Mechanistic and Therapeutic Studies of Autosomal Dominant Osteopetrosis

摘要 常染色体显性遗传性骨化症2型(ADO2)是一种由错义引起的骨硬化性疾病 氯通道7基因(CLCN7)突变导致破骨细胞性骨吸收缺陷 显性负向机制。疾病的严重程度差异很大，即使在同一个家庭中也是如此，三分之一的 携带突变基因的个体是无症状携带者。然而，在受影响的三分之二人中， 疾病(ADO2患者)，均有高骨密度，至少有一种临床表现，包括 骨折、骨坏死(尤其是颌骨和/或上颌骨)、骨髓炎、失明和/或骨髓 失败了。研究该病的病理生理学并进行“原则性证明”实验 治疗这种疾病我们创造了一种新型的ADO2“敲入”小鼠，这种疾病导致G213R突变 Clcn7基因。我们发现不同的遗传菌株和ADO2小鼠在129上的表型严重程度是不同的 背景(在本提案中使用)是一种很好的中度疾病模型[每总体积的骨量 (BV/TV)在12周龄时是WT的2倍，主要是由于破骨细胞功能障碍]。开始理解 机制，我们研究了WT和ADO2破骨细胞的细胞内运输，揭示了 早期内体转运，这是将内化的细胞表面蛋白分选到晚期所必需的 内小体和溶酶体，或将它们循环回到质膜。此外，我们发现， 药理药剂氯喹，它调节早期的内体转运，以及福司可林和罗氟司特， 通过不同的机制增加细胞内cAMP，增强ADO2的骨降解活性 破骨细胞。我们的假设是：1)ADO2破骨细胞的骨吸收活性可以是功能性的 通过调节内体转运或增加细胞内cAMP水平来恢复；2)氯喹 治疗将挽救幼年(6周)和成年(9个月)小鼠的ADO2表型； 在ADO2小鼠中，停止氯喹治疗将导致BV/TV的增加，如In Vivo？CT，但不是以加速的速度，也不是到与年龄匹配的治疗车辆相似的程度 4)骨髓移植(BMT)将逆转ADO2小鼠的骨骼缺陷，当 在病程中进行早期(6周龄)或晚期(9个月龄)。 成功完成拟议的目标可能提供原则证明，低剂量氯喹和 骨髓移植将显著改善或完全挽救模仿人类的小鼠模型中的ADO2表型 疾病。幼鼠和成年鼠将被用来更紧密地将我们的研究与发生在 ADO2患者的治疗。我们的研究也将为我们提供对潜在机制的重要见解 破骨细胞Clcn7功能障碍如何导致患者ADO2的发生。

Abstract Autosomal dominant osteopetrosis type 2 (ADO2) is an osteosclerotic disorder resulting from missense mutations in the Chloride Channel 7 gene (CLCN7), which cause defects in osteoclastic bone resorption by a dominant negative mechanism. Disease severity varies widely, even within the same family, and one third of individuals with mutations are asymptomatic carriers. However, of the two thirds who are affected by the disease (ADO2 patients), all have high bone mineral density with at least one clinical manifestation including fracture, osteonecrosis (particularly of the jaw and/or maxilla), osteomyelitis, blindness, and/or bone marrow failure. To investigate the pathophysiology of the disease and to perform “proof of principle” experiments to treat the disease we created a novel ADO2 “knock-in” mouse with a disease causing G213R mutation in the Clcn7 gene. We found that phenotypic severity differs among genetic strains and the ADO2 mouse on the129 background (used in this proposal) is an excellent model of moderate disease [bone volume per total volume (BV/TV) 2-fold over WT at 12 weeks of age, due primarily to osteoclast dysfunction]. To begin to understand the mechanism, we examined intracellular trafficking in WT and ADO2 osteoclasts which revealed defects in early endosomal trafficking, which is necessary for sorting internalized cell surface proteins into late endosomes and lysosomes, or recycling them back to the plasma membrane. In addition, we found that the pharmacologic agents, chloroquine, which modulates early endosomal trafficking, and forskolin and roflumilast, which increase intracellular cAMP by different mechanisms, enhance the bone-degrading activity of ADO2 osteoclasts. Our hypotheses are: 1) The bone-resorbing activity of ADO2 osteoclasts can be functionally restored by modulating either endosomal trafficking or increasing intracellular cAMP levels; 2) Chloroquine treatment will rescue the phenotype of the ADO2 mouse in young (6 weeks) and adult (9 months) mice; 3) Discontinuation of chloroquine therapy in ADO2 mice will result in an increase in BV/TV, as measured by in vivo µCT, but not at an accelerated rate and not to a degree similar to that of age-matched vehicle treated mutant mice; and 4) Bone marrow transplantation (BMT) will reverse the skeletal defects in ADO2 mice when performed either early (6 weeks of age) or late (9 months of age) in the disease course. Successful completion of the proposed aims may provide proof of principle that low dose chloroquine and BMT will substantially improve or fully rescue the ADO2 phenotype in a mouse model that mimics the human disease. Young and adult mice will be used to more closely align our studies with questions that occur in the treatment of ADO2 patients. Our studies will also provide important insight into the underlying mechanism of how Clcn7 dysfunction in osteoclasts leads to the development of ADO2 in patients.