Project Summary/Abstract Rett syndrome (RTT), a devastating neurodevelopmental disorder, is largely caused by loss-of-function mutations in the X-linked gene encoding the epigenetic regulator MeCP2. Our group was the first to show a detrimental role of microglial abnormalities in a MECP2 knockout (MECP2-KO) mouse model of RTT. Recent in vivo data suggest that correcting microglial abnormalities is sufficient to rectify major RTT-like symptoms in this model. Therefore, how MeCP2 deficiency causes microglial abnormalities and how the functional states of MeCP2-deficient microglia (RTT-MG) influence RTT pathology is a critical question. Recently we found that MeCP2 deficiency in RTT-MG resulted in enhanced mitochondrial reactive oxygen species (mtROS) production, which we hypothesize would lead to metabolic derangements and abnormal microglial functions. Interestingly, in vitro studies showed that several key RTT-MG abnormalities were rescued by mitochondria- targeted transgene mCAT (the antioxidant enzyme catalase being expressed only in mitochondria, which usually do not harbor catalase) and two FDA-approved mitoactive Nrf2 activators, opening a possibility for intervention. Our preliminary data further showed that global expression of mCAT (GL-mCAT) prolonged the lifespan and improved motor and respiratory functions of the MECP2-KO mice, supporting the promise of our approach in a whole animal setting. Here we propose to extend this line of research to gain a deeper understanding of how MeCP2 is related to functional and pathological states of microglia, and to generate preclinical proof of principle that is instrumental for developing novel therapies for RTT: Aim 1: Determine the pathological role of microglial mtROS in vivo: Several lines of evidence support that microglia abnormalities drive the progression of RTT. We have shown that global expression of mCAT ameliorates the RTT-like phenotype in MECP2-KO mice. Based on this encouraging result, we further hypothesize that quenching mtROS selectively in microglia to rectify microglial abnormalities will also ameliorate RTT-like deficits in MECP2-KO mice. For this aim, we will generate and analyze the phenotype and microglial pathology of MECP2-KO/MG-mCAT mice with microglia-targeted expression of mCAT. The result would support the role of microglial mtROS in the pathogenesis of RTT. Aim 2: Determine the role of mtROS in pathological characteristics and functional responsiveness of RTT microglia: Our previous data, mostly in vitro work, support the hypothesis that mtROS-related metabolic/molecular derangements lead to RTT-MG abnormalities including the loss of the “metabolic flexibility” required to drive their functional responsiveness. Now in this aim we will test this hypothesis in vivo, mainly by analyzing microglia acutely isolated from MECP2-KO and MECP2-KO/mCAT mice. We will determine if mCAT, expressed in vivo, is able to (a) rectify the metabolic/molecular derangements of RTT-MG, and (...