Neonatal microglia replacement in mice modulates seizure severity in adulthood

Abstract

Microglia replacement therapy, where endogenous brain macrophages are depleted and replaced by adoptively transferred surrogates, holds promise for treating pediatric neurologic diseases, but little is known about how early-life microglia replacement impacts the brain. We sought to investigate how early postnatal microglia depletion and adoptive macrophage transfer, essential components of microglia replacement, durably impact neural circuits in a mouse model. Using both pharmacologic and genetic models, postnatal microglia depletion worsened adult seizure severity, mortality, and neuropathology in a chemical seizure model. Replacement of endogenous microglia by adoptive transfer of monocytes rescued this effect, while transfer of authentic microglia from a donor mouse did not, and even worsened seizure phenotypes. RNA sequencing of transplanted microglia, monocyte-derived surrogates, and endogenous microglia revealed distinct state changes across groups in response to chemically induced seizures, demonstrating that both ontogeny and adoptive transfer significantly impact resident macrophage responses to the excitotoxic brain environment. In sum, we established models for neonatal microglia depletion and replacement, then applied them to identify durable impacts of depletion and reconstitution on the brain environment. We ultimately identified differential responses of macrophages to excitotoxic challenge based on their ontogeny, underscoring focus areas for ongoing preclinical development of microglia replacement therapies.

Keywords: cell therapies; leukodystrophies; microglia; microglia replacement; neuroinflammation; seizure.

Graphical abstract

Figure 1

Early postnatal treatment with PLX3397 increases seizure susceptibility in adulthood (A) Schematic of PLX3397 treatment paradigm. C57Bl/6 mice were given daily injections with PLX3397 from P1 to P7. Endogenous microglia were then allowed to repopulate 2–3 months before seizure induction. (B) Racine scores indicating seizure severity over time in male mice. No PLX, control C57Bl/6 mice; PLX Repop, C57Bl/6 mice with postnatal depletion and repopulation of microglia. No PLX n = 6, PLX Repop n = 9, data pooled from two independent experiments. (C) Seizure severity scores were determined as the total Racine score over the course of the experiment. No PLX n = 6, PLX Repop n = 9, unpaired Student’s t test. (D) Percent of male mice that died during status epilepticus in No PLX and PLX-repopulated groups. No PLX n = 6, PLX Repop n = 9. (E) Time to peak seizure severity in male mice. No PLX n = 6, PLX Repop n = 9, unpaired Student’s t test. (F) Representative traces showing fEPSP (local field potential) from the CA1 region of the hippocampus in No PLX (left, black) and PLX-repopulated (right, purple) male mice. Dotted line indicates the increased amplitude observed in PLX-repopulated mice. (G) Input-output curve showing increased slopes at higher stimulation in male PLX-repopulated animals. No PLX n = 8, PLX Repop n = 9, data combined from three independent cohorts, repeated measures ANOVA (with Bonferroni correction for multiple comparisons). All data represented as mean ± SEM. ns, not significant; ∗p < 0.05, ∗∗p < 0.01.

Figure 2

Neonatal treatment with PLX3397 leads to increased gliosis and neuropathology following seizures in adulthood (A) Representative IBA1 staining (magenta) showing morphology of untreated or PLX-repopulated microglia before seizures (baseline) or 5 days post-seizure (5dps) in the hippocampus (top row) and cortex (bottom row). Representative of n = 3 (No PLX-baseline), n = 4 (No PLX-5dps), n = 3 (PLX Repop-baseline), and n = 6 (PLX Repop-5dps), two independent experiments. Scale bar, 50 μm. (B) Microglia morphology typing before and after seizures. Microglia were assigned one of four morphology types: baseline/homeostatic with highly complex processes (type 0), short, thick, less complex processes (type 1), very large soma with many short, “fluffy”-appearing processes (type 2), and rounded shape with few to no processes (type 3). The percentage of cells of each type is shown before seizures (top) and after seizures (bottom) for No PLX and PLX Repop groups. No PLX (baseline) n = 2, PLX Repop (baseline) n = 3, No PLX (5dps) n = 4, PLX Repop (5dps) n = 5, data pooled from two independent experiments. (C) Representative astrocyte GFAP expression (white) in the cortex of mice with or without postnatal PLX3397 treatment before seizures (baseline) or 5dps. Scale bar, 50 μm. (D) Quantification of area covered by GFAP-expressing astrocytes. Ctx, cortex; Hipp, hippocampus; Stria, striatum; Thal, thalamus. No PLX-baseline n = 4, PLX Repop-baseline n = 4, No PLX-5dps n = 6, PLX Repop-5dps n = 9, data pooled from two independent experiments, one-way ANOVA with Tukey’s multiple comparisons. (E) Representative Fluorojade C staining (green) indicating neurodegeneration in the hippocampus, striatum, and thalamus of untreated mice (top) and PLX-repopulated mice (bottom) 5dps. Scale bar, 100 μm. (F) Quantification of Fluorojade C+ cells in the hippocampus, striatum, and thalamus. No PLX n = 5, PLX Repop n = 6, data pooled from two independent experiments, unpaired Student’s t test. All data represented as mean ± SEM. ns, not significant; ∗p < 0.05, ∗∗p < 0.01.

Figure 3

Postnatal repopulation with em-microglia, but not mo-microglia, results in worsened seizure outcomes (A) Schematic of microglia depletion and transplantation paradigm. Cx3cr1-CreER; Csf1r fl/fl mice were treated with tamoxifen on P1 and P2 to deplete endogenous microglia. On P3, donor GFP+ microglia (em-MG) or monocytes (mo-MG) were transplanted and allowed to engraft in the brain for 2–3 months. Some mice received a sham PBS injection that allowed endogenous microglia to repopulate the brain (Tam Repop). At age 2–3 months, kainic acid was used to induce seizures. (B) Representative dot renderings of engraftment levels for transplanted microglia (em-MG, top) and transplanted monocytes (mo-MG, bottom) at age 3 months. Representative of n = 9 (em-MG) and n = 13 (mo-MG) animals per group, three independent experiments (em-MG) and four independent experiments (mo-MG). (C) Racine scores indicating seizure severity over time in male mice. em-MG, transplanted microglia; mo-MG, transplanted monocytes; Endog-MG, unmanipulated endogenous microglia; Tam Repop = tamoxifen + sham injection. em-MG n = 19, mo-MG n = 16, Endog-MG n = 10, Tam Repop n = 12, data pooled from seven independent experiments. (D) Total seizure severity scores in male mice. Dotted line represents average seizure severity scores for PLX-repopulated male mice. em-MG n = 19, mo-MG n = 16, Endog-MG n = 10, Tam Repop n = 12, one-way ANOVA with Tukey’s multiple comparisons. (E) Percent of male mice in each group that died during status epilepticus. em-MG n = 19, mo-MG n = 16, Endog-MG n = 10, Tam Repop n = 12. (F) Time to peak seizure severity in male mice. em-MG n = 19, mo-MG n = 16, Endog-MG n = 10, Tam Repop n = 12, one-way ANOVA with Tukey’s multiple comparisons. All data represented as mean ± SEM. ns, not significant; ∗p < 0.05, ∗∗p < 0.01.

Figure 4

Mice with transplanted em-microglia or tamoxifen-repopulated em-microglia have more neuropathology following seizures compared with mo-MG-repopulated mice (A) Representative IBA1 staining (magenta) showing morphology of transplanted yolk sac-derived microglia (em-MG), transplanted monocyte-derived microglia (mo-MG), unmanipulated endogenous microglia (Endog-MG), and tamoxifen-repopulated microglia (Tam Repop) at baseline (top row) and 5 days post-seizure (5dps) (bottom row) in the hippocampus of male mice. Representative of n = 4 (em-MG-baseline), n = 4 (mo-MG-baseline), n = 4 (Endog-MG-baseline), n = 4 (Tam Repop-baseline), n = 6 (em-MG-5dps), n = 5 (mo-MG-5dps), n = 5 (Endog-MG-5dps), n = 5 (Tam Repop-5dps), three independent experiments (baseline) and four independent experiments (5dps). Scale bar, 50 μm. (B) Macrophage morphology typing before and after seizures. em-MG, Endog-MG, and Tam Repop microglia were assigned one of four morphology types as described in Figure 2. mo-MG were typed as follows: baseline/homeostatic defined as mo-microglia with fewer than five primary processes (type 0), five to seven primary processes with minimal secondary branching (type 1), and greater than eight primary processes with extensive secondary branching (type 2). The percentage of cells of each type is shown before seizures (top) and after seizures (bottom) for all groups. em-MG (baseline) n = 3, mo-MG (baseline) n = 3, Endog-MG (baseline) n = 2, Tam Repop (baseline) n = 3, em-MG (5dps) n = 7, mo-MG (5dps) n = 5, Endog-MG (5dps) n = 5, Tam Repop (5dps) n = 5, data pooled from animals from three independent experiments (baseline) and five independent experiments (5dps). (C) Representative astrocyte GFAP expression (white) in the hippocampus of male mice with em-MG, mo-MG, Endog-MG, and Tam Repop microglia 5dps. Scale bar, 150 μm. (D) Quantification of area covered by GFAP-expressing astrocytes in males. Hipp, hippocampus; Stria, striatum; Ctx, cortex; Thal, thalamus. em-MG n = 8, mo-MG n = 6, Endog-MG n = 6, Tam Repop n = 4, data pooled from four independent experiments, one-way ANOVA with Tukey’s multiple comparisons. (E) Representative Fluorojade C staining (green) indicating neurodegeneration in the hippocampus of mice 5dps. Scale bar, 150 μm. (F) Quantification of Fluorojade C+ cells in the hippocampus and striatum of male mice. em-MG n = 7, mo-MG n = 5, Endog-MG n = 5, Tam Repop n = 5, data pooled from two independent experiments, one-way ANOVA with Tukey’s multiple comparisons. All data represented as mean ± SEM. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

Ontogeny and transplantation modulate microglial responses to excitotoxic challenge (A) Total number of upregulated genes (red) and downregulated genes (blue) in KA-challenged as compared with saline populations. Total number of DEGs for each comparison is indicated above each bar. em-MG, transplanted microglia; mo-MG, transplanted monocytes; Endog-MG, unmanipulated endogenous microglia (cutoffs used: adjusted p < 0.05, logFC > 1 or <−1, and average TPM > 100 in at least one of the comparison groups). (B) PCA plot showing em-MG, mo-MG, and Endog-MG 5 days post-seizure. (C) Total number of DEGs (solid bars) and DEGs exclusive to post-seizure populations (dotted bars) between cell types 5 days post-seizure (cutoffs used as above). (D) Selected seizure-exclusive GO terms enriched in mo-MG as compared with em-MG after seizures. (E) Volcano plot showing DEGs in mo-MG versus em-MG after seizures. Dotted lines indicate cutoffs at log fold change equal to 1 and adjusted p value less than 0.05. (F) Gene expression values for selected DEGs in post-seizure mo-MG and em-MG. (G) Selected seizure-exclusive GO terms enriched in em-MG as compared with Endog-MG after seizures. (H) Volcano plot showing DEGs in em-MG versus Endog-MG after seizures. Dotted lines indicate cutoffs at log fold change equal to 1 and adjusted p value less than 0.05. (I) Gene expression values for selected DEGs in post-seizure em-MG and Endog-MG. For all RNA sequencing analysis, n = 2 (Endog-MG-saline), n = 3 (em-MG-saline), n = 3 (mo-MG-saline), n = 3 (Endog-MG-seizure), n = 4 (em-MG-seizure), n = 6 (mo-MG-seizure), data pooled from two independent experiments.