Plastin 3: Unravelling a novel pathomechanism for osteoporosis

骨质疏松症是老年人最常发生的十大疾病之一，尤其影响绝经后的妇女。骨质疏松症会导致骨折，尤其是上肢、股骨颈和椎体骨折。早期骨质疏松症伴骨折和骨生成障碍（OI）是单基因遗传性疾病，其发病年龄和严重程度具有高度变异性，由参与软骨形成和组装、骨生成或吸收的各种基因突变引起。在与Gerard帕尔斯（阿姆斯特丹）和Carola Zillikens（鹿特丹）的小组进行的富有成效的合作中，我们成功地鉴定了X连锁Plastin 3基因（PLS 3）中的家族性致病变异体，这些变异体存在于早期骨质疏松伴骨折的男性和轻度骨质疏松女性中。此外，在一个大的鹿特丹队列中，鉴定了与PLS 3中SNP的最显著关联，其与女性的低骨矿物质密度和骨质疏松症相关。PLS 3是一种钙离子依赖的F-肌动蛋白捆绑蛋白，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发挥重要作用。我们已经证明，在斑马鱼中，吗啉介导的pls 3敲低导致颅面骨骼和肌肉结构的显著畸形。这些通过共注射PLS 3 RNA而被完全拯救（货车Dijk等人，N Engl J Med 2013）。在另一个项目中，我们表明在女性中PLS 3的过表达是脊髓性肌萎缩症（SMA）的保护性修饰剂，SMA是一种频繁发生的神经肌肉疾病（Oprea等人，Science 2008）。为了阐明保护机制，我们产生了条件性PLS 3过表达小鼠，并且我们显示在神经肌肉接头水平的所有F-肌动蛋白捆绑过程都得到恢复（Ackermann等人，Mol Genet 2013）。该项目产生的初步数据显示，过表达PLS 3的小鼠具有更厚的皮质和骨小梁结构。PLS 3过表达可以在约5%的一般人群中发现，并且可能与针对骨质疏松症或OI的保护相关。在本申请的上下文中，我们旨在通过使用条件性敲除小鼠来阐明PLS 3耗竭的病理机制。我们将通过在成骨细胞/骨细胞或骨骼肌中耗尽Pls 3来产生普遍或细胞类型特异性缺乏Pls 3的小鼠。将进行一整套形态学、组织学、细胞、分子和生物化学的体外和离体实验，以确定由PLS 3缺失引起的骨质疏松症的潜在病理机制。我们确定了10个新的PLS 3相互作用的合作伙伴，这是有价值的资源，以解开分子机制。了解骨质疏松症的分子病理机制将为未来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.