Cell type-specific roles of lysosomal chloride/proton exchange

ClC-7是一种Cl-/H+交换剂，与其β亚基Ostm1一起定位于溶酶体和破骨细胞褶边。在小鼠和患者中，ClC-7/Ostm1功能障碍可导致骨质疏松症和神经退行性溶酶体贮积病。骨质疏松症的部分原因可能是破骨细胞皱褶边缘发育不全，这通常是由溶酶体胞吐作用形成的。事实上，缺乏clc -7的成纤维细胞和肥大细胞分别表现出明显减少的溶酶体胞吐或脱粒。溶酶体功能障碍，包括缓慢的蛋白质降解和自噬物质的积累，可能是由于溶酶体离子组成的改变。使用我们的各种小鼠模型中具有不同Clcn7基因突变的细胞，我们发现溶酶体被酸化到正常的低ph值。然而，我们发现溶酶体Cl-浓度急剧下降，并且在ClC-7缺乏或其转化为纯Cl-通道时，溶酶体离子稳态进一步改变。我们最近建立了一种新的小鼠模型，该模型携带一种导致人类骨质疏松的突变，可加速ClC-7的门控。令人惊讶的是，这些骨质疏松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功能获得突变的小鼠模型显示出严重的溶酶体疾病，但缺乏骨病理。这些由ClC-7改变引起的差异效应表明ClC-7具有细胞类型特异性作用。该项目研究了这些细胞类型特异性功能以及ClC-7在溶酶体离子稳态中的作用。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。我们将从我们各自的小鼠模型中研究和比较各种ClC-7突变对不同细胞类型（包括神经元和破骨细胞）中溶酶体离子浓度、形态和功能的影响。另一个方面是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，我们将在细胞和组织水平上研究ClC-7在特定免疫细胞溶酶体相关细胞器的胞吐、吞噬和微胞饮中的作用。最后，我们计划深入了解ClC-7活性的机制以及ClC-7功能障碍对溶酶体离子浓度的影响。我们的目标是开发一种光学传感器来监测ClC-7在溶酶体上的活性。此外，我们将进一步测量离子种类，并将研究其变化的原因与不同的致病改变的ClC-7。

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.