Molecular signaling during development and maturation of the nervous system

目标一：定义Nrg 1相互作用组（神经元中Nrg 1信号传导的转录靶点以及与Nrg 1物理相互作用的蛋白质的星系）。 子目标1：Nrg 1的轴突亚型参与轴突到核的信号传导。 为了鉴定Nrg 1核信号传导靶向的基因组，我们收集并分析了来自小鼠齿状回、丘脑核、基底外侧杏仁核、总海马和额叶皮质的大量RNAseq数据，这些小鼠携带Nrg 1跨膜结构域突变，该突变损害γ分泌酶加工和核信号传导（并且最初被鉴定为哥斯达黎加人群中的精神病风险基因）。 我们最初的研究集中在年轻人的齿状回。 齿状回中的神经元在整个生命过程中不断产生，因此通过观察齿状回，我们能够在神经祖细胞群体以及成熟神经元中寻找Nrg 1核信号中断的影响。已经完成了对这些突变动物及其非突变同窝仔的齿状回数据的分析（与NINDS生物信息学核心的Kory约翰逊博士合作; Rajebhosale等人，BioRxiv 2022;正在ELife审查）。 结果预测细胞周期进程和神经元成熟的改变。 这两种预测都得到了其他方法的证实。 RNAseq分析的第二个主要发现是小鼠突变数据与精神分裂症患者和对照之间的RNAseq差异之间的高度显著收敛。 在不存在Nrg 1核信号传导的情况下，其表达显著改变的基因群体与其表达改变与被诊断患有精神病的风险相关的基因群体显著重叠。 副目标2：Nrg 1如何在轴突或细胞核中启动信号传导，目前尚不清楚。 为了开始解开这个问题，我们正在使用邻近标记，耦合亲和纯化和蛋白质组学作为与Nrg 1瞬时或稳定相互作用的蛋白质的无偏筛选（与NINDS蛋白质组学核心设施主任Yan Li博士和NINDS病毒核心设施主任Ray Fields博士合作）。 目的二：研究胆碱能神经元的功能异质性。 副目标1：确定胆碱能神经元参与两种不同的BLA依赖行为，一种是对食欲刺激的反应，另一种是对学习威胁的反应。 这两项研究（与NINDS IRP的L Role博士和耶鲁大学的M Picciotto博士合作进行）鉴定了参与不同类型记忆的胆碱能神经元的特定群体，并首次证明胆碱能神经元直接参与记忆印迹（Crouse等人，ELife 2020和Rajebhosale等人，bioRxiv 2021）。 这些发现提供了重要的新的见解的方式，调节，胆碱能系统参与记忆的形成和检索，明确的含义，以了解年龄相关的认知能力下降，伴随着胆碱能脑核变性。 我们观察到，不同群体的胆碱能神经元所需的威胁响应行为取决于是否学习的威胁（线索依赖条件反射）或先天的（反应捕食者的气味）。 前者需要参与的胆碱能神经元在基底核，后者的胆碱能神经元在腹侧苍白球两个不同的基底前脑结构。 我们已经开始了一系列的调查，重点是腹侧苍白球胆碱能神经元的反应，无论是天生的食欲或天生厌恶嗅觉刺激。 这两种刺激广泛激活腹侧苍白球的胆碱能神经元。 使用交叉遗传学方法，我们发现，虽然两种刺激激活胆碱能神经元，激活的胆碱能神经元，代表两个不同的，不重叠的群体。 我们现在正着手在分子水平上了解这些不同种群的基本特性。 子目标2：深入了解胆碱能轴-轴突触的建立和功能的分子机制。 基底前脑胆碱能神经元轴突高度分支，估计在人脑中单个神经元轴突乔木可达100米。 关于这些终末场是如何在生命周期中建立或维持的，我们知之甚少。 乙酰胆碱在大脑中的大部分作用是通过激活突触前ACh受体而发生的。 这些轴突-轴突突触样相互作用是如何建立或调节的（甚至它们看起来像什么）是未知的。 以前，我们专注于轴突的信号机制，靶向烟碱乙酰胆碱受体（nAChRs）的轴突，并已证明，突触前α 7含ACh受体的水平在一定程度上调节轴突合成的α 7蛋白。 与Dr. Roles小组合作，我们最近开发了具有轴突nAChR的多巴胺能神经元与来自基底前脑胆碱能核的外植体之间的共培养系统，并证明了功能性胆碱能至多巴胺能轴突-轴突传递的建立（Zhong et al.，Frontiers in Neural Circuits，under review）。 我们预计，这种实验准备将显着扩大我们的能力，探测这些连接的建立背后的分子机制。

Objective 1: define the Nrg1 interactome (the transcriptional targets of Nrg1 signaling in neurons and the galaxy of proteins that physically interact with Nrg1). Subobjective 1: Axonal isoforms of Nrg1 participate in axon-to-nucleus signaling. In order to identify the suite of genes targeted by Nrg1 nuclear signaling, mechanism we collected and analyzed bulk RNAseq data from dentate gyrus, nucleus accumbens, basal lateral amygdala, total hippocampus and frontal cortices of mice carrying a mutation in the Nrg1 transmembrane domain that impairs gamma secretase processing and nuclear signaling (and which was initial identified as a psychosis risk gene in a Costa Rican population). Our initial study focused on the dentate gyrus from young adults. Neurons in the dentate are continually generated throughout life and therefore by looking at the dentate gyrus we were able to look for effects of disrupted Nrg1 nuclear signaling in neuroprogenitor populations as well as in mature neurons. Analyses of the dentate gyrus data from these mutant animals and their non-mutant littermates has been completed (in collaboration with Dr. Kory Johnson, NINDS bioinformatics core; Rajebhosale et al., BioRxiv 2022; under review at ELife). The results predict alterations in cell cycle progression and in neuronal maturation. Both predictions have been confirmed using other approaches. The second major finding of the RNAseq analyses is highly significant convergence between the mouse mutant data and RNAseq differences between schizophrenia patients and controls. The population of genes whose expression is significantly altered in the absence of Nrg1 nuclear signaling overlaps significantly with populations of genes whose altered expression is associated with risk of being diagnosed with psychotic illness. Subobjective 2: How Nrg1 initiates signaling, either in axons or in the nucleus, remains unknown. To begin to unravel this question we are using a proximity tagging, coupled affinity purification and proteomics as an unbiased screen for proteins that transiently or stably interact with Nrg1 (collaboration with Dr. Yan Li, Director of the NINDS proteomics core facility and Dr. Ray Fields, Director of the NINDS viral core facility). Objective 2: identify the functional heterogeneity of cholinergic neurons. Subobjective 1: Determine the involvement of cholinergic neurons in two distinct BLA dependent behaviors, one in response to an appetitive stimulus, the other in response to learned threat. These two studies (carried out in collaboration with Dr. L Role, NINDS IRP and Dr. M Picciotto, Yale Univ) identify specific populations of cholinergic neurons that participate in distinct types of memory and demonstrate for the first time that cholinergic neurons participate directly in memory engrams (Crouse et al. ELife 2020 and Rajebhosale et al., bioRxiv 2021). These findings provide significantly new insight into the ways that the modulatory, cholinergic system participates in memory formation and retrieval with clear implications to understanding age related cognitive decline that accompanies degeneration of cholinergic brain nuclei. We observed that distinct populations of cholinergic neurons were required for threat responsive behaviors depending on whether the threat was learned (cue-dependent conditioning) or was innate (response to predator odor). The former required engagement of cholinergic neurons in the nucleus Basalis, the latter cholinergic neurons in the ventral pallidum two distinct basal forebrain structures. We have initiated a line of investigation focusing on the response of ventral pallidal cholinergic neurons to either innately appetitive or innately aversive olfactory stimuli. Both stimuli broadly activate cholinergic neurons in the ventral pallidum. Using intersectional genetic approaches, we have found that although both stimuli activate cholinergic neurons, the cholinergic neurons activated, represent two distinct, non-overlapping populations. We are now embarking on efforts to understand, at the molecular level, the fundamental properties of these distinct populations. Subobjective 2: Gain insight into molecular mechanisms that regulate the establishment and function of cholinergic axo-axonal synapses. Basal forebrain cholinergic neuron axons are highly branched estimates are single neuronal axonal arbors are up to 100 meters in the human brain. Very little is known about how these terminal fields are established or maintained across the lifespan. Much of the action of acetylcholine in the brain occurs by activating pre-synaptic ACh receptors. How these axo-axonic synapse-like interactions are established or regulated (or even what they look like) is unknown. Previously we focused on axonal signaling mechanisms that targeted nicotinic acetylcholine receptors (nAChRs) to axons and have demonstrated that levels of presynaptic alpha 7 containing ACh receptors are in part regulated by axonal synthesis of alpha 7 protein. In collaboration with Dr. Roles group, we have recently developed a co-culture system between glutamatergic neurons with axonal nAChRs with explants from basal forebrain cholinergic nuclei and demonstrated the establishment of functional cholinergic to glutamatergic axo-axonal transmission (Zhong et al., Frontiers in Neural Circuits, under review). We anticipate that this experimental preparation will significantly expand our ability to probe molecular mechanisms that underlie the establishment of these connections.