Mechanisms of decreased bone formation with aging

项目摘要/摘要 骨量丢失是骨质疏松性骨折发病率增加的根本病理基础。 人口老龄化。老年小鼠骨骼中的细胞变化与老年时观察到的相似 人类。成骨细胞数量的减少和骨形成是两者骨骼老化的主要原因。 然而，潜在的机制仍不清楚。辅酶水平的降低 NAD+参与了多个组织的功能衰退，这是由于线粒体功能障碍和 依赖NAD的酶，如sirtuins(SIRT)和聚(ADP-核糖)的活性减弱 聚合酶(PAPS)。SIRT1和Parp5可以促进Wnt/β-连环蛋白途径的活性，这是 对于成骨细胞的形成是必不可少的。我们已经证明，成骨细胞的祖细胞数量和增殖是 这些变化与NAD+水平和烟酰胺表达降低有关 磷酸核糖转移酶(NAMPT)--NAD+挽救途径中的关键酶。相比之下，空气中的 NAD+消耗酶CD38随着年龄的增长而增加。NAD前体分子的给药 烟酰胺核苷(NR)对衰老小鼠的成骨细胞前体细胞和骨量的丢失具有拮抗作用。 此外，使用PRX1-CRE杂合性缺失间充质系细胞中的NAMPT模仿骨骼 衰老。我们还获得了证据，表明NAD耗竭对骨量的有害影响 由线粒体活性氧簇(ROS)和FoxO转录因子介导。值得注意的是， 成骨细胞中β-连环蛋白的蛋白水平和规范的WNT信号的刺激作用显著 依赖于NAD+。我们假设，随着年龄的增长，NAD+挽救途径的下降会减少 WNT信号转导，从而通过FoxO依赖和非依赖机制影响成骨细胞的数量。在……里面 目的1阐明NAD+挽救通路在成骨细胞系和CD38细胞中的作用 小白鼠和老年小白鼠的骨骼动态平衡。为此，我们将检查NAMPT删除或 成骨细胞系细胞的过度表达，以及CD38在骨骼老化中的影响。在《目标2》中我们将 确定骨祖细胞中线粒体ROS或FoxO在骨代谢不良骨骼效应中的作用 NAD+耗尽。在目标3中，我们将通过删除NAMPT和NAMPT来研究NAD+对Wnt信号的影响 过度表达WNT1。我们还将研究NAD+对β-连环蛋白的刺激作用是否依赖于 关于Parp5a和Parp5b。这些研究的成功完成应该有助于我们理解这些机制 介导NAD随增龄而降低及其在成骨细胞系细胞中的挽救途径 促进骨骼形成。这项工作也应该阐明ROS和ROS之间的相互作用或缺乏 NAD+，并可能建议更有针对性的治疗方法，以减缓或防止骨骼老化。

Project Summary/Abstract Loss of bone mass is the seminal pathology underlying the increased incidence of osteoporotic fractures in the aged population. The cellular changes in the skeleton of aged mice are similar to those observed in aged humans. A decrease in osteoblast number and bone formation are major contributors to skeletal aging in both humans and mice, however the underlying mechanisms remain unclear. A reduction in the levels of coenzyme NAD+ contributes to the functional decline of multiple tissues with age due to mitochondria dysfunction and attenuation of the activity of NAD-dependent enzymes such as the sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs). Sirt1 and Parp5 can promote the activity of the Wnt/β-catenin pathway, which is indispensable for osteoblast formation. We have shown that osteoblast progenitor number and proliferation are reduced in aged mice and that these changes correlate with lower levels of NAD+ and expression of nicotinamide phosphoribosyltransferase (Nampt) – a critical enzyme in the NAD+ salvage pathway. In contrast, the levels of the NAD+ consuming enzyme CD38 are increased with aging. Administration of the NAD precursor molecule nicotinamide riboside (NR) to aging mice counteracts the loss of osteoblast progenitors and bone mass. Moreover, heterozygous deletion of Nampt in cells of the mesenchymal lineage using Prx1-Cre mimics skeletal aging. We have also obtained evidence to suggest that the deleterious effects of NAD depletion on bone mass are mediated by mitochondrial reactive oxygen species (ROS) and the FoxO transcription factors. Notably, the protein levels of β-catenin and stimulation of the canonical Wnt signaling in osteoblastic cells are greatly dependent on NAD+. We hypothesize that a decline in the NAD+ salvage pathway with old age decreases Wnt signaling and thereby osteoblast number, via FoxO-dependent and -independent mechanisms. In Aim 1 we will elucidate the contribution of the NAD+ salvage pathway in cells of the osteoblast lineage and CD38 to skeletal homeostasis in young and old mice. To do this, we will examine the effects of Nampt deletion or overexpression in osteoblast lineage cells, as well as the impact of CD38 in skeletal aging. In Aim 2 we will determine the contribution of mitochondrial ROS or FoxOs in osteoprogenitors to the adverse skeletal effects of NAD+ depletion. In Aim 3 we will investigate the effects of NAD+ on Wnt signaling by deleting Nampt and overexpressing Wnt1. We will also examine whether the NAD+ stimulatory actions on β-catenin are dependent on Parp5a and Parp5b. Successful completion of these studies should help us understand the mechanisms mediating the decrease in NAD with aging and unravel how the NAD salvage pathway in osteoblast lineage cells promotes bone formation. This work should also elucidate the interaction, or lack thereof, between ROS and NAD+ and may suggest more targeted therapeutic approaches to slowing or preventing skeletal aging.