Microglia-astrocyte crosstalk regulates synapse remodeling via Wnt signaling

Abstract

Astrocytes and microglia are emerging key regulators of activity-dependent synapse remodeling that engulf and remove synapses in response to changes in neural activity. Yet, the degree to which these cells communicate to coordinate this process remains an open question. Here, we use whisker removal in postnatal mice to induce activity-dependent synapse removal in the barrel cortex. We show that astrocytes do not engulf synapses in this paradigm. Instead, astrocytes reduce contact with synapses prior to microglia-mediated synapse engulfment. We further show that the reduced astrocyte-synapse contact is dependent on the release of Wnts from microglia downstream of neuron-to-microglia fractalkine ligand-receptor (CX3CL1-CX3CR1) signaling. These results demonstrate an activity-dependent mechanism by which microglia instruct astrocyte-synapse interactions, providing a permissive environment for microglia to remove synapses. We further show that this mechanism is critical to remodel synapses in a changing sensory environment and that this signaling is upregulated in several disease contexts.

Keywords: Wnt; astrocyte; microglia; synapse remodeling.

Figure 1.. Microglia, but not astrocytes, engulf and remove synapses after whisker lesioning

(A) Experimental design for B-C. Layer IV barrel cortex, 6 days post-lesioning. (B) Representative images of anti-VGluT2⁺ synapses in control vs. deprived hemispheres (hems). Scale bars 10 μm. (C) VGluT2⁺ synapse density in control vs. deprived hems (n=5 mice. *p<0.05). (D) Experimental design for E-F. Layer IV barrel cortex, 5 days post-lesioning. (E) Representative max intensity projections of anti-VGluT2⁺ synapses, anti-CD68⁺ lysosomes, and anti-GFP⁺ microglia in control vs. deprived hems. Scale bars 5 μm. Inset: 3D render of anti-VGluT2⁺ synaptic material (arrow) within anti-CD68⁺ lysosomes inside anti-GFP⁺ microglia. Inset scale bar 2 μm. (F) VGluT2⁺ synaptic material within microglial lysosomes in control vs. deprived hems (n=4 mice. *p<0.05). (G) Experimental design for H-J. Layer IV barrel cortex, 5 days post-lesioning. (H-I) Representative max intensity projections (H, I top) and 3D renders (I bottom) of anti-VGluT2⁺ synapses, anti-LAMP2⁺ lysosomes, and anti-GFP⁺ astrocytes in control vs. deprived hems. Boxed region in H shown on left in I. 3D renders only show synaptic material within astrocyte lysosomes, examples indicated (arrows). Scale bar 5 μm. (J) VGluT2⁺ synaptic material within astrocyte lysosomes in control vs. deprived hems (n=6 mice). Data represent mean ± SEM. Circles (female) and squares (male) represent individual animals. Statistics: Ratio paired t test (C, F, J) See also Figure S1.

Figure 2.. Astrocytes decrease their association with thalamocortical synapses following whisker lesioning

(A) Top: experimental design for B-H. Layer IV barrel cortex, 4 days post-lesioning. Bottom: diagram of astrocyte-synapse nearest neighbor distance (NND) and astrocyte-synapse contact. (B-C) Representative images of anti-VGluT2⁺ synapses and anti-GFP⁺ astrocyte processes (B) before vs. after gel-expansion for expansion microscopy, (C) in control vs. deprived hemispheres (hems) post-expansion highlighting synapse-astrocyte contacts (asterisks) and synapse-astrocyte NNDs (dashed lines). Scale bars (B) 20 μm, (C) 10 μm (not adjusted by expansion index). (D-E) Representative 3D renders of control vs. deprived hems showing (D) anti-VGluT2⁺ synapses and anti-GFP⁺ astrocyte processes, (E) anti-VGluT2⁺ synapses pseudo-colored by synapse-astrocyte NND (corrected for expansion index). Astrocyte-contacted synapses (NND=0) are transparent. Scale bars 2 μm (corrected for expansion index). (F) Cumulative proportion plot of synapses’ synapse-astrocyte NND in control vs. deprived hems. Data pooled from n=5 mice. (G-H) Mean synapse-astrocyte NND (G) and percentage of synapses contacted by astrocytes (H), measured by expansion microscopy in control vs. deprived hems (n=5 mice. *p<0.05). (I) Experimental design for J-L. Layer IV barrel cortex, 4 days post-lesioning. (J) Representative electron micrographs of control vs. deprived hems. Pseudo-coloring highlights example synapses and astrocyte processes. Scale bars 1 μm. (K-L) Mean synapse-astrocyte NND (K) and percentage of synapses contacted by astrocytes (L), measured by electron microscopy in control vs. deprived hems (n=4 mice. *p<0.05). Bar graphs represent mean ± SEM. Circles (female) and squares (male) represent individual animals. Statistics: Ratio paired t test (G-H, K-L) See also Figures S2 and S3.

Figure 3.. Wnt receptor signaling is elicited in layer IV astrocytes following whisker removal

(A) Experimental design for B-C. Barrel cortex, 24 hours post-lesioning. (B) Volcano plot of differentially expressed genes (DEGs) in astrocytes in control vs. deprived hemispheres (hem). n=5 mice. (C) Plot of Ingenuity Pathway Analysis (IPA) root regulators of DEGs in control vs. deprived hem astrocytes. x-axis position indicates activation z-score. Dot size indicates −log₁₀ p-value. Wnt signaling pathway molecules: bold and blue. (D) Diagram of canonical Wnt siganling pathway. (E) Experimental design for F-I. Layer IV barrel cortex, 24 hours post-lesioning. (F) Representative images of control vs. deprived hems of TCF/Lef:H2B-GFP mice showing anti-SOX9⁺ astrocyte nuclei, DAPI⁺ nuclei, and anti-GFP⁺ nuclei. Right-most panels depict anti-GFP signal in astrocytes (SOX9⁺; examples outlined in insets) and non-astrocytes (DAPI⁺ SOX9-negative). Scale bars and inset scale bars 10 μm. (G) Average TCF/Lef:H2B-GFP mean fluorescence intensity (MFI) in control vs. deprived hem astrocytes (n=3 mice. ***p<0.001). (H) Representative images of anti-β-catenin, anti-SOX9⁺ astrocyte nuclei, and DAPI⁺ nuclei in control vs. deprived hems. Two right-most columns show anti-β-catenin in astrocyte nuclei (SOX9⁺; examples outlined in insets) and non-astrocytes (DAPI⁺ SOX9-negative). Scale bars and inset scale bars 10 μm. (I) Average nuclear β-catenin MFI in astrocytes and non-astrocytes in control vs. deprived hems (n=6 mice. *p<0.05). Bar graphs represent mean ± SEM. Circles (female) and squares (male) represent individual animals. Statistics: Ratio paired t test (G), Repeated measures 2-way ANOVA with Holm-Sidak post-hoc test (I). See also Figure S4, Tables S1–S3.

Figure 4.. Activation of Wnt signaling in astrocytes induces increased separation between synapses and astrocyte processes

(A) Diagram of Wnt signaling pathway and β-catenin destruction complex. (B) Diagram of Apc ^Flox allele. (C) Experimental design for D-J. Layer IV barrel cortex, P12. (D) Representative images of anti-β-catenin, anti-SOX9⁺ astrocytes, and DAPI⁺ nuclei in Aldh1l1^CreER/+; Apc^+/+; Rosa26^mTmG/+ (control) mice vs. Aldh1l1^CreER/+; Apc^Flox/Flox; Rosa26^mTmG/+ (APC cKO) mice. Right-most panels show anti-β-catenin in astrocyte nuclei (SOX9⁺; examples outlined in insets). Scale bars and inset scale bars 10 μm. (E) Average nuclear β-catenin MFI in astrocytes in control vs. APC cKO mice (n=5 control, 3 APC cKO mice. *p<0.05). (F) Representative 3D renders of anti-GFP⁺ astrocyte processes in control vs. APC cKO mice. Scale bars 2 μm (corrected for expansion index). (G) Total density of astrocyte processes in control vs. APC cKO mice (n=6 control, 5 APC cKO mice). (H) Representative 3D renders of anti-VGluT2⁺ synapses in control vs. APC cKO mice, pseudo-colored by synapse-astrocyte nearest neighbor distance (NND; corrected for expansion index). Astrocyte-contacted synapses (NND=0) are transparent. Insets: 3D renders of anti-VGluT2⁺ synapses and anti-GFP⁺ astrocyte processes. Scale bars and inset scale bars 2 μm (corrected for expansion index). (I-J) Percentage of synapses contacted by astrocytes (I) and mean synapse-astrocyte NND (J) in control vs. APC cKO mice (n=6 control, 5 APC cKO mice. *p<0.05). (K) Diagram of ligand-receptor analysis. (L) Heatmap of predicted ligand activities for Wnts in public astrocyte TRAP-Seq datasets. Superscripts: reference numbers. Bar graphs represent mean ± SEM. Circles (female) and squares (male) represent individual animals. Statistics: Student’s t test (E, G, I-J). See also Table S4.

Figure 5.. Whisker lesioning induces microglia-astrocyte crosstalk via Wnts

(A) Experimental design for microglia TRAP-Seq data. Barrel cortex, 24 hours post-lesioning. (B) Diagram of ligand-receptor analysis. (C) Predicted ligand activity vs. enrichment in microglia for top 20 ligands. Wnts bolded and blue. (D) Experimental design for E-F. 24 hours post-lesioning (E) Representative MERFISH sample highlighting barrel cortex layers. (F) Representative image of DAPI⁺ nuclei and anti-VGluT2⁺ synapses overlaid with MERFISH-detected transcripts for microglia marker genes. Layer IV outlined. Scale bar 250 μm. Inset highlights DAPI⁺ microglia expressing Wnt7a (arrow). Inset scale bar 5 μm. (G-H) Dot plots of gene expression in microglia in barrel cortex layers for (G) individual Wnt ligands and (H) the aggregate of all Wnt ligands. Data pooled from 2 males, 2 females. (I) Experimental design for J-M. (J) Representative images of GFP⁺ primary astrocytes with no treatment vs. exposure to WNT4, WNT5A, or WNT7A. Scale bars 50 μm. (K-M) Plots of astrocyte morphological complexity after no treatment vs. exposure to WNT4, WNT5A, or WNT7A (n=3 independent culture experiments. **p<0.01; ****p<0.0001; ns: p>0.05). Data in K-M represent the locally estimated scatterplot smoothing (LOESS) line of best fit (line) and 95% confidence interval (outline). Statistics: Mixed-model ANOVA with Tukey’s post-hoc test (K-M) See also Figure S5 and Tables S5–S7.

Figure 6.. Microglial Wnt release is necessary to induce decreased astrocyte-synapse interactions and synapse removal following whisker lesioning

(A) Diagram of microglia-astrocyte Wnt signaling. (B) Experimental design for C-E. Layer IV barrel cortex, 4 days post-lesioning. (C) Representative 3D renders of control vs. deprived hemispheres (hems) in Cx3cr1^Cre; Wls^+/+ (control) vs. Cx3cr1^Cre; Wls^Flox/Flox (WLS cKO) mice showing anti-VGluT2⁺ synapses pseudo-colored by synapse-astrocyte nearest neighbor distance (NND; corrected for expansion index). Astrocyte-contacted synapses (NND=0) are transparent. Insets: 3D renders of anti-VGluT2⁺ synapses and anti-GFP⁺ astrocyte processes. Scale bars and inset scale bars 2 μm (corrected for expansion index). (D-E) Mean synapse-astrocyte NND (D) and percentage of synapses contacted by astrocytes (E) in control vs. deprived hems of control and WLS cKO mice (n=4 control, 4 WLS cKO mice. *p<0.05). (F) Experimental design for G-H. Layer IV barrel cortex, 5 days post-lesioning. (G) Representative max intensity projections (left) and 3D renders (right) of anti-VGluT2⁺ synapses, anti-CD68⁺ lysosomes, and anti-IBA1⁺ microglia in control vs. deprived hems of control and WLS cKO mice. 3D renders only show synaptic material within microglia lysosomes, examples indicated by arrows. Scale bars 5 μm. (H) VGluT2⁺ synaptic material within microglial lysosomes in control vs. deprived hems of control and WLS cKO mice (n=4 control, 5 WLS cKO mice. *p<0.05). (I) Experimental design for J-K. Layer IV barrel cortex, 6 days post-lesioning. (J) Representative images of anti-VGluT2⁺ synapses in control vs. deprived hems of control and WLS cKO mice. Scale bars 10 μm. (K) VGluT2⁺ synapse density in control vs. deprived hems of control and WLS cKO mice (n=6 control, 6 WLS cKO mice. ***p<0.001). Data represent mean ± SEM. Circles (female) and squares (male) represent individual animals. Statistics: Repeated measures 2-way ANOVA with Holm-Sidak post-hoc test (D-E, H, K) See also Figure S6.

Figure 7:. CX3CR1-CX3CL1 signaling is upstream of microglia-astrocyte crosstalk via Wnts

(A) Experimental design for B-C. Barrel cortex, 24 hours post-lesioning. (B-C) Volcano plots of differentially expressed genes in microglia in the control vs. deprived hemispheres (hems) of (B) n=4 Cx3cr1^+/− mice and (C) n=5 Cx3cr1^−/− mice. (D) (Top) experimental design for E-F. Layer IV barrel cortex, 24 hours post-lesioning. (Bottom) diagram of Wnt signaling pathway. (E) Representative images of control vs. deprived hemispheres (hems) of TCF/Lef:H2B-GFP; Cx3cl1^−/− mice showing anti-SOX9⁺ astrocyte nuclei, DAPI⁺ nuclei, and anti-GFP⁺ nuclei. Right-most panels depict anti-GFP signal in astrocytes (SOX9⁺; examples outlined in insets). Scale bars and inset scale bars 10 μm. (F) Average TCF/Lef:H2B-GFP mean fluorescence intensity (MFI) in control vs. deprived hem astrocytes (n=4 mice. ns: p>0.05). (G) Experimental design for H-I. Layer IV barrel cortex, 24 hours post-lesioning. (H) Representative images of anti-β-catenin, anti-SOX9⁺ astrocyte nuclei, and DAPI⁺ nuclei in control vs. deprived hems of Cx3cr1^−/− mice. Right-most column shows anti-β-catenin in astrocyte nuclei (SOX9⁺; examples outlined in insets). Scale bars and inset scale bars 10 μm. (I) Average nuclear anti-β-catenin MFI in astrocytes in control vs. deprived hems of Cx3cr1^−/− mice (n=5 mice. ns: p>0.05). (J) Experimental design for K-M. Layer IV barrel cortex, 4 days post-lesioning. (K) Representative 3D renders of control vs. deprived hems in Aldh1l1^CreER/+; Rosa26^mTmG/+; Cx3cl1^−/− mice showing anti-VGluT2⁺ synapses pseudo-colored by synapse-astrocyte nearest neighbor distance (NND; corrected for expansion index). Astrocyte-contacted synapses (NND=0) are transparent. Insets: 3D renders of anti-VGluT2⁺ synapses and anti-GFP⁺ astrocyte processes. Scale bars and inset scale bars 2 μm (corrected for expansion index). (L-M) Mean synapse-astrocyte NND (L) and percentage of synapses contacted by astrocytes (M) in control vs. deprived hems of Aldh1l1^CreER/+; Rosa26^mTmG/+; Cx3cl1^−/− mice (n=4 mice. ns: p>0.05). Bar graphs represent mean ± SEM. Circles (female) and squares (male) represent individual animals. Statistics: Ratio paired t test (F, I, L-M). See also Figure S7.