In vivo Osteocyte Ca2+ Signaling in Aging Bone

摘要 骨对机械负荷的反应和适应能力随着年龄的增长而显著下降，这是一个主要的原因。 导致骨脆性。我们不知道骨细胞机械感应的改变是如何发生的 在老化的骨骼中。这是一个关键的知识差距，因为骨细胞在协调骨骼中起着核心作用 形成和再吸收反应。我导师实验室最近的突破性发现 揭示了活骨中的骨细胞如何感知和编码机械载荷信息。他们 创建了第一个具有骨细胞靶向遗传编码钙指示剂的报告小鼠， 允许测量骨细胞Ca 2+对体内负荷的反应。他们发现骨细胞 活骨中的种群对载荷大小进行数字编码，随着应变水平的增加， 在健康的年轻成人骨中，遵循稳健的线性响应曲线的Ca 2+响应骨细胞更多。 初步研究进一步表明，这种骨细胞负荷反应曲线随着骨细胞的生长而显著变化。 系统性挑战。拟议的F31训练补助金将确定骨细胞Ca 2+对骨钙素的反应。 体内力学载荷随年龄变化。在目标1中，我们将创建他莫昔芬诱导的GCaMP 6 f小鼠， 其中Ca 2+报告基因可以在整个成年期的选择性时间开启。这将解决 长期组成型GCaMP表达中固有的挑战，如在第一代中使用的 骨细胞Ca ~（2+）报告小鼠。跖骨将被加载 使用组合的加载和多光子成像通过生理应变水平的范围在体内 方法和皮质骨中的骨细胞Ca 2+反应将被测量。在目标2中，我们将使用这些 诱导型GCaMP 6 f小鼠评估骨细胞Ca 2+对年轻成年人、中年人 和年老的成年小鼠。最后，一系列通道（例如，T型和L型通道，Panx 1-P2 X7，压电 1，TRPV通道）参与体外骨细胞对负荷的Ca 2+响应。然而，体内 人们对这些渠道的重要性了解甚少。在目标3的探索性研究中，我们将使用 验证的选择性通道拮抗剂，以测试体内调节通道功能如何影响负荷- 响应曲线与F31赠款的相关性科学和培训目标：研究： 第一个检查骨细胞机械感应在整个成年生活中的体内。培训：建议 奖学金结合了广泛的实验和分析方法（小鼠模型，骨 生物力学，体内研究，多光子成像，细胞Ca 2+测量，骨和骨细胞 生理学，通道生物学），这将为研究职业发展提供一个强大的平台。

Abstract Bone’s ability to respond and adapt to mechanical loading declines significantly with age, which is a major contributor to bone fragility. We do not understand how alterations in osteocyte mechanical sensing occur in aging bone. This is a critical knowledge gap given the central role for osteocytes in orchestrating bone formation and resorption responses. Recent breakthrough discoveries from my mentor’s laboratory revealed how osteocytes in living bone perceive and encode mechanical loading information. They created the first ever reporter mice with an osteocyte-targeted genetically encoded calcium indicator to allow measurement of osteocyte Ca2+ responses to loading in vivo. They discovered that osteocyte populations in living bone numerically encode load magnitude, with increasing strain levels recruiting more Ca2+ responding osteocytes in healthy young adult bone following a robust, linear response curve. Preliminary studies further revealed that this osteocyte loading response curve changes markedly with systemic challenge. The proposed F31 training grant will determine how osteocyte Ca2+ responses to mechanical loading in vivo change with age. In Aim 1, we will create tamoxifen inducible GCaMP6f mice in which the Ca2+ reporter can be turned on at selective times throughout adulthood. This will address challenges inherent in long-term constitutive GCaMP expression as was used in the first-generation osteocyte Ca2+ reporter mice previously created by our laboratory. Metatarsal bones will be loaded in vivo through the range of physiological strain levels using a combined loading and multiphoton imaging approach and osteocyte Ca2+ responses in cortical bone will be measured. In Aim 2, we will use these inducible GCaMP6f mice to assess osteocyte Ca2+ responses to loading in young adult, middle aged, and aged adult mice. Finally, a range of channels (eg, T-type and L-type channels, Panx1-P2X7, Piezo 1, TRPV channels) are implicated in osteocyte Ca2+ response to loading in vitro. However, the in vivo importance of these channels is poorly understood. In the exploratory studies in Aim 3, we will use validated selective channel antagonists to test how modulating channel function in vivo influences load- response curves. Relevance to F31 grant Scientific and Training goals: Research: These studies will be the first to examine osteocyte mechano-sensing in vivo throughout adult life. Training: The proposed fellowship incorporates a broad range of experimental and analytical approaches (mouse models, bone biomechanics, in vivo studies, multiphoton imaging, cell Ca2+ measurements, bone and osteocyte physiology, channel biology) that will provide a robust platform for research career growth.