Wild-type microglia arrest pathology in a mouse model of Rett syndrome

Abstract

Rett syndrome is an X-linked autism spectrum disorder. The disease is characterized in most cases by mutation of the MECP2 gene, which encodes a methyl-CpG-binding protein. Although MECP2 is expressed in many tissues, the disease is generally attributed to a primary neuronal dysfunction. However, as shown recently, glia, specifically astrocytes, also contribute to Rett pathophysiology. Here we examine the role of another form of glia, microglia, in a murine model of Rett syndrome. Transplantation of wild-type bone marrow into irradiation-conditioned Mecp2-null hosts resulted in engraftment of brain parenchyma by bone-marrow-derived myeloid cells of microglial phenotype, and arrest of disease development. However, when cranial irradiation was blocked by lead shield, and microglial engraftment was prevented, disease was not arrested. Similarly, targeted expression of MECP2 in myeloid cells, driven by Lysm(cre) on an Mecp2-null background, markedly attenuated disease symptoms. Thus, through multiple approaches, wild-type Mecp2-expressing microglia within the context of an Mecp2-null male mouse arrested numerous facets of disease pathology: lifespan was increased, breathing patterns were normalized, apnoeas were reduced, body weight was increased to near that of wild type, and locomotor activity was improved. Mecp2(+/-) females also showed significant improvements as a result of wild-type microglial engraftment. These benefits mediated by wild-type microglia, however, were diminished when phagocytic activity was inhibited pharmacologically by using annexin V to block phosphatydilserine residues on apoptotic targets, thus preventing recognition and engulfment by tissue-resident phagocytes. These results suggest the importance of microglial phagocytic activity in Rett syndrome. Our data implicate microglia as major players in the pathophysiology of this devastating disorder, and suggest that bone marrow transplantation might offer a feasible therapeutic approach for it.

Figure 1. Wild type bone marrow transplantation at P28 arrests disease progression in Mecp2^−/y mice

a, Representative images of wild type and Mecp2^−/y littermates at P56. b, Lifespan of Mecp2^−/y mice receiving wild type bone marrow at P28 (Wild type→Mecp2^−/y; n = 15) is compared to naïve Mecp2^−/y (n = 17), Mecp2^−/y receiving Mecp2^−/y bone marrow (Mecp2^−/y→Mecp2^−/y; n = 9), and wild type mice receiving wild type bone marrow (Wild type→Wild type; n = 29) (***, p < 0.0001, Log Rank (Mantel-Cox)). c, Representative images of Wild type→Wild type as compared to Wild type→Mecp2^−/y are shown at P56 (4 weeks post bone marrow transplantation). d, Weights (mean ± s.e.m.) of Wild type→Wild type, Mecp2^−/y, Mecp2^−/y→Mecp2^−/y and Wild type→Mecp2^−/y mice (n = 15, 15, 7, 15 mice/group) are shown over time. e, Representative images of brains isolated from P56 Wild type→Wild type and Wild type→Mecp2^−/y mice transplanted at P28 and naïve Mecp2^−/y mice are presented; f, Brain weight (mean ± s.e.m.) per each group (***, p < 0.001; one-way ANOVA; n = 4 each group). g, Representative images of Nissl staining in hippocampal slices (CA1 area) of wild type and Mecp2^−/y mice are presented (bar equals 40 μm). h, Soma area (mean ± s.d.) of CA1 hippocampal cells. For each group of mice, a set of cells was created by randomly selecting 100 cells per mouse, 3 mice per group (***, p < 0.001; Two-way ANOVA with Bonferroni post-hoc test).

Figure 2. Bone marrow transplantation effects on general appearance, breathing, and locomotion of Mecp2^−/y and Mecp2^+/−mice

a, Neurological scores at P56 for Wild type→Wild type, Mecp2^−/y naïve, Mecp2^−/y→Mecp2^−/y and Wild type→Mecp2^−/y are presented. Behaviors (mean ± s.e.m.) are scored as indicated in Methods (*** p < 0.001; one-way ANOVA; n = 16, 16, 7, 16). b, On left are representative plethysmograph recordings of animals from each group. On right, expiratory time (T_E) for representative wild type, Mecp2^−/y, Mecp2^−/y→Mecp2^−/y and Wild type→Mecp2^−/y (transplantation at P28 and examination at indicated ages for all groups) as measured over a 5-minute period; T_E is normalized to mean breath duration for each mouse. c, Apneas (mean ± s.e.m.) per 30 min as measured in all four groups (***, p < 0.001; one-way ANOVA with Bonferroni post hoc test; n = 5 mice/group; for the entire figure all mice aged P56 except for Wild type→Mecp2^−/y at 12 weeks of age, i.e. 8 weeks post bone marrow transplantation). d, Interbreath irregularity (mean % ± s.e.m.) calculated as absolute [(TTOT_N – TTOT_N+1)/TTOT_N+1] for all four groups (**, p < 0.01; ***, p < 0.001; one-way ANOVA with Bonferroni post hoc test; n = 5 mice/group). e, Distance traveled (mean ± s.e.m.) in an open field (*, p < 0.05; ***, p < 0.001; one- way ANOVA, n = 5 mice/group). f, Representative traces of the path traveled by mice in an open field during 20 min test time. g–k, Mecp2^+/−mice were transplanted with wild type bone marrow at P56 and were examined for disease symptoms at 9 months of age. g, Weight (mean ± s.e.m.); h, latency to fall (mean ± s.e.m.) in the rotarod task; i, time (mean ± s.e.m.) spent in the center of the open field; j, apneas (mean ± s.e.m.) measured by whole body plethysmography in 30 min; and k, interbreath irregularity (mean % ± s.e.m.), all were improved in the treated mice as compared to non-treated controls (*, p < 0.05; ***, p < 0.001; one-way ANOVA, n = 6 mice/group; post-hoc Bonferroni test was used for individual comparisons).

Figure 3. Brain parenchymal engraftment of cells after bone marrow transplantation is required to arrest Rett syndrome

a, Representative confocal images of brain parenchyma from cerebellum of Wild type→Mecp2^−/y mice 8 weeks post transplantation (transplantation at P28), immunolabeled for CD11b and GFP (bar = 20 μm). b–e, Mecp2^−/y mice underwent bone marrow transplantation at P28 with their heads lead-protected. Mice were examined at their end-point, about 7 weeks after bone marrow transplantation. b, Representative dot plot of splenocytes obtained from bone marrow transplanted mouse with lead-protected head. c, Distribution of ‘peripheral chimerism’ in mice with lead-protected heads after bone marrow transplantation. d, Representative micrograph from mice with lead-protected heads after bone marrow transplantation, immunolabeled for GFP. Coronal cortical slice is presented showing sporadic cells found in meningeal spaces, but not in the parenchyma. e, Lifespan of Mecp2^−/y mice with wild type bone marrow transplantation with lead-covered heads compared to Wild type→Wild type lead-covered head controls (***, p < 0.0001, Log Rank (Mantel-Cox); n = 9 mice/group). f–k, Genetic approach for expressing Mecp2 protein in myeloid cells. Mecp2^lox-stop mice were bred to Lysm^Cre mice and their progeny (Mecp2^lox-stop/yLysm^Cre mice) were analyzed for disease progression. f, Representative image of mice at P56. g, Weights (mean ± s.e.m.) of mice as they progress with age. h, Lifespan for indicated groups (***, p < 0.0001, Log Rank (Mantel-Cox); n = 6 mice/group). i, Apneas (mean ± s.e.m.) measured by whole body plethysmography in 30 min for the three groups at 9 weeks. j, Interbreath irregularity (mean % ± s.e.m.) measured at 9 weeks. k, Distance traveled (mean ± s.e.m.) in an open field at 9 weeks (**, p < 0.01; ***, p < 0.001; one-way ANOVA, n = 5 mice/group; Bonferroni post hoc test was used for individual comparisons).

Figure 4. Microglial phagocytic activity is necessary for their beneficial effect in Mecp2^−/y mouse brains

a, Representative micrograph of phagocytosing microglia in orthogonal projections of confocal z-stacks. Scale bar = 25 μm. b, Wild type (i) or Mecp2^−/y (ii) microglia incubated for 2 or 5 hours with fluorescently-labeled UV-irradiated neural progenitor cells and stained with anti-CD11b. Scale bar = 50 μm. c, Bar graphs comparing surface area of UV-irradiated neural progenitor cells (NPC) to total surface area (mean± s.e.m) of wild type or Mecp2^−/y microglia are shown (**, p < 0.01; ***, p < 0.001; one-way ANOVA; representative experiment shown out of three independently performed). d, Representative micrograph of phagocytosing microglia in situ containing cleaved casapse-3 debris co-localized with lysosomal marker, Lamp-1. Scale bar = 50 μm. e–g, Mecp2^lox-stop/yLysm^Cre mice and the appropriate controls were treated with annexin V to pharmacologically inhibit phagocytic activity. e, Weights (mean± s.e.m) of Mecp2^+/yLysm^Cre, Mecp2^lox-stop/yLysm^Cre, Mecp2^lox-stop/y, Mecp2^+/yLysm^Cre treated with annexin V and Mecp2^lox-stop/yLysm^Cre treated with annexin V are shown at the end point for Mecp2^lox-stop/y and Mecp2^lox-stop/yLysm^Cre treated with annexin V groups (~P63). f, Neurological scores (mean± s.e.m) at P56 are presented (*** p < 0.001; one-way ANOVA; n = at least 7 mice/group; Bonferroni post hoc test was used for individual comparisons). g, Distance traveled (mean ± s.e.m.) in an open field by mice from all the above groups (***, p < 0.001; one-way ANOVA, n = 5 mice/group; Bonferroni post hoc test was used for individual comparisons).