Neuroimmune interactions and epigenetic mechanisms act at the interface of genetic and environmental risk factors that determine the severity and progression of both common and rare brain disorders. A prime example of a complex monogenic disease is Rett syndrome, an X-linked dominant neurodevelopmental disorder caused by mutations in MECP2. Rett syndrome is epigenetic at two levels: first in the regulation of MECP2 by X chromosome inactivation, and second because MECP2 encodes a known epigenetic regulator, methyl CpG binding protein 2. Girls with Rett syndrome are heterozygous for MECP2 mutations that are primarily germ-line paternal de novo events. Rett babies are born apparently normal, and then experience a regression in cognitive and motor functions in late infancy. Mouse models of Rett syndrome also recapitulate the delay in the onset of detectable neurological symptoms and motor deficits. While the MeCP2 protein is most highly expressed in neurons, both human Rett patients and mouse models exhibit system-wide immune, mitochondrial, and metabolic manifestations that are likely secondary to the causal mutation’s disruption of neuronal homeostasis. What is lacking in the Rett field is a temporal understanding of how the molecular signatures of disease progression in distinct cell types within the brain interact with the immune system’s responses inside and outside of the brain. We propose to investigate the molecular signatures of critical time points of neuroimmune pathogenesis in a novel Rett syndrome mouse model based on a human mutation. Epigenomic investigation of specific cell types in cortex, including microglia, excitatory neurons, and inhibitory neurons will be integrated with 1) single cell transcriptomics from hippocampus and hypothalamus, 2) measurements of immune dysfunction, and 3) metabolite and gut microbiota profiles. The objective of the first aim will be to characterize the time course of neuroimmune interactions in the context of symptom progression in Rett syndrome. Results from the first aim will reveal the molecular dynamics of how immune responses exacerbate neuronal dysfunction and vice versa. In the second aim, we propose to modulate the microglia prior to the onset of disease progression to directly test the role of microglia in the timing and severity of symptoms in this Rett mouse model. LPS injections in pre-symptomatic mice will be performed to activate microglia so as to test the hypothesis that microglia activated by a “second hit” will increase the severity and speed of onset of neurologic and motor symptoms in the Rett syndrome model. As a reciprocal experiment, microglia will be depleted in adolescent mice using the drug PLX5622 and either allowed to replenish after short term drug treatment or continuously depleted through adulthood to test the hypothesis that microglia are critical mediators of symptom progression. From these experiments, we expect to obtain an integrated molecular time course of events ex...