Modified Project Summary/Abstract Section The X-chromosome linked Neuroligin-4 (NLGN4) is a postsynaptic cell-adhesion molecule (CAM) abundantly expressed in human cerebral cortex, however, its cellular function and molecular properties remain relatively unclear. Human NLGN4 consists of a unique amino-acid sequence that is not evolutionarily well-conserved in conventional rodent models, limiting our ability to investigate how this human-specific gene impacts synapse organization. This inherent species differences between diverse NLGN4 orthologs underscore the immediate need to generate a human model system to uncover its human-specific mechanisms. Recent technological advances in the fields of genetic engineering and epigenetic reprogramming of pluripotent stem cells provide us with a unique opportunity to examine the mechanistic properties of NLGN4, while maintaining the fidelity of human cellular context. In this proposal, we aim to utilize neuronal subtypes derived from human stem cells to assess our central hypotheses that NLGN4 plays an instructive role in defining the input-output parameters of excitatory vs. inhibitory synapses. We anticipate that NLGN4 establishes molecular interactions with a subset of synaptic proteins via its intra- and extracellular domains, which collectively regulate its proper maturation, trafficking, and function. Both the amino-acid sequence of different NLGN4 motifs as well as post-translational modifications at some those critical residues might play significant roles in determining its functional specificity. In aim 1: To inquire how NLGN4 can modulate synaptic network activity, we will either completely eliminate its endogenous expression in human neurons or introduce loss-of-function mutation, and inspect adverse effects on synaptic morphology and transmission using confocal imaging and electrophysiological recording. In Aim 2: We will determine how distinct amino-acid residues of NLGN4 can differentially regulate its characteristics, by performing systematic structure-function and biochemical analyses. In Aim 3: We will investigate how NLGN4’s binding to other synaptic proteins may define its functional identity, using rigorous co-immunoprecipitation, cell-aggregation, and proximity-dependent biotinylation assay. This project will essentially provide a comprehensive knowledge about NLGN4 function, its similarities and differences with other NLGNs. Using NLGN4 as a model, this extensive set of complementary approaches would also allow us to acquire fundamental information about human synaptic environment and how pre- or postsynaptic CAMs modulate its composition and activity.