Replication Stress and Ribosome Biogenesis in Hematopoietic Stem Cell Aging

项目摘要/摘要 老年病是一个日益紧迫的社会问题，因为世界范围内 寿命。造血系统的老化是由造血干细胞(HSC)功能障碍和 以贫血、血小板增多和髓系细胞过度生产为特征 淋巴生成。这些缺陷共同在心血管疾病的发展中起着关键作用， 阻碍接种疫苗的获得性免疫，以及建立损害 组织，并导致脆弱。哺乳动物的HSC衰老是保守的，人类和小鼠的老年HSC(OHSC) 两者都表现出再生潜力降低，基因组不稳定，表观遗传漂移，代谢重新连接，以及 改变了细胞间的通讯。尽管这些明显的表型特征被广泛认为是重要的 OHSCs的特性，但我们对其潜在的分子机制和功能知之甚少 后果。这种知识上的差距阻碍了从根本上延缓或逆转HSC老化的努力。我们的实验室 发现复制应激是OHSC功能障碍和再生能力受损的有力驱动因素。这是 尤其是在脆弱的核糖体DNA基因座上，导致核糖体生物发生的丧失。该项目旨在 确定减少核糖体生物发生对oHSCs的功能后果，并确定计划 这种不充分的复制强调启动，目标是以它们为靶点恢复OHSC功能。我们的预赛 数据表明，oHSCs的蛋白质翻译能力是有缺陷的，即使它们有丝分裂。 信号通路过度活跃。他们还提示核仁应激反应(Nsr)的慢性激活。 作为oHSCs复制应激的结果。此外，我们有证据表明表观遗传改变和 细胞周期转录抑制与MCM下调和复制应激启动一致。在AIM 1，我们将使用体外实验确定静止和激活的oHSCs中缺陷蛋白翻译的程度 在活体内接近。我们还将询问驱动缺陷蛋白翻译的信号通路。 OHSCs特别关注使用小鼠遗传方法激活NSR。这些实验将 建立蛋白调控缺陷如何导致HSC老化，以及复制和 驱动OHSC的核仁应激损害了再生能力。在目标2中，我们将识别转录 负责复制应激启动的因子或细胞周期调节因子，也揭示了表观遗传学基础 来弥补这个缺陷。然后，我们将评估对特定表观遗传修饰物的药理学调整是否可以恢复 OHSC功能。这些实验将剖析复制压力的分子基础，并确定 纠正这一细胞固有的HSC老化标志是否会提高OHSC的再生能力。总而言之， 我们建议的研究是更好地理解和治疗HSC老化的有希望的途径。他们有 确定促进HSC功能寿命的可操作目标的令人兴奋的影响，这是一种合乎逻辑的战略 恢复老年人的血液和免疫功能。

PROJECT SUMMARY/ABSTRACT Diseases of the elderly are an increasingly urgent societal problem due to the worldwide increases in lifespan. The aging of the hematopoietic system is caused by dysfunction in hematopoietic stem cells (HSC) and is characterized by anemia, thrombocytosis, and overproduction of myeloid cells at the expense of lymphopoiesis. Together these defects play a key role in the development of cardiovascular diseases, loss of adaptive immunity that impedes vaccination, and establishment of chronic systemic inflammation that damages tissue and contributes to frailty. HSC aging is conserved in mammals, with human and murine old HSCs (oHSC) both exhibiting reduced regenerative potential, genomic instability, epigenetic drift, metabolic rewiring, and altered cell-cell communication. Although these overt phenotypic features are widely understood to be significant characteristics of oHSCs, we still know little about their underlying molecular mechanisms and functional consequences. This gap in knowledge has hindered efforts to delay or reverse HSC aging at its root. Our lab identified replication stress as a potent driver of oHSC dysfunction and impaired regenerative potential. This is especially severe at fragile ribosomal DNA loci, leading to loss of ribosome biogenesis. This project aims to determine the functional consequences of reduced ribosome biogenesis for oHSCs, and to identify the programs that underly replication stress initiation with a goal to target them to restore oHSC function. Our preliminary data suggest that oHSCs are defective in their capacity for protein translation, even though their mitogenic signaling pathways are overactive. They also suggest chronic activation of the Nucleolar Stress Response (NSR) as a consequence of replication stress in oHSCs. Furthermore, we have evidence for epigenetic alterations and cell cycle transcriptional repression consistent with Mcm downregulation and replication stress initiation. In Aim 1, we will determine the extent of defective protein translation in quiescent and activated oHSCs using in vitro and in vivo approaches. We will also interrogate the signaling pathways driving defective protein translation in oHSCs focusing in particular on NSR activation using a mouse genetic approach. These experiments will establish how defective proteostasis contribute to HSC aging, and the connection between replication and nucleolar stress in driving oHSC impaired regeneration potential. In Aim 2, we will identify the transcription factors or cell cycle regulators responsible for replication stress initiation, and also uncover the epigenetic basis for this defect. We will then assess whether pharmocological tuning of specific epigenetic modifiers can restore oHSC function. These experiments will dissect the molecular underpinnings of replication stress and determine whether correcting this cell-intrinsic hallmark of HSC aging will improve oHSC regenerative potential. Altogether, our proposed investigations are promising avenues to better understand and treat HSC aging. They have exciting implications for identifying actionable targets for promoting HSC functional longevity, a logical strategy towards restoring blood and immune function in the elderly.