Role of Mitochondrial Dynamics In Bone Homeostasis

项目总结/摘要 线粒体是细胞的重要组成部分，它们是能量生产（ATP）的中心。最近的报告 表明线粒体在细胞存活/死亡、钙稳态和活性氧中也起作用 物种（ROS）的产生/对氧化应激的反应。线粒体作为细胞器的一个独特特征是它们的 在细胞内组织成一个高度动态的网络，其特征是相互关联的过程， 束缚/融合、裂变和通过线粒体自噬去除。虽然还没有完全理解，但这种动态 线粒体网络的调节似乎对维持健康、适当的 线粒体功能在人类疾病以及小鼠模型中，线粒体缺陷导致 寿命缩短或“过早衰老”的表型，包括神经退行性变、肌肉萎缩、心脏 增大和骨质流失然而，线粒体功能、衰老和衰老之间的关系， 骨形成和再吸收的细胞活性在很大程度上是未知的。该提案的主要目标是 确定线粒体动力学在破骨细胞（OC）和成骨细胞（OB）功能中的作用， 从而随着年龄的增长保持骨量和强度。为了研究线粒体动力学，我们 将操纵线粒体融合蛋白2（Mfn 2），这是一种在线粒体束缚和 以前在骨细胞中没有检测到的线粒体自噬。Mfn 2是一个位于外部的GTTT， 线粒体膜，具有独特的帕金招募作用，促进线粒体自噬， 通过自噬途径破坏线粒体。mfn 2也有一个拴系功能，因为它面对的细胞质。 线粒体之间的束缚导致融合，产生更长的细胞器和/或连接更紧密的细胞器。 网络Mfn 2还可以将线粒体拴系到ER，影响这些结构之间的Ca++转移， 调节细胞死亡。Mfn 2的所有这些作用都可能导致细胞中的整体线粒体功能障碍 Mfn 2缺乏，导致ATP和ROS水平异常。线粒体含量增加，在这两个OC和 我们的初步数据显示，Mfn 2的表达在OB从早期前体细胞分化过程中起着重要的作用。 平行上调。此外，OC谱系细胞中Mfn 2的条件性缺失导致高骨量 和小鼠对刺激性骨质溶解的抵抗力。从体外间充质祖细胞中去除Mfn 2也 降低成骨细胞分化。在这个提议中，我们测试了Mfn 2控制线粒体的假设， OC（目标1）和OB（目标2）中的动力学，通过其在线粒体自噬和/或栓系中的作用，调节骨 体内稳态。总体方法是使用Mfn 2fl/fl小鼠确定Mfn 2消融的效果 与OC和OB谱系Cre系杂交，检查骨表型随年龄的变化以及对特定骨表型的影响。 使用体内和体外分析的细胞功能。通过利用分离主要效应子功能的突变体 的Mfn 2，我们将解决的具体机制，驱动表型在每个细胞谱系。

Project Summary/Abstract Mitochondria are critical components of cells, and they are the hub of energy production (ATP). Recent reports indicate that mitochondria also play a role in cell survival/death, calcium homeostasis, and reactive oxygen species (ROS) production/response to oxidative stress. A unique feature of mitochondria as organelles is their organization into a highly dynamic network within the cell characterized by the interrelated processes of tethering/fusion, fission and removal via mitophagy. Although still not completely understood, this dynamic regulation of the mitochondrial network seems to be important for the maintenance of healthy, properly functioning mitochondria. In human diseases as well as mouse models, mitochondrial defects lead to phenotypes of reduced lifespan or “premature aging”, including neural degeneration, muscle atrophy, heart enlargement, and bone loss. However, the relationship between mitochondrial function(s), aging, and the cellular activities of bone formation and resorption are largely unknown. The primary goal of this proposal is to establish the role of mitochondrial dynamics in the function of osteoclasts (OCs) and osteoblasts (OBs), and thereby in the maintenance of bone mass and strength with age. In order to study mitochondrial dynamics, we will manipulate mitofusin 2 (Mfn2), a molecule with important roles in both mitochondrial tethering and mitophagy that has not previously been examined in bone cells. Mfn2 is a GTPase located on the outer mitochondrial membrane, with a unique role in the recruitment of Parkin to promote mitophagy, the recycling of damaged mitochondria via the autophagy pathway. Mfn2 also has a tethering function as it faces the cytosol. Tethering between mitochondria leads to fusion, generating longer organelles and/or a more connected network. Mfn2 can also tether mitochondria to the ER, affecting Ca++ transfer between these structures and regulating cell death. All of these roles for Mfn2 can contribute to overall mitochondrial dysfunction in cells deficient in Mfn2, causing abnormal levels of ATP and ROS. Mitochondrial content increases in both OCs and OBs during differentiation from early precursors in vitro, and our preliminary data show that Mfn2 expression is upregulated in parallel. Further, conditional deletion of Mfn2 from OC lineage cells leads to high bone mass and resistance to stimulated osteolysis in mice. Removal of Mfn2 from mesenchymal progenitors in vitro also reduces osteoblastic differentiation. In this proposal, we test the hypothesis that Mfn2 controls mitochondrial dynamics in OCs (Aim 1) and OBs (Aim 2), via its role in mitophagy and/or tethering, to modulate bone homeostasis in vivo. The overall approach is to determine the effect of Mfn2 ablation using Mfn2fl/fl mice crossed to OC and OB lineage Cre lines, examining the bone phenotype with age and the effects on specific cell function using in vivo and in vitro analyses. By utilizing mutants that separate the major effector functions of Mfn2, we will address the specific mechanism that drives the phenotype in each cell lineage.