PROJECT SUMMARY AND CAREER DEVELOPMENT ABSTRACT Rett syndrome (RTT) is a devastating neurodevelopmental disorder for which there are currently no viable treatment strategies. The majority of RTT cases result from loss of function mutations in a transcription factor known as methyl CpG binding protein 2 (MECP2), and knockout of Mecp2 (Mecp2-KO) results in RTT- like phenotypes and pathologies in rodents. The brains of RTT-model mice are characterized by acquired microcephaly, which results not from neural atrophy, but rather a reduction in synapses and the subsequent simplification of the neuronal arbor. As such, the preclinical treatment strategies that have garnered the most attention are those designed to normalize synaptic density, often through the replacement of late response (LR) or direct modulation of their receptors. In this capacity RTT-mouse models are valuable tools to guide therapeutic design; however, given the challenges associated with translating data from mice to humans, we have recently adopted a different strategy. Using cortical and cerebellar tissue from 8 RTT patients, we performed RNA-sequencing (seq) analysis to screen for novel points of intervention that are rooted in human patient data, and then back-modeled relevant hits in mice. Excitingly, we identified the muscarinic acetylcholine receptor 4 (M4) as a gene significantly decreased in RTT patients. This finding is salient, as M4-modulators have been shown to rescue symptom domains in diseases like schizophrenia that overlap with RTT and M4- KO mice have cognitive and social deficits similar to Mecp2-KO mice. This raises the exciting possibility that attenuated M4 expression and signaling could be underlying cognitive and social phenotypes in RTT patients. In Aim 1, we propose to further develop preliminary data indicating that cognitive and social phenotypes in Mecp2-KO mice are responsive to M4 positive allosteric modulators (PAMs). In Aim 2, we propose to conclusively link M4-signaling to RTT in patients by perform an RNA-seq analysis on autopsy samples from patients that have been clinically diagnosed with RTT, but who do not have mutations in MECP2 (MECP2- mutation negative). Finally, in Aim 3 we propose co-administer our M4-PAM with modulators of glutamatergic and GABAergic signaling, to establish whether a complimentary symptom domains can be rescued. My career goal is to become the head of a lab with a research focus on pediatric diseases of the nervous system, and I have identified four areas of insufficient training that I propose to remedy to assist in achieving long term success in that role. The four areas are: 1) Training in electrophysiology 2) Training next generation sequencing analysis 3) Clinical exposure to RTT and 4) Mentor experience. To remove these technical and conceptual barriers, I have developed a training plan that integrates formal didactic training with hands-on mentorship, with the goal of turning these weaknesses into strengths. It is anticipated that completion of the proposed project and training plan will place me in an ideal position to receive a tenure track faculty position, and as such, I believe that this application is well suited for a K01 Career Development Award.