Modulating Neurogenesis to Counteract Aβ42-Induced Neurodegeneration

项目摘要 本补充提案扩展了资助赠款R 01 HD 100023，其目的是定量确定， 高时空分辨率，系统范围内的影响，必要的细胞信号通路对嗅觉 神经发生具体来说，父母补助金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insm 1a在脊椎动物嗅觉过程中的作用 发展到目前为止，我们的发现揭示了Notch信号传导之间的动态调节反馈回路 以及insm 1a，其充当开关以驱动嗅觉感觉神经元（OSN）的及时分化。在这里， 我们建议调查这种反馈回路与已确定但知之甚少的缺陷的相关性， 成年嗅觉神经发生是阿尔茨海默病（AD）的标志。如果成功，该项目将 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驱动的 神经变性 嗅觉功能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。AD驱动的神经退行性变具有严重的 神经系统的后果，特别是考虑到成年人神经元再生的罕见性。的 注意，人类成年神经发生的少数例子之一是在海马体的亚颗粒区发现的， 这被认为是记忆的关键，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也可以从基底干细胞再生，这是令人困惑的原因 当AD患者中的神经元群受损时，其不会简单地更新。 我们的亲本R 01驱动的结果表明，通过下游效应子her4.1（与受体HER4.1的正相关）的Notch信号传导可能是一个重要的信号传导途径。 人HES 5）在不同的时间点抑制Insm 1a，反之亦然，构成动态反馈回路， 调节嗅觉干细胞分化为感觉神经元的时间和空间限制。 斑马鱼胚胎发生人NOTCH 1受体及其效应子HES 5的水平已被证明是 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 这表明，在体外，淀粉样前体蛋白（APP）裂解产物Aβ42被广泛研究， AD中的异常聚集影响Notch信号传导和INSM 1调节的神经发生。因此，我们假设 1）嗅觉干细胞和OSNs之间的平衡在AD发病早期被破坏; 2）Aβ42 干扰驱动成年人嗅觉干细胞分化为神经元的遗传程序。 为了验证这些假设，我们将评估Aβ42和Notch信号转导1a反馈之间的联系。 在斑马鱼成年嗅觉神经发生过程中的体内回路。在目标1中，我们将定量确定单细胞 嗅觉干细胞中notch 1a、her4.1和insm 1a的表达水平对处理的响应变化 Aβ42肽接下来，在目标2中，我们将开发第一个空间和时间上易于处理的脊椎动物模型 过表达Aβ42肽，模拟AD病理，并用它来确定Aβ42的细胞类型特异性 对成年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 利用时空特异性CRISPR/Cas9介导的APP直向同源物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对嗅觉干细胞向OSN分化的直接影响，以及Aβ42在嗅觉干细胞分化过程中的作用。 Notch信号转导-insm 1a调节反馈回路的特定调制。

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.