transmembrane (TM) 5 and 6 of the chicken P2Y₆ (cP2Y₆) receptor. (B) Optimization of the UDP sensor occurred through a three-step process including insertion site variation, N- and C-terminal linker truncation, and point-mutation-based optimization of the linker sequence, yielding the UDP1.0 variant (see Figures S3A, S3B). (C) Representative images and corresponding ∆F/F trace from HEK293T cells expressing UDP1.0 under basal conditions, after the addition of 10 µM UDP, and then after 20 U/mL apyrase grade VII (apyr.) treatment. Scale bar, 20 µm. (D) Summary of peak UDP1.0 ∆F/F amplitude under basal, UDP, or UDP plus apyrase conditions. One-Way ANOVA with Tukey’s post-hoc test (n=90 total cellular ROIs). (E) One-photon excitation (Ex) and emission (Em) spectra for UDP1.0 in the presence of 10 µM UDP (solid lines), or 5 U/mL apyrase (dashed lines). (F and G) Selectivity of UDP1.0. (F) Comparison of UDP responses in the presence of non-selective P2Y receptor antagonists, or other purine agonists, neurotransmitters, and saline. Peak ∆F/F responses were normalized to UDP. One-Way ANOVA with post-hoc UDP comparison. (G) UDP1.0 fluorescent response curves to purinergic ligands with EC₅₀ values, see Star Methods for fitting (n=3–6 wells; 300–500 cells per well). (H) Wild-type cP2Y₆, but not UDP1.0, drives Gαq signaling measured using a luciferase complementation assay. Left, schematic of the complementation assay. Right, total luminescence emitted by cells co-transfected with LgBit-mGq alone (control), or LgBit-mGq with cP2Y₆-SmBit, or UDP1.0-SmBit, plotted against UDP concentration. One-Way ANOVA with Tukey’s post-hoc test (UDP1.0 vs. Control: p=0.3406; n=3 wells/group). (I) UDP1.0 fluorescence in HEK cells under basal conditions or with 100 µM UDP incubation for up to 2 hours. One-Way ANOVA with post-hoc comparison to the start of UDP incubation: P≥0.8934. Scale bar, 10 µm. (solid line: mean ± SEM; dashed line: one of three wells containing 300–500 cells). (J-L) Characterization of UDP1.0 in acute brain slice. (J) Schematic illustration of the slice experiments. An AAV2/9 expressing hSyn-UDP1.0 was injected into the cortex; 3 weeks later, acute brain slices were prepared and used for two-photon imaging. (K) UDP1.0 fluorescent responses to 250 µM UDP or 250 µM ATP. Left: example image showing the UDP1.0 response to UDP or ATP. Right: overall ∆F/F mean ± SEM response from n=4–6 trials. (L) Summary data showing the response of UDP1.0 to different UDP or ATP concentration in brain slice. UDP1.0 responses were calculated by the area under the curve shown in (K) over a 60s period. Summary data are presented as the mean ± SEM from 4–8 independent trials and N=3–4 slices.

Figure 2:. Enhanced UDP release occurs following status epilepticus

(A) In vivo 2-photon study timeline and image of UDP1.0 expression. Scale bar, 100µm. Examples of UDP sensor event fluorescence and corresponding ∆F/F traces. Scale bar, 20µm. (B) Examples of Type 1 sharp-peak events and Type 2 events with slower rise (∆F/F traces aligned to rise start time). (C) Modeling Type 1 and Type 2 rise kinetics (peak normalized) against transient or prolonged UDP application ex vivo (gray trace). (D) Example UDP sensor signals (top, ∆F/F) and sensor event size/location under the cranial window (bottom) at baseline and during epileptogenesis. The number of ∆F/F signals displayed (top) is meant to convey frequency (frequency box represents mean ± S.E.M events per 30 min recording). Event size/location displays all events aggregated across a longitudinal cohort of N=3–5. (E) Duration of UDP release events (dot: a single event). (F) The cumulative area covered by UDP sensor events over a 30 min period (dot: one imaging region). (G) The cumulative UDP ∆F/F∙s signal recorded over a 30 min period (dot: one imaging region). (H) Overlay of UDP sensor events ex vivo in the CA3 region of hippocampus. Scale bar, 70µm. Event scale as in (D). (I) Quantification of either UDP event frequency, cumulative UDP release area, or cumulative UDP signal area per 30 min. Welch’s T-test (dot: one or 2–3 CA3 regions surveyed from N=4 mice per time point). Data represent the mean ± SEM. D-G: Longitudinal study of 2 non-overlapping regions from N=3–5 mice. E-G: One-Way ANOVA with Fisher’s post-hoc testing vs. baseline for statistical comparison.

Figure 3:. Microglial calcium elevations during epileptogenesis are P2Y6 dependent

(A) Experimental timeline for in vivo, longitudinal calcium imaging of cortical microglia. Representative field of view. Scale bar, 40µm. (B) (Top) Examples of inactive, low, and high-level microglial calcium signaling used for segmenting activity. Scale bar: 1-fold ∆F/F and 60s. (Bottom) Microglial calcium activity distributions (∆F/F∙s) at baseline and 3 days after KA-SE. (C) Heatmaps of microglial GCaMP6s calcium activity (∆F/F) by genotype and time period (30 ROIs chosen to match inactive/low/high activity distributions—left color bar). (D) Percentage of total microglia ROIs exhibiting no, low, or high activity between genotypes and across periods of epileptogenesis. Two-way ANOVA with Sidak’s post-hoc test between activity levels (P-value compares high-level activity). (E) Overall calcium activity in P2ry6^+/+ and P2ry6^−/− microglia across two weeks of epileptogenesis. Two-way ANOVA with Sidak’s post-hoc test (dotted lines represent a longitudinal imaging region; dots represent group mean ± SEM). (F) (Top) 2P images of microglia in the CA1 region from acute brain slice. (Bottom) Spontaneous calcium activity in CA1 microglia across epileptogenesis. Two-Way ANOVA with Sidak’s post-hoc test (dots represent one ROI; survey of 2 slices per mouse; N=4 mice per time point and genotype). (G) As in (F), for microglia of the CA3 region. Scale bar, 50 µm. (H) In vivo average intensity images of microglia (overlay of start vs. 5 min) to indicate process motility. Scale bar, 15µm. (I) Overall percentage of microglial processes that extend outward, remain stable, or retract during in vivo imaging. Two-way ANOVA with Sidak’s post-hoc test: no significant motility differences between genotypes (p=0.5505–0.9965 by motility type). Bars represent the mean ± SEM. In vivo (A-E): N=4 P2ry6^+/+mice, N=6 P2ry6^−/− mice with 2 non-overlapping regions surveyed per mouse.

Figure 4:. P2Y6 activation regulates lysosome expression and facilitates neuronal soma engulfment

(A) CD68 lysosome expression across limbic regions 3 days after KA-SE. Scale bar, 100 µm. (B) Quantification of CD68 area by region and genotype 3- and 7-days after KA-SE. One-way ANOVA with Dunnett’s post-hoc test by region (dots: one region; two regions from N=4–5 mice/group). (C) IBA1 staining in the CA3 region by genotype and time point. Scale bar, 40µm. (D) (Left) CA3 microglia longest process analyses. Two-Way ANOVA effect of genotype: F_{(1, 1094)}=0.5979, p=0.4395 (dot: one cell). (Right) Sholl plots with representative cell morphologies. Scale bar, 5µm. Survey of 10–40 microglia per mouse; N=3–5 mice/group. (E) Correlations between initial seizure severity and CA3 microglia process length or CD68 area. Simple linear regression (dot: one mouse; aggregated from day 3 and day 7 time points). (F) Histological classification of NeuN neuron (Imaris surface rendering) and CD68 phagolysosome interactions in CA3 from 3D confocal microscopy, ranging from no/minimal interaction to full engulfment. Scale bar, 5µm. (G) (Left) Distribution of NeuN neurons based upon their CD68 coverage (survey of n=1500–1989 CA3 neurons from 10 CA3 subfields and N=5 mice/group). (Right) The number of CA3 neurons having different CD68 coverage. One-Way ANOVA with Sidak’s post-hoc test (dots: one CA3 subfield). (H) Representative images of ramified, ameboid, and neuron-engulfing microglia in CA3 brain slice (purple: transient intercalation of AF568 dye with a cell soma). Representative ∆F/F calcium responses to 500 µM UDP. (I) Comparison of CA3 microglia UDP calcium responses by morphology. One-way ANOVA with Tukey’s post-hoc test (dot: one microglial cell; survey of cells from WT brain slices prepared 3, 7, and 10 days post-SE).

Figure 5:. Attenuating calcium signaling through CalEx phenocopies lysosome impairments in microglia

(A) Illustration of ATP2B2 calcium extruder (“CalEx”) function. (B) Images of CalEx-mCherry recombination against an IBA1 co-stain in TMEM119^2A-CreER;R26^{LSL-CAG-ATP2B2-mCherry} mice treated with tamoxifen. Scale bar, 30 µm. (C) Quantification of mean recombination by region (dot: one mouse; N=5–6 mice per group). (D) Outline of experimental steps to validate CalEx function in microglia. (E) Example of mCherry fluorescence in an acute brain slice with corresponding ∆F/F calcium responses following 500 µM focal UDP application (“CalEx” tissue). Scale bar, 40 µm. Right ∆F/F traces: example UDP calcium responses in an acute slice prepared from the control mouse line. (F) Overall calcium response (∆F/F) to UDP application (lines represent the mean ± SEM; aggregate of n=69 responding cells in Control tissue and n=59 responding cells in CalEx tissue; survey of 2–4 slices from N=4–5 mice per group). (G) Quantification of 500 µM UDP calcium response duration, and signal area between Control and CalEx microglia. Student’s T-test (dot: one cell; n=69 control cells and n=59 CalEx-mCherry cells; 2–4 slices from N=4–5 mice/group). (H) Representative image of CalEx-mCherry and CD68 expression in CA1 SR one week after KA-SE. Scale bar, 25 µm. (I) Closer evaluation of IBA1 and CD68 expression in a representative mCherry⁺ and mCherry⁻ cell from (D). Scale bar, 7 µm. (J) Quantification of average lysosome size and overall CD68 area (normalized to IBA1 area) for mCherry⁺ and mCherry⁻ cells. Student’s T-test (dot: one cell; survey of n=412 mCherry⁺ microglia and n=568 mCherry⁻ microglia in CA1 SR from N=5 mice/group). (K) IBA1 morphology in CA1 SR with red and gray outlines denoting mCherry⁺ and mCherry⁻ cells, established from the mCherry channel. Scale bar, 20 µm. Sholl plots for mCherry⁺ and mCherry⁻ cells one week after KA-SE (n=88 mCherry⁺ and n=103 mCherry⁻ cells from N=5 mice/group). Bar or line graphs display the mean ± SEM.

Figure 6:. Enhanced pro-inflammatory signaling is coordinated with phagocytosis in P2ry6^+/+ microglia

(A) Outline of transcriptomics experiment using FACS-based microglia isolation 3 days after KA-SE from whole brain. (B) Top differentially expressed genes (DEGs) in microglia between WT-KO genotypes (log2FC>1.0 and BH-adjusted p<0.1). (C) Key Gene Ontology (GO) terms based upon the DEG set in (B). Details are provided in the Star Methods section. (D) Protein-level evaluation of hippocampal and cortical microglia using high-parameter flow cytometry 5 days after KA-SE. (E) Mode-normalized, representative histograms and intensity distributions for microglial P2Y12 and Cx3Cr1 expression. Two-Way ANOVA with Sidak’s post-hoc test (box plots display the mean and interquartile range with whiskers denoting min to max). (F) UMAPs of TNFα, Pro IL-1β, and CD68 expression, or NeuN/CD68 co-detection in hippocampal microglia (boxes highlight a distinct population between genotypes). (G) Frequency of marker expression by microglia in cortex and hippocampus. One-Way ANOVA with Tukey’s post-hoc test (normalized to mean naïve frequency per cohort/batch; dot: one mouse). (H) Pseudocolor plots displaying the NeuN gate, based upon an FMO, and application of that gate onto hippocampal microglia. (I) Psuedocolor plot with gates to isolate microglial populations which are CD68^Low, CD68^High, and CD68^High SSC^High. (J) Evaluation of key signal intensity differences between P2ry6^+/+ hippocampal microglia which are CD68^Low, CD68^High, and CD68^High SSC^High. One-Way ANOVA with Tukey’s post-hoc test (dot: one mouse, where dot shape denotes cohort; ****p<0.0001). (K) Representative immunofluorescent imaging of DAPI, IBA1, and IL-1β expression in WT CA1 one week after KA-SE. Scale bar, 20µm. (L) Correlations between Pro IL-1β intensity and NeuN intensity inside of microglia. Simple linear regression (dot: one mouse). (M) Comparison of different CD68 population frequency between genotypes in the hippocampus. Two-Way ANOVA with Sidak’s post-hoc test (dot: one mouse, where dot shape denotes cohort). Data were aggregated from N=6 naïve mice (4 WT, 2 KO) N=8 D5 P2ry6^+/+ mice and N=7 D5 P2ry6^−/− mice across two independent cohorts.

Figure 7:. P2Y₆ KO mice have improved CA3 neuronal survival and enhanced cognitive task performance

(A) Example of a NeuN intensity plot from day 7 post-SE WT tissue. Scale bar, 200 µm. (B) Representative NeuN intensity profiles from the CA3 region of P2ry6^+/+ and P2ry6^−/− tissue. Scale bar, 25 A.U. and 200 µm. (C) Quantification of NeuN cell density in the CA3 or CA1 pyramidal band. Two-way ANOVA with Sidak’s post-hoc test (dots: one region; bilateral survey from N=3–5 mice/group). (D) Timeline of Novel Object Recognition (NOR) task (repeated at baseline and day 7 post-SE). Outline of the task procedure. (E) Representative trial performance in the novel phase during the baseline (naïve) test. (Top) Distance moved with periods highlighted during novel or familiar object interaction. Scale bar, 1 min. (Bottom) Trace of mouse movement in the arena. (F) Discrimination index score by genotype and trial phase during the baseline (naïve) test. Two-Way ANOVA with Sidak’s post-hoc test between familiar and novel phase performance by genotype (dot: one mouse; ♦♦P<0.01, one-sample T-test comparison to chance: 0.50). (G) As in (E), representative trial performance 7 days after KA-SE. (H) As in (F), discrimination index 7 days after KA-SE. Two-Way ANOVA with Sidak’s post-hoc test between familiar and novel phase performance by genotype (dot: one mouse; ♦P<0.05, one-sample T-test comparison to chance). (I) Total distance moved by trial phase. Two-Way ANOVA with Sidak’s post-hoc test (dot: one mouse). P2ry6^+/+ and P2ry6^−/− mice used longitudinally at baseline and day 7 post-SE (N=6 mice/group at baseline; N=6/6 surviving P2ry6^+/+; N=5/6 surviving P2ry6^−/− at day 7 post-SE).