Genetic analyses of polygenic brain disorders, such as autism spectrum disorders (ASDs) and schizophrenia (SZ), have revealed “synaptic dysfunction” as a key cellular substrate for these disorders. Yet, translating this synaptic hypothesis to in vitro disease modeling and extracting disease-relevant biological information has been challenging. The PI has previously established a human neuronal model of SZ, combining isogenic genome engineering, patient-derived induced pluripotent stem cells (iPSCs), and induced neuronal differentiation (iN cells). Human neurons bearing mutations in the synaptic cell adhesion molecule Neurexin-1 (NRXN1, 2p16.3), a bona fide risk allele for SZ, display deficits in excitatory synaptic strength and neurotransmitter release probability as well as a consistent upregulation of calcium/calmodulin-dependent serine protein kinase (CASK) protein level, by which the mechanism is currently not understood. It remains unclear how these phenotypes arise and lead to synaptic pathology and abnormal neuronal networks implicated in the disease. In this grant, we aim to understand the cell type- and developmental-specific functions of NRXN1-CASK interaction in normal synapse development and their mechanistic contributions to SZ. Using synaptic molecules as a proxy, we will dissect how disruptions in the synaptic pathway can prime or actively participate in SZ. We will achieve this by studying aberrant CASK signaling in NRXN1 mutant background (Aim 1), investigating CASK’s normal function at human synapses using CASK KO induced neurons (Aim 2) and by revealing key cellular events mediated by NRXN1 and CASK during human forebrain development using KO cortical spheroid models (Aim 3). Our work integrates techniques in imaging, electrophysiology, biochemistry and single cell RNA-seq with rigorous experimental designs using isogenic engineered and patient-derived iPSCs from multiple genetic and gender backgrounds, differentiation of pure induced neuronal subtypes with defined synaptic characteristics and differentiation of 3-D cortical spheroids with characterized cellular features. Findings of this grant will provide mechanistic understanding of the molecular and cellular underpinnings of neuropsychiatric disorders and such information will translate to other disorders of the synapse, including intellectual disability, ASDs, and bipolar and mood disorders.