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

基本信息

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
项目类别：
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

项目摘要

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硬化症 2 型 (ADO2) 是一种由错义引起的骨硬化性疾病氯离子通道 7 基因 (CLCN7) 突变，导致破骨细胞骨吸收缺陷显性负性机制。疾病严重程度差异很大，即使在同一家庭内，也有三分之一。然而，三分之二的人中，有突变的人是无症状携带者。疾病（ADO2 患者），所有人都具有高骨密度，并且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/或骨髓研究该疾病的病理生理学并进行“原理证明”实验以失败。治疗这种疾病我们创造了一种新型 ADO2“敲入”小鼠，该小鼠患有导致 G213R 突变的疾病我们发现遗传品系和 ADO2 小鼠的表型严重程度不同。背景（在本提案中使用）是中度疾病的优秀模型[骨量/总体积 (BV/TV) 12 周龄时是 WT 的 2 倍，主要是由于破骨细胞功能障碍]。为了了解这一机制，我们检查了 WT 和 ADO2 破骨细胞的细胞内运输，发现了早期内体运输，这对于将内化的细胞表面蛋白分类到晚期是必需的内体和溶酶体，或将它们回收回质膜此外，我们发现药物，调节早期内体运输的氯喹，以及毛喉素和罗氟司特，通过不同机制增加细胞内cAMP，增强ADO2的骨降解活性我们的假设是：1）ADO2 破骨细胞的骨吸收活性可以发挥功能。通过调节内体运输或增加细胞内 cAMP 水平来恢复 2) 氯喹；治疗将挽救幼年（6 周）和成年（9 个月）小鼠中 ADO2 小鼠的表型 3)； ADO2 小鼠停止氯喹治疗将导致 BV/TV 增加，如通过测量体内 µCT，但速度不加快，且程度不及年龄匹配的载体治疗突变小鼠；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个月龄）进行。成功完成拟议目标可能会提供低剂量氯喹和 BMT 将显着改善或完全挽救模仿人类的小鼠模型中的 ADO2 表型将使用年轻和成年小鼠来使我们的研究与发生在疾病中的问题更加紧密地结合起来。我们的研究也将为了解 ADO2 患者的潜在机制提供重要的见解。破骨细胞中的 Clcn7 功能障碍如何导致患者出现 ADO2。