Mechanistic and Therapeutic Studies of Autosomal Dominant Osteopetrosis

摘要 常染色体显性骨硬化症2型（ADO2）是一种由错义 氯离子通道7基因（CLCN 7）的突变，通过一种新的途径导致骨吸收缺陷。 显性负性机制疾病的严重程度差异很大，即使在同一个家庭中， 具有突变的个体是无症状携带者。然而，在受影响的三分之二的人中， ADO2患者的骨密度均较高，至少有一种临床表现，包括 骨折、骨坏死（特别是颌骨和/或上颌骨）、骨髓炎、失明和/或骨髓 失败研究疾病的病理生理学，并进行"原理证明"实验， 为了治疗这种疾病，我们创造了一种新的ADO2 "基因敲入"小鼠，这种小鼠的基因组中存在导致疾病的G213R突变。 Clcn7基因。我们发现，表型严重程度不同的遗传品系和ADO2小鼠对129 背景（用于本建议）是一个极好的中度疾病模型[骨体积/总体积 (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，我们检查了WT和ADO2破骨细胞的细胞内运输，发现 早期内体运输，这对于将内化的细胞表面蛋白分选到晚期内体运输是必需的。 内体和溶酶体，或将它们再循环回质膜。此外，我们发现， 药理学试剂，氯喹，其调节早期内体运输，以及毛喉素和罗氟司特， 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在功能上被调节， 通过调节内体运输或增加细胞内cAMP水平恢复; 2）氯喹 治疗将挽救年轻（6周）和成年（9个月）小鼠中ADO2小鼠的表型; 3） 在ADO2小鼠中停止氯喹治疗将导致BV/TV增加，如通过以下方法测量的： 体内µ CT，但不是以加速速率，并且与年龄匹配的溶剂处理的程度不相似 4）骨髓移植（BMT）将逆转ADO2小鼠的骨骼缺陷， 在病程早期（6周龄）或晚期（9月龄）进行。 成功地完成拟议的目标可能会提供低剂量氯喹和 BMT将显著改善或完全挽救模拟人ADO2表型的小鼠模型中的ADO2表型。 疾病年轻和成年小鼠将被用来更密切地调整我们的研究与问题，发生在 ADO2患者的治疗。我们的研究还将为深入了解 破骨细胞中Clcn7功能障碍如何导致患者发生ADO2。

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.