Wild-type microglia do not reverse pathology in mouse models of Rett syndrome.

We first sought to replicate BMT-mediated rescue of male mice derived from the same Mecp2tm1.1Jae/y colony from the original report (4), implementing established standards for conducting preclinical studies (2,6). Mice were maintained on C57Bl/6J background, which was confirmed in recipient animals by genome scanning (data available upon request). Four week-old Mecp2tm1.1Jae/y mice and wild type littermates were subjected to the same protocol of lethal split-dose γ-irradiation and randomized to receive tail vein injection of bone marrow from Mecp2-deficient male littermates or bone marrow from Mecp2-proficient animals including C57Bl/6J male mice ubiquitously expressing GFP and Mecp2+/y littermates of the recipients. All animals achieved multilineage peripheral blood engraftment judged by the fraction of donor-derived GFP-expressing cells in peripheral blood 4 and 8 weeks post-transplant (Extended Data Figure 1a). PCR analysis of blod and tail tissue 4 weeks after transplant also confirmed expression of the appropriate mutant or WT variant of Mecp2 in blood in all groups (Extended Data Figure 1b). Microglial engaftment in brain parenchyma, 30 and 90 days post-transplant was similar in mutant and WT recipients engrafted with marrow from WT mice ubiquitously expressing a GFP transgene, (Fig. 1, A and B, and Extended Data Figure 1c), and comparable to engraftment observed by Derecki et al. (4) and others (7). Figure 1 Early transplantation of wild-type microglia into the brain does not rescue Mecp2-null mice Contrary to our expectation, Mecp2tm1.1Jae/y mice that received Mecp2+/y marrow had no extension of lifespan compared to Mecp2tm1.1Jae/y marrow recipients (Fig. 1C). No difference in survival was observed in mutant animals that received Mecp2+/y marrow from WT littermates or C57Bl/6J animals ubiquitously expressing GFP (Extended Data Figure 1d). We also observed no benefit in outcome measures at 12 weeks of age, 8 weeks after transplant, including weight, breathing, locomotion, general condition, walking gait, tremor, hindlimb clasping or neurological score (Figure 1i). Thus, the same BMT procedure with substantially greater numbers of animals,randomly assigned to treatment group, from the same Mecp2tm1.1Jae/y mouse colony did not replicate any aspects of protection reported by Derecki et al (4). Furthermore,histologic analysis blind to genotype and treatment group showed no neuropathologic evidence of differential apoptosis, microglial response, or tissue degeneration between experimental groups (Extended Data Figure 1e). No protective effect on survival was noted in two additional mouse models of Rett syndrome as well (Figure 1, e and g): Mecp2LucHyg/y mice (Extended Data Figure 2), and Mecp2R168X/y mice (8), despite excellent engraftment after BMT (Extended Data Figure 2). Experiments with these two models were performed in independent laboratories following the same BMT protocol (4). In all models, WT mice transplanted with WT bone marrow showed no mortality, indicating the procedure was well tolerated (Figure 1, c, e, and g). Likewise, BMT was well-tolerated by mutant animals, as Mecp2 mutant animals receiving mutant marrow exhibited either no change (Mecp2LucHyg/y and Mecp2R168X/y mice), or, surprisingly, slightly reduced mortality (Mecp2tm1.1Jae/y mice) compared to naive mice not subjected to BMT (Figure 1, d, f and h). The small survival extension may be related to a salutary effect of post-irradiation antibiotic treatment of transplanted animals, to which naive animals were not exposed, or to differences in animal handling (9). To further address the role for microglia in RTT reported by Derecki et al (4), we used the Cre/lox system and a lox-stop-lox allele of Mecp2 (Mecp2LSL) to examine the effect of genetically-driven expression of Mecp2 in microglia during development. First, we analyzed the suitability of the LysM-Cre transgene, which was used by Derecki et al (4) in their genetic Mecp2LSL/y rescue experiments (4), to drive efficient microglia-specific gene restoration. As previously reported (10), LysM-Cre driven dTomato reporter cells account for less than 25% of microglia, as assessed using flow cytometry of microglia derived from mice containing the LysM-Cre transgene and a transgene expressing Cre-dependent dTomato (Extended Data Figure 3a). Furthermore, when we generated LysM-Cre; Mecp2LSL/Y mice, we observed MeCP2 expression in neurons (large NeuN+ cells) in many brain regions (Extended Data Figure 3b). To identify a Cre transgenic line that drives efficient expression within microglia, we next evaluated Vav1-Cre transgene, which selectively expresses throughout the hematopoietic compartment (11). In contrast to LysM-Cre, Vav1-Cre transgene targeted microglia with high efficiency (Figure 2a) and specificity (Figure 2b). As Vav1-Cre-driven expression in brain proved to be efficient and restricted to microglia, we applied this system to test whether expression of Mecp2 in microglia rescues Mecp2-null mice. To quantify Mecp2 restoration in microglia, we utilized the fms-GFP transgene, expression of which within brain is restricted to microglia, for flow sorting (11) (Extended Data Figure 3c). Microglia derived from Vav1-Cre; Mecp2LSL/Y animals expressed Mecp2 mRNA at 75% of the level of Mecp2 mRNA in microglia derived from Mecp2+/Y animals (Figure 2c). Similar to other Mecp2-null mouse models, Mecp2LSL/Y animals showed hypoactivity, poor motor coordination on parallel rod walking, increased basal and hypoxia breathing rate, increased apneas, and early death, none of which were improved by Mecp2 expression in microglia of Vav1-Cre; Mecp2LSL/Y animals (Figure 2, d–h). We thus conclude that, in contrast to the data reported by Derecki et al. (4), driving Mecp2 expression developmentally in microglia does not ameliorate the phenotype of MeCP2-null mice. Figure 2 Genetic reconstitution of Mecp2 in microglia does not rescue Mecp2-null mice In conclusion, we observe no benefit of BMT-mediated delivery of WT microglia into the brains of three different preclinical models of RTT, nor do we observe a causative role of microglia in the disease process. Our BMT studies included large numbers of mice derived from the same parent colony used in the original report (4), with treatment assigned randomly and analysis conducted blind to genotype and treatment group. Finally, we showed that even early and highly efficient genetically-driven Mecp2 expression in microglia of Mecp2-null mice conferred no protective effect. Restoration of MeCP2 in microglia using bone marrow transplantation or genetics does not rescue the major observed phenotypes in RTT, which argues against the previously proposed therapeutic potential of BMT in patients with RTT (4).