Astrocyte Gi-GPCR signaling corrects compulsive-like grooming and anxiety-related behaviors in Sapap3 knockout mice - PubMed (original) (raw)
Astrocyte Gi-GPCR signaling corrects compulsive-like grooming and anxiety-related behaviors in Sapap3 knockout mice
Joselyn S Soto et al. Neuron. 2024.
Abstract
Astrocytes are morphologically complex cells that serve essential roles. They are widely implicated in central nervous system (CNS) disorders, with changes in astrocyte morphology and gene expression accompanying disease. In the Sapap3 knockout (KO) mouse model of compulsive and anxiety-related behaviors related to obsessive-compulsive disorder (OCD), striatal astrocytes display reduced morphology and altered actin cytoskeleton and Gi-G-protein-coupled receptor (Gi-GPCR) signaling proteins. Here, we show that normalizing striatal astrocyte morphology, actin cytoskeleton, and essential homeostatic support functions by targeting the astrocyte Gi-GPCR pathway using chemogenetics corrected phenotypes in Sapap3 KO mice, including anxiety-related and compulsive behaviors. Our data portend an astrocytic pharmacological strategy for rescuing phenotypes in brain disorders that include compromised astrocyte morphology and tissue support.
Keywords: GPCR; OCD; RNA sequencing; astrocyte; behavior; glia; proteomics; striatum.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.
Conflict of interest statement
Declaration of interests UCLA filed a US provisional patent (no. 63/658,760) based on this work. B.S.K. is on the editorial advisory board of Neuron.
Figures
Figure 1.. Behavioral improvements following in vivo astrocyte hM4Di activation.
(A) (i) Hypergeometric analysis heat map shows enrichment of astrocyte territory-related genes within those associated with human brain disorders; analyzed from Ref. * indicates FDR < 0.05. (ii) Heat map shows percentage of genes related to OCD and HD shared with mouse striatal scRNA-seq data; analyzed from Ref. OPC, oligodendrocyte precursor cell. (iii) Cartoon showing predicted astrocyte morphology changes in OCD. (B) Map of Sapap3 interacting astrocyte proteins related to the actin cytoskeleton and G-protein signaling; data from Ref . (C) Schematic showing the experimental design for wild-type (WT) and Sapap3 KO mice. (D) WT and Sapap3 KO mice treated with in vivo hM4Di activation or control AAV. (E) Traces of elevated plus maze recordings for each experimental group. (F) (i) Graphs show grooming behavior metrics: area of open lesions, number of lesions, grooming bouts, and time spent grooming. (n = 11–12 mice per group, area of lesions: Kruskal-Wallis Test; number of lesions: Kruskal-Wallis Test; number of grooming bouts: Two-way ANOVA with Tukey’s post hoc test [hM4Di effect p-value = 0.06]; time spent grooming: Two-way ANOVA with Tukey’s post hoc test [hM4Di effect p-value = 0.02]). (ii) Graphs show anxiety-like behaviors by two metrics: time spent in the open arms of the elevated plus maze and time spent in the center of the open field. (n = 11–12 mice per group, time spent in open arms: Two-way ANOVA with Tukey’s post hoc test [hM4Di effect p-value = 3.5 × 10−4]; time in center: Two-way ANOVA with Tukey’s post hoc test [hM4Di effect p-value = 0.01]). (G) as in (D) but for hM3Dq. (H) Elevated plus maze recordings as in (E) but for hM3Dq. (I) (i) Grooming behavior measurements as in (F) but for hM3Dq (n = 7–9 mice per group, area of lesions: Kruskal-Wallis Test; number of lesions: Kruskal-Wallis Test; number of grooming bouts: Two-way ANOVA with Tukey’s post hoc test [hM3Dq effect p-value = 0.23]; time spent grooming: Two-way ANOVA with Tukey’s post hoc test [hM3Dq main effect p-value = 0.61]). (ii) Anxiety behavior measurements as in (F) but for hM3Dq (n = 7–9 mice per group, time spent in open arms: Two-way ANOVA with Tukey’s post hoc test [hM3Dq effect p-value = 0.44]; time in center: Kruskal-Wallis Test). (J) Summary heat map of behavioral Z-scores in Sapap3 KO mice with each experimental treatment, or with astrocyte-selective Sapap3 AAV from Ref. (K) Summary heat map shows percent recovery of behaviors for each astrocyte-specific treatment in Sapap3 KO mice versus Sapap3 KO mice treated with 10 mg/kg fluoxetine from Ref. Data are represented as mean ± SEM.
Figure 2.. Restoration of astrocyte morphology in Sapap3 KO mice with hM4Di DREADDs.
(A) Images showing LifeAct-GFP in WT and Sapap3 KO astrocytes with tdTomato control AAV or in vivo hM4Di activation. (B) Left, LifeAct GFP mean actin intensity as a function of distance from the soma. Points represent the mean intensity from 15 cells per group from 4 mice per group. (Two-way repeated ANOVA with Bonferroni post hoc test; P < 0.05 at 20–40 μm). Right, graphs show the astrocyte actin territory area and the roundness of the actin signal (n = 15 astrocytes from 4 animals per group; Actin area: Kruskal-Wallis Test; Roundness: Kruskal-Wallis Test). (C) Summary of astrocyte morphology z-scores in each experimental treatment. Far right column shows morphology changes with astrocyte-selective Sapap3 AAV from Ref. (D) Images of single WT or Sapap3 KO astrocytes from each experimental group after fractal dimension (Df) analysis. Graph shows the Df. (n = 15 astrocytes from 4 animals per group; Two-way ANOVA with Tukey’s post hoc test). (E) Left, zoom-in images of S100β+ striatal astrocytes in each experimental treatment group. Right, graphs show the S100β somata area and branch number (n = 3–4 mice per group; Two-way ANOVA with Tukey’s post hoc test). Data are represented as mean ± SEM.
Figure 3.. Neuronal properties in Sapap3 KO mice following in vivo astrocyte hM4Di activation.
(A) Heat map of coronal brain sections show the total number of ΔFosB+ cells in WT and Sapap3 KO mice treated with in vivo hM4Di activation or control AAV. Heat maps show the number of ΔFosB+ cells in the striatum and two cortical areas. Blue and yellow heat maps depict the resultant z-scores. (B) Images of ΔFosB+ neurons in the striatum of each experimental group. Graphs show the percentage of NeuN+ neurons expressing ΔFosB. (n = 8 FOVs from 4 mice per group; Two-way ANOVA with Tukey’s post hoc test). DStr, dorsal striatum; VStr, ventral striatum. (C) Images of ΔFosB and D1 receptor mRNA+ neurons in the striatum of each experimental group. Percentage of D1+ neurons with ΔFosB expression. (n = 8 FOVs from 4–5 mice per group; Two-way ANOVA with Tukey’s post hoc test). (D) ΔFosB and D2 receptor mRNA+ neurons in the striatum of each experimental group. Graphs show the percentage of D2+ neurons with ΔFosB expression (n = 8 FOVs from 4–5 mice per group; Kruskal-Wallis Test). (E) Percent of total D1+ and D2+ neurons expressing ΔFosB in the striatum of Sapap3 KO mice treated with in vivo hM4Di activation or control AAV. (n = 8 FOVs from 4–5 mice per group; Unpaired t-test). (F) Representative traces of MSN evoked action potentials from each experimental group. (G) Relationship of injected current to number of action potentials for WT mice treated with in vivo hM4Di activation or control AAV. (n = 16–18 cells from 5–7 mice per group; One-way ANOVA). (H) As in (G) but for Sapap3 KO mice (n = 12–23 cells from 4–8 mice per group; One-way ANOVA). (I) Number of evoked MSN action potentials at 480 pA current in each experimental group. (n = 12–23 cells from 4–8 mice per group; Two-way ANOVA with Tukey’s post hoc test). (J) MSN rheobase in each experimental group. (n = 12–23 cells from 4–8 mice per group; Two-way ANOVA with Tukey’s post hoc test). Data are represented as mean ± SEM.
Figure 4.. Molecular mechanisms following astrocytic hM4Di activation in Sapap3 KO mice.
(A) Summary of findings from Figs 1–3. (B) Top, differentially expressed proteins (DEPs) in Sapap3 KO mice versus WT (FDR < 0.05). Center, differentially expressed genes (DEGs) in Sapap3 KO mice versus WT (FDR < 0.05). Bottom, list of 31 genes/proteins that were differentially expressed in both proteomic and transcriptomic datasets. (C) (i) Significant molecular and biological function Enrichr gene ontology (GO) terms for the 209 DEPs in Sapap3 KO striata. (ii) PANTHER protein classifications of the 209 DEPs in Sapap3 KO striata. (D) Left, Venn diagram shows the comparison of the 209 DEPs in Sapap3 KO striata and 271 DEPs (FDR < 0.05) when Sapap3 KO + hM4Di mice was compared to Sapap3 KO + tdTomato mice. Right, top heat map shows 43 reciprocally changed proteins upon activation of hM4Di in astrocytes. Bottom heat map shows the 20 most enriched and depleted proteins that were unique to the hM4Di DEP dataset. The color legend corresponds to both heat maps. (E) Top: significant GO terms for the 43 recovered proteins from **(D)**. Bottom: significant GO terms for the 228 hM4Di unique proteins from **(D)**. (F) Left, Venn diagram of the 43 hM4Di recovered proteins and 2,869 DEGs (P-value < 0.05) in human caudate OCD subjects from Ref . Right, heat map shows possible shared reciprocal changes upon activation of astrocytic hM4Di from the 15 shared genes. (G) Left, Venn diagram of the 228 hM4Di unique proteins and 2,869 human DEGs from **(F)**. Right, heat map of the 51 shared genes in human OCD and Sapap3 KO + hM4Di mice. (H) Cartoon of Gi-GPCR signaling pathways related to actin polymerization regulation. (I) List of highly expressed actin polymerization effectors in striatal astrocytes (FPKM > 10). Astrocyte specific RNA-seq was obtained from Ref . Heat map and color scale (i) shows the log2fold change at the mRNA level of the 38 effectors in human caudate of OCD subjects compared to controls. Heat map and color scale (ii) shows the mRNA abundance (FPKM) of the effectors in our astrocyte specific mouse RNA-seq datasets. Heat maps and color scale (iii) shows the log2fold change of the actin effectors in Sapap3 KO mice. Arrowheads show effectors that display similar direction changes in human and mouse and were recovered with hM4Di. (J) Dot plot shows baseline expression of striatal enriched Gαi-coupled protein receptors from mouse astrocyte data overlapped with human striatum single-nucleus RNA sequencing from Ref. (K) Cartoon: in Sapap3 KO mice, decreased astrocyte morphology leads to decreased tissue homeostasis and decreased astrocyte-neuronal contacts. In Sapap3 KO mice with astrocytic activation of hM4Di, tissue homeostasis is restored.
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