Modulating Neurogenesis to Counteract Aβ42-Induced Neurodegeneration

调节神经发生以抵消 Aβ42 诱导的神经变性

• 批准号: 10287125
• 负责人: Ankur Saxena
• 金额: $ 39.28万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10287125
• 关键词: Adult; Affect; Afferent Neurons; Alzheimer' s Disease; Alzheimer' s disease pathology; Alzheimer' s disease patient; Amyloid beta-42; Amyloid beta-Protein Precursor; Behavior; Biological Models; CRISPR/Cas technology; Cells; Chemicals; Cleaved cell; Clinical; Complement; Data; Development; ES05; Embryo; Embryonic Development; Equilibrium; Feedback; Functional disorder; Funding; Genetic; Genetic Programming; Grant; Hippocampus (Brain); Human; Image; Impaired cognition; In Vitro; Induced pluripotent stem cell derived neurons; Injury; Investigation; Life; Mediating; Memory; Modeling; NOTCH1 gene; Natural regeneration; Nerve Degeneration; Nerve Regeneration; Nervous system structure; Neurodegenerative Disorders; Neurons; Olfactory Epithelium; Olfactory dysfunction; Parents; Pathway interactions; Peptides; Population; Production; Resolution; Role; Signal Pathway; Signal Transduction; System; Testing; Time; WNT Signaling Pathway; Work; Zebrafish; adult neurogenesis; cell type; diadenosine pyrophosphate; experimental study; in vivo; neurogenesis; notch protein; olfactory neurogenesis; olfactory sensory neurons; overexpression; parent grant; receptor; spatiotemporal; stem cell differentiation; stem cells; time use; transcription factor; treatment response

Project Summary This supplemental proposal expands upon funded grant R01HD100023, which aims to quantitatively determine, with high spatiotemporal resolution, the system-wide effects of essential cell signaling pathways on olfactory neurogenesis. Specifically, the parent grant makes use of time-lapse imaging of zebrafish embryos, quantitative tracking of multicellular behavior in vivo, and genetic and chemical perturbation to elucidate the interconnected effects of Wnt signaling, Notch signaling, and the transcription factor insm1a during vertebrate olfactory development. Our findings thus far have revealed a dynamic regulatory feedback loop between Notch signaling and insm1a that acts as a switch to drive the timely differentiation of olfactory sensory neurons (OSNs). Here, we propose to investigate the relevance of this feedback loop to identified but poorly understood deficiencies in adult olfactory neurogenesis that are a hallmark of Alzheimer’s disease (AD). If successful, this project would create a unique in vivo platform to interrogate the role of adult neurogenesis in possibly mitigating AD-driven neurodegeneration. Olfactory dysfunction is often one of the earliest clinical indicators of several neurodegenerative diseases, but the mechanisms underlying this pathophysiology are unclear. AD-driven neurodegeneration has grave consequences across the nervous system, particularly given the rarity of neuronal regeneration in adults. Of note, one of the few examples of human adult neurogenesis is found in the subgranular zone of the hippocampus, which is thought to be critical for memory and is severely impacted in AD patients, contributing to memory-related cognitive decline. OSNs, meanwhile, also regenerate throughout life from basal stem cells, and it is puzzling why this neuronal population, when damaged in AD patients, does not simply renew. Our parent R01-driven results suggest that Notch signaling via the downstream effector her4.1 (orthologous to human HES5) inhibits insm1a and vice-versa at distinct time points, constituting a dynamic feedback loop that regulates the timed, spatially-restricted differentiation of olfactory stem cells into sensory neurons during zebrafish embryogenesis. Levels of human NOTCH1 receptor and its effector HES5 have been shown to increase in induced pluripotent stem cell-derived neurons from AD patients, and our own preliminary data suggest that in vitro, amyloid precursor protein (APP)’s cleaved product Aβ42, studied extensively for its abnormal aggregation in AD, affects Notch signaling and INSM1-regulated neurogenesis. Thus, we hypothesize that 1) the balance between olfactory stem cells and OSNs is disrupted early in the onset of AD; 2) Aβ42 interferes with the genetic programming that drives olfactory stem cell differentiation into neurons in adults. To test these hypotheses, we will evaluate connections between Aβ42 and the Notch signaling-insm1a feedback loop in vivo during zebrafish adult olfactory neurogenesis. In Aim 1, we will quantitatively ascertain single-cell level changes in the expression of notch1a, her4.1, and insm1a in olfactory stem cells in response to treatment with Aβ42 peptide. Next, in Aim 2, we will develop the first spatially- and temporally-tractable vertebrate model that overexpresses Aβ42 peptide, mimicking AD pathology, and use it to determine Aβ42’s cell type-specific effects on adult olfactory neurogenesis. Finally, in Aim 3, we will complement the Aβ42 overexpression experiments with spatiotemporally-specific CRISPR/Cas9-mediated targeting of APP orthologues appa and appb’s C-termini to inhibit Aβ42 production in olfactory stem cells post-injury. With these selective approaches, we will ascertain the direct effects of Aβ42 on olfactory stem cell differentiation into OSNs and the context- specific modulation of the Notch signaling-insm1a regulatory feedback loop.

项目概要 该补充提案扩展了资助赠款 R01HD100023，旨在定量确定， 具有高时空分辨率，重要细胞信号通路对嗅觉的全系统影响 神经发生。具体来说，家长资助利用斑马鱼胚胎的延时成像、定量 跟踪体内多细胞行为以及遗传和化学扰动以阐明相互关联的 Wnt信号、Notch信号和转录因子insm1a对脊椎动物嗅觉的影响 发展。迄今为止，我们的研究结果揭示了 Notch 信号传导之间的动态调节反馈循环 insm1a 充当开关，驱动嗅觉感觉神经元 (OSN) 的及时分化。这里， 我们建议调查这一反馈循环与已识别但知之甚少的缺陷的相关性 成人嗅觉神经发生是阿尔茨海默病 (AD) 的标志。如果成功的话，这个项目将 创建一个独特的体内平台来探究成人神经发生在可能缓解 AD 驱动的过程中的作用 神经变性。 嗅觉功能障碍往往是几种神经退行性疾病最早的临床指标之一，但是 这种病理生理学的机制尚不清楚。 AD 驱动的神经退行性疾病具有严重的 整个神经系统的后果，特别是考虑到成人神经元再生的罕见性。的 请注意，人类成人神经发生的少数例子之一是在海马体的颗粒下区发现的， 它被认为对记忆至关重要，并且在 AD 患者中受到严重影响，导致记忆相关的 认知能力下降。与此同时，OSN 也在整个生命周期中从基底干细胞再生，令人费解的是为什么 当 AD 患者的这种神经元群受损时，它不会简单地更新。 我们的亲本 R01 驱动的结果表明，Notch 信号通过下游效应器 her4.1（与 人类HES5）在不同的时间点抑制insm1a，反之亦然，构成一个动态反馈循环 调节嗅觉干细胞向感觉神经元的定时、空间限制的分化 斑马鱼胚胎发生。人类 NOTCH1 受体及其效应子 HES5 的水平已被证明 AD 患者诱导多能干细胞衍生神经元的增加，以及我们自己的初步数据 表明在体外，淀粉样前体蛋白 (APP) 的裂解产物 Aβ42，对其进行了广泛的研究 AD 中的异常聚集会影响 Notch 信号传导和 INSM1 调节的神经发生。因此，我们假设 1) 嗅觉干细胞和 OSN 之间的平衡在 AD 发病早期就被破坏； 2) Aβ42 干扰驱动成人嗅觉干细胞分化为神经元的遗传编程。 为了检验这些假设，我们将评估 Aβ42 和 Notch 信号传导-insm1a 反馈之间的联系 斑马鱼成年嗅神经发生期间的体内循环。在目标 1 中，我们将定量确定单细胞 嗅觉干细胞中 notch1a、her4.1 和 insm1a 的表达水平因治疗而变化 与 Aβ42 肽。接下来，在目标 2 中，我们将开发第一个空间和时间可处理的脊椎动物模型 过度表达 Aβ42 肽，模仿 AD 病理学，并用它来确定 Aβ42 的细胞类型特异性 对成人嗅觉神经发生的影响。最后，在目标 3 中，我们将补充 Aβ42 过表达 时空特异性 CRISPR/Cas9 介导的 APP 直向同源物靶向实验 appa 和 appb 的 C 末端抑制损伤后嗅觉干细胞中 Aβ42 的产生。通过这些选择性的方法， 我们将确定 Aβ42 对嗅觉干细胞分化为 OSN 的直接影响以及背景- Notch 信号传导-insm1a 调节反馈环路的特定调节。