Induced neuronal cells: A novel tool to study neuropsychiatric diseases

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Abstract The goal of this project is to describe the function of synaptic adhesion molecules of the Neuroligin family (Nlgns) in the mouse brain and in human neurons. Recent single cell expression studies have highlighted the obversation that Nlgns are expressed also in non-neuronal cells, in particular oligodendrocyte precursors cells (OPCs) and astrocytes who express Nlgns to even higher levels than neurons. Since little is known about the function of Nlgns in glia and their effect on neurons and neural circuits, we propose to specifically delete Nlgns in OPCs and astrocytes using our triple conditional Nlgn1-3 knock-out strain. Brains will be characterized morphologically, electrophysiologically on the cellular and circuit level, and mutant mice will be characterized by behavior. Next, we will perform an in-depth molecular characterization of the Neuroligin proteins by characterizing the molecular mechanisms underlying the surprising functional diversity of Nlgns. We will map their functional domains in mouse neurons by expressing various domain-mutant proteins in Nlgn1-4 quadruple knock-out cells. We will explore whether Nlgn sequence relates to functional specificity and investigate the notion of a synaptic Neurexin “code” that may determine Nlgn specificity. To complement our mouse studies and explore human-specific Neuroligin function as well as human disease-associated mutations, we will capitalize on our previous human stem cell and reprogramming work in which we have developed human induced neuronal (iN) cells that exhibit all principal functional properties of primary mouse neurons including robust synapse formation. We propose to utilize this system to investigate the so far obscure function of NLGN4Y, a Y chromosomal gene closely related to NLGN4 on the X- chromosome and a member of the family not present in mouse. We will assess subcellular targeting by tagging the endogenous locus and assess the functional consequences of genetic deletion. Another frequently mutated Nlgn gene is NLGN3. Unlike NLGN4 it is better conserved in mice, but almost nothing is known about its function in human cells. In addition to generate loss-of-function alleles, we will study the functional consequences of distinct ASD-associated mutations introduced into the human NLGN3 gene. We will use a conditional mutagenesis approach as we have successfully done in the past, as it allows the generation of a perfect control conidition derived from the identical cell line as the experimental condition. Mutant human neurons and controls will be characterized biochemically, morphologically, by gene expression, and electrophysiologically. Finally, we propose to investigate the role of the proposed Nlgns-modulators MDGAs which are also found mutated in ASD and other neurodevelopmental disorders. We will assess their requirement for proper synapse formation and function by generating loss-of-function alleles in human neurons. We will further probe their function as Neuroligin modulators as competitive Nlgn binding molecules.