PROJECT SUMMARY/ABSTRACT Autism spectrum and related neurodevelopmental disorders are thought to arise from widespread mild synaptic dysfunction that leads to an altered trajectory of brain network development (Johnson et al., 2015). In Rett syndrome (RTT), a debilitating neurodevelopmental disorder, loss-of-function mutations in MECP2 on the X chromosome lead to a devastating loss of language, motor and visual function at the behavioral level. Yet no therapy exists to stop or reverse the cognitive decline. Previous work in RTT mouse models reveal synaptic- and network-level defects that precede symptom onset. Mecp2-deficiency alters the timing of excitatory synaptic maturation in excitatory and inhibitory neurons in the developing cortex (Mierau et al, 2016). Microelectrode array (MEA) recordings and calcium imaging of neuronal activity in cultured murine cortical neurons reveal delays in the development of functional connectivity in the Mecp2-deficient cortical networks including smaller network size, weaker strength of connectivity and impairments in the development of network topology including features that predict local and global efficiency of the networks (Dunn et al, unpublished). These findings reveal cellular- scale deficits in information processing that likely underlie the cognitive impairment in RTT and could be targeted with novel therapies to rescue cognitive function. In my proposed K02 and subsequent R01 research, we will translate this approach to a human in vitro cellular model of RTT. In Aim 1, air-liquid interface cortical organoids (ALI-COs) will be generated from RTT patient-derived induced pluripotent stem cells (iPSCs) and allogenic controls to first identify network-level defects in the development of cortical networks. The ALI-CO model shows further cortical maturation than previous organoid models and avoids the necrotic core (Giandomenico et al., 2019). In Aim 2, we will target excitatory synaptic maturation in a specific type of inhibitory neuron in the MeCP2- deficient ALI-COs using novel cell-type specific modulators we identified (Mierau et al, unpublished). Our goal is to prevent early synaptic defects and ensure the formation of network activity and topology necessary to support cellular-scale information processing. This strategy could be used in our future R01 research to prevent the cognitive decline in RTT. In Aim 3, we will develop novel tools for detecting and modulating network dynamics in the MeCP2-deficient ALI-COs as a platform for testing strategies to rescue network function in RTT after the cognitive decline has occurred. Re-expressing Mecp2 in adult Mecp2-deficient mice rescues many behavioral and synaptic features, raising the hope that neuronal function can be rescued in RTT even after the cognitive decline. We will next test dynamic electric, optogenetic, and pharmacologic approaches for modulating cognitive function. This will support the subsequent R01 aims in Year 4 and 5 of the K02 award to...