Modulating Neurogenesis to Counteract Aβ42-Induced Neurodegeneration

项目摘要 这项补充提案在资助赠款R01HD100023的基础上进行了扩展，旨在定量确定， 在高时空分辨率下，基本细胞信号通路对嗅觉的全系统影响 神经发生。具体地说，父母的资助利用了斑马鱼胚胎的时间推移成像，数量 体内多细胞行为的跟踪，以及遗传和化学扰动来阐明相互关联的 Wnt信号、Notch信号和转录因子insm1a在脊椎动物嗅觉中的作用 发展。到目前为止，我们的发现揭示了Notch信号之间的动态调节反馈环路 以及insm1a，作为开关推动嗅觉感觉神经元(OSN)的及时分化。这里, 我们建议调查这种反馈环路与已发现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，这个项目将 创建一个独特的体内平台，以询问成人神经发生在可能减轻AD驱动方面的作用 神经退行性变。 嗅觉障碍通常是几种神经退行性疾病的最早临床指标之一，但 这种病理生理学的机制尚不清楚。广告驱动的神经退行性变很严重 对神经系统的影响，特别是考虑到成年人神经元再生的稀有情况。的 注意，人类成年神经发生的为数不多的例子之一是在海马体颗粒下区域发现的， 它被认为对记忆至关重要，在AD患者中受到严重影响，导致与记忆相关的 认知能力下降。与此同时，OSNs在整个生命过程中也会从基础干细胞中再生，这是令人费解的原因 当AD患者受损时，这种神经元群体不会简单地再生。 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，构成了一个动态反馈环， 调节嗅觉干细胞在时间和空间上受限的分化为感觉神经元 斑马鱼胚胎发生。人类NOTCH1受体及其效应物HES5的水平已被证明 阿尔茨海默病患者诱导的多能干细胞来源的神经元增加，以及我们自己的初步数据 提示在体外，淀粉样前体蛋白(APP)S切割产物Aβ42，对其进行了广泛的研究 AD时聚集异常，影响Notch信号转导和INSM1调节的神经发生。因此，我们假设 1)嗅觉干细胞和嗅觉神经元之间的平衡在阿尔茨海默病早期被破坏；2)Aβ42 干扰了促使成年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β42和Notch Signal-Insm1a反馈之间的联系 斑马鱼成体嗅觉神经发生过程中的活体环路。在目标1中，我们将定量确定单细胞 嗅觉干细胞对治疗的反应中notch1a、her4.1和insm1a表达水平的变化 与Aβ42肽结合。接下来，在目标2中，我们将开发第一个在空间上和时间上都可处理的脊椎动物模型 过度表达Aβ42肽，模拟AD病理，并用它来确定Aβ42‘S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的过度表达 时空特异性CRISPR/Cas9介导的APP同源基因APPA和APP靶向的实验 APPB的C末端抑制嗅觉干细胞损伤后Aβ42的产生。有了这些有选择性的方法， 我们将确定Aβ42在嗅觉干细胞分化为OSN中的直接作用，以及上下文- Notch信号的特定调制-insm1a调节反馈回路。

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.