Plastin 3: Unravelling a novel pathomechanism for osteoporosis

骨质疏松症是老年人最常见的十种疾病之一，尤其影响绝经后的妇女。骨质疏松症会导致骨折，尤其是在上肢、股骨颈和椎体。早期骨质疏松伴骨折和成骨不全（OI）是单基因遗传性疾病，在发病年龄和严重程度上具有高度可变性，由参与软骨形成和组装、骨生成或再吸收的各种基因突变引起。在与Gerard Pals（阿姆斯特丹）和Carola Zillikens（鹿特丹）小组的富有成效的合作工作中，我们成功地确定了X-linked Plastin 3基因（PLS3）在早期骨质疏松症合并骨折的男性和轻度骨质疏松症的女性中的家族致病性变异。此外，在鹿特丹的一项大型队列研究中，发现了PLS3中SNP与低骨密度和女性骨质疏松症的最显著关联。PLS3是一种Ca2+依赖的f -肌动蛋白结合蛋白，在细胞迁移、内吞作用和所有f -肌动蛋白依赖的细胞过程中起重要作用。我们已经证明，斑马鱼中morpholino介导的pls3敲低会导致颅面骨骼和肌肉结构的严重畸形。这些细胞通过共注射PLS3 RNA完全获救（van Dijk等）。中华医学杂志（2013）。在另一个项目中，我们发现，在女性中，PLS3的过表达是脊髓性肌萎缩症（SMA）的保护性修饰因子，这是一种常见的神经肌肉疾病（Oprea等）。科学2008)。为了揭示保护机制，我们生成了一个条件PLS3过表达的小鼠，我们发现神经肌肉连接水平的所有F-actin捆绑过程都恢复了（Ackermann et al Hum Mol Genet 2013）。本项目的初步数据显示，过表达PLS3的小鼠具有较厚的皮质和骨小梁结构。大约5%的普通人群中存在PLS3过表达，这可能与预防骨质疏松症或成骨不全症有关。在本应用的背景下，我们的目标是通过使用条件敲除小鼠来揭示PLS3耗竭的病理机制。我们将通过在成骨细胞/骨细胞或骨骼肌中消耗Pls3来产生普遍或细胞类型特异性缺乏Pls3的小鼠。我们将通过形态学、组织学、细胞、分子和生化等一系列的体外和离体实验来确定PLS3丢失导致骨质疏松的潜在病理机制。我们发现了10个新的pls3相互作用伙伴，它们为揭示分子机制提供了宝贵的资源。了解骨质疏松症的分子病理机制将为今后的治疗开辟新的途径。

Osteoporosis represents one of the ten most frequently occurring diseases in elderly people, particularly affecting women after menopause. Osteoporosis causes fractures especially in the upper limbs, femoral neck and vertebral bodies. Early osteoporosis with fractures and osteogenesis imperfecta (OI) are monogenic inherited diseases with a high variability in age of onset and severity, caused by mutations in various genes involved in cartilage formation and assembly, bone generation or resorption. In a productive collaborative work with the groups of Gerard Pals (Amsterdam) and Carola Zillikens (Rotterdam), we succeeded in identifying familial pathogenic variants in the X-linked Plastin 3 gene (PLS3) in men with early osteoporosis with fractures and women with mild osteoporosis. Furthermore, in a large Rotterdam cohort the most significant association with a SNP in PLS3 was identified, which correlates with low bone mineral density and osteoporosis in women. PLS3 is a Ca2+-dependent F-actin bundling protein with an essential role in cell migration, endocytosis und all F-actin-dependent cellular processes. We have shown that morpholino-mediated pls3 knockdown in zebrafish causes dramatic deformities in craniofacial skeletal and muscle structures. These were fully rescued by co-injection of PLS3 RNA (van Dijk et al. N Engl J Med 2013). In another project we showed that in women overexpression of PLS3 is a protective modifier for spinal muscular atrophy (SMA), a frequently occurring neuromuscular disease (Oprea et al. Science 2008). To unravel the protective mechanism, we generated a conditional PLS3 overexpressing mouse and we showed that all F-actin bundling processes at the neuromuscular junction level are restored (Ackermann et al Hum Mol Genet 2013). Preliminary data generated in this project showed that mice overexpressing PLS3 have thicker cortical and trabecular bone structures. PLS3 overexpression can be found in about 5% of the general population and might correlate with protection against osteoporosis or OI. In the context of this application, we aim at unravelling the pathomechanism underlying PLS3 depletion by using conditional knock-out mice. We will generate mice lacking Pls3 either ubiquitously or cell-type -specifically by depleting Pls3 in osteoblasts/osteocytes or in skeletal muscle. A full battery of morphological, histological, cellular, molecular and biochemical in vitro and ex vivo experiments will be carried out to determine the underlying pathomechanism of osteoporosis caused by loss of PLS3. We identified 10 novel PLS3-interacting partners, which represent valuable resources to unravel the molecular mechanism. Understanding the molecular pathomechanism of osteoporosis will open new avenues for future therapies.