Mechanistic and Therapeutic Studies of Autosomal Dominant Osteopetrosis

常染色体显性骨石症的机制和治疗研究

• 批准号: 9236909
• 负责人: Michael J Econs
• 金额: $ 34.51万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9236909
• 关键词: Adenylate Cyclase; Adult; Affect; Age; Age-Months; Albers-Schonberg disease; Back; Blindness; Bone Density; Bone Diseases; Bone Marrow Transplantation; Bone Resorption; Bone necrosis; Cell Surface Proteins; Cell membrane; Cells; Chloride Channels; Chloroquine; Clinical; Cyclic AMP; Cyclophosphamide; Defect; Development; Disease; Disease model; Dominant-Negative Mutation; Dose; Family; Forskolin; Fracture; Functional disorder; Gene Mutation; Genes; Human; Impairment; In Vitro; Individual; Injectable; Jaw; Knock-in Mouse; Lead; Lysosomes; Maxilla; Measures; Missense Mutation; Modeling; Monitor; Mus; Mutant Strains Mice; Mutation; Osteoclasts; Osteomyelitis; Pancytopenia; Pathway interactions; Patients; Pharmacology; Phenotype; Recycling; Serum; Severities; Severity of illness; Sorting - Cell Movement; Testing; Therapeutic; Therapeutic Effect; Therapeutic Studies; bone; bone resorbing activity; disease phenotype; experimental study; genetic strain; human disease; improved; in vivo; inhibitor/antagonist; insight; late endosome; mouse model; mutant; novel; phosphodiesterase IV; skeletal; success; trafficking; young adult

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.

抽象的 常染色体显性骨质疏松术2型（ADO2）是骨硬膜硬膜硬化疾病，导致错义导致 氯化物通道7基因的突变（CLCN7），该突变通过A造成破骨骨的缺陷。 主要的负面机制。疾病的严重程度甚至在同一家庭内，三分之一 突变的个体是不对称的载体。但是，在受到影响的三分之二 疾病（ADO2患者），均具有高骨矿物质密度，至少一种临床表现 骨折，骨坏死（部分是颌骨和/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执行“原则证明”实验 治疗该疾病，我们创建了一种新型的ADO2“敲入”小鼠，疾病引起了G213R突变 CLCN7基因。我们发现遗传菌株和129的ADO2小鼠之间的表型严重程度差异 背景（该提案中使用）是中度疾病的出色模型[总体积的骨头体积 （BV/TV）在12周龄时WT超过2倍，这是由于破骨细胞功能障碍的一级]。开始理解 我们检查了WT和ADO2破骨细胞中细胞内运输的机制，这些贩运揭示了缺陷 早期内体贩运，这对于将内部细胞表面蛋白分类为晚期是必不可少的 内体和溶酶体，或将其回收回质膜。此外，我们发现 药理学剂，氯喹，调节早期内体贩运，福斯科蛋白和roflumilast， 通过不同的机制增加细胞内cAMP，增强了ADO2的骨降解活性 破骨细胞。我们的假设是：1）ADO2破骨细胞的骨呈现活性可以在功能上 通过调节内体贩运或增加细胞内营地水平来恢复； 2）氯喹 治疗将挽救年轻（6周）和成年（9个月）小鼠的Ado2小鼠的表型； 3） 在ADO2小鼠中停用氯喹疗法将导致BV/TV的增加，如IN 体内µCT，但不以加速速率，不在类似于年龄匹配的车辆的程度 突变小鼠； 4）当骨髓移植（BMT）将逆转ADO2小鼠的骨骼缺陷 在疾病病程中进行了早期（6周龄）或晚期（9个月大）。 成功完成拟议的目标可能会提供原则证明，低剂量的氯喹和 BMT将在模仿人类的小鼠模型中基本上改善或充分挽救ADO2表型 疾病。年轻小鼠和成年小鼠将被用来更紧密地使我们的研究与发生的问题相结合 ADO2患者的治疗。我们的研究还将提供有关对的基本机制的重要见解 破骨细胞中的CLCN7功能障碍如何导致患者的ADO2发展。