Human microglial cells as a therapeutic target in a neurodevelopmental disease model

Abstract

Although microglia are macrophages of the central nervous system, their involvement is not limited to immune functions. The roles of microglia during development in humans remain poorly understood due to limited access to fetal tissue. To understand how microglia can impact human neurodevelopment, the methyl-CpG binding protein 2 (MECP2) gene was knocked out in human microglia-like cells (MGLs). Disruption of the MECP2 in MGLs led to transcriptional and functional perturbations, including impaired phagocytosis. The co-culture of healthy MGLs with MECP2-knockout (KO) neurons rescued synaptogenesis defects, suggesting a microglial role in synapse formation. A targeted drug screening identified ADH-503, a CD11b agonist, restored phagocytosis and synapse formation in spheroid-MGL co-cultures, significantly improved disease progression, and increased survival in MeCP2-null mice. These results unveil a MECP2-specific regulation of human microglial phagocytosis and identify a novel therapeutic treatment for MECP2-related conditions.

Keywords: ADH-503; CD11b; MECP2; chromatin; iPSC; integrin; microglia; neurodevelopment; neurons; phagocytosis; stem cells; synaptogenesis.

Graphical abstract

Figure 1

The absence of MECP2 leads to decreased cell viability and dysregulations in cell migration, integrin, and phagosome formation pathways in MGLs (A) Representative images of CTRL, KO, and KO^R MGLs stained with CD68 and MeCP2 antibodies. Scale bar 10 μm. (B) The percentage of cell viability was compared between different cell lines. Each data point shows the mean ± SEM, as indicated. Significance was tested using a two-way ANOVA with the Tukey’s multiple comparison test (^∗p = 0.0304 and ^∗p = 0.0247 for day 24 and 29, respectively) (three lines for CTRL MGLs and KO and two isogenic rescue lines for KO^R MGLs, n = 3 independent experiments for each sample at each time point). (C) Bright-field images of CTRL, KO, and KO^R MGLs at day 7 of differentiation, scale bar 200 μm. (D) Bright-field images of CTRL MGLs with (right) or without (left) masks that marked round (in green), elongated (in blue), or cell clumps (in red), scale bar 200 μm. (E–H) Percentage of round cells, elongated cells, and cell clumps, respectively. (H) Area fold change compared to the control of CTRL, KO, and KO^R. One-way ANOVA with the Tukey’s multiple comparison test was performed (^∗p = 0.0323, ^∗∗p = 0.0022, and n.s. for E to G, respectively, for ^∗p = 0.0149 and ^∗∗p = 0.0021 for H (Each bar shows the mean ± SEM, each dot represents one sample, two different isogenic pairs, two different clones for CTRL with three independent experiments per sample). (I) Heatmap showing the 39 differentially expressed genes (greater than 1.25-fold) between these two groups (KO n = 5 from two independent cell lines, two to three biological replicates, CTRL n = 5 from two independent cell lines, two to three biological replicates and KO^Rn = 3 from two independent cell lines, one to two independent experiments p < 0.05, data were analyzed for statistical significance using Rosalind onramp software, see supplemental information, methods' section). Increasing fold changes compared to CTRL are marked in red while decreasing ones are in blue. (J) Top canonical pathways were obtained with 39 differentially expressed genes (DEGs) using Ingenuity Pathway Analysis (IPA), where significance was calculated by the right-tailed Fisher’s exact test (p < 0.05). (K) The top network involving the majority of DEG obtained through IPA, genes that are downregulated are in green and upregulated are in red. (L) Top 5 diseases and biological functions were obtained with IPA.

Figure 2

MeCP2 chromatin immunoprecipitation sequencing and proteomic assays on MGL (A) Explanatory schematic of CTRL, KO^R, and KO MGLs regarding the role of MeCP2. (B) Venn diagram of MeCP2 occupation peaks between CTRL and KO^R. (C) Annotation distribution of MeCP2 target peaks. (D) Top canonical pathways for the promoter-TSS region obtained using IPA, where significance was calculated by the right-tailed Fisher’s exact test (p < 0.05). (E) A table highlighting several MECP2 targets of interest in the genome. (F and G) Top 5 known and de novo motifs obtained with MeCP2 ChIP-seq in MGLs. (H) LC-MS proteomic analyses conducted on MGL shows 480 overlapping candidates with MeCP2 ChIP-seq in the promoter region. (I) Top canonical pathways of the intersecting candidates between proteomics and ChIP-seq obtained through IPA (p < 0.05).

Figure 3

KO MGLs have increased glutamate, and ROS release decreased phagocytosis but the globally similar inflammatory response to LPS as CTRL and KO^R MGL (A) Glutamate release by MGLs. Bars represent mean ± SEM. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗p = 0.0329, two different KO lines, one rescue line and two independent CTRL MGL lines with two clones). (B) Measurement of reactive oxygen species (ROS) released in the conditioned media by MGLs. One-way ANOVA with Tukey’s multiple comparison test was used to assess the significance (^∗∗∗∗p < 0.0001). Bars represent mean ± SEM. Two independent cell lines for CTRL and KO MGLs and one isogenic rescue line with three independent experiments per sample were used. (C) Migration assays using transwell chambers using ATP or CXC3L1. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗p = 0.0261), two independent cell lines for CTRL and KO MGLs, and one isogenic rescue line with three independent experiments per sample. (D) Heatmap showing the means for each cytokine released in the conditioned media for a given genotype (blue to red, low to high concentration in the media in pg/mL), three independent CTRL and KO MGL lines and two isogenic rescue lines were used. (E and F) MIP-1 alpha and GM-CSF released in the conditioned media by MGLs was measured, respectively (in pg/mL). Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗∗∗∗p < 0.0001 and ^∗p = 0.0179, three independent CTRL and KO MGL lines and two isogenic rescue lines were used). Bars represent mean ± SD. (G) Bright-field images of CTRL, KO, and KO^R MGLs engulfing zymosan particles. Once engulfed, the zymosan particles fluoresce in red, scale bar 200 μm. Blue is a live nuclear stain (Nucleo Blue). (H) Phagocytosis percentage of zymosan particles compared to CTRL MGLs. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗∗∗∗p < 0.0001 and ^∗p = 0.0289, two different isogenic KO/KO^R pairs, at least four biological replicates each, two independent CTRL MGL lines with three clones). Bars represent mean ± SEM. Each dot represents one sample. (I) Phagocytosis of pHrodo-conjugated brain organoid-derived synaptosomes-enriched fractions measured as red fluorescent area normalized to hour = 0 and to CTRL baseline phagocytosis using incucyte. Significance was tested by one-way ANOVA (^∗p < 0.001) using one control and one KO isogenic pair with 5 independent experiments each. Bars represent mean ± SEM. (^∗p = 0.0184 and ^∗p = 0.0269, respectively).

Figure 4

KO^R or CTRL MGL rescues the synaptic defects of KO neurons in long-term co-culture experiments (A) Bright-field images of neuron-MGL (labeled with long-term stable membrane stain PKH26 in red) co-culture on day 2 and 50 of neuronal differentiation, scale bar 400 μm. (B) Representative images of co-culture neurons (stained with MAP2 and HOMER1) and MGLs (stained with CD68) for 2 months. The last panel shows a magnification of the square area shown. Note that MGL is nearby of MAP2⁺ neurons and synapses, scale bar 50 μm. (C) Representative images of synaptic puncta co-localization with or without CTRL, KO, or KO^R MGLs, scale bar 20 μm. (D) Quantification of the number of synaptic puncta (KO vs. CTRL neurons without MGL ^∗∗p = 0.0013; KO neurons without MGL vs. KO neurons with CTRL MGLs ^∗∗p = 0.0023; KO neurons with CTRL MGLs vs. KO neurons with KO MGLs ^∗∗∗p = 0.0007, one isogenic KO/KO^R pair [KO^R results shown in Figure.S5d], and one CTRL MGL line was used [related to KO], synaptic puncta from 10 neurons were counted, the experiment was run in two independent batches). (E) Quantification of the number of co-localized synaptic puncta (CM, conditioned media from CTRL or KO MGLs) (^∗p = 0.0482 one isogenic KO/KO^R pair and one CTRL MGLs line was used, synaptic puncta from 10 neurons were counted). (F) Quantification of the number of co-localized synaptic puncta without healthy human primary fibroblasts (Fibro), (^∗∗p = 0.0084, one isogenic KO/KO^R pair, and one CTRL MGLs line was used, synaptic puncta from 10 to 15 neurons were counted). (G) Bright-field images of spheroids co-cultured with MGL (in red) before and after plating on MEA plates, scale bar 400 μm. (H) Graph showing the spike rate compared to CTRL spheroids without any MGLs, recorded in 5 min emerging from spheroids with or without CTRL or KO^R MGLs. ^∗∗p = 0.0021, ^∗p = 0.0150, n.s. not significant, one isogenic rescue line and two different CTRL and KO lines were used, two to three independent experiments per genotype were used. Significance is assessed by one-way ANOVA with Tukey’s multiple comparison test for the experiments in D, E, F, and H (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001). The number of neurons counted for synaptic puncta is represented by one data point in each graph. All synaptic puncta quantification was calculated as a percentage compared to CTRL neurons. Bars represent mean ± SEM.

Figure 5

ADH-503 rescues phagocytosis in KO MGLs and synaptic defects in KO spheroids and improves disease score and survival in mouse (A) Schematic of the compounds used in the screening; agonist compounds are in red and inhibitors in blue. (B) Drug screening using phagocytosis assay. Phagocytosis percentage of zymosan particles compared to CTRL MGLs. Significance was tested by two-way ANOVA (^∗∗∗∗p < 0.001, ^∗p = 0.0109, ^∗∗p = 0.0041), experiment run in two batches, n = 12 to 24 biological replicates. Bars represent mean ± SEM. (C) MECP2 binding events to ITGAM (CD11b) promoter, baseline binding events detected (untreated) or at the promoter region of ITGAM. Significance was tested by two-way ANOVA followed by Sidak multiple comparison test (^∗∗∗p = 0.0004, ^∗p = 0.0236, CTRL vs. KO ITGAM -190 bp ^∗∗p = 0.0013, untreated KO^R vs. ITGAM -190 bp ^∗∗p = 0.0019), n = 3 independent experiments. Bars represent mean ± SEM. (D) Peaks detected in the promoter region of ITGAM in CTRL and KO^R MGLs by MECP2 ChIP-seq. (E) Quantification of colocalized synaptic (VGLUT/HOMER1) puncta in KO spheroids co-cultured with KO or CTRL MGLs and treated with 1 μM of ADH-503 using Imaris software. The dotted line indicates the number of colocalized synaptic puncta in CTRL spheroids co-cultured with CTRL MGLs. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (KO no MGL vs. KO + CTRL MGLs ^∗p = 0.0163, KO no MGL vs. KO + KO MGLs+ ADH-503 ^∗p = 0.0109, KO + KO MGLs vs. KO + CTRL MGLs ^∗p = 0.0344, KO + KO MGLs vs. KO + KO + ADH-503 ^∗p = 0.0236, KO + CTRL MGLs vs. KO + KO MGLs+ ADH-503, and KO no MGL vs. KO + KO MGLs were non-significant). (F) Schematic of ADH-503 treatment of MeCP2-KO male mice. (G) Average total symptom score was calculated in each group at indicated time points. MeCP2-KO mice treated with ADH-503 showed a significant reduction in total score compared with vehicle-treated Mecp2-KO mice. N = KO-vehicle: 11, KO-ADH-503: 10. Significance was tested with Student’s t test, week 8; ^∗∗∗p = 0.0004, week 9, ^∗∗∗p = 0.0007. (H) Kaplan-Meier survival curves. MeCP2-KO mice treated with ADH-503 survived significantly longer than non-treated control MeCP2-KO mice (^∗p = 0.0244; log rank test), with median survival of 63.5 days (for control mice) and 91 days (for ADH-503-treated mice). N = KO: 20, KO-ADH-503: 10. (I) Cryosections of WT or MeCP2-KO mouse brains treated with vehicle or ADH-503 stained with NeuN (scale bar 100 μm, on the left) and measurement of soma size was plotted on a bar graph. Significance was tested by one-way ANOVA with Tukey’s multiple comparison test (^∗∗p = 0.0078, ^∗∗∗p = 0.0004), n = 6 mice/group.