Modulating Neurogenesis to Counteract Aβ42-Induced Neurodegeneration

项目总结

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.