Mechanisms of bone fragility in Autosomal Dominant Osteopetrosis type II: from human to mouse and back

摘要 骨吸收破骨细胞和成骨细胞之间的偶联或通讯 在骨重建过程中，是骨重建周期中的关键步骤。虽然令人信服的证据表明骨头 破骨细胞随着年龄的增长而丢失和脆弱，以及骨质疏松是由损坏的偶联造成的，破骨细胞的确切机制- 成骨细胞的通讯仍不清楚。因此，尽管激励性耦合在理论上是有吸引力的 治疗目标，目前治疗骨质疏松症的方法，骨质疏松症是一种常见的疾病，影响着 美国仅限于抑制破骨细胞性骨吸收或刺激成骨细胞性骨吸收 队形。我们的长期目标是确定破骨细胞-成骨细胞通讯的关键机制 生理性骨重建中骨形成与骨吸收的偶联，使用罕见的骨质疏松性疾病 作为一种工具。罕见的骨骼疾病为了解典型的骨骼生理学和所获得的知识提供了重要的见解 这些疾病带来了治疗骨质疏松症的新疗法。 在这项建议中，我们利用了来自常染色体显性遗传性II型骨化症(ADOII)患者的样本， 一种罕见的遗传性骨化病，以高骨量和骨骼脆性为特征，与人类 用小鼠模型进行研究。ADOII是由CLCN7基因的杂合突变引起的，该基因编码 破骨细胞性骨吸收所必需的ClC-7Cl-/H+交换器。虽然缺乏破骨细胞的吸收 显然有助于骨表型，骨形成也不适当的高。在初步数据中 研究了ADOII骨的骨结构单元(BSU)组成，发现骨形成主要是 以重塑为基础，过度的骨形成称为溢流重塑。破骨细胞丰富， 扇形水泥线暗示着一种间歇性的坑状再吸收模式，与溢流结合在一起 重塑导致了一种典型的拼图状骨骼结构。我们假设合成代谢活跃但 ADOII中吸收不良的破骨细胞过度表达合成代谢偶联因子，不适当地刺激骨 形成过度填充吸收的空洞，并导致散乱的拼图样骨和脆性。 我们将结合对ADOII患者和小鼠模型的体内和体外研究来验证这一假设， 多模式和多尺度成像、生物力学和空间/单核转录学。具体来说，我们会： 1)测试人和小鼠ADOII的高骨量和脆性是否由破骨细胞介导； 探讨ADOII单核异常高骨形成机制 物理相邻的破骨细胞和成骨细胞的转录分析。 拟议的研究利用了现存的髂骨骨活检标本的独特资源。 来自一个携带CLCN7(G215R)突变的丹麦家庭的15名ADOII患者，年龄和性别匹配 对照组和两种ADOII小鼠模型，包括类似的Clcn7G213R/+突变。透过…的镜头 ADOII，这些研究将为破骨细胞-成骨细胞通讯提供独特的见解。

SUMMARY Coupling, or communication between the bone-resorbing osteoclasts and bone-forming osteoblasts during bone remodeling, is a key step in the bone remodeling cycle. While compelling evidence shows that bone loss and fragility with age and osteoporosis result from corrupted coupling, the precise mechanism of osteoclast- osteoblast communication remains unclear. Thus, although stimulating coupling is a theoretically attractive therapeutic target, current treatments for osteoporosis, a common disorder affecting 54 million of the elderly in the US alone, are limited to either inhibiting osteoclastic bone resorption or stimulating osteoblastic bone formation. Our long-term goal is to determine the mechanism of osteoclast-osteoblast communication critical for coupling of bone formation to resorption during physiological bone remodeling, using rare osteopetrotic diseases as a tool. Rare bone diseases provide important insights into typical bone physiology and knowledge gained from these diseases has already led to new therapies for osteoporosis. In this proposal, we utilize samples from patients with autosomal dominant osteopetrosis type II (ADOII), a rare inheritable osteopetrosis characterized by high bone mass and skeletal fragility and complement human studies with mouse models. ADOII results from heterozygous mutations in the CLCN7 gene, which encodes the ClC-7 Cl-/H+ exchanger essential for osteoclastic bone-resorption. While the lack of osteoclastic resorption clearly contributes to bone phenotype, bone formation is also inappropriately high. In preliminary data investigating the bone structural unit (BSU) composition of ADOII bones, we found bone formation is primarily remodeling based, with excess bone formation called overflow remodeling. Osteoclasts are abundant and scalloped cement lines suggest an intermittent pit-like resorption mode, which in combination with overflow remodeling results in a characteristic puzzle-like bone structure. We hypothesize that the anabolically active but poorly resorptive osteoclasts in ADOII overexpress anabolic coupling factors, inappropriately stimulating bone formation to overfill the resorbed cavities and leading to disorganized puzzle-like bone and fragility. We will test this hypothesis by combining in vivo and in vitro studies of ADOII patients and mouse models, multimodal and multiscale imaging, biomechanics and spatial/single-nuclei transcriptomics. Specifically, we will: 1) test if the high bone mass and fragility in human and mouse ADOII is osteoclast-mediated; and 2) investigate the mechanism of inappropriately high bone formation in ADOII by single nuclei transcriptomic analysis of physically adjacent osteoclasts and osteoblasts. The proposed studies take advantage of the unique resource of extant iliac crest bone biopsy specimens from 15 ADOII patients from a Danish family carrying the CLCN7 (G215R) mutation and age and sex matched controls, and two mouse models of ADOII, including the analogous Clcn7G213R/+ mutation. Through the lens of ADOII, these studies will provide unique insights into osteoclast-osteoblast communication.