PROJECT SUMMARY Deficits in neuronal development are observed in multiple neurodevelopmental disorders (NDs), and while research on NDs has historically focused on intrinsic changes within neurons, recent work has identified that alterations to astrocytes also occur and have functional impact. Unbiased proteomic approaches have identified a dysregulation in astrocyte protein secretion occurs in astrocytes isolated and cultured from Rett Syndrome (RTT), Down Syndrome (DS), and Fragile X Syndrome (FXS) model mice when compared to wild-type (WT), with strong overlap across disorders in protein secretion alterations, suggesting convergent dysregulation. This proposal focuses on altered protein secretion from astrocytes in Rett Syndrome, a disorder caused by a loss-of- function mutation of the methyl-CpG binding protein 2 (Mecp2) gene. In mouse models of RTT astrocyte-specific restoration of Mecp2 improves key phenotypes including altered neuronal morphology and motor behavior, showing that correcting astrocyte dysfunction is beneficial. The goal of the current proposal is to determine if correcting altered release of specific astrocyte-secreted proteins that impact neuronal development is sufficient to ameliorate RTT phenotypes, with a focus on the class 3 semaphorin, Sema3c, which shows increased release from astrocytes in all three NDs. Class 3 semaphorins regulate axon guidance, dendritic spines, and synapse formation, leading to the hypothesis that increased Sema3c causes dysregulation of these developmental process in RTT. Preliminary experiments demonstrate RTT mice with reduced levels of astrocyte Sema3c show phenotypic improvement including increased body weight and corrected anxiety-like behavior. Aim 1 addresses how this occurs on a cellular level by asking if Sema3c reduction impacts neuronal morphology, synaptic balance, and synaptic function. In vitro experiments show that increasing the level of Sema3c is sufficient to inhibit neurite outgrowth, suggesting upregulation of Sema3c contributes to reduced dendritic arbor size and spine density that are observed in RTT. To identify how Sema3c signals to neurons to inhibit their development in RTT, proximity labeling with TurboID is used to identify the in vivo Sema3c protein interactome, analyzed by mass spectrometry. This Aim will provide mechanistic insight into how increased Sema3c in RTT drives neurodevelopmental dysregulation, and how reducing Sema3c may improve RTT phenotypes. Prior work that identified altered protein secretion from astrocytes in RTT studied early postnatal astrocytes in isolation from other cell types. To ask how astrocyte-neuron interaction is altered in RTT in vivo at later ages when phenotypes emerge, Aim 2 uses TurboID proximity labeling to identify the secreted and plasma membrane proteins of astrocytes and neurons in the cortex of RTT and WT mice. These datasets will identify further potential therapeutic targets for neurodevelopmental disorders. Together these...