Induced neuronal cells: A novel tool to study neuropsychiatric diseases

摘要 这个项目的目标是描述神经连接蛋白家族的突触黏附分子的功能 (Nlgns)在小鼠大脑和人类神经元中。最近的单细胞表达研究强调了 观察到NIGN也在非神经细胞，特别是少突胶质前体细胞中表达 (OPC)和星形胶质细胞表达Nlgns的水平甚至高于神经元。由于人们对此知之甚少 Nlgns在胶质细胞中的功能及其对神经元和神经回路的影响，我们建议具体删除Nlgns 在OPC和星形胶质细胞中使用我们的三重条件NLGN1-3基因敲除株。大脑将被定性为 形态上，电生理上的细胞和电路水平，突变的小鼠将被描述为 通过行为。接下来，我们将通过以下方式对神经连接蛋白进行深入的分子表征 描述了Nlgns令人惊讶的功能多样性背后的分子机制。我们将绘制地图 通过在NLGN1-4中四重表达不同结构域突变蛋白在小鼠神经元中的功能域 敲除细胞。我们将探索NLGN序列是否与功能特异性有关，并研究 可能决定NLGN特异性的突触神经尿毒素“代码”的概念。 以补充我们的小鼠研究并探索人类特有的神经连接蛋白功能 疾病相关突变，我们将利用我们以前的人类干细胞和重新编程工作 我们已经开发出了人类诱导的神经元(IN)细胞，这种细胞表现出所有的主要功能特性 原代小鼠神经元包括强健的突触形成。我们建议利用这一系统来调查 NLGN4Y是一个Y染色体基因，与NLGN4在X-染色体上有密切的亲缘关系。 染色体和该家族中不存在的小鼠的成员。我们将通过标记来评估亚细胞靶向 并评估基因缺失的功能后果。另一个频繁突变的基因 NLGN基因为NLGN3。与NLGN4不同的是，它在小鼠身上保存得更好，但对其几乎一无所知 在人类细胞中发挥作用。除了产生功能缺失的等位基因外，我们还将研究功能性 人类NLGN3基因中引入不同的ASD相关突变的后果。我们将使用 条件突变方法，就像我们在过去成功地做的那样，因为它允许产生一个 以同一细胞系衍生的完美对照分生孢子为实验条件。突变人类 神经元和对照组将通过生物化学、形态、基因表达和 电生理学的。最后，我们建议研究所提出的Nlgns-调制器MDGA的作用 在自闭症和其他神经发育障碍中也发现了突变。我们将评估他们的 人类产生功能缺失等位基因对正常突触形成和功能的要求 神经元。我们将进一步探讨它们作为竞争性NLGN结合分子作为神经连接蛋白调节剂的功能。

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.