Cell type-specific roles of lysosomal chloride/proton exchange

溶酶体氯化物/质子交换的细胞类型特异性作用

• 批准号: 388470839
• 负责人: Professor Dr. Thomas J. Jentsch
• 金额: --
• 依托单位: Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/388470839?language=en
• 关键词: Cell; type; specific; roles; lysosomal

ClC-7 is a Cl-/H+ exchanger that, together with its beta-subunit Ostm1, localizes to lysosomes and to the osteoclast ruffled border. Dysfunction of ClC-7/Ostm1 leads to osteopetrosis and a neurodegenerative lysosomal storage disease in mice and patients. The osteopetrosis may in part be caused by the underdevelopment of the ruffled border of osteoclasts, which is normally formed by lysosomal exocytosis. Indeed, ClC-7-deficient fibroblasts and mast cells showed significantly reduced lysosomal exocytosis or degranulation, respectively. The lysosomal dysfunction, which involves a slowed protein degradation and the accumulation of autophagic material, is likely due to alterations of the lysosomal ion composition. Using cells derived from our various mouse models with different mutations in the Clcn7 gene, we found that lysosomes were acidified to a normally low pH. However, we found a drastic decrease in the lysosomal Cl- concentration and further alterations of the lysosomal ion homeostasis upon ClC-7 deficiency or its conversion into a pure Cl- channel. We recently generated a novel mouse model carrying a human osteopetrosis-causing mutation that accelerates the gating of ClC-7. Surprisingly, these osteopetrotic mice did not show any signs of lysosomal dysfunction or neurodegeneration. In contrast, recently reported patients and another mouse model with a gain-of-function mutation in ClC-7 displayed a severe lysosomal disease but lacked bone pathology. These differential effects by ClC-7 alteration suggest cell type-specific roles for ClC-7. This project addresses these cell type-specific functions and the involvement of ClC-7 in lysosomal ion homeostasis in general. One focus lies on the differential roles of ClC-7 in bone resorption and basic lysosomal function. We will study and compare the effects of various ClC-7 mutations on the ion concentrations, morphology and function of lysosomes in different cell types, including neurons and osteoclasts, from our respective mouse models. Another aspect is the function of ClC-7 in the immune system. Using cell type-specific ClC-7 KO mice, we will investigate the roles of ClC-7 in the exocytosis of lysosome-related organelles, phagocytosis and micropinocytosis by specialized immune cells on a cellular and organismal level. Lastly, we plan to gain mechanistic insight into ClC-7 activity and the alterations of lysosomal ion concentrations by ClC-7 dysfunction. We aim at developing an optical sensor to monitor ClC-7 activity on lysosomes. Moreover, we will measure further ion species and we will investigate the reason for alterations thereof with the diverse disease-causing alterations of ClC-7.

ClC-7是一种Cl-/H+交换剂，与其β亚基Ostm 1一起定位于溶酶体和破骨细胞皱褶边缘。ClC-7/Ostm 1的功能障碍导致小鼠和患者的骨硬化症和神经退行性溶酶体贮积病。骨硬化症可能部分由破骨细胞的皱褶边缘发育不全引起，其通常由溶酶体胞吐作用形成。事实上，ClC-7缺陷的成纤维细胞和肥大细胞分别显示出显著减少的溶酶体胞吐作用或脱粒作用。溶酶体功能障碍涉及蛋白质降解减慢和自噬物质积累，可能是由于溶酶体离子组成的改变。使用细胞来自我们的各种小鼠模型中的Clcn 7基因的不同突变，我们发现，溶酶体酸化到一个正常的低pH值。然而，我们发现了一个急剧下降的溶酶体Cl-浓度和进一步改变的溶酶体离子稳态后ClC-7缺乏或转换成一个纯Cl-通道。 我们最近产生了一种新的小鼠模型，该模型携带了一种导致人类骨硬化症的突变，该突变加速了ClC-7的门控。令人惊讶的是，这些骨硬化小鼠没有表现出任何溶酶体功能障碍或神经变性的迹象。相比之下，最近报道的患者和另一种具有ClC-7功能获得性突变的小鼠模型显示出严重的溶酶体疾病，但缺乏骨病理学。ClC-7改变的这些差异效应表明ClC-7的细胞类型特异性作用。该项目解决了这些细胞类型特异性功能和一般的溶酶体离子稳态中ClC-7的参与。一个焦点在于ClC-7在骨吸收和基本溶酶体功能中的不同作用。我们将研究和比较各种ClC-7突变对不同细胞类型（包括神经元和破骨细胞）中溶酶体的离子浓度、形态和功能的影响。另一方面是ClC-7在免疫系统中的功能。使用细胞类型特异性的ClC-7 KO小鼠，我们将研究ClC-7在溶酶体相关细胞器的胞吐作用，细胞和生物体水平上的专门免疫细胞的吞噬作用和微胞饮作用中的作用。最后，我们计划获得CLC-7活性和CLC-7功能障碍引起的溶酶体离子浓度改变的机理性见解。我们的目标是开发一种光学传感器来监测溶酶体上的ClC-7活性。此外，我们将测量进一步的离子种类，并且我们将研究其改变的原因以及ClC-7的多种致病改变。