Molecular signaling during development and maturation of the nervous system

神经系统发育和成熟过程中的分子信号传导

• 批准号: 10708643
• 负责人: David Talmage
• 金额: $ 217.94万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708643
• 关键词: Acetylcholine; Affinity Chromatography; Age-associated memory impairment; Alzheimer' s Disease; Amygdaloid structure; Animals; Attention; Axon; Basal Nucleus of Meynert; Behavior; Behavioral; Bioinformatics; Brain; Cell Cycle Progression; Cell Nucleus; Cells; Central Nervous System; Cholinergic Receptors; Coculture Techniques; Collaborations; Complex; Core Facility; Costa Rican; Coupled; Cues; Data; Desire for food; Development; Diagnosis; Diffuse; Event; Family; Functional disorder; Galaxy; Gene Targeting; Genes; Genetic Transcription; Globus Pallidus; Glutamates; Heterogeneity; Hippocampus (Brain); Human; Impairment; Individual; Intervention; Investigation; Lateral; Learning; Life; Longevity; Maintenance; Memory; Mental disorders; Molecular; Molecular Probes; Mus; Mutant Strains Mice; Mutation; NRG1 gene; National Institute of Neurological Disorders and Stroke; Nervous System; Neuregulin 1; Neurodegenerative Disorders; Neurons; Nicotine; Nicotinic Receptors; Nuclear; Nucleus Accumbens; Odors; Parkinson Disease; Patients; Phenotype; Population; Post-Traumatic Stress Disorders; Preparation; Property; Prosencephalon; Protein Isoforms; Proteins; Proteomics; Psychotic Disorders; Research; Retrieval; Risk; Role; Schizophrenia; Series; Signal Transduction; Signaling Protein; Stimulus; Structure; Synapses; Synaptic plasticity; System; Time; Transmembrane Domain; Viral; age related; basal forebrain; basal forebrain cholinergic neurons; cholinergic; cholinergic neuron; conditioning; dentate gyrus; flexibility; frontal lobe; frontier; gamma secretase; genetic approach; human population genetics; insight; loss of function mutation; meter; mutant; nerve stem cell; nervous system development; neural circuit; neuropsychiatric disorder; neurotransmission; olfactory stimulus; presynaptic; psychosis risk; resilience; response; risk variant; scaffold; targeted treatment; transcriptome sequencing; transmission process; young adult

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.

目的1：确定Nrg1相互作用组(Nrg1信号在神经元中的转录靶点以及与Nrg1物理相互作用的一系列蛋白质)。 亚目的1：Nrg1的轴突亚型参与轴突到核的信号转导。为了确定Nrg1核信号的靶向基因，我们收集并分析了大量的RNAseq数据，这些数据来自小鼠的齿状回、伏隔核、杏仁核、全海马区和额叶皮质，这些小鼠携带Nrg1跨膜域的突变，该突变损害了伽马分泌酶处理和核信号传递(最初在哥斯达黎加人群中被确定为精神病风险基因)。我们最初的研究集中在年轻人的齿状回上。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，我们能够在神经前体群体中以及在成熟神经元中寻找中断的Nrg1核信号的影响。对这些突变动物及其非突变后代的齿状回数据的分析已经完成(与NINDS生物信息学核心的Kory Johnson博士合作；Rajebhosale等人，BioRxiv 2022；正在eLife进行审查)。结果预测了细胞周期进程和神经元成熟的变化。这两个预测都用其他方法得到了证实。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组之间的RNAseq差异之间存在高度显著的趋同。在缺乏Nrg1核信号的情况下，其表达显著改变的基因群体与其表达改变与被诊断为精神疾病的风险相关的基因群体显著重叠。 亚目的2：Nrg1是如何启动信号的，无论是在轴突还是在核内，目前还不清楚。为了开始揭开这个问题，我们正在使用邻近标记、结合亲和纯化和蛋白质组学作为对与Nrg1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李燕博士和NINDS病毒核心设施主任Ray Fields博士合作)。 目的：鉴定胆碱能神经元的功能异质性。 亚目的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，另一种是对习得的威胁的反应。这两项研究(与NINDS IRP的L博士和耶鲁大学的M Picciotto博士合作进行)确定了参与不同类型记忆的特定胆碱能神经元群体，并首次证明了胆碱能神经元直接参与记忆印记(Crouse等人)。ELife 2020和Rajebhosale等人，BioRxiv 2021)。这些发现为调节胆碱能系统参与记忆形成和提取的方式提供了重要的新见解，对于理解伴随着胆碱能脑核退化的年龄相关性认知衰退具有明确的意义。 我们观察到，威胁反应行为需要不同的胆碱能神经元群体，这取决于威胁是后天获得的(线索依赖的条件反射)还是天生的(对捕食者气味的反应)。前者需要基底核的胆碱能神经元参与，后者需要苍白球腹侧的胆碱能神经元参与两种不同的基底前脑结构。我们已经开始了一系列的研究，重点是腹侧苍白球胆碱能神经元对天生嗜好或天生厌恶的嗅觉刺激的反应。这两种刺激都广泛激活了苍白球腹侧的胆碱能神经元。利用交叉遗传方法，我们发现，尽管两种刺激都激活了胆碱能神经元，但激活的胆碱能神经元代表了两个不同的、不重叠的群体。我们现在正在努力在分子水平上了解这些不同群体的基本属性。 亚目的2：深入了解调节胆碱能轴突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枝，估计是单个神经元轴突乔木在人脑中长达100米。关于这些终端场是如何在整个生命周期内建立或维护的，人们知之甚少。乙酰胆碱在大脑中的大部分作用是通过激活突触前ACh受体来实现的。这些轴突-轴突类似的相互作用是如何建立或调节的(甚至是它们看起来是什么样子)，目前尚不清楚。在此之前，我们专注于针对烟碱型乙酰胆碱受体(NAChRs)至轴突的轴突信号机制，并已证明含有ACh受体的突触前α7水平部分受轴突合成的α7蛋白调节。最近，我们与Dr.Roles小组合作，开发了谷氨酸能神经元与轴突nAChRs与基底前脑胆碱能核团外植体的共培养系统，并演示了功能性胆碱能到谷氨酸能轴突传递的建立(钟等，神经电路的前沿，正在审查中)。我们预计，这一实验准备工作将极大地扩展我们探索建立这些联系的分子机制的能力。