Mechanisms of spine and synaptic remodeling in the prefrontal cortex (PFC) underlie critical functions of working memory, cognitive flexibility, and sociability yet are incompletely understood. Impaired spine elimina- tion during brain development likely contributes to increased spine and synapse density in autism spectrum disorders (ASD). We identified a novel mechanism of selective spine pruning in which Semaphorin-3 receptor complexes, comprising L1 adhesion molecules, Neuropilins, and PlexinAs, promote activity-dependent spine elimination in the juvenile brain through Rho family GTPases. All L1-CAMs bind Ankyrin B (AnkB), an actin- spectrin adaptor encoded by the high confidence ASD gene Ank2. A role for AnkB in spine regulation has not been explored, because mouse models with germline deletion of the Ank2 gene are lethal. We generated a novel, inducible Ank2 mouse line (Nex1Cre-ERT2:Ank2flox:EGFPflox) in which Ank2 can be deleted in postmitot- ic pyramidal neurons (PNs) during postnatal development, circumventing lethality. The AnkB conditional mu- tant resembles multiple human de novo ASD variants lacking both principal AnkB isoforms (220 and 440 kDa). Preliminary results reveal elevated spine density and altered excitatory neurotransmission in AnkB mutant PFC. We will investigate the hypothesis that AnkB regulates spine pruning and synapse elimination in develop- ing PFC networks, and that genetic deficiency of AnkB disrupts these mechanisms at sensitive postnatal stag- es, impacting cortical excitability and behaviors relevant to ASD. In Aim 1 Nex1Cre-ERT2:Ank2flox:EGFPflox mice will be analyzed to delineate how AnkB gene dosage in de- veloping and adult PNs of each sex alters dendritic spine regulation and levels of excitatory and inhibitory syn- apses in the PFC. A new mouse model Nex1Cre-ERT2:Ank2 exon37flox:EGFPflox deleting only AnkB-440 will be generated to distinquish isoform-specific roles. In Aim 2 a novel molecular rescue assay in Ank2-deficient neuronal cultures will interrogate AnkB function in Semaphorin-3 induced spine remodeling, receptor clustering and intracellular signaling. Selected human de novo Ank2 missense mutations will be analyzed for impaired L1-CAM and β-spectrin binding and signaling leading to spine pruning. Super-resolution microscopy will be employed to explore a potentially new role for AnkB in organizing synaptic nanodomains in spines. In Aim 3 the hypothesis that AnkB deficiency increases cortical excitability will be tested by electrophysiological record- ing of layer 2/3 PNs in cortical slices from normal and AnkB-deficient mice of each sex. Excitatory and inhibito- ry transmission will be compared, and synaptic inputs mapped by laser scanning photo-stimulation. Behavioral testing of Ank2 mutant mice will address roles for AnkB in sociability, reversal learning, and working memory. This project is expected to have significant impact, because it will illuminate novel molecular mechanisms for regulating...