Mecp2 knock-out astrocytes affect synaptogenesis by interleukin 6 dependent mechanisms

Abstract

Synaptic abnormalities are a hallmark of several neurological diseases, and clarification of the underlying mechanisms represents a crucial step toward the development of therapeutic strategies. Rett syndrome (RTT) is a rare neurodevelopmental disorder, mainly affecting females, caused by mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, leading to a deep derangement of synaptic connectivity. Although initial studies supported the exclusive involvement of neurons, recent data have highlighted the pivotal contribution of astrocytes in RTT pathogenesis through non-cell autonomous mechanisms. Since astrocytes regulate synapse formation and functionality by releasing multiple molecules, we investigated the influence of soluble factors secreted by Mecp2 knock-out (KO) astrocytes on synapses. We found that Mecp2 deficiency in astrocytes negatively affects their ability to support synaptogenesis by releasing synaptotoxic molecules. Notably, neuronal inputs from a dysfunctional astrocyte-neuron crosstalk lead KO astrocytes to aberrantly express IL-6, and blocking IL-6 activity prevents synaptic alterations.

Keywords: Cell biology; Immunology; Neuroscience; Omics; Transcriptomics.

Graphical abstract

Figure 1

Soluble factors secreted by Mecp2 KO astrocytes affect synaptogenesis, with cortical astrocytes showing the most detrimental effects (A) Experimental design overview. (B, D, and F) Representative images of primary branches from WT neurons (DIV14) immunostained for Synapsin1/2 (green), Shank2 (red) and their merge with MAP2 (white), in co-culture with cortical (B), hippocampal (D) and cerebellar astrocytes (F). Scale bar = 5 μm. (C, E, and G) Violin plots indicate the median (dashed line) and 25^th and 75^th percentiles (dotted lines) of Synapsin1/2, Shank2 and colocalized puncta density. Values for puncta number are expressed as percentages compared to WT-WT co-cultures (set at 100%). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by Mann-Whitney test. Analyses were performed on n > 60 neurons (in C), on n > 66 neurons (in E) and on n > 46 neurons (in G) per experimental group from N > 7 (in C and E) or N > 5 (in G) biological replicates. All data derived from at least 2 independent experiments. (H) Representative traces of miniature excitatory post-synaptic currents (mEPSCs) recorded in neurons cultured with either WT or KO astrocytes. (I) Quantitative analysis of frequency and amplitude of mEPSCs. Data are represented as mean ± SEM. ∗p < 0.05 by Student’s t test. WT n = 27; KO n = 35.

Figure 2

Mecp2 KO astrocytes affect the expression of genes involved in synaptic maturation in WT co-cultured neurons (A) Experimental groups included in RNA-seq analysis: neurons cultured alone (CTRL) (n = 5) and neurons matured in co-culture with WT astrocytes (+aWT) (n = 7) or Mecp2 KO astrocytes (+aKO) (n = 8). Replicate samples were derived from 2 independent experiments. (B) Volcano plots of DEGs with p.adj<0.05 from the different comparisons, plotted as log₂FC against -log₁₀ adjusted p value. Each dot represents a gene: top-right sector highlights genes that are significantly upregulated, top-left sector genes that are significantly downregulated, with p.adj < 0.05. (C and D) Enrichment analysis of downregulated DEGs at p.adj < 0.05 in +aKO vs. CTRL (C) and +aWT vs. CTRL (D) comparisons, showing the top 20 significant GO terms (biological processes). Size and color of dots represent number of genes associated with each term and FDR adjusted p value, respectively, according to the scale indicated in the figure. (E) Enrichment plots from preranked GSEA of the comparison +aKO vs. +aWT. Three of the top ten negatively correlated gene sets are reported, together with their normalized enrichment score (NES) and significance (FDR) (see Table S6 for complete results).

Figure 3

Inflammatory pathways are activated in WT neurons in culture with Mecp2 KO astrocytes (A and B) Metascape analyses of upregulated pathways (see Tables S7 and S8) from +aWT vs. CTRL and +aKO vs. CTRL. Top 20 most represented biological processes together with their statistical value (represented as -log10 p value) (A) and enrichment analysis of transcriptional regulators using the TRRUST database (B) are shown. (C) The histogram reports the mRNA levels of a panel of genes associated with inflammatory responses. WT neurons (DIV14) cultured with KO astrocytes (+aKO, n = 6–7) were compared to WT neurons cultured with WT astrocytes (+aWT, n = 7–8). Data are represented as mean ± SEM and expressed as percentage of +aWT. ∗p < 0.05, by Mann-Whitney test. (D and E) Western blot analysis of NF-kB p65 protein levels (D) and Irak1 (E) in WT neurons (at DIV14) cultured with Mecp2 KO or WT astrocytes (n = 10 +aWT and n = 9–10 +aKO). Data are represented as mean ± SEM and expressed as percentage of +aWT. Representative bands for p65 and Irak1 are reported above the corresponding graph. p65 and Irak1 signals were normalized using Gapdh and total protein content, respectively. Samples derived from 3 independent experiments.

Figure 4

Mecp2 KO astrocytes secrete excessive IL-6, when in culture with WT neurons (A) The graph depicts the mRNA expression of selected astrocyte genes in Mecp2 KO cortical astrocytes cultured for 14 days with WT neurons. Data are expressed as percentages of +aWT condition (n = 10) and represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by Student’s t test or Mann-Whitney test in accordance with data distribution. Samples derived from at least 3 independent experiments. (B) Cytokines’ quantification in the co-culture medium by LEGENDplex assay kit. Data are represented as mean ± SEM and expressed as percentage of +aWT condition (n = 11). ∗p < 0.05 by Student’s t test. Samples derived from at least 3 independent experiments. (C) Western blot analysis of phosphorylated Stat3 over Stat3 protein levels in WT neurons treated for 24 h with the medium derived from the co-cultures of neurons with WT (WT CCM) or KO astrocytes (KO CCM). Data are represented as mean ± SEM and expressed as percentage of +aWT (n = 9/10). Representative bands are reported. (D) Immunofluorescence staining for GFAP in acutely sorted astrocytes from P7 cortices, confirming the purity of astrocyte isolation. Scale bar = 30 μm. (E) The graph shows the mRNA expression of IL-6 in HET cortical astrocytes, compared to WT astrocytes. Data, expressed as percentage of WT samples, are reported as mean ± SEM. Samples (n = 10 WT and n = 9 HET) derived from 3 independent litters. ∗p < 0.05 by Student’s t test.

Figure 5

IL-6 secretion by Mecp2 KO astrocytes depends on neuron-derived inputs (A) The graph depicts the mRNA levels of selected astrocyte genes in Mecp2 KO cortical astrocytes cultured alone, expressed as percentages of WT astrocytes (n = 10). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 by Student’s t test or Mann-Whitney test in accordance with data distribution. (B) Western blot analysis of phosphorylated Stat3 over Stat3 protein levels in neurons exposed to WT or KO ACM. Data, represented as mean ± SEM, are expressed as percentages of neurons treated with WT ACM (n = 12); representative bands for phosphorylated and total Stat3 are reported. (C) The histograms report the IL-6 concentrations detected by ELISA assay in the medium of KO astrocytes at different days after removal of neurons (0, 4 and 8 days). Data are also compared to those analyzed in the medium of KO astrocytes in mono-culture. Data are represented as mean ± SEM. ∗p < 0.05, ∗∗ p < 0.01 by one-way ANOVA test, followed by Kruskal-Wallis test. (D) mRNA expression levels of IL-6 in WT and KO astrocytes when in culture with WT or KO neurons are reported as mean ± SEM, and expressed as percentage of WT-WT co-cultures (n = 9–11). ∗∗p < 0.01 by two-way ANOVA test, followed by Tukey’s post hoc test.

Figure 6

IL-6 released by Mecp2 KO astrocytes causes dendritic and synaptic defects in WT neurons (A) Neutralizing anti-IL6 antibody, or an isotypic antibody, was added to co-cultures and analysis of dendrites and synaptic puncta density was conducted at DIV6 and DIV14, respectively. (B) Representative images of primary branches from WT neurons (DIV14) immunostained for Synapsin1/2 (green), Shank2 (red) and their merge with MAP2 (white). Scale bar = 5 μm. (C‒E) Violin plots indicate the median (dashed line) and 25^th and 75^th percentiles (dotted lines) of Synapsin1/2 (C), Shank2 (D) and colocalized puncta number (E) in neurons co-cultured with WT (+aWT) or KO (+aKO) astrocytes seeded on transwell inserts. Values for puncta number are expressed as percentages of +aWT untreated condition (NT). ∗∗p < 0.01, ∗∗∗p < 0.001 by two-way ANOVA test followed by Tukey’s post hoc test. #p < 0.001 denotes the statistical comparison between +aWT condition treated with anti-IL-6 antibody versus untreated (NT) or IgG-treated +aWT neurons. Analyses were performed on n > 50 neurons per experimental group from N > 4 biological replicates. (F) Representative images of Map2 positive neurons (DIV6), when cultured with WT or KO astrocytes and treated with anti-IL6 antibody or left untreated (NT). Scale bar, 20 μm. (G) Violin plot depicts the total dendritic length in WT neurons cultured with WT (+aWT) or KO (+aKO) astrocytes, following treatment with anti-IL6 antibody or left untreated (NT). Analysis was performed on n > 40 neurons per experimental group from N = 5 replicates. ∗∗p < 0.01 by two-way ANOVA test, followed by Tukey’s post hoc test. (H) Recombinant IL-6 (200 pg/mL) was added to WT cortical neurons every 2 days, starting at DIV2. The arrowheads indicate the time of IL-6 treatment. (I) Representative images of primary branches from WT neurons (DIV14) immunostained for Synapsin1/2 (green), Shank2 (red) and their merge with MAP2 (white). Scale bar, 5 μm. (J‒L) Violin plots indicate the median (dashed line) and 25^th and 75^th percentiles (dotted lines) of Synapsin1/2 puncta density (J), Shank2 puncta density (K) and puncta colocalization (L) of WT neurons treated with recombinant IL-6. Data are indicated as mean ± SEM. Analyses were performed on n = 40 NT and n = 41 treated neurons from N = 4 biological replicates. ∗∗∗p < 0.001 by Student's t test.